Compositions and methods for inducing maturation of oocytes

Abstract

Methods, compositions and devices characterized by in vitro maturation of oocytes. In particular, the present disclosure features methods of inducing oocyte maturation in vitro by co-culturing oocytes of a female subject with an ex vivo composition comprising a plurality of ovarian supporting cells (e.g., granular cells). Additional methods of administering a follicle triggering agent and extracting oocytes from a female subject are also provided. Such methods, compositions, and devices are particularly useful for assisted reproductive technology (ART) procedures.

Description

Technical Field

[0001] The present disclosure relates to the field of in vitro maturation of oocytes. Background Art

[0002] One in ten women suffer from infertility and require assisted reproductive technology (ART) such as in vitro fertilization (IVF). Maintaining the health of oocytes during culture remains a challenge, resulting in poor oocyte quality, which in turn leads to poor embryo quality. In addition, immature oocytes are often discarded, limiting the pool of oocytes available for IVF. In vitro maturation (IVM) is expected to mature oocytes in vitro after egg extraction, allowing all retrieved eggs to be utilized. The current IVM method is inefficient, using follicle-stimulating hormone (FSH) spiked into the culture medium, showing a maturity of 5-40% for immature eggs. Worse still, this method produces many unhealthy eggs, with an embryo survival rate of less than 17%, far lower than standard IVF. Therefore, there is still a need to promote oocyte maturation for female subjects undergoing ART procedures. Summary of the invention

[0003] In one aspect, the disclosure features a method of inducing oocyte maturation in vitro, the method comprising co-culturing one or more oocytes previously removed from a human subject with a population of ovarian supporting cells.

[0004] In another aspect, the disclosure features a method of preparing one or more oocytes previously removed from a human subject for use in an assisted reproductive technology (ART) procedure, the method comprising co-culturing the one or more oocytes with a population of ovarian supporting cells.

[0005] In another aspect, the disclosure features a method of producing mature oocytes for use in an ART procedure, the method comprising co-culturing one or more oocytes previously removed from a human subject with a population of ovarian support cells.

[0006] In some embodiments, one or more follicle triggering agents are administered to a subject during the follicle triggering phase prior to retrieval of one or more oocytes from the subject.

[0007] In some embodiments, a follicle triggering agent is not administered to the subject during the follicle triggering phase prior to retrieval of one or more oocytes from the subject.

[0008] In some embodiments, the duration of the follicle triggering phase is no more than 8 days. In some embodiments, the duration of the follicle triggering phase is no more than 7 days. In some embodiments, the duration of the follicle triggering phase is no more than 6 days. In some embodiments, the duration of the follicle triggering phase is no more than 5 days. In some embodiments, the duration of the follicle triggering phase is no more than 4 days. In some embodiments, the duration of the follicle triggering phase is no more than 3 days. In some embodiments, the duration of the follicle triggering phase is no more than 2 days. In some embodiments, the duration of the follicle triggering phase is no more than 1 day. In some embodiments, the duration of the follicle triggering phase is 1 day to 8 days. In some embodiments, the duration of the follicle triggering phase is 1 day to 7 days. In some embodiments, the duration of the follicle triggering phase is 1 day to 6 days. In some embodiments, the duration of the follicle triggering phase is 1 day to 5 days. In some embodiments, the duration of the follicle triggering phase is 1 day to 4 days. In some embodiments, the duration of the follicle triggering phase is 1 day to 3 days. In some embodiments, the duration of the follicle triggering phase is 2 days to 8 days. In some embodiments, the duration of the follicle triggering phase is 2 days to 7 days. In some embodiments, the duration of the follicle triggering phase is 2 days to 6 days. In some embodiments, the duration of the follicle triggering phase is 2 days to 5 days. In some embodiments, the duration of the follicle triggering phase is 2 days to 4 days. In some embodiments, the duration of the follicle triggering phase is 3 days to 8 days. In some embodiments, the duration of the follicle triggering phase is 3 days to 7 days. In some embodiments, the duration of the follicle triggering phase is 3 days to 6 days. In some embodiments, the duration of the follicle triggering phase is 3 days to 5 days.

[0009] In some embodiments, the one or more follicle triggering agents include follicle stimulating hormone (FSH), clomiphene citrate and / or human chorionic gonadotropin (hCG). In some embodiments, the one or more follicle triggering agents include FSH.

[0010] In some embodiments, FSH is administered to the subject in one or more doses per day. In some embodiments, FSH is administered to the subject once per day.

[0011] In some embodiments, FSH is administered to a subject in an amount of about 100 international units (IU) to about 1,000 IU per day. In some embodiments, FSH is administered to a subject in an amount of about 200 IU to about 800 IU per day. In some embodiments, FSH is administered to a subject in an amount of about 300 IU to about 700 IU per day. In some embodiments, FSH is administered to a subject in an amount of about 300 IU to about 600 IU per day, about 300 IU to about 500 IU per day, or about 300 IU to about 400 IU per day.

[0012] In some embodiments, the duration of FSH administration is equal to the duration of the follicular triggering phase. In some embodiments, the duration of FSH administration is less than the duration of the follicular triggering phase. In some embodiments, during the follicular triggering phase, the duration of FSH administration is 1, 2, 3, 4 or 5 days, optionally wherein during the follicular triggering phase, the subject is administered FSH in an amount of about 200 IU per day for 1, 2, 3, 4 or 5 days, optionally wherein during the follicular triggering phase, the subject is administered FSH in an amount of about 200 IU per day for 3 days.

[0013] In some embodiments, the one or more follicle triggering agents include clomiphene citrate. In some embodiments, clomiphene citrate is administered to the subject in one or more doses per day. In some embodiments, clomiphene citrate is administered to the subject once per day.

[0014] In some embodiments, clomiphene citrate is administered to a subject in an amount of about 50 mg to about 100 mg per day. In some embodiments, clomiphene citrate is administered to a subject in an amount of about 50 mg per day.

[0015] In some embodiments, the duration of clomiphene citrate administration is equal to the duration of the follicular triggering phase. In some embodiments, the duration of clomiphene citrate administration is less than the duration of the follicular triggering phase. In some embodiments, the duration of clomiphene citrate administration during the follicular triggering phase is 1, 2, 3, 4, or 5 days.

[0016] In some embodiments, the one or more follicle triggering agents include hCG. In some embodiments, one or more doses of hCG are administered to the subject daily. In some embodiments, 1, 2, or 3 doses of hCG are administered to the subject during follicle triggering.

[0017] In some embodiments, hCG is administered to a subject in an amount of about 200 μg to about 700 μg per dose. In some embodiments, hCG is administered to a subject in an amount of about 200 μg to about 500 μg per dose, about 300 μg to about 600 μg per dose, about 400 μg to about 700 μg per dose, about 200 μg to about 300 μg per dose, about 300 μg to about 400 μg per dose, about 400 μg to about 500 μg per dose, about 500 μg to about 600 μg per dose, or about 600 μg to about 700 μg per dose. In some embodiments, hCG is administered to a subject in an amount of about 500 μg per dose. In some embodiments, hCG is administered to a subject in an amount of about 2,500 IU to about 10,000 IU per dose.

[0018] In some embodiments, the subject is a subject who has completed oral contraceptive treatment within 28 days of the start of the follicular triggering phase. In some embodiments, the follicular triggering phase begins at least 5 days after stopping the contraceptive treatment.

[0019] In some embodiments, the subject has not undergone oral contraceptive treatment within 28 days after the onset of the follicular triggering phase.

[0020] In some embodiments, the follicular triggering phase begins on day 2 of the subject's menstrual cycle.

[0021] In some embodiments, the contraceptive treatment comprises administering a gonadotropin-releasing hormone (GnRH) agonist to the subject.

[0022] In some embodiments, it has been determined that the subject exhibits a follicle size of about 6 mm to about 8 mm before the onset of the follicle triggering phase. In some embodiments, it has been determined that the subject exhibits a follicle size of about 6 mm to about 8 mm before the final follicle triggering agent is administered. In one embodiment, follicle size is determined using a scoring index (e.g., following ultrasound imaging or other follicle size determination methods known in the art).

[0023] In some embodiments, a biological sample isolated from a subject prior to retrieval of one or more oocytes has been determined to have an anti-Mullerian hormone (AMH) concentration of about 0.1 ng / ml to about 1 ng / ml or about 1 ng / ml to about 6 ng / ml.

[0024] In some embodiments, it is determined that the sample has an AMH concentration of about 1ng / ml to about 6ng / ml, optionally wherein it is determined that the sample has an AMH concentration of about 2.5ng / ml to about 3.0ng / ml. In some embodiments, it is determined that the biological sample has an AMH concentration of about 2ng / ml to about 5ng / ml. In some embodiments, it is determined that the biological sample has an AMH concentration of about 2.5ng / ml to about 3.0ng / ml. In some embodiments, it is determined that the biological sample separated from the subject before taking out one or more oocytes has an AMH concentration of at least 1ng / ml. In some embodiments, it is determined that the biological sample separated from the subject before taking out one or more oocytes has an AMH concentration of not more than 6ng / ml. In some embodiments, it is determined that the biological sample separated from the subject before taking out one or more oocytes has an AMH concentration of about 0.1ng / ml to about 1ng / ml. In some embodiments, the sample is a blood sample.

[0025] In some embodiments, the subject is between 18 and 48 years of age at the time of retrieval of the one or more oocytes. In some embodiments, the subject is between 25 and 45 years of age at the time of retrieval of the one or more oocytes. In some embodiments, the subject is less than 35 years of age at the time of retrieval of the one or more oocytes. In some embodiments, the subject is greater than 35 years of age at the time of retrieval of the one or more oocytes.

[0026] In some embodiments, prior to removing one or more oocytes from the subject, it was determined that the subject exhibited a follicle size of about 6 mm to about 14 mm. In some embodiments, prior to removing one or more oocytes from the subject, it was determined that the subject exhibited a follicle size of about 8 mm to about 12 mm. In some embodiments, prior to removing one or more oocytes from the subject, it was determined that the subject exhibited a follicle size of about 8 mm to about 9 mm. In some embodiments, prior to removing the one or more oocytes from the subject, it was determined that the subject exhibited a follicle size of no greater than 14 mm.

[0027] In some embodiments, follicle size has been assessed by ultrasound image analysis.

[0028] In some embodiments, a total of 20 or fewer oocytes are removed from a subject. In some embodiments, 15 or fewer oocytes are removed from a subject. In some embodiments, 10 or fewer oocytes are removed from a subject. In some embodiments, 9 or fewer oocytes are removed from a subject. In some embodiments, 8 or fewer oocytes are removed from a subject. In some embodiments, 7 or fewer oocytes are removed from a subject. In some embodiments, 6 or fewer oocytes are removed from a subject. In some embodiments, 5 or fewer oocytes are removed from a subject. In some embodiments, multiple oocytes are removed from a subject.

[0029] In some embodiments, 10% to 100% of the oocytes removed from the subject are germinal vesicle (GV) stage or meiosis I (MI) stage oocytes. In some embodiments, 20% to 100% of the oocytes removed from the subject are GV stage or MI stage oocytes. In some embodiments, 30% to 100% of the oocytes removed from the subject are GV stage or MI stage oocytes. In some embodiments, 40% to 100% of the oocytes removed from the subject are GV stage or MI stage oocytes. In some embodiments, 50% to 100% of the oocytes removed from the subject are GV stage or MI stage oocytes. In some embodiments, 60% to 100% of the oocytes removed from the subject are GV stage or MI stage oocytes. In some embodiments, 70% to 100% of the oocytes removed from the subject are GV stage or MI stage oocytes. In some embodiments, 80% to 100% of the oocytes removed from the subject are oocytes at the GV stage or the MI stage. In some embodiments, 90% to 100% of the oocytes removed from the subject are oocytes at the GV stage or the MI stage. In some embodiments, 100% of the oocytes removed from the subject are oocytes at the GV stage or the MI stage.

[0030] In some embodiments, the ovarian supporting cell population comprises ovarian granulosa cells and / or ovarian stromal cells, optionally wherein the ovarian granulosa cells are forkhead box protein L2 (FOXL2) positive and / or wherein the ovarian stromal cells are nuclear receptor subfamily 2 group F member 2 (NR2F2) positive.

[0031] In some embodiments, the ovarian support cell group comprises about 50,000 to about 100,000 ovarian support cells. In some embodiments, the ovarian support cell group comprises about 50,000 to about 60,000 ovarian support cells, about 60,000 to about 70,000 ovarian support cells, about 70,000 to about 80,000 ovarian support cells, about 80,000 to about 90,000 ovarian support cells, about 90,000 to about 100,000 ovarian support cells or about 100,000 to about 150,000, optionally wherein the ovarian support cell group comprises about 125,000 ovarian support cells. In some embodiments, the population of ovarian support cells comprises about 50,000 ovarian support cells, about 55,000 ovarian support cells, about 60,000 ovarian support cells, about 65,000 ovarian support cells, about 70,000 ovarian support cells, about 75,000 ovarian support cells, about 80,000 ovarian support cells, about 85,000 ovarian support cells, about 90,000 ovarian support cells, about 95,000 ovarian support cells, about 100 ,000 ovarian supporting cells, about 105,000 ovarian supporting cells, about 110,000 ovarian supporting cells, about 115,000 ovarian supporting cells, about 120,000 ovarian supporting cells, about 125,000 ovarian supporting cells, about 130,000 ovarian supporting cells, about 135,000 ovarian supporting cells, about 140,000 ovarian supporting cells, about 145,000 ovarian supporting cells or about 150,000 ovarian supporting cells.

[0032] In some embodiments, the ovarian supporting cell population includes a mixture of cell types (e.g., granulosa cells, stromal cells, and other possible cell types). In some embodiments, the ovarian supporting cell population includes a cell mixture such that the mixture contains a 1:1 distribution of cell types. In some embodiments, the ovarian supporting cell population includes a mixture of multiple cell types such that the mixture contains an uneven distribution of cell types (e.g., 2:1, 3:1, 4:1, 5:1, and other possible population distributions).

[0033] In some embodiments, the ovarian supporting cells include steroidogenic granulosa cells. In some embodiments, the steroidogenic granulosa cells produce estradiol.

[0034] In some embodiments, the ovarian supporting cells are obtained by differentiation of an induced pluripotent stem cell (iPSC) population.

[0035] In some embodiments, the ovarian support cells are obtained by modifying iPSC to express one or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express two or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express three or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express four or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express all five of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2.

[0036] In some embodiments, ovarian support cells are cryopreserved and thawed before co-cultivation with one or more oocytes. In some embodiments, ovarian support cells are thawed about 24 hours to about 120 hours before co-cultivation with one or more oocytes. In some embodiments, ovarian support cells are thawed about 24 hours to about 48 hours, about 48 hours to about 72 hours, about 72 hours to about 96 hours or about 96 hours to about 120 hours before co-cultivation with one or more oocytes. In some embodiments, ovarian support cells are thawed about 24 hours to about 36 hours, about 30 hours to about 40 hours, about 36 hours to about 48 hours, about 48 hours to about 56 hours, about 56 hours to about 72 hours, about 72 hours to about 84 hours, about 80 hours to about 96 hours, about 90 hours to about 100 hours, about 96 hours to about 108 hours, or about 108 hours to about 120 hours before co-cultivation with one or more oocytes.

[0037] In some embodiments, one or more oocytes are co-cultured with a population of ovarian supporting cells for about 12 hours to about 120 hours. In some embodiments, one or more oocytes are co-cultured with a population of ovarian supporting cells for about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 24 hours to about 48 hours, about 36 hours to about 60 hours, about 54 hours to about 72 hours, about 68 hours to about 96 hours, or about 96 hours to about 120 hours. In some embodiments, one or more oocytes are co-cultured with a population of ovarian supporting cells for about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, about 54 hours, about 56 hours, about 58 hours, about 60 hours, about 62 hours , about 64 hours, about 66 hours, about 68 hours, about 70 hours, about 72 hours, about 74 hours, about 76 hours, about 78 hours, about 80 hours, about 82 hours, about 84 hours, about 86 hours, about 88 hours, about 90 hours, about 92 hours, about 94 hours, about 96 hours, about 98 hours, about 100 hours, about 102 hours, about 104 hours, about 106 hours, about 108 hours, about 110 hours, about 112 hours, about 114 hours, about 116 hours, about 118 hours or about 120 hours.

[0038] In some embodiments, the co-cultivation is performed in an adherent co-cultivation system. In some embodiments, the co-cultivation is performed in a suspension co-cultivation system.

[0039] In some embodiments, before and / or after co-cultivation, the parameters of one or more oocytes are assessed, the parameters being selected from the group consisting of: total oocyte score, oocyte maturation rate from GV stage to MII stage, oocyte maturation rate from GV stage to MI stage, oocyte maturation rate from MI stage to MII stage, average oocyte shape, average oocyte size, average egg quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality. In some embodiments, after co-cultivation, one or more oocytes are stripped.

[0040] In some embodiments, the method further comprises isolating one or more meiosis II (MII) stage oocytes from a mixture produced by co-culturing one or more oocytes removed from a subject with a population of ovarian support cells.

[0041] In some embodiments, the subject is undergoing an autologous ART procedure, and wherein the method further comprises contacting each of the one or more MII stage oocytes with a mature sperm cell.

[0042] In some embodiments, one or more MII stage oocytes are cryopreserved and thawed prior to contacting. In some embodiments, one or more MII stage oocytes are not cryopreserved and are not thawed prior to contacting.

[0043] In some embodiments, contacting comprises in vitro fertilization (IVF) of one or more MII stage oocytes. In some embodiments, contacting comprises intracytoplasmic sperm injection (ICSI) into one or more MII stage oocytes.

[0044] In some embodiments, contacting results in the formation of an embryo. In some embodiments, the embryo is transferred to the uterus of a subject. In some embodiments, about 3 days after one or more MII stage oocytes contact with mature sperm cells, the embryo is transferred to the uterus of a subject. In some embodiments, about 5 days after one or more MII stage oocytes contact with mature sperm cells, the embryo is transferred to the uterus of a subject. In some embodiments, the embryo transferred to the uterus of a subject is a blastocyst stage embryo.

[0045] In some embodiments, the methods result in the formation of a plurality of embryos having a survival rate greater than 20% (e.g., a survival rate of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more).

[0046] In another aspect, the present disclosure features a method for producing mature oocytes for use in an ART procedure, the method comprising: (a) administering one or more follicle triggering agents to a human subject during the follicle triggering phase; (b) removing one or more oocytes from the subject after the follicle triggering phase; and (c) culturing the one or more oocytes with a population of ovarian supporting cells to produce one or more mature oocytes.

[0047] In another aspect, the present disclosure features a method for promoting oocyte maturation in a subject who is undergoing an ART procedure and who has previously been administered one or more follicle triggering agents during a follicle triggering phase, the method comprising: (a) removing one or more oocytes from the subject; (b) culturing the one or more oocytes with a population of ovarian supporting cells to produce one or more mature oocytes; and (c) isolating the one or more mature oocytes.

[0048] In some embodiments, the duration of the follicle triggering phase is no more than 8 days. In some embodiments, the duration of the follicle triggering phase is no more than 7 days. In some embodiments, the duration of the follicle triggering phase is no more than 6 days. In some embodiments, the duration of the follicle triggering phase is no more than 5 days. In some embodiments, the duration of the follicle triggering phase is no more than 4 days. In some embodiments, the duration of the follicle triggering phase is no more than 3 days. In some embodiments, the duration of the follicle triggering phase is no more than 2 days. In some embodiments, the duration of the follicle triggering phase is no more than 1 day. In some embodiments, the duration of the follicle triggering phase is 1 day to 8 days. In some embodiments, the duration of the follicle triggering phase is 1 day to 7 days. In some embodiments, the duration of the follicle triggering phase is 1 day to 6 days. In some embodiments, the duration of the follicle triggering phase is 1 day to 5 days. In some embodiments, the duration of the follicle triggering phase is 1 day to 4 days. In some embodiments, the duration of the follicle triggering phase is 1 day to 3 days. In some embodiments, the duration of the follicle triggering phase is 2 days to 8 days. In some embodiments, the duration of the follicle triggering phase is 2 days to 7 days. In some embodiments, the duration of the follicle triggering phase is 2 days to 6 days. In some embodiments, the duration of the follicle triggering phase is 2 days to 5 days. In some embodiments, the duration of the follicle triggering phase is 2 days to 4 days. In some embodiments, the duration of the follicle triggering phase is 3 days to 8 days. In some embodiments, the duration of the follicle triggering phase is 3 days to 7 days. In some embodiments, the duration of the follicle triggering phase is 3 days to 6 days. In some embodiments, the duration of the follicle triggering phase is 3 days to 5 days.

[0049] In some embodiments, the one or more follicle triggering agents include FSH, clomiphene citrate, and / or hCG. In some embodiments, the one or more follicle triggering agents include FSH.

[0050] In some embodiments, FSH is administered to the subject in one or more doses per day. In some embodiments, FSH is administered to the subject once per day.

[0051] In some embodiments, FSH is administered to a subject in an amount of about 100 IU to about 1,000 IU per day. In some embodiments, FSH is administered to a subject in an amount of about 200 IU to about 800 IU per day. In some embodiments, FSH is administered to a subject in an amount of about 300 IU to about 700 IU per day. In some embodiments, FSH is administered to a subject in an amount of about 300 IU to about 600 IU per day, about 300 IU to about 500 IU per day, or about 300 IU to about 400 IU per day.

[0052] In some embodiments, the duration of FSH administration is equal to the duration of the follicular triggering phase. In some embodiments, the duration of FSH administration is less than the duration of the follicular triggering phase. In some embodiments, during the follicular triggering phase, the duration of FSH administration is 1, 2, 3, 4 or 5 days, optionally wherein during the follicular triggering phase, the subject is administered FSH in an amount of about 200 IU per day for 1, 2, 3, 4 or 5 days, optionally wherein during the follicular triggering phase, the subject is administered FSH in an amount of about 200 IU per day for 3 days.

[0053] In some embodiments, the one or more follicle triggering agents include clomiphene citrate.

[0054] In some embodiments, clomiphene citrate is administered to a subject in one or more doses per day. In some embodiments, clomiphene citrate is administered to a subject once per day.

[0055] In some embodiments, clomiphene citrate is administered to a subject in an amount of about 50 mg to about 100 mg per day. In some embodiments, clomiphene citrate is administered to a subject in an amount of about 50 mg per day.

[0056] In some embodiments, the duration of clomiphene citrate administration is equal to the duration of the follicular triggering phase. In some embodiments, the duration of clomiphene citrate administration is less than the duration of the follicular triggering phase. In some embodiments, the duration of clomiphene citrate administration during the follicular triggering phase is 1, 2, 3, 4, or 5 days.

[0057] In some embodiments, one or more follicle triggering agents include hCG. In some embodiments, hCG is administered to the subject once or more per day. In some embodiments, hCG is administered to the subject in 1, 2 or 3 doses during the follicle triggering phase.

[0058] In some embodiments, hCG is administered to a subject in an amount of about 200 μg to about 700 μg per dose. In some embodiments, hCG is administered to a subject in an amount of about 200 μg to about 500 μg per dose, about 300 μg to about 600 μg per dose, about 400 μg to about 700 μg per dose, about 200 μg to about 300 μg per dose, about 300 μg to about 400 μg per dose, about 400 μg to about 500 μg per dose, about 500 μg to about 600 μg per dose, or about 600 μg to about 700 μg per dose. In some embodiments, hCG is administered to a subject in an amount of about 500 μg per dose. In some embodiments, hCG is administered to a subject in an amount of about 2,500 IU to about 10,000 IU per dose.

[0059] In some embodiments, the subject is a subject who has completed oral contraceptive treatment within 28 days of the start of the follicular triggering phase. In some embodiments, the follicular triggering phase begins at least 5 days after stopping the contraceptive treatment.

[0060] In some embodiments, the subject has not undergone oral contraceptive treatment within 28 days after the onset of the follicular triggering phase.

[0061] In some embodiments, the follicular triggering phase begins on day 2 of the subject's menstrual cycle.

[0062] In some embodiments, the contraceptive treatment comprises administering a GnRH agonist to the subject.

[0063] In some embodiments, it has been determined that the subject exhibits a follicle size of about 6 mm to about 8 mm prior to the onset of the follicular triggering phase.

[0064] In some embodiments, it has been determined that the subject exhibited a follicle size of about 6 mm to about 8 mm prior to administration of the final follicle triggering agent.

[0065] In some embodiments, it is determined that the biological sample separated from the subject before taking out one or more oocytes has an AMH concentration of about 1ng / ml to about 6ng / ml. In some embodiments, it is determined that the sample has an AMH concentration of about 2ng / ml to about 5ng / ml. In some embodiments, it is determined that the sample has an AMH concentration of about 2.5ng / ml to about 3.0ng / ml. In some embodiments, it is determined that the biological sample separated from the subject before taking out one or more oocytes has an AMH concentration of at least 1ng / ml. In some embodiments, it is determined that the biological sample separated from the subject before taking out one or more oocytes has an AMH concentration of not more than 6ng / ml.

[0066] In some embodiments, the sample is a blood sample.

[0067] In some embodiments, the subject is between 18 and 48 years of age at the time of retrieval of the one or more oocytes. In some embodiments, the subject is between 20 and 45 years of age. In some embodiments, the subject is between 25 and 45 years of age at the time of retrieval of the one or more oocytes. In some embodiments, the subject is less than 35 years of age at the time of retrieval of the one or more oocytes. In some embodiments, the subject is greater than 35 years of age at the time of retrieval of the one or more oocytes.

[0068] In some embodiments, prior to removing one or more oocytes from the subject, it was determined that the subject exhibited a follicle size of about 6 mm to about 14 mm. In some embodiments, prior to removing one or more oocytes from the subject, it was determined that the subject exhibited a follicle size of about 8 mm to about 12 mm. In some embodiments, prior to removing one or more oocytes from the subject, it was determined that the subject exhibited a follicle size of about 8 mm to about 9 mm. In some embodiments, prior to removing one or more oocytes from the subject, it was determined that the subject exhibited a follicle size of no greater than 14 mm.

[0069] In some embodiments, follicle size has been assessed by ultrasound image analysis.

[0070] In some embodiments, a total of 20 or fewer oocytes are removed from a subject. In some embodiments, 15 or fewer oocytes are removed from a subject. In some embodiments, 10 or fewer oocytes are removed from a subject. In some embodiments, 9 or fewer oocytes are removed from a subject. In some embodiments, 8 or fewer oocytes are removed from a subject. In some embodiments, 7 or fewer oocytes are removed from a subject. In some embodiments, 6 or fewer oocytes are removed from a subject. In some embodiments, 5 or fewer oocytes are removed from a subject. In some embodiments, multiple oocytes are removed from a subject.

[0071] In some embodiments, 10% to 100% of the oocytes removed from the subject are GV or MI stage oocytes. In some embodiments, 20% to 100% of the oocytes removed from the subject are GV or MI stage oocytes. In some embodiments, 30% to 100% of the oocytes removed from the subject are GV or MI stage oocytes. In some embodiments, 40% to 100% of the oocytes removed from the subject are GV or MI stage oocytes. In some embodiments, 50% to 100% of the oocytes removed from the subject are GV or MI stage oocytes. In some embodiments, 60% to 100% of the oocytes removed from the subject are GV or MI stage oocytes. In some embodiments, 70% to 100% of the oocytes removed from the subject are GV or MI stage oocytes. In some embodiments, 80% to 100% of the oocytes removed from the subject are oocytes at the GV stage or the MI stage. In some embodiments, 90% to 100% of the oocytes removed from the subject are oocytes at the GV stage or the MI stage. In some embodiments, 100% of the oocytes removed from the subject are oocytes at the GV stage or the MI stage.

[0072] In some embodiments, the ovarian support cell group includes ovarian granulosa cells and / or ovarian stromal cells, optionally wherein the ovarian granulosa cells are FOXL2 positive and / or wherein the ovarian stromal cells are NR2F2 positive. In some embodiments, the ovarian support cell group includes a mixture of ovarian granulosa cells and ovarian stromal cells. In some embodiments, the ovarian support cell group includes a cell mixture so that the mixture includes ovarian granulosa cells and ovarian stromal cells distributed in about 1: 1, wherein the colony contains or does not contain one or more other cell types. In some embodiments, the ovarian support cell group includes a mixture of cell types so that the mixture includes one or more cell types more abundant than another cell type (e.g., relative distribution is 2: 1, 3: 1, 4: 1, 5: 1, and other possible population distributions). In some embodiments, the ovarian supporting cell population comprises a mixture of ovarian granulosa cells and ovarian stromal cells, such that one cell type is more abundant in the mixture (e.g., 90% ovarian granulosa cells and 10% ovarian stromal cells, 80% ovarian granulosa cells and 20% ovarian stromal cells, 70% ovarian granulosa cells and 30% ovarian stromal cells, 60% ovarian granulosa cells and 40% ovarian stromal cells, 40% ovarian granulosa cells and 60% ovarian stromal cells, 30% ovarian granulosa cells and 70% ovarian stromal cells, 20% ovarian granulosa cells and 80% ovarian stromal cells, or 10% ovarian granulosa cells and 90% ovarian stromal cells, as well as other possible distributions). In some embodiments, the ovarian supporting cell population comprises a mixture of ovarian granulosa cells and ovarian stromal cells and one or more additional cell types.

[0073] In some embodiments, the population of ovarian Sertoli cells comprises ovarian granulosa cells.

[0074] In some embodiments, the ovarian support cell population comprises about 50,000 to about 100,000 ovarian support cells. In some embodiments, the ovarian support cell population comprises about 50,000 to about 60,000 ovarian support cells, about 60,000 to about 70,000 ovarian support cells, about 70,000 to about 80,000 ovarian support cells, about 80,000 to about 90,000 ovarian support cells, or about 90,000 to about 100,000 ovarian support cells. In some embodiments, the population of ovarian support cells includes about 50,000 ovarian support cells, about 55,000 ovarian support cells, about 60,000 ovarian support cells, about 65,000 ovarian support cells, about 70,000 ovarian support cells, about 75,000 ovarian support cells, about 80,000 ovarian support cells, about 85,000 ovarian support cells, about 90,000 ovarian support cells, about 95,000 ovarian support cells, or about 100,000 ovarian support cells.

[0075] In some embodiments, the ovarian supporting cells include steroidogenic granulosa cells. In some embodiments, the steroidogenic granulosa cells produce estradiol.

[0076] In some embodiments, ovarian supporting cells are obtained by inducing differentiation of an iPSC population.

[0077] In some embodiments, the ovarian support cells are obtained by modifying iPSC to express one or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express two or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express three or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express four or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express all five of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2.

[0078] In some embodiments, ovarian support cells are cryopreserved and thawed before co-cultivation with one or more oocytes. In some embodiments, ovarian support cells are thawed about 24 hours to about 120 hours before co-cultivation with one or more oocytes. In some embodiments, ovarian support cells are thawed about 24 hours to about 48 hours, about 48 hours to about 72 hours, about 72 hours to about 96 hours or about 96 hours to about 120 hours before co-cultivation with one or more oocytes. In some embodiments, ovarian support cells are thawed about 24 hours to about 36 hours, about 30 hours to about 40 hours, about 36 hours to about 48 hours, about 48 hours to about 56 hours, about 56 hours to about 72 hours, about 72 hours to about 84 hours, about 80 hours to about 96 hours, about 90 hours to about 100 hours, about 96 hours to about 108 hours, or about 108 hours to about 120 hours before co-cultivation with one or more oocytes.

[0079] In some embodiments, one or more oocytes are co-cultured with a population of ovarian supporting cells for about 12 hours to about 120 hours. In some embodiments, one or more oocytes are co-cultured with a population of ovarian supporting cells for about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 24 hours to about 48 hours, about 36 hours to about 60 hours, about 54 hours to about 72 hours, about 68 hours to about 96 hours, or about 96 hours to about 120 hours. In some embodiments, one or more oocytes are co-cultured with a population of ovarian supporting cells for about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, about 54 hours, about 56 hours, about 58 hours, about 60 hours, about 62 hours , about 64 hours, about 66 hours, about 68 hours, about 70 hours, about 72 hours, about 74 hours, about 76 hours, about 78 hours, about 80 hours, about 82 hours, about 84 hours, about 86 hours, about 88 hours, about 90 hours, about 92 hours, about 94 hours, about 96 hours, about 98 hours, about 100 hours, about 102 hours, about 104 hours, about 106 hours, about 108 hours, about 110 hours, about 112 hours, about 114 hours, about 116 hours, about 118 hours or about 120 hours.

[0080] In some embodiments, the co-cultivation is performed in an adherent co-cultivation system. In some embodiments, the co-cultivation is performed in a suspension co-cultivation system.

[0081] In some embodiments, before and / or after co-culture, one or more parameters of the oocytes are assessed, the parameters being selected from the group consisting of: total oocyte score, oocyte maturation rate from GV to MII, oocyte maturation rate from GV to MI, oocyte maturation rate from MI to MII, average oocyte shape, average oocyte size, average egg quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality.

[0082] In some embodiments, one or more oocytes are denuded following co-cultivation.

[0083] In some embodiments, the method further comprises isolating one or more MII stage oocytes from a mixture produced by co-culturing one or more oocytes removed from a subject with a population of ovarian support cells.

[0084] In some embodiments, the subject is undergoing an autologous ART procedure, and wherein the method further comprises contacting each of the one or more MII stage oocytes with a mature sperm cell.

[0085] In some embodiments, one or more MII stage oocytes are cryopreserved and thawed prior to contacting. In some embodiments, one or more MII stage oocytes are not cryopreserved and are not thawed prior to contacting.

[0086] In some embodiments, the contacting comprises IVF of one or more MII stage oocytes. In some embodiments, the contacting comprises ICSI of one or more MII stage oocytes.

[0087] In some embodiments, contacting results in the formation of an embryo. In some embodiments, the embryo is transferred to the uterus of a subject. In some embodiments, about 3 days after one or more MII stage oocytes contact with mature sperm cells, the embryo is transferred to the uterus of a subject. In some embodiments, about 5 days after one or more MII stage oocytes contact with mature sperm cells, the embryo is transferred to the uterus of a subject. In some embodiments, the embryo transferred to the uterus of a subject is a blastocyst stage embryo.

[0088] In some embodiments, the methods result in the formation of a plurality of embryos having a survival rate greater than 20% (e.g., a survival rate of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more).

[0089] In another aspect, the disclosure features an ex vivo composition that includes a population of ovarian support cells and one or more diluents or excipients.

[0090] In some embodiments, the ovarian support cell population comprises about 50,000 to about 100,000 ovarian support cells. In some embodiments, the ovarian support cell population comprises about 50,000 to about 60,000 ovarian support cells, about 60,000 to about 70,000 ovarian support cells, about 70,000 to about 80,000 ovarian support cells, about 80,000 to about 90,000 ovarian support cells, or about 90,000 to about 100,000 ovarian support cells. In some embodiments, the population of ovarian support cells includes about 50,000 ovarian support cells, about 55,000 ovarian support cells, about 60,000 ovarian support cells, about 65,000 ovarian support cells, about 70,000 ovarian support cells, about 75,000 ovarian support cells, about 80,000 ovarian support cells, about 85,000 ovarian support cells, about 90,000 ovarian support cells, about 95,000 ovarian support cells, or about 100,000 ovarian support cells.

[0091] In some embodiments, the ovarian supporting cells include steroidogenic granulosa cells. In some embodiments, the steroidogenic granulosa cells produce estradiol.

