Compositions and methods for inducing oocyte maturation

Abstract

Methods, compositions, and devices are featured for in vitro maturation of oocytes. In particular, the present disclosure features methods for inducing oocyte maturation in vitro by co-culturing oocytes from a female subject with an in vitro composition containing a plurality of ovarian support cells (e.g., granulosa cells). Additional methods are provided for administering a follicle-inducing agent and for harvesting oocytes from a female subject. 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 oocyte maturation. [Background technology]

[0002] One in ten women suffer from infertility and require assisted reproductive technologies (ART) such as in vitro fertilization (IVF). Maintaining oocyte health in culture presents challenges, resulting in poor oocyte quality and reduced embryo quality. Furthermore, developmentally immature oocytes are routinely discarded, narrowing the pool of oocytes available for IVF. In vitro maturation (IVM) offers the potential to mature oocytes outside the body after egg retrieval, allowing for the utilization of all harvested eggs. Current IVM methods, which involve spike-in of follicle-stimulating hormone (FSH) into the culture medium, are inefficient, with only 5–40% of immature eggs achieving maturity. Worse yet, this method results in many unhealthy eggs and embryo survival rates of less than 17%, far lower than standard IVF. Therefore, there remains a need in the art to promote oocyte maturation for women undergoing ART procedures. Summary of the Invention

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

[0004] In a further aspect, the present disclosure provides a method of preparing one or more oocytes previously collected 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 support cells.

[0005] In a further aspect, the disclosure features a method of generating mature oocytes for use in ART treatment, the method including co-culturing one or more oocytes previously collected from a human subject with a population of ovarian support cells.

[0006] In some embodiments, prior to harvesting one or more oocytes from a subject, the subject is administered one or more follicle-inducing agents during a follicle-inducing period.

[0007] In some embodiments, prior to collection of one or more oocytes from a subject, the subject is not administered a follicle-inducing agent during the follicle-inducing period.

[0008] In some embodiments, the follicular induction period has a duration of 8 days or less. In some embodiments, the follicular induction period has a duration of 7 days or less. In some embodiments, the follicular induction period has a duration of 6 days or less. In some embodiments, the follicular induction period has a duration of 5 days or less. In some embodiments, the follicular induction period has a duration of 4 days or less. In some embodiments, the follicular induction period has a duration of 3 days or less. In some embodiments, the follicular induction period has a duration of 2 days or less. In some embodiments, the follicular induction period has a duration of 1 day or less. In some embodiments, the follicular induction period has a duration of 1 to 8 days. In some embodiments, the follicular induction period has a duration of 1 to 7 days. In some embodiments, the follicular induction period has a duration of 1 to 6 days. In some embodiments, the follicular induction period has a duration of 1 to 5 days. In some embodiments, the follicular induction period has a duration of 1 to 4 days. In some embodiments, the follicular induction period has a duration of 1 to 3 days. In some embodiments, the follicular induction period has a duration of 2 to 8 days. In some embodiments, the follicular induction period has a duration of 2 to 7 days. In some embodiments, the follicular induction period has a duration of 2 to 6 days. In some embodiments, the follicular induction period has a duration of 2 to 5 days. In some embodiments, the follicular induction period has a duration of 2 to 4 days. In some embodiments, the follicular induction period has a duration of 3 to 8 days. In some embodiments, the follicular induction period has a duration of 3 to 7 days. In some embodiments, the follicular induction period has a duration of 3 to 6 days. In some embodiments, the follicular induction period has a duration of 3 to 5 days.

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

[0010] In some embodiments, FSH is administered to the subject one or more times 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 follicle induction period.In some embodiments, the duration of FSH administration is shorter than the duration of follicle induction period.In some embodiments, the duration of FSH administration is 1, 2, 3, 4 or 5 days during follicle induction period, and optionally, during follicle induction period, FSH is administered to subject in an amount of about 200 IU per day for 1, 2, 3, 4 or 5 days, and optionally, during follicle induction period, FSH is administered to subject in an amount of about 200 IU per day for 3 days.

[0013] In some embodiments, the one or more follicle-inducing agents include clomiphene citrate. In some embodiments, the clomiphene citrate is administered to the subject one or more times per day. In some embodiments, the 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 induction period. In some embodiments, the duration of clomiphene citrate administration is shorter than the duration of the follicular induction period. In some embodiments, the duration of clomiphene citrate administration is 1, 2, 3, 4, or 5 days during the follicular induction period.

[0016] In some embodiments, the one or more follicle-inducing agents include hCG. In some embodiments, the hCG is administered to the subject one or more times per day. In some embodiments, the hCG is administered to the subject one, two, or three times during the follicle-inducing period.

[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 has completed oral contraceptive therapy within 28 days of the start of the follicular induction period. In some embodiments, the follicular induction period begins at least 5 days after cessation of contraceptive therapy.

[0019] In some embodiments, the subject has not received oral contraceptive therapy within 28 days of the start of the follicular induction period.

[0020] In some embodiments, the follicular induction period 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, the subject is determined to exhibit a follicle size of about 6 mm to about 8 mm prior to the start of the follicle induction period. In some embodiments, the subject is determined to exhibit a follicle size of about 6 mm to about 8 mm prior to administration of the final follicle induction agent. In one embodiment, follicle size is determined using a scoring metric (e.g., according to ultrasound imaging or other follicle size determination methods known in the art).

[0023] In some embodiments, the biological sample isolated from the subject prior to harvesting one or more oocytes is determined to have an anti-Müllerian 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, the sample is determined to have an AMH concentration of about 1 ng / ml to about 6 ng / ml, and optionally, the sample is determined to have an AMH concentration of about 2.5 ng / ml to about 3.0 ng / ml. In some embodiments, the sample is determined to have an AMH concentration of about 2 ng / ml to about 5 ng / ml. In some embodiments, the sample is determined to have an AMH concentration of about 2 ng / ml to about 3.0 ng / ml. In some embodiments, the biological sample isolated from the subject prior to harvesting one or more oocytes is determined to have an AMH concentration of at least 1 ng / ml. In some embodiments, the biological sample isolated from the subject prior to harvesting one or more oocytes is determined to have an AMH concentration of 6 ng / ml or less. In some embodiments, the biological sample isolated from the subject prior to harvesting one or more oocytes is determined to have an AMH concentration of about 0.1 ng / ml to about 1 ng / ml. In some embodiments, the sample is a blood sample.

[0025] In some embodiments, the subject is between 18 and 48 years old at the time of collection of one or more oocytes. In some embodiments, the subject is between 25 and 45 years old at the time of collection of one or more oocytes. In some embodiments, the subject is under 35 years old at the time of collection of one or more oocytes. In some embodiments, the subject is over 35 years old at the time of collection of one or more oocytes.

[0026] In some embodiments, prior to collection of one or more oocytes from the subject, the subject is determined to exhibit a follicle size of about 6 mm to about 14 mm. In some embodiments, prior to collection of one or more oocytes from the subject, the subject is determined to exhibit a follicle size of about 8 mm to about 12 mm. In some embodiments, prior to collection of one or more oocytes from the subject, the subject is determined to exhibit a follicle size of about 8 mm to about 9 mm. In some embodiments, prior to collection of one or more oocytes from the subject, the subject is determined to exhibit a follicle size of 14 mm or less.

[0027] In some embodiments, follicle size is assessed by ultrasound imaging.

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

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

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

[0031] In some embodiments, the population of ovarian support cells comprises about 50,000 to about 100,000 ovarian support cells. In some embodiments, 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, and optionally, the population of ovarian support cells comprises about 125,000 ovarian support cells. In some embodiments, the population of ovarian support cells is 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 105,000 ovarian support cells, about 110,000 ovarian support cells, about 115,000 ovarian support cells, or about 120,000 ovarian support cells. , about 125,000 ovarian support cells, about 130,000 ovarian support cells, about 135,000 ovarian support cells, about 140,000 ovarian support cells, about 145,000 ovarian support cells, or about 150,000 ovarian support cells.

[0032] In some embodiments, the population of ovarian support cells comprises a mixture of multiple cell types (e.g., granulosa cells, stromal cells, among other possible cell types). In some embodiments, the population of ovarian support cells comprises a mixture of cells where the cell types are distributed 1:1. In some embodiments, the population of ovarian support cells comprises a mixture of cell types where the cell types are distributed unevenly (e.g., 2:1, 3:1, 4:1, 5:1, among other possible population distributions).

[0033] In some embodiments, the ovarian supportive cells comprise steroidogenic granulosa cells. In some embodiments, the steroidogenic granulosa cells produce estradiol.

[0034] In some embodiments, the ovarian support cells are obtained by differentiation of a population of induced pluripotent stem cells (iPSCs).

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

[0036] In some embodiments, the ovarian support cells are cryopreserved and thawed prior to co-culture with one or more oocytes. In some embodiments, the ovarian support cells are thawed about 24 hours to about 120 hours prior to co-culture with one or more oocytes. In some embodiments, the 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 prior to co-culture with one or more oocytes. In some embodiments, the 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 prior to co-culture with one or more oocytes.

[0037] In some embodiments, one or more oocytes are co-cultured with the population of ovarian support cells for about 12 hours to about 120 hours, hi some embodiments, one or more oocytes are co-cultured with the population of ovarian support cells for about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 24 hours to about 48 hours, 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 incubated with the population of ovarian support cells for about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, 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 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about 96 hours, about 97 hours, about 98 hours, about 99 hours, about 100 hours, about 101 hours, about 102 hours, about 103 hours, about 104 hours, about 105 hours, about 106 hours, about 107 hours, about 108 hours, about 109 hours, about 110 hours, about 111 hours, about 112 hours, about 113 hours, about 114 hours, about 115 hours, about 116 hours, about 117 hours, about 1 The cells are co-cultured for 4 hours, about 66 hours, about 68 hours, about 70 hours, about 72 hours, about 74 hours, 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-culture is performed in an adherent co-culture system. In some embodiments, the co-culture is performed in a suspension co-culture system.

[0039] In some embodiments, before and / or after co-culture, one or more oocytes are assessed for a parameter selected from the group consisting of total oocyte score, GV to MII oocyte maturation rate, GV to MI oocyte maturation rate, MI to MII oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality. In some embodiments, one or more oocytes are denuded after co-culture.

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

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

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

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

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

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

[0046] In a further aspect, the disclosure features a method of generating mature oocytes for use in ART treatment, the method including: (a) administering one or more follicle-inducing agents to a human subject during a follicle-inducing period; (b) collecting one or more oocytes from the subject after the follicle-inducing period; and (c) culturing the one or more oocytes with a population of ovarian support cells, thereby generating one or more mature oocytes.

[0047] In a further aspect, the disclosure features a method of promoting oocyte maturation in a subject undergoing ART treatment who has previously been administered one or more follicle-inducing agents during a follicle-inducing period, the method including: (a) collecting one or more oocytes from the subject; (b) culturing the one or more oocytes with a population of ovarian support cells, thereby producing one or more mature oocytes; and (c) isolating the one or more mature oocytes.

[0048] In some embodiments, the follicular induction period has a duration of 8 days or less. In some embodiments, the follicular induction period has a duration of 7 days or less. In some embodiments, the follicular induction period has a duration of 6 days or less. In some embodiments, the follicular induction period has a duration of 5 days or less. In some embodiments, the follicular induction period has a duration of 4 days or less. In some embodiments, the follicular induction period has a duration of 3 days or less. In some embodiments, the follicular induction period has a duration of 2 days or less. In some embodiments, the follicular induction period has a duration of 1 day or less. In some embodiments, the follicular induction period has a duration of 1 to 8 days. In some embodiments, the follicular induction period has a duration of 1 to 7 days. In some embodiments, the follicular induction period has a duration of 1 to 6 days. In some embodiments, the follicular induction period has a duration of 1 to 5 days. In some embodiments, the follicular induction period has a duration of 1 to 4 days. In some embodiments, the follicular induction period has a duration of 1 to 3 days. In some embodiments, the follicular induction period has a duration of 2 to 8 days. In some embodiments, the follicular induction period has a duration of 2 to 7 days. In some embodiments, the follicular induction period has a duration of 2 to 6 days. In some embodiments, the follicular induction period has a duration of 2 to 5 days. In some embodiments, the follicular induction period has a duration of 2 to 4 days. In some embodiments, the follicular induction period has a duration of 3 to 8 days. In some embodiments, the follicular induction period has a duration of 3 to 7 days. In some embodiments, the follicular induction period has a duration of 3 to 6 days. In some embodiments, the follicular induction period has a duration of 3 to 5 days.

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

[0050] In some embodiments, FSH is administered to the subject one or more times 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 follicle induction period.In some embodiments, the duration of FSH administration is shorter than the duration of follicle induction period.In some embodiments, the duration of FSH administration is 1, 2, 3, 4 or 5 days during follicle induction period, and optionally, during follicle induction period, FSH is administered to subject in an amount of about 200 IU per day for 1, 2, 3, 4 or 5 days, and optionally, during follicle induction period, FSH is administered to subject in an amount of about 200 IU per day for 3 days.

[0053] In some embodiments, the one or more follicle-inducing agents comprises clomiphene citrate.

[0054] In some embodiments, clomiphene citrate is administered to the subject one or more times per day. In some embodiments, clomiphene citrate is administered to the 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 induction period. In some embodiments, the duration of clomiphene citrate administration is shorter than the duration of the follicular induction period. In some embodiments, the duration of clomiphene citrate administration is 1, 2, 3, 4, or 5 days during the follicular induction period.

[0057] In some embodiments, the one or more follicle-inducing agents include hCG. In some embodiments, the hCG is administered to the subject one or more times per day. In some embodiments, the hCG is administered to the subject one, two, or three times during the follicle-inducing period.

[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 has completed oral contraceptive therapy within 28 days of the start of the follicular induction period. In some embodiments, the follicular induction period begins at least 5 days after cessation of contraceptive therapy.

[0060] In some embodiments, the subject has not received oral contraceptive therapy within 28 days of the start of the follicular induction period.

[0061] In some embodiments, the follicular induction period 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, the subject is determined to exhibit a follicle size of about 6 mm to about 8 mm prior to the start of the follicular induction period.

[0064] In some embodiments, the subject is determined to exhibit a follicle size of about 6 mm to about 8 mm prior to the final administration of the follicle-inducing agent.

[0065] In some embodiments, a biological sample isolated from a subject prior to collection of one or more oocytes is determined to have an AMH concentration of about 1 ng / ml to about 6 ng / ml. In some embodiments, the sample is determined to have an AMH concentration of about 2 ng / ml to about 5 ng / ml. In some embodiments, the sample is determined to have an AMH concentration of about 2.5 ng / ml to about 3.0 ng / ml. In some embodiments, a biological sample isolated from a subject prior to collection of one or more oocytes is determined to have an AMH concentration of at least 1 ng / ml. In some embodiments, a biological sample isolated from a subject prior to collection of one or more oocytes is determined to have an AMH concentration of 6 ng / ml or less.

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

[0067] In some embodiments, the subject is between 18 and 48 years old at the time of collection of one or more oocytes. In some embodiments, the subject is between 20 and 45 years old. In some embodiments, the subject is between 25 and 45 years old at the time of collection of one or more oocytes. In some embodiments, the subject is under 35 years old at the time of collection of one or more oocytes. In some embodiments, the subject is over 35 years old at the time of collection of one or more oocytes.

[0068] In some embodiments, prior to collection of one or more oocytes from the subject, the subject is determined to exhibit a follicle size of about 6 mm to about 14 mm. In some embodiments, prior to collection of one or more oocytes from the subject, the subject is determined to exhibit a follicle size of about 8 mm to about 12 mm. In some embodiments, prior to collection of one or more oocytes from the subject, the subject is determined to exhibit a follicle size of about 8 mm to about 9 mm. In some embodiments, prior to collection of one or more oocytes from the subject, the subject is determined to exhibit a follicle size of about 14 mm or less.

[0069] In some embodiments, follicle size is assessed by ultrasound imaging.

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

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

[0072] In some embodiments, the population of ovarian support cells comprises ovarian granulosa cells and / or ovarian stromal cells, optionally wherein the ovarian granulosa cells are FOXL2-positive and / or the ovarian stromal cells are NR2F2-positive. In some embodiments, the population of ovarian support cells comprises a mixture of ovarian granulosa cells and ovarian stromal cells. In some embodiments, the population of ovarian support cells comprises a mixture of cells having an approximately 1:1 distribution of ovarian granulosa cells to ovarian stromal cells, with or without one or more additional cell types in the population. In some embodiments, the population of ovarian support cells comprises a mixture of cells having a distribution of multiple cell types, wherein one or more cell types are more prevalent than other cell types (e.g., having a relative distribution of 2:1, 3:1, 4:1, 5:1, among other possible population distributions). In some embodiments, the population of ovarian support cells comprises a mixture of ovarian granulosa cells and ovarian stromal cells, with one cell type predominant 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, among other possible distributions). In some embodiments, the population of ovarian support cells comprises a mixture of ovarian granulosa cells and ovarian stromal cells, along with one or more additional cell types.

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

[0074] In some embodiments, the population of ovarian support cells comprises about 50,000 to about 100,000 ovarian support cells. In some embodiments, 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. 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, or about 100,000 ovarian support cells.

[0075] In some embodiments, the ovarian supportive cells comprise steroidogenic granulosa cells. In some embodiments, the steroidogenic granulosa cells produce estradiol.

[0076] In some embodiments, the ovarian support cells are obtained by differentiation of a population of iPSCs.

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

[0078] In some embodiments, the ovarian support cells are cryopreserved and thawed prior to co-culture with one or more oocytes. In some embodiments, the ovarian support cells are thawed about 24 hours to about 120 hours prior to co-culture with one or more oocytes. In some embodiments, the 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 prior to co-culture with one or more oocytes. In some embodiments, the 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 prior to co-culture with one or more oocytes.

[0079] In some embodiments, one or more oocytes are co-cultured with the population of ovarian support cells for about 12 hours to about 120 hours, hi some embodiments, one or more oocytes are co-cultured with the population of ovarian support cells for about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 24 hours to about 48 hours, 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 incubated with the population of ovarian support cells for about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, 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 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about 96 hours, about 97 hours, about 98 hours, about 99 hours, about 100 hours, about 101 hours, about 102 hours, about 103 hours, about 104 hours, about 105 hours, about 106 hours, about 107 hours, about 108 hours, about 109 hours, about 110 hours, about 111 hours, about 112 hours, about 113 hours, about 114 hours, about 115 hours, about 116 hours, about 117 hours, about 1 The cells are co-cultured for 4 hours, about 66 hours, about 68 hours, about 70 hours, about 72 hours, about 74 hours, 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-culture is performed in an adherent co-culture system. In some embodiments, the co-culture is performed in a suspension co-culture system.

[0081] In some embodiments, before and / or after co-culture, one or more oocytes are assessed for a parameter selected from the group consisting of total oocyte score, GV to MII oocyte maturation rate, GV to MI oocyte maturation rate, MI to MII oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm 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 after co-culture.

[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 collected from the subject with a population of ovarian feeder cells.

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

[0085] In some embodiments, the one or more MII stage oocytes are cryopreserved and thawed prior to contacting. In some embodiments, the one or more MII stage oocytes are not cryopreserved and 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, the contacting results in the formation of an embryo. In some embodiments, the embryo is transferred to the subject's uterus. In some embodiments, the embryo is transferred to the subject's uterus about 3 days after contacting one or more MII stage oocytes with mature sperm cells. In some embodiments, the embryo is transferred to the subject's uterus about 5 days after contacting one or more MII stage oocytes with mature sperm cells. In some embodiments, the embryo transferred to the subject's uterus is a blastocyst stage embryo.

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

[0089] In a further aspect, the disclosure features an ex vivo composition including a population of ovarian support cells and one or more diluents or additives.

[0090] In some embodiments, the population of ovarian support cells comprises about 50,000 to about 100,000 ovarian support cells. In some embodiments, 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. 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, or about 100,000 ovarian support cells.

[0091] In some embodiments, the ovarian supportive cells comprise steroidogenic granulosa cells. In some embodiments, the steroidogenic granulosa cells produce estradiol.

[0092] In some embodiments, the ovarian support cells are obtained by differentiation of a population of iPSCs.

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

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

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

[0096] In some embodiments, the population of ovarian support cells comprises about 50,000 to about 100,000 ovarian support cells. In some embodiments, 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. 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, or about 100,000 ovarian support cells.

[0097] In some embodiments, the ovarian supportive cells comprise steroidogenic granulosa cells. In some embodiments, the steroidogenic granulosa cells produce estradiol.

[0098] In some embodiments, the ovarian support cells are obtained by differentiation of a population of iPSCs.

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

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

[0101] In a further aspect, the disclosure features a composition of any one of the preceding aspects, or a cell culture medium of any one of the preceding aspects, for use in practicing a method of any one of the preceding aspects.

[0102] In a further aspect, the disclosure features a kit including the composition of any one of the preceding aspects and an accompanying instruction, the instruction instructing a user of the kit to co-culture the population of ovarian support cells with one or more oocytes according to the method of any one of the preceding aspects.

[0103] In a further aspect, the disclosure features a kit including the cell culture medium of any one of the preceding aspects and an accompanying instruction, wherein the instruction instructs a user of the kit to co-culture the population of ovarian support cells with one or more oocytes according to the method of any one of the preceding aspects.

[0104] The accompanying drawings are included to illustrate and further understand embodiments of the present disclosure. [Brief explanation of the drawings]