[0092] In some embodiments, ovarian supporting cells are obtained by inducing differentiation of an iPSC population.

[0093] In some embodiments, the ovarian support cells are obtained by modifying iPSC to express one or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express two or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express three or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express four or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express all five of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2.

[0094] In some embodiments, the ovarian Sertoli cells are cryopreserved.

[0095] In another aspect, the disclosure features a cell culture medium including a population of ovarian Sertoli cells.

[0096] In some embodiments, the ovarian support cell population comprises about 50,000 to about 100,000 ovarian support cells. In some embodiments, the ovarian support cell population comprises about 50,000 to about 60,000 ovarian support cells, about 60,000 to about 70,000 ovarian support cells, about 70,000 to about 80,000 ovarian support cells, about 80,000 to about 90,000 ovarian support cells, or about 90,000 to about 100,000 ovarian support cells. In some embodiments, the population of ovarian support cells includes about 50,000 ovarian support cells, about 55,000 ovarian support cells, about 60,000 ovarian support cells, about 65,000 ovarian support cells, about 70,000 ovarian support cells, about 75,000 ovarian support cells, about 80,000 ovarian support cells, about 85,000 ovarian support cells, about 90,000 ovarian support cells, about 95,000 ovarian support cells, or about 100,000 ovarian support cells.

[0097] In some embodiments, the ovarian supporting cells include steroidogenic granulosa cells. In some embodiments, the steroidogenic granulosa cells produce estradiol.

[0098] In some embodiments, ovarian supporting cells are obtained by inducing differentiation of an iPSC population.

[0099] In some embodiments, the ovarian support cells are obtained by modifying iPSC to express one or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express two or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express three or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express four or more of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2. In some embodiments, the ovarian support cells are obtained by modifying iPSC to express all five of FOXL2, NR5A1, GATA4, RUNX1 and RUNX2.

[0100] In some embodiments, the cell culture medium is stored cryopreserved.

[0101] In another aspect, the disclosure features a composition of any preceding aspect or a cell culture medium of any preceding aspect for use in performing a method of any preceding aspect.

[0102] In another aspect, the disclosure features a kit including the composition of any of the preceding aspects and a package insert, wherein the package insert instructs a user of the kit to co-culture a population of ovarian support cells with one or more oocytes according to the method of any of the preceding aspects.

[0103] In another aspect, the disclosure features a kit including the cell culture medium of any of the preceding aspects and a package insert, wherein the package insert instructs a user of the kit to co-culture a population of ovarian support cells with one or more oocytes according to the method of any of the preceding aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] The accompanying drawings are used to illustrate embodiments of the present disclosure and to provide a further understanding of the implementation thereof.

[0105] Figure 1A is a block diagram of an embodiment and apparatus for assisted in vitro maturation of human oocytes.

[0106] Figure 1B is an exemplary embodiment of a device 100 for in vitro rescue of oocytes after assisted stimulation.

[0107] Figure 2A is a block diagram of an exemplary implementation of a machine learning module.

[0108] Figure 2B is an exemplary table showing training data used to train a machine learning model.

[0109] Figure 2C is an exemplary table showing additional training data for training a machine learning model.

[0110] Figure 3A is an exemplary flow chart of a microstimulation protocol.

[0111] Figure 3B is an exemplary flow chart of oocyte denudation.

[0112] Figure 4 is an exemplary table of metabolite formulations.

[0113] Figure 5 is an exemplary flow chart for preparing granulosa cell co-cultures.

[0114] Fig. 6A is an exemplary embodiment of a co-cultured second biological sample.

[0115] Figure 6Bis an exemplary embodiment of a control group culture of a second biological sample.

[0116] Figure 6C is an exemplary embodiment of co-cultured oocytes.

[0117] Fig.6D is an exemplary embodiment of a control culture of immature oocytes.

[0118] Fig. 7A is a flow chart of an exemplary method for inducing human oocyte maturation in vitro.

[0119] Figure 7B is an exemplary flow chart illustrating a method for in vitro rescue of oocytes after stimulation.

[0120] Figure 8 is a block diagram of a computing system that can be used to implement any one or more of the methods disclosed herein, and any one or more portions thereof.

[0121] Fig. 9 The experimental workflow for ovarian organoid (ovaroid) formation is shown. First, barcoded transcription factor (TF) expression vectors were integrated into FOXL2-T2A-tdTomato reporter induced pluripotent stem cells (hiPSCs). After inducing TF expression, cells positive for tdTomato and granulosa cell-related surface markers were sorted and the barcodes were sequenced. The top TFs based on barcode enrichment were selected and further characterized by combinatorial screening and bulk RNA sequencing. Next, monoclonal hiPSC lines that can induce expression of top TFs and efficiently generate granulosa-like cells were generated. Granulosa-like cells from these cell lines were further evaluated for estradiol production in response to follicle-stimulating hormone (FSH) stimulation. Finally, they aggregated with human primordial germ cell-like cells (hPGCLCs) to form ovarian organoids. As measured by immunofluorescence microscopy and scRNA-seq, these ovarian organoids produce estradiol and progesterone, form follicle-like structures, and support hPGCLC maturation.

[0122] Fig. 10A Schematic diagram of the experimental co-culture IVM method. hiPSCs are differentiated to form ovarian supporting cells (OSCs) using inducible transcription factor overexpression. Immature human cumulus-oocyte complexes (COCs) are obtained from donors clinically after brief gonadotropin stimulation. In the laboratory, embryology dishes are prepared, including seeding OSCs as needed, and COCs are introduced for IVM co-culture. After 24-28 hours of IVM co-culture, oocyte maturity and morphological quality are assessed, and samples are stored for analysis or for embryogenesis.

[0123] Fig. 10B Representative images of co-culture devices containing human COCs (n=5) and 100,000 OSCs at the time of plating. Scale bar 100 μm. In suspension cultures, COCs with expanded and unexpanded cumulus are seen with surrounding OSCs.

[0124] Fig.11A Shown are the oocyte maturation rates after 24–28 h of IVM experiments in Experiment 1, including oocytes co-cultured with OSCs, or in medium control. n indicates the number of individual oocytes per culture condition. Error bars indicate mean ± SEM. p values ​​are from unpaired t-tests comparing OSC-IVM to medium control conditions.

[0125] Fig. 11B Total oocyte scores (TOS) obtained by imaging analysis of MII oocytes after 24-28 hours of IVM experiments are shown. n represents the number of individual MII oocytes analyzed. The median (dashed line) and quartiles (dotted line) are indicated. Unpaired t-tests showed no significant differences between the means (p=0.2909). Due to the small number of oocytes retrieved from each donor, it was not possible to consistently distribute oocytes under the two conditions analyzed. The oocytes included in the groups were mainly from non-overlapping donor cohorts, and paired comparisons were not used.

[0126] Fig. 12A Shown are the maturation rates of oocytes after 28 h of IVM experiments in Experiment 2, including co-culture of oocytes with OSCs or commercial IVM controls. n represents the number of individual oocytes per culture condition. Error bars represent mean ± SEM. p values ​​are derived from paired t-tests comparing experimental OSC-IVM with control conditions (commercial IVM control).

[0127] Fig. 12B Total oocyte score (TOS) obtained by imaging analysis of MII oocytes after 28 hours of IVM experiment is shown. n represents the number of individual MII oocytes analyzed. The median (dashed line) and quartiles (dotted line) are indicated. Unpaired t-test showed no significant difference between the means (p=0.9420). COCs of each donor were randomly and evenly distributed between the control group and the intervention group for pairwise statistical comparison.

[0128] Fig.13AThe results of embryogenesis after 28 hours of IVM experiments in oocyte subgroups used for embryogenesis in experiment 2 are shown, including oocyte co-culture with OSC or in commercially available IVM controls. Error bars represent mean ± SEM. Results are shown as the percentage of the total COC treated in this group. For two IVM conditions, the results of fertilization, cleavage, blastocyst formation, high-quality blastocyst formation and euploid blastocyst formation were evaluated.

[0129] Fig. 13B Representative images of embryogenesis under OSC-IVM and commercial IVM conditions at cleavage day 3 and blastocyst formation days 5, 6, and 7 are shown. Embryos with suitable vitrification quality were labeled "usable quality embryos" and used for trophectoderm biopsy.

[0130] Fig.14A is a schematic diagram of the experimental co-culture IVM method. hiPSCs were differentiated using inducible transcription factor overexpression to form ovarian supporting cells (OSCs). Human oocytes were obtained from donors in the clinic after standard gonadotropin stimulation, and immature oocytes (GV and MI) identified after denudation were allocated to this study. In the embryology laboratory, culture dishes were prepared, including seeding of OSCs as needed, and immature oocytes were introduced for IVM co-culture. After 24-28 hours of IVM co-culture, oocyte maturity and health were assessed, and oocyte samples were stored for further analysis.

[0131] Fig. 14B Representative images of co-culture setups containing immature human oocytes (n=3) and OSCs at the time of plating. Scale bar: 200 μm. Denuded GV oocytes can be seen with surrounding OSCs in suspension culture.

[0132] Fig.15A Maturation rates of oocytes after 24-28 h IVM experiments are shown, including oocytes co-cultured with OSCs (OSC-IVM), or in culture medium controls (culture medium-IVM). n represents the number of individual oocytes per culture condition. Error bars represent mean ± SEM. p values ​​are derived from unpaired t-tests comparing experimental OSC-IVM with control culture medium-IVM. Due to the small number of oocytes retrieved per donor, each group of oocytes came from a predominantly non-overlapping donor group, so no pairwise comparisons were performed.

[0133] Fig. 15BTotal oocyte score (TOS) obtained by imaging analysis of MII oocytes after 24-28 hours of IVM experiments is shown. n represents the number of individual MII oocytes analyzed. The median (dashed line) and quartiles (dotted line) are indicated. Unpaired t-test showed no significant (ns, p=0.5725) differences between the means.

[0134] Fig.16A Shown are representative images of MII oocytes co-cultured with OSC for 28 hours, stained with fluorescent α-tubulin dye (cyan) to show meiotic spindles. The blue line across the middle of PB1 and the spindle assembly from the center of the oocyte is used to derive the PB1-spindle angle. The PB1 spindle angle range is as shown above. Culture medium-IVM conditions provide examples of MII lacking spindles.

[0135] Fig. 16B Quantification of the angle between PB1 and the spindle obtained by oocyte fluorescence imaging analysis (as shown in A) is shown. n = represents the number of individual oocytes analyzed under each condition. The number of MII oocytes in which spindle assembly was not observed is also indicated below the axis label. The median (dashed line) and quartiles (dotted line) are indicated. ANOVA statistical analysis found no significant difference between the mean values ​​of each condition (ns, p = 0.1155).

[0136] Fig.17A UMAP projections of oocyte transcriptomes are shown, with symbols colored by experimental batch, experimental condition (OSC-IVM, medium-IVM, IVF-MII), oocyte maturation state, and Leiden cluster. Each symbol represents one oocyte. n=81 oocytes.

[0137] Fig. 17B UMAP projections colored by the score for each gene marker set (GV and IVF MII) are shown.

[0138] Fig. 17C UMAP projections generated from each cell score in two signature marker sets (GV vs. IVF MII) are shown, colored according to experimental condition, oocyte maturation state, and Leiden clustering.

[0139] Fig.17D Quantification of oocytes in each maturation outcome (GV, MI, and MII) by experimental condition (IVM or IVF) is shown, with color distribution indicating the percentage of the population in each Leiden cluster. Striped bars are used to indicate clusters with predominantly IVF-like characteristics.

[0140] Fig.18AShown are immunofluorescence images of sections of human ovarian organoids (F66 / N.R1.GF#4 granulosa-like cells + hPGCLCs) stained for FOXL2 (granulosa cells), OCT4 (germ cells / pluripotency), and DAZL (mature germ cells) at culture days 2, 4, 14, and 32. Scale bar, 40 μm.

[0141] Fig.18B Shows Fig.18A Mouse ovarian organoid (embryonic mouse ovarian somatic cells + hPGCLC) sections stained as indicated. Scale bar, 40 μm.

[0142] Fig.18C The fraction of OCT4+ and DAZL+ cells relative to the total (DAPI+) in human ovarian organoids and mouse xenogeneic ovarian organoids is shown over time. Images from 2 replicates of human ovarian organoids (F66 / N.R1.GF#4 and F66 / N.R2#1 granulosa-like cells+hPGCLCs) and 1 replicate of mouse xenogeneic ovarian organoids were counted at 11 time points.

[0143] Fig.18D Shown are immunofluorescence images of sections of human ovarian organoids (F66 / N.R2#1 granulosa-like cells + hPGCLCs) stained for SOX17 (germ cells), TFAP2C (early germ cells), and AMHR2 (granulosa cells) at culture days 4 and 8. Scale bar, 40 μm.

[0144] Fig.18E DAZL and OCT4 expression in day 16 ovarian organoids as observed by immunofluorescence is shown. Some DAZL+OCT4- cells (magenta arrows) as well as DAZL+OCT4+ cells (cyan arrows) can be seen. Ovarian organoids also begin to form follicle-like morphology (yellow arrows). Scale bar = 40 μm.

[0145] Fig.19A FOXL2, OCT4, and AMHR2 staining of human ovarian organoid (F66 / N.R1.G#7+hPGCLC) sections at day 35 are shown. Scale bar is 40 μm. Follicle-like structures are marked with yellow triangles.

[0146] Fig.19B A whole ovarian organoid view of follicle-like structures in human ovarian organoid (F66 / N.R1.G#7) is shown. Scale bar = 1 μm.

[0147] Fig.19CA section of a human ovarian organoid (F66 / N.R1.GF#4+hPGCLC) at day 70 of culture is shown, stained with FOXL2, NR2F2, and AMHR2, showing multiple small follicles (yellow triangles) composed of a single layer of FOXL2+AMHR2+ cells. NR2F2+ cells are interspersed between these cells. Scale bar = 100 μm.

[0148] Fig.19D Shown is a section of a human ovarian organoid (F66 / N.R2#1+hPGCLC) at day 70 of culture, stained with FOXL2, NR2F2, and AMHR2, showing antral follicles composed of FOXL2+AMHR2+ granulosa-like cells arranged in multiple layers surrounding a central cavity. NR2F2 staining is visible outside the follicle (labeled "stroma"). Scale bar 100 μm.

[0149] Fig. 20A Expression (log2 CPM) of selected granulosa cell (FOXL2), stromal / theca cell (NR2F2), and germ cell (PRDM1) markers is shown. Expression scRNA-seq analysis from ovarian organoids (F66 / N.R1.GF#4 granulosa-like cells + hPGCLCs). Data from all samples (days 2, 4, 8, and 14) were merged for joint dimensionality reduction and clustering.

[0150] Fig. 20B Leiden clustering showing four main clusters; Fig. 20A Figure 4 scRNA-seq analysis of ovarian organoids, with marker gene expression (log2 CPM) plotted for each cluster.

[0151] Fig. 20C The mapping of cells to the human fetal ovary reference atlas (Garcia-Alonso et al., 2022) and the Fig. 20A Cell type assignment for the scRNA-seq analysis described in .

[0152] Fig.20D Shows the Fig. 20A The proportions of somatic cell types, germ cells, DAZL+ cells, and DDX4+ cells in ovarian organoids at each day of scRNA-seq analysis as described in.

[0153] Fig.21A Denuded oocytes following standard care are shown.

[0154] Fig.21B COCs produced with minimal stimulation are shown.

[0155] Fig. 21C OSC-IVM statistically significantly improved the oocyte maturation rate.

[0156] Fig.22A Shown are the morphological qualities of oocytes grown in OSC-IVM culture.

[0157] Fig. 22B The angle between the spindle of PB1 and oocytes grown in OSC-IVM culture is shown.

[0158] Fig. 22C It was shown that oocytes grown in OSC-IVM culture had a high degree of similarity to MII oocytes in vivo.

[0159] Fig.22D It was shown that oocytes grown in OSC-IVM culture had a high degree of similarity to MII oocytes in vivo.

[0160] Fig.23A Oocyte degradation rates for OSC-IVM product toxicity assessment are shown.

[0161] Fig. 23B Fertilization and blastocyst generation of OSC-IVM outgrowths are shown.

[0162] Fig.24 Shown compared with culture medium-IVM oocyte, OSC-IVM oocyte shows similar stress and cell cycle-related differential gene expression relative to IVF-MII control.Gene expression values ​​of oocytes of different developmental states (GV, MI or MII) under each experimental condition (OSC-IVM, culture medium-IVM, IVF-MII) are analyzed in groups, and each row represents a specific group.Shown relative (upper figure) and absolute (lower figure) gene expression of specific genes with known effects in cell cycle, stress and meiosis in each group, and each column represents a specific gene.Samples are sorted on the y-axis using unsupervised hierarchical clustering (UHC) for selected genes, as a measure of relative similarity.Accompanying drawings are not necessarily drawn to scale, and can be illustrated with dotted lines, diagrams and partial views.In some cases, details unnecessary to understanding the embodiment or details that make other details difficult to understand may be omitted.

[0163] definition

[0164] Unless otherwise defined herein, the scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. If there is any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In addition, unless the context otherwise requires, singular terms should include plural terms and plural terms should include the singular. Unless otherwise indicated, the use of "or" means "and / or". The use of the term "including" and other forms such as "includes" and "included" is not restrictive.

[0165] As used herein, the term "about" refers to a value that is 10% (10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less) above or below the value being described. For example, the phrase "about 50 mg" refers to a value between 45 mg and 55 mg (including 45 mg and 55 mg).

[0166] The term "assisted reproductive technology" or "ART" as used herein refers to a fertility treatment in which one or more female gametes (oocytes) or gametes (eggs) are manipulated in vitro to promote the formation of embryos, which can then be implanted into a subject to achieve pregnancy. For example, in some embodiments, oocytes removed from a subject undergoing an ART procedure can be matured in vitro using a co-culture method such as described herein. In some embodiments, after the formation of mature oocytes (eggs), the eggs can be treated with sperm cells to promote the formation of fertilized eggs and ultimately promote the formation of embryos. The embryo can then be transferred to the uterus of a female subject, for example, using compositions and methods of the art. Exemplary ART procedures include in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) techniques described herein and known in the art.

[0167] The term "subject" as used herein refers to an organism that is treated for a particular disease or disorder described herein. Examples of one or more subjects include mammals, such as humans (e.g., female humans), who are being treated for a disease or disorder corresponding to diminished ovarian reserve or immature oocyte release.

[0168] As used herein, the term "controlled ovarian hyperstimulation" refers to a procedure in which ovulation is induced in a subject, such as a human subject, followed by retrieval of an oocyte or egg for embryo formation, e.g., by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). A controlled ovarian hyperstimulation procedure may involve administering follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG), and / or a gonadotropin-releasing hormone (GnRH) antagonist to the subject to promote follicle maturation. Controlled ovarian hyperstimulation methods are known in the art and are described herein as they relate to methods of inducing follicle maturation and ovulation in conjunction with assisted reproductive techniques.

[0169] As used herein, in the context of cells from a subject, the term "derived from" refers to a cell isolated from a subject, such as a mammalian egg, or a cell obtained from one or more cells isolated from a subject by amplification, division, maturation, or manipulation (e.g., ex vivo amplification, division, maturation, or manipulation). For example, if the egg is isolated directly from a subject or is obtained from the maturation of an oocyte isolated from a subject, such as an oocyte isolated from a subject about 1 day to about 5 days after the subject undergoes an ovarian hyperstimulation procedure (e.g., an oocyte isolated from a subject about 2 days to about 4 days after the ovarian hyperstimulation procedure), the egg is "derived from" a subject or an oocyte described herein.

[0170] The term "dose" as used herein refers to the amount of a therapeutic agent (e.g., a follicle stimulating agent as described herein), which is administered to a subject to treat a disease or illness, such as enhancing oocyte maturation and / or releasing and promoting the removal and in vitro maturation of viable oocytes. The therapeutic agent as described herein can be administered in a single dose or multiple doses. In each case, the therapeutic agent can be administered using one or more unit dosage forms of the therapeutic agent. For example, a therapeutic agent of, for example, two 50mg unit dosage forms can be used to administer a 100mg single dose of the therapeutic agent. Similarly, a therapeutic agent of, for example, six 50mg unit dosage forms or a therapeutic agent of two 50mg unit dosage forms and a therapeutic agent of a 200mg unit dosage form, and other combinations can be used to administer a single dose of 300mg of the therapeutic agent. Similarly, a therapeutic agent of, for example, 6 50mg unit dosage forms and a therapeutic agent of 3 200mg unit dosage forms, or a therapeutic agent of 10 50mg unit dosage forms and a therapeutic agent of 2 200mg unit dosage forms, and other combinations can be used to administer a single dose of 900mg of the therapeutic agent.

[0171] The term "follicle triggering phase" as used herein refers to the time point at which the follicle triggering agent is administered. The time point at which the follicle triggering agent is administered to a female subject (i.e., the follicle triggering phase) is day 1, day 2, or day 3 of her menstrual cycle, preferably day 2 of the menstrual cycle. However, if the female subject is taking hormonal contraceptives, the time point at which the follicle triggering agent is administered is 4-6 days (e.g., 4 days, 5 days, or 6 days) after taking the last oral contraceptive pill, preferably 5 days after taking the last oral contraceptive pill.

[0172] The term "follicle stimulating hormone" (FSH) as used herein refers to a biologically active heterodimeric human fertility hormone that can induce ovulation in a subject. FSH can be purified from postmenopausal human urine or produced as a recombinant protein product. Exemplary recombinant FSH products include follicle stimulating hormone alpha (GONAL-F, Merck Serono / EMD Serono) and follicle stimulating hormone beta (PUR EGON / FOLLISTIM, MSD / Scherig-Plough).

[0173] As used herein, the term "human chorionic gonadotropin" (hCG) refers to a polypeptide hormone that interacts with the luteinizing hormone chorionic gonadotropin receptor (LHCGR) to induce follicle maturation and ovulation. hCG can be purified from pregnant women's urine or produced as a recombinant protein product. Exemplary recombinant hCG products include chorionic gonadotropin alpha ( Merck Serono / EMD Serono).

[0174] The term "in vitro fertilization" (IVF) used herein refers to the process in which an egg (such as a human egg) is contacted with one or more sperm cells in vitro to promote egg fertilization and fertilized oval formation. The egg can come from a subject such as a human subject who has undergone various ARTs known in the art. For example, one or more oocytes can be obtained from a subject after a controlled ovarian hyperstimulation program is performed by injecting a follicle-stimulating agent, for example, about 1 day to about 5 days after the injection of the agent (for example, about 1 day to about 4 days after injecting a follicle-stimulating agent to the subject). The egg can also be taken out directly from the subject, for example, by a transvaginal egg removal method known in the art.

[0175] The term "intracytoplasmic sperm injection" (ICSI) as used herein refers to the process of injecting sperm cells directly into eggs (such as human eggs) to promote fertilization of the eggs and the formation of fertilized oval. The sperm cells can be injected into the eggs, for example, by piercing the egg membrane with a microinjector, thereby delivering the sperm cells directly into the cytoplasm of the egg. ICSI procedures used in conjunction with the compositions and methods described herein are known in the art and are described, for example, in WO 2013 / 158658, WO 2008 / 051620, and WO 2000 / 009674, etc., the disclosures of which are incorporated herein by reference because they relate to compositions and methods for performing intracytoplasmic sperm injection.

[0176] The terms "ovum" and "oocyte" used herein refer to haploid female reproductive cells or gametes. In the context of assisted reproductive technology described herein, ovum can be produced in vitro by maturation of one or more oocytes isolated from a subject undergoing ART. Ovum can also be directly isolated from the subject, for example, by a transvaginal egg retrieval method described herein or known in the art. Ovum or oocyte used in the present disclosure may refer to multiple oocytes. Oocytes may form a complex with surrounding cells, such as a cumulus-oocyte complex (COC).

[0177] As used herein, the terms "mature egg" and "mature oocyte" refer to one or more eggs or oocytes that are in the metaphase II (MII) stage of meiosis and typically have morphological or structural features consistent with metaphase II, such as polar bodies and other features described herein.

[0178] The terms "immature ovum" and "immature oocyte" as used herein refer to one or more eggs or oocytes that have not yet reached the MII stage of meiosis. In some embodiments, the immature oocyte can be an oocyte that includes an oocyte in the germinal vesicle (GV) stage and / or metaphase I (MI) stage, as determined by morphological characteristics and / or other indications known in the art.

[0179] The term "oocyte maturation" as used herein refers to the process by which an immature oocyte develops into a mature oocyte. Oocyte maturation occurs when an immature oocyte undergoes a cell signaling event caused by external and internal stimuli. External stimuli may be produced by adjacent cells or supporting cells as described herein. Oocyte maturation may occur before the oocyte is released and removed from a subject. Oocyte maturation may occur in vitro due to the culture methods and culture components described herein.

[0180] As used herein, "ovarian supporting cells" (OSC) or "supporting cells" refer to one or more cells that promote the maturation of one or more oocytes. OSC can be an ovarian granulosa cell (e.g., a granulosa cell described herein). Additionally or alternatively, OSC can be an ovarian stromal cell (e.g., a stromal cell described herein). OSC can form a cumulus-oocyte complex (COC) with an oocyte. OSC can be produced by an exogenous source, such as produced by induced pluripotent stem cells (iPSC), such as human induced pluripotent stem cells (hiPSC), as described herein. OSC can be applied to the oocyte removed using the in vitro cell culture method and composition described herein. OSC can be a mixture of two or more cell types. OSC can be a mixture of stromal cells and granulosa cells, so that the mixture is a stromal cell and granulosa cell group of about 1:1. OSC can be a mixture of stromal cells and granulosa cells, so that a cell type has a higher relative abundance than one or more cell types, so that the mixture is about 2:1 populations, 3:1 populations, 4:1 populations, 5:1 populations, and other possible population distributions. OSCs can be a mixture of stromal cells and granular cells such that one cell type is more abundant in the mixture (e.g., 90% stromal cells and 10% granular cells, 80% stromal cells and 20% granular cells, 70% stromal cells and 30% granular cells, 60% stromal cells and 40% granular cells, 40% stromal cells and 60% granular cells, 30% stromal cells and 70% granular cells, 20% stromal cells and 80% granular cells, or 10% stromal cells and 90% granular cells, among other possible distributions). In some embodiments, OSCs can be a mixture of stromal cells and granular cells with one or more other cell types.

[0181] "Ovarian stromal cells" or "stromal cells" used herein are cumulus cells surrounding oocytes to ensure oocyte health and subsequent embryonic development. Ovarian stromal cells can form COCs with oocytes. Ovarian stromal cells can express markers consistent with matrix subtypes, such as nuclear receptor subfamily 2 group F member 2 (NR2F2), which can be detected by methods known in the art. Ovarian stromal cells can be steroidogenic stromal cells. Ovarian stromal cells can be produced by differentiated hiPSCs as described herein.

[0182] "Steroidogenic stromal cells" as used herein are stromal cells that can produce one or more steroids (such as estradiol, progesterone, or a combination thereof). One or more steroids can be produced in response to hormonal stimulation, such as produced by FSH, androstenedione, or a combination thereof. One or more steroids can be secreted.

[0183] "Ovarian granulosa cells" or "granulosa cells" as used herein are cumulus cells surrounding the oocyte to ensure oocyte health and subsequent embryonic development. Ovarian granulosa cells can form a COC with the oocyte. Ovarian granulosa cells can express markers consistent with granulosa cell subtypes, such as FOXL2, CD82 and / or follicle stimulating hormone receptor (FSHR), which can be detected by methods known in the art. Ovarian granulosa cells can be steroidogenic granulosa cells. Ovarian granulosa cells can be produced by differentiated hiPSCs as described herein.

[0184] As used herein, "steroidogenic granulosa cells" are granulosa cells that can produce one or more steroids (such as estradiol, progesterone, or a combination thereof). One or more steroids can be produced in response to hormonal stimulation, such as by FSH, androstenedione, or a combination thereof. One or more steroids can be secreted.

[0185] As used herein, the term "biological sample" or "sample" refers to a specimen isolated from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or skin), pancreatic juice, chorionic villus sample, hair, oocyte, egg and / or cells.

[0186] As used herein, the terms "oral contraceptive treatment," "oral contraception," "contraception," or "the birth control pill" refer to hormonal treatments typically used to prevent pregnancy. Oral contraceptive treatments may prevent the ovaries from releasing an egg cell and may contain hormones including estrogen and progestin.

[0187] The term "ovarian reserve" used herein refers to the number of oocytes in the subject's ovaries and the quality of the oocytes. Ovarian reserve naturally decreases with age and / or medical conditions described herein. Subjects with reduced ovarian reserve may seek IVF or other ART to achieve successful pregnancy. As described herein, anti-mullerian hormone (AMH) levels may indicate the subject's ovarian reserve.

[0188] As used herein, the term "stimulation regimen" refers to the process of administering one or more follicle triggering agents to a subject during the follicle triggering phase.

[0189] The term "follicle trigger" or "trigger" as used herein refers to a chemical or biological composition that stimulates the ovary to release an oocyte during ovulation. Follicle triggers may include hormones such as human chorionic gonadotropin and follicle stimulating hormone. The term "induced pluripotent stem cell" (iPSC) as used herein refers to artificial stem cells derived from reprogrammed and otherwise manipulated harvested somatic cells. iPSC can be differentiated into other cell types, including ovarian supporting cells or granulosa cells, by methods known in the art and methods described herein. iPSC can be human (hiPSC) or iPSC from, for example, other mammalian sources.

[0190] As used herein, the term "cell culture" refers to laboratory procedures that achieve in vitro cell proliferation and / or cultivation of prokaryotic or eukaryotic cell types.

[0191] As used herein, the term "co-culture" refers to a cell culture method in which more than one cell type or cell population is cultured in some degree of contact. In a typical co-culture system, two or more cell types may share an artificial growth medium.

[0192] As used herein, the term "adherent co-culture system" or "adherent cell culture" refers to a cell culture arrangement in which cells attach to a surface for proper growth and proliferation.

[0193] As used herein, the term "suspension co-culture system" or "suspension cell culture" refers to a cell culture device by which cells are cultured dispersed in a liquid culture medium for proper growth and proliferation. DETAILED DESCRIPTION

[0194] Devices, compositions and methods for assisted reproductive technology (ART) are described herein. For example, the devices, compositions and methods described herein are directed to follicle stimulation to induce the ovaries to release oocytes and in vitro maturation of oocytes after follicle stimulation (ie, after stimulation).

[0195] Advantageously, the method described herein is carried out in vitro maturation of immature oocytes by co-culturing with ovarian supporting cells (e.g., ovarian granulosa cells and / or stromal cells), so that previously discarded oocytes can be harvested and used for the purpose of traditional in vitro fertilization (IVF). The described in vitro maturation method improves the ability of using these usually discarded immature oocytes in IVF procedures, and can lead to more cost-effective treatment strategies and reduce the risk of treated subjects. For example, compared with current IVF treatment plans, the method can reduce the risk of systemic ovarian hyperstimulation in subjects seeking IVF surgery by requiring less hormone injections and / or lower hormone injection doses. Aspects of the present disclosure can be used to increase the overall number of healthy oocytes that women can use for IVF. Aspects of the present disclosure can also be used to significantly reduce the hormone dosage of subjects during egg retrieval and improve the quality of cultured oocytes. This may greatly expand the opportunity to obtain reproductive technology, significantly shorten the duration of a single cycle, and generally require fewer cycles to achieve pregnancy.

[0196] I. Methods of stimulating oocyte release

[0197] A. Subject Selection

[0198] The method for stimulating oocyte release described herein is directed to the experimenter seeking IVF treatment selection. Generally speaking, the experimenter is a woman with low oocyte removal quantity or an experimenter with many immature oocytes. The age of the experimenter may be between 20 and 45 years old, and the age of the experimenter is usually 35 years old or more. Due to age increase and / or the heredity or medical condition (for example, polycystic ovary syndrome (PCOS)) that causes ovarian reserve reduction, the experimenter may have ovarian reserve reduction. The ovarian reserve of experimenter can be 20 or less oocytes, so that experimenter has 1 to 5 oocytes, 4 to 10 oocytes, 8 to 16 oocytes or 15 to 20 oocytes, for example experimenter has 1 oocyte, 2 oocytes, 3 oocytes, 4 oocytes, 5 oocytes, 6 oocytes, 7 oocytes, 8 oocytes, 9 oocytes, 10 oocytes, 11 oocytes, 12 oocytes, 13 oocytes, 14 oocytes, 15 oocytes, 16 oocytes, 17 oocytes, 18 oocytes, 19 oocytes or 20 oocytes.Experimenter's anti-mullerian hormone (AMH) level may be consistent with ovarian reserve reduction.Experimenter's AMH level can be measured by blood test and additive methods known in the art. The AMH level of the subject can be between 1 and 6 ng / mL (e.g., 1-2 ng / mL, 2-4 ng / mL, or 4-6 ng / mL; e.g., 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, or 6 ng / mL). The subject may have measured an estradiol level between 20 and 50 pg / mL (e.g., 20-30 pg / mL, 25-35 pg / mL, 30-40 pg / mL, 35-45 pg / mL, or 40-50 pg / mL; e.g., 20 pg / mL, 21 pg / mL, 22 pg / mL, 23 pg / mL, 24 pg / mL, 25 pg / mL, 30 pg / mL, 35 pg / mL, 40 pg / mL, 45 pg / mL, or 50 pg / mL).

[0199] A physician or skilled practitioner can assess the method of stimulating oocyte release by collecting biological samples from a subject. Biological samples may include laboratory samples stored in a biobank for research. In some embodiments, biological samples may include body fluids, including blood, saliva, urine, semen (seminal fluid), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage fluid), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, optic nerve fluid, gastric juice, breast milk, cell culture supernatant, etc. Biological samples may include medical diagnosis, user input describing user feelings and / or symptom complaints, information collected from wearable devices related to the user, etc. For example, biological samples may include information obtained from a medical professional visit, such as a health history. In another non-limiting example, biological samples may include information such as data collected from a wearable device worn by a user, which is intended to collect information related to the user's sleep pattern, exercise pattern, etc. In one embodiment, biological samples are collected on a specific date and / or time of the user's menstrual cycle. For example, but not limited to, biological samples can be collected on the second day of the user's menstrual cycle to assess one or more hormone levels. The biological sample can be used to determine the experimenter's ovarian reserve marker, which can be measured by the experimenter's AMH level and / or other hormone levels or other indications. 1ng / mL or lower AMH levels can be used to indicate low ovarian reserve. The AMH level measured by the experimenter of low ovarian reserve can be 1.0ng / mL, 0.9ng / mL, 0.8ng / mL, 0.7ng / mL, 0.6ng / mL, 0.5ng / mL, 0.4ng / mL, 0.3ng / mL, 0.2ng / mL or 0.1ng / mL. Other biological samples that can be used to determine one or more markers of a subject's overall health include, but are not limited to, menstrual cycle progression, and / or monitoring circulating hormone levels such as estradiol (E2), luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone (P4), estrone (E1), estriol (E3), testosterone, androgens, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), adrenaline levels, and other hormones.