[0105] [Figure 1A] 1 is a block diagram illustrating an embodiment and device for supporting human oocyte maturation in vitro. [Figure 1B] FIG. 1 shows an exemplary embodiment of a device 100 for assisting in vitro oocyte rescue after stimulation. [Figure 2A] FIG. 1 is a block diagram illustrating an example embodiment of a machine learning module. [Figure 2B] FIG. 1 is a diagram of an example table showing training data for training a machine learning model. [Figure 2C] FIG. 10 is an exemplary table illustrating further training data for training a machine learning model. [Figure 3A] 1 is an exemplary flow chart of a mini-stimulation protocol. [Figure 3B] 1 is an exemplary flow chart of oocyte denudation. [Figure 4] FIG. 1 shows an exemplary table of metabolite formulations. [Figure 5] 1 is an exemplary flow chart for preparing a granulosa cell co-culture. [Figure 6A] FIG. 1 shows an exemplary embodiment of a co-cultured second biological sample. [Figure 6B] FIG. 10 shows an exemplary embodiment of a control group culture of a second biological sample. [Figure 6C] FIG. 1 shows an exemplary embodiment of co-cultured oocytes. [Figure 6D] FIG. 1 shows an exemplary embodiment of a control culture of immature oocytes. [Figure 7A] 1 is a flow chart showing an exemplary method for inducing human oocyte maturation in vitro. [Figure 7B] 1 is an exemplary flow chart showing a method for in vitro oocyte rescue after stimulation. [Figure 8] FIG. 1 is a block diagram illustrating 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. [Figure 9] This diagram shows the experimental workflow for ovaloid formation. First, barcoded transcription factor (TF) expression vectors were integrated into FOXL2-T2A-tdTomato reporter human induced pluripotent stem cells (hiPSCs). After induction of TF expression, cells positive for tdTomato and granulosa-associated surface markers were sorted and barcodes were sequenced. Top TFs based on barcode enrichment were selected for further characterization by combinatorial screening and bulk RNA-seq analysis. Next, monoclonal hiPSC lines were generated that inducibly express the top TFs and generate granulosa-like cells with high efficiency. Granulosa-like cells from these lines were further evaluated for estradiol production in response to follicle-stimulating hormone (FSH). Finally, they were combined with human primordial germ cell-like cells (hPGCLCs) to form ovaloids. These ovarian lamina propria produced estradiol and progesterone, formed follicle-like structures, and supported hPGCLC maturation as measured by immunofluorescence microscopy and scRNA-seq analysis. [Figure 10A] Schematic diagram of the experimental co-culture IVM approach. hiPSCs are differentiated using overexpression of inducible transcription factors to form ovarian support cells (OSCs). Immature human cumulus-oocyte complexes (COCs) are obtained from in-clinic donors after brief gonadotropin stimulation. In the laboratory, developmental dishes containing seeded OSCs are prepared as needed, and COCs are introduced for IVM co-culture. Oocyte maturation and morphological quality are assessed after 24–28 h of IVM co-culture, and samples are stored for analysis or used for embryogenesis. [Figure 10B] Representative images showing a co-culture configuration containing human COCs (n=5) and 100,000 OSCs at the time of plating. Scale bar is 100 μm. In suspension culture, COCs with spread and non-spread cumulus are seen with surrounding OSCs. [Figure 11A]Figure 1 shows the oocyte maturation rate after 24-28 h of IVM experiments in experiment 1 involving oocyte co-culture with OSCs or medium control. n indicates the number of individual oocytes in each culture condition. Error bars indicate the mean ± SEM. p-values are obtained from unpaired t-tests comparing OSC-IVM with medium control conditions. [Figure 11B] Figure 1 shows the total oocyte score (TOS) generated from imaging analysis of MII oocytes 24-28 hours after IVM experiments. n indicates the number of individual MII oocytes analyzed. Median (dashed line) and quartiles (dotted lines) are shown. An unpaired t-test showed no significant difference between means (p=0.2909). Due to the low number of oocytes collected per donor, oocytes could not be consistently divided between both conditions analyzed. Groups primarily contain oocytes from non-overlapping donor cohorts, and pairwise comparisons were not used. [Figure 12A] Figure 1 shows the oocyte maturation rate after 28 hours of IVM experiments in Experiment 2 involving oocyte co-culture with OSCs or commercial IVM control. n indicates the number of individual oocytes in each culture condition. Error bars indicate the mean ± SEM. p-values were obtained from paired t-tests comparing experimental OSC-IVM with the control condition (commercial IVM control). [Figure 12B] Figure 1 shows the total oocyte score (TOS) generated from imaging analysis of MII oocytes after a 28-hour IVM experiment. n indicates the number of individual MII oocytes analyzed. Median (dashed line) and quartiles (dotted lines) are shown. An unpaired t-test showed no significant difference between means (p=0.9420). COCs from each donor were randomly and equally distributed between control and treatment to allow pairwise statistical comparisons. [Figure 13A]Figure 1 shows embryogenesis results after a 28-hour IVM experiment in a subset of oocytes used for embryogenesis in experiment 2 involving oocyte co-culture with OSCs or a commercial IVM control. Error bars represent the mean ± SEM. Results are shown as a percentage of the total COCs treated in that group. Fertilization, cleavage, blastocyst formation, quality blastocyst formation, and euploid blastocyst formation outcomes were assessed for both IVM conditions. [Figure 13B] Representative images of embryogenesis in OSC-IVM and commercial IVM conditions at day 3 cleavage and days 5, 6, and 7 of blastocyst formation. Embryos with adequate vitrification quality were labeled "usable quality blasts" and used for trophectoderm biopsy. [Figure 14A] Schematic diagram of the experimental co-culture IVM approach. hiPSCs were differentiated using overexpression of inducible transcription factors to form ovarian support cells (OSCs). Human oocytes were obtained from in-clinic donors after standard gonadotropin stimulation, and immature oocytes (GV and MI) identified after denudation were assigned to this study. In the embryology laboratory, dishes containing OSC seeding were prepared as needed, and immature oocytes were introduced for IVM co-culture. After 24–28 h of IVM co-culture, oocyte maturation and health were assessed, and oocyte samples were stored for further analysis. [Figure 14B] Representative images showing the co-culture setup containing immature human oocytes (n=3) and OSCs at the time of plating. Scale bar: 200 μm. Denuded GV oocytes are seen in suspension culture with surrounding OSCs. [Figure 15A] Figure 1 shows the oocyte maturation rate after 24-28 h IVM experiments in experiments involving oocyte co-culture with OSCs (OSC-IVM) or medium control (medium-IVM). n indicates the number of individual oocytes in each culture condition. Error bars indicate the mean ± SEM. p values are from unpaired t-tests comparing experimental OSC-IVM with control medium-IVM. Due to the low number of oocytes collected per donor, each group contains oocytes from primarily non-overlapping donor groups, and pairwise comparisons were not utilized. [Figure 15B] Figure 1 shows the total oocyte score (TOS) generated from imaging analysis of MII oocytes 24-28 hours after IVM experiments. n indicates the number of individual MII oocytes analyzed. Median (dashed line) and quartiles (dotted line) are shown. An unpaired t-test showed no significant differences between the means (ns, p=0.5725). [Figure 16A] Representative images of MII oocytes after 28 hours of IVM coculture with OSCs are shown, stained with a fluorescent alpha-tubulin dye (cyan) to visualize the meiotic spindle. The blue line from the oocyte center across the spindle assembly and the center of PB1 was used to determine the PB1-spindle angle. The range of PB1-spindle angles is indicated above. An example of an MII with no spindle is obtained from the medium-IVM condition. [Figure 16B] Quantification of the angle between PB1 and the spindle obtained from oocyte fluorescence imaging analysis (shown in A). n= indicates the number of individual oocytes analyzed from each condition. The number of MII oocytes that did not exhibit spindle assembly is also shown below the axis labels. Median (dashed line) and quartiles (dotted lines) are shown. ANOVA (analysis of variance) statistical analysis revealed no significant differences between the means of each condition (ns, p=0.1155). [Figure 17A] UMAP projection of oocyte transcriptomes with symbols colored by experimental batch, experimental condition (OSC-IVM, medium-IVM, IVF-MII), oocyte maturation status, and Leiden cluster. Each symbol represents one oocyte. n=81 oocytes. [Figure 17B] Shown is a UMAP projection colored by the score for each of the genetic marker sets (GV and IVF MII). [Figure 17C] UMAP projections generated from the scores of cells for each of the two signature marker sets (GV vs. IVF MII) and colored by experimental condition, oocyte maturation status, and Leiden cluster. [Figure 17D]Figure 1 shows quantification of oocytes in each maturation outcome (GV, MI, and MII) by experimental condition (IVM or IVF). Color distribution indicates the percentage of the population in each Leiden cluster. Striped bars are used to indicate clusters with predominantly IVF-like features. [Figure 18A] Immunofluorescence images of human obaloid (F66 / N.R1.GF#4 granulosa-like cells + hPGCLC) sections stained for FOXL2 (granulosa), OCT4 (germ cell / pluripotency), and DAZL (mature germ cell) at days 2, 4, 14, and 32 of culture are shown. Scale bar is 40 μm. [Figure 18B] A mouse ovarian (mouse fetal ovarian somatic cells + hPGCLC) section stained as in Figure 18A is shown. The scale bar is 40 μm. [Figure 18C] Figure 1 shows the percentage of OCT4+ and DAZL+ cells relative to total (DAPI+) cells over time in human and mouse xenogeneic ovarian nuclei. Counts were performed at 11 time points on images from two replicates of human ovarian nuclei (F66 / N.R1.GF#4 and F66 / N.R2#1 granulosa-like cells + hPGCLCs) and one replicate of mouse xenogeneic ovarian nuclei. [Figure 18D] Immunofluorescence images of human oballoid (F66 / N.R2#1 granulosa-like cells + hPGCLC) sections stained for SOX17 (germ cells), TFAP2C (early germ cells), and AMHR2 (granulosa) on days 4 and 8 of culture are shown. Scale bar is 40 μm. [Figure 18E] Immunofluorescence analysis of DAZL and OCT4 expression in day 16 ovaloids shows several DAZL+OCT4- cells (magenta arrows) and DAZL+OCT4+ cells (cyan arrows). The ovaloids are also beginning to form follicle-like morphologies (yellow arrows). Scale bar = 40 μm. [Figure 19A]Shown is a section of a day 35 human ovarian follicular ... [Figure 19B] This shows the overall appearance of the follicle-like structure in a human obaloid (F66 / N.R1.G#7). The scale bar is 1 mm. [Figure 19C] Sections of human ovarian follicular ... [Figure 19D] Sections of human ovarian follicular follicular nuclei (F66 / N.R2#1+hPGCLC) at day 70 in culture stained for FOXL2, NR2F2, and AMHR2 are shown. This shows an antral follicle composed of FOXL2+AMHR2+ granulosa-like cells arranged in several layers around a central cavity. NR2F2 staining is visible outside the follicle (marked "stroma"). Scale bar is 100 μm. [Figure 20A] Expression (log2 CPM) of selected granulosa (FOXL2), stromal / thecal (NR2F2), and germ cell (PRDM1) markers. Expression is from scRNA-seq analysis of ovarian (F66 / N.R1.GF#4 granulosa-like cells + hPGCLCs). Data from all samples (days 2, 4, 8, and 14) were combined for joint dimensionality reduction and clustering. [Figure 20B] Leiden clustering of the four major clusters. Marker gene expression (log2 CPM) is plotted for each cluster obtained from scRNA-seq analysis of ovaloids as in Figure 20A. [Figure 20C]Figure 20A shows the mapping of cells to the Human Fetal Ovary Reference Atlas (Garcia-Alonso et al., 2022) and cell type assignment based on scRNA-seq analysis shown in Figure 20A. [Figure 20D] FIG. 20B shows the percentages of somatic cell types, germ cells, DAZL+ cells, and DDX4+ cells in the ovarian nuclei on each day based on the scRNA-seq analysis shown in FIG. 20A. [Figure 21A] FIG. 1 shows denuded oocytes from standard treatment. [Figure 21B] FIG. 1 shows COC from minimal stimulation. [Figure 21C] FIG. 1 shows that OSC-IVM statistically significantly improves oocyte maturation rate. [Figure 22A] FIG. 1 shows the morphological quality of oocytes grown in culture with OSC-IVM. [Figure 22B] FIG. 1 shows the angle between PB1 and the spindle in oocytes grown in culture with OSC-IVM. [Figure 22C] FIG. 1 shows that oocytes grown in culture with OSC-IVM show high similarity to in vivo MII oocytes. [Figure 22D] FIG. 1 shows that oocytes grown in culture with OSC-IVM show high similarity to in vivo MII oocytes. [Figure 23A] FIG. 1 shows the oocyte degradation rate from toxicity assessment of OSC-IVM products. [Figure 23B] FIG. 1 shows fertilization and blastocyst formation of OSC-IVM products. [Figure 24]This figure demonstrates that OSC-IVM oocytes, compared to medium-IVM oocytes, exhibit similar stress- and cell cycle-related differential gene expression relative to IVF-MII controls. For each experimental condition (OSC-IVM, medium-IVM, IVF-MII), gene expression values for oocytes at different developmental states (GV, MI, or MII) are grouped for analysis. Each row represents a specific group. For each group, relative gene expression (upper panel) and absolute gene expression (lower panel) for specific genes with known roles in cell cycle, stress, and meiosis are shown, with each column representing a specific gene. Samples are ordered on the y-axis using unsupervised hierarchical clustering (UHC) of selected genes as a measure of relative similarity. Figures are not necessarily drawn to scale and may be shown in phantom, schematic, and fragmentary form. In certain instances, certain details may be omitted if they are not necessary for understanding the embodiments or if they obscure other details. DETAILED DESCRIPTION OF THE INVENTION

[0106] definition Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. In the event of any possible ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise stated. The use of the term "including," and variations thereof, such as "include" and "included," is meant to be open-ended.

[0107] As used herein, the term "about" means that a value is within 10% above or below the stated value (10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less). For example, the phrase "about 50 mg" refers to a value between 45 mg and 55 mg, inclusive.

[0108] As used herein, the term "assisted reproductive technology" or "ART" refers to infertility treatments in which one or more female gametocytes (oocytes) or gametes (eggs) are manipulated in vitro to promote the formation of an embryo, and the embryo is then implanted into a subject seeking pregnancy. For example, in some embodiments, oocytes collected from a subject undergoing ART treatment can be matured in vitro using, for example, the co-culture methods described herein. In some embodiments, upon formation of mature oocytes (eggs), the eggs can be treated with sperm cells to promote the formation of a zygote and ultimately an embryo. The embryo can then be transferred into the uterus of a female subject using, for example, compositions and methods known in the art. Exemplary ART treatments include in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), which are known in the art and described herein.

[0109] As used herein, the term "subject" refers to a living organism being treated for a particular disease or condition described herein. Examples of subjects include mammals, such as humans (e.g., female humans), being treated for a disease or condition corresponding to a decline in ovarian reserve or the release of immature oocytes.

[0110] As used herein, the term "controlled ovarian stimulation" refers to a technique for inducing ovulation in a subject (e.g., a human subject) prior to collection of oocytes or eggs for use in embryogenesis, e.g., by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Controlled ovarian stimulation may include 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 stimulation is known in the art, but is also described herein as a method for inducing follicle maturation and ovulation in conjunction with assisted reproductive technology.

[0111] As used herein, the term "derived from," with respect to a cell derived from a subject, means that the cell (e.g., a mammalian egg) is isolated from the subject or is obtained by growth, division, maturation, or manipulation (e.g., in vitro growth, division, maturation, or manipulation) of one or more cells isolated from the subject. For example, when an egg is "derived from" a subject or an oocyte as described herein, the egg is either directly isolated from the subject or obtained by maturation of an oocyte isolated from the subject (e.g., an oocyte isolated from a subject about 1 to about 5 days after ovarian stimulation (e.g., an oocyte isolated from a subject about 2 to about 4 days after ovarian stimulation)).

[0112] As used herein, the term "dose" refers to the amount of a therapeutic agent (e.g., a follicle-stimulating agent described herein) administered to a subject for the treatment of a disease or condition (e.g., to promote oocyte maturation and / or release viable oocytes and promote the collection and in vitro maturation of viable oocytes). The therapeutic agents described herein can be administered one or more times. In each case, the therapeutic agent can be administered using one or more unit dosage forms. For example, a 100 mg dose of the therapeutic agent may be administered using, for example, two 50 mg unit dosage forms of the therapeutic agent. Similarly, a 300 mg dose of the therapeutic agent may be administered using, for example, six 50 mg unit dosage forms of the therapeutic agent, or two 50 mg unit dosage forms of the therapeutic agent and one 200 mg unit dosage form of the therapeutic agent. Similarly, a 900 mg dose of the therapeutic agent may be administered using, for example, six 50 mg unit dosage forms of the therapeutic agent and three 200 mg unit dosage forms of the therapeutic agent, or ten 50 mg unit dosage forms of the therapeutic agent and two 200 mg unit dosage forms of the therapeutic agent.

[0113] As used herein, the term "follicle induction period" refers to the time point for administering a follicle-inducing agent. The time point for administering a follicle-inducing agent to a female subject (i.e., the follicle induction period) is day 1, day 2, or day 3 of the subject's menstrual cycle, with day 2 of the menstrual cycle being preferred. However, if the female subject is taking hormonal contraceptives, the time point for administering the follicle-inducing agent is 4 to 6 days (e.g., 4, 5, or 6 days) after taking the last oral contraceptive, with 5 days after taking the last oral contraceptive being preferred.

[0114] As used herein, the term "follicle-stimulating hormone" (FSH) refers to a biologically active heterodimeric human reproductive hormone capable of inducing ovulation in a subject. FSH may be purified from the urine of postmenopausal humans or produced as a recombinant protein product. Exemplary recombinant FSH products include follitropin alfa (GONAL-F, Merck Serono / EMD Serono) and follitropin beta (PUREGON / FOLLISTIM, MSD / Scherig-Plough).

[0115] 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 ovarian follicle maturation and ovulation. hCG can be purified from the urine of pregnant women or produced as a recombinant protein product. Exemplary recombinant hCG products include choriogonadotropin alpha (Ovidrel®, Merck Serono / EMD Serono).

[0116] As used herein, the term "in vitro fertilization" (IVF) refers to a process in which eggs, such as human eggs, are contacted with one or more sperm cells outside of a living organism to promote fertilization of the eggs and the formation of a zygote. The eggs may be derived from a subject, such as a human subject undergoing various ARTs known in the art. For example, one or more oocytes may be obtained from a subject after injection of a follicle maturation stimulating agent for controlled ovarian stimulation, e.g., about 1 to about 5 days after injection of the agent (e.g., about 4 days after injection of the follicle maturation stimulating agent into the subject). Eggs may also be collected directly from a subject, e.g., by transvaginal egg collection methods known in the art.

[0117] As used herein, the term "intracytoplasmic sperm injection" (ICSI) refers to the process of directly injecting sperm cells into an egg, such as a human egg, to promote fertilization of the egg and the formation of a zygote. For example, sperm cells can be injected into an egg by perforating the egg membrane with a microinjector to deliver the sperm cells directly into the egg's cytoplasm. ICSI techniques useful in combination with the compositions and methods described herein are known in the art and are described, for example, in WO2013 / 158658, WO2008 / 051620, and WO2000 / 009674, the disclosures of which are incorporated herein by reference, particularly with respect to compositions and methods for performing intracytoplasmic sperm injection.

[0118] As used herein, the terms "egg" and "oocyte" refer to haploid female germ cells or gametes. With respect to the assisted reproductive technologies described herein, eggs can be produced in vitro by maturation of one or more oocytes isolated from a subject undergoing ART. Eggs can also be isolated directly from a subject, for example, by transvaginal egg collection methods described herein or known in the art. As used in this disclosure, an egg or oocyte may refer to multiple oocytes. An oocyte may form a complex with surrounding cells (e.g., a cumulus-oocyte complex (COC)).

[0119] As used herein, the terms "mature egg" and "mature oocyte" refer to one or more eggs or oocytes at the metaphase II (MII) stage of meiosis, typically having morphological or structural features consistent with metaphase II, such as polar bodies and other features described herein.

[0120] As used herein, the terms "immature egg" and "immature oocyte" refer to one or more eggs or oocytes that have not reached the meiosis MII stage. In some embodiments, an immature oocyte can be an oocyte, including an oocyte at the germinal vesicle (GV) and / or metaphase I (MI) stage, as determined by morphological characteristics and / or other indicators known in the art.

[0121] As used herein, the term "oocyte maturation" refers to the process by which an immature oocyte develops into a mature oocyte. Oocyte maturation occurs as the immature oocyte undergoes cell signaling events triggered by external and internal stimuli. External stimuli can occur from adjacent cells or support cells as described herein. Oocyte maturation can occur prior to oocyte release and retrieval from a subject. Oocyte maturation can occur in vitro as a result of the culture methods and culture compositions described herein.

[0122] As used herein, "ovarian support cells" (OSCs) or "support cells" refer to one or more cells that promote the maturation of one or more oocytes. OSCs can be ovarian granulosa cells (e.g., a type of granulosa cell described herein). Additionally or alternatively, OSCs can be ovarian stromal cells (e.g., a type of stromal cell described herein). OSCs can form cumulus-oocyte complexes (COCs) with oocytes. OSCs can be generated from exogenous sources, such as induced pluripotent stem cells (iPSCs), e.g., human induced pluripotent stem cells (hiPSCs), as described herein. OSCs can be applied to harvested oocytes using the in vitro cell culture methods and compositions described herein. OSCs can be a mixture of two or more cell types. OSCs can be a mixture of stromal cells and granulosa cells, such that the stromal cells and granulosa cells are present in an approximately 1:1 ratio. OSCs may be a mixture of stromal cells and granulosa cells, in which one cell type is more abundant than one or more other cell types, for example, about a 2:1 population, about a 3:1 population, about a 4:1 population, or about a 5:1 population, among other possible population distributions. OSCs may also be a mixture of stromal cells and granulosa cells, in which one cell type is more abundant (for example, 90% stromal cells and 10% granulosa cells, 80% stromal cells and 20% granulosa cells, 70% stromal cells and 30% granulosa cells, 60% stromal cells and 40% granulosa cells, 40% stromal cells and 60% granulosa cells, 30% stromal cells and 70% granulosa cells, 20% stromal cells and 80% granulosa cells, or 10% stromal cells and 90% granulosa cells, among other possible distributions). In some embodiments, the OSCs can be a mixture of stromal and granulosa cells in combination with one or more additional cell types.

[0123] As used herein, "ovarian stromal cells" or "stromal cells" refer to cumulus cells that surround oocytes to ensure healthy oocytes and subsequent embryonic development. Ovarian stromal cells may form COCs with oocytes. Ovarian stromal cells may express markers consistent with stromal subtypes, such as nuclear receptor subfamily 2 group F member 2 (NR2F2). NR2F2 can be detected by methods known in the art. Ovarian stromal cells may be steroidogenic stromal cells. Ovarian stromal cells can be generated from differentiated hiPSCs as described herein.

[0124] As used herein, "steroidogenic stromal cells" are stromal cells that can produce one or more steroids, such as estradiol, progesterone, or a combination thereof. The one or more steroids can be produced in response to hormonal stimulation, such as with FSH, androstenedione, or a combination thereof. The one or more steroids can be secreted.

[0125] As used herein, "ovarian granulosa cells" or "granulosa cells" refer to cumulus cells that surround oocytes to ensure healthy oocytes and subsequent embryonic development. Ovarian granulosa cells can form COCs with oocytes. Ovarian granulosa cells can express markers consistent with the granulosa subtype, such as FOXL2, CD82, and / or follicle-stimulating hormone receptor (FSHR). Such markers can be detected by methods known in the art. Ovarian granulosa cells can be steroidogenic granulosa cells. Ovarian granulosa cells can be generated from hiPSCs differentiated as described herein.

[0126] As used herein, "steroidogenic granulosa cells" are granulosa cells capable of producing one or more steroids, such as estradiol, progesterone, or a combination thereof. The one or more steroids may be produced in response to hormonal stimulation, such as with FSH, androstenedione, or a combination thereof. The one or more steroids may be secreted.

[0127] As used herein, the term "biological sample" or "sample" refers to a specimen (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., from the placenta or dermis), pancreatic juice, chorionic villus sample, hair, oocytes, eggs, and / or cells) isolated from a subject.

[0128] As used herein, the terms "oral contraceptive therapy," "oral contraception," "contraception," or "contraceptives" refer to hormonal treatments typically used to prevent pregnancy. Oral contraceptive therapy can prevent the release of oocytes from the ovaries and can include hormones, including estrogen and progestin.

[0129] As used herein, the term "ovarian reserve" refers to the number of oocytes in a subject's ovaries and the quality of the oocytes. Ovarian reserve naturally decreases with age and / or medical conditions as described herein. Subjects with decreased ovarian reserve may seek IVF or other ART to achieve successful pregnancy. As described herein, the level of anti-Mullerian hormone (AMH) can indicate a subject's ovarian reserve.

[0130] As used herein, the term "stimulation protocol" refers to the process of administering one or more follicle-inducing agents to a subject during a follicle-inducing period.

[0131] As used herein, the term "follicle-inducing agent" or "inducing agent" refers to a chemical or biological composition that stimulates the release of oocytes from the ovaries during ovulation. Follicle-inducing agents can include hormones such as human chorionic gonadotropin and follicle-stimulating hormone. As used herein, the term "induced pluripotent stem cells" (iPSCs) refers to artificial stem cells derived from harvested somatic cells that have been reprogrammed or manipulated. iPSCs can be differentiated into other cell types, including ovarian feeder cells or granulosa cells, by methods known in the art and described herein. iPSCs can be human (hiPSCs) or can be iPSCs from other mammalian sources, for example.

[0132] As used herein, the term "cell culture" refers to a laboratory method that allows for the growth of cells in vitro and / or the cultivation of prokaryotic or eukaryotic cell species.

[0133] As used herein, the term "co-culture" refers to a type of cell culture method in which multiple cell types or cell populations are cultured with some degree of contact between them. In a typical co-culture system, two or more cell types may share an artificial growth medium.

[0134] As used herein, the term "adherent co-culture system" or "adherent cell culture" refers to the formation of cell culture by attaching cells to a surface for proper growth and proliferation.

[0135] As used herein, the term "suspension co-culture system" or "suspension cell culture" refers to a cell culture configuration in which cells are cultured by dispersion in a liquid medium for suitable growth and proliferation.

[0136] Detailed Description Described herein are devices, compositions, and methods for use in assisted reproductive technologies (ART), including those related to follicle stimulation for oocyte release in the ovaries, as well as in vitro maturation of oocytes following follicle stimulation (i.e., post-stimulation).

[0137] Advantageously, the methods described herein achieve in vitro maturation of immature oocytes through co-culture with ovarian support cells (e.g., ovarian granulosa cells and / or stromal cells), thereby enabling the collection and use of oocytes that would previously have been discarded for typical in vitro fertilization (IVF) purposes. The described in vitro maturation methods improve the ability to use such normally discarded immature oocytes in IVF procedures, potentially resulting in more cost-effective treatment strategies and reduced risks to the treated subject. For example, the methods may reduce the risk of in vivo ovarian hyperstimulation by requiring subjects seeking IVF treatment to receive fewer hormone injections and / or lower doses of hormone injections than current IVF treatment options. Use of embodiments of the present disclosure can increase a woman's overall pool of available, healthy oocytes for use in IVF. Use of embodiments of the present disclosure can also significantly reduce the amount of hormone administered to a subject during egg collection and improve the quality of oocytes in culture. This could greatly expand access to reproductive technologies, significantly shorten the duration of each cycle, and potentially require fewer cycles overall to achieve pregnancy.

[0138] I. Methods for stimulating oocyte release A. Target Selection The methods of stimulating oocyte release described herein are directed to subjects seeking an IVF treatment option. Typically, the subjects are women with low oocyte collection numbers or subjects with a high number of immature oocytes. The subjects may be between 20 and 45 years of age, and typically are 35 years of age or older. The subjects may have a diminished ovarian reserve due to aging and / or may have a genetic or medical condition that leads to diminished ovarian reserve (e.g., polycystic ovary syndrome (PCOS)). The subject may have an ovarian reserve of 20 or fewer oocytes, such as having 1-5 oocytes, 4-10 oocytes, 8-16 oocytes, or 15-20 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, or 20 oocytes). The subject may have anti-Müllerian hormone (AMH) levels consistent with reduced ovarian reserve. The subject may have had AMH levels measured by blood tests and other methods known in the art. The subject may have an AMH level of 1-6 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 a measured estradiol level of 20-50 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).

[0139] A physician or practitioner can evaluate a subject for a method of stimulating oocyte release by collecting a biological sample from the subject. The biological sample may include laboratory specimens maintained by a biorepository for testing. In some embodiments, the biological sample may include bodily fluids such as blood, saliva, urine, semen (semenal plasma), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal discharge, ocular fluid, gastric fluid, breast milk, cell culture supernatant, etc. The biological sample may include a medical diagnosis, user input describing the user's mood and / or symptom complaints, information collected from a wearable device related to the user, etc. For example, the biological sample may include information obtained from a visit to a medical professional, such as a medical history. In yet another non-limiting example, the biological sample may include information such as data collected from a wearable device worn by the user, which is designed to collect information regarding the user's sleep patterns, exercise patterns, etc. In some embodiments, the biological sample is collected on a specific day and / or time of the user's menstrual cycle. For example, without limitation, the biological sample may be collected on day 2 of the user's menstrual cycle to assess one or more hormone levels. The biological sample can be used to determine the subject's AMH level and / or other hormone levels or markers of the subject's ovarian reserve, which can be measured by other indicators. An AMH level of 1 ng / mL or less can be used to indicate low ovarian reserve. A subject with low ovarian reserve can have a measured AMH level of 1.0 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, or 0.1 ng / 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, monitoring menstrual cycle progression and / or 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.