[0200] Other biological sample data collected from the subject include at least one oocyte. "Biological sample data" used in the present disclosure is the data providing the biological, genetic, biochemical and / or physiological characteristics, composition or activity characteristics of the biological sample. In some embodiments, the oocyte can be an immature oocyte. "Immature oocyte" used in the present disclosure is one or more immature germ cells derived from the ovary. In some embodiments, the immature oocyte can be an oocyte including GV and / or MI oocyte. In some embodiments, the immature oocyte can be a plurality of oocytes. The immature oocyte can be an immature cumulus-oocyte complex (COC) taken from the subject. As used in the present disclosure, "cumulus-oocyte complex" is an oocyte surrounded by specialized granulosa cells. As used in the present disclosure, "specialized granulosa cells" are cumulus cells that surround the oocyte to ensure the healthy development of the oocyte and the embryo. In some embodiments, the immature oocyte can include an oocyte, wherein specialized granulosa cells are added to mature the oocyte in cell culture (e.g., co-culture), thereby producing a COC.

[0201] In some embodiments of the method, a biological sample can be extracted from a user by an extraction device. An "extraction device" is a device and / or tool that can obtain, record and / or determine the measured values ​​associated with a sample. The extraction device may include a needle, a syringe, a vial, a lancet, a vacuum collection tube (ECT), a tourniquet, a vacuum extraction tube system, any combination thereof, etc. For example, the extraction device may include a butterfly needle set. Data from a biological sample may include, for example, measured values ​​of serum calcium, phosphate, electrolytes, blood urea nitrogen and creatinine, uric acid, etc.

[0202] In an embodiment of the method, biological sample information of a subject can be obtained by ultrasound. "Ultrasound" used in the present disclosure is any program that uses sound waves to generate one or more images of a user's body. For example, ultrasound can be used to obtain images of the reproductive organs and / or tissues of a subject. In one embodiment, an ultrasound examination can be performed at a specific time of the subject's menstrual cycle. For example, a subject may receive an ultrasound examination on the 2nd day of its cycle, which can be used to determine follicle size and / or follicle count. This information can be used to select a stimulation regimen and / or adjust the stimulation regimen. For example, an ultrasound examination shows that a subject with PCOS may need to adjust the dosage of one or more drugs received and / or used during the stimulation regimen. In addition, the length of her stimulation regimen can be modified based on her PCOS diagnosis. In one embodiment, ultrasound can be repeated once or multiple times throughout the subject's entire stimulation regimen, and the information obtained can be used to adjust her stimulation regimen in real time.

[0203] B. Oocyte Stimulation Protocol

[0204] A physician or skilled practitioner can use the described biological parameters to determine a stimulation regimen for the release of an oocyte from a subject. Such biological parameters include hormone levels (e.g., baseline hormone levels and / or hormone levels due to the use of contraceptives), subject anatomy (e.g., follicle size, follicle count, ovarian morphology and / or uterine morphology), and other biological parameters known to skilled practitioners. A skilled practitioner can implement a stimulation regimen using any one or more triggers described herein or compositions intended to stimulate follicle maturation and oocyte release.

[0205] The concentration of hormone levels or other related compounds of biological samples may include estradiol (E2), luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone (P4), estrone (E1), estriol (E3), testosterone, androgen, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), adrenaline levels, etc. In some embodiments, the measurement of hormone levels can be based on blood analysis of biological samples. For example, blood analysis can include plasma hormone analysis technology. In some embodiments, the measurement of hormone levels can be based on salivary hormone testing technology. The measurement of hormone levels can be based on other forms of analysis, such as hair, urine, and any other forms of biological samples described in the present disclosure. Prior to the follicular triggering phase, the subject's baseline serum estradiol level can be about 30 pg / mL to about 60 pg / mL (e.g., about 30 pg / mL to about 45 pg / mL, about 40 pg / mL to about 55 pg / mL, or about 45 pg / mL to about 60 pg / mL; e.g., about 30 pg / mL, about 35 pg / mL, about 40 pg / mL, about 45 pg / mL, about 50 pg / mL, about 55 pg / mL, or about 60 pg / mL). Prior to the follicular triggering phase, the subject's baseline serum progesterone level can be about 0.5 ng / mL to about 2.5 ng / mL (e.g., about 0.5 ng / mL to about 1.0 ng / mL, about 1.0 ng / mL to about 1.5 ng / mL, about 1.5 ng / mL to about 2.0 ng / mL, or about 2.0 ng / mL to about 2.5 ng / mL; e.g., about 1.0 ng / mL, about 1.5 ng / mL, about 2.0 ng / mL, or about 2.5 ng / mL).

[0206] In addition, the use of the experimenter's contraceptive measures (for example, hormonal contraception) may affect the distribution of the stimulation scheme.Consider that contraceptive measures may help determine the follicle triggering phase in the female menstrual cycle.For example, but not limited to, the experimenter who does not use any contraceptive form can start using the stimulation scheme of recombinant follicle stimulating hormone (rFSH) between the first day and the third day of the menstrual cycle, preferably on the second day of the menstrual cycle.In another non-limiting example, the experimenter who is using contraceptives can start using the stimulation scheme of rFSH 4-6 days (for example, 4 days, 5 days or 6 days) after she takes the last oral contraceptive pill, preferably 5 days after she takes the last oral contraceptive pill.In one embodiment, rFSH can be utilized to stimulate 2 to 3 days (for example, 2 days or 3 days), specifically depending on the tolerance, follicle size and / or growth dynamics of the experimenter.After this 2 or 3 day time window, the buffer period (coasting period) of 1 to 3 days (for example 1 day, 2 days or 3 days) can be utilized to monitor follicle size and allow follicle to further mature and develop. "Buffer period" as used in the present disclosure refers to any period of time during which the drugs used in the entire stimulation protocol are not administered and / or consumed. If medically necessary, the buffer period can last for example 1 day, 2 days, 3 days or longer. During the buffer period, the subject can continue to receive one or more ultrasound examinations to monitor his progress.

[0207] Once the follicle size reaches any value between about 8-10mm (e.g., 7.5mm, 8mm, 8.5mm, 9mm, 9.5mm, 10mm, 10.5mm or greater), a dose of a triggering agent (such as human chorionic gonadotropin (hCG)) can be used to trigger the subject. "Folliculometry" used in the present disclosure is any measurement of follicles. Follicles can include any sac containing unfertilized eggs found in the ovaries. Follicle measurement values ​​can be obtained using any method described herein including, for example, ultrasound, manual measurement, automatic measurement, etc. In one embodiment, double hCG injection can be used to induce follicle maturation in preparation for taking out one or more follicles. Double hCG injection can be two or three hCG injections. On the triggering day of double dose hCG injection, blood tests of one or more hormone levels (such as E2, P4 and LH) can be performed to monitor hormone levels. After the double dose of hCG injection day, one or more hormone levels can be measured, such as by blood tests to determine and check the levels of E2, P4 and LH.

[0208] A "trigger" is a chemical that can trigger the production of ovarian cells. Triggers (e.g., follicle triggers) can include any substance, including any over-the-counter and / or prescription products. Triggers (e.g., follicle triggers) can include any one or a combination of the following non-limiting examples, such as LUPRON (Abbott Laboratories, North Chicago, IL), Ganirelix (Ferring Pharmaceuticals, Saint-Prex, Switzerland), Cetrotide (Merck Global, Readington Township, NJ), (Merck Global), (Merck Global), (Ferring Pharmaceuticals), (Patheon Pharmaceuticals Inc., Waltham, MA), Serephene (Teva, Tel Aviv-Yafo, Israel), (Merck Global), (Mylan,Canonsburg,PA), (Schering Plow,Kenilworth,NJ)、 (Ferring Laboratories, Parsippany, NJ), Repronex (Ferring Pharmaceuticals), (Zoetis Canada Inc., Kirkland, Canada), (Ferring Pharmaceuticals) and other drugs that are suitable for inducing ovarian cell production understood by those skilled in the art. Triggers (such as follicle triggers) may include human serum albumin, FSH, hCG, androstenedione and doxycycline and other triggers known in the art.

[0209] In one embodiment, the subject may not receive a trigger (e.g., a follicle trigger) to stimulate the production of oocytes. In one embodiment, in a preferred stimulation regimen, the subject may receive multiple injections of a trigger within 1 to 4 days (e.g., 1 day, 2 days, 3 days, or 4 days) but no more than 5 days. The subject may receive multiple injections over multiple days so that the subject receives five doses of one or more triggers. For example, the subject may receive three days of stimulation, with each injection using 300 IU to 700 IU of rFSH (e.g., 300-500 IU, 400-600 IU, 500-700 IU, 300-350 IU, 350-400 IU, 400-450 IU, 450-500 IU, 500-550 IU, 550-600 IU, 60 0-650 IU, 650-700 IU; for example, 300 IU, 325 IU, 350 IU, 375 IU, 400 IU, 425 IU, 450 IU, 475 IU, 500 IU, 525 IU, 550 IU, 575 IU, 600 IU, 625 IU, 650 IU, 675 IU or 700 IU), injected once or more per day. The subject may receive an injection of hCG as a trigger (e.g., a follicle trigger) using 200-700 μg or 2,500-10,000 IU hCG (e.g., 200-500 μg, 300-600 μg, 400-700 μg, 200-300 μg, 300-400 μg, 400-500 μg, 500-600 μg, or 600-700 μg), with a preferred stimulating dose of 500 μg. The subject may receive one or more injections of clomiphene citrate in combination with other triggering agents, with each injection of clomiphene citrate at a dose of 50-150 mg (e.g., 50-75 mg, 60-80 mg, 75-100 mg, 90-115 mg, 110-130 mg, 125-150 mg; for example, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg).

[0210] Prior to receiving the trigger, the level of hormones or other related compounds in the subject's serum can be assessed. Prior to receiving the trigger, the subject's serum estradiol level can be about 250 pg / mL to about 400 pg / mL (e.g., about 250 pg / mL to about 275 pg / mL, about 275 pg / mL to about 300 pg / mL, about 300 pg / mL to about 325 pg / mL, about 325 pg / mL to about 350 pg / mL, about 350 pg / mL to about 375 pg / mL, or about 375 pg / mL to about 400 pg / mL). for example, about 250 pg / mL, about 260 pg / mL, about 270 pg / mL, about 280 pg / mL, about 290 pg / mL, about 300 pg / mL, about 310 pg / mL, about 320 pg / mL, about 330 pg / mL, about 340 pg / mL, about 350 pg / mL, about 360 pg / mL, about 370 pg / mL, about 380 pg / mL, about 390 pg / mL, or about 400 pg / mL). Prior to receiving the trigger, the subject's serum progesterone level can be from about 0.25 ng / mL to about 0.75 ng / mL (e.g., from about 0.25 ng / mL to about 0.35 ng / mL, from about 0.35 ng / mL to about 0.45 ng / mL, from about 0.45 ng / mL to about 0.55 ng / mL, from about 0.55 ng / mL to about 0.65 ng / mL, or from about 0.65 ng / mL to about 0.75 ng / mL; e.g., about 0.25 ng / mL, about 0.30 ng / mL, about 0.35 ng / mL, about 0.40 ng / mL, about 0.45 ng / mL, about 0.50 ng / mL, about 0.55 ng / mL, about 0.60 ng / mL, about about 0.65 ng / mL, about 0.70 ng / mL, or about 0.75 ng / mL). Prior to receiving the trigger, the subject's serum LH level may be about 1.0 mIU / mL to about 2.5 mIU / mL (e.g., about 1.0 mIU / mL to about 1.5 mIU / mL, about 1.5 mIU / mL to about 2.0 mIU / mL, or about 2.0 mIU / mL to about 2.5 mIU / mL; e.g., about 1.0 mIU / mL, about 1.25 mIU / mL, about 1.5 mIU / mL, about 1.75 mIU / mL, about 2 mIU / mL, about 2.25 mIU / mL, or about 2.5 mIU / mL). Prior to receiving the trigger, the subject's serum FSH level may be about 11 mIU / mL to about 14 mIU / mL (e.g., about 11 mIU / mL to about 12 mIU / mL, about 12 mIU / mL to about 13 mIU / mL, or about 13 mIU / mL to about 14 mIU / mL; e.g., about 11 mIU / mL, about 12 mIU / mL, about 13 mIU / mL, or about 14 mIU / mL).

[0211] A triggering agent (e.g., a follicle triggering agent) may be administered over a period of time to produce a follicle stimulation protocol that is a minimal stimulation protocol. The minimal stimulation protocol is configured by a skilled practitioner to trigger the release of cells in about 3 days. A "minimal stimulation protocol" is a stimulation process that is shorter in duration than a typical in vitro fertilization (IVF) stimulation protocol to help induce the ovaries to produce oocytes. Typically, the average time span using a standard IVF stimulation protocol is about 8-14 days. A minimal stimulation regimen may induce cell release in 8 days or less (e.g., 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day; e.g., between 1-3 days, between 2-4 days, between 3-5 days, between 4-6 days, between 5-7 days, or between 6-8 days; e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days), which is a shorter period of time compared to the 8-14 days of a standard IVF stimulation regimen. The average time to perform a minimal stimulation regimen may be 2 days. The average time to perform a minimal stimulation regimen may be 3 days. The average time to perform a minimal stimulation regimen may be 4 days. The average time to perform a minimal stimulation regimen may be 5 days. The average time to perform a minimal stimulation regimen may be 6 days. In one embodiment, a minimal stimulation regimen may not require the administration of a follicle trigger to successfully retrieve and subsequently mature the oocytes. In one embodiment, a minimal stimulation protocol may include selecting a first trigger (eg, a follicle trigger) and selecting a second trigger (eg, a follicle trigger) based on follicle measurement and / or other biological sample data.

[0212] C. Oocyte retrieval

[0213] After follicular stimulation, an oocyte (or a group of cells comprising an oocyte) is removed from the subject. "Oocyte" as used in the present disclosure is a reproductive cell derived from the ovary. About 24-48 hours (e.g., between 24-32 hours, between 32-40 hours, between 40-48 hours; for example, about 24 hours, about 28 hours, about 32 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours) after the last dose of triggering agent (e.g., follicle triggering agent) is administered, the subject can undergo oocyte retrieval. On the day of oocyte retrieval, blood tests for one or more hormone levels (such as E2, LH, FSH and / or P4) can be performed to ensure quality indicators, hormone levels are within range and / or hCG doses have been ingested. On the day of oocyte retrieval, the hormone level of E2 can be about 300 pg / mL to about 450 pg / mL (e.g., about 300 pg / mL to about 350 pg / mL, about 350 pg / mL to about 400 pg / mL, or about 400 pg / mL to about 450 pg / mL; for example, about 300 pg / mL, about 325 pg / mL, about 350 pg / mL, about 375 pg / mL, about 400 pg / mL, about 425 pg / mL, or about 450 pg / mL). On the day of oocyte retrieval, the hormone level of LH can be about 3 mIU / mL to about 6 mIU / mL (e.g., about 3 mIU / mL to about 4 mIU / mL, about 4 mIU / mL to about 5 mIU / mL, or about 5 mIU / mL to about 6 mIU / mL; for example, about 3 mIU / mL, about 3.5 mIU / mL, about 4 mIU / mL, about 4.5 mIU / mL, about 5 mIU / mL, about 5.5 mIU / mL, or about 6 mIU / mL). On the day of oocyte retrieval, the hormone level of FSH can be about 6 mIU / mL to about 9 mIU / mL (e.g., about 6 mIU / mL to about 7 mIU / mL, about 7 mIU / mL to about 8 mIU / mL, or about 8 mIU / mL to about 9 mIU / mL; for example, about 6 mIU / mL, about 6.5 mIU / mL, about 7 mIU / mL, about 7.5 mIU / mL, about 8 mIU / mL, about 8.5 mIU / mL, or about 9 mIU / mL). On the day of oocyte retrieval, the hormone level of P4 can be about 0.5 ng / mL to about 1.5 ng / mL (e.g., about 0.5 ng / mL to about 1.0 ng / mL, about 0.75 ng / mL to about 1.0 ng / mL, about 1.0 ng / mL to about 1.5 ng / mL, or about 1.25 ng / mL to about 1.5 ng / mL; for example, about 0.5 ng / mL, about 0.75 ng / mL, about 1.0 ng / mL, about 1.25 ng / mL, or about 1.5 ng / mL).

[0214] Oocytes (or groups of cells comprising oocytes) can be removed from the subject using methods known in the art. For example, oocytes can be removed by aspiration using transvaginal ultrasound, wherein a needle guide on the probe aspirates the released follicular contents. The follicular aspirate can then be examined using a dissecting microscope and washed with HEPES medium (G-MOPS Plus, ) and filtered through a 70 μm cell strainer ( Corning) filtration. The oocytes and / or COCs are then transferred to a culture dish and culture medium to begin co-culture and appropriate controls, as described herein. Other retrieval methods may include extraction equipment such as a needle, syringe, vial, lancet, vacuum collection tube (ECT), tourniquet, vacuum extraction tube system, any combination thereof, etc. For example, the extraction equipment may include a butterfly needle set.

[0215] The oocytes retrieved may include, but are not limited to, immature oocytes, mature oocytes, groups of one or more oocytes, groups of one or more cells (such as cumulus-oocyte complexes), etc. "Cumulus-oocyte complexes" (COCs) used in the present disclosure are oocytes containing one or more surrounding cumulus cells. A COC may contain immature oocytes. A COC may contain mature oocytes.

[0216] " Immature oocyte " used in the present disclosure is one or more immature germ cells derived from the ovary. In some embodiments, the immature oocyte can be an oocyte including but not limited to a germ vesicle stage (GV) and a metaphase I stage (MI) oocyte, as further described below. In some embodiments, the immature oocyte can be a plurality of oocytes. The immature oocyte can be an immature cumulus-oocyte complex (COC) taken from a subject. " Mature oocyte " used in the present disclosure can be one or more mature oocytes in the metaphase II stage (MII). Once taken out, COC can be left standing for 1 hour, 2 hours, 3 hours or longer to allow equilibrium to in vitro conditions, to achieve in vitro maturation.

[0217] When taking out, any one or more oocytes or cells taken out as described herein can be used to freeze and store suitably for future use, analysis or experiment using methods known in the art. In addition, any one or more oocytes or cells taken out as described herein can be used freshly (i.e., can be used immediately, such as for in vitro maturation or any one or more analysis or experiment as described herein).

[0218] II. Oocyte rescue method

[0219] A. Oocyte denudation

[0220] According to the above-mentioned method for taking out oocytes, one or more COCs may need oocyte stripping. As described in the present disclosure, "oocyte stripping" refers to removing cumulus cells or other cell types from oocytes by mechanical separation, chemical separation or its combination. Several methods of oocyte stripping are known in the art. In some embodiments, stripping can be carried out in IVM holes, and most cumulus and / or granulosa cells are removed by pipetting to gently mechanically separate cells. If enzymolysis is required, cells can be transferred to a separate culture dish for hyaluronidase treatment. COCs can be stripped using a stripper tip, and then washed in IVM culture medium or MOPS plus culture medium to clean oocytes for imaging, and hyaluronidase is inactivated when needed. The stripper tip can include 200 microns and / or 400 microns for fine cleaning. In some embodiments, oocytes in reproductive vesicle (GV) stage) and metaphase I (MI) stage can be prepared and used for cultivation after COC stripping. The stripped COC can be transferred to a separate culture dish for imaging.

[0221] B. Co-culture of oocytes for in vitro maturation

[0222] i. Co-culture contents and duration

[0223] In the methods described herein, oocytes can be co-cultured with specialized granulosa cells and / or specialized matrix cells. "Specialized granulosa cells" and "specialized matrix cells" refer to cumulus cells surrounding oocytes to ensure the healthy development of oocytes and embryos. In some embodiments, granulosa cells and / or matrix co-culture cells are derived from human induced pluripotent stem cells (hiPSC). "Co-culture" used in the present disclosure is a cell culture device in which two or more different cell groups grow with a certain degree of contact between them. In some embodiments, steroidogenic granulosa cells derived from human induced pluripotent stem cells (hiPSC) can be co-cultured with immature oocytes (COCs) to reconstruct the follicle niche in vitro, promoting rapid and effective oocyte maturation in a manner that enhances oocyte health and developmental capacity. "Steroidogenic granulosa cells" used in the present disclosure are granulosa cells expressing high levels of steroidogenic enzymes that produce estradiol. For example, steroidogenic granulosa cells can be parietal granulosa cells extracted from antral follicles. The use of steroidogenic granulosa cells in co-culture with COCs can increase oocyte in vitro maturation after egg / oocyte retrieval, allowing all retrieved eggs / oocytes to be utilized by providing nutrients, raw materials, and mechanical support directly to the oocytes during gametogenesis and folliculogenesis. Steroidogenic granulosa cells can be grown in standard IVF and IVM culture media and perform oocyte maturation of immature COCs, as described below. This can increase the total amount of healthy oocytes available for IVF and reduce the number of egg / oocyte retrieval procedures that users undergo.

[0224] In some embodiments of the method, a cell culture can be formed by combining an immature oocyte with a specialized granulosa cell and / or a specialized stromal cell, which is added to the cell culture to mature the oocyte following a minimal stimulation protocol, thereby producing a COC. In one embodiment, one or more specialized granulosa cells and / or specialized stromal cells can be thawed during the resting period of one or more COCs. In one embodiment, 50,000-150,000 specialized granule cells (e.g., 50,000-60,000 cells, 60,000-70,000 cells, 70,000-80,000 cells, 80,000-90,000 cells, 90,000-100,000 cells, 100,000-110,000 cells, 110,000-120,000 cells, 120,000-130,000 cells, 130,000-140,000 cells, or 140,000-150,000 cells; e.g., 50,000 cells In some embodiments, 50,000 cells, 20,000 cells, 30,000 cells, 40,000 cells, 55,000 cells, 60,000 cells, 65,000 cells, 70,000 cells, 75,000 cells, 80,000 cells, 85,000 cells, 90,000 cells, 95,000 cells, 100,000 cells, 105,000 cells, 110,000 cells, 115,000 cells, 120,000 cells, 125,000 cells, 130,000 cells, 135,000 cells, 140,000 cells, 145,000 cells, or 150,000 cells) can be combined with a COC during culture. In one embodiment, the thawed specialized granulosa cells can be placed in culture prior to COC removal, including any time from about 24-120 hours in advance (e.g., about 24-48 hours, about 48-72 hours, about 72-96 hours, about 96-120 hours; for example, about 24-36 hours, about 30-40 hours, about 36-48 hours, about 48-56 hours, about 56-72 hours, about 72-84 hours, about 80-96 hours, about 90-100 hours, about 96-108 hours, about 108-120 hours; for example, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 56 hours, about 60 hours, about 65 hours, about 72 hours, about 78 hours, about 86 hours, about 92 hours, about 96 hours, about 102 hours, about 110 hours, about 115 hours, about 120 hours). COCs can be transferred into medium containing thawed specialized granulosa cells to form swarm cultures as described in more detail below.In one embodiment, the swarm culture can be cultured in an incubator for any time between 12 and 48 hours (e.g., 12 to 16 hours, 12 to 20 hours, 18 to 24 hours, 18 to 36 hours, 24 to 36 hours, 36 to 48 hours; e.g., 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours). Co-cultivation can be performed at a biologically suitable temperature, e.g., 37°C.

[0225] In some embodiments of the method, the recovered oocytes (including immature cumulus-oocyte complexes) can be subjected to group culture. "Group culture" is a combination of extracted COCs and one or more additional cells. Additional cells may include any cells grown with the extracted COCs. Additional cells may include specialized stromal cells. Additional cells may include specialized granulosa cells. In one embodiment, group cultures can be cultured and / or incubated for a specific length of time, such as between 12-120 hours (e.g., 12-24 hours, 12-36 hours, 24-48 hours, 36-60 hours, 54-72 hours, 68-96 hours, 96-120 hours; for example, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours, 110 hours, 112 hours, 114 hours, 116 hours, 118 hours or 120 hours). For example, the colony culture may include culturing the COC with a co-culture of granulosa cells, as further described below. In some embodiments, the colony culture may include culturing a control group of COCs without co-culture, as further described below. In some embodiments, the user can donate immature oocytes, such as the oocytes in the GV phase and the MI phase, which can be used in culture medium as a part of colony culture to help COC growth. Oocyte donation can follow the oocyte removal process as described above. The experimenter participating in oocyte donation can be different or identical with the experimenter related to the second biological sample containing immature COC. In some embodiments, the oocyte donation experimenter can experience stimulation scheme as described above.

[0226] In some embodiments, the maturity of the oocytes removed from the subject can determine the length of time that the oocytes are co-cultured with ovarian supporting cells (e.g., specialized granulosa cells and / or specialized stromal cells). For example, oocytes with lower maturity (e.g., GV oocytes) may require a longer co-culture period than oocytes at a more advanced stage of meiosis (e.g., MI oocytes).

[0227] In some embodiments for oocyte culture, the cell culture medium may include LAG medium (Medicult, ). For example, LAG medium can be used for incubation after removal of oocytes and / or COCs from a minimal stimulation protocol. For example, modified MediCult IVM medium can be used as a baseline control during culture. In some embodiments, the cell culture medium may include Figure 4 Metabolites exemplified in . For example, the modified Medicourt IVM culture medium may include human serum albumin, FSH, hCG, androstenedione, doxycycline, or any combination thereof. The culture medium may be pre-cultured in a standard sterile 37°C incubator balanced for about 18 to 24 hours (e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours), wherein O2 (e.g., with 1-10% O2 atmosphere, such as 4-8% O2 or 5-7% O2, e.g., 6% O2) and appropriate CO2 levels, as known in the art. Co-culture and specialized granular cell cultures may be adherent cell cultures in cell culture dishes or flasks. Co-cultures and specialized granular cell cultures may be suspension cell cultures in cell culture flasks. Cell culture materials and methods include standard sterile cell culture methods known in the art. Cell morphology and cell viability may be assessed by one or more established methods known in the art.

[0228] In some embodiments, co-cultivation is carried out according to Figure 5 The steps outlined in . For example, a population of ovarian support cells (e.g., ovarian granulosa cells) can be cryopreserved and thawed. In some embodiments, the ovarian support cells are centrifuged to form a cell pellet and then resuspended in a culture medium suitable for in vitro maturation. In some embodiments, the ovarian support cells are additionally centrifuged one or more times and each time resuspended in an in vitro maturation culture medium. The ovarian support cells can then be co-cultured with oocytes obtained from a subject undergoing an ART procedure to induce oocyte maturation.

[0229] ii. Granulosa cells from hiPSCs

[0230] The specialized granulocytes used in the methods described herein can be produced from hiPSC using a scheme guided by a transcription factor (TF). In some embodiments, hiPSC can be transformed with any one or more plasmids encoding one or more transcription factors. In some embodiments, hiPSC can be transformed by electroporation, liposome-mediated transformation, virus-mediated gene transfer, and other cell transformation methods known in the art. In some embodiments, the gene expression of the desired transcription factor can be induced in a doxycycline-dependent manner. In some embodiments, the transcription factor is constitutively expressed. In some embodiments, the plasmid or expression vector for reprogramming hiPSC may have a reporter gene, such as a fluorescent protein. In some embodiments, hiPSC can be differentiated into stromal cells and induce the expression of transcription factors (including GATA4, FOXL2, or a combination thereof). In some embodiments, hiPSC can be differentiated into granulocytes, and the transcription factors induced to be expressed include FOXL2, NR5A1, GATA4, RUNX1, RUNX2, or a combination thereof. hiPSCs can also be differentiated into granulosa cells by expressing KLF2, TCF21, NR2F2, or a combination thereof in addition to one or more transcription factors among FOXL2, NR5A1, GATA4, RUNX1, and / or RUNX2.

[0231] hiPSC reprogramming to stromal cells and / or granulosa cells can be determined by genotyping methods known in the art. hiPSC reprogramming to granulosa cells can be determined by protein expression using one or more methods known in the art. The differentiation of hiPSC to stromal cells can be determined by the relative expression of typical biomarkers of stromal cell types, including NR2F2, etc. known in the art. The differentiation of hiPSC to granulosa cells can be determined by the relative expression of typical biomarkers of granulosa cell types, including granulosa cell biomarkers such as AMHR2, CD82, FOXL2, FSHR, IGFBP7, KRT19, STAR, WNT4, or a combination thereof known in the art. In some embodiments, hiPSC reprogramming to granulosa cells can be determined by the production of growth factors and / or hormones (including estradiol and progesterone), which can fully support the in vitro maturation of the recovered oocytes through paracrine and juxtacrine cell signaling. In some embodiments, the resulting granulosa cells produce estradiol under the stimulation of androstenedione and FSH or forskolin. In some embodiments, the granulosa cells described herein can be produced in multiple batches. In some embodiments, granulosa cells can be frozen and thawed before the co-culture method. In some embodiments, granulosa cells are newly differentiated before the in vitro maturation method. In some embodiments, granulosa cells can be inoculated and balanced for 2-8 hours (e.g., 2-3 hours, 2-4 hours, 3-4 hours, 4-6 hours, 5-7 hours, 6-8 hours; For example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours) before adding oocytes for in vitro maturation.

[0232] In some embodiments, a subject may donate hiPSCs. hiPSC donation may follow the oocyte retrieval process as described above. The subject participating in hiPSC donation may be different or the same as the subject from which the oocytes were retrieved. In some embodiments, the hiPSC donor may undergo a stimulation regimen as described above.

[0233] In some embodiments, hiPSC, granulosa cells, cumulus cells, oocytes, GV oocytes, MI oocytes, MII oocytes, and all other types of cells described in the present disclosure can be lysed, genomic material extracted, and quick-frozen for further manipulation and / or analysis (e.g., as part of the omics data collection technology described in Section II (C) (iii) below). For example, cells can be enzymatically lysed using enzymes such as lysozyme, lysostaphylococcus enzymes, lysosomal enzymes, cellulases, proteases, or polysaccharidases. Other lysis methods can be applied, such as chemical lysis, detergent lysis, alkaline lysis, mechanical lysis, thermal lysis, acoustic lysis, physical lysis, non-mechanical lysis, and other lysis methods known in the art. In some embodiments, the culture medium can be quick-frozen. The freezing method may include the use of a cryoprotectant (such as dimethyl sulfoxide) and / or any other freezing method known in the art.

[0234] iii. Transgenic granulosa cells

[0235] The specialized transgenic granulosa cells utilized in the methods described herein can be produced using clustered regularly interspaced short palindromic repeats (CRISPR) technology. "CRISPR" is a programmable technology that edits DNA at precise locations for specific genetic code fragments. CRISPR technology may include CRISPR-CAS 9. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses a CRISPR sequence as a guide to identify and cut a specific DNA strand complementary to a CRISPR sequence to allow exogenous nucleic acids to be inserted into the genome of a cell. For example, CRISPR-based gene editing technology can be used to introduce one or more genes encoding factors that induce differentiation into ovarian supporting cells (e.g., ovarian granulosa cells) into the iPSC genome. These factors include, for example, FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

[0236] Exemplary CRISPR systems include systems utilizing Cas9 enzymes. Cas9 enzymes together with CRISPR sequences form the basis of CRISPR-Cas9 technology, which can be used to edit genes in organisms. CRISPR technology may include 1 type of CRISPR system, including type I (cas3), type III (cas10), type IV, and 12 subtypes. CRISPR technology may include 2 types of CRISPR systems, including type II (cas9), type V (cas12), type VI (cas13), and 9 subtypes. In some embodiments, CRISPR technology may involve CRISPR-Cas design tools, which are computer software platforms and bioinformatics tools for facilitating the design of guide RNA (gRNA) for CRISPR / Cas gene editing systems. For example, CRISPR-Cas design tools may include: CRISPRon, CRISPRoff, Invitrogen TrueDesign Genome Editor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPOR, sgRNA Designer, Synthego Design Tool, etc. CRISPR technology can also be used as a diagnostic tool. For example, CRISPR-based diagnostics can be combined with enzymatic processes such as SHERLOCK-based in vitro transcription analysis (SPRINT). SPRINT can be used for high-throughput or portable point-of-care diagnostic devices to detect a variety of substances, such as metabolites in subject samples or pollutants in environmental samples.

[0237] C. Oocyte rescue

[0238] i. Oocyte Scoring

[0239] In any phase of in vitro maturation or after in vitro maturation, oocytes and / or granulosa cells can be appropriately frozen and stored for future analysis, experiment or for oocyte maturation. According to the morphology of the oocyte determined by image analysis, scoring indicators can be adopted to score it. In some embodiments, the distribution of scoring indicators may include imaging the group culture, and analyzing the image of one or two of the co-culture and non-co-culture growth only culture medium control groups. In some embodiments, oocytes are scored and compared and analyzed at any stage of in vitro maturation. For example, the colony culture image may include pre-culture group COC image, group COC image after cultivation, and oocyte image stripped after cultivation. In some embodiments, the oocytes receiving scoring have never been frozen. In some embodiments, the oocytes scored by image analysis can be thawed after frozen storage. In some embodiments, the oocytes scored can be taken out without in vitro maturation as described above. In some embodiments, the oocytes scored can be cultivated without the granulosa cells. In some embodiments, images can be sent to qualified third parties, such as embryologists, developmental biologists or other related technology practitioners, to perform scoring distribution.

[0240] In some embodiments of the methods described herein, oocytes can be assessed and subsequently classified according to their maturation state according to the following criteria:

[0241] GV - Germline vesicles are present within the oocyte and usually contain nucleoli.

[0242] There is no germinal vesicle in the MI-oocyte, and no polar body is present in the perivitelline space between the oocyte and the zona pellucida.

[0243] There is no germinal vesicle in the MII-oocyte, and no polar body is present in the perivitelline space between the oocyte and the zona pellucida.

[0244] In some embodiments of the method, the scoring index may include a total oocyte score (TOS), as a function of the colony culture imaged via correlation microscopy or imaging analysis software analysis. The methods and methods of TOS have been described in the art (Lazzaroni-Tealdi et al., PLoS One 10: e0143632, 2015). Oocyte scoring may include indicators such as shape, size, egg quality characteristics, perivitelline space (PVS) structure, zona pellucida (ZP), polar body (PB) morphology and other possible limiting conditions. The total oocyte score on the oocyte image before and after the culture for generating the TOS index can be based on a scale system of -6 to +6.

[0245] For oocyte shape, a value of -1 can be assigned if the oocyte morphology is poor (oocyte overall color is dark and / or oval), a value of 0 can be assigned if it is almost normal (oocyte overall color is not so dark and shape is not too oval), and a value of +1 can be assigned if it is judged to be normal. For oocyte size: if oocyte size is defined as abnormally small or large, a value of -1 can be assigned if the size is below 120 μm or greater than 160 μm. A value of 0 can be assigned if the size is almost normal, that is, the deviation from the normal value does not exceed 10 μm, and a value of +1 can be assigned if the oocyte size is within the normal range of >130 μm and <150 μm. Regarding the characteristics of the ooplasm, a value of -1 can be assigned if the ooplasm is very granular and / or very vacuolated and / or shows multiple inclusions. A value of 0 can be assigned if it is only slightly granular and / or shows only a few inclusions. Lack of granularity and inclusions may result in a value of +1. Regarding the structure of the perivitelline space (PVS), if the PVS is abnormally large, absent, or very granular, the PVS can be defined as -1. For a moderately enlarged PVS and / or a small PVS and / or a PVS with small granularity, it can be assigned a value of 0. A normal-sized PVS without granules can be assigned a value of +1. Regarding the zona pellucida (ZP), if the ZP is very thin or thick (<10 μm or >20 μm), the oocyte can be assigned a value of -1. If the deviation of the ZP from the normal value does not exceed 2 μm, it can be assigned a value of 0. A normal zona (>12 μm and <18 μm) can be assigned a value of +1. Regarding the polar body (PB) morphology, the PB morphology is defined as follows: flat and / or multiple PBs or zero PBs, granular and / or abnormally small or large PBs are assigned a value of -1. A PB judged as fair but not excellent can be assigned a value of 0, while a PB of normal size and shape can be assigned a value of +1. In some embodiments, the MII oocyte PB score may not be summarized in the TOS.

[0246] In some embodiments of the method, the scoring metric may include: Figure 1A The results analysis is performed using the TOS defined and exemplified in . Parametric or nonparametric tests can be used to determine the significance of the findings found during the analysis. The results analysis can be used to determine the GV to MII oocyte maturation rate; GV to MI oocyte maturation rate; MI to MII oocyte maturation rate; mean total oocyte score; mean oocyte shape; mean oocyte size; mean egg quality; mean PVS quality; mean ZP quality; mean polar body quality, etc. In some embodiments, these results can be reported as means, medians, and deviations.

[0247] ii. In vitro fertilization and embryo culture

[0248] In some embodiments of the present methods, the quality or maturation state of any one or more of the eggs or oocytes described herein can be assessed, such as by the scoring criteria described herein, to determine whether they are ready for in vitro fertilization and embryogenesis.

[0249] In some embodiments of the method, the egg or oocyte can be matured by in vitro maturation and then used for IVF and / or ART described herein. Any one or more oocytes can be used for intracytoplasmic sperm injection (ICSI). After the egg is fertilized by contact with one or more sperm cells, the fertilized egg subsequently formed can be matured in vitro to produce an embryo, such as a morula or blastocyst (e.g., a mammalian blastocyst), which can then be transferred to the uterus of a subject (e.g., a subject from whom oocytes were initially harvested) to be implanted in the endometrium. Embryo transplantation that can be performed using the methods described herein includes fresh embryo transplantation, in which the egg or oocyte used for embryo generation is removed from the subject, and the subsequent embryo is transferred to the subject in the same menstrual cycle. Alternatively, embryos can also be produced first, then cryopreserved for long-term storage, and then transplanted into the subject.

[0250] iii. Omics data collection and analysis of oocytes, cells and culture media

[0251] In some embodiments of the method, the scoring index may include an analysis based on oocytes. For example, frozen cell lysates and cell culture medium may be analyzed to carry out batch RNA sequencing, whole genome sulfite sequencing (WGBS), proteomics and metabolomics based on mass spectrometry. Cell culture medium may be used for metabolomics analysis to determine the change of molecular content in culture medium after co-cultivation compared with the pre-culture culture medium control. This may be used to describe the dynamic changes of paracrine signals between granulosa cells and oocytes. The data collected may then be summarized to carry out downstream analysis to determine the change of epigenetic state, metabolite presence and gene expression between different co-cultivation conditions and controls.

[0252] In some embodiments of the method, the analysis based on omics can include genomics, proteomics, transcriptomics, pharmacogenomics, epigenomics, microbiome, lipidomics, glycomics, transcriptomics, culture group and / or any other applicable omics understood by those skilled in the art. In some embodiments, after cultivation, the oocytes that failed to mature, showed GV or MI features can be harvested together with the relevant granulosa cells in their culture for single cell RNA sequencing. For this reason, oocytes and granulosa cells can be quick-frozen and library preparation can be performed. In the oocytes showing MII oocyte development, half of the oocytes and their relevant granulosa cells can be harvested, and single cell RNA sequencing is performed using the above-mentioned quick-freezing method described in the present disclosure. The remaining half of the MII oocytes can be used for proteomic research. The culture medium under all conditions can be quick-frozen and used for metabolomics and proteomics, to identify the production of cholesterol metabolite levels and paracrine proteins. For example, frozen cell lysates and cell culture medium can be analyzed to perform batch RNA sequencing, whole genome sulfite sequencing (WGBS), proteomics and metabolomics based on mass spectrometry. Cell culture media can be used for metabolomics analysis to determine changes in the content of molecules in the culture media after co-culture compared to pre-culture culture media controls to analyze the dynamic changes in paracrine signaling between granulosa cells and oocytes. Because the culture media components are snap-frozen, the samples are effectively quenched and can be used for metabolic assessment. The collected data can then be aggregated for downstream analysis to determine changes in epigenetic state, metabolite presence, and gene expression between different co-culture conditions and controls.

[0253] III. Ex Vitro Compositions and Cell Culture Media

[0254] A. Granulosa cells from hiPSCs

[0255] The specialized granulocytes used in the methods described herein can be produced from hiPSC using a scheme guided by a transcription factor (TF). In some embodiments, hiPSC can be transformed with any one or more plasmids encoding one or more transcription factors. In some embodiments, hiPSC can be transformed by electroporation, liposome-mediated transformation, virus-mediated gene transfer, and other cell transformation methods known in the art. In some embodiments, the gene expression of the desired transcription factor can be induced in a doxycycline-dependent manner. In some embodiments, the transcription factor is constitutively expressed. In some embodiments, the plasmid or expression vector for reprogramming hiPSC may have a reporter gene, such as a fluorescent protein. In some embodiments, hiPSC can be differentiated into granulocytes, and the transcription factors induced to be expressed include FOXL2, NR5A1, GATA4, RUNX1, RUNX2, or a combination thereof. The reprogramming of hiPSC into granulocytes can be determined by genotyping methods known in the art. The reprogramming of hiPSC into granulocytes can be determined by protein expression using one or more methods known in the art. The differentiation of hiPSC to granulosa cells can be determined by the relative expression of typical biomarkers of granulosa cell types, including granulosa cell biomarkers such as AMHR2, CD82, FOXL2, FSHR, IGFBP7, KRT19, STAR, WNT4 or a combination thereof known in the art. In some embodiments, hiPSC reprogramming to granulosa cells can be determined by the production of growth factors and / or hormones (including estradiol and progesterone), which can fully support the in vitro maturation of recovered oocytes through paracrine and near-crine cell signaling. In some embodiments, the resulting granulosa cells produce estradiol under the stimulation of androstenedione and FSH or forskolin. In some embodiments, the granulosa cells described herein can be produced in multiple batches. In some embodiments, granulosa cells can be frozen and thawed before the co-culture method. In some embodiments, granulosa cells are newly differentiated before the in vitro maturation method. In some embodiments, granulosa cells can be plated and equilibrated for 2-8 hours (e.g., 2-3 hours, 2-4 hours, 3-4 hours, 4-6 hours, 5-7 hours, 6-8 hours; e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours) prior to adding oocytes for in vitro maturation.

[0256] In some embodiments, the subject can donate hiPSC. hiPSC donation can follow the oocyte retrieval process as described above. The subject participating in hiPSC donation can be different or the same as the subject of the oocyte retrieval. In some embodiments, the hiPSC donor can undergo a stimulation scheme as described above. In some embodiments, hiPSC, granulosa cells, cumulus cells, oocytes, GV stage oocytes, MI stage oocytes, MII stage oocytes and all other types of cells described in the present disclosure can be lysed, genomic materials are extracted and quick-frozen as the last step of the culture process. For example, cells can be enzymatically lysed using enzymes such as lysozyme, lysostaphylococcus enzymes, lysosomal enzymes, cellulases, proteases or polysaccharidases. Other lysis methods can be applied, such as chemical lysis, detergent lysis, alkaline lysis, mechanical lysis, thermal lysis, acoustic lysis, physical lysis, non-mechanical lysis and other lysis methods known in the art. In some embodiments, the culture medium can be quick-frozen. The freezing method may include the use of a cryoprotectant (such as dimethyl sulfoxide) and / or any other freezing method known in the art.

[0257] B. Transgenic granulosa cells

[0258] Specialized transgenic granulosa cells can be generated using clustered regularly interspaced short palindromic repeats (CRISPR) technology. "CRISPR" is a programmable technology that targets specific genetic code segments to edit DNA at precise locations. CRISPR technology may include CRISPR-CAS 9. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses a CRISPR sequence as a guide to recognize and cut a specific DNA strand that is complementary to the CRISPR sequence. The Cas9 enzyme, together with the CRISPR sequence, forms the basis of a technology called CRISPR-Cas9, which can be used to edit genes in organisms. CRISPR technology may include Class 1 CRISPR systems, including Type I (cas3), Type III (cas10), Type IV, and 12 subtypes. CRISPR technology may include Class 2 CRISPR systems, including Type II (cas9), Type V (cas12), Type VI (cas13), and 9 subtypes. In some embodiments, CRISPR technology may involve CRISPR-Cas design tools, which are computer software platforms and bioinformatics tools for facilitating the design of guide RNA (gRNA) for CRISPR / Cas gene editing systems. For example, CRISPR-Cas design tools may include: CRISPRon, CRISPRoff, Invitrogen TrueDesign Genome Editor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPOR, sgRNA Designer, SynthegoDesign Tool, etc. CRISPR technology can also be used as a diagnostic tool. For example, CRISPR-based diagnosis can be combined with an enzymatic process such as SHERLOCK-based in vitro transcription analysis (SPRINT). SPRINT can be used for high-throughput or portable point-of-care diagnostic devices to detect a variety of substances, such as metabolites in subject samples or pollutants in environmental samples.

[0259] C. Cell culture medium

[0260] Granulocytes, such as granulocytes derived from iPSCs (e.g., hiPSCs) or transgenic granulocytes (as described above), can be provided as a composition that also contains a cell culture medium (e.g., IVF, IVM, (e.g., MediCult IVM medium) or LAG medium). The cell culture medium can include human serum albumin (e.g., about 5-15 mg / mL, such as 10 mg / mL), FSH (e.g., about 70-80 mIU / mL, such as 75 mIU / mL), hCG (e.g., about 95-105 mIU / mL, such as 100 mIU / mL), androstenedione (e.g., about 495-505 ng / mL, such as 500 ng / mL), doxycycline (e.g., 0.5-1.5 μg / mL, such as 1 μg / mL) and other compounds, such as hyaluronidase and / or dPBS.

[0261] IV. Apparatus and Related Methods for Oocyte Maturation

[0262] The present disclosure provides devices for assisted reproductive technology (e.g., IVF). FIG. 1 depicts two exemplary devices (e.g., see Figure 1A and Figure 1B ). refer to Figure 1A , shows an exemplary embodiment of a device 100 for inducing human oocyte maturation in vitro. Figure 1B , showing an exemplary embodiment of a device 100 for in vitro rescue of oocytes after assisted stimulation.

[0263] A. Computing equipment

[0264] In the device of the present disclosure (for example, Figure 1A and Figure 1B), the apparatus 100 includes a computing device 104. The computing device 104 includes a processor 108 and a memory 112 communicatively connected to the processor 108, wherein the memory 112 contains instructions for configuring the processor 108 to perform a linking process. The processor 108 and the memory 112 are contained in the computing device 104. The computing device 104 may include any computing device described in the present disclosure, including but not limited to a microcontroller, a microprocessor, a digital signal processor (DSP) and / or a system on a chip (SoC) described in the present disclosure. The computing device may include, be included in and / or communicate with a mobile device (such as a mobile phone or a smart phone). The computing device 104 may include a single computing device operating independently, or may include two or more computing devices that operate in cooperation, in parallel, sequentially, etc.; the two or more computing devices may be included together in a single computing device or in two or more computing devices. The computing device 104 may interface or communicate with one or more additional devices via a network interface device, as described in further detail below. The network interface device may be used to connect the computing device 104 to one or more of various networks and one or more devices. Examples of network interface devices include, but are not limited to, network interface cards (e.g., mobile network interface cards, LAN cards), modems, and any combination thereof. Examples of networks include, but are not limited to, wide area networks (e.g., the Internet, enterprise networks), local area networks (e.g., networks associated with offices, buildings, campuses, or other relatively small geographic spaces), telephone networks, data networks associated with telephone / voice providers (e.g., mobile communications provider data and / or voice networks), direct connections between two computing devices, and any combination thereof. The network may employ wired and / or wireless modes of communication. In general, any network topology may be used. Information (e.g., data, software, etc.) may be transmitted to or from a computer and / or computing device. Computing device 104 may include, but is not limited to, for example, a computing device or computing device cluster located at a first location and a second computing device or computing device cluster located at a second location. Computing device 104 may include one or more computing devices dedicated to data storage, security, load-balanced traffic distribution, and the like. The computing device 104 may distribute one or more computing tasks as described below to multiple computing devices of the computing device, which may operate in parallel, serially, redundantly, or in any other manner for allocating tasks or memory between computing devices. The computing device 104 may be implemented using a "shared nothing" architecture, where data is cached at the worker, which, in one embodiment, may enable scalability of the system 100 and / or computing device.

[0265] Continuing with reference to the apparatus of the present disclosure (e.g., Figure 1A and Figure 1B), the computing device 104 can be designed and / or configured to perform any method, method step, or sequence of method steps in any embodiment described in the present disclosure in any order and with any degree of repetition. For example, the computing device 104 can be configured to repeatedly perform a single step or sequence until the desired or commanded result is achieved; the repetition of a step or sequence of steps can be performed iteratively and / or recursively, using the output of a previous repetition as the input of a subsequent repetition, aggregating the input and / or output of a repetition to produce an aggregated result, reducing or recursively one or more variables (such as global variables), and / or dividing a larger processing task into a set of smaller processing tasks that are processed iteratively. The computing device 104 can perform any step or sequence of steps described in the present disclosure in parallel, such as using two or more parallel threads, processor cores, etc. to perform a step two or more times simultaneously and / or substantially simultaneously; the division of tasks between parallel threads and / or processes can be performed according to any scheme suitable for dividing tasks between iterations. After reading the entire contents of the present disclosure, those skilled in the art will recognize the various ways in which steps, sequences of steps, processing tasks, and / or data can be subdivided, shared, or otherwise processed using iteration, recursion, and / or parallel processing.

[0266] Continuing with reference to the apparatus of the present disclosure (e.g., Figure 1A and Figure 1B ), in addition, the computing device 104 may use a machine learning process 116 to perform the determination, classification and / or analysis steps, methods, processes, etc. described in the present disclosure. As used in the present disclosure, a "machine learning process" is a process that automatically uses a body of data called "training data" and / or "training set" to generate an algorithm that will be executed by a computing device / module to produce an output based on the data provided as input; this is in contrast to non-machine learning software programs in which the commands to be executed are predetermined by the user and written in a programming language. The machine learning process 116 may utilize supervised, unsupervised, lazy learning processes and / or neural networks.

[0267] i. Oocyte maturation

[0268] Specific reference Figure 1AThe computing device 104 is configured to promote rapid and effective maturation of oocytes in a manner that enhances the health and developmental capacity of oocytes, thereby helping human oocytes to mature in vitro. The computing device 104 is configured to receive first biological sample data from a first biological sample 120 associated with a user. The "biological sample data" used in the present disclosure is data that provides the characteristics of the biological, genetic, biochemical and / or physiological characteristics, composition or activity of a biological sample. "Biological sample" is any information associated with a user. The biological sample may include laboratory samples stored in a biobank for research. In some embodiments, the first biological sample 120 may include body fluids, including blood, saliva, urine, semen (seminal fluid), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage fluid), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, optic nerve fluid, gastric juice, breast milk, cell culture supernatant, etc. The biological sample may include medical diagnosis, user input describing user feelings and / or symptom complaints, information collected from wearable devices associated with the user, etc. For example, the biological sample may include information obtained from a medical professional visit, such as a health history. In another non-limiting example, the biological sample may include information such as data collected from a wearable device worn by the user, which is intended to collect information related to the user's sleep pattern, exercise pattern, etc. In one embodiment, the first biological sample 120 is collected on a specific date and / or time of the user's menstrual cycle. For example, but not limited to, the first biological sample 120 can be collected on the second day of the user's menstrual cycle to assess one or more hormone levels, such as E2, FSH, LH, P4 and / or AMH. The first biological sample 120 can be used to determine one or more indicators of the user's overall health, including but not limited to ovarian reserve health and / or circulating hormone levels. For example, a healthcare professional can use this information to monitor cycle progress and inform regimens and / or drug selection.

[0269] All "users" of the present disclosure are organisms, such as humans, plants, animals, and all other organisms composed of cells. In some embodiments, biological samples can be extracted from users by extraction equipment. "Extraction equipment" is a device and / or tool that can obtain, record and / or determine the measured values ​​related to the sample. The extraction equipment may include a needle, a syringe, a vial, a lancet, a vacuum collection tube (ECT), a tourniquet, a vacuum extraction tube system, any combination thereof, etc. For example, the extraction equipment may include a butterfly needle group. The computing device 104 can receive the biological sample in the form of data uploaded to the memory. The data may include, for example, the measured values ​​of serum calcium, phosphate, electrolytes, blood urea nitrogen and creatinine, uric acid, etc.

[0270] Still refer to Figure 1A, the computing device 104 may receive the first biological sample data from the biological sample database 124. A "biological sample database" is a database containing all data related biological samples of a user including analytical information.

[0271] ii. Oocyte rescue

[0272] Specific reference Figure 1B The computing device 104 is configured to assist in the rescue of oocytes in vitro after stimulation. "Oocyte rescue" is the process of maturing immature oocytes in vitro, which are usually ignored in standard in vitro maturation procedures. The "oocyte" used in the present disclosure is a reproductive cell derived from the ovary. After the simulation can refer to a standard in vitro fertilization IVF stimulation protocol performed on a user. As used in the present disclosure, a "stimulation protocol" is a drug injection process spanning a specific period of time to induce the ovary to produce one or more oocytes. All "users" in the present disclosure are organisms, such as humans, plants, animals, and all other organisms composed of cells. In some embodiments, after stimulation, it can refer to a minimum stimulation protocol. As used in the present disclosure, a "minimum stimulation protocol" is a stimulation process with a shorter duration than a general in vitro fertilization (IVF) stimulation protocol to help induce the ovary to produce eggs. Typically, the average time span using a standard IVF stimulation protocol is about 8-14 days. The minimum stimulation protocol can induce cell release within 2-6 days, which is a shortened time period compared to 8-14 days. The average time to execute the minimum stimulation protocol 132 can be 3 days. The longest time is 6 days and the shortest time is 2 days.

[0273] Still refer to Figure 1B, the computing device 104 is configured to receive biological sample data 120 from a biological sample associated with the user, the biological sample including at least one oocyte. "Biological sample data" used in the present disclosure is data that provides the characteristics of the biological, genetic, biochemical and / or physiological characteristics, composition or activity of the biological sample. "Biological sample" used in the present disclosure is a biological laboratory sample (e.g., a blood sample or other body fluid sample) obtained from a subject. In some embodiments, the oocyte can be an immature oocyte. "Immature oocyte" used in the present disclosure is one or more immature reproductive cells derived from the ovary. In some embodiments, the immature oocyte can be an oocyte including a GV stage and / or a MI stage oocyte. In some embodiments, the immature oocyte can be a plurality of oocytes. The immature oocyte can be an immature cumulus-oocyte complex (COC) taken from the mother. As used in the present disclosure, "cumulus-oocyte complex" is an oocyte surrounded by specialized granulosa cells. As used in the present disclosure, "specialized granulosa cells" are cumulus cells that surround the oocyte to ensure the healthy development of the oocyte and the embryo. In some embodiments, the immature oocyte may comprise an oocyte to which a specialized granulosa cell is added to mature the oocyte in cell culture, thereby producing a COC. In some embodiments, the biological sample may comprise a body fluid, including blood, saliva, urine, semen (seminal fluid), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage fluid), pericardial fluid, peritoneal fluid, amniotic fluid, nasal fluid, optic nerve fluid, gastric juice, breast milk, cell culture supernatant, etc.

[0274] Still refer to Figure 1B , the computing device 104 can receive the biological sample data 120 of the biological sample related to the user after stimulation from the biological sample database 124. The "biological sample database" used in the present disclosure is a database containing all data-related biological samples of the user containing analysis information. In some embodiments, the biological sample database 124 can include a stimulation scheme index. The "stimulation scheme index" used in the present disclosure is a data structure associated with the user information completed about the stimulation scheme, such as: user age, user BMI, COC quantity taken out, mature MII stage oocyte quantity, immature MI oocyte quantity, immature GV oocyte quantity, AMH level (μg / L), antral follicle count (AFC) at the time of the last ultrasound examination, user oocyte retrieval day E2 level (ng / L), user oocyte retrieval day P4 level (ng / L), user oocyte retrieval day LH (IU / L), user oocyte retrieval day FSH (IU / L), stimulation days, gonadotropin used and total injection dose (IU).

[0275] iii. Biological sample database

[0276] In some embodiments of the device (e.g., Figure 1A and Figure 1B ), the biological sample database 124 may include a systemic hormone index. The "systemic hormone index" used in the present disclosure is a data structure related to medical knowledge related to systemic hormone therapy. For example, the systemic hormone index may include side effects and risks, the correct hormone administration method, the correlation between E2, LH, FSH and / or P4 deficiency and systemic hormones, etc. In some embodiments, the biological sample database 124 may be communicatively connected to the computing device 104. The "communication connection" used in the present disclosure refers to a connection through a connection, attachment or connection between two or more related objects, thereby allowing information to be received and / or transmitted between them. For example, but not limited to, such a connection may be wired or wireless, direct or indirect, and may be located between two or more components, circuits, devices, systems, etc., thereby allowing data and / or signals to be received and / or transmitted between them. The data and / or signals between them may include, but are not limited to, electrical, electromagnetic, magnetic, video, audio, radio and microwave data and / or signals, combinations thereof, and the like. The communication connection may be achieved by, for example, but not limited to, wired or wireless electronics, digital or analog communications, directly or through one or more intermediate devices or components. In addition, a communicative connection may include electrically coupling or connecting at least one output of one device, component, or circuit to at least one input of another device, component, or circuit. For example, but not limited to, mutual communication between components of a computing device is achieved through a bus or other facility. A communicative connection may also include an indirect connection, for example, but not limited to, through a wireless connection, radio communication, low-power wide area network, optical communication, magnetic coupling, capacitive coupling, or optical coupling. In some cases, the term "communicative coupling" may be used in this disclosure instead of a communicative connection.

[0277] The biological sample database 124 and all other databases described in the present disclosure may be implemented, without limitation, as a relational database, a key-value retrieval database (such as a NOSQL database), or any other format or structure deemed appropriate by a person skilled in the art after reading the entire disclosure. The biological sample database 124 may alternatively or additionally be implemented using a distributed data storage scheme and / or data structure (such as a distributed hash table, etc.). The biological sample database 124 may include multiple data entries and / or records. The data entries in the database may be marked with or linked to one or more additional information elements, which may be reflected in data entry units and / or linked tables, such as tables associated by one or more indexes in a relational database. After reading the entire disclosure, a person skilled in the art will recognize that the data entries in the database may store, retrieve, organize and / or reflect the various ways in which the data and / or records used herein, as well as data categories and / or populations consistent with the present disclosure.

[0278] iv. Stimulus Package

[0279] In the device of the present disclosure (see, for example, Figure 1A ), the computing device 104 is configured to assign a stimulation protocol 132 to the user based on the first biological sample 120. In some embodiments, the stimulation protocol 132 can be assigned based on the hormone levels of the measured biological sample. A "stimulation protocol" is a course of drug (e.g., trigger) injections spanning a specific period of time (e.g., follicular triggering period) to induce the ovaries to produce one or more oocytes.

[0280] As used in the present disclosure, "measured hormone levels" are quantitative values ​​representing one or more hormone levels of a user. The measured hormone levels of a biological sample may include estradiol (E2), luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone (P4), estrone (E1), estriol (E3), testosterone, androgen, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), adrenaline levels, etc. In some embodiments, the measurement of hormone levels can be based on blood analysis of biological samples. For example, blood analysis can include plasma hormone analysis technology. In some embodiments, the measurement of hormone levels can be based on salivary hormone testing technology. The measurement of hormone levels can be based on other forms of analysis, such as hair, urine, and any other forms of biological samples described in the present disclosure.

[0281] In one embodiment (see, e.g., Figure 1A), the selection of a stimulation regimen can be accomplished using information obtained from ultrasound. "Ultrasound" as used in the present disclosure is any procedure that uses sound waves to generate one or more images of a user's body. For example, ultrasound can be used to obtain images of a subject's reproductive organs and / or tissues. In one embodiment, an ultrasound examination can be performed at a specific time during the subject's menstrual cycle. For example, a subject may undergo an ultrasound examination on day 2 of their cycle, which can be used to determine follicle size and / or follicle count. This information can be used to select a stimulation regimen and / or adjust the stimulation regimen. For example, a subject whose ultrasound examination shows polycystic ovary syndrome (PCOS) may need to adjust the dosage of one or more drugs received and / or used during the stimulation regimen. In addition, the length of her stimulation regimen can be modified based on her PCOS diagnosis. In one embodiment, ultrasound can be repeated one or more times throughout the subject's entire stimulation regimen, and the information obtained can be used to adjust her stimulation regimen in real time.

[0282] In addition, the use of contraceptive measures by the subject may also affect the allocation of the stimulation scheme. "Contraception" used in the present disclosure refers to any method and / or device for preventing pregnancy due to sexual intercourse. This may include, but is not limited to, any drug, technology, equipment and / or contraceptive measures used by the subject. Whether the subject uses contraceptive measures may help determine at which stage of the menstrual cycle she should start the stimulation scheme. For example, but not limited to, a subject who does not use any contraceptive measures can start using recombinant follicle stimulating hormone (rFSH) to carry out the stimulation scheme on the second day of the menstrual cycle. In another non-limiting example, a subject who is taking contraceptive measures can start using rFSH to carry out the stimulation scheme 5 days after taking the last oral contraceptive pill. In one embodiment, rFSH can be used to stimulate 2 to 3 days, depending on the tolerance, follicle size and / or growth dynamics of the subject. After this 2 to 3 day time window, a 1 to 2 day buffer period can be used to monitor the follicle size and allow the follicle to further mature and develop. "Buffer period" used in the present disclosure refers to any time period during which the drugs used in the entire stimulation scheme are not applied and / or consumed. The buffer period can last for example 1 day, 2 days, 3 days, etc. During the buffer period, subjects may continue to undergo one or more ultrasound examinations to monitor their progress.

[0283] Once the follicle size reaches 8-10mm, the subject can be triggered by injecting a dose of human chorionic gonadotropin (hCG). In one embodiment, a double hCG injection can be used to induce follicle maturation to prepare one or more follicles for removal. Blood tests for one or more hormone levels such as E2, P4 and LH can be performed on the triggering day of the double dose of human chorionic gonadotropin (hCG) injection to monitor hormone levels. After the double dose of hCG injection day, one or more hormone levels can be measured, such as by blood tests to determine and check the doses of E2, P4 and LH.

[0284] Approximately 24-48 hours after the hCG dose is administered, the subject may undergo oocyte retrieval. On the day of oocyte retrieval, blood tests may be performed to detect the level of one or more hormones such as E2, LH and / or P4 to ensure that quality indicators, hormone levels are within range and / or the hCG dose has been taken in. The assigned stimulation scheme 132 may include a minimum stimulation scheme configured to trigger cell release within a 3-day time span. "Minimum stimulation scheme" is a stimulation process with a shorter duration compared to a general in vitro fertilization (IVF) stimulation scheme to help induce ovaries to produce eggs. Typically, the average time span using a standard IVF stimulation scheme is about 8-14 days. The minimum stimulation scheme can induce cell release within 2-6 days, which is a shortened time period compared to 8-14 days. The average time to perform the minimum stimulation scheme 132 can be 3 days. The longest time is 6 days and the shortest time is 2 days. In one embodiment, the minimal stimulation protocol may include selecting a first trigger (e.g., a follicle trigger) based on the first biological sample 120, and selecting a second trigger (e.g., a follicle trigger) based on a follicle measurement, which is disclosed in more detail below. "Folliculometry" as used in the present disclosure is any measurement of a follicle. A follicle may include any sac containing an unfertilized egg found in an ovary. Follicle measurements may be obtained using any method described herein including, for example, ultrasound, manual measurement, automatic measurement, etc.

[0285] A "trigger" is a chemical that triggers the production of ovarian cells. A trigger (e.g., a follicle trigger) can include any substance, including any over-the-counter and / or prescription product. Triggers (e.g., follicle triggers) may include, for example, Lupron, manufactured by Abbott Laboratories, headquartered in North Chicago, Illinois; Ganirelix, manufactured by Ferring Pharmaceuticals, headquartered in Saint-Pres, Switzerland; Cetrotide, manufactured by Merck Global, headquartered in Whitehouse Station, Readington Township, New Jersey; Gonal-F, manufactured by Merck Global, Follistim, manufactured by Merck Global; Bravelle, manufactured by Ferring Pharmaceuticals, headquartered in Saint-Pres, Switzerland, Clomid, manufactured by Patheon Pharmaceuticals Inc., headquartered in Waltham, Massachusetts; Serephene, manufactured by Teva, headquartered in Tel Aviv-Yafo, Israel; Glucophage, manufactured by Merck Global; Fortamet, manufactured by Mylan, headquartered in Canonsburg, Pennsylvania; Pregnyl, manufactured by Schering Plough, headquartered in Kenilworth, New Jersey; and Novarel produced by Ferring Laboratories; Repronex produced by Ferring Pharmaceuticals, Inc.; Factrel produced by Zoetis Canada Inc. headquartered in Kirkland, Canada; Menopur produced by Ferring Pharmaceuticals, and other drugs that are applicable to induce ovarian cell production as understood by those skilled in the art. Triggers (e.g., follicle triggers) may include human serum albumin, FSH, hCG, androstenedione, and doxycycline. The computing device 104 can assign the trigger used based on the measured hormone level of the first biological sample 120. In some embodiments, the computing device 104 can use a machine learning process to generate and / or train a machine learning model including a classifier. In one embodiment, continue to refer to Figure 1A , a machine learning model can be used to assign a specific stimulation regimen to a user based on the first biological sample 120.

[0286] v. Classifier

[0287] As used in the present disclosure, a "classifier" is a machine learning model, such as a mathematical model, a neural network, or a program generated by a machine learning algorithm called a "classification algorithm," as described in further detail below, which classifies input into data categories or groups and outputs data categories or groups and / or labels associated therewith. A classifier can be configured to output at least one data that labels or otherwise identifies a group of data that are clustered together, are found to be close under a distance metric as described below, and so on.

[0288] In the device of the present disclosure (for example, Figure 1A and Figure 1B ), may be classified using (but not limited to) linear classifiers (such as but not limited to logistic regression and / or naive Bayes classifiers), nearest neighbor classifiers (such as k nearest neighbor classifiers), support vector machines, least squares support vector machines, Fisher linear discriminant, quadratic classifiers, decision trees, boosted trees, random forest classifiers, learning vector quantization, and / or neural network-based classifiers. The computing device 104 may be configured to generate a classifier using a naive Bayes classification algorithm. The naive Bayes classification algorithm generates a classifier by assigning a class label to a problem instance, represented as a vector of element values. The class labels are drawn from a finite set. The naive Bayes classification algorithm may include generating a set of algorithms that assume that given a class variable, the value of a particular element is independent of the value of any other element. The naive Bayes classification algorithm can be expressed as P(A / B)=P(B / A)P(A)÷P(B) based on Bayes' theorem, where P(A / B) is the probability of assuming A given the data B, also known as the posterior probability; P(B / A) is the probability of data B assuming A is true; P(A) is the probability that A is true regardless of the data, also known as the prior probability of A; and P(B) is the probability of the data regardless of the assumption. The naive Bayes algorithm can be generated by first converting the training data into a frequency table. Then, the computing device 104 can calculate the likelihood table by calculating the probabilities of different data entries and classification labels. The computing device 104 can calculate the posterior probability of each category using the naive Bayes equation. The category containing the highest posterior probability is the predicted result. The naive Bayes classification algorithm may include a Gaussian model that follows a normal distribution. The naive Bayes classification algorithm may include a polynomial model for discrete counting. The Naive Bayes classification algorithm may include a Bernoulli model, which may be utilized when the vector is binary.

[0289] Continue to refer Figure 1A and Figure 1B, the computing device 104 can be configured to generate a classifier using a K-nearest neighbor (KNN) algorithm. The "K-nearest neighbor algorithm" used in the present disclosure includes a classification method that uses feature similarity to analyze the similarity of features outside the sample to the training data to classify the input data into one or more feature clusters and / or categories represented by the training data; this can be performed by representing the training data and the input data in vector form, and using one or more vector similarity metrics to identify the classification within the training data, and determining the classification of the input data. The K-nearest neighbor algorithm may include specifying a K value or guiding the classifier to select the number of K entries that are most similar to the training data of a given sample, determining the most common classifier for the entry in the database, and classifying the known samples; this can be performed recursively and / or iteratively to generate a classifier that can be used to classify the input data into further samples. For example, a set of initial samples can be performed to cover the initial heuristics and / or "first guesses" of the output and / or relationship, which can be seeded without restriction using expert input received according to any process described herein. As a non-limiting example, the initial heuristic method may include a sorting of the associations between the input and training data elements. Heuristics may include selecting a few highest-numbered associations and / or training data elements.

[0290] Continue to refer Figure 1A and Figure 1B, generating a k-nearest neighbor algorithm can generate a first vector output containing a cluster of data entries, generate a second vector output containing input data, and calculate the distance between the first vector output and the second vector output using any suitable norm (such as cosine similarity, Euclidean distance measurement, etc.). Each vector output can be represented as an n-tuple value, but not limited to this, where n is at least two values. Each value in the n-tuple value can represent a measurement or other quantitative value related to a given data or attribute category, examples of which will be provided in further detail below; the vector can be represented in n-dimensional space using the axis of each value category represented in the n-tuple value (but not limited to this), so that the vector has a geometric direction that characterizes the relative number of attributes in the n-tuple compared to each other. When the direction of two vectors and / or the relative number of values ​​in each vector relative to each other are the same, they can be considered equivalent; therefore, as a non-limiting example, for the purposes of this disclosure, a vector represented as [5,10,15] can be considered equivalent to a vector represented as [1,2,3]. Vectors may be more similar when their directions are more similar; vectors may be more different when their directions are more divergent; however, vector similarity may alternatively or additionally be determined using an average of similarities between similar attributes, or any other similarity measure applicable to any n-tuple of values, or aggregating numerical similarity measures for the purposes of loss functions. Any of the vectors described herein may be scaled so that each vector represents each attribute along an equivalent scale of values. Each vector may be "normalized," or divided by a "length" attribute, such as a length attribute l derived using the Pythagorean norm: where a i is the attribute number i of the vector. Scaling and / or normalization can make vector comparisons independent of the absolute number of attributes while preserving any dependence on attribute similarity; this can be advantageous, for example, when the cases represented in the training data are represented by different numbers of samples, which may result in scale-equivalent vectors with different values.

[0291] refer to Figure 1AThe computing device 104 and / or another device may generate a regimen classifier 128 using a classification algorithm. A "regimen classifier" is a classifier that is trained to ingest biological samples associated with a user and output / assign a stimulation regimen 132 to the associated user based on the received training data. The training data may consist of inputs and / or outputs including systemic hormone index data, feedback on past stimulation regimen 132 assignments, and any other data described in the present disclosure. The training data may be received from a biological sample database 124. In some embodiments, the training data may include multiple data entries, including biological samples associated with outputs including assigned stimulation regimens. In some embodiments, the training data may include inputs, such as assigned stimulation regimens associated with outputs, such as pregnancy success rates or scoring indicators described in the present disclosure. In some embodiments, the training data may include correlations between stimulation regimens and associated side effects. In some embodiments, the training data may include methods and procedures for preventing triggers from overstimulating the ovaries. For example, the training data may include the number of injections of multiple doses of a specific trigger (e.g., a follicle trigger) that a user can accept before overstimulation occurs.