[0140] Other biological sample data collected from a subject includes at least one oocyte. As used in this disclosure, "biological sample data" refers to data that characterize the biological, genetic, biochemical, and / or physiological properties, composition, or activity of a biological sample. In some embodiments, the oocyte may be an immature oocyte. As used in this disclosure, an "immature oocyte" refers to one or more immature germ cells that develop in the ovary. In some embodiments, the immature oocyte may be an oocyte, including a GV oocyte and / or an MI oocyte. In some embodiments, the immature oocyte may be a plurality of oocytes. The immature oocyte may be an immature cumulus-oocyte complex (COC) collected from a subject. As used in this disclosure, a "cumulus-oocyte complex" refers to an oocyte surrounded by specific granulosa cells. As used in this disclosure, "specific granulosa cells" refer to the cumulus cells that surround the oocyte to ensure healthy oocyte and embryo development. In some embodiments, immature oocytes may include oocytes to which certain granulosa cells are added to mature the oocyte in cell culture (e.g., co-culture) and create a COC.

[0141] In some embodiments of the present methods, a biological sample may be collected from a user via a collection device. A "collection device" is a device and / or tool capable of obtaining, recording, and / or verifying measurements associated with a sample. Collection devices may include needles, syringes, vials, lancets, evacuated collection tubes (ECTs), tourniquets, evacuated collection tube systems, any combination thereof, and the like. For example, a collection device may include a butterfly needle set. Data from the biological sample may include measurements of, for example, serum calcium, phosphate, electrolytes, blood urea nitrogen and creatinine, uric acid, and the like.

[0142] In one embodiment of the method, the subject's biological sample information may be obtained from an ultrasound examination. As used in this disclosure, "ultrasound examination" refers to any technique that utilizes sound waves to generate one or more images of a user's body. For example, an ultrasound examination may be used to obtain images of the subject's reproductive organs and / or tissues. In certain embodiments, the ultrasound examination may be performed at a particular time during the subject's menstrual cycle. For example, a subject may undergo an ultrasound examination on day 2 of their cycle, which may be used to identify follicle size and / or follicle number. Selection of a stimulation protocol and / or adjustments to the stimulation protocol may be made using such information. For example, for a subject whose ultrasound examination indicates PCOS, dosage adjustments may be made to one or more medications taken and / or utilized during the stimulation protocol. Furthermore, the length of the subject's stimulation protocol may be modified based on the subject's diagnosis of PCOS. In one embodiment, the ultrasound examination may be repeated one or more times throughout the subject's stimulation protocol, and the information obtained may be used to adjust the subject's stimulation protocol in real time.

[0143] B. Oocyte Stimulation Protocol A physician or a skilled practitioner can use the described biological parameters to determine the stimulation protocol for oocyte release for a subject. Such biological parameters include, for example, hormone levels (e.g., baseline hormone levels and / or hormone levels due to the use of contraceptives), physical characteristics of the subject (e.g., follicle size, follicle number, ovarian morphology, and / or uterine morphology), among other biological parameters known to those skilled in the art. A skilled practitioner may implement a stimulation protocol using any one or a combination of the induction agents or compositions intended to stimulate follicle maturation and oocyte release described herein.

[0144] Hormone levels or concentrations of other relevant compounds in a biological sample can include levels of 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, and the like. In some embodiments, measuring hormone levels can be based on blood analysis of the biological sample. For example, blood analysis can include plasma hormone analysis. In some embodiments, measuring hormone levels can be based on saliva hormone testing. Measuring hormone levels can also be based on other forms of analysis (e.g., hair, urine, and any other form of biological sample described throughout this disclosure). Prior to the follicular induction period, the subject's baseline serum level of estradiol 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 induction period, the subject's baseline serum level of progesterone can be from about 0.5 ng / mL to about 2.5 ng / mL (e.g., from about 0.5 ng / mL to about 1.0 ng / mL, from about 1.0 ng / mL to about 1.5 ng / mL, from about 1.5 ng / mL to about 2.0 ng / mL, or from 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).

[0145] Furthermore, a subject's use of contraception (e.g., hormonal contraception) can influence the design of the stimulation protocol. Contraception considerations can help determine the follicle induction period in a woman's menstrual cycle. For example, and without limitation, a subject not using any form of contraception can begin a stimulation protocol using recombinant follicle-stimulating hormone (rFSH) between days 1 and 3 of their menstrual cycle, preferably on day 2 of their menstrual cycle. In yet another non-limiting example, a subject using contraception can begin a stimulation protocol using rFSH 4 to 6 days (e.g., 4, 5, or 6) after taking their last oral contraceptive, preferably 5 days after administering their last oral contraceptive. In one embodiment, rFSH stimulation can be used for 2 to 3 days (e.g., 2 or 3 days), depending on the subject's tolerance, follicle size, and / or growth dynamics. After this two or three day period, a one to three day (e.g., one, two, or three day) coasting period can be utilized to monitor follicle size and allow for further follicle maturation and development. As used in this disclosure, a "coasting period" is any period during which the agents used throughout the stimulation protocol are not administered and / or consumed. The coasting period can last one, two, three, or more days, for example, if medically necessary. During the coasting period, the subject can continue to undergo one or more ultrasound examinations to monitor their progress.

[0146] When the follicle size reaches anywhere between approximately 8 and 10 mm (e.g., 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, or greater), the subject may be triggered by administration of an inducer such as human chorionic gonadotropin (hCG). As used herein, a "follicle measurement" is any measurement of a follicle. A follicle may include any sac present in the ovary that contains an unfertilized egg. Follicle measurements can be obtained using any method described herein, such as ultrasound, manual measurement, or automated measurement. In one embodiment, a double hCG injection can be used to induce follicular maturation and prepare one or more follicles for collection. The double hCG injection can be two or three injections of hCG. On the first day of the double hCG injection, blood tests for one or more hormone levels, such as E2, P4, and LH, may be performed to monitor hormone levels. After the two doses of hCG, one or more hormone levels may be measured, for example, with blood tests to determine and test levels of E2, P4, and LH.

[0147] An "inducing agent" is a chemical that causes cell development in the ovaries. Inducing agents (e.g., follicle-inducing agents) can include any substance, including any non-prescription and / or prescription drugs. Induction agents (e.g., follicle-inducing agents) include Lupron Depot® (Abbott Laboratories, North Chicda, IL), Ganirelix (Ferring Pharmaceuticals, Saint-Prex, Switzerland), Cetrotide (Merck Global, Readington Township, NJ), Gonal-F® (Merck Global), Follistim® (Merck Global), Bravelle® (Ferring Pharmaceuticals), Clomid® (Patheon Pharmaceuticals Inc., Waltham, MA), Serephene (Teva, Tel Aviv-Yafo, Israel), Glucophage® (Merck Global), Fortamet® (Mylan, Canonsburg, PA), Pregnyl® (Schering Plough, Kenilworth, NJ), Novarell® (Ferring Pharmaceuticals), Laboratories, Parsippany, NJ), Repronex (Ferring Pharmaceuticals), Factrel® (Zoetis Canada Inc., Kirkland, Canada), Menopr® (Ferring Pharmaceuticals), and other drugs that induce cell development in the ovaries that one of skill in the art would understand to be applicable. Inducing agents (e.g., follicle-inducing agents) can include human serum albumin, FSH, hCG, androstenedione, and doxycycline, among other inducers known in the art.

[0148] In one embodiment, the subject may not receive an induction agent (e.g., a follicle-inducing agent) to stimulate oocyte production. In one embodiment, the subject may receive multiple injections of induction agent over a period of 1-4 days (e.g., 1, 2, 3, or 4 days), but not more than 5 days, in a preferred stimulation protocol. The subject may receive multiple injections over multiple days, such as 5 injections of one or more induction agents. For example, the subject may receive 300 IU-700 IU (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, e.g., 30 They may undergo 3 days of stimulation (one or more injections per day) with rFSH at 0 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). The subject may receive an injection of 200-700 μg or 2,500-10,000 IU of 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) (preferably a 500 μg stimulation dose) as an inducer. The subject may receive one or more injections of clomiphene citrate in combination with other induction agents at a dose of 50-150 mg per injection (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).

[0149] Prior to administration of the inducer, the subject's serum may be assessed for levels of hormones or other suitable compounds. Prior to administration of the inducer, the subject's serum level of estradiol may be between about 250 pg / mL and about 400 pg / mL (e.g., between about 250 pg / mL and about 275 pg / mL, between about 275 pg / mL and about 300 pg / mL, between about 300 pg / mL and about 325 pg / mL, between about 325 pg / mL and about 350 pg / mL, between about 350 pg / mL and about 375 pg / mL, or between about 375 pg / mL and 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 administration of the inducer, the subject's progesterone serum level can be about 0.25 ng / mL to about 0.75 ng / mL (e.g., about 0.25 ng / mL to about 0.35 ng / mL, about 0.35 ng / mL to about 0.45 ng / mL, about 0.45 ng / mL to about 0.55 ng / mL, about 0.55 ng / mL to about 0.65 ng / mL, or 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 0.65 ng / mL, about 0.70 ng / mL, or about 0.75 ng / mL). Prior to administration of the inducer, the subject's serum level of LH can 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 administration of the inducer, the subject's serum level of FSH can 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).

[0150] Inducing agents (e.g., follicle-inducing agents) may be administered sequentially to produce a minimal stimulation protocol, a follicle stimulation protocol. A minimal stimulation protocol is configured by a skilled artisan to induce cell release over a span of approximately three days. A "minimal stimulation protocol" is a stimulation process over a shorter period of time than an average in vitro fertilization (IVF) stimulation protocol, serving to induce the ovaries to produce oocytes. Typically, the average duration for a stimulation protocol using standard IVF is approximately 8 to 14 days. A minimal stimulation protocol may induce cell release over a period of 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., 1 to 3 days, 2 to 4 days, 3 to 5 days, 4 to 6 days, 5 to 7 days, or 6 to 8 days, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days). This is a shorter period of time than the 8 to 14 days of a standard IVF stimulation protocol. The average time to perform a minimal stimulation protocol may be two days. The average time to perform a minimal stimulation protocol may be three days. The average time to perform a minimal stimulation protocol may be four days. The average time to perform a minimal stimulation protocol may be five days. The average time to perform a minimal stimulation protocol may be six days. In one embodiment, a minimal stimulation protocol may not require administration of a follicle-inducing agent for successful oocyte retrieval and subsequent maturation. In one embodiment, a minimal stimulation protocol may include selecting a first inducing agent (e.g., a follicle-inducing agent) and selecting a second inducing agent (e.g., a follicle-inducing agent) depending on follicle measurements and / or other biological sample data.

[0151] C. Oocyte CollectionAfter follicular stimulation, oocytes (or a group of cells containing oocytes) are harvested from the subject. As used in this disclosure, "oocytes" refer to germ cells derived from the ovaries. Approximately 24 to 48 hours (e.g., 24 to 32 hours, 32 to 40 hours, or 40 to 48 hours, e.g., about 24 hours, about 28 hours, about 32 hours, about 36 hours, about 40 hours, about 44 hours, or about 48 hours) after the final administration of an induction agent (e.g., a follicle-inducing agent), the subject can undergo oocyte harvest. On the day of oocyte harvest, blood tests for one or more hormone levels, such as E2, LH, FSH, and / or P4, can be performed to assess quality, ensure hormone levels are within range, and / or confirm that the hCG dose was taken. E2 hormone levels 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, e.g., 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 collection. The LH hormone level can be about 3mIU / mL to about 6mIU / mL (e.g., about 3mIU / mL to about 4mIU / mL, about 4mIU / mL to about 5mIU / mL, or 5mIU / mL to about 6mIU / mL, e.g., about 3mIU / mL, about 3.5mIU / mL, about 4mIU / mL, about 4.5mIU / mL, about 5mIU / mL, about 5.5mIU / mL, or about 6mIU / mL) on the day of oocyte collection. The FSH hormone level can be about 6mIU / mL to about 9mIU / mL (e.g., about 6mIU / mL to about 7mIU / mL, about 7mIU / mL to about 8mIU / mL, or about 8mIU / mL to about 9mIU / mL, e.g., about 6mIU / mL, about 6.5mIU / mL, about 7mIU / mL, about 7.5mIU / mL, about 8mIU / mL, about 8.5mIU / mL, or about 9mIU / mL) on the day of oocyte collection.P4 hormone levels 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, e.g., 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) on the day of oocyte collection.

[0152] Oocytes (or a group of cells containing oocytes) are collected from a subject using methods known in the art. For example, oocytes can be collected by aspiration using transvaginal ultrasound with a needle guide on the probe to aspirate the released follicular contents. The follicular aspirate can then be examined using a dissecting microscope, washed with HEPES medium (G-MOPS Plus, Vitrolife®), and filtered through a 70-micron cell strainer (Falcon®, Corning). The oocytes and / or COCs are then transferred to a culture dish and medium to initiate co-culture and appropriate controls as described herein. Other collection methods can include collection devices such as needles, syringes, vials, lancets, evacuated collection tubes (ECTs), tourniquets, evacuated collection tube systems, or any combination thereof. For example, the collection device can include a butterfly needle set.

[0153] The collected oocytes may include, for example, 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. As used in this disclosure, a "cumulus oocyte complex" (COC) is an oocyte that includes one or more surrounding cumulus cells. A COC may include an immature oocyte. A COC may include a mature oocyte.

[0154] As used in this disclosure, an "immature oocyte" refers to one or more immature germ cells that develop in the ovary. In some embodiments, an immature oocyte can be an oocyte, including but not limited to, an oocyte at the germinal vesicle stage (GV) and metaphase I (MI) stage, as further described below. In some embodiments, an immature oocyte can be a plurality of oocytes. An immature oocyte can be an immature cumulus oocyte complex (COC) collected from a subject. As used in this disclosure, a "mature oocyte" can be one or more mature oocytes at the metaphase II stage (MII). Once collected, the COC can be allowed to rest for 1 hour, 2 hours, 3 hours, or more to equilibrate to the in vitro conditions for in vitro maturation.

[0155] Upon collection, any one or more of the collected oocytes or cells described herein can be suitably frozen and stored using methods known in the art for future use, analysis, or experimentation. Additionally, any one or more of the collected oocytes or cells described herein can be used fresh (i.e., suitable for immediate use, such as for in vitro maturation or for any one or more of the analyses or experiments described herein).

[0156] II. Oocyte rescue methods A. Oocyte exposure Following the oocyte collection methods described above, one or more COCs may require oocyte denudation. As used herein, "oocyte denudation" refers to the removal of cumulus cells or other cell types from oocytes by mechanical separation, chemical separation, or a combination thereof. Several methods of oocyte denudation are known in the art. In some embodiments, denudation can occur within the IVM well by gently mechanically dissociating the cells with pipetting, removing most cumulus and / or granulosa cells. If enzymatic dissociation is required, the cells may be transferred to a separate dish for hyaluronidase treatment. COCs can be stripped with a stripper tip and washed with IVM medium or MOPS plus medium to clean the oocytes for imaging and, if necessary, inactivate the hyaluronidase. Stripper tips may include 200- and / or 400-micron tips for fine washing. In some embodiments, germinal vesicle (GV) and metaphase I (MI) stage oocytes can be prepared and used for culture after COC denudation. Denuded COCs may be transferred to a separate culture dish for imaging.

[0157] B. Co-culture of oocytes for in vitro maturation i. Contents and timing of co-culture In the methods described herein, oocytes can be mixed with specific granulosa cells and / or specific stromal cells in co-culture. "Specific granulosa cells" and "specific stromal cells" refer to the cumulus cells that surround oocytes to ensure healthy oocyte and embryo development. In some embodiments, the granulosa co-culture cells and / or stromal co-culture cells are derived from human induced pluripotent stem cells (hiPSCs). As used herein, "co-culture" refers to a cell culture technique in which two or more distinct cell populations are grown with some degree of contact between them. In some embodiments, steroidogenic granulosa cells derived from human induced pluripotent stem cells (hiPSCs) can be co-cultured with immature oocytes (COCs), thereby reconstituting the follicular microenvironment in vitro and promoting oocyte maturation rapidly and efficiently in a manner that enhances oocyte health and developmental competence. As used herein, "steroidogenic granulosa cells" are granulosa cells that express high levels of steroidogenic enzymes that produce estradiol. For example, steroidogenic granulosa cells can be mural granulosa cells harvested from cystic follicles. Applying steroidogenic granulosa cells to co-culture COCs can increase in vitro oocyte maturation after egg / oocyte harvest, enabling utilization of all harvested eggs / oocytes by directly providing nutrients, materials, and mechanical support to oocytes throughout gametogenesis and folliculogenesis. As described further below, steroidogenic granulosa cells can be grown in standard IVF and IVM media to achieve oocyte maturation of immature COCs. This can increase the overall pool of available healthy oocytes for use in IVF and reduce the number of egg / oocyte harvest procedures users undergo.

[0158] In some embodiments of the method, a cell culture can be formed by mixing immature oocytes with specific granulosa cells and / or specific stromal cells, which can be added to mature the oocytes in cell culture and form COCs after harvesting one or more oocyte-COCs following a minimal stimulation protocol. In one embodiment, one or more specific granulosa cells and / or specific stromal cells can be thawed during the resting period of one or more COCs. In one embodiment, any number of specific granulosa cells between 50,000 and 150,000 (e.g., 50,000 to 60,000 cells, 60,000 to 70,000 cells, 70,000 to 80,000 cells, 80,000 to 90,000 cells, 90,000 to 10,000 cells, 100,000 to 110,000 cells, 110,000 to 120,000 cells, 120,000 to 130,000 cells, 130,000 to 140,000 cells, or 140,000 to 150,000 cells, e.g., 50,000 cells ... 5,000 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 COCs during culture.In one embodiment, thawed granulosa cells can be placed in culture medium about 24 to 120 hours (e.g., about 24 to 48 hours, about 48 to 72 hours, about 72 to 96 hours, about 96 to 120 hours, e.g., about 24 to 36 hours, about 30 to 40 hours, about 36 to 48 hours, about 48 to 56 hours, about 56 to 72 hours, about 72 to 84 hours, about 80 to 96 hours, about 90 to 100 hours, about 96 to 108 hours, about 108 to 120 hours, e.g., 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) prior to COC harvest. COCs can be transferred to medium containing thawed specific granulosa cells to form group cultures, as described in more detail below. In one embodiment, group cultures can be cultured in an incubator for any time period between 12 and 48 hours (e.g., 12-16 hours, 12-20 hours, 18-24 hours, 18-36 hours, 24-36 hours, 36-48 hours, e.g., 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours). Co-culture can be performed at a biologically appropriate temperature, e.g., 37°C.

[0159] In some embodiments of the method, the harvested oocytes containing immature cumulus-oocyte complexes can be cultured in group culture. "Group culture" refers to combining the harvested COC with one or more additional cells. The additional cells can include any cells that grow with the harvested COC. The additional cells can include certain stromal cells. The additional cells can include certain granulosa cells. In one embodiment, the group cultures are incubated for a specified time, e.g., 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, e.g., 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, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 hours, 95 hours, 96 hours, 97 hours, 98 hours, 99 hours, 100 hours, 101 hours, 102 hours, 103 hours, 104 hours, 105 hours, 106 hours, 107 hours, 108 hours, 109 hours, 110 hours, 111 hours, 112 hours, 113 hours, 114 hours, The COCs can be cultured and / or incubated for a period of time (e.g., 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 group culture can include culturing COCs with a granulosa cell co-culture, as described further below. In some embodiments, the group culture can include culturing a control group of COCs without co-culture, as described further below. In some embodiments, the user can provide immature oocytes, such as GV and MI stage oocytes. Such immature oocytes can be used in culture as part of a group culture to support the growth of COCs. Oocyte donation can be performed according to the oocyte collection process described above. The subject participating in the oocyte donation can be different from or the same as the subject involved in the second biological sample containing the immature COC. In some embodiments, the subject donating the oocytes can undergo a stimulation protocol as disclosed above.

[0160] In some embodiments, the maturity of oocytes collected from a subject may determine the length of time the oocytes are co-cultured with ovarian support cells (e.g., certain granulosa cells and / or certain stromal cells). For example, less mature oocytes (e.g., GV oocytes) may require a longer co-culture period than oocytes at more advanced stages of meiosis (e.g., MI oocytes).

[0161] In some embodiments related to oocyte culture, the cell culture medium can include LAG medium (Medicult, CooperSurgical®). For example, LAG medium can be used for incubation of oocytes and / or COCs after collection from a minimal stimulation protocol. For example, modified Medicult IVM medium can be used as a baseline control during the culture process. In some embodiments, the cell culture medium can include metabolic substances such as those illustrated in FIG. 4. For example, modified Medicult IVM medium can include human serum albumin, FSH, hCG, androstenedione, doxycycline, or any combination thereof. The medium can be pre-incubated for about 18-24 hours (e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours) in a standard 37°C sterile incubator containing O2 (e.g., 1-10% O2 atmosphere, e.g., 4-8% O2 or 5-7% O2, e.g., 6% O2) and an appropriate CO2 level. This is known in the art. The co-culture and certain granulosa cell cultures may be adherent cell cultures in cell culture dishes or flasks. The co-culture and certain granulosa 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 using one or more established methods known in the art.

[0162] In some embodiments, the co-culture is performed according to the steps outlined in Figure 5. 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, which is then resuspended in a medium suitable for in vitro maturation. In some embodiments, the ovarian support cells are centrifuged one or more additional times, each time resuspended in an in vitro maturation medium. The ovarian support cells can then be co-cultured with oocytes obtained from a subject undergoing ART treatment, thereby inducing oocyte maturation.

[0163] ii.hiPSC-derived granulosa cells Certain granulosa cells for use in the methods described herein can be generated from hiPSCs using a transcription factor (TF) induction protocol. In some embodiments, hiPSCs can be transformed with any one or more plasmids encoding one or more transcription factors. In some embodiments, hiPSCs can be transformed by electroporation, liposome-mediated transformation, or viral-mediated gene transfer, among other cell transformation methods known in the art. In some embodiments, 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 used to reprogram hiPSCs can carry a reporter gene, such as a fluorescent protein. In some embodiments, hiPSCs can be differentiated into stromal cells by inducible expression of transcription factors, including GATA4, FOXL2, or a combination thereof. In some embodiments, hiPSCs can be differentiated into stromal cells by inducible expression of transcription factors, including FOXL2, NR5A1, GATA4, RUNX1, RUNX2, or a combination thereof. In addition to a combination of one or more of the transcription factors FOXL2, NR5A1, GATA4, RUNX1, and / or RUNX2, hiPSCs can differentiate into granulosa cells via expression of KLF2, TCF21, NR2F2, or a combination thereof.

[0164] Reprogramming of hiPSCs into stromal cells and / or granulosa cells can be determined by genotyping methods known in the art. Reprogramming of hiPSCs into granulosa cells can be determined by protein expression using any one or more methods known in the art. Differentiation of hiPSCs into stromal cells can be determined by the relative expression of biomarkers typical of stromal cell types, including NR2F2, among others known in the art. Differentiation of hiPSCs into granulosa cells can be determined by the relative expression of biomarkers typical of granulosa cell types, such as AMHR2, CD82, FOXL2, FSHR, IGFBP7, KRT19, STAR, WNT4, or combinations thereof, among others known in the art. In some embodiments, reprogramming of hiPSCs into granulosa cells can be determined by the production of growth factors and / or hormones, including estradiol and progesterone, which can appropriately support in vitro maturation of harvested oocytes via paracrine and juxtacrine cell signaling. In some embodiments, the resulting granulosa cells produce estradiol upon stimulation with androstenedione and FSH or forskolin. In some embodiments, the granulosa cells described herein can be produced in multiple batches. In some embodiments, the granulosa cells can be frozen and thawed prior to the co-culture method. In some embodiments, the granulosa cells are newly differentiated prior to the in vitro maturation method. In some embodiments, the granulosa cells can be seeded 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) before adding oocytes for in vitro maturation.

[0165] In some embodiments, a subject can donate hiPSCs. The donation of hiPSCs can be performed after the oocyte collection process described above. The subject participating in the donation of hiPSCs can be different from or the same as the subject from whom the oocytes were collected. In some embodiments, the hiPSC donor can undergo a stimulation protocol as disclosed above.

[0166] In some embodiments, hiPSCs, granulosa cells, cumulus cells, oocytes, GV stage oocytes, MI stage oocytes, MII stage oocytes, and all other cell types described throughout this disclosure can be lysed, extracted for genomic material, and flash-frozen to prepare them for further manipulation and / or analysis (e.g., for analysis as part of the omics data collection methods described in Section II(C)(iii) below). For example, cells can be subjected to enzymatic cell lysis using enzymes such as lysozyme, lysostaphin, zymolase, cellulose, protease, or glycanase. Other lysis methods, such as chemical lysis, detergent lysis, alkaline lysis, mechanical lysis, heat lysis, ultrasonic lysis, physical lysis, non-mechanical lysis, and other lysis methods known in the art, may also be applied. In some embodiments, the medium may be flash-frozen. Freezing methods may include using cryoprotectants such as dimethyl sulfoxide and / or any other freezing methods known in the art.