[0292] In any device of the present disclosure (e.g., Figure 1A and Figure 1B ), the computing device 104 can use the training data to train any classifier or other machine learning model. In some embodiments, the training data may include biological sample data and / or correlations between biological samples extracted from users and maturity levels, scores, or other numerical and / or quantitative fields associated with oocytes, such as but not limited to the form of training examples. The training examples can be input by one or more experts. "Expert" used in the present disclosure refers to a person skilled in the art or organization. Expert knowledge can be retrieved from the feedback index in the biological sample database 124.

[0293] A medical professional can remove a biological sample containing an oocyte from a user after simulation, such as a doctor inserting an extraction device into an ovarian follicle containing an egg and extracting the egg and surrounding fluid. An "extraction device" is a device and / or tool capable of obtaining, recording, and / or determining measurements associated with a sample. The extraction device may include a needle, a syringe, a vial, a lancet, a vacuum collection tube (ECT), a tourniquet, a vacuum extraction tube system, any combination thereof, and the like. For example, the extraction device may include a butterfly needle set. Oocyte retrieval may include obtaining immature oocytes, mature oocytes, COCs, and any other cell types found in the ovaries that participate in reproduction.

[0294] refer to Figure 1BThe computing device 104 is configured to determine the maturity level 128 of at least one oocyte. The "maturity level" used in the present disclosure is data representing an assessment of the oocyte stage of oogenesis; the maturity level may include quantitative elements and / or fields, such as days, hours, etc. of maturity, the stage of maturity, and / or a score representing the degree of maturity. In some embodiments, the maturity level 128 may be an assessment of the oocyte maturation stage of oogenesis. Oocyte maturation refers to the release of meiotic arrest, allowing the oocyte to progress from meiotic prophase I to meiotic metaphase II. Determining the maturity level 128 of the oocyte may include stripping the oocyte. Oocyte stripping refers to removing the somatic cell layer surrounding the oocyte. For example, the COC can be stripped to remove the granulosa cell layer surrounding the oocyte in order to determine the nuclear maturity of the oocyte. Oocyte stripping may include enzymatic and mechanical methods with the aid of hyaluronidase and sterile glass pipettes, as described below.

[0295] Still refer to Figure 1B In some embodiments, determining the maturity level 128 may include determining the maturity level 128 using a machine learning process 116. For example, but not limited to, the machine learning process 116 may be used to generate and train a machine learning model comprising a classifier; the classifier may classify oocytes into maturity levels based on the biological sample data.

[0296] Still refer to Figure 1BThe computing device 104 and / or another device may generate a maturity classifier 132 using a classification algorithm. The "maturity classifier" used in the present disclosure is a classifier that is trained to ingest biological sample data 120 and output a maturity level 128. Additionally or alternatively, the maturity classifier 132 may be trained to ingest oocyte stripping data to output a maturity level 128. The "oocyte stripping data" used in the present disclosure is data that provides characteristics of biological, genetic, biochemical and / or physiological properties, composition or activity of a stripped oocyte. The computing device 104 may receive oocyte stripping data from a biological sample database. In some embodiments, the computing device 104 may train the maturity classifier 132 using training data (including correlations between oocytes and maturity levels, such as but not limited to the form of training examples). The training examples may be derived from a maturity level index and a feedback index contained in a biological sample database. The "maturity level index" used in the present disclosure is a data structure associated with biological knowledge associated with an oocyte oogenesis stage (such as an oocyte maturation stage). The "feedback index" used in the present disclosure is a data structure that associates past maturity level 128 evaluations performed by a computing device with communications from a third party. The "third party" used in the present disclosure is a qualified individual or organization, such as an embryologist, a statistician, etc. In some embodiments, the maturity classifier 132 training data can be received from a biological sample database. For example, the biological sample data 120 may include data related to an immature COC that has been stripped, and the training data is used to determine that the maturity level 128, and the oocyte produced by the stripping is a GV and / or MI oocyte. In some embodiments, the machine learning model comprising the classifier is trained to classify the oocyte into maturity levels according to the biological sample data, and the machine learning process 116 can also be used to calculate the maturity level, score or other numerical value and / or quantitative field associated with the oocyte.

[0297] vi. Second Biological Sample

[0298] The devices described herein (see, e.g., Figure 1A ) can be configured to receive a second biological sample. Figure 1AIn the present invention, the computing device 104 is configured to receive second biological sample data from a second biological sample 136 related to the user, wherein the second biological sample 136 includes at least one oocyte. The "oocyte" used in the present disclosure is a reproductive cell derived from an ovary. The oocyte may include, but is not limited to, an immature oocyte, a mature oocyte, a group of one or more oocytes, a group of one or more cells, a cumulus-oocyte complex, etc. The "cumulus-oocyte complex" used in the present disclosure is an oocyte containing one or more surrounding cumulus cells. COC may contain immature oocytes. COC may contain mature oocytes. The "immature oocyte" used in the present disclosure is one or more immature reproductive cells derived from the ovary. In some embodiments, the immature oocyte may be an oocyte including, but not limited to, a reproductive vesicle (GV) and a metaphase 1 (M1) oocyte, as further described below. In some embodiments, the immature oocyte may be a plurality of oocytes. The immature oocyte may be an immature cumulus-oocyte complex (COC) taken from a subject. "Mature oocyte" used in the present disclosure is one or more mature germ cells derived from the ovary. Once taken out, COC can be left to stand for 2-3 hours to allow balance to in vitro conditions. In one embodiment, oocyte can be combined with specialized granulosa cells. "Specialized granulosa cells" are cumulus cells that can surround oocytes to ensure the healthy development of oocytes and embryos. In some embodiments, immature oocytes can include oocytes, wherein specialized granulosa cells are added to mature oocytes in cell culture (e.g., co-culture), thereby producing COC after extracting the second biological sample 136. The second biological sample 136 may include body fluids as described above. In one embodiment, one or more specialized granulosa cells can be thawed in the resting period of one or more COCs. In one embodiment, in the culture process, any number of specialized granulosa cells between 50,000 and 100,000 can be combined with COC. In one embodiment, the thawed specialized granulosa cells can be put into culture medium before COC is taken out (including any time of 24 to 120 hours in advance). COC can be transferred to the culture medium containing the specialized granulosa cells thawed to form a group culture as described in more detail below. In one embodiment, the group culture can be cultured in an incubator for a period of 12 to 48 hours. The computing device 104 can receive the second biological sample data from the biological sample database 124 as described above. The second biological sample 136 can be extracted using an extraction device and received as described above. In some embodiments, the computing device 104 can record the measured hormone levels of the second biological sample 136 using the methods disclosed above. Specialized granulosa cells can be produced using clustered regularly spaced short palindromic repeats (CRISPR) technology."CRISPR" is a programmable technology that targets specific genetic code fragments to edit DNA at precise locations. CRISPR technology may include CRISPR-CAS 9. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses a CRISPR sequence as a guide to recognize and cut a specific DNA chain complementary to the CRISPR sequence. The Cas9 enzyme, together with the CRISPR sequence, forms the basis of a technology called CRISPR-Cas9, which can be used to edit genes in organisms. CRISPR technology may include Class 1 CRISPR systems, including Type I (cas3), Type III (cas10), Type IV, and 12 subtypes. CRISPR technology may include Class 2 CRISPR systems, including Type II (cas9), Type V (cas12), Type VI (cas13), and 9 subtypes. In some embodiments, CRISPR technology may involve CRISPR-Cas design tools, which are computer software platforms and bioinformatics tools for facilitating the design of guide RNA (gRNA) for CRISPR / Cas gene editing systems. For example, CRISPR-Cas design tools may include: CRISPRon, CRISPRoff, Invitrogen TrueDesign Genome Editor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPOR, sgRNA Designer, Synthego Design Tool, etc. CRISPR technology can also be used as a diagnostic tool. For example, CRISPR-based diagnosis can be combined with an enzymatic process such as SHERLOCK-based in vitro transcription analysis (SPRINT). SPRINT can be used for high-throughput or portable point-of-care diagnostic devices to detect a variety of substances, such as metabolites in subject samples or pollutants in environmental samples.

[0299] In some embodiments regarding the culture of the second biological sample 136, the cell culture medium may include LAG medium. For example, LAG medium may be used for incubation after the COC is removed from the stimulation protocol 132. The packaging size may be a 10 mL vial. It may be stored at 2-8°C in the dark for up to one month. The culture medium equilibration may be pre-cultured for 18 to 24 hours, including 100 ul of seed droplets and placed at 37°C in a 6% O 2 and appropriate CO 2incubator. In some embodiments, the cell culture medium may include IVM culture medium (e.g., 1 mL to 100 mL of culture medium, such as 1 mL, 5 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL, may be used for each co-culture). For example, a modified MediCult IVM culture medium may be used as a baseline control during culture. The packaging size may be a 10 mL vial. It may be stored at 2-8 ° C in the dark for up to one month. In some embodiments, the cell culture medium may include metabolites. For example, a modified MediCult IVM culture medium may include human serum albumin, FSH, hCG, androstenedione, doxycycline, and other compounds. Other cell culture materials and equipment may include: liquid nitrogen, hyaluronidase, dPBS, IVF qualified mineral oil, universal GPS culture dishes, G-NOPS plus medium, micropipettes, stripping pipettes, inverted ICSI microscope with camera, dry injection benchtop incubator, saturated humidity incubator, embryoscope, microcentrifuge, refrigerator, -20°C freezer to 100°C freezer, liquid nitrogen storage dehydrator, 35mm stripping culture dishes, stripping pipette tips, and other components included in the cell culture process as understood by those skilled in the art.

[0300] Still refer to Figure 1ADevice, in some embodiments, culturing the second biological sample 136 may include culturing immature cumulus-oocyte complex (COC) in colony culture. "Colony culture" is a combination of extracted COC and one or more other cells. Other cells may include any cells grown together with the extracted COC. Other cells may include specialized granulosa cells. In one embodiment, colony culture may be cultured and / or incubated for a specific time length, such as 12 to 120 hours. For example, colony culture may include culturing cumulus-oocyte complex with granulosa cell co-culture, as further described below. "Co-culture" is a cell culture device in which two or more different cell groups grow with a certain degree of contact. In some embodiments, colony culture may include culturing a COC control group without co-culture, as further described below. In some embodiments, a user may donate immature oocytes, such as reproductive vesicles (GV) and metaphase 1 (M1) oocytes, which may be used in culture medium as part of colony culture to help COC growth. Oocyte donation may follow the oocyte removal process as described above. The user who participates in oocyte donation can be different or identical with the experimenter relevant to the second biological sample containing immature COC.In some embodiments, oocyte donation user can experience stimulation scheme as above.In some embodiments, granulosa cell, cumulus cell, oocyte, GV oocyte, MI, oocyte and the cell of all other types described by the disclosure can be cracked, extract genomic material and quick-frozen.For example, cell can utilize enzymes such as lysozyme, lysostaphylococcus enzyme, lysosomal enzyme, cellulase, protease or polysaccharidase to carry out enzymatic cell lysis.Other lysis methods can be applied, such as chemical lysis, detergent lysis, alkaline lysis, mechanical lysis, thermal lysis, sonication, physical lysis, non-mechanical lysis etc.In some embodiments, culture medium can be quick-frozen.Freezing method can comprise and use any other freezing method described in cryoprotectant (such as dimethyl sulfoxide) and / or the disclosure.

[0301] Still refer to Figure 1A In the apparatus, computing device 104 is configured to assign a scoring indicator 152 to second biological sample 136 based on culturing second biological sample 136.

[0302] vii. Training Program

[0303] In the devices described herein (see, for example, Figure 1B), the computing device 104 is configured to assign a culture protocol 136 to the oocyte according to the maturity level 128. In some embodiments, the assigned oocyte can be a denuded oocyte. A "culture protocol" as used in the present disclosure is a cell culture process for growing cells under controlled conditions. The culture protocol 136 may include cell culture metabolites selected according to the maturity level 128; and a cell culture medium selected according to the maturity level 128. A "cell culture metabolite" as used in the present disclosure is a substance involved in cell metabolism that optimizes the synthesis of new molecules in cell culture. For example, a cell culture metabolite may include doxycycline. The computing device 104 may generate and train a culture classifier 140 using training data, which may include correlations between oocytes and / or maturity levels and cell culture protocols, such as but not limited to in the form of training examples. The training examples may be derived from a metabolite index, a cell culture medium index, a protocol index, a feedback index contained in a culture database. For example, the culture classifier 140 can take the determined GV oocyte from the maturity classifier 132 and assign the oocyte a culture protocol 136 containing IVM culture medium and 500ng / mL androstenedione. The training data for the culture classifier 140 can come from the culture database 144 and the biological sample reception. The "culture database 144" used in the present disclosure is a database related to scientific knowledge related to the cell culture process. The culture database 144 can be communicatively connected to the computing device 104 and implemented as described above. The culture database 144 may include a metabolite index, a cell culture medium index, and a protocol index. The "metabolite index" used in the present disclosure is a data structure related to scientific knowledge of metabolites. The metabolite index may contain data about the effects of specific metabolites in cell culture because it is related to the maturity level 128 of the oocyte and the dosage requirements and formulation. The "cell culture medium index" used in the present disclosure is a data structure related to scientific knowledge of cell culture medium. For example, the cell culture medium index may contain data about the best culture medium and preparation / storage method used in cell culture. The "protocol index" used in the present disclosure is a data structure related to scientific knowledge related to IVM cell culture procedures. For example, the protocol index can include co-culture and population culture methods used in IVM.

[0304] refer to Figure 1BDevice, in some embodiments, culture scheme 136 may include culturing oocytes with co-cultures containing granulosa cells produced by human induced pluripotent stem cells (hiPSC). The cultured oocytes may be stripped oocytes. "Co-culture" used in the present disclosure is a cell culture device in which two or more different cell groups grow in contact to a certain extent. hiPSC can be produced using clustered regularly spaced short palindromic repeats (CRISPR) technology. "CRISPR" is a programmable technology that edits DNA at precise locations for specific genetic code fragments. In some embodiments, CRISPR-based gene editing technology can be used to introduce one or more genes encoding factors that induce differentiation into ovarian supporting cells (e.g., ovarian granulosa cells) into the iPSC genome. These factors include, for example, FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

[0305] CRISPR technology may include CRISPR-CAS 9. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses a CRISPR sequence as a guide to recognize and cut a specific DNA strand that is complementary to the CRISPR sequence. The Cas9 enzyme, together with the CRISPR sequence, forms the basis of a technology called CRISPR-Cas9, which can be used to edit genes in organisms. CRISPR technology may include Class 1 CRISPR systems, including Type I (cas3), Type III (cas10), Type IV, and 12 subtypes. CRISPR technology may include Class 2 CRISPR systems, including Type II (cas9), Type V (cas12), Type VI (cas13), and 9 subtypes. In some embodiments, CRISPR technology may involve CRISPR-Cas design tools, which are computer software platforms and bioinformatics tools for facilitating the design of guide RNAs (gRNAs) for CRISPR / Cas gene editing systems. For example, CRISPR-Cas design tools may include: CRISPRon, CRISPRoff, Invitrogen TrueDesign GenomeEditor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPOR, sgRNADesigner, Synthego Design Tool, etc. CRISPR technology can also be used as a diagnostic tool. For example, CRISPR-based diagnosis can be combined with an enzymatic process such as SHERLOCK-based in vitro transcription analysis (SPRINT). SPRINT can be used for high-throughput or portable point-of-care diagnostic devices to detect a variety of substances, such as metabolites in subject samples or pollutants in environmental samples.

[0306] Still reference Figure 1B Device, in some embodiments, the user can donate hiPSC. The donation of hiPSC can follow the oocyte retrieval process as described above. The user participating in the hiPSC donation can be different or the same as the user associated with the biological sample. In some embodiments, the hiPSC donation user can undergo the stimulation scheme as described above. In some embodiments of the culture scheme 136 about the allocation, hiPSC, granulosa cells, cumulus cells, oocytes, GV stage oocytes, MI stage oocytes, MII stage oocytes and all other types of cells described in the present disclosure can be lysed, genomic materials can be extracted and quick-frozen as the last step of the culture process. For example, cells can be enzymatically lysed using enzymes such as lysozyme, lysostaphylococcus enzymes, lysosomal enzymes, cellulases, proteases or polysaccharidases. Other lysis methods can be applied, such as chemical lysis, detergent lysis, alkaline lysis, mechanical lysis, thermal lysis, sonication, physical lysis, non-mechanical lysis, etc. In some embodiments, the culture medium can be quick-frozen. The freezing method may include the use of a cryoprotectant (such as dimethyl sulfoxide) and / or any other freezing method described in the present disclosure.

[0307] Still refer to Figure 1B In the embodiment of oocyte culture, the cell culture medium may include LAG medium. For example, LAG medium can be used for incubation after COC is removed from the stimulation protocol. The packaging size can be a 10 mL vial. It can be stored at 2-8°C in the dark for up to one month. The culture medium balance can be pre-cultured for 18 to 24 hours, including a 100 μl seed droplet and placed in a 37°C incubator containing 6% O 2 and appropriate CO 2. In some embodiments, the cell culture medium may include IVM medium. For example, the modified MediCult IVM medium may be used as a baseline control during the culture process. The packaging size may be a 10 mL vial. It can be stored at 2-8°C in the dark for up to one month. In some embodiments, the cell culture medium may include metabolites. For example, the modified MediCult IVM medium may include human serum albumin, FSH, hCG, androstenedione, doxycycline, and other compounds. Other cell culture materials and equipment may include: liquid nitrogen, hyaluronidase, dPBS, IVF qualified mineral oil, universal GPS culture dishes, G-NOPS plus culture medium, micropipettes, stripping pipettes, inverted ICSI microscope with camera, dry injection tabletop incubator, saturated humidity incubator, embryoscope, microcentrifuge, refrigerator, -20°C freezer to 100°C freezer, liquid nitrogen storage dehydrator, 35mm stripping culture dishes, stripping pipette tips, and other components included in the cell culture process as understood by those skilled in the art.

[0308] viii. Training data

[0309] In some of the devices described herein (see, for example, Figure 1A ), computing device 104 is configured to receive the cultivation data 140 relevant to the second biological sample 136. "Cultivation data" is the data of the biological, genetic, biochemical and / or physiological characteristics, composition or active features of the biological sample of cell culture. Cultivation data 140 may include record data and identification of the growth trend of the COC formed due to the addition of specialized granulocytes to the second biological extraction. In one embodiment, the second biological sample 136 can be left to stand for 2-3 hours in the culture medium after taking out, to allow equilibrium to in vitro conditions. Computing device 104 may receive cultivation data 140 from a culture sample database 144. "Cultivation sample database" is a database of analytical data including the cultivation process and method of the relevant second biological sample 136. For example, cultivation data 140 may be an image of the second biological sample 136 of cultivation, wherein computing device 104 may be configured to analyze the image to obtain results, targets, etc. In some embodiments, computing device 104 may receive data from a culture sample database 144, such as a record of the process, results, targets, etc. of an embryologist about the second biological sample 136 of cultivation. The culture sample database 144 can be communicatively connected to the computing device 104 and implemented as described above. In some embodiments, the culture sample database 144 can include an oocyte analysis index. An "oocyte analysis index" is a data structure that contains rules, analysis methods, and methods for analyzing cell culture media. For example, the oocyte analysis index can include methods for oocyte scoring, result analysis, confounding variable analysis, etc.

[0310] In some of the devices described herein (see, for example, Figure 1B ), the computing device 104 is configured to receive culture data 148 according to the culture scheme 136. "Cultivation data 148" is data that provides the characteristics of the biological, genetic, biochemical and / or physiological characteristics, composition or activity of the biological sample of the cell culture. The culture data 148 may include recording data and identifying the growth trend of the COC formed by adding specialized granulosa cells (such as hiPSC) to the stripped or unstripped oocyte. The culture data 148 can be received from the oocyte index contained in the culture database 144. The "culture sample index" used in the present disclosure is a database containing analysis data of the culture process and method of the oocyte. For example, the culture data 148 can be an image of the oocyte cultured, wherein the computing device 104 can be configured to analyze the image to obtain results, goals, etc. In some embodiments, the culture database 144 may include an oocyte index. "Oocyte index" is a data structure that contains rules, analysis methods and methods for analyzing cell culture media. For example, the oocyte analysis index may include methods for scoring oocytes, result analysis, confounding variable analysis, etc. In some embodiments, computing device 104 can receive data from the feedback index in the cultivation database 144, such as the record of the embryologist about the process, result, target etc. of the oocyte of cultivation.Computing device 104 can train classifier or be configured to use other machine learning models of training data calculation scoring index.In some embodiments, training data can comprise the correlation between cultivation data and oocyte scoring, result analysis, confounding variable analysis, such as but not limited to the form of training examples.Training examples can be derived from the data in the oocyte index retrieved from the cultivation database 114 and the feedback index.

[0311] ix. Rating

[0312] "Scoring index" used in the present disclosure is a quantitative assessment measure used to compare and track the performance or generation of oocyte maturity. In one embodiment, the scoring index can be calculated after stripping. "Stripping" used in the present disclosure is any process of removing cells from an oocyte. Stripping can include any mechanical and / or enzymatic process. For example, but not limited to, stripping can include removing granulosa cells and / or cumulus cells from an oocyte. This can be performed mechanically and / or using one or more chemicals (such as enzymes) to help separate.

[0313] In some of the devices described herein (see, for example, Figure 1A), computing device 104 can receive subject information about the completion of stimulation scheme 132, such as: subject age, subject BMI, COC quantity taken out, AMH level (μg / L), antral follicle count (AFC) at the last ultrasound examination, subject oocyte retrieval day E2 level (ng / L), subject oocyte retrieval day P4 level (ng / L), subject oocyte retrieval day LH (IU / L), subject oocyte retrieval day FSH (IU / L), stimulation days, gonadotropin used and total injection dose (IU). Distribution scoring index 152 may include computing device 104 analyzing one or two image population cultures of co-culture and non-co-culture growth groups. Computing device 104 may receive: pre-culture group COC image, cultured group COC image, and cultured stripped oocyte image. In some embodiments, the image may be an image of a frozen lysate and a cell culture medium. In one embodiment, scoring index may include evaluating the presence of polar bodies in mature oocytes by microscopy. If polar bodies are found, oocytes can be selected and used for intracytoplasmic sperm injection (ICSI) fertilization and / or oocyte cryopreservation.

[0314] In some embodiments of the devices described herein (e.g., see Figure 1A and Figure 1B ), the image can be sent to a third party for scoring distribution. "Third party" refers to a qualified individual or organization, such as an embryologist, who analyzes the colony culture and formulates / distributes the scoring index 152. In addition, the computing device 104 can perform any determination, classification and / or analysis steps, methods, processes performed by the third party. In some embodiments, the scoring index 152 may include a total oocyte score (TOS) as a function of the analyzed imaging colony culture. The oocyte scoring may include indicators such as shape, size, egg quality characteristics, perivitelline space (PVS) structure, zona pellucida (ZP), polar body (PB) morphology. The "oocyte scoring" used in the present disclosure is a grading system for assessing the yield and quality of mature human oocytes. For example, the computing device 104 can be configured to perform a total oocyte score to the oocyte images before and after the culture, to generate a TOS index on a scale system of -6 to +6. The computing device 104 can generate and / or train a machine learning model including a classification algorithm (image classifier 148) to perform a total oocyte score. The training data may include any data described in the present disclosure, such as subject information, follicle dynamic information, oocyte scoring index 152, research sample table (e.g., oocyte scoring index 152 instruction set). The image classifier 148 can take the population culture image as input and output the total oocyte score using the training data. The training data may include data from the culture sample database 144 as described above.

[0315] The detailed disclosure of the machine learning model will be described in further detail below. For oocyte shape, if the oocyte morphology is poor (the overall color of the oocyte is dark and / or oval), a value of -1 can be assigned; if it is almost normal (the overall color of the oocyte is not so dark and the shape is not too oval), a value of 0 can be assigned; if it is judged to be normal, a value of +1 can be assigned. For oocyte size: If the oocyte size is defined as abnormally small or large, a value of -1 can be assigned if the size is less than 120μ or greater than 160μ. If the size is almost normal, that is, the deviation from the normal value does not exceed 10μ, a value of 0 can be assigned, and if the oocyte size is within the normal range>130μ and <150μ, a value of +1 can be assigned. Regarding the characteristics of the ooplasm, if the ooplasm is very granular and / or very vacuolated and / or shows multiple inclusions, a value of -1 can be assigned. If it is only slightly granular and / or shows only a small number of inclusions, a value of 0 can be assigned. Lack of granularity and inclusions may result in a value of +1. Regarding the structure of the perivitelline space (PVS), if the PVS is abnormally large, absent, or very granular, the PVS can be defined as -1. For a moderately enlarged PVS and / or a small PVS and / or a PVS with small granularity, it can be assigned a value of 0. A normal-sized PVS without granules can be assigned a value of +1. Regarding the zona pellucida (ZP), if the ZP is very thin or thick (<10 μm or >20 μm), the oocyte can be assigned a value of -1. If the deviation of the ZP from the normal value does not exceed 2 μm, it can be assigned a value of 0. A normal zona (>12 μm and <18 μm) can be assigned a value of +1. Regarding the polar body (PB) morphology, the PB morphology is defined as follows: flat and / or multiple PBs or zero PBs, granular and / or abnormally small or large PBs are assigned a value of -1. A PB judged as fair but not excellent can be assigned a value of 0, while a PB of normal size and shape can be assigned a value of +1. In some embodiments, the MII oocyte PB score may not be summarized in the TOS. In some embodiments, the TOS calculated by the computing device 104 can be cross-checked with an embryologist or similar technician in the field to confirm whether the quality score is biased by image quality. Feedback related to professional corrections, adjustments, and correlations can be added to the training data of the machine learning model.

[0316] In some embodiments of the devices described herein (e.g., see Figure 1A and Figure 1B), the computing device 104 can train a classifier or other machine learning model configured to calculate TOS using the training data. In some embodiments, the training data may include a correlation between culture data and image quality and a 6-point qualitative scale, such as but not limited to the form of training examples. The training examples can be derived from data in the oocyte index and feedback index retrieved from the culture database 144. The scoring index 152 may include performing result analysis according to TOS. The "result analysis" used in the present disclosure can be: 1.) measuring the maturation rate and oocyte quality score between group cultures; or 2.) measuring the maturation rate and oocyte quality score between control cultures and co-cultures. Parametric or non-parametric tests can be used to determine the significance of the findings during the analysis. The computing device 104 can use a classification algorithm using the above method to determine the GV to MII oocyte maturation rate; GV to MI oocyte maturation rate; MI to MII oocyte maturation rate; average total oocyte score; average oocyte shape; average oocyte size; average egg quality; average PVS quality; average ZP quality; average polar body quality, etc. In some embodiments, the results may be reported as mean, median, and deviation.

[0317] In some embodiments, and with continued reference to Figure 1A or Figure 1B , the computing device 104 can use the machine learning process 116 and / or models described in this disclosure to perform result analysis.

[0318] In with Figure 1AIn a relevant example, computing device 104 can train machine learning model to cultivate image output result analysis based on the group of input, wherein training data comprises oocyte scoring index, research sample table, subject information, feedback from computing device 104 programmers / third parties, data from the stimulation scheme of distribution and data of all other forms described by the present disclosure.Training data can come from biological sample database 124 and cultivation database 144.In some embodiments, communication from third parties can be input in machine learning process 116, to create machine learning model to generate scoring index.For example, third-party communication can comprise embryologist's note relevant to oocyte total score, wherein these notes are input in the machine learning model comprising classifier, to use training data, biological sample database 124 received and cultivation database 144 to generate result analysis, wherein comprise subject information, data from image classifier 148, data from the stimulation scheme of distribution, research sample table and data of any other form described in the present disclosure.Additionally or alternatively, machine learning process 116 can be used to send the communication relevant to the scoring index generated by computing device to third parties. For example, the oocyte scoring metric may be sent to a remote computing device operated by a third party in communication with the computing device 104, where the third party may perform further analysis, such as analysis of the results. Further to this example, third-party responses to communications generated by the computing device 104 may be uploaded to a database in communication with the computing device 104 and used as feedback in the training data.

[0319] Still refer to Figure 1A In some embodiments, the scoring index 152 may include an omics-based analysis. For example, frozen cell lysates and cell culture media can be analyzed for batch RNA sequencing, whole genome sulfite sequencing (WGBS), mass spectrometry-based proteomics and metabolomics. Cell culture media can be used for metabolomics analysis to determine the changes in molecular content in the culture medium after co-culture compared to the pre-culture culture medium control. This can be used by the computing device 104 to describe the dynamic changes of paracrine signals between granulosa cells and oocytes. The collected data can then be summarized for downstream analysis to determine the changes in epigenetic state, metabolite presence, and gene expression between different co-culture conditions and controls.

[0320] In with Figure 1BIn another relevant example, computing device 104 can train machine learning model to output result analysis based on the culture image of input, wherein training data comprises oocyte scoring index, research sample table, subject information, feedback from computing device 104 programmer / third party, data from stimulation scheme and data of all other forms described by the present disclosure.Training data can come from biological sample database and culture database 144.In some embodiments, communication from third party can be input in machine learning process 116, to create machine learning model to generate scoring index 152.For example, third party communication can comprise embryologist note relevant to oocyte total score, wherein these notes are input in the machine learning model comprising classifier, to use training data, biological sample database received and culture database 144 to generate result analysis, wherein comprise subject information, data from image classifier, data from stimulation scheme, research sample table and data of any other form described in the present disclosure.Additionally or alternatively, machine learning process 116 can be used to send the communication relevant to the scoring index generated by computing device to third party. For example, the oocyte scoring metric may be sent to a remote computing device operated by a third party in communication with the computing device 104, where the third party may perform further analysis, such as analysis of the results. Further to this example, third-party responses to communications generated by the computing device 104 may be uploaded to a database in communication with the computing device 104 and used as feedback in the training data.

[0321] Still refer to Figure 1BIn some embodiments, the scoring index may include an analysis based on omics. Omics is a novel, comprehensive method for analyzing the complete genes or molecular features of humans and other organisms. For example, unlike genetics, which focuses on a single gene, genomics focuses on all genes (genomes) and their interrelationships. In some embodiments, analysis based on omics may include genomics, proteomics, transcriptomics, pharmacogenomics, epigenomics, microbiome, lipidomics, glycomics, transcriptomics, culture omics and / or any other applicable omics understood by those skilled in the art. In some embodiments, after cultivation, oocytes that fail to mature and show GV or MI features can be harvested together with the associated granulosa cells in their culture for single-cell RNA sequencing. For this reason, oocytes and granulosa cells can be quick-frozen and library preparation can be performed. In oocytes that show MII oocyte development, half of the oocytes and their associated granulosa cells can be harvested, and single-cell RNA sequencing is performed using the above-mentioned quick-freezing method described in the present disclosure. The remaining half of the MII oocytes can be used for proteomics research. The culture medium under all conditions can be quick-frozen and used for metabolomics and proteomics, to identify the production of cholesterol metabolite levels and paracrine proteins. For example, frozen cell lysates and cell culture medium can be analyzed to carry out batch RNA sequencing, whole genome sulfite sequencing (WGBS), proteomics and metabolomics based on mass spectrometry. Cell culture medium can be used for metabolomics analysis, to determine the change of molecular content compared with the pre-culture culture medium control in the culture medium after co-cultivation. This can be used to describe the dynamic changes of paracrine signals between granulosa cells and oocytes by computing device 104. Because the culture medium components are quick-frozen, the sample is effectively quenched and can be used for metabolic assessment. The data collected can then be summarized to carry out downstream analysis, to determine the change of epigenetic state, metabolite presence and gene expression between different co-cultivation conditions and controls.

[0322] B. Machine Learning Module

[0323] Reference now Figure 2A , showing an exemplary embodiment of a machine learning module 200 that can perform one or more machine learning processes described in the present disclosure. The machine learning module can use a machine learning process to perform the determination, classification and / or analysis steps, methods, processes, etc. described in the present disclosure. A "machine learning process" as used in the present disclosure is a process that automatically uses training data 204 to generate an algorithm that will be executed by a computing device / module to produce an output 208 based on data provided as input 212; this is in contrast to a non-machine learning software program in which the commands to be executed are predetermined by the user and written in a programming language.

[0324] i. Training Data

[0325] Still reference Figure 2A The machine learning module of the present invention, "training data" as used herein is data containing correlations, which can be used by the machine learning process to model the relationship between two or more categories of data elements. For example, but not limited to, the training data 204 may include multiple data entries, each entry representing a group of data elements recorded, received and / or generated together; the data elements may be associated by shared presence in a given data entry, proximity in a given data entry, etc. Multiple data entries in the training data 204 may show one or more trends of correlations between data element categories; for example, but not limited to, a higher value of a first data element belonging to a first category of data elements may tend to be associated with a higher value of a second data element belonging to a second category of data elements, which indicates that there may be a ratio or other mathematical relationship between the values ​​belonging to the two categories. Data elements of multiple categories may be associated in the training data 204 according to various correlations; correlations may represent causal and / or predictive relationships between data element categories, which can be modeled by the machine learning process as relationships such as mathematical relationships, as described in further detail below. The training data 204 can be formatted and / or organized by category of data elements, for example by associating data elements with one or more descriptors corresponding to the categories of data elements. As a non-limiting example, training data 204 may include data entered by a person or process in a standardized form so that an entry of a given data element in a given field in the form can be mapped to one or more category descriptors. Elements in training data 204 may be linked to descriptors of a category through labels, tokens, or other data elements; for example, but not limited to, training data 204 may be provided in a fixed-length format, a format that links data locations to categories (e.g., a comma-separated value (CSV) format), and / or a self-describing format (such as Extensible Markup Language (XML), JavaScript Object Notation (JSON), etc.), thereby enabling a process or device to detect the category of the data.

[0326] Alternatively or additionally, continue to refer to Figure 2A, the training data 204 may include one or more unclassified elements; that is, the training data 204 may not be formatted or contain descriptors for certain data elements. The machine learning algorithm and / or other process may use, for example, natural language processing algorithms, tokenization, detecting relevant values ​​in the raw data, etc., to sort the training data 204 according to one or more classifications; correlation and / or other processing algorithms may be used to generate categories. As a non-limiting example, in a text corpus, phrases consisting of "n" compound words (e.g., nouns modified by other nouns) may be identified based on the statistically significant prevalence of n-grams containing compound words in a particular order; such n-grams may be classified as language elements (such as "words") to be tracked like individual words, thereby generating new categories based on statistical analysis. Similarly, in a data entry containing some text data, a person's name may be identified by a reference list, dictionary, or other glossary, thereby allowing temporary classification by a machine learning algorithm, and / or automatically associating the data in the data entry with a descriptor or a given format. The ability to automatically classify data entries can make the same training data 204 suitable for two or more different machine learning algorithms, as described in further detail below. The training data 204 used by the machine learning module 200 may associate any input data described in this disclosure with any output data described in this disclosure.

[0327] ii. Training Data Classifier

[0328] Further references Figure 2A, one or more supervised and / or unsupervised machine learning processes and / or models may be used to filter, sort and / or select training data, as described in further detail below; such models may include, but are not limited to, training data classifiers 216. Training data classifiers 216 may include a "classifier", which, as used in the present disclosure, is a machine learning model as defined below, such as a mathematical model, a neural network, or a program generated by a machine learning algorithm referred to as a "classification algorithm", as described in further detail below, which classifies inputs into data categories or groups and outputs data categories or groups and / or labels associated therewith. A classifier may be configured to output at least one data that labels or otherwise identifies a group of data that are clustered together, are found to be close under a distance metric as described below, and the like. The machine learning module 200 may generate a classifier using a classification algorithm, which is defined as a process by which a computing device and / or any module and / or component operating thereon derives a classifier from the training data 204. Classification can be performed using (but not limited to) linear classifiers (such as but not limited to logistic regression and / or naive Bayes classifiers), nearest neighbor classifiers (such as k-nearest neighbor classifiers), support vector machines, least squares support vector machines, Fisher linear discriminant, quadratic classifiers, decision trees, boosted trees, random forest classifiers, learning vector quantization and / or neural network-based classifiers.

[0329] iii. Lazy learning process

[0330] Still refer to Figure 2A , the machine learning module 200 may be configured to perform a lazy learning process 220 and / or protocol, which may alternatively be referred to as a "lazy loading" or "call on demand" process and / or protocol, which may be a process of machine learning by combining the input and the training set to derive an algorithm for generating output on demand after receiving the input to be converted to the output. For example, a set of initial simulations may be performed to cover initial heuristics and / or "preliminary guesses" of the output and / or relationship. As a non-limiting example, the initial heuristic method may include a ranking of the associations between the input and elements of the training data 204. The heuristic method may include selecting some of the highest number of associations and / or training data 204 elements. Lazy learning may implement any suitable lazy learning algorithm, including but not limited to the K nearest neighbor algorithm, the lazy naive Bayes algorithm, etc.; those skilled in the art will realize that various lazy learning algorithms can be applied to generate the outputs described in this disclosure after reading the entire contents of this disclosure, including but not limited to the lazy learning application of the machine learning algorithm as described in further detail below.

[0331] iv. Machine Learning Model

[0332] Alternatively or additionally, continue to refer to Figure 2A, the machine learning process described in the present disclosure can be used to generate a machine learning model 224. A "machine learning model" as used in the present disclosure is a mathematical and / or algorithmic representation of the relationship between input and output, which is generated and stored in a memory using any machine learning process (including but not limited to any of the above processes); once the input is created, it is submitted to the machine learning model 224, which generates an output based on the derived relationship. For example (but not limited to), a linear regression model generated using a linear regression algorithm can calculate the output data using the coefficients derived in the machine learning process to calculate the linear combination of the input data. As a further non-limiting example, a machine learning model 224 can be generated by creating an artificial neural network, such as a convolutional neural network including a layer of input nodes, one or more intermediate layers, and an output node. Connections between nodes can be created through a process of "training" the network, in which elements from a set of training data 204 are applied to the input nodes, and then a suitable training algorithm (such as Levenberg-Marquardt, conjugate gradient, simulated annealing, or other algorithms) is used to adjust the connections and weights between nodes in adjacent layers of the neural network to produce the desired value at the output node. This process is sometimes referred to as deep learning.

[0333] Still refer to Figure 2A , the machine learning algorithm may include at least one supervised machine learning process 228. As defined herein, at least one supervised machine learning process 228 includes an algorithm that receives a training set that associates multiple inputs with multiple outputs and attempts to find one or more mathematical relationships that associate the inputs with the outputs, wherein each of the one or more mathematical relationships is optimal according to certain criteria specified for the algorithm using certain scoring functions. For example, the supervised learning algorithm may include inputs as described above, outputs as described above, and a scoring function that represents the form of the desired relationship to be detected between the inputs and the outputs; for example, the scoring function may seek to maximize the probability that a given input and / or a combination of element inputs is associated with a given output to minimize the probability that a given input is not associated with a given output. The scoring function may be represented as a risk function that represents the "expected loss" of the algorithm that associates the inputs with the outputs, wherein the loss is calculated as an error function that represents the degree to which the prediction generated by the relationship is incorrect when compared to a given input-output pair provided in the training data 204. A person skilled in the art, after reading the entire contents of this disclosure, will recognize various possible variations of at least one supervised machine learning process 228 that can be used to determine the relationship between the inputs and the outputs. The supervised machine learning process may include a classification algorithm as described above.

[0334] Further references Figure 2A, the machine learning process may include at least one unsupervised machine learning process 232. As used herein, an unsupervised machine learning process is a process that draws inferences from a data set without regard to labels; thus, the unsupervised machine learning process is free to discover any structure, relationship, and / or correlation provided in the data. An unsupervised process may not require a response variable; an unsupervised process may be used to discover interesting patterns and / or inferences between variables, to determine the degree of correlation between two or more variables, etc.

[0335] Still refer to Figure 2A , the machine learning module 200 can be designed and configured to create a machine learning model 224 using linear regression model development techniques. The linear regression model may include ordinary least squares regression, which aims to minimize the square of the difference between the predicted outcome and the actual outcome according to an appropriate norm that measures this difference (e.g., a vector space distance norm); the coefficients of the resulting linear equation can be modified to improve the minimization. The linear regression model may include a ridge regression method, in which the function to be minimized includes a least squares function plus a term that multiplies the square of each coefficient by a scalar to penalize larger coefficients. The linear regression model may include a least absolute shrinkage and selection operator (LASSO) model, in which ridge regression is combined with multiplying the least squares term by 1 and dividing it by twice the number of samples. The linear regression model may include a multi-task lasso model, in which the norm applied in the least squares term of the lasso model is a Frobenius norm equal to the square root of the sum of the squares of all terms. The linear regression model may include an elastic network model, a multi-task elastic network model, a minimum angle regression model, a LARS lasso model, an orthogonal matching pursuit model, a Bayesian regression model, a logistic regression model, a stochastic gradient descent model, a perceptron model, a passive attack algorithm, a robust regression model, a Huber regression model, or any other suitable model that may occur to a person skilled in the art after reading the entire disclosure. In one embodiment, the linear regression model may be generalized to a polynomial regression model, thereby seeking a polynomial equation (e.g., a quadratic, cubic, or higher order equation) that provides the best predicted output / actual output fit; a method similar to the above method may be applied to minimize the error function, which will be apparent to a person skilled in the art after reading the entire disclosure.

[0336] Continue to refer Figure 2A, the machine learning algorithm may include, but is not limited to, linear discriminant analysis. The machine learning algorithm may include quadratic discriminant analysis. The machine learning algorithm may include kernel ridge regression. The machine learning algorithm may include support vector machines, including, but not limited to, regression processes based on support vector classification. The machine learning algorithm may include stochastic gradient descent algorithms, including classification and regression algorithms based on stochastic gradient descent. The machine learning algorithm may include a nearest neighbor algorithm. The machine learning algorithm may include various forms of latent space regularization, such as variational regularization. The machine learning algorithm may include a Gaussian process, such as Gaussian process regression. The machine learning algorithm may include a crossover decomposition algorithm, including partial least squares and / or canonical correlation analysis. The machine learning algorithm may include a naive Bayes method. The machine learning algorithm may include a decision tree-based algorithm, such as a decision tree classification or regression algorithm. The machine learning algorithm may include an integrated method, such as a bagging meta-estimator, a random tree forest, AdaBoost, a gradient tree boosting, and / or a voting classifier method. The machine learning algorithm may include a neural network algorithm, including a convolutional neural network process.

[0337] C. Training Data

[0338] Reference now Figure 2B , an exemplary table 236 of training data 204 is shown. Training data 204 may include any data described in the present disclosure. For example, training data 204 may include de-identified user information. In the present disclosure, a user may be referred to as a subject. De-identified subject information may include subject age, subject BMI, number of COCs removed, AMH level (μg / L), antral follicle count (AFC) at the last ultrasound examination, subject oocyte retrieval day E2 level (ng / L), subject oocyte retrieval day P4 level (ng / L), subject oocyte retrieval day LH (IU / L), subject oocyte retrieval day FSH (IU / L), stimulation days, gonadotropin used, total injection dose (IU), etc. Additionally or alternatively, the training data 204 may include pre-culture group COC images, post-culture group COC images, post-culture denuded oocyte images, third-party annotations (such as embryologist annotations), machine learning feedback, follicle dynamics information, research sample tables, frozen oocyte lysate data, frozen granulosa cell lysate data, frozen cell culture medium data, data from a systemic hormone index, data from an oocyte analysis index, data from a biological sample database 124, data from a culture database 144, and the like.

[0339] Reference now Figure 2C, an exemplary table 236 of training data 204 is shown. Training data 204 may include any data described in the present disclosure. For example, training data 204 may include de-identified user information. In the present disclosure, a user may be referred to as a subject. De-identified subject information may include subject age, subject BMI, number of COCs removed, AMH level (μg / L), antral follicle count (AFC) at the last ultrasound examination, subject oocyte retrieval day E2 level (ng / L), subject oocyte retrieval day P4 level (ng / L), subject oocyte retrieval day LH (IU / L), subject oocyte retrieval day FSH (IU / L), stimulation days, gonadotropin used, total injection dose (IU), etc. Additionally or alternatively, the training data 204 may include a pre-co-culture group of COC images, a post-co-culture group of COC images, images of denuded oocytes after culture, third-party annotations (such as embryologist annotations), machine learning feedback, follicle dynamics information, a research sample table, frozen oocyte lysate data, frozen granulosa cell lysate data, frozen cell culture medium data, data from a systemic hormone index, data from a maturity index, data from a biological sample database 124, data from a culture database 144, and the like.

[0340] D. Mini-stimulation program

[0341] Reference now Figure 3A, an exemplary flow chart of a micro-stimulation scheme 300 is shown. In step 305, the minimum stimulation scheme may include selecting a first trigger (e.g., a follicle trigger) based on a first biological sample to be injected into a user. The selected first trigger may be selected based on the measured hormone level of the user. The first trigger may include human recombinant follicle stimulating hormone (rFSH). The rFSH trigger may include, for example, Gonal-F produced by Merck Global, Follistim produced by Merck Global; Follitropin Alfa produced by Teva headquartered in Tel Aviv-Yafo, Israel; and Glucophage produced by Merck Global. rFSH or any other trigger described in the present disclosure (e.g., follicle trigger) may be injected into the user multiple times in different increments. In some embodiments, a trigger may not be administered to the subject. In one embodiment, the time at which the subject can initiate the minimum stimulation may be determined by the subject's contraceptive status, as described in more detail above. For example, a subject who does not take contraceptive measures may start using rFSH stimulation on the second day of the menstrual cycle. In another non-limiting example, a subject who is taking birth control pills, such as combined oral contraceptive (COC) pills, can start stimulation 5 days after taking the last pill. Medication dosage and selection can be determined by one or more laboratory tests, such as a blood test performed on the second day of the subject's menstrual cycle to determine blood levels of E2, FSH, LH, p4, and / or AMH. One or more measurements can be used to determine ovarian reserve health, circulating hormone levels, and / or fertility status.

[0342] In step 310, the scheme 300 may include stimulating the user with the first trigger over a period of time (such as 3 days). For example, 100-200 IU of rFSH may be injected three or more times during a stimulation period of 1-4 days. For example, the stimulation period may last for 3 days. After the injection of the first trigger (e.g., follicle trigger), an ultrasound examination may be performed to determine the average follicle size of cells (such as oocytes). In step 315, the scheme may include a buffer period of one day. The buffer period includes any buffer period as described above, as described in more detail. The buffer period may include stopping the use of the second trigger until serum estradiol (E2) is reduced to a safe level that a person skilled in the art considers to prevent the onset of ovarian hyperstimulation syndrome. In some embodiments, an ultrasound examination may be performed during the buffer period after the 3-day mini-stimulation scheme 300 to determine the average follicle size of the cells. In some embodiments, the buffer period may span 2 days. Determining the average follicle size of the cells may include identifying when the average follicle size is between 8-12 nm. In step 320, the second trigger may be injected into the user based on the average follicle size of the determined cells. The second trigger may include human chorionic gonadotropin (hCG). The dosage of the second trigger may be determined based on one or more factors related to the user, including follicle size, previous diagnosis of any medical condition, ultrasound imaging, drug allergies, subject tolerance to specific drugs, etc. rFSH triggers may include, for example, Pregnyl produced by Schering Plough, headquartered in Kenilworth, New Jersey; Novarel produced by Ferring Laboratories, headquartered in Parsippany, New Jersey; Chorex produced by Encocam, headquartered in Huntington, UK; and Profasi produced by Serum Institute of India Ltd, headquartered in Pune, India. In some embodiments, the second trigger may be any trigger described in the present disclosure. Similar to the first trigger, the second trigger may be injected into the user multiple times in different increments. For example, in an amount of 200 μg-700 μg, injected once or multiple times within a 3-day stimulation period. In step 325, after the second trigger is injected, cells can be removed for the user, wherein the cells include oocytes and / or COCs. For example, after the buffer period, when the follicle size is 8-9 mm, 500 μg of hCG trigger can be administered, and the oocyte can be taken out 36 hours after administration. Oocyte retrieval can include inserting an extraction device into the follicle containing the egg and extracting the egg and surrounding fluid by a medical professional (such as a doctor). Oocyte retrieval can include obtaining immature oocytes, mature oocytes, COCs, and any other cell types found in the ovaries that participate in reproduction. Oocyte retrieval can occur in any time range between 12 and 96 hours after hCG administration.In one embodiment, blood tests can be measured to check the levels of hormones such as E2, LH and / or P4 to ensure one or more quality indicators and to check whether the subject is taking hCG as prescribed. This also helps to determine whether the hormone levels are within the standard predicted values.

[0343] E. Oocyte denudation

[0344] Reference now Figure 3B , an exemplary flow chart of oocyte stripping is shown. In step 305, method 300 may include receiving a COC from a biological sample. The COC may be received according to the oocyte retrieval method disclosed above. In step 310, method 300 may include oocyte stripping of the COC. In some embodiments, stripping may be performed in an IVM well, and the cells may be gently mechanically separated by removing most cumulus and / or granulosa cells by pipetting. If enzymatic hydrolysis is required, the cells may be transferred to a separate culture dish for hyaluronidase treatment. In step 315, the COC may be stripped using a stripper tip and then washed in IVM culture medium or MOPS plus culture medium to clean the oocyte for imaging and inactivate hyaluronidase when necessary. The stripper tip may include 200 microns and or 400 microns for fine cleaning. In some embodiments, due to the stripping of the COC, GV and MI oocytes may be prepared and used for culture. In step 320, method 300 may include transferring the stripped COC to a culture dish for imaging.

[0345] F. Co-culture

[0346] Reference now Figure 4, shows an exemplary table 400 of metabolite formulations that may be included in the cultures described herein. The metabolite 404 column lists exemplary metabolites that may be used as triggers and / or cell culture metabolites. The "cell culture metabolites" used in the present disclosure are substances that participate in cellular metabolism and optimize the synthesis of new molecules in cell culture. The stock solution preparation concentration 408 column lists exemplary concentrations of cell culture metabolites. The final concentration 412 column in the IVM medium lists exemplary concentrations of cell culture metabolites in the IVM medium for population culture or co-culture of the first or second biological sample 136. For example, 10 mg / mL of HSA may be added to the IVM medium for any cell culture described herein (e.g., population culture or co-culture). About 75 mUI / mL of FSH may be added to the IVM medium for any cell culture described herein. About 100 mUI / mL of hCG may be added to the IVM medium for any cell culture described herein. About 500ng / mL of androstenedione can be added to IVM culture medium for any cell culture described herein (e.g., colony culture or co-culture). About 1μg / mL of doxycycline can be added to IVM culture medium for any cell culture described herein. In some embodiments, steroidogenic granulosa cells derived from human induced pluripotent stem cells (hiPSC) can be co-cultured with immature oocytes (e.g., COC) that are stripped or not stripped, thereby reconstructing the follicle niche in vitro to promote rapid and effective maturation of oocytes, thereby enhancing the health and developmental capacity of oocytes. "Steroidogenic granulosa cells" used in the present disclosure are granulosa cells expressing high levels of steroidogenic enzymes such as estradiol. For example, steroidogenic granulosa cells can be parietal granulosa cells extracted from antral follicles. The use of steroidogenic granulosa cells in the co-culture of oocytes (e.g., COCs) can increase the in vitro maturation of oocytes after egg / oocyte retrieval, thereby allowing the utilization of all retrieval eggs / oocytes by providing nutrition, raw materials, and mechanical support directly to oocytes during gametogenesis and folliculogenesis. Steroidogenic granulosa cells can grow in standard IVF and IVM culture media and carry out the maturation of immature oocytes. This can increase the total amount of healthy oocytes that can be used for IVF, and reduce the number of egg / oocyte retrieval procedures that users undergo.

[0347] i. Preparation of granulosa cell co-cultures

[0348] Reference now Figure 5, depicting an exemplary flow chart 500 for preparing a granulosa cell co-culture. Granulosa cells in the subject's ovaries play a key role in the female reproductive system. These cells release estrogen, progesterone, and other hormones that promote the maturation of oocytes in the ovaries, making them a reasonable tool for IVM applications. In addition, granulosa cells provide a developmental environment for the development of follicles and oocytes, directly providing nutrients, raw materials, and mechanical support to oocytes during gametogenesis and folliculogenesis. In step 505, the liquid nitrogen-frozen granulosa cell cryovial can be thawed and incubated. In some embodiments, thawing and incubation can occur up to 72 hours before the user attempts the oocyte again. The granulosa cell cryovial can be a 1 mL cryovial containing 50,000 to 500,000 granulosa cells. In some embodiments, the granulosa cell cryovial can be a 1 mL cryovial containing 100,000 granulosa cells. The granulosa cell cryovial can be thawed by placing the granulosa cell cryovial in a water bath or a dry bead bath. The granulosa cell cryovial can be incubated for 3 to 5 minutes. In step 510, IVM culture medium can be added to the cryovial for cell suspension. In some embodiments, 0.5ml ICM culture medium can be added to induce cell suspension. In step 515, the cell suspension is transferred to the tube and centrifuged. In some embodiments, 1mL cell suspension can be transferred to a 1.5mL tube, wherein the tube is centrifuged at 300xg for 5 minutes. In step 520, a pipette can be used to remove the supernatant. The pipette can be a p1000 pipette. In step 525, the cell pellet formed according to the centrifuge tube containing the cell suspension can be resuspended in IVM culture medium and centrifuged. The IVM culture medium can be 1mL IVM culture medium. The tube can be centrifuged as described above. In step 530, the supernatant can be removed using a pipette. The pipette can be a p1000 pipette. In step 535, the cell pellet can be resuspended in IVM culture medium. The IVM culture medium can be 0.1mL IVM culture medium. In this step, the pellet cells can now be 1,000 cells per 1ul. In some embodiments, 10 ul of cell suspension may be used for each oocyte in the second biological sample 136 associated with the user.

[0349] Reference now Fig. 6A , depicts an exemplary embodiment of a co-cultured second biological sample 136 including immature COCs associated with a user. In some embodiments, COCs received after removal of oocytes from a follicle aspirate associated with a user can be randomly divided into two halves into a culture medium, such as Lag medium for a granulosa cell plate, while the other half can enter LAG medium without a co-culture plate. The COCs can be incubated in LAG medium at 37C for 2 hours. Granulosa cells can be cultured as described in Example 1. Figure 5Prepare as described in . Prepared granulosa cells can be added to the right center well containing IVM medium, 10,000 granulosa cells per culturable COC. The culture dish containing granulosa cells can then be returned to the incubator until use. After a 2 hour incubation period, COCs in LAG medium can be transferred to IVM medium in the granulosa cell dish using a Pasteur pipette.

[0350] Reference now Figure 6B , depicts an exemplary embodiment of a control culture of a second biological sample 136 including immature COCs associated with a user. The COCs can be incubated in LAG medium at 37° C. for 2 hours. After the 2-hour incubation period, the COCs in the LAG medium can be transferred to the IVM medium in the control dish using a Pasteur pipette.

[0351] Reference now Figure 6C , depicts an exemplary embodiment of co-cultured oocytes including immature oocytes associated with a user. In some embodiments, the oocytes can be denuded oocytes. Oocytes received after retrieval of oocytes from a follicle aspirate associated with a user can be randomly divided into two halves and enter a culture medium, such as LAG medium for a granulosa cell plate, while the other half can enter LAG medium without a co-culture plate. Alternatively, some of the oocytes for culture can be pre-frozen, in which case they may be thawed prior to placement in culture. The oocytes can be incubated in LAG medium at 37°C for 2 hours. Granulosa cells can be placed in the culture medium according to Figure 5 Prepare as described in . The prepared granulosa cells can be added to the surrounding 50 μl wells containing IVM medium, with 10,000 granulosa cells added for each culturable oocyte. The culture dish containing the granulosa cells can then be returned to the incubator until use. After a 2-hour incubation period, the oocytes in the LAG medium can be transferred to the IVM medium in the granulosa cell dish using a Pasteur pipette. In the granulosa cell co-culture, after 18 to 48 hours of incubation, the culture plate can be removed and the oocytes and granulosa cells can be imaged in their respective wells. In some embodiments, the culture can be imaged after 24 hours.

[0352] Reference now Fig.6D , depicting an exemplary embodiment of a control culture of immature oocytes relevant to the user. The oocytes can be incubated in LAG medium at 37°C for 2 hours. After the 2-hour incubation period, the oocytes in the LAG medium can be transferred to the IVM medium in the control culture dish using a Pasteur pipette. In the control culture, after 10 to 48 hours of incubation, the culture plate can be removed and the oocytes can be imaged in their respective wells. In some embodiments, the culture can be imaged after 24 hours.

[0353] ii. In vitro induction of human oocyte maturation

[0354] Reference now Fig. 7A , depicts a flow chart of an exemplary method for inducing in vitro maturation of human oocytes. Method 700 may include using computing device 104 (e.g., Figure 1A ) to perform the steps to be listed. In step 705, method 700 includes receiving a first biological sample associated with a user. The first biological sample can be any form of biological sample defined and exemplified in the present disclosure. For example, the first biological sample can include at least a blood sample of the user as shown in FIG. 1. The user can be Figure 1A In some embodiments, the user can be defined as a person. Figure 1A The method 700 comprises, in step 710, assigning a stimulation regimen to the user based on the first biological sample. The stimulation regimen is at least Figure 1A In some embodiments, a stimulation regimen may be assigned based on the hormone level measured in a biological sample. Figure 1A As defined, the measured hormone levels may include E2, LH, FSH, and / or P4 levels. The assigned stimulation protocol may include a minimum stimulation protocol configured to trigger the release of cells within 3 days, at least as defined and exemplified in FIG. 1. In one embodiment, the minimum stimulation protocol may include selecting a first trigger based on a first biological sample and selecting a second trigger based on a follicle measurement. Figure 1A The triggering agents defined in may include human serum albumin, FSH, hCG, androstenedione and doxycycline in the formulations described in Figures 1-6 of the present disclosure.

[0355] Still referring to step 710, in some embodiments, the minimal stimulation protocol may include injecting a first trigger into the user; performing ultrasound to determine the average follicle size of the cells; injecting a second trigger into the user; and removing the cells, wherein the cells include oocytes from the user. The first trigger may include human recombinant follicle stimulating hormone (rFSH). The rFSH may be injected into the user multiple times in different increments. For example and with reference to Figure 1A , 100-200 IU of rFSH can be injected three or more times over a 3-day stimulation period. After the first trigger is injected, an ultrasound examination can be performed to determine the average follicle size of cells (such as oocytes). In some embodiments, such as Figure 1AAs defined, an ultrasound examination may be performed during a 2 day buffer period following a 3 day stimulation regimen. Determining the average follicle size of the cells may include identifying when the average follicle size is between 8-12 nm. Based on determining the average follicle size of the cells, a second trigger may be injected into the user. The second trigger may include human chorionic gonadotropin (hCG). Similar to the first trigger, the second trigger may be injected into the user multiple times in different increments. For example, referring to Figure 1A The amount of the second triggering agent can be in the range of 200 μg-700 μg, and injected once or multiple times during the 3-day stimulation period. After the injection of the second triggering agent, cells can be removed for the user, wherein the cells include oocytes. For example, referring to Figure 1A After a buffer period, when the follicle size is 8-9 mm, 500 μg hCG trigger can be administered and the oocyte retrieved 36 hours after administration. Oocyte retrieval may include inserting an extraction device into the follicle containing the egg and extracting the surrounding fluid by a medical professional (such as a physician).

[0356] Still refer to Fig. 7A The method 700 includes, at step 715, receiving a second biological sample associated with the user, wherein the second biological sample includes Figure 1A The second biological sample may include a body fluid as described above. The second biological sample may be extracted using an extraction device and received as described above. In step 720, method 700 includes culturing the second biological sample. In some embodiments, culturing the second biological sample may include culturing cumulus-oocyte complexes in a colony culture, such as Figure 1A As defined. For example, referring to Figures 1-6, the population culture may include culturing the cumulus-oocyte complex with a granulosa cell co-culture and culturing the control group COC with no co-culture. In some embodiments, the cell culture medium may include LAG medium. For example, referring to Figures 1-6, LAG medium may be used for incubation after COC is removed from the stimulation protocol. The packaging size may be a 10 mL vial. It can be stored at 2-8°C in the dark for up to one month. The culture medium balance may be pre-cultured for 18 to 24 hours, including a 100 μl seed droplet and placed in a 37°C incubator containing 6% O 2 and appropriate CO 2. In some embodiments, the cell culture medium may include IVM medium. For example, referring to Figures 1-6, the modified MediCult IVM medium can be used as a baseline control during the culture process. The packaging size can be a 10 mL vial. It can be stored at 2-8°C in the dark for up to one month. In some embodiments, the cell culture medium may include metabolites. For example, the modified MediCult IVM medium may include human serum albumin, FSH, hCG, androstenedione, doxycycline and other compounds. Other cell culture materials and equipment may include: liquid nitrogen, hyaluronidase, dPBS, IVF qualified mineral oil, universal GPS culture dishes, G-NOPS plus culture medium, micropipettes, stripping pipettes, inverted ICSI microscopes equipped with cameras, dry injection tabletop incubators, saturated humidity incubators, embryoscopes, microcentrifuges, 4°C refrigerators, -20°C freezers, -80°C freezers, liquid nitrogen storage dehydrators, 35mm stripping culture dishes, stripping pipette tips, and other components included in the cell culture process as understood by those skilled in the art.

[0357] Still refer to Fig. 7A In step 725, method 700 includes assigning a scoring indicator to the second biological sample based on culturing the second biological sample. The assignment can be based on subject information about the completion of the stimulation regimen, such as: subject age, subject BMI, number of COCs retrieved, AMH level (μg / L), antral follicle count (AFC) at the last ultrasound examination, subject E2 level on the day of oocyte retrieval (ng / L), subject P4 level on the day of oocyte retrieval (ng / L), subject LH on the day of oocyte retrieval (IU / L), subject FSH on the day of oocyte retrieval (IU / L), number of days of stimulation, gonadotropin used, and total injected dose (IU). In some embodiments, the assignment of scoring indicators may include imaging the group culture and analyzing images of one or both of the co-cultured and non-co-cultured growth groups. For example, the group culture image may include a pre-culture group COC image, a post-culture group COC image, and a post-culture stripped oocyte image. In some embodiments, the images can be sent to a third party, such as Figure 1A In some embodiments, the scoring metric 152 may include a total oocyte score (TOS) as a function of analyzing the imaged population culture. The oocyte score may include at least Figure 1A The indicators described in detail in the , such as shape, size, ooplasm characteristics, perivitelline space (PVS) structure, zona pellucida (ZP), polar body (PB) morphology, etc. The total oocyte score on the pre- and post-culture oocyte images used to generate the TOS indicators can be based on a scale system of -6 to +6.

[0358] In some embodiments, the scoring criteria may include Figure 1AThe results analysis is performed using TOS defined and exemplified in . Parametric or non-parametric tests can be used to determine the significance of the findings found during the analysis. The results analysis can be used to determine the GV to MII oocyte maturation rate; the GV to MI oocyte maturation rate; the MI to MII oocyte maturation rate; the average total oocyte score; the average oocyte shape; the average oocyte size; the average egg quality; the average PVS quality; the average ZP quality; the average polar body quality, etc. In some embodiments, these results can be reported as means, medians, and deviations. Still referring to step 725, in some embodiments, the scoring indicators may include omics-based analysis. For example, referring to Figure 1A , frozen cell lysates and cell culture media can be analyzed for bulk RNA sequencing, whole-genome bisulfite sequencing (WGBS), mass spectrometry-based proteomics, and metabolomics.

[0359] In any of the devices described herein (e.g., see Figure 1A and Figure 1B ), the cell culture medium can be used for metabolomics analysis to determine changes in the content of molecules in the culture medium after co-culture compared to the pre-culture medium control. This can be used to describe the dynamic changes in paracrine signaling between granulosa cells and oocytes. The collected data can then be aggregated for downstream analysis to determine changes in epigenetic state, metabolite presence, and gene expression between different co-culture conditions and controls.

[0360] iii. Oocyte rescue after stimulation

[0361] Reference now Figure 7B , depicts an exemplary flow chart illustrating a method of in vitro rescue of oocytes after stimulation, for example, and with reference to FIGS. 1-6 . Method 700 may include using a computing device 104 (e.g., Figure 1B ) or any computing device described in this disclosure (e.g., see Figure 1A and Figure 1B In some embodiments, method 700 may include using Figure 1BA third party as defined and described in . In step 705, the method includes receiving a biological sample associated with the user, including at least one oocyte, for example, with reference to Figures 1-6. In some embodiments, the oocyte may be an immature oocyte. In some embodiments, the immature oocyte may be a plurality of oocytes. The immature oocyte may be an immature cumulus-oocyte complex (COC) taken from the mother. In some embodiments, the immature oocyte may include an oocyte to which specialized granulosa cells are added to mature the oocyte in cell culture, thereby producing a COC. In some embodiments, the biological sample may include body fluids, including blood, saliva, urine, semen (seminal fluid), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage fluid), pericardial fluid, peritoneal fluid, amniotic fluid, nasal fluid, optic nerve fluid, gastric juice, breast milk, cell culture supernatant, etc. In some embodiments, with reference to Figure 1B , a medical professional can remove a biological sample containing an oocyte from a user after simulation, such as a doctor inserting an extraction device into an ovarian follicle containing an egg and extracting the egg and surrounding fluid. The extraction device may include a needle, a syringe, a vial, a lancet, a vacuum collection tube (ECT), a tourniquet, a vacuum extraction tube system, any combination thereof, and the like. For example, the extraction device may include a butterfly needle set. Oocyte retrieval may include obtaining immature oocytes, mature oocytes, COCs, and any other cell types found in the ovaries that are involved in reproduction.

[0362] Still refer to Figure 7B In step 710, method 700 includes determining a maturity level of at least one oocyte, for example, by reference to Figure 1B and Figure 3B In some embodiments, the maturity level can be an assessment of the oocyte maturation stage of oogenesis. Determining the maturity level of an oocyte can include denuding the oocyte, e.g., Figure 1B Oocyte denudation can include enzymatic and mechanical methods using hyaluronidase and sterile glass pipettes, such as Figure 1B and Figure 3B In step 715, method 700 includes assigning a culture regimen to the oocytes based on the maturity level. The culture regimen may include cell culture metabolites selected based on the maturity level; and cell culture medium selected based on the maturity level, such as reference Figure 1B. In some embodiments, the culture scheme may include culturing the oocyte with a granulosa cell co-culture containing granulosa cells derived from human induced pluripotent stem cells (hiPSC). The cultured oocyte may be an exfoliated oocyte. hiPSC can be produced using the clustered regularly interspaced short palindromic repeats (CRISPR) technology as described above. In some embodiments, the user can donate hiPSC. hiPSC donation can follow the oocyte retrieval process as described above. The user participating in hiPSC donation may be different or the same as the user associated with the biological sample. In some embodiments, the hiPSC donation user may undergo a stimulation scheme as described above. In some embodiments of the culture scheme for distribution, hiPSC, granulosa cells, cumulus cells, oocytes, GV stage oocytes, MI oocytes, and all other types of cells described in the present disclosure may be lysed, genomic material extracted, and snap frozen as the last step of the culture process, for example and with reference to Figures 1-6.

[0363] Still refer to Figure 7B In step 720, method 700 includes culturing at least one oocyte according to a culture protocol, such as with reference to FIGS. 1-6. In embodiments related to oocyte culture, the cell culture medium may include LAG medium. In some embodiments, the cell culture medium may include IVM medium. In some embodiments, the cell culture medium may include metabolites. In step 725, method 700 includes calculating a scoring indicator based on the cultured oocytes, such as with reference to Figure 1B. Calculating the scoring index may include analyzing the co-culture and control culture of the image. The image may include: pre-cultured oocyte images, oocyte images after culture, and oocyte images stripped after culture. In some embodiments, the image may be an image of a frozen lysate and a cell culture medium. In some embodiments, the scoring index may include a total oocyte score (TOS) as a function of the culture analyzed for imaging. For example, each oocyte image may be subjected to an overall oocyte score (TOS) system that measures oocyte health by a 6-point qualitative scale as described above. In some embodiments, the scoring index may include oocyte scoring. Oocyte scoring may include indicators such as shape, size, egg quality characteristics, perivitelline space (PVS) structure, zona pellucida (ZP), polar body (PB) morphology. In some embodiments, the scoring index may include performing result analysis according to TOS. The "result analysis" used in the present disclosure is a measurement of the maturation rate and oocyte quality score between the control culture and the co-culture. Parametric or non-parametric tests may be used to determine the importance of the findings during the analysis. The result analysis can determine the oocyte maturation rate from GV to MII; the oocyte maturation rate from GV to MI; the oocyte maturation rate from MI to MII; the average total oocyte score; the average oocyte shape; the average oocyte size; the average egg quality; the average PVS quality; the average ZP quality; the average polar body quality, etc. In some embodiments, these results can be reported as mean values, medians, and deviations. In some embodiments, the scoring index may include an analysis based on omics. In some embodiments, after culture, oocytes that fail to mature and show GV or MI characteristics can be harvested together with the associated granulosa cells in their culture for single-cell RNA sequencing. To this end, oocytes and granulosa cells can be snap-frozen and library preparation can be performed. In oocytes that exhibit MII oocyte development, half of the oocytes and their associated granulosa cells can be harvested and single-cell RNA sequencing can be performed using the above-mentioned snap-frozen method described in the present disclosure. The remaining half of the MII oocytes can be used for proteomics research. Media from all conditions can be snap frozen and used for metabolomics and proteomics to identify cholesterol metabolite levels and paracrine protein production. Cell culture media can be used for metabolomics analysis to determine changes in the content of molecules in the media after co-culture compared to the pre-culture media control. This can be used to describe the dynamic changes in paracrine signaling between granulosa cells and oocytes. The collected data can then be pooled for downstream analysis to determine changes in epigenetic state, metabolite presence, and gene expression between different co-culture conditions and controls.

[0364] G. Software

[0365] It should be noted that any one or more aspects and embodiments described herein may be conveniently implemented using one or more machines (e.g., one or more computing devices used as user computing devices for electronic documents, one or more server devices (such as document servers), etc.) programmed according to the teachings of this specification, as will be apparent to one of ordinary skill in the computer arts. A skilled programmer may readily write appropriate software coding based on the teachings of this disclosure, as will be apparent to one of ordinary skill in the software arts. The aspects and implementations discussed above using software and / or software modules may also include appropriate hardware to assist in implementing the machine executable instructions of the software and / or software modules.