[0167] iii. Transgenic granulosa cells Certain transgenic granulosa cells for use in the methods described herein can be generated using the clustered regularly interspaced short palindromic repeats (CRISPR) method. CRISPR is a programmable technology that targets specific strands of the genetic code and edits DNA at precise locations. CRISPR methods can include CRISPR-CAS9. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cut specific strands of DNA complementary to the CRISPR sequence, allowing for the insertion of exogenous nucleic acids into the cell genome. For example, CRISPR-based gene editing methods can be used to introduce one or more genes encoding factors that induce differentiation into ovarian support cells (e.g., ovarian granulosa cells) into the iPSC genome. These factors include, for example, FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

[0168] Exemplary CRISPR systems include those that utilize the Cas9 enzyme. The Cas9 enzyme, along with CRISPR sequences, is the basis for a technology known as CRISPR-Cas9, which can be used to edit genes in organisms. CRISPR methods can include Class 1 CRISPR systems, including Type I (cas3), Type III (cas10), and Type IV and 12 subtypes. CRISPR methods can include Class 2 CRISPR systems, including Type II (cas9), Type V (cas12), Type VI (cas13), and 9 subtypes. In some embodiments, CRISPR methods can include a CRISPR-Cas design tool, a computer software platform, and bioinformatics tools used to facilitate the design of guide RNAs (gRNAs) for use with CRISPR / Cas gene editing systems. For example, CRISPR-Cas design tools include CRISPRon, CRISPRoff, Invitrogen TrueDesign Genome Editor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPOR, sgRNA Designer, and Synthego Design Tool. CRISPR methods can also be used as diagnostic tools. For example, CRISPR-based diagnostics can be coupled with enzymatic processes such as SHERLOCK-based in vitro transcriptional profiling (SPRINT). SPRINT can be used in high-throughput or portable point-of-care devices to detect various substances, such as metabolites in subject samples or pollutants in environmental samples.

[0169] C. Oocyte Rescue i. Oocyte Score At any stage of in vitro maturation, or immediately after in vitro maturation, oocytes and / or granulosa cells can be appropriately frozen and stored for future analysis, experimentation, or use in oocyte maturation. Oocytes can be scored using a scoring metric based on their morphology as determined by image analysis. In some embodiments, establishing a scoring metric can include acquiring images of the group culture and analyzing the images of one or both of the co-culture group and the control group without co-culture growth medium. In some embodiments, oocytes are scored and comparatively analyzed during any such stage of in vitro maturation. For example, the images of the group culture can include images of the group COC before culture, images of the group COC after culture, and images of the denuded oocytes after culture. In some embodiments, the oocytes used for scoring have never been frozen. In some embodiments, the oocytes used for scoring by image analysis can be thawed after cryopreservation. In some embodiments, the oocytes used for scoring can be collected without in vitro maturation as described. In some embodiments, the oocytes subjected to scoring may be cultured without granulosa cells as described. In some embodiments, images may be sent to a qualified third party (e.g., an embryologist, developmental biologist, or other relevant expert) for scoring to be performed.

[0170] In some embodiments of the methods described herein, oocytes can be evaluated and then classified according to their maturation state according to the following criteria:

[0171] GV - Typically, there is a germinal vesicle within the oocyte that contains a single nucleolus.

[0172] MI - no germinal vesicle is present in the oocyte and no polar body is present in the perivitelline space between the oocyte and the zona pellucida.

[0173] MII - The germinal vesicle is absent in the oocyte and the polar body is present in the perivitelline space between the oocyte and the zona pellucida.

[0174] In some embodiments of the present methods, the scoring metric can include a total oocyte score (TOS) based on analysis of the imaged group cultures using appropriate microscopy or image analysis software. TOS methods and approaches have been described in the art (Lazzaroni-Tealdi et al., PLoS One 10:e0143632, 2015). Oocyte scores can include metrics such as shape, size, ooplasmic characteristics, perivitelline space structure (PVS), zona pellucida (ZP), and polar body (PB) morphology, among other possible qualitative indicators. The total oocyte score for both pre- and post-culture oocyte images for generation of the TOS metric can be based on a -6 to +6 scale system.

[0175] Regarding oocyte shape, if the oocyte morphology is poor (overall oocyte dark and / or ovoid), a value of -1 can be assigned; if it is approximately normal (overall oocyte coloration is not very dark and not very ovoid), a value of 0 can be assigned; and if it is determined to be normal, a value of +1 can be assigned. Regarding oocyte size, if the oocyte size is determined to be abnormally small or large, a value of -1 can be assigned if the size is less than 120 μm or more than 160 μm. If the size is approximately normal, i.e., does not deviate from normal by more than 10 μm, a value of 0 can be assigned; if the oocyte size is within the normal range of more than 130 μm to less than 150 μm, a value of +1 can be assigned. Regarding ooplasm characteristics, if the ooplasm is very granular and / or very vacuolated and / or shows some inclusions, a value of -1 can be assigned. If it is only slightly granular and / or shows few inclusions, a value of 0 can be assigned. The absence of granularity and inclusions can be assigned a value of +1. Regarding the structure of the perivitelline space (PVS), abnormally large, absent, or highly granular PVS can be defined as -1. A value of 0 can be assigned for moderately large and / or moderately small and / or less granular PVS. A value of +1 can be assigned to non-granular, normally sized PVS. Regarding the zona pellucida (ZP), -1 can be assigned to oocytes with very thin or thick ZPs (<10 μm or >20 μm). If the ZP does not deviate from normal by more than 2 μm, the ZP can be assigned a value of 0. A normal zona pellucida (>12 μm and <18 μm) can be assigned a value of +1. Regarding polar body (PB) morphology, PB morphology is defined as follows: Flat and / or multiple PBs or no PBs, granular and / or abnormally small or large PBs are designated as -1. A PB that is judged to be fair but not excellent can be designated as 0, and a PB of normal size and shape can be designated as +1.In some embodiments, the PB scores of MII oocytes may not be counted in the TOS.

[0176] In some embodiments of the present methods, the scoring metric may include performing outcome analysis according to the TOS, as defined and illustrated in FIG. 1A. Parametric or non-parametric tests may be applied to determine the significance of findings during analysis. Outcome analysis may be used to determine 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 oocyte quality, mean PVS quality, mean ZP quality, mean polar body quality, etc. In some embodiments, these results may be reported as mean, median, and standard deviation.

[0177] ii. In vitro fertilization and embryo culture In some embodiments of the methods, any one or more of the eggs or oocytes described herein can be assessed for quality or maturity state, such as by the scoring metrics described herein, to determine whether they can be used for in vitro fertilization and embryogenesis.

[0178] In some embodiments of the methods, eggs or oocytes can be matured through in vitro maturation and then used for IVF and / or ART as described herein. Any one or more oocytes can be used for intracytoplasmic sperm injection (ICSI). Following fertilization of the eggs by contact with one or more sperm cells, the resulting zygote 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., the subject from whom the oocytes were originally collected) for implantation into the uterine lining. Embryo transfers that can be performed using the methods described herein include fresh embryo transfers, in which eggs or oocytes used for embryo development are collected from a subject and the subsequent embryo is implanted into the subject during the same menstrual cycle. Alternatively, embryos can be generated and cryopreserved for long-term storage prior to implantation into a subject.

[0179] iii. Omics data collection and analysis of oocytes, cells, and media In some embodiments of the present method, the scoring metric may include omics-based analysis. For example, frozen cell lysates and cell culture medium can be analyzed using bulk RNA sequencing, whole-genome bisulfite sequencing (WGBS), mass spectrometry-based proteomics, and metabolomics. Cell culture medium can be subjected to metabolomic analysis to determine changes in the molecular content of the medium after co-culture compared to a pre-culture medium control. This can be used to profile dynamic changes in paracrine signaling between granulosa cells and oocytes. The collected data can then be aggregated for subsequent analysis to identify changes in epigenetic status, metabolite presence, and gene expression between various co-culture conditions and the control.

[0180] In some embodiments of the present methods, the omics-based analysis can include genomics, proteomics, transcriptomics, pharmacogenomics, epigenomics, microbiomics, lipidomics, glycomics, transcriptomics-culturomics, and / or any other omics that are apparent to one of skill in the art as being applicable. In some embodiments, after culture, oocytes that fail to mature and exhibit characteristics of GV or MI can be harvested, along with associated granulosa cells from the culture, for single-cell RNA-seq analysis. To this end, the oocytes and granulosa cells can be snap-frozen for library preparation. Half of the oocytes that exhibit MII oocyte development can be harvested and, along with their associated granulosa cells, subjected to single-cell RNA-seq analysis using the snap-freezing method described throughout this disclosure. The remaining half of the MII oocytes can be used for proteomics analysis. Culture media from all conditions can be further rapidly frozen and utilized for metabolomics and proteomics to identify cholesterol metabolite levels and paracrine protein production. For example, frozen cell lysates and cell culture media can be analyzed using bulk RNA sequencing, whole-genome bisulfite sequencing (WGBS), mass spectrometry-based proteomics, and metabolomics. Cell culture media can be used for metabolomics analysis to determine changes in the molecular content of the media after co-culture compared to the pre-culture medium control to profile dynamic changes in paracrine signaling between granulosa cells and oocytes. Rapid freezing of the media components effectively quenches the samples, making them suitable for metabolic assessment. Collected data can then be aggregated for subsequent analysis to identify changes in epigenetic status, metabolite presence, and gene expression between various co-culture conditions and the control.

[0181] III. In Vitro Compositions and Cell Culture Media A. hiPSC-derived granulosa cells Certain granulosa cells for use in the methods described herein can be generated from hiPSCs using a transcription factor (TF) induction protocol. In some embodiments, hiPSCs can be transformed with any one or more plasmids encoding one or more transcription factors. In some embodiments, hiPSCs can be transformed by electroporation, liposome-mediated transformation, or viral-mediated gene transfer, among other cell transformation methods known in the art. In some embodiments, 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 used to reprogram hiPSCs can carry a reporter gene, such as a fluorescent protein. In some embodiments, hiPSCs can be differentiated into stromal cells by inducible expression of transcription factors, including FOXL2, NR5A1, GATA4, RUNX1, RUNX2, or a combination thereof. Reprogramming of hiPSCs into granulosa cells can be determined by genotyping methods known in the art. Reprogramming of hiPSCs into granulosa cells can be determined by protein expression using any one or more methods known in the art. Differentiation of hiPSCs into granulosa cells can be determined by the relative expression of biomarkers typical of the granulosa cell type, such as AMHR2, CD82, FOXL2, FSHR, IGFBP7, KRT19, STAR, WNT4, or combinations thereof, among other granulosa cell biomarkers known in the art. In some embodiments, reprogramming of hiPSCs into granulosa cells can be determined by the production of growth factors and / or hormones, including estradiol and progesterone, which can adequately support in vitro maturation of harvested oocytes through paracrine and juxtacrine cell signaling. In some embodiments, the resulting granulosa cells produce estradiol upon stimulation with androstenedione and FSH or forskolin. In some embodiments, the granulosa cells described herein can be produced in multiple batches.In some embodiments, the granulosa cells can be frozen and thawed prior to the co-culture procedure. In some embodiments, the granulosa cells are newly differentiated prior to the in vitro maturation procedure. In some embodiments, the granulosa cells can be seeded 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) before adding the oocytes for in vitro maturation.

[0182] In some embodiments, a subject can donate hiPSCs. The donation of hiPSCs can occur after the oocyte collection process described above. The subject participating in the hiPSC donation can be different from or the same as the subject from whom the oocytes were collected. In some embodiments, the hiPSC donor can undergo a stimulation protocol as disclosed above. In some embodiments, hiPSCs, granulosa cells, cumulus cells, oocytes, GV stage oocytes, MI stage oocytes, MII stage oocytes, and all other cell types described throughout this disclosure can be lysed, extracted for genomic material, and flash-frozen as a final step in the culture process. For example, cells can be subjected to enzymatic cell lysis using enzymes such as lysozyme, lysostaphin, zymolase, cellulose, protease, or glycanase. Other lysis methods, such as chemical lysis, detergent lysis, alkaline lysis, mechanical lysis, heat lysis, ultrasonic lysis, physical lysis, non-mechanical lysis, and other lysis methods known in the art, can also be applied. In some embodiments, the culture medium can be flash-frozen. The freezing method may include using a cryoprotectant such as dimethyl sulfoxide and / or any other freezing method known in the art.

[0183] B. Transgenic granulosa cells Specific transgenic granulosa cells can be generated using the clustered regularly interspaced short palindromic repeats (CRISPR) technology. CRISPR is a programmable technology that targets specific strands of the genetic code and edits DNA at precise locations. CRISPR technology can include CRISPR-CAS9. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cut specific strands of DNA complementary to the CRISPR sequence. The Cas9 enzyme, along with CRISPR sequences, is the basis for a technology known as CRISPR-Cas9, which can be used to edit genes in organisms. CRISPR technology can include class 1 CRISPR systems, including type I (cas3), type III (cas10), and type IV and 12 subtypes. CRISPR technology can also include class 2 CRISPR systems, including type II (cas9), type V (cas12), type VI (cas13), and nine subtypes. In some embodiments, the CRISPR method can include a CRISPR-Cas design tool, which is a computer software platform, and a bioinformatics tool used to facilitate the design of guide RNAs (gRNAs) for use with the CRISPR / Cas gene editing system. For example, CRISPR-Cas design tools can include CRISPRon, CRISPRoff, Invitrogen TrueDesign Genome Editor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPOR, sgRNA Designer, Synthego Design Tool, and the like. The CRISPR method can also be used as a diagnostic tool. For example, CRISPR-based diagnostics can be coupled with enzymatic processes such as SHERLOCK-based in vitro transcriptional profiling (SPRINT). SPRINT can be used to detect various substances, such as metabolites in subject samples or pollutants in environmental samples, in a high-throughput or portable point-of-care device.

[0184] C. Cell culture medium Granulosa cells, such as granulosa cells derived from iPSCs (e.g., hiPSCs) or transgenic granulosa cells (as described above), can be provided in a composition further comprising a cell culture medium (e.g., IVF medium, IVM medium (e.g., MediCult IVM medium), or LAG medium). The cell culture medium may contain human serum albumin (e.g., about 5-15 mg / mL, e.g., 10 mg / mL), FSH (e.g., about 70-80 mIU / mL, e.g., 75 mIU / mL), hCG (e.g., about 95-105 mIU / mL, e.g., 100 mIU / mL), androstenedione (e.g., about 495-505 ng / mL, e.g., 500 ng / mL), doxycycline (e.g., 0.5-1.5 μg / mL, e.g., 1 μg / mL), and other compounds such as hyaluronidase and / or dPBS.

[0185] IV. DEVICES AND RELATED METHODS FOR EGG MATURATION The present disclosure provides devices for use in assisted reproductive technologies (e.g., IVF). Figure 1 shows two exemplary devices (see, e.g., Figures 1A and 1B). Referring to Figure 1A, an exemplary embodiment of a device 100 for inducing human oocyte maturation in vitro is shown. Referring to Figure 1B, an exemplary embodiment of a device 100 for assisting in ex vivo oocyte rescue after stimulation is shown.

[0186] A. Computing Devices In an apparatus of the present disclosure (e.g., FIGS. 1A and 1B), the apparatus 100 includes a computing device 104. The computing device 104 includes a processor 108 and a memory 112 communicatively coupled to the processor 108, the memory 112 including instructions that configure the processor 108 to perform a linking process. The processor 108 and the memory 112 are included in the computing device 104. The computing device 104 may include any computing device described in this disclosure. Such computing devices include, but are not limited to, microcontrollers, microprocessors, digital signal processors (DSPs), and / or systems-on-chips (SoCs) described in this disclosure. The computing device may include, be included in, and / or communicate with a mobile device such as a mobile phone or smartphone. The computing device 104 may include a single computing device operating independently or may include two or more computing devices operating, for example, cooperatively, in parallel, or serially. The two or more computing devices may be included together in a single computing device or two or more computing devices. The computing device 104 can interact with or communicate with one or more additional devices, as described in more detail below, via a network interface device. The network interface device can 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, a network interface card (e.g., a mobile network interface card, a LAN card), a modem, 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 an office, building, campus, or other relatively small geographic space), 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 can use wired and / or wireless communication modes. In general, any network topology can be used. Information (e.g., data, software, etc.) can be sent to and / or received from computers and / or computing devices. Computing devices 104 can include, but are not limited to, a computing device or cluster of computing devices at a first location and a second computing device or cluster of computing devices at a second location, for example. Computing devices 104 can include one or more computing devices specialized for data storage, security, traffic distribution for load balancing, etc. Computing device 104 may distribute one or more computing tasks described below across multiple computing devices of the computing device, which may operate in parallel, serially, overlapping, or any other manner used to distribute tasks or memory among computing devices. Computing device 104 may be implemented using a "fully loosely coupled" architecture in which data is cached by workers, which in one embodiment may enable scalability of system 100 and / or the computing devices.

[0187] With continued reference to the apparatus of the present disclosure (e.g., FIGS. 1A and 1B), the computing device 104 may be designed and / or configured to perform any method, method step, or sequence of method steps in any embodiment described herein, in any order and to any degree of iteration. For example, the computing device 104 may be configured to repeatedly perform a single step or sequence until a desired or ordered result is achieved. Repeating a step or series of steps may be performed iteratively and / or recursively, using the output of a previous iteration as input to a subsequent iteration, aggregating the inputs and / or outputs of an iteration to generate an aggregate result, decreasing or decrementing one or more variables, such as a global variable, and / or dividing a larger processing task into a set of smaller processing tasks that are addressed iteratively. The computing device 104 may perform any step or sequence of steps described herein in parallel, e.g., performing a step two or more times simultaneously and / or substantially simultaneously using two or more parallel threads, processor cores, etc. The division of tasks among parallel threads and / or processes may be performed according to any protocol suitable for dividing tasks among iterations. Those skilled in the art will recognize, upon reviewing this disclosure in its entirety, various methods in which steps, order of steps, processing tasks, and / or data may be subdivided, shared, or processed using iterations, recursion, and / or parallel processing.

[0188] Continuing with reference to the apparatus of the present disclosure (e.g., FIGS. 1A and 1B), the computing device 104 can further employ a machine learning process 116 to perform the determining, classifying, and / or analyzing steps, methods, processes, etc. described herein. As used in this disclosure, a “machine learning process” is a process that automatically uses a large amount of data, known as “training data” and / or a “training set,” to generate an algorithm that is executed by the computing device / module given data provided as input and generates an output. This is in contrast to a non-machine learning software program, in which the commands to execute are predetermined by a user and written in a programming language. The machine learning process 116 can utilize a supervised process, an unsupervised process, a lazy learning process, and / or a neural network.

[0189] i. Egg maturation With specific reference to the apparatus of FIG. 1A , the computing device 104 is configured to support in vitro human oocyte maturation by promoting rapid and efficient oocyte maturation in a manner that enhances oocyte health and developmental potential. The computing device 104 is configured to acquire first biological sample data from a first biological sample 120 associated with a user. As used in this disclosure, “biological sample data” is data that characterizes the biological, genetic, biochemical, and / or physiological properties, composition, or activity of a biological sample. A “biological sample” is any information associated with a user. A biological sample may include a laboratory specimen held by a biorepository for testing. In some embodiments, the first biological sample 120 may include a bodily fluid, such as blood, saliva, urine, semen (seminal plasma), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal secretions, ocular fluid, gastric juice, breast milk, cell culture supernatant, etc. The biological sample may include a medical diagnosis, user input describing the user's mood and / or symptom complaints, information collected from a wearable device related to the user, etc. For example, the biological sample may include information obtained from a visit to a medical professional, such as a medical history. In yet another non-limiting example, the biological sample may include information such as data collected from a wearable device worn by the user. The wearable device is designed to collect information regarding the user's sleep patterns, exercise patterns, etc. In one embodiment, the first biological sample 120 is collected on a particular day and / or time of the user's menstrual cycle. For example, the first biological sample 120 may be collected on day two of the user's menstrual cycle to assess one or more hormone levels, such as, but not limited to, E2, FSH, LH, P4, and / or AMH. The first biological sample 120 may be used to measure one or more markers of the user's overall health. Such health conditions include, but are not limited to, ovarian reserve health and / or circulating hormone levels. This information can be used, for example, by a medical professional to monitor the progress of the cycle and inform protocol and / or drug selection.

[0190] As used in this disclosure, a "user" refers to a living organism, such as a human, plant, animal, or any other living organism composed of cells. In some embodiments, a biological sample can be collected from a user via a collection device. A "collection device" is a device and / or tool capable of obtaining, recording, and / or verifying measurements associated with a sample. Collection devices may include needles, syringes, vials, lancets, evacuated collection tubes (ECTs), tourniquets, evacuated collection tube systems, any combination thereof, and the like. For example, a collection device may include a butterfly needle set. The computing device 104 may receive the biological sample in the form of data uploaded to memory. The data may include measurements of, for example, serum calcium, phosphate, electrolytes, blood urea nitrogen and creatinine, uric acid, and the like.

[0191] 1A, the computing device 104 may receive the first biological sample data from a biological sample database 124. A "biological sample database" is a database that contains all data related to a user's biological sample, including analytical information.

[0192] ii. Oocyte rescue With specific reference to the apparatus of FIG. 1B , the computing device 104 is configured to support ex vivo oocyte rescue after stimulation. "Oocyte rescue" refers to the process of ex vivo maturation of immature oocytes, which are typically ignored in standard in vitro maturation methods. As used herein, "oocytes" refer to germ cells derived from the ovaries. Post-stimulation may refer to a standard in vitro fertilization (IVF) stimulation protocol performed on a user. As used herein, a "stimulation protocol" refers to a drug infusion process over a specific period of time to induce the ovaries to produce one or more oocytes. As used herein, a "user" refers to a living organism, such as a human, plant, animal, or any other organism composed of cells. In some embodiments, post-stimulation may refer to a minimal stimulation protocol. As used herein, a "minimal stimulation protocol" refers to a stimulation process that is shorter than the average in vitro fertilization (IVF) stimulation protocol and helps induce the ovaries to produce eggs. Typically, the average duration for a stimulation protocol using standard IVF is approximately 8 to 14 days. The minimal stimulation protocol can induce cell release in 2-6 days, a shorter period compared to 8-14 days. The average time to perform the minimal stimulation protocol 132 can be 3 days. The maximum time can be 6 days, and the minimum time can be 2 days.

[0193] With further reference to FIG. 1B , the computing device 104 is configured to acquire biological sample data 120 from a biological sample associated with a user, the biological sample including at least one oocyte. As used in this disclosure, “biological sample data” is data that characterizes the biological, genetic, biochemical, and / or physiological properties, composition, or activity of a biological sample. As used in this disclosure, a “biological sample” is a biological laboratory specimen (e.g., a blood sample or other bodily fluid sample) obtained from a subject. In some embodiments, an oocyte may be an immature oocyte. As used in this disclosure, an “immature oocyte” is one or more immature germ cells that develop in the ovary. In some embodiments, an immature oocyte may be an oocyte, including a GV stage oocyte and / or an MI stage oocyte. In some embodiments, an immature oocyte may be multiple oocytes. An immature oocyte may be an immature cumulus-oocyte complex (COC) collected from a mother. As used in this disclosure, a “cumulus-oocyte complex” is an oocyte surrounded by specific granulosa cells. As used in this disclosure, "certain granulosa cells" are cumulus cells that surround an oocyte to ensure healthy oocyte and embryo development. In some embodiments, immature oocytes may include oocytes when certain granulosa cells are added to mature the oocyte in cell culture and create a COC. In some embodiments, biological samples may include bodily fluids such as blood, saliva, urine, semen (seminal plasma), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, ascites, amniotic fluid, nasal secretions, ocular fluid, gastric juice, breast milk, cell culture supernatant, and the like.

[0194] 1B , computing device 104 can receive biological sample data 120 derived from a user's biological sample after stimulation from a biological sample database 124. As used in this disclosure, a "biological sample database" is a database that includes all data related to a user's biological sample, including analytical information. In some embodiments, biological sample database 124 can include a stimulation protocol index. As used in this disclosure, a "stimulation protocol index" is a data structure that correlates user information regarding the completion of a stimulation protocol. Such user information may be, for example, the user's age, the user's BMI, the number of COCs collected, the number of mature MII stage oocytes, the number of immature MI oocytes, the number of immature GV oocytes, the AMH level (μg / L), the antral follicle count (AFC) at the last ultrasound, the E2 level (ng / L) on the user's day of oocyte collection, the P4 level (ng / L) on the user's day of oocyte collection, the LH (IU / L) on the user's day of oocyte collection, the FSH (IU / L) on the user's day of oocyte collection, the number of days of stimulation, the amount of gonadotropin used, and the total injected dose (IU).

[0195] iii. Biospecimen database In some embodiments of the apparatus (e.g., FIGS. 1A and 1B), the biological sample database 124 may include a systemic hormone index. As used herein, a "systemic hormone index" is a data structure relating medical knowledge related to systemic hormone therapy. For example, the systemic hormone index may include side effects and risks, appropriate methods for administering hormones, correlations between E2, LH, FSH, and / or P4 deficiencies and systemic hormones, etc. In some embodiments, the biological sample database 124 may be communicatively connected to the computing device 104. As used herein, "communicatively connected" means a connection made by a connection, attachment, or coupling that allows for the reception and / or transmission of information between two or more related elements. For example, without limitation, the connection may be a wired or wireless connection, a direct or indirect connection, and may be a connection between two or more components, circuits, devices, systems, etc., that allows for the reception and / or transmission of data and / or signal(s) therebetween. The data and / or signals therebetween include, but are not limited to, electrical, electromagnetic, magnetic, video, audio, radio, and microwave data and / or signals, combinations thereof, and the like, among others. A communicative connection can be achieved, for example, without limitation, by wired or wireless electronic, digital, or analog communication, either directly or via one or more intervening devices or components. Furthermore, 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, without limitation, via a bus or other facility for intercommunication between elements of a computing device. A communicative connection may also include an indirect connection, for example, without limitation, via a wireless connection, wireless communication, a low-power wide area network, optical communication, magnetic coupling, capacitive coupling, or optical coupling, etc. In some examples, the term “communicatively coupled” may be used in place of “communicatively connected” in this disclosure.