[0366] Such software can be a computer program product using a machine-readable storage medium. A machine-readable storage medium can be any medium capable of storing and / or encoding a sequence of instructions for a machine (e.g., a computing device) to execute and causing the machine to execute any of the methods and / or embodiments described herein. Machine-readable storage medium examples include, but are not limited to: disks, optical disks (e.g., CD, CD-R, DVD, DVD-R, etc.), magneto-optical disks, read-only memory "ROM" devices, random access memory "RAM" devices, magnetic cards, optical cards, solid-state memory devices, EPROM, EEPROM, and any combination thereof. As used herein, machine-readable media is intended to include a single medium and a set of physically separated media, such as a set of optical disks or one or more hard disk drives in conjunction with a computer memory. As used herein, a machine-readable storage medium does not include a temporary form of signal transmission.

[0367] Such software may also include information (e.g., data) carried as a data signal on a data carrier (such as a carrier wave). For example, machine executable information may include a data-bearing signal embodied in a data carrier, in which the signal encodes a sequence of instructions or a portion thereof and any associated information (e.g., data structures and data) executed by a machine (e.g., a computing device), thereby causing the machine to perform any of the methods and / or embodiments described herein.

[0368] Examples of computing devices include, but are not limited to, electronic book reading devices, computer workstations, terminal computers, server computers, handheld devices (e.g., tablet computers, smart phones, etc.), network appliances, network routers, network switches, network bridges, any machine capable of executing a sequence of instructions that specifies that the machine is to take action, and any combination thereof. In one example, the computing device may include and / or be included in a kiosk.

[0369] Figure 8A schematic diagram of one embodiment of a computing device in the exemplary form of a computer system 800 is shown within which an instruction set for causing a control system to perform one or more aspects and / or methods of the present disclosure may be executed. It is also contemplated that a plurality of computing devices may be utilized to implement a specifically configured instruction set for performing any one or more of the aspects and / or methods of the present disclosure in a distributed computing transaction. The computer system 800 includes a processor 804 and a memory 808 that communicate with each other and with other components via a bus 812. The bus 812 may include any of several types of bus structures, including but not limited to a memory bus, a memory controller, a peripheral bus, a local bus, and any combination thereof using any of a variety of bus architectures.

[0370] The processor 804 may include any suitable processor, such as, but not limited to, a processor including logic circuits for performing arithmetic and logic operations, such as an arithmetic and logic unit (ALU), which may be regulated by a state machine and controlled by operational inputs from memory and / or sensors; as non-limiting examples, the processor 804 may be organized according to a Von Neumann and / or Harvard architecture. The processor 804 may include, incorporate, and / or be incorporated into (but not limited to) a microcontroller, a microprocessor, a digital signal processor (DSP), a field programmable gate array (FPGA), a complex programmable logic device (CPLD), a graphics processing unit (GPU), a general-purpose GPU, a tensor processing unit (TPU), an analog or mixed signal processor, a trusted platform module (TPM), a floating point unit (FPU), and / or a system on a chip (SoC).

[0371] The memory 808 may include various components (e.g., machine-readable media), including, but not limited to, random access memory components, read-only components, and any combination thereof. In one example, a basic input / output system 816 (BIOS), including basic routines that facilitate the transfer of information between elements within the computer system 800 during startup, may be stored in the memory 808. The memory 808 may also include instructions (e.g., software) 820 (e.g., stored on one or more machine-readable media) that implement any one or more of the aspects and / or methods of the present disclosure. In another example, the memory 808 may also include any number of program modules, including, but not limited to, an operating system, one or more application programs, other program modules, program data, and any combination thereof.

[0372] The computer system 800 may also include a storage device 824. Examples of storage devices (e.g., storage devices 824) include, but are not limited to, hard disk drives, disk drives, optical disk drives combined with optical media, solid-state memory devices, and any combination thereof. The storage device 824 may be connected to the bus 812 (not shown) via an appropriate interface. Exemplary interfaces include, but are not limited to, SCSI, Advanced Technology Attachment (ATA), Serial ATA, Universal Serial Bus (USB), IEEE 1394 (FIREWIRE), and any combination thereof. In one example, the storage device 824 (or one or more components thereof) may be removably docked with the computer system 800 (e.g., via an external port connector (not shown)). In particular, the storage device 824 and the associated machine-readable medium 828 may provide non-volatile and / or volatile storage of machine-readable instructions, data structures, program modules, and / or other data for the computer system 800. In one example, the software 820 may reside in whole or in part in the machine-readable medium 828. In another example, the software instructions 820 may reside in whole or in part in the processor 804.

[0373] The computer system 800 may also include an input device 832. In one example, a user of the computer system 800 may input commands and / or other information into the computer system 800 via the input device 832. Examples of the input device 832 include, but are not limited to, an alphanumeric input device (e.g., a keyboard), a pointer device, a joystick, a game controller, an audio input device (e.g., a microphone, a voice response system, etc.), a cursor control device (e.g., a mouse), a touch pad, an optical scanner, a video capture device (e.g., a still camera, a video camera), a touch screen, and any combination thereof. The input device 832 may be docked to the bus 812 via any of a plurality of interfaces (not shown), including, but not limited to, a serial interface, a parallel interface, a game port, a USB interface, a FIREWIRE interface, a direct interface to the bus 812, and any combination thereof. The input device 832 may include a touch screen interface, which may be part of or separate from the display 836, as discussed further below. The input device 832 may be used as a user selection device for selecting one or more graphical representations in the graphical interface as described above.

[0374] The user may also input commands and / or other information to the computer system 800 through a storage device 824 (e.g., a removable disk drive, a flash drive, etc.) and / or a network interface device 840. A network interface device (such as network interface device 840) may be used to connect the computer system 800 to one or more of a plurality of networks (such as network 844) and one or more remote devices 848 to which the network is connected. Examples of network interface devices include, but are not limited to, network interface cards (e.g., mobile network interface cards, LAN cards), modems, and any combination thereof. Examples of networks include, but are not limited to, wide area networks (e.g., the Internet, enterprise networks), local area networks (e.g., networks associated with offices, buildings, campuses, or other relatively small geographic spaces), telephone networks, data networks associated with telephone / voice providers (e.g., mobile communications provider data and / or voice networks), direct connections between two computing devices, and any combination thereof. Networks (such as network 844) may employ wired and / or wireless modes of communication. In general, any network topology may be used. Information (eg, data, software instructions 820 , etc.) may be communicated to and / or from computer system 800 via network interface device 840 .

[0375] The computer system 800 may also include one or more display adapters 852 for transmitting displayable images to one or more display devices, such as display device 836. Display device examples include, but are not limited to, liquid crystal displays (LCDs), cathode ray tubes (CRTs), plasma displays, light emitting diode (LED) displays, and any combination thereof. Display adapter 852 and display device 836 may be used in combination with processor 804 to provide a graphical representation of aspects of the present disclosure. In addition to display devices, computer system 800 may include one or more other peripheral output devices, including, but not limited to, audio speakers, printers, and any combination thereof. Such peripheral output devices may be connected to bus 812 via peripheral interface 856. Examples of peripheral interfaces include, but are not limited to, serial ports, USB connections, FIREWIRE connections, parallel connections, and any combination thereof.

[0376] The illustrative embodiments of the present invention have been described in detail above. Various modifications and additions may be made without departing from the spirit and scope of the present invention. The features of the various embodiments described above may be appropriately combined with the features of the other described embodiments so as to provide a variety of feature combinations in related new embodiments. In addition, although a plurality of separate embodiments have been described above, what is described herein is merely an illustration of the application of the principles of the present invention. In addition, although the particular methods herein may be shown and / or described as being performed in a particular order, the order is highly variable within the capabilities of the ordinary technician to implement the methods, devices, systems, and software disclosed herein. Therefore, this description is only by way of example and is not intended to limit the scope of the present invention.

[0377] Exemplary embodiments have been disclosed above and illustrated in the accompanying drawings. It will be appreciated by those skilled in the art that various changes, deletions and additions may be made to those embodiments specifically disclosed herein without departing from the spirit and scope of the invention.

[0378] Example

[0379] Example 1. Method for follicle stimulation of ovarian oocyte release

[0380] This example illustrates how to provide minimally limited stimulation to a subject undergoing an ART procedure using a trigger that reduces the subject's hormonal burden.

[0381] A 35-year-old female subject with polycystic ovary syndrome (PCOS) undergoing an ART procedure is examined by a clinician on menstrual cycle day 2. The clinician's ultrasound analysis determines that the subject's ovaries produce fewer than or equal to 20 oocytes (e.g., 1 to 5 oocytes, 4 to 10 oocytes, 8 to 16 oocytes, or 15 to 20 oocytes, e.g., 1 oocyte, 2 oocytes, 3 oocytes, 4 oocytes, 5 oocytes, 6 oocytes, 7 oocytes, 8 oocytes, 9 oocytes, 10 oocytes, 11 oocytes, 12 oocytes, 13 oocytes, 14 oocytes, 15 oocytes, 16 oocytes, 17 oocytes, 18 oocytes, 19 oocytes, 20 oocytes); therefore, it is determined that she has a diminished ovarian reserve.

[0382] A trigger (e.g., 100-200 IU of human recombinant follicle stimulating hormone (rFSH)) is administered to the subject to stimulate follicle maturation and oocyte release. The administration of the trigger begins on day 2 ± 1 of the menstrual cycle (e.g., day 1, day 2, or day 3), and continues for 1 to 4 days (e.g., 1 day, 2 days, 3 days, or 4 days) every day. The size of the follicles of the subject is monitored by ultrasound until the average follicle size reaches about 8-10 mm (e.g., 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, or larger), and then the oocyte (or a group of cells comprising the oocyte, such as the cumulus-oocyte complex (COC)) is removed from the subject by a suction-based method. For example, the oocyte can be removed by transvaginal ultrasound and a needle guide on a probe to aspirate the contents of the released follicle. The follicle contents (e.g., follicle aspirates) containing the oocyte are aspirated with a HEPES medium (G-MOPS Plus, ) and then filtered through a 70 μm cell strainer ( Corning) and examined on a dissecting microscope. The oocyte (or a group of cells containing an oocyte, such as a COC) is transferred to a culture dish and culture medium to initiate co-culture with the granulosa cells.

[0383] Example 2. Method for follicle stimulation of ovarian oocytes and in vitro maturation of oocytes

[0384] This example illustrates minimal follicular stimulation in subjects with low ovarian reserve, followed by oocyte retrieval and in vitro maturation.

[0385] i. Follicle stimulation for the release of oocytes from the ovaries

[0386] A 30-year-old female subject underwent a blood test that detected an anti-Mullerian hormone (AMH) level of less than or equal to 6 ng / mL (e.g., 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, or 6 ng / mL). Therefore, it was determined that her ovarian reserve was diminished. Additional blood tests showed that her estradiol level was between 20 and 50 pg / mL (e.g., 20-30 pg / mL, 25-35 pg / mL, 30-40 pg / mL, 35-45 pg / mL, or 40-50 pg / mL; for example, 20 pg / mL, 21 pg / mL, 22 pg / mL, 23 pg / mL, 24 pg / mL, 25 pg / mL, 30 pg / mL, 35 pg / mL, 40 pg / mL, 45 pg / mL, or 50 pg / mL), which again confirmed the determination of diminished ovarian reserve.

[0387] Trigger (e.g., 50 mg clomiphene citrate) is administered to the subject to stimulate follicle maturation and oocyte release. Since the subject is taking hormonal contraceptives, the trigger is administered starting at about the 5th ± 1st day (e.g., the 4th, 5th, or 6th day) after taking the last dose of contraceptives, and continues to be administered daily for 1 to 4 days (e.g., 1 day, 2 days, 3 days, or 4 days). The size of the follicles of the subject is monitored by ultrasound until the average follicle size reaches about 8-10 mm (e.g., 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, or larger), and then the oocyte (or cell group comprising the oocyte, such as COC) is removed from the subject by aspiration-based method. For example, oocyte removal can utilize transvaginal ultrasound and a needle guide on a probe to aspirate the release follicle contents. The follicle contents (e.g., follicle aspirate) containing the oocyte is aspirated with a HEPES medium (G-MOPS Plus, ) and then filtered through a 70 μm cell strainer ( Corning) filtration, and examine on a dissecting microscope. The oocyte (or cell group comprising the oocyte, such as COC) is transferred to a culture dish containing a cell culture medium (such as IVM, IVF or LAG culture medium) for about 1 to 3 hours (such as 1 hour, 2 hours or 3 hours), and then the granulosa cells are introduced for co-cultivation.

[0388] ii. In vitro maturation of oocytes

[0389] If present, the COCs of cultivation can be separated from their cumulus cells (and any other non-oocytes) in the process referred to herein as oocyte stripping. Oocyte stripping is carried out by mechanically separating cells with removal of cumulus cells and / or granulosa cells on the COCs in the IVM holes by a pipette. Other oocyte stripping can be carried out by enzymolysis (such as hyaluronidase treatment). COCs can be stripped using a stripper tip, then cleaned in IVM culture medium or MOPS plus culture medium, imaged with clean oocytes, and deactivating hyaluronidase when needed. The stripper tip includes 200 microns and / or 400 microns for fine cleaning.

[0390] Next, the germinal vesicle (GV) and metaphase I (MI) oocytes are mixed with about 50,000-100,000 (e.g., 50,000-60,000 cells, 60,000-70,000 cells, 70,000-80,000 cells, 80,000-90,000 cells, or 90,000-100,000 cells; e.g., 50,000 cells, 55,000 cells, 60,000 cells, 65,000 cells, 70,000 cells, 75,000 cells, 80,000 cells, 85,000 cells, 90,000 cells, 95,000 cells or 100,000 cells) granulosa cells (e.g., specialized granulosa cells, hiPSC-derived granulosa cells or steroidogenic granulosa cells, as described herein) are co-cultured. Metaphase II (MII) oocytes (e.g., oocytes with polar bodies in the perivitelline space) can be appropriately cryopreserved. Co-culture of oocytes and granulosa cells continues for about 12-120 hours (e.g., 12-24 hours, 12-36 hours, 24-48 hours, 36-60 hours, 54-72 hours, 68-96 hours, 96-120 hours; for example, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, 50 hours, Hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours, 110 hours, 112 hours, 114 hours, 116 hours, 118 hours or 120 hours).

[0391] After co-culture, any one or more oocytes are used in assisted reproductive technology (ART) procedures. For example, the oocytes can be used for intracytoplasmic sperm injection (ICSI).

[0392] Example 3. Administration of follicle triggering agents

[0393] This example illustrates administering a trigger to a subject.

[0394] A 30-year-old female subject undergoes a blood test and detects an estradiol level between 20 and 50 pg / mL (e.g., 20-30 pg / mL, 25-35 pg / mL, 30-40 pg / mL, 35-45 pg / mL, or 40-50 pg / mL; for example, 20 pg / mL, 21 pg / mL, 22 pg / mL, 23 pg / mL, 24 pg / mL, 25 pg / mL, 30 pg / mL, 35 pg / mL, 40 pg / mL, 45 pg / mL, or 50 pg / mL). The subject is administered multiple injections of a triggering agent within 1 to 4 days (e.g., 1 day, 2 days, 3 days, or 4 days), but not more than 5 days. The subject may receive multiple injections over multiple days, such that the subject receives five injections of one or more triggering agents. For example, subjects receive three days of stimulation with 300 IU to 700 IU of rFSH per injection (e.g., 300-500 IU, 400-600 IU, 500-700 IU, 300-350 IU, 350-400 IU, 400-450 IU, 450-500 IU, 500-550 IU, 550-600 IU, 600 -650 IU, 650-700 IU; for example, 300 IU, 325 IU, 350 IU, 375 IU, 400 IU, 425 IU, 450 IU, 475 IU, 500 IU, 525 IU, 550 IU, 575 IU, 600 IU, 625 IU, 650 IU, 675 IU or 700 IU), injected once or more per day. In another example, the subject receives an injection of hCG as a trigger using 200-700 μg or 2,500-10,000 IU hCG (e.g., 200-500 μg, 300-600 μg, 400-700 μg, 200-300 μg, 300-400 μg, 400-500 μg, 500-600 μg, or 600-700 μg). In yet another example, the subject receives one or more (e.g., 1, 2, 3, 4, or 5) doses of clomiphene citrate, each dose of 50-150 mg (e.g., 50-75 mg, 60-80 mg, 75-100 mg, 90-115 mg, 110-130 mg, 125-150 mg; e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg).

[0395] Example 4. Device for follicle stimulation and in vitro maturation of oocytes

[0396] In this embodiment, reference Figure 8, which shows a schematic diagram of an embodiment of a computing device in the exemplary form of a computer system 800, within which an instruction set for causing a control system to perform any one or more aspects and / or methods of the present disclosure may be executed. It is also contemplated that a plurality of computing devices may be utilized to implement a specifically configured instruction set for performing any one or more of the aspects and / or methods of the present disclosure in a distributed computing transaction. The computer system 800 includes a processor 804 and a memory 808, which communicate with each other and with other components via a bus 812. The bus 812 may include any of several types of bus structures, including but not limited to a memory bus, a memory controller, a peripheral bus, a local bus, and any combination thereof using any of a variety of bus architectures.

[0397] The processor 804 may include any suitable processor, such as, but not limited to, a processor including logic circuits for performing arithmetic and logic operations, such as an arithmetic and logic unit (ALU), which may be regulated by a state machine and controlled by operational inputs from memory and / or sensors; as non-limiting examples, the processor 804 may be organized according to a von Neumann and / or Harvard architecture. The processor 804 may include, incorporate, and / or be incorporated into (but not limited to) a microcontroller, a microprocessor, a digital signal processor (DSP), a field programmable gate array (FPGA), a complex programmable logic device (CPLD), a graphics processing unit (GPU), a general-purpose GPU, a tensor processing unit (TPU), an analog or mixed signal processor, a trusted platform module (TPM), a floating point unit (FPU), and / or a system on a chip (SoC).

[0398] The memory 808 may include various components (e.g., machine-readable media), including, but not limited to, random access memory components, read-only components, and any combination thereof. In one example, a basic input / output system 816 (BIOS), including basic routines that facilitate the transfer of information between elements within the computer system 800 during startup, may be stored in the memory 808. The memory 808 may also include instructions (e.g., software) 820 (e.g., stored on one or more machine-readable media) that implement any one or more of the aspects and / or methods of the present disclosure. In another example, the memory 808 may also include any number of program modules, including, but not limited to, an operating system, one or more application programs, other program modules, program data, and any combination thereof.

[0399] The computer system 800 may also include a storage device 824. Examples of storage devices (e.g., storage devices 824) include, but are not limited to, hard disk drives, disk drives, optical disk drives combined with optical media, solid-state memory devices, and any combination thereof. The storage device 824 may be connected to the bus 812 (not shown) via an appropriate interface. Exemplary interfaces include, but are not limited to, SCSI, Advanced Technology Attachment (ATA), Serial ATA, Universal Serial Bus (USB), IEEE 1394 (FIREWIRE), and any combination thereof. In one example, the storage device 824 (or one or more components thereof) may be removably docked with the computer system 800 (e.g., via an external port connector (not shown)). In particular, the storage device 824 and the associated machine-readable medium 828 may provide non-volatile and / or volatile storage of machine-readable instructions, data structures, program modules, and / or other data for the computer system 800. In one example, the software 820 may reside in whole or in part in the machine-readable medium 828. In another example, the software instructions 820 may reside in whole or in part in the processor 804.

[0400] The computer system 800 may also include an input device 832. In one example, a user of the computer system 800 may input commands and / or other information into the computer system 800 via the input device 832. Examples of the input device 832 include, but are not limited to, an alphanumeric input device (e.g., a keyboard), a pointer device, a joystick, a game controller, an audio input device (e.g., a microphone, a voice response system, etc.), a cursor control device (e.g., a mouse), a touch pad, an optical scanner, a video capture device (e.g., a still camera, a video camera), a touch screen, and any combination thereof. The input device 832 may be docked to the bus 812 via any of a plurality of interfaces (not shown), including, but not limited to, a serial interface, a parallel interface, a game port, a USB interface, a FIREWIRE interface, a direct interface to the bus 812, and any combination thereof. The input device 832 may include a touch screen interface, which may be part of or separate from the display 836, as discussed further below. The input device 832 may be used as a user selection device for selecting one or more graphical representations in the graphical interface as described above.

[0401] The user may also input commands and / or other information to the computer system 800 through a storage device 824 (e.g., a removable disk drive, a flash drive, etc.) and / or a network interface device 840. A network interface device (such as network interface device 840) may be used to connect the computer system 800 to one or more of a plurality of networks (such as network 844) and one or more remote devices 848 to which the network is connected. Examples of network interface devices include, but are not limited to, network interface cards (e.g., mobile network interface cards, LAN cards), modems, and any combination thereof. Examples of networks include, but are not limited to, wide area networks (e.g., the Internet, enterprise networks), local area networks (e.g., networks associated with offices, buildings, campuses, or other relatively small geographic spaces), telephone networks, data networks associated with telephone / voice providers (e.g., mobile communications provider data and / or voice networks), direct connections between two computing devices, and any combination thereof. Networks (such as network 844) may employ wired and / or wireless modes of communication. In general, any network topology may be used. Information (eg, data, software instructions 820 , etc.) may be communicated to and / or from computer system 800 via network interface device 840 .

[0402] The computer system 800 may also include one or more display adapters 852 for transmitting displayable images to one or more display devices, such as display device 836. Display device examples include, but are not limited to, liquid crystal displays (LCDs), cathode ray tubes (CRTs), plasma displays, light emitting diode (LED) displays, and any combination thereof. Display adapter 852 and display device 836 may be used in combination with processor 804 to provide a graphical representation of aspects of the present disclosure. In addition to display devices, computer system 800 may include one or more other peripheral output devices, including, but not limited to, audio speakers, printers, and any combination thereof. Such peripheral output devices may be connected to bus 812 via peripheral interface 856. Examples of peripheral interfaces include, but are not limited to, serial ports, USB connections, FIREWIRE connections, parallel connections, and any combination thereof.

[0403] Example 5: Materials and methods for Examples 6 to 8

[0404] We have developed human ovarian supporting cells (OSCs) derived from human induced pluripotent stem cells (hiPSCs) with the ability to recapitulate dynamic ovarian function in vitro. Here, we investigated the potential of these OSCs as a co-culture system for IVM to improve maturation of human oocytes retrieved from abbreviated gonadotropin-stimulated cycles. We reveal that OSC-IVM significantly improves maturation rates compared with existing IVM systems. Most importantly, we demonstrate that OSC-assisted IVM oocytes are able to form robust euploid blastocysts, a key hallmark of their clinical utility. Collectively, these findings demonstrate a novel IVM approach that can be widely applied in modern IVF practice.

[0405] Specifically, to determine whether in vitro maturation (IVM) of human oocytes could be improved by co-culture with ovarian supporting cells (OSCs) derived from human induced pluripotent stem cells (hiPSCs), oocyte donors were recruited to undergo abbreviated gonadotropin stimulation with or without an hCG trigger, and cumulus-oocyte complexes (COCs) were allocated between OSC-IVM conditions and medium-only IVM controls.

[0406] Oocyte donors aged between 19 and 37 years were recruited with informed consent, and anti-Mullerian hormone (AMH) values ​​greater than 1 ng / mL were used as inclusion criteria. OSC-IVM culture conditions consisted of 100,000 OSCs in suspension culture supplemented with human chorionic gonadotropin (hCG), recombinant follicle-stimulating hormone (rFSH), androstenedione, and doxycycline. IVM controls lacked OSCs and contained the same supplements or only FSH and hCG.

[0407] Primary endpoints included metaphase II (MII) formation rate and morphological quality assessment. Fertilization and blastocyst formation and PGT-A analysis were also performed using a limited cohort of oocytes. OSC-IVM resulted in a statistically significant improvement in the MII formation rate compared to a media-only control. OSC-IVM resulted in a statistically significant improvement in the MII formation rate compared to a commercially available IVM control. There was no significant difference in oocyte morphological quality between OSC-IVM and the control group. OSC-IVM improved maturation, fertilization, cleavage, blastocyst formation, high-quality blastocyst formation, and euploid blastocyst formation compared to a commercially available IVM control.

[0408] In conclusion, the novel OSC-IVM platform is an effective tool to mature human oocytes obtained from shortened gonadotropin-stimulated cycles, thereby improving blastocyst formation. OSC-IVM showed broad utility for different stimulation protocols, including hCG-triggered truncated IVF and untriggered conventional IVM cycles, making it a very useful tool in modern fertility treatment.

[0409] i. Collection of Cumulus-oocyte Complex (COC)

[0410] Subject age, IRB, and informed consent

[0411] Subjects participated in the study with informed consent through the Ruber Clinic (Madrid, Spain), Spring Fertility Clinic (New York, USA), and Pranor Clinic (Lima, Peru) (CNRHA47 / 428973.9 / 22, IRB#20225832, Western IRB, and protocol #GC-MSP-01, respectively). The subjects' ages ranged from 19 to 37 years. Oocytes retrieved from the Ruber and Pranor clinics were used only for the maturation analysis endpoint, while oocytes retrieved from Spring Fertility were used for the embryogenesis endpoint.

[0412] Stimulus characteristics

[0413] Twenty-five subjects received 3–4 days of stimulation with 300–600 IU rFSH and an hCG trigger in preparation for immature oocyte aspiration in Experiment 1, with AMH values ​​>1 ng / mL (see below). Twenty-one subjects received 200 IU rFSH and an hCG trigger for three consecutive days in preparation for immature oocyte aspiration in Experiment 2, with AMH values ​​>1.5 ng / mL used as an inclusion criterion to enrich for donors that produced more oocytes (see below). Six subjects received three to five doses of clomiphene citrate (100 mg) and an additional one to two doses of 150 IU rFSH (with or without an hCG trigger) in Experiment 2, with the goal of subsequent embryo formation, and AMH values ​​>2.0 ng / mL were used as an inclusion criterion (see below). Gonadotropin injections were initiated on day 2 of the natural cycle or on the fifth day after stopping oral contraceptive pills. Table 1 below provides a complete table of the donor stimulation regimen for each donor in the study.

[0414] Table 1: Donor stimulation protocol

[0415]

[0416]

[0417]

[0418] ii. Aspiration of small follicles to remove immature cumulus-oocyte complexes

[0419] 36 hours after the injection of the trigger (10,000 IU hCG), aspiration was performed using transvaginal ultrasound with needle guidance on the probe to retrieve oocytes for co-culture experiments. Aspiration was performed with a double-lumen 19-gauge needle (the double-lumen needle was chosen because the second channel inside the needle provided additional rigidity) without flushing the follicle. The follicular contents were collected into a 15-mL round-bottom polystyrene centrifuge tube through the aspiration needle and tubing using vacuum pump suction (100 mm Hg). For cases where the final outcome was embryogenesis, aspiration was performed 36 hours after the trigger injection (10,000 IU hCG) or 48 hours after the last rFSH injection in untriggered cycles. Aspiration was performed with a single-lumen 19- or 20-gauge needle and vacuum pump suction (approximately 200 mmHg) without flushing the follicle, and the follicular contents were collected into a 15-mL round-bottom polystyrene centrifuge tube through the aspiration needle and tubing. In all cases, when the follicle collapsed, the aspiration needle was rapidly rotated about its long axis to create a scraping effect to help release the COC into the aspirate fluid. Although the follicles were not flushed, the aspiration needle was removed from the subject and flushed frequently throughout the oocyte retrieval process to limit clotting and needle clogging.

[0420] The follicular aspirate is examined in the laboratory using a dissecting microscope. The aspirate tends to contain more blood than a typical IVF follicular aspirate and should therefore be washed with HEPES medium (G-MOPS Plus, ) to minimize condensation. Typically, a 70 μm cell strainer ( The aspirate was additionally filtered to improve the oocyte search process. Using a sterile Pasteur pipette, the COCs were transferred to a culture medium containing LAG medium (Medicult, ) in a culture dish until used for the IVM procedure. The number of COCs aspirated was approximately equal to 40% of the antral follicles seen in the subject's ovaries on the starting day.

[0421] iii. Preparation of Ovarian Support Cells (OSC)

[0422] OSCs were generated from human induced pluripotent stem cells (hiPSCs) according to a previously described transcription factor (TF)-guided protocol. OSCs were produced in multiple batches and cryopreserved in vials containing 120,000 to 150,000 viable cells each and maintained in CryoStor CS10 Cell Freezing Medium (StemCell ) and stored in liquid nitrogen.

[0423] Culture dishes for oocyte maturation experiments (4+8 dishes, BIRR) were prepared with culture medium and additional components in 100 μL drops under mineral oil the day before oocyte collection. On the morning of oocyte collection, cryopreserved OSCs were thawed at 37°C for 2-3 minutes (in a heated bead or water bath), resuspended in OSC-IVM medium, and washed twice using centrifugation and sedimentation to remove residual cryoprotectant. Equilibrated OSC-IVM medium was used for the final resuspension. The 50 μL drop was then replaced with 50 μL OSC suspension 2-4 hours before the addition of oocytes, and OSCs were plated at a concentration of 100,000 OSCs per 100 μL drop to allow culture equilibration and culture medium conditioning ( Figure 2A ).

[0424] iv. In vitro maturation

[0425] After COC was taken out, it was pre-incubated in LAG medium (Medicult, Coope) at 37°C. ) for 2-3 hours before introducing in vitro maturation conditions. Two different sets of experiments were compared to achieve the following...

Claims

1. A method for preparing one or more oocytes that have been previously removed from a human subject for use in an assisted reproductive technology (ART) procedure, the method comprising co-culturing the one or more oocytes with a population of ovarian supporting cells.

2. A method for producing mature oocytes for use in an ART procedure, the method comprising co-culturing one or more oocytes previously removed from a human subject with a population of ovarian supporting cells.

3. A method for inducing oocyte maturation in vitro, the method comprising co-culturing one or more oocytes previously removed from a human subject with a population of ovarian supporting cells, wherein the co-culture is performed for a period of about 6 hours to about 120 hours.

4. The method of any one of claims 1-3, wherein a follicle triggering agent is not administered to the subject prior to removal of the one or more oocytes from the subject.

5. The method of any one of claims 1-3, wherein one or more follicle triggering agents are administered to the subject during the follicle triggering phase prior to removal of the one or more oocytes from the subject.

6. The method of claim 5, wherein the duration of the follicle triggering phase does not exceed 8 days.

7. The method of claim 6, wherein the duration of the follicle triggering phase does not exceed 7 days.

8. The method of claim 7, wherein the duration of the follicle triggering phase does not exceed 6 days.

9. The method of claim 8, wherein the duration of the follicle triggering phase does not exceed 5 days.

10. The method of claim 9, wherein the duration of the follicle triggering phase does not exceed 4 days.

11. The method of claim 10, wherein the duration of the follicle triggering phase does not exceed 3 days.

12. The method of claim 11, wherein the duration of the follicle triggering phase does not exceed 2 days.

13. The method of claim 12, wherein the duration of the follicle triggering phase does not exceed 1 day.

14. The method of claim 5, wherein the duration of the follicle triggering phase is 1 to 8 days.

15. The method of claim 14, wherein the duration of the follicle triggering phase is 1 day to 7 days.

16. The method of claim 15, wherein the duration of the follicle triggering phase is 1 to 6 days.

17. The method of claim 16, wherein the duration of the follicle triggering phase is 1 to 5 days.

18. The method of claim 17, wherein the duration of the follicle triggering phase is 1 to 4 days.

19. The method of claim 18, wherein the duration of the follicle triggering phase is 1 to 3 days.

20. The method of claim 5, wherein the duration of the follicle triggering phase is 2 to 8 days.

21. The method of claim 20, wherein the duration of the follicle triggering phase is 2 to 7 days.

22. The method of claim 21, wherein the duration of the follicle triggering phase is 2 to 6 days.

23. The method of claim 22, wherein the duration of the follicle triggering phase is 2 to 5 days.

24. The method of claim 23, wherein the duration of the follicle triggering phase is 2 to 4 days.

25. The method of claim 5, wherein the duration of the follicle triggering phase is 3 to 8 days.

26. The method of claim 25, wherein the duration of the follicle triggering phase is 3 to 7 days.

27. The method of claim 26, wherein the duration of the follicle triggering phase is 3 to 6 days.

28. The method of claim 27, wherein the duration of the follicle triggering phase is 3 to 5 days.

29. The method of any one of claims 5-28, wherein the one or more follicle triggering agents comprises follicle stimulating hormone (FSH), clomiphene citrate and / or human chorionic gonadotropin (hCG).

30. The method of claim 29, wherein the one or more follicle triggering agents comprises FSH.

31. The method of claim 30, wherein the FSH is administered to the subject in one or more doses per day.

32. The method of claim 31, wherein the FSH is administered to the subject once daily.

33. The method of any one of claims 30-32, wherein the FSH is administered to the subject in an amount of about 100 International Units (IU) to about 1,000 IU per day.

34. The method of claim 33, wherein the FSH is administered to the subject in an amount of about 200 IU to about 800 IU per day.

35. The method of claim 34, wherein the FSH is administered to the subject in an amount of about 300 IU to about 700 IU per day.

36. The method of claim 35, wherein the FSH is administered to the subject in an amount of about 300 IU to about 600 IU per day, about 300 IU to about 500 IU per day, or about 300 IU to about 400 IU per day.

37. The method of any one of claims 30-36, wherein the duration of FSH administration is equal to the duration of the follicular triggering phase.

38. The method of any one of claims 30-36, wherein the duration of the FSH administration is less than the duration of the follicular triggering phase.

39. The method of claim 38, wherein the duration of FSH administration during the follicular triggering phase is 1, 2, 3, 4 or 5 days, optionally wherein FSH is administered to the subject in an amount of about 200 IU per day for 1, 2, 3, 4 or 5 days during the follicular triggering phase, optionally wherein FSH is administered to the subject in an amount of about 200 IU per day for 3 days during the follicular triggering phase.

40. The method of any one of claims 29-39, wherein the one or more follicle triggering agents comprises clomiphene citrate.

41. The method of claim 40, wherein the clomiphene citrate is administered to the subject in one or more doses per day.

42. The method of claim 41, wherein the clomiphene citrate is administered to the subject once daily.

43. The method of any one of claims 40-42, wherein the clomiphene citrate is administered to the subject in an amount of about 50 mg to about 100 mg per day.

44. The method of claim 43, wherein the clomiphene citrate is administered to the subject in an amount of about 50 mg per day.

45. The method of any one of claims 40-44, wherein the duration of clomiphene citrate administration is equal to the duration of the follicular triggering phase.

46. ​​The method of any one of claims 40-44, wherein the duration of clomiphene citrate administration is less than the duration of the follicular triggering phase.

47. The method of claim 46, wherein the duration of clomiphene citrate administration during the follicular triggering phase is 1, 2, 3, 4 or 5 days.

48. The method of any one of claims 29-47, wherein the one or more follicle triggering agents comprises hCG.

49. The method of claim 48, wherein the hCG is administered to the subject in one or more doses per day.

50. The method of claim 49, wherein the hCG is administered to the subject in 1, 2 or 3 doses during the follicular triggering phase.

51. The method of any one of claims 48-50, wherein the hCG is administered to the subject in an amount of about 200 g to about 700 g per dose.

52. The method of claim 51, wherein the hCG is administered to the subject in an amount of about 200 μg to about 500 μg per dose, about 300 μg to about 600 μg per dose, about 400 μg to about 700 μg per dose, about 200 μg to about 300 μg per dose, about 300 μg to about 400 μg per dose, about 400 μg to about 500 μg per dose, about 500 μg to about 600 μg per dose, or about 600 μg to about 700 μg per dose.

53. The method of claim 52, wherein the hCG is administered to the subject in an amount of about 500 μg per dose.

54. The method of any one of claims 48-50, wherein the hCG is administered to the subject in an amount of about 2,500 IU to about 10,000 IU per dose.