[0196] The biological sample database 124, and all other databases described throughout this disclosure, can be implemented, without limitation, as a relational database, a key-value lookup database such as a NOSQL database, or any other format or structure for a database that one of ordinary skill in the art would recognize as suitable after reviewing this entire disclosure. The biological sample database 124 can alternatively or additionally be implemented using a distributed data storage protocol and / or data structure, such as a distributed hash table. The biological sample database 124 can include multiple data entries and / or data records. Data entries in the database may be flagged with or linked to one or more additional elements of information, which may be reflected in the data entry cells and / or in linked tables, such as tables related by one or more indexes in a relational database. Those skilled in the art will recognize, upon review of this disclosure in its entirety, various ways in which data entries in a database can store, retrieve, organize, and / or reflect data and / or records used in this disclosure, and can further store, retrieve, organize, and / or reflect categories and / or groupings of data consistent with this disclosure.

[0197] iv. Stimulation Protocol In an apparatus of the present disclosure (see, e.g., FIG. 1A ), the computing device 104 is configured to assign a stimulation protocol 132 to a user in response to the first biological sample 120. In some embodiments, the stimulation protocol 132 can be assigned based on measured hormone levels in the biological sample. A “stimulation protocol” is a process of injecting agents (e.g., induction agents) over a specific period of time (e.g., a follicular induction period) to induce the ovaries to produce one or more oocytes.

[0198] As used in this disclosure, a "measured hormone level" is a quantitative value representing the level of one or more hormones in a user. Measured hormone levels in a biological sample can include levels of 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, and the like. In some embodiments, the measurement of hormone levels can be based on a blood analysis of the biological sample. For example, the blood analysis can include a plasma hormone assay. In some embodiments, the measurement of hormone levels can be based on a saliva hormone assay. The measurement of hormone levels can also be based on other forms of analysis (e.g., hair, urine, and any other form of biological sample described throughout this disclosure).

[0199] In one embodiment (see, e.g., FIG. 1A ), selection of a stimulation protocol can be made using information obtained from an ultrasound examination. As used in this disclosure, “ultrasound examination” refers to any technique that uses sound waves to generate one or more images of a user's body. For example, ultrasound examination can be used to obtain images of the subject's reproductive organs and / or tissues. In certain embodiments, the ultrasound examination can be performed at a particular time during the subject's menstrual cycle. For example, a subject can undergo an ultrasound examination on day 2 of their cycle, which can be used to identify follicle size and / or follicle number. Selection of a stimulation protocol and / or adjustments to the stimulation protocol can be made using such information. For example, for a subject whose ultrasound examination indicates polycystic ovary syndrome (PCOS), dosage adjustments can be made to one or more medications taken and / or utilized during the stimulation protocol. Furthermore, the length of a subject's stimulation protocol can be modified based on the subject's diagnosis of PCOS. In one embodiment, ultrasound examination can be repeated one or more times throughout the subject's stimulation protocol, and the information obtained can be used to adjust the subject's stimulation protocol in real time.

[0200] Additionally, a subject's use of contraception may influence the assignment of a stimulation protocol. As used herein, "contraception" refers to any method and / or means used to prevent pregnancy as a result of sexual activity. This may include, without limitation, any medications, techniques, means, and / or birth control utilized by a subject. A subject's use of contraception may help determine at what point in the subject's menstrual cycle a stimulation protocol should be initiated. For example, without limitation, a subject not using any form of contraception may begin a stimulation protocol using recombinant follicle-stimulating hormone (rFSH) on day 2 of their menstrual cycle. In yet another non-limiting example, a subject using contraception may begin a stimulation protocol using rFSH five days after taking their last oral contraceptive. In one embodiment, rFSH stimulation may be used for 2-3 days, depending on the subject's tolerance, follicle size, and / or growth dynamics. After this 2-3 day period, a 1-2 day coasting period may be utilized to monitor follicle size and allow for further follicle maturation and development. As used in this disclosure, a "coasting period" is any period during which the medications used throughout the stimulation protocol are not administered and / or consumed. The coasting period may last, for example, 1 day, 2 days, 3 days, etc. During the coasting period, the subject may continue to undergo one or more ultrasound examinations to monitor their progress.

[0201] When the follicle size reaches anywhere between 8 and 10 mm, the subject can be triggered with an administration of human chorionic gonadotropin (hCG). In one embodiment, double hCG injections can be used to induce follicular maturation and prepare one or more follicles for harvest. To monitor hormone levels, blood tests for one or more hormone levels, such as E2, P4, and LH, can be performed on the first day of the second dose of human chorionic gonadotropin (hCG) injection. After the second dose of hCG, one or more hormone levels can be measured, such as blood tests to measure and test the amount of E2, P4, and LH.

[0202] Approximately 24-48 hours after administration of the hCG dose, the subject may undergo oocyte retrieval. On the day of oocyte retrieval, blood testing of one or more hormone levels, such as E2, LH, and / or P4, may be performed to verify quality criteria, that hormone levels are within range, and / or that the hCG dose was administered. The assigned stimulation protocol 132 may include a minimum stimulation protocol configured to induce cell release over a three-day period. A "minimal stimulation protocol" is a stimulation process that lasts for a shorter period of time than the average in vitro fertilization (IVF) stimulation protocol, and serves to induce the ovaries to produce eggs. Typically, the average duration for a stimulation protocol using standard IVF is approximately 8-14 days. A minimum stimulation protocol can induce cell release over a shorter period of time, 2-6 days, compared to 8-14 days. The average time for performing the minimum stimulation protocol 132 may be 3 days. The maximum time may be 6 days, and the minimum time may be 2 days. In one embodiment, the minimal stimulation protocol may include selecting a first inducing agent (e.g., a follicle-inducing agent) in response to the first biological sample 120 and selecting a second inducing agent (e.g., a follicle-inducing agent) in response to the follicle measurement. This is disclosed in more detail below. As used in this disclosure, a "follicle measurement" is any measurement of a follicle. A follicle may include any sac present in an ovary that contains an unfertilized egg. The follicle measurement may be obtained using any method described herein, such as, for example, ultrasound, manual measurement, automated measurement, etc.

[0203] An "inducing agent" is a chemical that causes cell development in the ovaries. Inducing agents (e.g., follicle-inducing agents) can include any substance, including any non-prescription and / or prescription drugs. Examples of inducers (e.g., follicle inducers) include Lupron, manufactured by Abbott Laboratories (headquartered in North Chicago, IL), Ganirelix, manufactured by Ferring Pharmaceuticals (headquartered in Saint-Prex, Switzerland), Cetrotide, manufactured by Merck Global (headquartered in Whitehouse Station, Readington Township, NJ), Gonal-F, manufactured by Merck Global, Follistim, manufactured by Merck Global, Bravelle, manufactured by Ferring Pharmaceuticals (headquartered in Saint-Prex, Switzerland), Clomid, manufactured by Patheon Pharmaceuticals Inc. (headquartered in Waltham, MA), Serephene, manufactured by Teva (headquartered in Tel Aviv-Yafo, Israel), Glucophage, manufactured by Merck Global, Fortamet, manufactured by Mylan (headquartered in Canonsburg, PA), Pregnyl, manufactured by Schering Plough (headquartered in Kenilworth, NJ), Ferring Examples of suitable agents include Novarel, manufactured by Pharmacists Laboratories (headquartered in Parsippany, NJ), Repronex, manufactured by Ferring Pharmaceuticals, Inc., Factrel, manufactured by Zoetis Canada Inc. (headquartered in Kirkland, Canada), Menopur, manufactured by Ferring Pharmaceuticals, and other agents that induce cell production in the ovaries that would be apparent to one skilled in the art. Inducing agents (e.g., follicle-inducing agents) include human serum albumin, FSH, hCG, androstenedione, and doxycycline.The computing device 104 can assign an inducer to use based on the measured hormone levels in 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, with further reference to FIG. 1A , a machine learning model can be utilized to assign a particular stimulation protocol to a user depending on the first biological sample 120.

[0204] v.classifier As used in this disclosure, a "classifier" is a machine learning model, such as a mathematical model, neural net, or program generated by a machine learning algorithm known as a "classification algorithm," as described in more detail below, that sorts inputs into categories or bins of data and outputs the categories or bins of data and / or their associated labels. A classifier may be configured to at least output data that labels or identifies sets of data that are known to be close and clustered together under some distance metric, for example, as described below.

[0205] In the apparatus of the present disclosure (e.g., FIGS. 1A and 1B ), classification can be performed using, without limitation, a linear classifier, such as, without limitation, a logistic regression classifier and / or a naive Bayes classifier, a nearest neighbor classifier, such as, without limitation, a k-nearest neighbor classifier, a support vector machine, a least-squares support vector machine, Fisher's linear discriminant, a quadratic classifier, a decision tree, a boosted tree, a random forest classifier, learning vector quantization, and / or a neural network-based classifier. The computing device 104 may be configured to generate the classifier using a naive Bayes classification algorithm. The naive Bayes classification algorithm generates the classifier by assigning class labels to problem instances represented as vectors of element values. The class labels are drawn from a finite set. The naive Bayes classification algorithm includes generating a family of algorithms that assume that, given a class variable, the value of a particular element is independent of the values of other elements. The Naive Bayes classification algorithm can be based on Bayes' theorem, expressed as P(A / B) = P(B / A) P(A) ÷ P(B), where P(A / B) is the probability of hypothesis A given data B, also known as the posterior probability; P(B / A) is the probability of data B if hypothesis A is true; P(A) is the probability that hypothesis 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 hypothesis. The Naive Bayes algorithm can be generated by first converting training data into a frequency table. Then, the computing device 104 can calculate a likelihood table by calculating the probabilities of different data entries and classification labels. The computing device 104 can calculate the posterior probability of each class using the Naive Bayes equation. The class with the highest posterior probability is the predicted outcome. The Naive Bayes classification algorithm can include a Gaussian model that follows a normal distribution. The Naive Bayes classification algorithm can include a polynomial model used for discrete counts. Naive Bayes classification algorithms may include Bernoulli models that can be utilized when the vectors are binary.

[0206] 1A and 1B, the computing device 104 may be configured to generate a classifier using a K-nearest neighbor (KNN) algorithm. As used in this disclosure, a "K-nearest neighbor algorithm" includes a classification method that uses feature similarity to analyze how similar out-of-sample features are to training data to classify input data into one or more clusters and / or categories of features represented in the training data. This can be performed by representing both the training data and the input data in vector form and using one or more measures of vector similarity to identify classifications in the training data and determine the classification of the input data. The K-nearest neighbor algorithm may include specifying a K value, or a number that instructs the classifier to select the training data of the k entries most similar to a given sample, determining the most common classifiers for entries in the database, and classifying the known sample. This can be performed recursively and / or iteratively to generate a classifier that can be used to classify input data as additional samples. For example, an initial set of samples can be run to cover initial heuristics and / or "first guesses" on outputs and / or relationships. Without limitation, a seed value can be provided using expert input received according to any process described herein. As a non-limiting example, an initial heuristic can include ranking associations between the input and elements of training data. The heuristic can include selecting a few of the highest-ranking associations and / or training data elements.

[0207] 1A and 1B, a k-nearest neighbor algorithm can be generated to generate a first vector output containing data entry clusters, a second vector output containing input data, and any suitable norm, such as cosine similarity or a Euclidean distance measure, can be used to calculate the distance between the first and second vector outputs. Each vector output can be represented, without limitation, as an n-tuple of values, where n is 2 or greater. Each value in the n-tuple of values can represent a measurement or other quantitative value associated with a given category of data or attribute. Examples are provided in more detail below. Vectors can be represented in n-dimensional space, without limitation, using axes for each category of values represented in the n-tuple of values, such that the vectors have a geometric direction that characterizes the relative amounts of attributes in the n-tuple compared to each other. Two vectors can be considered equivalent if their directions and / or the relative amounts of values in each vector compared to each other are the same. Thus, as a non-limiting example, a vector represented as [5, 10, 15] can be treated as equivalent to a vector represented as [1, 2, 3] for purposes of this disclosure. Vectors may be more similar if their directions are more similar, and more dissimilar if their directions are more dissimilar. However, vector similarity may alternatively or additionally be determined using the average similarity between similar attributes, or any other measure of similarity appropriate for any n-tuple of values, or an aggregation of numerical similarity measures for purposes of a loss function. 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 scaled using a "length" attribute (e.g., the Pythagorean norm

number

[0208] Referring to the apparatus of FIG. 1A , the computing device 104 and / or another device may use a classification algorithm to generate the protocol classifier 128. A “protocol classifier” is a classifier trained to acquire a biological sample associated with a user and output / assign a stimulation protocol 132 to the associated user based on received training data. The training data may consist of inputs and / or outputs including systemic hormone index data, feedback from previous stimulation protocol 132 assignments, and any other data described throughout this disclosure. The training data may be received from the biological sample database 124. In some embodiments, the training data may include multiple data entries including a biological sample associated with an output including the assigned stimulation protocol. In some embodiments, the training data may include inputs such as an assigned stimulation protocol associated with an output such as a pregnancy success rate or a scoring metric, as described throughout this disclosure. In some embodiments, the training data may include correlations between stimulation protocols and associated side effects. In some embodiments, the training data may include methods and procedures for preventing ovarian hyperstimulation with an induction agent. For example, the training data may include the number of injections a user can receive containing a particular induction agent (e.g., a follicle-inducing agent) before hyperstimulation occurs.

[0209] In any apparatus of the present disclosure (e.g., FIGS. 1A and 1B), the computing device 104 can use training data to train any classifier or other machine learning model. In some embodiments, the training data can include, without limitation, biological sample data collected from a user and / or correlations of biological samples to maturity levels, scores, or other numerical and / or quantitative fields related to oocytes in the form of training examples. The training examples can be input by one or more experts. As used in this disclosure, an "expert" is a person or organization skilled in the art. Expert knowledge can be obtained from feedback indexes in the biological sample database 124.

[0210] A biological sample containing oocytes can be obtained from a user after stimulation by a medical professional, such as a physician, inserting a collection device into an egg-containing follicle and collecting the egg and surrounding bodily fluids. A "collection device" is a device and / or tool capable of obtaining, recording, and / or verifying measurements associated with a sample. Collection devices may include needles, syringes, vials, lancets, evacuated collection tubes (ECTs), tourniquets, evacuated collection tube systems, any combination thereof, and the like. For example, a collection device may include a butterfly needle set. Oocyte collection may include collecting immature oocytes, mature oocytes, COCs, and any other type of cell involved in reproduction present in the ovaries.

[0211] Referring to the apparatus of FIG. 1B , the computing device 104 is configured to determine the maturity level 128 of at least one oocyte. As used in this disclosure, a "maturity level" is data representing an assessment of an oocyte stage of oogenesis. The maturity level may include quantitative elements and / or fields, such as days, hours, etc., a stage of maturity, and / or a score representing maturity. In some embodiments, the maturity level 128 may be an assessment value of an oocyte maturation stage of oogenesis. Oocyte maturation refers to the release of meiotic arrest, allowing an oocyte to progress from meiotic prophase I to metaphase II. Determining the maturity level 128 of an oocyte may include denuding the oocyte. Oocyte denudation refers to the removal of the somatic cell layer surrounding the oocyte. For example, to determine the nuclear maturity of an oocyte, a COC can be denuded to remove the layer of granulosa cells surrounding the oocyte. Oocyte denudation may include enzymatic and mechanical methods using hyaluronidase and a sterile glass pipette, as further described below.

[0212] 1B , in some embodiments, determining the maturity level 128 may include using a machine learning process 116 to determine the maturity level 128. For example, without limitation, the machine learning process 116 may be used to generate and train a machine learning model, including a classifier. The classifier may classify the oocyte into a maturity level according to the biological sample data.

[0213] With further reference to the apparatus of FIG. 1B , the computing device 104 and / or another device may use a classification algorithm to generate the maturity classifier 132. As used herein, a “maturity classifier” is a classifier trained to take biological sample data 120 and output a maturity level 128. Additionally or alternatively, the maturity classifier 132 can be trained to take oocyte denudation data and output a maturity level 128. As used herein, “oocyte denudation data” is data that characterizes the biological, genetic, biochemical, and / or physiological properties, composition, or activity of a denuded oocyte. The computing device 104 can receive the oocyte denudation data from a biological sample database. In some embodiments, the computing device 104 can train the maturity classifier 132 with training data including, for example, without limitation, correlations between maturity levels and oocytes in the form of training examples. The training examples can be obtained from maturity level indexes and feedback indexes included in the biological sample database. As used in this disclosure, a "maturity level index" is a data structure that associates biological knowledge regarding the stage of oocyte oogenesis, such as the stage of oocyte maturation. As used in this disclosure, a "feedback index" is a data structure that associates past maturity level 128 assessments performed by a computing device and communication from a third party. As used in this disclosure, a "third party" is a qualified individual or organization, such as an embryologist, statistician, etc. In some embodiments, training data for the maturity classifier 132 can be received from a biological sample database. For example, the biological sample data 120 can include data related to denuded immature COCs, which, together with the training data, determine as a maturity level 128 that oocytes resulting from denudation are GV oocytes and / or MI oocytes.In some embodiments, the machine learning model, including a classifier trained to classify oocytes into maturity levels according to biological sample data, can also use the machine learning process 116 to calculate maturity levels, scores, or other numerical and / or quantitative fields associated with the oocytes.

[0214] vi. Second biological sample An apparatus described herein (see, e.g., FIG. 1A ) may be configured to acquire a second biological sample. In FIG. 1A , for example, a computing device 104 is configured to acquire second biological sample data from a second biological sample 136 associated with a user, where the second biological sample 136 includes at least one oocyte. As used in this disclosure, an “oocyte” is a germ cell derived from an ovary. An 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, and the like. As used in this disclosure, a “cumulus-oocyte complex” is an oocyte including one or more surrounding cumulus cells. A COC may include an immature oocyte. A COC may include a mature oocyte. As used in this disclosure, an “immature oocyte” is one or more immature germ cells that develop in the ovary. In some embodiments, the immature oocyte may be an oocyte, including, but not limited to, a germinal vesicle (GV) oocyte and a metaphase I (MI) 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) collected from a subject. As used in this disclosure, a "mature oocyte" refers to one or more mature germ cells that develop in the ovary. Once collected, the COC may be allowed to rest for 2-3 hours to equilibrate to in vitro conditions. In one embodiment, the oocyte may be combined with specific granulosa cells. As used in this disclosure, "specific granulosa cells" refer to cumulus cells that can surround the oocyte to ensure healthy oocyte and embryo development. In some embodiments, the immature oocyte may include an oocyte to which specific granulosa cells are added to mature the oocyte in cell culture and create a COC after collecting the second biological sample 136. The second biological sample 136 may include a bodily fluid as disclosed above. In one embodiment, one or more specific granulosa cells may be thawed during the resting period of one or more COCs. In one embodiment, 50,000 to 100,000 specific granulosa cells may be combined with COCs during culture.In one embodiment, thawed specific granulosa cells can be placed in culture medium, for example, 24 to 120 hours prior to COC harvest. COCs can be transferred to the medium containing the thawed specific granulosa cells to form a group culture, as described in more detail below. In some embodiments, the group culture can be a culture in an incubator for any period between 12 and 48 hours. The computing device 104 can receive second biological sample data from the biological sample database 124, as described above. The second biological sample 136 can be collected and acquired using a collection device, as disclosed above. In some embodiments, the computing device 104 can record hormone levels in the second biological sample 136 measured using the methods disclosed above. The specific granulosa cells can be generated using the clustered regularly interspaced short palindromic repeats (CRISPR) method. CRISPR is a programmable technology that targets specific strands of genetic code and edits DNA at precise locations. The CRISPR method can include CRISPR-CAS9. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA complementary to the CRISPR sequence. The Cas9 enzyme, along with CRISPR sequences, is the basis of a technology known as CRISPR-Cas9, which can be used to edit genes in organisms. CRISPR methods can include class 1 CRISPR systems, including type I (cas3), type III (cas10), and type IV and 12 subtypes. CRISPR methods can include class 2 CRISPR systems, including type II (cas9), type V (cas12), type VI (cas13), and 9 subtypes. In some embodiments, CRISPR methods can include a computer software platform, the CRISPR-Cas design tool, and bioinformatics tools used to facilitate the design of guide RNAs (gRNAs) for use with CRISPR / Cas gene editing systems.For example, CRISPR-Cas design tools include CRISPRon, CRISPRoff, Invitrogen TrueDesign Genome Editor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPOR, sgRNA Designer, and Synthego Design Tool. CRISPR methods can also be used as diagnostic tools. For example, CRISPR-based diagnostics can be coupled with enzymatic processes such as SHERLOCK-based in vitro transcriptional profiling (SPRINT). SPRINT can be used in high-throughput or portable point-of-care devices to detect various substances, such as metabolites in subject samples or pollutants in environmental samples.

[0215] In some embodiments relating to the culture of the second biological sample 136, the cell culture medium may include LAG medium. For example, LAG medium can be used for the incubation of COCs after harvest from the stimulation protocol 132. The package size may be a 10 mL vial. Storage may be for up to one month at 2-8°C away from light. Medium equilibration may be 18-24 hours prior to culture, which may include a 100 μl seed droplet and be placed in a 37°C incubator with 6% O2 and moderate CO2. In some embodiments, the cell culture medium may include IVM medium (e.g., 1 mL-100 mL of medium per co-culture, e.g., 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 of medium can be used). For example, modified MediCult IVM medium may be used as a baseline control during the culture process. The package size may be a 10 mL vial. Storage is permitted at 2-8°C away from light for up to one month. In some embodiments, cell culture media may contain metabolic substances. For example, modified MediCult IVM medium may contain human serum albumin, FSH, hCG, androstenedione, doxycycline, and other compounds. Other cell culture materials and equipment may include liquid nitrogen, hyaluronidase, dPBS, IVF-compatible mineral oil, universal GPS dishes, G-NOPS plus medium, micropipettes, stripper pipettes, an inverted ICSI microscope with a camera, a dry injection tabletop incubator, a saturated humidity incubator, an embryoscope, a microcentrifuge, a refrigerator, a -20°C to 100°C freezer, a liquid nitrogen storage container, 35 mm denuded dishes, stripper pipette tips, and other components readily apparent to those skilled in the art that are involved in cell culture processes.

[0216] 1A , in some embodiments, culturing the second biological sample 136 can include culturing immature cumulus-oocyte complexes (COCs) in group culture. "Group culture" refers to combining a harvested COC with one or more additional cells. The additional cells can include any cells that grow with the harvested COC. The additional cells can include certain granulosa cells. In some embodiments, the group culture can be cultured and / or incubated for a specific length of time, e.g., 12-120 hours. For example, group culture can include culturing cumulus-oocyte complexes with granulosa cell co-cultures, as described further below. "Co-culture" is a cell culture technique in which two or more distinct populations of cells are grown with some degree of contact between them. In some embodiments, group culture can include culturing a control group of COCs without co-culture, as described further below. In some embodiments, a user can provide immature oocytes, such as germinal vesicle (GV) oocytes and metaphase 1 (M1) oocytes. Such immature oocytes can be used in media as part of a group culture to support the growth of COCs. Oocyte donation can be performed according to the oocyte collection process described above. The user participating in the oocyte donation can be different from or the same as the user of the second biological sample containing the immature COC. In some embodiments, the user donating the oocytes can undergo a stimulation protocol as disclosed above. In some embodiments, granulosa cells, cumulus cells, oocytes, GV oocytes, MI oocytes, and all other cell types described throughout this disclosure can be lysed, extracted for genomic material, and flash-frozen. For example, cells can be subjected to enzymatic cell lysis using enzymes such as lysozyme, lysostaphin, zymolase, cellulose, protease, or glycanase. Other lysis methods, such as chemical lysis, detergent lysis, alkaline lysis, mechanical lysis, heat lysis, ultrasonic lysis, physical lysis, non-mechanical lysis, etc., can also be applied. In some embodiments, the media can be flash-frozen.Freezing methods may include using a cryoprotectant such as dimethyl sulfoxide and / or any other freezing method described throughout this disclosure.

[0217] With further reference to the apparatus of FIG. 1A, the computing device 104 is configured to assign a scoring metric 152 to the second biological sample 136 in response to culturing the second biological sample 136.

[0218] vii. Culture Protocol In the apparatus described herein (see, e.g., FIG. 1B ), the computing device 104 is configured to assign oocytes to a culture protocol 136 according to the maturity level 128. In some embodiments, the assigned oocytes may be denuded oocytes. As used in this disclosure, a “culture protocol” is a cell culture process in which cells are grown under controlled conditions. The culture protocol 136 may include a cell culture metabolite selected according to the maturity level 128 and a cell culture medium selected according to the maturity level 128. As used in this disclosure, a “cell culture metabolite” is a substance involved in cellular metabolism that optimizes the synthesis of new molecules in a cell culture. For example, the cell culture metabolite may include doxycycline. The computing device 104 can generate and train the culture classifier 140 using training data. The training data can include, for example, without limitation, correlations of oocytes or maturity levels to cell culture protocols in the form of training examples. The training examples can be obtained from a metabolite index, a cell medium index, a protocol index, and a feedback index contained in a culture database. For example, the culture classifier 140 can select GV oocytes determined from the maturity classifier 132 and assign the oocytes a culture protocol 136 including IVM medium and 500 ng / mL androstenedione. Training data for the culture classifier 140 can be obtained from a culture database 144 and a biological sample. As used in this disclosure, a "culture database 144" is a database that associates scientific knowledge about cell culture processes. The culture database 144 can be communicatively connected to the computing device 104 and can be implemented as described above. The culture database 144 can include a metabolite index, a cell culture medium index, and a protocol index. As used in this disclosure, a "metabolite index" is a data structure related to scientific knowledge about metabolites.A metabolite index may include data regarding the effect of specific metabolites in cell culture as they relate to oocyte maturity levels 128 along with dosing requirements and formulations. As used in this disclosure, a "cell media index" is a data structure relating scientific knowledge about cell culture media. For example, a cell media index may include data regarding optimal media and preparation / storage methods used in cell culture. As used in this disclosure, a "protocol index" is a data structure relating scientific knowledge about IVM cell culture procedures. For example, a protocol index may include methods for co-culture and group culture used in IVM.