55. The method of any one of claims 5-54, wherein the subject is a subject who has completed oral contraceptive treatment within 28 days of the start of the follicular triggering phase.

56. The method of claim 55, wherein the follicular triggering phase begins at least 5 days after cessation of contraceptive treatment.

57. The method of any one of claims 5-54, wherein the subject has not undergone oral contraceptive treatment within 28 days of the start of the follicular triggering phase.

58. The method of claim 57, wherein the follicular triggering phase begins on day 2 of the subject's menstrual cycle.

59. The method of any one of claims 55-58, wherein the contraceptive treatment comprises administering a gonadotropin-releasing hormone (GnRH) agonist to the subject.

60. The method of any one of claims 5-59, wherein the subject has been determined to exhibit a follicle size of about 6 mm to about 8 mm prior to the onset of the follicular triggering phase.

61. The method of any one of claims 5-59, wherein the subject has been determined to exhibit a follicle size of about 6 mm to about 8 mm prior to administration of the final follicle trigger.

62. The method of any one of claims 1-61, wherein a biological sample isolated from the subject prior to retrieval of the one or more oocytes has been determined to have an anti-Mullerian hormone (AMH) concentration of about 0.1 ng / mL to about 1 ng / mL or about 1 ng / mL to about 6 ng / mL.

63. The method of claim 62, wherein the biological sample has been determined to have an AMH concentration of about 1 ng / mL to about 6 ng / mL, optionally wherein the biological sample has been determined to have an AMH concentration of about 2.5 ng / mL to about 3.0 ng / mL.

64. The method of any one of claims 1-61, wherein a biological sample isolated from the subject prior to removal of the one or more oocytes has been determined to have an AMH concentration of at least 1 ng / mL.

65. The method of any one of claims 1-61, wherein a biological sample isolated from the subject prior to removal of the one or more oocytes has been determined to have an AMH concentration of no greater than 6 ng / mL.

66. The method of any one of claims 1-61, wherein a biological sample isolated from the subject prior to removal of the one or more oocytes has been determined to have an AMH concentration of about 0.1 ng / mL to about 1 ng / mL.

67. The method of any one of claims 62-66, wherein the biological sample is a blood sample.

68. The method of any one of claims 1-67, wherein the subject is between 18 and 48 years old at the time of retrieval of the one or more oocytes.

69. The method of claim 68, wherein the subject is between 20 and 45 years old at the time of retrieval of the one or more oocytes.

70. The method of claim 68, wherein the subject is less than 35 years old at the time of retrieval of the one or more oocytes.

71. The method of claim 68, wherein the subject is older than 35 years of age at the time of retrieval of the one or more oocytes.

72. The method of any one of claims 1-71, wherein prior to removing the one or more oocytes from the subject, it is determined that the subject exhibits a follicle size of about 6 mm to about 14 mm.

73. The method of claim 72, wherein prior to removing the one or more oocytes from the subject, it is determined that the subject exhibits a follicle size of about 8 mm to about 12 mm.

74. The method of claim 73, wherein prior to removing the one or more oocytes from the subject, it is determined that the subject exhibits a follicle size of about 8 mm to about 9 mm.

75. The method of any one of claims 1-71, wherein prior to removing the one or more oocytes from the subject, it is determined that the subject exhibits a follicle size of no greater than 14 mm.

76. The method of any one of claims 72-75, wherein the follicle size has been assessed by ultrasound image analysis.

77. The method of any one of claims 1-76, wherein a total of 20 or fewer oocytes are removed from the subject.

78. The method of claim 77, wherein 15 or fewer oocytes are removed from the subject.

79. The method of claim 78, wherein 10 or fewer oocytes are removed from the subject.

80. The method of claim 79, wherein 9 or fewer oocytes are removed from the subject.

81. The method of claim 80, wherein 8 or fewer oocytes are removed from the subject.

82. The method of claim 81, wherein 7 or fewer oocytes are removed from the subject.

83. The method of claim 82, wherein 6 or fewer oocytes are removed from the subject.

84. The method of claim 83, wherein 5 or fewer oocytes are removed from the subject.

85. The method of any one of claims 1-84, wherein a plurality of oocytes are removed from the subject.

86. The method of claim 85, wherein between 10% and 100% of the oocytes removed from the subject are germinal vesicle (GV) stage or meiosis I (MI) stage oocytes.

87. The method of claim 86, wherein 20% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

88. The method of claim 87, wherein 30% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

89. The method of claim 88, wherein 40% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

90. The method of claim 89, wherein 50% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

91. The method of claim 90, wherein 60% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

92. The method of claim 91, wherein 70% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

93. The method of claim 92, wherein 80% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

94. The method of claim 93, wherein 90% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

95. The method of claim 94, wherein 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

96. The method of any one of claims 1-95, wherein the ovarian supporting cell population comprises ovarian granulosa cells and / or ovarian stromal cells, optionally wherein the ovarian granulosa cells are forkhead box protein L2 (FOXL2) positive and / or wherein the ovarian stromal cells are nuclear receptor subfamily 2 group F member 2 (NR2F2) positive.

97. The method of any one of claims 1-96, wherein the population of ovarian support cells comprises about 50,000 to about 500,000 ovarian support cells.

98. The method of any one of claims 1-97, wherein the population of ovarian support cells comprises about 50,000 to about 60,000 ovarian support cells, about 60,000 to about 70,000 ovarian support cells, about 70,000 to about 80,000 ovarian support cells, about 80,000 to about 90,000 ovarian support cells, about 90,000 to about 100,000 ovarian support cells, or about 100,000 to about 150,000 ovarian support cells, optionally wherein the population of ovarian support cells comprises about 125,000 ovarian support cells.

99. The method of any one of claims 1-98, wherein the population of ovarian support cells comprises about 50,000 ovarian support cells, about 55,000 ovarian support cells, about 60,000 ovarian support cells, about 65,000 ovarian support cells, about 70,000 ovarian support cells, about 75,000 ovarian support cells, about 80,000 ovarian support cells, about 85,000 ovarian support cells, about 90,000 ovarian support cells, about 95,000 ovarian support cells, about 100,000 ovarian support cells, about 110,000 ovarian support cells, about 120,000 ovarian support cells, about 130,000 ovarian support cells, about 140,000 ovarian support cells, about 150,000 ovarian support cells, about 160,000 ovarian support cells, about 170,000 ovarian support cells, about 180,000 ovarian support cells, about 190,000 ovarian support cells, about 200,000 ovarian support cells, about 210,000 ovarian support cells, about 220,000 ovarian support cells, about 230,000 ovarian support cells, about 240,000 ovarian support cells, about 250,000 ovarian support cells, about 260,000 ovarian support cells, about 270,000 ovarian support cells, about 280,000 ovarian support cells, about 290,000 ovarian support cells, about 300,000 ovarian support cells, about 310,000 ovarian support cells, about 320,000 ovarian support cells Sertoli cells, about 100,000 ovarian Sertoli cells, about 105,000 ovarian Sertoli cells, about 110,000 ovarian Sertoli cells, about 115,000 ovarian Sertoli cells, about 120,000 ovarian Sertoli cells, about 125,000 ovarian Sertoli cells, about 130,000 ovarian Sertoli cells, about 135,000 ovarian Sertoli cells, about 140,000 ovarian Sertoli cells, about 145,000 ovarian Sertoli cells, or about 150,000 ovarian Sertoli cells.

100. The method of any one of claims 96-99, wherein the ovarian supporting cells comprise steroidogenic granulosa cells.

101. The method of claim 100, wherein the steroidogenic granulosa cells produce estradiol.

102. The method of any one of claims 1-101, wherein the ovarian supporting cells are obtained by differentiation of an induced pluripotent stem cell (iPSC) population.

103. The method of claim 102, wherein the ovarian supporting cells are obtained by modifying iPSCs to express one or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1 and RUNX2.

104. The method of claim 103, wherein the ovarian supporting cells are obtained by modifying iPSCs to express two or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

105. The method of claim 104, wherein the ovarian supporting cells are obtained by modifying iPSCs to express three or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

106. The method of claim 105, wherein the ovarian supporting cells are obtained by modifying iPSCs to express four or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

107. The method of claim 106, wherein the ovarian supporting cells are obtained by modifying iPSCs to express all five of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

108. The method of any one of claims 1-107, wherein the ovarian supporting cells are cryopreserved and thawed prior to co-culturing with the one or more oocytes.

109. The method of claim 108, wherein the ovarian supporting cells are thawed about 24 hours to about 120 hours prior to co-culturing with the one or more oocytes.

110. The method of claim 108, wherein the ovarian supporting cells are thawed about 24 hours to about 48 hours, about 48 hours to about 72 hours, about 72 hours to about 96 hours, or about 96 hours to about 120 hours prior to co-culturing with the one or more oocytes.

111. The method of claim 108, wherein the ovarian supporting cells are thawed about 24 hours to about 36 hours, about 30 hours to about 40 hours, about 36 hours to about 48 hours, about 48 hours to about 56 hours, about 56 hours to about 72 hours, about 72 hours to about 84 hours, about 80 hours to about 96 hours, about 90 hours to about 100 hours, about 96 hours to about 108 hours, or about 108 hours to about 120 hours prior to co-culturing with the one or more oocytes.

112. The method of any one of claims 1-111, wherein the one or more oocytes are co-cultured with the ovarian supporting cell population for about 12 hours to about 120 hours.

113. The method of any one of claims 1-111, wherein the one or more oocytes are co-cultured with the ovarian supporting cell population for about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 24 hours to about 48 hours, about 36 hours to about 60 hours, about 54 hours to about 72 hours, about 68 hours to about 96 hours, or about 96 hours to about 120 hours.

114. The method of any one of claims 1-111, wherein the one or more oocytes are co-cultured with the population of ovarian supporting cells for about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, about 54 hours, about 56 hours, about 58 hours, about 60 hours, about 62 hours, about 64 hours, about 66 hours, about 68 hours, about 70 hours, about 72 hours, about 74 hours, about 76 hours, about 78 hours, about 80 hours, about 82 hours, about 84 hours, about 86 hours, about 88 hours, about 90 hours, about 92 hours, about 94 hours, about 96 hours, about 98 hours, about 100 hours, about 102 hours, about 104 hours, about 106 hours, about 108 hours, about 110 hours, about 112 hours, about 114 hours, about 116 hours, about 118 hours or about 120 hours.

115. The method of any one of claims 1-114, wherein the co-cultivation is performed in an adherent co-cultivation system.

116. The method of any one of claims 1-114, wherein the co-cultivation is performed in a suspension co-cultivation system.

117. The method of any one of claims 1-116, wherein before and / or after the co-culture, parameters of the one or more oocytes are evaluated, the parameters being selected from the group consisting of: total oocyte score, oocyte maturation rate from GV to MII, oocyte maturation rate from GV to MI, oocyte maturation rate from MI to MII, average oocyte shape, average oocyte size, average egg quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality.

118. The method of any one of claims 1-117, wherein the one or more oocytes are denuded following the co-cultivation.

119. The method of any one of claims 1-118, further comprising isolating one or more meiosis II (MII) stage oocytes from a mixture produced by co-culturing the one or more oocytes removed from the subject with a population of ovarian supporting cells.

120. The method of claim 119, wherein the subject is undergoing an autologous ART procedure, and wherein the method further comprises contacting each of the one or more MII stage oocytes with a mature sperm cell.

121. A method as claimed in claim 120, wherein the one or more MII stage oocytes are cryopreserved and thawed prior to contacting.

122. The method of claim 120, wherein the one or more MII stage oocytes are not cryopreserved and are not thawed prior to contacting.

123. The method of any one of claims 120-122, wherein the contacting comprises in vitro fertilization (IVF) of one or more MII stage oocytes.

124. The method of any one of claims 120-122, wherein the contacting comprises intracytoplasmic sperm injection (ICSI) into the one or more MII stage oocytes.

125. The method of any one of claims 120-124, wherein the contacting results in the formation of an embryo.

126. The method of claim 125, wherein the embryo is transferred to the uterus of the subject.

127. A method as described in claim 126, wherein the embryo is transferred to the subject's uterus approximately 3 days after the one or more MII stage oocytes are contacted with mature sperm cells.

128. The method of claim 126, wherein the embryo is transferred to the subject's uterus approximately 5 days after the one or more MII stage oocytes are contacted with mature sperm cells.

129. The method of claim 126, wherein the embryo transferred to the subject's uterus is a blastocyst stage embryo.

130. A method of producing a mature oocyte for use in an ART procedure, the method comprising: (a) administering one or more follicle triggering agents to a human subject during the follicle triggering phase; (b) removing one or more oocytes from the subject after the follicular triggering period; as well as (c) culturing the one or more oocytes with a population of ovarian supporting cells to produce one or more mature oocytes.

131. A method of promoting oocyte maturation in a subject who is undergoing an ART procedure and who has previously been administered one or more follicle triggering agents during the follicle triggering phase, the method comprising: (a) removing one or more oocytes from the subject; (b) culturing the one or more oocytes with a population of ovarian supporting cells to produce one or more mature oocytes; and (c) isolating the one or more mature oocytes.

132. The method of claim 130 or 131, wherein the duration of the follicle triggering phase does not exceed 8 days.

133. The method of claim 132, wherein the duration of the follicle triggering phase does not exceed 7 days.

134. The method of claim 133, wherein the duration of the follicle triggering phase does not exceed 6 days.

135. The method of claim 134, wherein the duration of the follicle triggering phase does not exceed 5 days.

136. The method of claim 135, wherein the duration of the follicle triggering phase does not exceed 4 days.

137. The method of claim 136, wherein the duration of the follicle triggering phase does not exceed 3 days.

138. The method of claim 137, wherein the duration of the follicle triggering phase does not exceed 2 days.

139. The method of claim 138, wherein the duration of the follicle triggering phase does not exceed 1 day.

140. The method of claim 130 or 131, wherein the duration of the follicle triggering phase is 1 to 8 days.

141. The method of claim 140, wherein the duration of the follicle triggering phase is 1 to 7 days.

142. The method of claim 141, wherein the duration of the follicle triggering phase is 1 to 6 days.

143. The method of claim 142, wherein the duration of the follicle triggering phase is 1 to 5 days.

144. The method of claim 143, wherein the duration of the follicle triggering phase is 1 to 4 days.

145. The method of claim 144, wherein the duration of the follicle triggering phase is 1 to 3 days.

146. The method of claim 145, wherein the duration of the follicle triggering phase is 2 to 8 days.

147. The method of claim 146, wherein the duration of the follicle triggering phase is 2 to 7 days.

148. The method of claim 147, wherein the duration of the follicle triggering phase is 2 to 6 days.

149. The method of claim 148, wherein the duration of the follicle triggering phase is 2 to 5 days.

150. The method of claim 149, wherein the duration of the follicle triggering phase is 2 to 4 days.

151. The method of claim 130 or 131, wherein the duration of the follicle triggering phase is 3 to 8 days.

152. The method of claim 151, wherein the duration of the follicle triggering phase is 3 to 7 days.

153. The method of claim 152, wherein the duration of the follicle triggering phase is 3 to 6 days.

154. The method of claim 153, wherein the duration of the follicle triggering phase is 3 to 5 days.

155. The method of any one of claims 130-154, wherein the one or more follicle triggers comprise FSH, clomiphene citrate and / or hCG.

156. The method of claim 155, wherein the one or more follicle triggering agents comprises FSH.

157. The method of claim 156, wherein the FSH is administered to the subject in one or more doses per day.

158. The method of claim 157, wherein the FSH is administered to the subject once daily.

159. The method of any one of claims 156-158, wherein the FSH is administered to the subject in an amount of about 100 IU to about 1,000 IU per day.

160. The method of claim 159, wherein the FSH is administered to the subject in an amount of about 200 IU to about 800 IU per day.

161. The method of claim 160, wherein the FSH is administered to the subject in an amount of about 300 IU to about 700 IU per day.

162. The method of claim 161, wherein the FSH is administered to the subject in an amount of about 300 IU to about 600 IU per day, about 300 IU to about 500 IU per day, or about 300 IU to about 400 IU per day.

163. The method of any one of claims 156-162, wherein the duration of FSH administration is equal to the duration of the follicular triggering phase.

164. The method of any one of claims 156-162, wherein the duration of FSH administration is less than the duration of the follicular triggering phase.

165. The method of claim 164, wherein the duration of FSH administration during the follicular triggering phase is 1, 2, 3, 4 or 5 days, optionally wherein FSH is administered to the subject in an amount of about 200 IU per day for 1, 2, 3, 4 or 5 days during the follicular triggering phase, optionally wherein FSH is administered to the subject in an amount of about 200 IU per day for 3 days during the follicular triggering phase.

166. The method of any one of claims 155-165, wherein the one or more follicle triggering agents comprises clomiphene citrate.

167. The method of claim 166, wherein the clomiphene citrate is administered to the subject in one or more doses per day.

168. The method of claim 167, wherein the clomiphene citrate is administered to the subject once daily.

169. The method of any one of claims 166-168, wherein the clomiphene citrate is administered to the subject in an amount of about 50 mg to about 100 mg per day.

170. The method of claim 169, wherein the clomiphene citrate is administered to the subject in an amount of about 50 mg per day.

171. The method of any one of claims 166-170, wherein the duration of clomiphene citrate administration is equal to the duration of the follicular triggering phase.

172. The method of any one of claims 166-170, wherein the duration of clomiphene citrate administration is less than the duration of the follicular triggering phase.

173. The method of claim 172, wherein the duration of clomiphene citrate administration during the follicular triggering phase is 1, 2, 3, 4 or 5 days.

174. The method of any one of claims 155-173, wherein the one or more follicle triggers comprises hCG.

175. The method of claim 174, wherein the hCG is administered to the subject in one or more doses per day.

176. The method of claim 175, wherein the hCG is administered to the subject in 1, 2 or 3 doses during the follicular triggering phase.

177. The method of any one of claims 174-176, wherein the hCG is administered to the subject in an amount of about 200 g to about 700 g per dose.

178. The method of claim 177, wherein the hCG is administered to the subject in an amount of about 200 μg to about 500 μg per dose, about 300 μg to about 600 μg per dose, about 400 μg to about 700 μg per dose, about 200 μg to about 300 μg per dose, about 300 μg to about 400 μg per dose, about 400 μg to about 500 μg per dose, about 500 μg to about 600 μg per dose, or about 600 μg to about 700 μg per dose.

179. The method of claim 178, wherein the hCG is administered to the subject in an amount of about 500 μg per dose.

180. The method of any one of claims 174-176, wherein the hCG is administered to the subject in an amount of about 2,500 IU to about 10,000 IU per dose.

181. The method of any one of claims 130-180, wherein the subject is a subject who has completed oral contraceptive treatment within 28 days of the start of the follicular triggering phase.

182. The method of claim 181, wherein the follicle triggering phase begins at least 5 days after cessation of contraceptive treatment.

183. The method of any one of claims 130-180, wherein the subject has not undergone oral contraceptive treatment within 28 days of the start of the follicular triggering phase.

184. The method of claim 183, wherein the follicular triggering phase begins on day 2 of the subject's menstrual cycle.

185. The method of any one of claims 181-184, wherein the contraceptive treatment comprises administering a GnRH agonist to the subject.

186. The method of any one of claims 130-185, wherein the subject has been determined to exhibit a follicle size of about 6 mm to about 8 mm prior to the onset of the follicular triggering phase.

187. The method of any one of claims 130-185, wherein the subject has been determined to exhibit a follicle size of about 6 mm to about 8 mm prior to administration of the final follicle trigger.

188. The method of any one of claims 130-187, wherein a biological sample isolated from the subject prior to removal of the one or more oocytes has been determined to have an AMH concentration of about 1 ng / mL to about 6 ng / mL.

189. The method of claim 188, wherein the biological sample has been determined to have an AMH concentration of about 2 ng / mL to about 5 ng / mL.

190. The method of claim 189, wherein the biological sample has been determined to have an AMH concentration of about 2.5 ng / mL to about 3.0 ng / mL.

191. The method of any one of claims 130-187, wherein a biological sample isolated from the subject prior to removal of the one or more oocytes has been determined to have an AMH concentration of at least 1 ng / mL.

192. The method of any one of claims 130-187, wherein a biological sample isolated from the subject prior to removal of the one or more oocytes has been determined to have an AMH concentration of no greater than 6 ng / mL.

193. The method of any one of claims 188-192, wherein the biological sample is a blood sample.

194. The method of any one of claims 130-193, wherein the subject is between 18 and 48 years old at the time of retrieval of the one or more oocytes.

195. The method of claim 194, wherein the subject is between 20 and 45 years old at the time of retrieval of the one or more oocytes.

196. The method of claim 194, wherein the subject is less than 35 years old at the time of retrieval of the one or more oocytes.

197. The method of claim 194, wherein the subject is older than 35 years of age at the time of retrieval of the one or more oocytes.

198. The method of any one of claims 130-197, wherein prior to removing the one or more oocytes from the subject, it is determined that the subject exhibits a follicle size of about 6 mm to about 14 mm.

199. The method of claim 198, wherein prior to removal of the one or more oocytes from the subject, it is determined that the subject exhibits a follicle size of about 8 mm to about 12 mm.

200. The method of claim 199, wherein prior to removing the one or more oocytes from the subject, it is determined that the subject exhibits a follicle size of about 8 mm to about 9 mm.

201. The method of any one of claims 130-197, wherein prior to removal of the one or more oocytes from the subject, it is determined that the subject exhibits a follicle size of no greater than 14 mm.

202. The method of any one of claims 198-201, wherein the follicle size has been assessed by ultrasound image analysis.

203. The method of any one of claims 130-202, wherein a total of 20 or fewer oocytes are removed from the subject.

204. The method of claim 203, wherein 15 or fewer oocytes are removed from the subject.

205. The method of claim 204, wherein 10 or fewer oocytes are removed from the subject.

206. The method of claim 205, wherein 9 or fewer oocytes are removed from the subject.

207. The method of claim 206, wherein 8 or fewer oocytes are removed from the subject.

208. The method of claim 207, wherein 7 or fewer oocytes are removed from the subject.

209. The method of claim 208, wherein 6 or fewer oocytes are removed from the subject.

210. The method of claim 209, wherein 5 or fewer oocytes are removed from the subject.

211. The method of any one of claims 130-210, wherein a plurality of oocytes are removed from the subject.

212. The method of claim 211, wherein 10% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

213. The method of claim 212, wherein 20% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

214. The method of claim 213, wherein 30% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

215. The method of claim 214, wherein 40% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

216. The method of claim 215, wherein 50% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

217. The method of claim 216, wherein 60% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

218. The method of claim 217, wherein 70% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

219. The method of claim 218, wherein 80% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

220. The method of claim 219, wherein 90% to 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

221. The method of claim 220, wherein 100% of the oocytes retrieved from the subject are GV or MI stage oocytes.

222. The method of any one of claims 130-221, wherein the ovarian supporting cell population comprises ovarian granulosa cells and / or ovarian stromal cells, optionally wherein the ovarian granulosa cells are FOXL2 positive and / or wherein the ovarian stromal cells are NR2F2 positive.

223. The method of any one of claims 130-222, wherein the population of ovarian support cells comprises about 50,000 to about 100,000 ovarian support cells.

224. The method of any one of claims 130-222, wherein the population of ovarian support cells comprises about 50,000 to about 60,000 ovarian support cells, about 60,000 to about 70,000 ovarian support cells, about 70,000 to about 80,000 ovarian support cells, about 80,000 to about 90,000 ovarian support cells, or about 90,000 to about 100,000 ovarian support cells.

225. The method of any one of claims 130-222, wherein the population of ovarian support cells comprises about 50,000 ovarian support cells, about 55,000 ovarian support cells, about 60,000 ovarian support cells, about 65,000 ovarian support cells, about 70,000 ovarian support cells, about 75,000 ovarian support cells, about 80,000 ovarian support cells, about 85,000 ovarian support cells, about 90,000 ovarian support cells, about 95,000 ovarian support cells, or about 100,000 ovarian support cells.

226. The method of any one of claims 222-225, wherein the ovarian granulosa cells comprise steroidogenic granulosa cells.

227. The method of claim 226, wherein the steroidogenic granulosa cells produce estradiol.

228. The method of any one of claims 130-227, wherein the ovarian supporting cells are obtained by differentiation of an iPSC population.

229. The method of claim 228, wherein the ovarian supporting cells are obtained by modifying iPSCs to express one or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1 and RUNX2.

230. The method of claim 229, wherein the ovarian supporting cells are obtained by modifying iPSCs to express two or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

231. The method of claim 230, wherein the ovarian supporting cells are obtained by modifying iPSCs to express three or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

232. The method of claim 231, wherein the ovarian supporting cells are obtained by modifying iPSCs to express four or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

233. The method of claim 232, wherein the ovarian supporting cells are obtained by modifying iPSCs to express all five of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

234. The method of any one of claims 130-233, wherein the ovarian supporting cells are cryopreserved and thawed prior to co-culturing with the one or more oocytes.

235. The method of claim 234, wherein the ovarian supporting cells are thawed about 24 hours to about 120 hours prior to co-culturing with the one or more oocytes.

236. The method of claim 234, wherein the ovarian supporting cells are thawed about 24 hours to about 48 hours, about 48 hours to about 72 hours, about 72 hours to about 96 hours, or about 96 hours to about 120 hours prior to co-culturing with the one or more oocytes.

237. The method of claim 234, wherein the ovarian supporting cells are thawed about 24 hours to about 36 hours, about 30 hours to about 40 hours, about 36 hours to about 48 hours, about 48 hours to about 56 hours, about 56 hours to about 72 hours, about 72 hours to about 84 hours, about 80 hours to about 96 hours, about 90 hours to about 100 hours, about 96 hours to about 108 hours, or about 108 hours to about 120 hours prior to co-culturing with the one or more oocytes.

238. The method of any one of claims 130-237, wherein the one or more oocytes are co-cultured with the ovarian supporting cell population for about 12 hours to about 120 hours.

239. The method of any one of claims 130-237, wherein the one or more oocytes are co-cultured with the ovarian supporting cell population for about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 24 hours to about 48 hours, about 36 hours to about 60 hours, about 54 hours to about 72 hours, about 68 hours to about 96 hours, or about 96 hours to about 120 hours.

240. The method of any one of claims 130-237, wherein the one or more oocytes are co-cultured with the population of ovarian supporting cells for about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, about 54 hours, about 56 hours, about 58 hours, About 60 hours, about 62 hours, about 64 hours, about 66 hours, about 68 hours, about 70 hours, about 72 hours, about 74 hours, about 76 hours, about 78 hours, about 80 hours, about 82 hours, about 84 hours, about 86 hours, about 88 hours, about 90 hours, about 92 hours, about 94 hours, about 96 hours, about 98 hours, about 100 hours, about 102 hours, about 104 hours, about 106 hours, about 108 hours, about 110 hours, about 112 hours, about 114 hours, about 116 hours, about 118 hours or about 120 hours.

241. The method of any one of claims 130-240, wherein the co-cultivation is carried out in an adherent co-cultivation system.

242. The method of any one of claims 130-240, wherein the co-cultivation is carried out in a suspension co-cultivation system.

243. A method as described in any one of claims 130-242, wherein before and / or after the co-culture, parameters of the one or more oocytes are evaluated, and the parameters are selected from the group consisting of: total oocyte score, oocyte maturation rate from GV stage to MII stage, oocyte maturation rate from GV stage to MI stage, oocyte maturation rate from MI stage to MII stage, average oocyte shape, average oocyte size, average egg quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality and average polar body quality.

244. The method of any one of claims 130-243, wherein the one or more oocytes are denuded following the co-cultivation.

245. The method of any one of claims 130-244, further comprising isolating one or more MII stage oocytes from the mixture, the mixture being produced by co-culturing the one or more oocytes removed from the subject with a population of ovarian supporting cells.

246. A method as claimed in claim 245, wherein the subject is undergoing an autologous ART procedure, and wherein the method further comprises contacting each of the one or more MII stage oocytes with a mature sperm cell.

247. A method as claimed in claim 246, wherein the one or more MII stage oocytes are cryopreserved and thawed prior to contacting.

248. The method of claim 246, wherein the one or more MII stage oocytes are not cryopreserved and are not thawed prior to contacting.

249. The method of any one of claims 246-248, wherein the contacting comprises IVF of one or more MII stage oocytes.

250. The method of any one of claims 246-248, wherein said contacting comprises ICSI into said one or more MII stage oocytes.

251. The method of any one of claims 246-250, wherein said contacting results in the formation of an embryo.

252. The method of claim 251, wherein the embryo is transferred to the uterus of the subject.

253. A method as described in claim 252, wherein the embryo is transferred to the subject's uterus approximately 3 days after the one or more MII stage oocytes are contacted with mature sperm cells.

254. A method as described in claim 252, wherein the embryo is transferred to the subject's uterus approximately 5 days after the one or more MII stage oocytes are contacted with mature sperm cells.

255. The method of claim 252, wherein the embryo transferred to the subject's uterus is a blastocyst stage embryo.

256. An ex vivo composition comprising a population of ovarian support cells and one or more diluents or excipients, optionally wherein the population comprises from about 10,000 to about 100,000 ovarian support cells.

257. The composition of claim 256, wherein the population of ovarian support cells comprises about 50,000 to about 100,000 ovarian support cells.

258. The composition of claim 256, wherein the population of ovarian support cells comprises about 50,000 to about 60,000 ovarian support cells, about 60,000 to about 70,000 ovarian support cells, about 70,000 to about 80,000 ovarian support cells, about 80,000 to about 90,000 ovarian support cells, or about 90,000 to about 100,000 ovarian support cells.

259. The composition of claim 256, wherein the population of ovarian support cells comprises about 50,000 ovarian support cells, about 55,000 ovarian support cells, about 60,000 ovarian support cells, about 65,000 ovarian support cells, about 70,000 ovarian support cells, about 75,000 ovarian support cells, about 80,000 ovarian support cells, about 85,000 ovarian support cells, about 90,000 ovarian support cells, about 95,000 ovarian support cells, or about 100,000 ovarian support cells.

260. The composition of any one of claims 256-259, wherein the ovarian supporting cells comprise steroidogenic granulosa cells.

261. The composition of claim 260, wherein the steroidogenic granulosa cells produce estradiol.

262. A composition as described in any one of claims 256-261, wherein the ovarian supporting cells are obtained by differentiation of an iPSC population.

263. The composition of claim 262, wherein the ovarian supporting cells are obtained by modifying iPSCs to express one or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1 and RUNX2.

264. The composition of claim 263, wherein the ovarian supporting cells are obtained by modifying iPSCs to express two or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

265. The composition of claim 264, wherein the ovarian supporting cells are obtained by modifying iPSCs to express three or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

266. The composition of claim 265, wherein the ovarian supporting cells are obtained by modifying iPSCs to express four or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

267. The composition of claim 266, wherein the ovarian supporting cells are obtained by modifying iPSCs to express all five of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

268. The composition of any one of claims 256-267, wherein the ovarian supporting cells are cryopreserved.

269. A cell culture medium comprising a population of ovarian support cells, optionally wherein the population comprises about 10,000 to about 150,000 ovarian support cells.

270. The cell culture medium of claim 269, wherein the population of ovarian support cells comprises about 50,000 to about 150,000 ovarian support cells.

271. The cell culture medium of claim 269, wherein the population of ovarian support cells comprises about 50,000 to about 60,000 ovarian support cells, about 60,000 to about 70,000 ovarian support cells, about 70,000 to about 80,000 ovarian support cells, about 80,000 to about 90,000 ovarian support cells, about 90,000 to about 100,000 ovarian support cells, about 100,000 to about 110,000 ovarian support cells, about 110,000 to about 120,000 ovarian support cells, about 120,000 to about 130,000 ovarian support cells, about 130,000 to about 140,000 ovarian support cells, or about 140,000 to about 150,000 ovarian support cells.

272. The cell culture medium of claim 269, wherein the population of ovarian support cells comprises about 50,000 ovarian support cells, about 55,000 ovarian support cells, about 60,000 ovarian support cells, about 65,000 ovarian support cells, about 70,000 ovarian support cells, about 75,000 ovarian support cells, about 80,000 ovarian support cells, about 85,000 ovarian support cells, about 90,000 ovarian support cells, about 95,000 ovarian support cells, about 10 ... In some embodiments, the present invention relates to a plurality of ovarian supporting cells, about 100,000 ovarian supporting cells, about 105,000 ovarian supporting cells, about 110,000 ovarian supporting cells, about 115,000 ovarian supporting cells, about 120,000 ovarian supporting cells, about 125,000 ovarian supporting cells, about 130,000 ovarian supporting cells, about 135,000 ovarian supporting cells, about 140,000 ovarian supporting cells, about 145,000 ovarian supporting cells, or about 150,000 ovarian supporting cells.

273. The cell culture medium of any one of claims 269-272, wherein the ovarian supporting cells comprise steroidogenic granulosa cells.

274. The cell culture medium of claim 273, wherein the steroidogenic granulosa cells produce estradiol.

275. The cell culture medium of any one of claims 269-274, wherein the ovarian supporting cells are obtained by differentiation of an iPSC population.

276. The cell culture medium of claim 275, wherein the ovarian supporting cells are obtained by modifying iPSCs to express one or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1 and RUNX2.

277. The cell culture medium of claim 276, wherein the ovarian supporting cells are obtained by modifying iPSCs to express two or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

278. The cell culture medium of claim 277, wherein the ovarian supporting cells are obtained by modifying iPSCs to express three or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

279. The cell culture medium of claim 278, wherein the ovarian supporting cells are obtained by modifying iPSCs to express four or more of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

280. The cell culture medium of claim 279, wherein the ovarian supporting cells are obtained by modifying iPSCs to express all five of FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

281. The cell culture medium of any one of claims 269-280, wherein the cell culture medium is cryopreserved.

282. The composition of any one of claims 256-268 or the cell culture medium of any one of claims 269-281 for use in performing the method of any one of claims 1-255.

283. A kit comprising the composition of any one of claims 256-268 and a package insert, wherein the package insert instructs a user of the kit to co-culture the ovarian support cell population with one or more oocytes according to the method of any one of claims 1-255.

284. A kit comprising the cell culture medium of any one of claims 269-281 and a package insert, wherein the package insert instructs a user of the kit to co-culture the ovarian support cell population with one or more oocytes according to the method of any one of claims 1-255.

285. A device for assisting in vitro maturation of human oocytes, the device comprising: A computing device, wherein the computing device comprises: at least one processor; and A memory, which is communicatively connected to the at least one processor, the memory comprising instructions, the instructions being used to configure the at least one processor to: receiving first biological sample data from a first biological sample associated with a user; assigning a stimulation regimen to the user based on the first biological sample; receiving second biological sample data from a second biological sample associated with the user, wherein the second biological sample includes at least one oocyte; receiving culture data associated with the second biological sample; and A scoring index is assigned to the second biological sample according to the culture data of the second biological sample.

286. A device for in vitro rescue of oocytes after assisted stimulation, the device comprising: A computing device, wherein the computing device comprises: at least one processor; and A memory, which is communicatively connected to the at least one processor, the memory comprising instructions, the instructions being used to configure the at least one processor to: receiving biological sample data from a biological sample associated with a user, wherein the biological sample includes at least one oocyte; determining a maturity level of the at least one oocyte based on the biological sample data; assigning a culture regimen to the at least one oocyte according to the maturity level; receiving culture data associated with at least one oocyte according to the culture protocol; and A scoring index is calculated based on the culture data.

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