[0219] Referring to the device of FIG. 1B, in some embodiments, the culture protocol 136 can include culturing oocytes with a co-culture containing granulosa cells generated from human induced pluripotent stem cells (hiPSCs). The cultured oocytes can be denuded oocytes. As used in this disclosure, "co-culture" refers to a cell culture method in which two or more different populations of cells are grown with some degree of contact between them. hiPSCs can be generated using the clustered regularly interspaced short palindromic repeats (CRISPR) method. CRISPR is a programmable technology that targets specific strands of the genetic code and edits DNA at precise locations. In some embodiments, CRISPR-based gene editing can be used to introduce one or more genes into the iPSC genome that encode factors that induce differentiation into ovarian support cells (e.g., ovarian granulosa cells). These factors include, for example, FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

[0220] The CRISPR method may include CRISPR-CAS9. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA complementary to the CRISPR sequence. The Cas9 enzyme, together with CRISPR sequences, is the basis of a technology known as CRISPR-Cas9, which can be used to edit genes in organisms. The CRISPR method may include class 1 CRISPR systems, including type I (cas3), type III (cas10), and type IV and 12 subtypes. The CRISPR method may include class 2 CRISPR systems, including type II (cas9), type V (cas12), type VI (cas13), and 9 subtypes. In some embodiments, the CRISPR method may include a computer software platform, the CRISPR-Cas design tool, and bioinformatics tools used to facilitate the design of guide RNAs (gRNAs) for use with the CRISPR / Cas gene editing system. For example, CRISPR-Cas design tools include CRISPRon, CRISPRoff, Invitrogen TrueDesign Genome Editor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPOR, sgRNA Designer, and Synthego Design Tool. CRISPR methods can also be used as diagnostic tools. For example, CRISPR-based diagnostics can be coupled with enzymatic processes such as SHERLOCK-based in vitro transcriptional profiling (SPRINT). SPRINT can be used in high-throughput or portable point-of-care devices to detect various substances, such as metabolites in subject samples or pollutants in environmental samples.

[0221] With further reference to the device of FIG. 1B , in some embodiments, a user may provide hiPSCs. The provision of hiPSCs can be performed following the oocyte collection process, as described above. The user participating in the provision of hiPSCs may be the same as or different from the user of the biological sample. In some embodiments, the user providing the hiPSCs may undergo a stimulation protocol, as disclosed above. In some embodiments, the assigned culture protocol 136 involves hiPSCs, granulosa cells, cumulus cells, oocytes, GV oocytes, MI oocytes, and all other cell types described throughout this disclosure, which can be lysed, extracted for genomic material, and flash-frozen as a final step in the culture process. For example, cells may be subjected to enzymatic cell lysis using enzymes such as lysozyme, lysostaphin, zymolase, cellulose, protease, or glycanase. Other lysis methods, such as chemical lysis, detergent lysis, alkaline lysis, mechanical lysis, heat lysis, ultrasonic lysis, physical lysis, non-mechanical lysis, etc., may also be applied. In some embodiments, the culture medium can be flash-frozen. Freezing methods may include using a cryoprotectant such as dimethyl sulfoxide and / or any other freezing method described throughout this disclosure.

[0222] With further reference to the device of FIG. 1B, in embodiments relating to oocyte culture, the cell culture medium may include LAG medium. For example, LAG medium can be used for incubation of COCs after collection resulting from a stimulation protocol. Package size may be a 10 mL vial. Storage may be for up to one month at 2-8°C away from light. Medium equilibration may be 18-24 hours prior to culture, including a 100 μl seed droplet, and placed in a 37°C incubator with 6% O2 and moderate CO2. In some embodiments, the cell culture medium may include IVM medium. For example, modified MediCult IVM medium may be used as a baseline control during the culture process. Package size may be a 10 mL vial. Storage may be for up to one month at 2-8°C away from light. In some embodiments, the cell culture medium may include metabolic substances. For example, 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-compatible mineral oil, universal GPS dishes, G-NOPS plus medium, micropipettes, stripper pipettes, an inverted ICSI microscope with camera, a dry injection tabletop incubator, a saturated humidity incubator, an embryoscope, a microcentrifuge, a refrigerator, a -20°C to 100°C freezer, a liquid nitrogen storage container, 35mm denuded dishes, stripper pipette tips, and other components that would be apparent to one of skill in the art that would be involved in a cell culture process.

[0223] viii. Culture data In some apparatuses described herein (see, e.g., FIG. 1A ), the computing device 104 is configured to receive culture data 140 related to the second biological sample 136. "Culture data" refers to data characterizing the biological, genetic, biochemical, and / or physiological properties, composition, or activity of a cell-cultured biological sample. The culture data 140 may include recording data and identifying the growth trends of COCs formed as a result of adding specific granulosa cells to the second biological collection. In one embodiment, the second biological sample 136 may be allowed to rest in culture medium for 2-3 hours after collection to allow equilibration to in vitro conditions. The computing device 104 may receive the culture data 140 from a culture sample database 144. The "culture sample database" is a database containing analytical data related to the culture process and method of the second biological sample 136. For example, the culture data 140 may be images of the cultured second biological sample 136, and the computing device 104 may be configured to analyze the images for results, goals, etc. In some embodiments, the computing device 104 can receive data, such as an embryologist's annotations regarding the process, results, purpose, etc. of the second biological sample 136 being cultured, from a culture sample database 144. The culture sample database 144 can be communicatively connected to the computing device 104 and can be 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 includes rubrics, analytical methods, and approaches for analyzing cell culture media. For example, the oocyte analysis index can include methods for oocyte scoring, outcome analysis, confounding variable analysis, etc.

[0224] In some apparatuses described herein (see, e.g., FIG. 1B ), the computing device 104 is configured to receive culture data 148 in response to the culture protocol 136. "Culture data 148" is data that characterizes the biological, genetic, biochemical, and / or physiological properties, composition, or activity of a cell culture biological specimen. The culture data 148 may include recording data and identifying the growth trends of COCs formed as a result of adding specific granulosa cells, such as hiPSCs, to denuded or non-denuded oocytes. The culture data 148 may be obtained from an oocyte index included in the culture database 144. As used in this disclosure, a "culture specimen index" is a database that includes analytical data related to oocyte culture processes and methods. For example, the culture data 148 may be images of oocytes being cultured, and the computing device 104 may be configured to analyze the images for results, goals, etc. In some embodiments, the culture database 144 may include an oocyte index. An "oocyte index" is a data structure that includes rubrics, analytical methods, and approaches for analyzing cell culture media. For example, an oocyte analysis index may include methods for oocyte scoring, outcome analysis, confounding variable analysis, etc. In some embodiments, computing device 104 may receive data, such as embryologist annotations regarding the process, results, objectives, etc. of culturing oocytes, from a feedback index in culture database 114. Computing device 104 may train a classifier or other machine learning model configured to calculate scoring metrics using training data. In some embodiments, training data may include, for example, without limitation, correlations of culture data with oocyte scoring, outcome analysis, and confounding variable analysis in the form of training examples. The training examples may be obtained from data in the oocyte index and feedback index obtained from culture database 114.

[0225] ix. Scoring As used in this disclosure, a "scoring metric" is a quantitative evaluation scale used to compare and track the performance or outcome of oocyte maturation. In one embodiment, a scoring metric can be calculated after denudation. As used in this disclosure, "denudation" is any process by which cells can be removed from an oocyte. Denudation can include any mechanical and / or enzymatic process. For example, and without limitation, denudation can include removing granulosa cells and / or cumulus cells from an oocyte. This can be done mechanically and / or with one or more chemicals, such as enzymes, that aid in the separation.

[0226] In some apparatuses described herein (see, e.g., FIG. 1A ), the computing device 104 can receive subject information regarding the completion of the stimulation protocol 132. Such subject information can include, for example, the subject's age, the subject's BMI, the number of COCs collected, the AMH level (μg / L), the antral follicle count (AFC) at the last ultrasound, the subject's E2 level (ng / L) on the day of oocyte collection, the subject's P4 level (ng / L) on the day of oocyte collection, the subject's LH (IU / L) on the day of oocyte collection, the subject's FSH (IU / L) on the day of oocyte collection, the number of days of stimulation, the amount of gonadotropin used, and the total injected dose (IU). Assigning the scoring metric 152 can include the computing device 104 analyzing images of one or both of the group cultures, the co-culture growth group and the non-co-culture growth group. The computing device 104 can receive images of the group COCs before culture, images of the group COCs after culture, and images of the denuded oocytes after culture. In some embodiments, the images may be images of cryolysates and cell culture media. In one embodiment, the scoring metric may include evaluating developmentally mature oocytes by microscopic examination for the presence of polar bodies. If polar bodies are present, the oocytes may be selected and used for intracytoplasmic sperm injection (ICSI) fertilization and / or oocyte freezing.

[0227] In some embodiments of the apparatus described herein (see, e.g., FIGS. 1A and 1B), the images may be sent to a third party for scoring. A "third party" is a qualified individual or organization (e.g., an embryologist) that analyzes the group cultures and develops / assigns a scoring metric 152. Additionally, the computing device 104 may perform any determination, classification, and / or analysis steps, methods, or processes performed by the third party. In some embodiments, the scoring metric 152 may include a total oocyte score (TOS) in response to analysis of the group culture images. The oocyte score may include indicators such as shape, size, ooplasmic characteristics, perivitelline space (PVS) structure, zona pellucida (ZP), polar body (PB) morphology, etc. As used in this disclosure, "oocyte scoring" refers to a grading system that evaluates the production and quality of mature human oocytes. For example, the computing device 104 may be configured to determine a total oocyte score for both pre-culture and post-culture oocyte images to generate a TOS indicator 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) for determining an overall oocyte score. The training data can include any data described throughout this disclosure, such as subject information, follicle dynamics information, oocyte scoring metrics 152, test sample sheets (e.g., sets of oocyte scoring metric 152 instructions), etc. The image classifier 148 can take group culture images as input and, utilizing the training data, output an overall oocyte score. The training data can include data from the culture sample database 144, as described above.

[0228] A detailed disclosure of the machine learning model is provided in further detail below. Regarding oocyte shape, if the oocyte morphology is poor (overall oocyte dark and / or ovoid), a value of -1 can be assigned; if it is approximately normal (overall oocyte coloration is not very dark and not very ovoid), a value of 0 can be assigned; and if determined to be normal, a value of +1 can be assigned. Regarding oocyte size, if the oocyte size is determined to be abnormally small or large, a value of -1 can be assigned if the size is less than 120 μm or more than 160 μm. If the size is approximately normal, i.e., does not deviate from normal by more than 10 μm, a value of 0 can be assigned; if the oocyte size is within the normal range of more than 130 μm to less than 150 μm, a value of +1 can be assigned. Regarding ooplasm characteristics, if the ooplasm is very granular and / or very vacuolated and / or shows some inclusions, a value of -1 can be assigned. If it is only slightly granular and / or shows few inclusions, a value of 0 can be assigned. The absence of granularity and inclusions can be assigned a value of +1. Regarding the structure of the perivitelline space (PVS), abnormally large, absent, or highly granular PVS can be defined as -1. A value of 0 can be assigned for moderately large and / or moderately small and / or less granular PVS. A value of +1 can be assigned to non-granular, normally sized PVS. Regarding the zona pellucida (ZP), -1 can be assigned to oocytes with very thin or thick ZPs (<10 μm or >20 μm). If the ZP does not deviate from normal by more than 2 μm, the ZP can be assigned a value of 0. A normal zona pellucida (>12 μm and <18 μm) can be assigned a value of +1. Regarding polar body (PB) morphology, PB morphology is defined as follows: Flat and / or multiple PBs or no PBs, granular and / or abnormally small or large PBs are designated as -1. A PB that is judged to be fair but not excellent can be designated as 0, and a PB of normal size and shape can be designated as +1.In some embodiments, the PB scores of MII oocytes may not be aggregated into the TOS. In some embodiments, the TOS calculated by the computing device 104 may be cross-checked by an embryologist or similarly skilled artisan to ensure that the quality scoring is not biased by the quality of the images. Expert feedback regarding corrections, adjustments, and correlations may be added to the training data for the machine learning model.

[0229] In some embodiments of the apparatus described herein (see, e.g., FIGS. 1A and 1B), the computing device 104 can train a classifier or other machine learning model configured to calculate the TOS using training data. In some embodiments, the training data can include, for example, without limitation, a six-point qualitative scale in the form of training examples and correlation of culture data to image quality. The training examples can be obtained from data in an oocyte scoring index and feedback index retrieved from the culture database 144. The scoring metric 152 can include performing an outcome analysis as a function of the TOS. As used in this disclosure, an "outcome analysis" can be 1.) a measure of maturation rate and oocyte quality score between cultures in a group culture, or 2.) a measure of maturation rate and oocyte quality score between a control culture and a co-culture. Parametric or non-parametric tests can be applied to determine the significance of findings under analysis. The computing device 104 can use classification algorithms using the methods described above to determine 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 oocyte quality, mean PVS quality, mean ZP quality, mean polar body quality, etc. In some embodiments, these results can be reported as mean, median, and variance.

[0230] In some embodiments, with continued reference to FIG. 1A or FIG. 1B, the computing device 104 may perform the results analysis using machine learning processes 116 and / or models as described throughout this disclosure.

[0231] 1A , the computing device 104 may train a machine learning model to output an outcome analysis based on input group culture images, where the training data includes oocyte scoring metrics, test sample sheets, subject information, feedback from the computing device 104 programmer / third party, data from the assigned stimulation protocol, and any other form of data described throughout this disclosure. The training data may be obtained from the biological specimen database 124 and the culture database 144. In some embodiments, communication from a third party can be input into the machine learning process 116 to create a machine learning model that generates the scoring metrics. For example, the third party communication can include an embryologist's annotations regarding the total oocyte score, where the annotations are input into the machine learning model. The machine learning model includes a classifier that generates an outcome analysis using the training data received from the biological specimen database 124 and the culture database 144 (including subject information, data from the image classifier 148, data from the assigned stimulation protocol, test sample sheets, and any other form of data described throughout this disclosure). Additionally or alternatively, a communication regarding the scoring metrics generated by the computing device may be sent to a third party using the machine learning process 116. For example, the oocyte scoring metrics may be sent to a remote computing device operated by a third party communicatively connected to the computing device 104, where the third party may perform further analysis, such as results analysis. To further illustrate this example, the third party's response to the communication generated by the computing device 104 may be uploaded to a database communicatively connected to the computing device 104 and used as feedback in the training data.

[0232] 1A , in some embodiments, the scoring metric 152 can include omics-based analyses. For example, 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. The cell culture media can be subjected to metabolomic analysis to determine changes in the molecular content of the media after co-culture compared to a pre-culture media control. This can be used by the computing device 104 to profile dynamic changes in paracrine signaling between granulosa cells and oocytes. The collected data can then be compiled for subsequent analysis to identify changes in epigenetic state, metabolite presence, and gene expression between various co-culture conditions and controls.

[0233] In another example related to FIG. 1B , the computing device 104 may train a machine learning model to output an outcome analysis based on input culture images, where the training data includes oocyte scoring metrics, test sample sheets, subject information, feedback from a programmer / third party of the computing device 104, data from the stimulation protocol, and any other form of data described throughout this disclosure. The training data may be obtained from the biological specimen database and culture database 144. In some embodiments, communication from a third party can be input into the machine learning process 116 to create a machine learning model that generates the scoring metric 152. For example, the communication by the third party can include an embryologist's annotations regarding the total oocyte score, where the annotations are input into the machine learning model. The machine learning model includes a classifier that generates an outcome analysis using training data received from the biological specimen database and culture database 144 (including subject information, data from the image classifier, data from the stimulation protocol, test sample sheets, and any other form of data described throughout this disclosure). Additionally or alternatively, communication regarding the scoring metric generated by the computing device may be sent to the third party using the machine learning process 116. For example, the oocyte scoring metrics can be transmitted to a remote computing device operated by a third party communicatively connected to the computing device 104, where the third party can perform further analysis, such as results analysis. To further illustrate this example, the third party's responses to communications generated by the computing device 104 can be uploaded to a database communicatively connected to the computing device 104 and used as feedback in the training data.

[0234] With further reference to FIG. 1B, in some embodiments, the scoring metric can include omics-based analysis. Omics is a novel and comprehensive approach for analyzing the complete genetic or molecular profile of humans and other organisms. For example, in contrast to genetics, which focuses on single genes, genomics focuses on all genes (genomes) and their interrelationships. In some embodiments, omics-based analysis can include genomics, proteomics, transcriptomics, pharmacogenomics, epigenomics, microbiomics, lipidomics, glycomics, transcriptomics-culturomics, and / or any other omics approach apparent to those skilled in the art as applicable. In some embodiments, after culture, oocytes that fail to mature and exhibit characteristics of GV or MI can be harvested, along with associated granulosa cells from the culture, for single-cell RNA-seq analysis. To this end, oocytes and granulosa cells can be flash-frozen for library preparation. Half of the oocytes showing MII oocyte development can be collected and, along with their associated granulosa cells, subjected to single-cell RNA sequencing analysis using the rapid freezing method described throughout this disclosure. The remaining half of the MII oocytes can be used for proteomic analysis. Culture media from all conditions can be further rapidly frozen and utilized for metabolomics and proteomics to identify cholesterol metabolite levels and paracrine protein production. For example, 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. Cell culture media can be utilized for metabolomics analysis to determine changes in the molecular content of the media after co-culture compared to pre-culture media controls. This can be used to profile dynamic changes in paracrine signaling between granulosa cells and oocytes. Rapid freezing of the media components effectively quenches the samples, making them suitable for metabolic assessment.The collected data can then be compiled for subsequent analysis to identify changes in epigenetic status, metabolite presence, and gene expression between the various co-culture conditions and controls.

[0235] B. Machine Learning Module Referring now to the machine learning module of FIG. 2A , an exemplary embodiment of a machine learning module 200 is shown, which can perform one or more machine learning processes described in this disclosure. The machine learning module can perform the determining, classifying, and / or analyzing steps, methods, processes, etc., as described in this disclosure, using a machine learning process. As used in this disclosure, a “machine learning process” is a process that automatically uses training data 204 to generate an algorithm, which is executed by a computing device / module given data provided as input 212 and generates output 208. This is in contrast to a non-machine learning software program, where the commands to execute are predetermined by a user and written in a programming language.

[0236] i. Training Data With further reference to the machine learning module of FIG. 2A , as used herein, “training data” refers to data containing correlations that a machine learning process can use to model relationships between two or more categories of data elements. For example, without limitation, training data 204 may include multiple data entries, each representing a set of data elements recorded, received, and / or generated together. Data elements may be related by common presence in a given data entry, proximity in a given data entry, etc. The multiple data entries in training data 204 may reveal one or more trends in correlations between categories of data elements. For example, without limitation, higher values of a first data element belonging to a first category of data elements tend to correlate with higher values of a second data element belonging to a second category of data elements, indicating a possible proportional or other mathematical relationship linking values belonging to the two categories. Multiple categories of data elements may be related in training data 204 according to various correlations. Correlations can indicate causal and / or predictive associations between categories of data elements, which can be modeled as relationships, such as mathematical relationships, by machine learning processes, as described in further detail below. Training data 204 can be formatted and / or organized by categories of data elements, for example, by associating the data elements with one or more descriptors that correspond to the categories of the data elements. As a non-limiting example, training data 204 can include data entered by a person or process in a standardized format, whereby entry of a given data element into a predetermined field of a format can be mapped to one or more descriptors of a category. Elements in training data 204 can be associated with descriptors of a category by tags, tokens, or other data elements.For example, without limitation, the training data 204 may be provided in a fixed-length format, a format that associates data locations with categories (e.g., comma-separated value format (CSV)), and / or a self-describing format (e.g., Extensible Markup Language (XML), JavaScript Object Notation (JSON), etc.) that allows a process or device to detect the category of the data.

[0237] Alternatively or additionally, and with further reference to FIG. 2A , the training data 204 may include one or more uncategorized elements. That is, the training data 204 may be unformatted or may not include descriptors for some elements of the data. Machine learning algorithms and / or other processes may sort the training data 204 according to one or more categorizations, for example, using natural language processing algorithms, tokenization, detecting correlated values in the raw data, etc. Correlation algorithms and / or other processing algorithms may be used to generate categories. As a non-limiting example, in a corpus of text, phrases that comprise “n” compounds (e.g., nouns modified by other nouns) may be identified by a statistically significant prevalence of n-grams containing such words in a particular order, and such n-grams may be classified as elements of language such as “words” that are tracked similarly to single words, generating new categories as a result of the statistical analysis. Similarly, in a data entry that includes some text data, a person's name can be identified by referencing a list of terms, a dictionary, or other glossary, allowing for ad-hoc classification by a machine learning algorithm and / or allowing for automatic association of data in the data entry with a descriptor or predefined format. The ability to automatically classify data entries allows the same training data 204 to be applied to two or more different machine learning algorithms, as described in more detail below. The training data 204 used by the machine learning module 200 can associate any of the input data described in this disclosure with any of the output data described in this disclosure.

[0238] ii. Training Data Classifier 2A , the training data can be filtered, sorted, and / or selected using one or more supervised and / or unsupervised machine learning processes and / or models, as described in more detail below. Such models may include, without limitation, the training data classifier 216. A “classifier,” as used in this disclosure, that may be included in the training data classifier 216, is a machine learning model, as defined below (e.g., a mathematical model, neural net, or program generated by a machine learning algorithm known as a “classification algorithm,” as described in more detail below). It sorts input into categories or bins of data and outputs the categories or bins of data and / or their associated labels. The classifier may be configured to at least output data that labels or identifies sets of data that are known to be close and clustered together under a certain distance metric, as described below, for example. The machine learning module 200 can 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 is not limited to, linear classifiers such as, but not limited to, a logistic regression classifier and / or a naive Bayes classifier, a nearest neighbor classifier such as a k-nearest neighbor classifier, a support vector machine, a least squares support vector machine, Fisher's linear discriminant, a quadratic classifier, a decision tree, a boosted tree, a random forest classifier, learning vector quantization, and / or a neural network-based classifier.

[0239] iii. Lazy learning process With further reference to FIG. 2A , the machine learning module 200 may be configured to execute a lazy learning process 220 and / or lazy learning protocol. This may alternatively be referred to as a “lazy load” process and / or protocol or a “call-when-needed” process and / or protocol, which performs machine learning by receiving input to be converted into an output and deriving an algorithm used to combine the input with a training set and generate the output on demand. For example, an initial set of simulations may be run to cover initial heuristics and / or “first guesses” on the output and / or relationships. As a non-limiting example, the initial heuristic may include ranking associations between the input and elements of the training data 204. The heuristic may include selecting several top-ranked associations and / or training elements of the data 204. The lazy learning may implement any suitable lazy learning algorithm, including, but not limited to, a K-nearest neighbor algorithm, a lazy Naive Bayes algorithm, etc. Upon reviewing this disclosure in its entirety, those skilled in the art will be aware of a variety of lazy learning algorithms that can be applied to generate outputs as described in this disclosure, including, but not limited to, lazy learning applications of machine learning algorithms, such as those described in more detail below.

[0240] iv. Machine Learning Models Alternatively or additionally, and with further reference to FIG. 2A , the machine learning model 224 may be generated using the machine learning process described herein. As used herein, a “machine learning model” is a mathematical and / or algorithmic representation of a relationship between inputs and outputs generated using any machine learning process stored in memory, including, but not limited to, any of the processes described above. Once created, the inputs are sent to the machine learning model 224, which generates an output based on the resulting relationship. For example, but not limited to, a linear regression model generated using a linear regression algorithm may calculate a linear combination of the input data using coefficients derived during the machine learning process to calculate the output data. As a further non-limiting example, the machine learning model 224 may be generated by creating an artificial neural network. The artificial neural network may be, for example, a convolutional neural network including an input layer of nodes, one or more hidden layers, and an output layer of nodes. Connections between the nodes may be created by a process of “training” the network. In this process, elements from a set of training data 204 are applied to input nodes, and then an appropriate training algorithm (e.g., Levenberg-Marquardt, conjugate gradient, simulated annealing, or other algorithm) is used to adjust the connections and weights between nodes in adjacent layers of the neural network in order to produce desired values at output nodes. This process is sometimes referred to as deep learning.

[0241] 2A , the machine learning algorithm may include at least one supervised machine learning process 228. The at least one supervised machine learning process 228, as defined herein, includes an algorithm that receives a training set relating some inputs to some outputs and seeks to find one or more mathematical relationships relating the inputs to the outputs, where each of the one or more mathematical relationships is optimal according to some criteria specified to the algorithm using a scoring function. For example, the supervised learning algorithm may include the outputs as described above as inputs, and may further include a scoring function that represents the desired form of relationship to be found between the inputs and outputs. The scoring function may seek, for example, to maximize the probability that a given input and / or combination of elemental inputs is associated with a given output and minimize the probability that a given input is not associated with a given output. The scoring function may be expressed as a risk function that represents the “expected loss” of the algorithm relating inputs to outputs, where the loss is calculated as an error function that represents the degree to which the predictions generated by the relationships are inaccurate when compared to the given input-output pairs provided in the training data 204. Those skilled in the art will, upon reviewing this disclosure in its entirety, be aware of the various possible variations of at least one supervised machine learning process 228 that can be used to determine the relationship between inputs and outputs. The supervised machine learning process may include a classification algorithm as defined above.

[0242] 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 on a dataset without regard to labels. As a result, an unsupervised machine learning process is free to discover any structure, relationships, and / or correlations provided in the data. An unsupervised process may not require a response variable. An unsupervised process may be used, for example, to discover interesting patterns and / or inferences between variables or to determine the degree of correlation between two or more variables.

[0243] With further reference to FIG. 2A , the machine learning module 200 can be designed and configured to create the machine learning model 224 using techniques for developing linear regression models. The linear regression model can include ordinary least squares regression, which aims to minimize the squared difference between predicted and actual results according to an appropriate norm for measuring such 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 can include ridge regression, in which the function to be minimized includes a least-squares function plus a term that penalizes large coefficients by multiplying the square of each coefficient by a scalar. The linear regression model can include a least absolute shrinkage and selection operator (LASSO) model, which combines ridge regression with multiplying the least-squares term by a coefficient equal to 1 divided by twice the number of samples. The linear regression model can include a multitask LASSO model. In a multi-task LASSO model, the norm applied to the least-squares terms of the LASSO model is the Frobenius norm, which takes the square root of the sum of the squares of all terms. Linear regression models include elastic net models, multi-task elastic net models, minimum angle regression models, LARS lasso models, orthogonal matching pursuit models, Bayesian regression models, logistic regression models, stochastic gradient descent models, perceptron models, passive-aggressive algorithms, robustness regression models, Huber regression models, or any other suitable models that may occur to one skilled in the art upon reviewing this disclosure in its entirety. In one embodiment, the linear regression model can be generalized to a polynomial regression model, which finds a polynomial (e.g., quadratic, cubic, or higher order) that provides the best predicted output / actual output fit. Similar methods to those described above may be applied to minimize the error function, as will be apparent to one skilled in the art upon reviewing this disclosure in its entirety.

[0244] With further reference to FIG. 2A , the machine learning algorithm may include, but is not limited to, linear discriminant analysis. The machine learning algorithm may include, but is not limited to, quadratic discriminant analysis. The machine learning algorithm may include, but is not limited to, kernel ridge regression. The machine learning algorithm may include, but is not limited to, support vector machines, including support vector classification-based regression processes. 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 nearest neighbor algorithms. The machine learning algorithm may include various forms of latent space regularization, such as variational regularization. The machine learning algorithm may include Gaussian processes, such as Gaussian process regression. The machine learning algorithm may include cross-decomposition algorithms, including partial least squares and / or canonical correlation analysis. The machine learning algorithm may include naive Bayes methods. The machine learning algorithm may include decision tree-based algorithms, such as decision tree classification or regression algorithms. The machine learning algorithm may include ensemble methods, such as bagging meta-estimators, forests of randomized trees, AdaBoost, gradient tree boosting, and / or voting classifier methods. The machine learning algorithm may include a neural net algorithm, including a convolutional neural net process.

[0245] C. Training Data 2B , an exemplary table 236 of training data 204 is shown. The training data 204 may include any data described throughout this disclosure. For example, the training data 204 may include de-identified user information. In this disclosure, a user may be referred to as a subject. The de-identified subject information may include the subject's age, the subject's BMI, the number of COCs collected, the AMH level (μg / L), the antral follicle count (AFC) at the last ultrasound, the E2 level (ng / L) on the subject's day of oocyte collection, the P4 level (ng / L) on the subject's day of oocyte collection, the LH level (IU / L) on the subject's day of oocyte collection, the FSH level (IU / L) on the subject's day of oocyte collection, the number of days of stimulation, the amount of gonadotropin used, the total injected dose (IU), etc. Additionally or alternatively, the training data 204 may include group COC images before culture, group COC images after culture, images of denuded oocytes after culture, third party annotations such as embryologist annotations, machine learning feedback, follicle dynamics information, test sample sheets, frozen oocyte lysate data, frozen granulosa cell lysate data, frozen cell culture media 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, etc.

[0246] 2C , an exemplary table 236 of training data 204 is shown. The training data 204 may include any data described throughout this disclosure. For example, the training data 204 may include de-identified user information. In this disclosure, a user may be referred to as a subject. The de-identified subject information may include the subject's age, the subject's BMI, the number of COCs collected, the AMH level (μg / L), the antral follicle count (AFC) at the last ultrasound, the E2 level (ng / L) on the subject's day of oocyte collection, the P4 level (ng / L) on the subject's day of oocyte collection, the LH level (IU / L) on the subject's day of oocyte collection, the FSH level (IU / L) on the subject's day of oocyte collection, the number of days of stimulation, the amount of gonadotropin used, the total injected dose (IU), etc. Additionally or alternatively, the training data 204 may include COC images before co-culture, COC images after co-culture, images of denuded oocytes after culture, third party annotations such as embryologist annotations, machine learning feedback, follicle dynamics information, test sample sheets, frozen oocyte lysate data, frozen granulosa cell lysate data, frozen cell culture media data, data from a systemic hormone index, data from a maturity index, data from the biological sample database 124, data from the culture database 144, etc.

[0247] D. Mini-Stimulation Protocol Referring now to FIG. 3A, an exemplary flowchart of a mini-stimulation protocol 300 is shown. In step 305, the mini-stimulation protocol may include selecting a first induction agent (e.g., a follicle-inducing agent) to be injected into the user for the first biological sample. The first induction agent may be selected based on the user's measured hormone levels. The first induction agent may include human recombinant follicle-stimulating hormone (rFSH). Examples of rFSH induction agents include Gonal-F, manufactured by Merck Global; Follistim, manufactured by Merck Global; Follitropin Alfa, manufactured by Teva (headquartered in Tel Aviv-Yafo, Israel); and Glucophage, manufactured by Merck Global. rFSH, or any other induction agent (e.g., a follicle-inducing agent) described throughout this disclosure, may be injected into the user multiple times in varying increments. In some embodiments, no induction agent may be administered to the subject. In one embodiment, the timing of when a subject can begin minimal stimulation can be determined by the subject's contraceptive status, as described in more detail above. For example, a subject not taking contraceptives can begin stimulation with rFSH on day 2 of the subject's menstrual cycle. In yet another non-limiting example, a subject taking contraceptives, such as a combined oral contraceptive (COC), can begin stimulation five days after taking the last medication. The dosage and selection of medication can be determined by one or more laboratory tests (e.g., blood tests performed on day 2 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 the health of ovarian reserve, circulating hormone levels, and / or fertility status.

[0248] In step 310, protocol 300 may include stimulating the user with a first induction agent for a period of time (e.g., three days). For example, rFSH may be injected three or more times at a dose of 100-200 IU over a one- to four-day stimulation period. For example, the stimulation period may span three days. After injection of the first induction agent (e.g., a follicle-inducing agent), an ultrasound examination may be performed to determine the average follicle size of the cells, such as oocytes. In step 315, the protocol may include a one-day coasting period. The coasting period may include any of the coasting periods described above, as described in more detail. The coasting period may include a period during which the second induction agent is withheld until serum estradiol (E2) levels have decreased to a level considered safe by those skilled in the art to prevent the onset of ovarian hyperstimulation syndrome. In some embodiments, an ultrasound examination may be performed during the coasting period after the three-day mini-stimulation protocol 300 to determine the average follicle size of the cells. In some embodiments, the coasting period may span two days. Determining the average follicle size of the cells may include identifying a time when the average follicle size is between 8 and 12 mm. In step 320, a second inducer may be injected into the user in response to determining the average follicle size of the cells. The second inducer may include human chorionic gonadotropin (hCG). The second inducer may be administered based on one or more factors related to the user, including follicle size, previous diagnoses of any medical conditions, ultrasound imaging, drug allergies, the subject's tolerance to certain medications, etc. Examples of rFSH inducers include Pregnyl, manufactured by Schering Plough (headquartered in Kenilworth, NJ), Novarel, manufactured by Ferring Laboratories (headquartered in Parsippany, NJ), Chorex, manufactured by Encocam (headquartered in Huntingdon, England), and Profasi, manufactured by Serum Institute of India Ltd (headquartered in Pune, India).In some embodiments, the second inducer can be any inducer as described throughout this disclosure. Like the first inducer, the second inducer can be injected into the user multiple times in varying amounts. For example, it can be injected one or more times over a three-day stimulation period in amounts ranging from 200 μg to 700 μg. After the injection of the second inducer, cells can be collected for the user in step 325, where the cells include oocytes and / or COCs. For example, after a coasting period, 500 μg of hCG inducer can be administered to follicles measuring 8-9 mm, with oocytes collected 36 hours after administration. Oocyte collection can involve a medical professional, such as a physician, inserting a collection device into an egg-containing follicle and collecting the eggs and surrounding fluids. Oocyte collection can include collecting immature oocytes, mature oocytes, COCs, and any other type of cell involved in reproduction present in the ovaries. Oocyte collection may occur within any time frame ranging from 12 to 96 hours after hCG administration. In one embodiment, blood tests for hormone levels, such as E2, LH, and / or P4, may be evaluated to ensure one or more quality metrics and confirm that the subject has taken hCG as prescribed. This may also help determine whether hormone levels are within normal expected ranges.

[0249] E. Oocyte denudation Referring now to FIG. 3B, an exemplary flowchart for oocyte denudation is shown. At step 305, method 300 may include obtaining a COC from a biological sample. The COC may be obtained according to the oocyte collection method disclosed above. At step 310, method 300 may include oocyte denudation of the COC. In some embodiments, denudation may occur in an IVM well by gently mechanically dissociating the cells with pipetting to remove most cumulus and / or granulosa cells. If enzymatic dissociation is required, the cells may be transferred to a separate dish for hyaluronidase treatment. At step 315, the COC may be stripped with a stripper tip and washed with IVM medium or MOPS plus medium to clean the oocytes for imaging and, if necessary, inactivate the hyaluronidase. The stripper tip may include 200 micron and / or 400 micron tips for fine cleaning. In some embodiments, GV oocytes and MI oocytes may be prepared and used for culture as a result of COC denudation. At step 320, the method 300 may include transferring the denuded COCs to a culture dish for imaging.

[0250] F. Co-culture Referring now to FIG. 4, an exemplary table 400 of metabolite formulations that can be included in the cultures described herein is shown. The Metabolite 404 column lists exemplary metabolites that can be used as inducers and / or cell culture metabolites. As used in this disclosure, a "cell culture metabolite" is a substance involved in cellular metabolism that optimizes the synthesis of new molecules in cell culture. The Stock Solution Prep Concentration 408 column lists exemplary concentrations for the cell culture metabolites. The Final Concentration in IVM Medium 412 column lists exemplary concentrations of the cell culture metabolites in IVM medium for group or co-culture cultures of the first or second biological sample 136. For example, 10 mg / mL of HSA can be added to the IVM medium for any cell culture (e.g., group or co-culture) described herein. Approximately 75 mUI / mL of FSH can be added to the IVM medium for any cell culture described herein. Approximately 100 mUI / mL of hCG can be added to the IVM medium for any cell culture described herein. Approximately 500 ng / mL of androstenedione can be added to IVM medium for any cell culture (e.g., group culture or co-culture) described herein. Approximately 1 μg / mL of doxycycline can be added to IVM medium for any cell culture described herein. In some embodiments, steroidogenic granulosa cells derived from human induced pluripotent stem cells (hiPSCs) can be co-cultured with denuded or non-denuded immature oocytes (e.g., COCs), thereby reconstituting the follicular microenvironment in vitro and rapidly and efficiently promoting oocyte maturation to enhance oocyte health and developmental potential. As used in this disclosure, "steroidogenic granulosa cells" are granulosa cells that express high levels of steroidogenic enzymes, such as estradiol. For example, steroidogenic granulosa cells can be mural granulosa cells harvested from cystic follicles.The application of steroidogenic granulosa cells to co-culture with oocytes (e.g., COCs) can enhance in vitro oocyte maturation after egg / oocyte collection, enabling utilization of all collected eggs / oocytes by directly providing nutrients, materials, and mechanical support to oocytes throughout gametogenesis and folliculogenesis. Steroidogenic granulosa cells can be grown in standard IVF and IVM media to achieve oocyte maturation of immature oocytes. This can increase the overall pool of healthy oocytes available for use in IVF and reduce the number of egg / oocyte collection procedures users undergo.

[0251] i. Preparation of granulosa cell co-cultures Referring now to FIG. 5, an exemplary flowchart 500 for preparing a granulosa cell co-culture is illustrated. Granulosa cells in a subject's ovaries play an important role in the female reproductive system. These cells release estrogen, progesterone, and other hormones that promote oocyte maturation in the ovaries, making them a logical tool for IVM. Furthermore, granulosa cells provide a developmental microenvironment for follicle and oocyte development, directly supplying nutrients, materials, and mechanical support to oocytes throughout gametogenesis and folliculogenesis. In step 505, a cryovial of liquid nitrogen-frozen granulosa cells can be thawed and incubated. In some embodiments, thawing and incubation can occur within 72 hours prior to oocyte collection from the user. 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 may be a 1 mL cryovial containing 100,000 granulosa cells. Thawing can be accomplished by placing the granulosa cell cryovial in a water bath or a dry bead bath. The granulosa cell cryovial may be incubated for 3 to 5 minutes. In step 510, IVM medium may be added to the cryovial for cell suspension. In some embodiments, 0.5 mL of ICM medium may be added to induce cell suspension. In step 515, the cell suspension is transferred to a tube and centrifuged. In some embodiments, 1 mL of the cell suspension may be transferred to a 1.5 mL tube, and the tube is centrifuged at 300 x g for 5 minutes. In step 520, the supernatant may be removed using a pipette. The pipette may be a p1000 pipette. In step 525, the cell pellet formed in the centrifuge tube containing the cell suspension may be resuspended in IVM medium and centrifuged. The IVM medium may be 1 mL of IVM medium. The tube may be centrifuged as described above. The supernatant can be removed using a pipette at step 530. The pipette can be a p1000 pipette.In step 535, the cell pellet can be resuspended in IVM medium, which can be 0.1 mL of IVM medium. In this step, the granulosa cells can be 1,000 cells per 1 ul. In some embodiments, 10 ul of cell suspension can be used for the oocytes in the user's second biological sample 136.

[0252] Referring now to FIG. 6A, an exemplary embodiment of a co-cultured second biological sample 136 containing immature COCs according to a user is shown. In some embodiments, COCs obtained after oocyte collection from a user's follicular aspirate can be randomly split in half into a medium, such as LAG medium, in a granulosa cell plate, and the other half can be placed in LAG medium in a plate without co-culture. The COCs can be incubated in LAG medium at 37° C. for 2 hours. Granulosa cells may be prepared as shown in FIG. 5. The prepared granulosa cells can be added to the right-center well containing IVM medium, with 10,000 granulosa cells added per COC to be cultured. The dish containing the granulosa cells can then be placed back into the incubator until use. After the 2-hour incubation period, the COCs in the LAG medium can be transferred to the IVM medium in the granulosa cell dish using a Pasteur pipette.

[0253] 6B, an exemplary embodiment of a control group culture of a second biological sample 136 containing immature COCs according to a user is shown. 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 IVM medium in a control dish using a Pasteur pipette.

[0254] Referring now to FIG. 6C, an exemplary embodiment of a co-cultured oocyte containing a user's immature oocyte is shown. In some embodiments, the oocytes can be denuded oocytes. In some embodiments, oocytes obtained after oocyte collection from a user's follicle aspirate can be randomly split in half into a medium, such as LAG medium, in a granulosa cell plate, and the other half can be placed in LAG medium in a plate without co-culture. Alternatively, some oocytes for culture can be pre-frozen, in which case the oocytes can be thawed before culturing. The oocytes can be incubated in LAG medium at 37° C. for 2 hours. Granulosa cells can be prepared as shown in FIG. 5. The prepared granulosa cells can be added to surrounding 50 μl wells containing IVM medium, and 10,000 granulosa cells can be added per oocyte to be cultured. The dish containing the granulosa cells can then be placed back into the incubator until use. After a 2-hour incubation period, oocytes in LAG medium can be transferred to IVM medium in the granulosa cell dish using a Pasteur pipette. In granulosa cell co-cultures, after 18-48 hours of incubation, the culture plate can be removed and the oocytes and granulosa cells can be imaged in their individual wells. In some embodiments, cultures can be imaged after 24 hours.

[0255] Referring now to FIG. 6D, an exemplary embodiment of a user-configured control culture of immature oocytes is shown. 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 IVM medium in a control dish using a Pasteur pipette. After 10-48 hours of incubation in the control culture, the culture plate can be removed and the oocytes can be imaged in their individual wells. In some embodiments, the cultures can be imaged after 24 hours.

[0256] ii. Induction of human oocyte maturation in vitro Referring now to FIG. 7A, a flow diagram of an exemplary method for inducing human oocyte maturation in vitro is shown. Method 700 may include performing the enumerated steps using computing device 104 (e.g., of FIG. 1A). At step 705, method 700 includes obtaining a first biological sample from a user. The first biological sample may be any form of biological sample defined and illustrated throughout this disclosure. For example, the first biological sample may include a blood sample from the user, as illustrated at least in FIG. 1. The user may be a user (e.g., an individual) as defined in FIG. 1A. In some embodiments, the biological sample may be collected from the user by a collection device, as defined and illustrated at least in FIG. 1A. For example, the collection device may include a medical syringe for collecting blood from the user. The biological sample may also include systemic hormones. At step 710, method 700 includes assigning a stimulation protocol to the user in response to the first biological sample. The stimulation protocol is a drug infusion process, as defined and illustrated at least in FIG. 1A. In some embodiments, a stimulation protocol can be assigned based on measured hormone levels in the biological sample. The measured hormone levels can include E2, LH, FSH, and / or P4 levels, as defined in FIG. 1A. The assigned stimulation protocol can include a minimal stimulation protocol configured to induce cell release over a three-day period, as defined and illustrated in at least FIG. 1. In one embodiment, the minimal stimulation protocol can include selecting a first induction agent in response to the first biological sample and selecting a second induction agent in response to the follicle measurements. The induction agents can include human serum albumin, FSH, hCG, androstenedione, and doxycycline, as defined in FIG. 1A, according to the formulations shown in FIGS. 1-6.

[0257] Still referring to step 710, in some embodiments, the minimum stimulation protocol may include injecting a first induction agent into the user, performing an ultrasound examination to determine the average follicle size of the cells, injecting a second induction agent into the user, and harvesting cells containing oocytes from the user. The first induction agent may include human recombinant follicle-stimulating hormone (rFSH). The rFSH may be injected into the user multiple times in varying increments. For example, with reference to FIG. 1A, the rFSH may be injected in an amount of 100-200 IU three or more times during a three-day stimulation period. After injection of the first induction agent, an ultrasound examination may be performed to determine the average follicle size of the cells, such as oocytes. In some embodiments, the ultrasound examination may be performed during a two-day coasting period after the three-day stimulation protocol, as defined in FIG. 1A. Determining the average follicle size of the cells may include identifying when the average follicle size is between 8-12 mm. Depending on the average follicle size of the cells, a second inducer can be injected into the user. The second inducer can include human chorionic gonadotropin (hCG). Like the first inducer, the second inducer can be injected into the user multiple times in varying amounts. For example, referring to FIG. 1A, the second inducer can be injected once or multiple times over a three-day stimulation period in an amount ranging from 200 μg to 700 μg. After the injection of the second inducer, cells can be collected for the user, where the cells include oocytes. For example, referring to FIG. 1A, after a coasting period, 500 μg of hCG inducer can be administered for a follicle size of 8 to 9 mm, and the oocytes can be collected 36 hours after administration. Oocyte collection can involve a medical professional, such as a physician, inserting a collection device into an egg-containing follicle and collecting the eggs and surrounding body fluids.

[0258] 7A , method 700 includes, at step 715, obtaining a second biological sample from a user, where the second biological sample includes at least immature cumulus-oocyte complexes (COCs), as defined in FIG. 1A . The second biological sample may include a bodily fluid, as described above. The second biological sample may be collected and obtained using a collection device, as disclosed above. At step 720, method 700 includes culturing the second biological sample. In some embodiments, culturing the second biological sample may include culturing the cumulus-oocyte complexes in a group culture, as defined in FIG. 1A . For example, with reference to FIGS. 1-6 , the group culture may include culturing the cumulus-oocyte complexes with a granulosa cell co-culture and culturing a control group of COCs without co-culture. In some embodiments, the cell culture medium may include LAG medium. For example, with reference to FIGS. 1-6 , LAG medium may be used for incubation of COCs after collection from a stimulation protocol. The package size may be a 10 mL vial. Storage may be at 2-8°C away from light for up to one month. Medium equilibration may be performed for 18-24 hours prior to culture, containing a 100 μl seed droplet and placed in a 37°C incubator with 6% O2 and moderate CO2. In some embodiments, the cell culture medium may include IVM medium. For example, see Figures 1-6. Modified MediCult IVM medium may be used as a baseline control during the culture process. The package size may be a 10 mL vial. Storage may be at 2-8°C away from light for up to one month. In some embodiments, the cell culture medium may include metabolic substances. For example, 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-compatible mineral oil, universal GPS dishes, G-NOPS plus medium, micropipettes, stripper pipettes, an inverted ICSI microscope with camera, a dry injection tabletop incubator, a saturated humidity incubator, an embryoscope, a microcentrifuge, a 4° C. refrigerator, a −20° C. freezer, a −80° C. freezer, a liquid nitrogen storage container, 35 mm denuded dishes, stripper pipette tips, and other components that would be apparent to one of skill in the art that would be involved in a cell culture process.

[0259] 7A , at step 725, method 700 includes assigning a scoring metric to the second biological sample depending on the culture of the second biological sample. The assignment can be based on subject information regarding the completion of the stimulation protocol. Such subject information can include, for example, the subject's age, the subject's BMI, the number of COCs collected, the AMH level (μg / L), the antral follicle count (AFC) at the last ultrasound, the subject's E2 level (ng / L) on the day of oocyte collection, the subject's P4 level (ng / L) on the day of oocyte collection, the subject's LH (IU / L) on the day of oocyte collection, the subject's FSH (IU / L) on the day of oocyte collection, the number of days of stimulation, the amount of gonadotropin used, and the total injected dose (IU). In some embodiments, assigning a scoring metric can include acquiring images of the group cultures and analyzing the images of one or both of the co-culture and non-co-culture groups. For example, the group culture images may include a group COC image before culture, a group COC image after culture, and a denuded oocyte image after culture. In some embodiments, the images may be sent to a third party for score assignment, as defined in FIG. 1A. In some embodiments, the scoring metric 152 may include a total oocyte score (TOS) in response to an analysis of the group culture images. The oocyte score may include indicators such as shape, size, ooplasmic characteristics, perivitelline space (PVS) structure, zona pellucida (ZP), and polar body (PB) morphology, as described in detail at least in FIG. 1A. The total oocyte score for both pre-culture and post-culture oocyte images for generation of the TOS metric may be based on a -6 to +6 scale system.

[0260] In some embodiments, the scoring metric may include performing a result analysis according to the TOS, as defined and illustrated in FIG. 1A. Parametric or non-parametric tests may be applied to determine the significance of findings during the analysis. The result analysis may 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 mean total oocyte score, the mean oocyte shape, the mean oocyte size, the mean oocyte cytoplasm quality, the mean PVS quality, the mean ZP quality, the mean polar body quality, and the like. In some embodiments, these results may be reported as means, medians, and deviations. Further referring to step 725, in some embodiments, the scoring metric may include omics-based analyses. For example, referring to FIG. 1A, frozen cell lysates and cell culture media may be analyzed for bulk RNA sequencing, whole-genome bisulfite sequencing (WGBS), mass spectrometry-based proteomics, and metabolomics.

[0261] In any of the devices described herein (see, e.g., Figures 1A and 1B), cell culture medium can be subjected to metabolomic analysis to determine changes in the molecular content of the medium after co-culture compared to the pre-culture medium control. This can be used to profile dynamic changes in paracrine signaling between granulosa cells and oocytes. The collected data can then be compiled for subsequent analysis to identify changes in epigenetic state, metabolite presence, and gene expression between various co-culture conditions and the control.

[0262] iii. In vitro oocyte rescue after stimulation Referring now to FIG. 7B, an exemplary flow diagram illustrating a method for oocyte rescue, e.g., after in vitro stimulation, is shown, and further reference is made to FIGS. 1-6. Method 700 may include using computing device 104 (e.g., of FIG. 1B) or any computing device described throughout this disclosure (e.g., see FIGS. 1A and 1B). In some embodiments, method 700 may include utilizing a third party as defined and described in FIG. 1B. At step 705, the method includes, e.g., with reference to FIGS. 1-6, obtaining a biological sample from a user, the biological sample including at least one oocyte. In some embodiments, the oocyte may be an immature oocyte. In some embodiments, the immature oocyte may be a multiple oocyte. The immature oocyte may be an immature cumulus-oocyte complex (COC) collected from a mother. In some embodiments, the immature oocyte may include an oocyte to which certain granulosa cells are added to mature the oocyte in cell culture and create a COC. In some embodiments, the biological sample may include bodily fluids such as blood, saliva, urine, semen (seminal plasma), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, peritoneal fluid, amniotic fluid, nasal discharge, ocular fluid, gastric fluid, breast milk, cell culture supernatant, etc. In some embodiments, with reference to FIG. 1B , a biological sample containing oocytes can be obtained from a user after stimulation by a medical professional, such as a physician, inserting a collection device into an ovarian follicle containing an egg and collecting the egg and surrounding bodily fluid. The collection device may include a needle, syringe, vial, lancet, evacuated collection tube (ECT), tourniquet, evacuated collection tube system, any combination thereof, etc. For example, the collection device may include a butterfly needle set. Collection of oocytes may include collecting immature oocytes, mature oocytes, COCs, and any other type of cell involved in reproduction present in the ovary.

[0263]

[0041] Still referring to FIG. 7B, at step 710, method 700 includes determining a maturity level of at least one oocyte. See, e.g., FIG. 1B and FIG. 3B. In some embodiments, the maturity level can be an estimate of the oocyte maturation stage of oogenesis. Determining the maturity level of an oocyte can include, e.g., with reference to FIG. 1B, denuding the oocyte. Denuding the oocyte can include enzymatic methods using hyaluronidase and a sterile glass pipette, as described in FIGS. 1B and 3B, and mechanical methods. At step 715, method 700 includes assigning a culture protocol to the oocyte according to the maturity level. The culture protocol includes selecting cell culture metabolites according to the maturity level and selecting a cell culture medium according to the maturity level (e.g., see FIG. 1B). In some embodiments, the culture protocol can include culturing the oocyte with a granulosa cell co-culture including granulosa cells derived from human induced pluripotent stem cells (hiPSCs). The cultured oocytes may be denuded oocytes. hiPSCs can be generated using clustered regularly interspaced short palindromic repeats (CRISPR) technology. In some embodiments, a user may provide hiPSCs. Donation of hiPSCs can occur according to the oocyte collection process described above. The user participating in the hiPSC donation may be the same as or different from the user of the biological sample. In some embodiments, the user providing the hiPSCs may undergo a stimulation protocol as disclosed above. In some embodiments of the assigned culture protocol, hiPSCs, granulosa cells, cumulus cells, oocytes, GV oocytes, MI oocytes, and all other cell types described throughout this disclosure can be lysed, extracted for genomic material, and flash-frozen as a final step in the culture process. See, e.g., Figures 1-6.

[0264]

[0041] With further reference to FIG. 7B, at step 720, method 700 includes culturing at least one oocyte according to a culture protocol. See, e.g., further FIGS. 1-6. In embodiments relating to culturing oocytes, 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 a metabolic agent. At step 725, method 700 includes calculating a scoring metric for the cultured oocyte. See, e.g., further FIG. 1B. Calculating the scoring metric may include analyzing images of the co-culture and the control culture. The images may include images of the oocyte before culture, images of the oocyte after culture, and images of the denuded oocyte after culture. In some embodiments, the images may be images of the cryolysate and cell culture medium. In some embodiments, the scoring metric may include a total oocyte score (TOS) according to analysis of the images of the cultures. For example, each oocyte image can be subjected to a total oocyte score (TOS) determination system, which evaluates the health of the oocyte according to a six-point qualitative scale as described above. In some embodiments, the scoring metric can include an oocyte score. The oocyte score can include indicators such as shape, size, ooplasmic characteristics, perivitelline space (PVS) structure, zona pellucida (ZP), polar body (PB) morphology, etc. In some embodiments, the scoring metric can include performing outcome analysis according to the TOS. As used in this disclosure, "outcome analysis" refers to measurements of maturation rates and oocyte quality scores between control cultures and co-cultures. Parametric or non-parametric tests can be applied to determine the significance of findings during analysis. Outcome analysis may determine 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 oocyte quality, mean PVS quality, mean ZP quality, mean polar body quality, etc. In some embodiments, these results may be reported as mean, median, and variance.In some embodiments, the scoring metric may include omics-based analysis. In some embodiments, after culture, oocytes that fail to mature and exhibit characteristics of GV or MI can be collected for single-cell RNA-seq analysis along with associated granulosa cells from the culture. To this end, the oocytes and granulosa cells can be snap-frozen for library preparation. Half of the oocytes that exhibit MII oocyte development can be collected and subjected to single-cell RNA-seq analysis along with their associated granulosa cells using the snap-freezing method described throughout this disclosure. The remaining half of the MII oocytes can be used for proteomic analysis. Culture media for all conditions can be further snap-frozen and subjected to 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 molecular content of the media after co-culture compared to pre-culture media controls. This can be used to profile dynamic changes in paracrine signaling between granulosa cells and oocytes. The collected data can then be compiled for subsequent analysis to identify changes in epigenetic status, metabolite presence, and gene expression between the various co-culture conditions and controls.

[0265] G. Software It should also be noted that any one or more of the aspects and embodiments described herein can be conveniently implemented using one or more machines (e.g., one or more computing devices utilized as user computing devices for electronic documents, one or more server devices, such as document servers, etc.) programmed in accordance with the teachings herein, as will be apparent to those skilled in the computer arts. Appropriate software code can be readily produced by skilled programmers based on the teachings of the present disclosure, as will be apparent to those skilled in the software arts. The above-described aspects and embodiments utilizing 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.

[0266] Such software may be a computer program product using a machine-readable storage medium. A machine-readable storage medium may be any medium capable of storing and / or encoding a sequence of instructions for execution by a machine (e.g., a computing device), causing the machine to perform the methods and / or embodiments described herein. Examples of machine-readable storage media include, but are not limited to, magnetic disks, optical disks (e.g., CDs, CD-Rs, DVDs, DVD-Rs, etc.), magneto-optical disks, read-only memory "ROM" devices, random-access memory "RAM" devices, magnetic cards, optical cards, solid-state memory devices, EPROMs, EEPROMs, and any combination thereof. As used in this disclosure, machine-readable medium is intended to include a single medium as well as a collection of physically separate media, such as, for example, a collection of compact discs or one or more hard disk drives in combination with computer memory. As used herein, machine-readable storage media does not include transitory forms of signal transmission.

[0267] Such software may also include information (e.g., data) carried in a data signal on a data carrier such as a carrier wave. For example, machine-executable information may be included in a data carrier signal embodied in a data carrier, the signal encoding a sequence of instructions, or portions thereof, for execution by a machine (e.g., a computing device), and any associated information (e.g., data structures and data) that cause the machine to perform any one of the methods and / or embodiments described herein.

[0268] Examples of computing devices include, but are not limited to, e-book reading devices, computer workstations, terminal computers, server computers, handheld devices (e.g., tablet computers, smartphones, etc.), web appliances, network routers, network switches, network bridges, any machine capable of executing a sequence of instructions that specify actions to be taken by that machine, and any combination thereof. In one example, a computing device may include and / or be included in a kiosk.

[0269] 8 is a schematic diagram illustrating one embodiment of a computing device, an exemplary form of computer system 800, that can cause a control system to execute a set of instructions to perform any one or more of the aspects and / or methods of the present disclosure. It is further contemplated that multiple computing devices can be used to implement a specially configured set of instructions to cause one or more of the devices to perform any one or more of the aspects and / or methods of the present disclosure. Computer system 800 includes a processor 804 and a memory 808, which communicate with each other and with other components via a bus 812. Bus 812 can be 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.

[0270] Processor 804 may include any suitable processor, such as, but not limited to, a processor incorporating logic circuitry for performing arithmetic and logical operations, such as an arithmetic logic unit (ALU), which may be regulated by a state machine and directed by operational input from memory and / or sensors. Processor 804 may be organized according to, by way of non-limiting example, a von Neumann architecture and / or a Harvard architecture. Processor 804 may include, and / or be incorporated into, a microcontroller, a microprocessor, a digital signal processor (DSP), a field programmable gate array (FPGA), a complex programmable logic device (CPLD), a graphical 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-chip (SoC), without limitation.

[0271] Memory 808 may include a variety of components (e.g., machine-readable media). Such components include, but are not limited to, random-access memory components, read-only components, and any combination thereof. In one example, a basic input / output system 816 (BIOS), containing the basic routines that help transfer information between elements within computer system 800, may be stored in memory 808, such as during start-up. Memory 808 may also include instructions 820 (e.g., software) (e.g., stored on one or more machine-readable media) that embody any one or more of the aspects and / or methods of the present disclosure. In other examples, memory 808 may further include any number of program modules. Such program modules include, but are not limited to, an operating system, one or more application programs, other program modules, program data, and any combination thereof.

[0272] Computer system 800 may also include a storage device(s) 824. Examples of a storage device (e.g., storage device 824) include, but are not limited to, a hard disk drive, a magnetic disk drive, an optical disk drive combined with optical media, a solid-state memory device, and any combination thereof. Storage device 824 may be connected to bus 812 by an appropriate interface (not shown). 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, storage device 824 (or one or more components thereof) may be removably interfaced with computer system 800 (e.g., via an external port connector (not shown)). In particular, storage device 824 and associated machine-readable media 828 may provide non-volatile and / or volatile storage of machine-readable instructions, data structures, program modules, and / or other data for computer system 800. In one example, the software 820 may reside completely or partially within the machine-readable medium 828. In other examples, the software 820 may reside completely or partially within the processor 804.

[0273] Computer system 800 may also include input devices 832. In one example, a user of computer system 800 can input commands and / or other information into computer system 800 via input devices 832. Examples of input devices 832 include, but are not limited to, alphanumeric input devices (e.g., keyboards), pointing devices, joysticks, gamepads, audio input devices (e.g., microphones, voice response systems, etc.), cursor control devices (e.g., mice), touchpads, optical scanners, video capture devices (e.g., still cameras, video cameras), touch screens, and any combination thereof. Input devices 832 may be interfaced to bus 812 via any of a variety of interfaces (not shown). Such interfaces include, but are not limited to, a serial interface, a parallel interface, a game port, a USB interface, a FIREWIRE® interface, a direct interface to bus 812, and any combination thereof. Input devices 832 may include a touchscreen interface, which may be part of or separate from display 836, as described further below. The input device 832 may be utilized as a user selection device for selecting one or more graphical representations in the graphical interface, as described above.

[0274] A user may also input commands and / or other information into computer system 800 via storage device 824 (e.g., a removable disk drive, a flash drive, etc.) and / or network interface device 840. A network interface device, such as network interface device 840, may be utilized to connect computer system 800 to one or more of various networks, such as network 844, and one or more remote devices 848 connected thereto. Examples of network interface devices include, but are not limited to, a network interface card (e.g., a mobile network interface card, a LAN card), a modem, and any combination thereof. Examples of networks include, but are not limited to, a wide area network (e.g., the Internet, an enterprise network), a local area network (e.g., a network associated with an office, building, campus, or other relatively small geographic space), a telephone network, a data network associated with a telephone / voice provider (e.g., a mobile communications provider data network and / or voice network), a direct connection between two computing devices, and any combination thereof. A network, such as network 844, may use wired and / or wireless communication modes. In general, any network topology may be used. Information (eg, data, software 820 , etc.) can be sent to and / or received from computer system 800 via network interface device 840 .

[0275] Computer system 800 may further include a video display adapter 852 for communicating images displayable on a display device, such as display device 836. Examples of display devices include, but are not limited to, a liquid crystal display (LCD), a cathode ray tube (CRT), a plasma display, a light emitting diode (LED) display, and any combination thereof. Display adapter 852 and display device 836 may be utilized in combination with processor 804 to provide graphical representations of aspects of the present disclosure. In addition to a display device, computer system 800 may include one or more other peripheral output devices, including, but not limited to, audio speakers, a printer, 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, a serial port, a USB connection, a FIREWIRE® connection, a parallel connection, and any combination thereof.

[0276] The foregoing has been a detailed description of exemplary embodiments of the present invention. Various modifications and additions may be made without departing from the spirit and scope of the present invention. Features of each of the various embodiments described above may be combined with features of other described embodiments as needed to provide multiple feature combinations in related new embodiments. Moreover, while several separate embodiments have been described above, what has been described herein is merely illustrative of the application of the principles of the present invention. Furthermore, while certain methods presented herein may be illustrated and / or described as being performed in a particular order, that order can be varied considerably within the ordinary skill of the art to achieve methods, apparatus, systems, and software according to the present disclosure. Accordingly, this description is intended to be illustrative only, and not to otherwise limit the scope of the present invention.

[0277] Exemplary embodiments have been disclosed above and illustrated in the accompanying drawings. Those skilled in the art will recognize that various modifications, omissions, and additions can be made to what is specifically disclosed herein without departing from the spirit and scope of the invention. [Example]

[0278] Example 1. Method of follicle stimulation for ovarian release of oocytes This example demonstrates how subjects undergoing ART treatment can be minimally stimulated with inducers, which reduces the hormonal burden on the subject.

[0279] A 35-year-old female subject with polycystic ovary syndrome (PCOS) undergoing ART is examined by a clinician on day 2 of her menstrual cycle. Ultrasound analysis by the clinician determines that the subject's ovaries are producing 20 or fewer oocytes (e.g., 1-5 oocytes, 4-10 oocytes, 8-16 oocytes, or 15-20 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, or 20 oocytes). The subject is therefore determined to have reduced ovarian reserve.

[0280] To stimulate follicular maturation and oocyte release, a subject is administered an inducer (e.g., 100-200 IU of recombinant human follicle-stimulating hormone (rFSH)). Administration of the inducer begins on day 2±1 (e.g., day 1, 2, or 3) of the subject's menstrual cycle and continues daily for 1-4 days (e.g., day 1, 2, 3, or 4). The subject's follicle size is monitored by ultrasound until the average follicle size reaches approximately 8-10 mm (e.g., 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, or greater), after which oocytes (or a group of cells containing oocytes, e.g., cumulus-oocyte complexes (COCs)) are collected from the subject by aspiration. For example, transvaginal ultrasound can be used to collect oocytes using a needle guide attached to a probe to aspirate and expel the follicular contents. Oocyte-containing follicular contents (e.g., follicular aspirates) are washed with HEPES medium (G-MOPS Plus, Vitrolife®), then filtered through a 70-micron cell strainer (Falcon®, Corning®) and examined under a dissecting microscope. Oocytes (or oocyte-containing cell clusters, e.g., COCs) are transferred to culture dishes and medium to initiate co-culture with granulosa cells.

[0281] Example 2. Methods for ovarian release of oocytes and follicle stimulation for in vitro maturation of oocytes This example demonstrates minimal follicular stimulation in subjects with low ovarian reserve, followed by oocyte collection and in vitro maturation.

[0282] i. Follicular stimulation for ovarian release of oocytes A 30-year-old female subject undergoes a blood test that detects an anti-Mullerian hormone (AMH) level of 6 ng / mL or less (e.g., 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, or 6 ng / mL), and the subject is therefore determined to have low ovarian reserve. Further blood testing reveals that the subject's estradiol level is 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), reaffirming the determination of low ovarian reserve.

[0283] To stimulate follicular maturation and oocyte release, the subject is administered an induction agent (e.g., 50 mg of clomiphene citrate). Since the subject is taking hormonal contraceptives, administration of the induction agent begins on or about day 5±1 (e.g., day 4, day 5, or day 6) after the last dose of contraceptives and continues daily for 1-4 days (e.g., day 1, day 2, day 3, or day 4). The subject's follicle size is monitored by ultrasound until the average follicle size reaches approximately 8-10 mm (e.g., 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, or more), after which oocytes (or clusters of cells containing oocytes, e.g., COCs) are harvested from the subject by aspiration. For example, transvaginal ultrasound can be used to harvest oocytes using a needle guide attached to a probe to aspirate and expel the follicular contents. Oocyte-containing follicular contents (e.g., follicular aspirates) are washed with HEPES medium (G-MOPS Plus, Vitrolife®) and then filtered through a 70-micron cell strainer (Falcon®, Corning) and examined under a dissecting microscope. Oocytes (or oocyte-containing cell clusters, e.g., COCs) are transferred to a culture dish containing cell culture medium (e.g., IVM medium, IVF medium, or LAG medium) for approximately 1–3 h (e.g., 1 h, 2 h, or 3 h), after which granulosa cells are introduced for co-culture.

[0284] ii. In vitro maturation of oocytes If present, cultured COCs can be separated from their cumulus cells (and any other non-oocyte cells) in a process referred to herein as oocyte denudation. Oocyte denudation is performed on COCs in IVM wells by mechanically dissociating the cells with pipetting and removing cumulus and / or granulosa cells. Further oocyte denudation may be performed by enzymatic dissociation (e.g., hyaluronidase treatment). COCs can be stripped with stripper tips and washed with IVM medium or MOPS plus medium to wash oocytes for imaging and to inactivate hyaluronidase, if necessary. Stripper tips include 200 micron and / or 400 micron tips for fine washing.

[0285] Next, germinal vesicle stage (GV) and metaphase I (MI) oocytes are transfected with approximately 50,000 to 100,000 granulosa cells (e.g., 50,000 to 60,000 cells, 60,000 to 70,000 cells, 70,000 to 80,000 cells, 80,000 to 90,000 cells, or 90,000 to 100,000 cells, e.g., 50,000 cells, 55,000 cells, 60,000 to 70,000 cells, 70,000 to 80,000 cells, 80,000 to 90,000 cells, or 90,000 to 100,000 cells). The oocytes are co-cultured with 0, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 cells (e.g., specific granulosa cells, hiPSC-derived granulosa cells, or steroidogenic granulosa cells described herein). Metaphase II (MII) oocytes (e.g., oocytes with polar bodies in the perivitelline space) can be appropriately frozen for storage. Co-culture of oocytes and granulosa cells is performed for approximately 12 to 120 hours (e.g., 12 to 24 hours, 12 to 36 hours, 24 to 48 hours, 36 to 60 hours, 54 to 72 hours, 68 to 96 hours, 96 to 120 hours, e.g., 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).

[0286] Following co-cultivation, any one or more of the oocytes may be used in an assisted reproductive technology (ART) procedure, for example, the oocytes may be used for intracytoplasmic sperm injection (ICSI).

[0287] Example 3. Administration of follicle-inducing agents This example demonstrates the administration of an inducer to a subject.

[0288] A 30-year-old female subject undergoes a blood test that detects an estradiol level of 20-50 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). The subject receives multiple injections of an induction agent over a period of 1-4 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, so as to receive up to 5 injections of one or more induction agents. For example, a subject may receive between 300 IU and 700 IU 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, e.g., , 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) of rFSH for 3 days of stimulation (one or more injections per day). In other examples, the subject receives an injection of 200-700 μg or 2,500-10,000 IU (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) of hCG as an inducer. In yet other examples, the subject receives one or more administrations (e.g., 1, 2, 3, 4, or 5 administrations) of clomiphene citrate at 50-150 mg per administration (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).

[0289] Example 4. Device for performing follicle stimulation and in vitro maturation of oocytes In this example, reference is made to FIG. 8. FIG. 8 is a schematic diagram illustrating one embodiment of a computing device, an exemplary form of computer system 800, that may cause a control system to execute a set of instructions to perform any one or more of the aspects and / or methods of the present disclosure. It is further contemplated that multiple computing devices may be used, implementing a specially configured set of instructions to cause one or more of the devices to perform any one or more of the aspects and / or methods of the present disclosure. Computer system 800 includes a processor 804 and a memory 808 that communicate with each other and with other components via a bus 812. Bus 812 may be 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.

[0290] Processor 804 may include any suitable processor, such as, but not limited to, a processor incorporating logic circuitry for performing arithmetic and logical operations, such as an arithmetic logic unit (ALU), which may be regulated by a state machine and directed by operational input from memory and / or sensors. Processor 804 may be organized according to, by way of non-limiting example, a von Neumann architecture and / or a Harvard architecture. Processor 804 may include, and / or be incorporated into, a microcontroller, a microprocessor, a digital signal processor (DSP), a field programmable gate array (FPGA), a complex programmable logic device (CPLD), a graphical 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-chip (SoC), without limitation.

[0291] Memory 808 may include a variety of components (e.g., machine-readable media). Such components include, but are not limited to, random-access memory components, read-only components, and any combination thereof. In one example, a basic input / output system 816 (BIOS), containing the basic routines that help transfer information between elements within computer system 800, may be stored in memory 808, such as during start-up. Memory 808 may also include instructions 820 (e.g., software) (e.g., stored on one or more machine-readable media) that embody any one or more of the aspects and / or methods of the present disclosure. In other examples, memory 808 may further include any number of program modules. Such program modules include, but are not limited to, an operating system, one or more application programs, other program modules, program data, and any combination thereof.

[0292] Computer system 800 may also include a storage device(s) 824. Examples of a storage device (e.g., storage device 824) include, but are not limited to, a hard disk drive, a magnetic disk drive, an optical disk drive combined with optical media, a solid-state memory device, and any combination thereof. Storage device 824 may be connected to bus 812 by an appropriate interface (not shown). 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, storage device 824 (or one or more components thereof) may be removably interfaced with computer system 800 (e.g., via an external port connector (not shown)). In particular, storage device 824 and associated machine-readable media 828 may provide non-volatile and / or volatile storage of machine-readable instructions, data structures, program modules, and / or other data for computer system 800. In one example, the software 820 may reside completely or partially within the machine-readable medium 828. In other examples, the software 820 may reside completely or partially within the processor 804.

[0293] Computer system 800 may also include input devices 832. In one example, a user of computer system 800 can input commands and / or other information into computer system 800 via input devices 832. Examples of input devices 832 include, but are not limited to, alphanumeric input devices (e.g., keyboards), pointing devices, joysticks, gamepads, audio input devices (e.g., microphones, voice response systems, etc.), cursor control devices (e.g., mice), touchpads, optical scanners, video capture devices (e.g., still cameras, video cameras), touch screens, and any combination thereof. Input devices 832 may be interfaced to bus 812 via any of a variety of interfaces (not shown). Such interfaces include, but are not limited to, a serial interface, a parallel interface, a game port, a USB interface, a FIREWIRE® interface, a direct interface to bus 812, and any combination thereof. Input devices 832 may include a touchscreen interface, which may be part of or separate from display 836, as described further below. The input device 832 may be utilized as a user selection device for selecting one or more graphical representations in the graphical interface, as described above.

[0294] A user may also input commands and / or other information into computer system 800 via storage device 824 (e.g., a removable disk drive, a flash drive, etc.) and / or network interface device 840. A network interface device, such as network interface device 840, may be utilized to connect computer system 800 to one or more of various networks, such as network 844, and one or more remote devices 848 connected thereto. Examples of network interface devices include, but are not limited to, a network interface card (e.g., a mobile network interface card, a LAN card), a modem, and any combination thereof. Examples of networks include, but are not limited to, a wide area network (e.g., the Internet, an enterprise network), a local area network (e.g., a network associated with an office, building, campus, or other relatively small geographic space), a telephone network, a data network associated with a telephone / voice provider (e.g., a mobile communications provider data network and / or voice network), a direct connection between two computing devices, and any combination thereof. A network, such as network 844, may use wired and / or wireless communication modes. In general, any network topology may be used. Information (eg, data, software 820 , etc.) can be sent to and / or received from computer system 800 via network interface device 840 .

[0295] Computer system 800 may further include a video display adapter 852 for communicating images displayable on a display device, such as display device 836. Examples of display devices include, but are not limited to, a liquid crystal display (LCD), a cathode ray tube (CRT), a plasma display, a light emitting diode (LED) display, and any combination thereof. Display adapter 852 and display device 836 may be utilized in combination with processor 804 to provide graphical representations of aspects of the present disclosure. In addition to a display device, computer system 800 may include one or more other peripheral output devices, including, but not limited to, audio speakers, a printer, 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, a serial port, a USB connection, a FIREWIRE® connection, a parallel connection, and any combination thereof.

[0296] Example 5: Materials and Methods for Examples 6-8 We have developed human ovarian support cells (OSCs) generated from human induced pluripotent stem cells (hiPSCs) that have the ability to recapitulate dynamic ovarian function in vitro. Here, we investigate the potential of such OSCs, harvested from simplified gonadotropin-stimulated cycles, as a coculture system for IVM to enhance human oocyte maturation. We demonstrate that OSC-IVM significantly improves maturation rates compared with available IVM systems. Most importantly, we demonstrate that OSC-supported IVM oocytes are capable of robust euploid blastocyst formation, a key marker of their clinical utility. Taken together, these findings demonstrate a novel approach to IVM that is broadly applicable to modern IVF practices.

[0297] Specifically, to determine whether in vitro maturation (IVM) of human oocytes could be improved by co-culture with ovarian feeder cells (OSCs) derived from human induced pluripotent stem cells (hiPSCs), oocyte donors were recruited and subjected to brief gonadotropin stimulation with and without hCG induction, and cumulus-oocyte complexes (COCs) were assigned to OSC-IVM conditions or medium-only IVM controls.

[0298] Oocyte donors aged 19–37 years were recruited for donation with informed consent, using an anti-Müllerian hormone (AMH) level of greater than 1 ng / mL as an inclusion criterion. OSC-IVM culture conditions consisted of 100,000 OSCs in suspension culture with human chorionic gonadotropin (hCG), recombinant follicle-stimulating hormone (rFSH), androstenedione, and doxycycline. IVM controls contained the same supplements without OSCs or only FSH and hCG.

[0299] Primary endpoints consisted of metaphase II (MII) formation rate and morphological quality assessment. A limited cohort of oocytes was further used for fertilization and blastocyst formation using PGT-A analysis. OSC-IVM resulted in a statistically significant improvement in MII formation rate compared with the medium-only control. OSC-IVM resulted in a statistically significant improvement in MII formation rate compared with the commercial IVM control. There was no significant difference in oocyte morphological quality between OSC-IVM and the control. OSC-IVM improved maturation, fertilization, cleavage, blastocyst formation, high-quality blastocyst formation, and euploid blastocyst formation compared with the commercial IVM control.

[0300] In conclusion, the novel OSC-IVM platform is an effective tool for the maturation of human oocytes obtained from simplified gonadotropin-stimulated cycles, resulting in improved blastocyst formation. OSC-IVM shows broad utility for various stimulation regimens, including hCG-induced simplified IVF and uninduced conventional IVM cycles, making it a valuable tool for modern infertility treatment.

[0301] i. Collection of cumulus-oocyte complexes (COCs) Subject age, IRB, and informed consent Subjects were enrolled in the study through the Ruber Clinic (Madrid, Spain), Spring Fertility Clinic (New York, USA), and Pranor Clinic (Lima, Peru) under informed consent (CNRHA 47 / 428973.9 / 22, IRB number 20225832, Western IRB, and protocol number GC-MSP-01, respectively). Subjects' ages ranged from 19 to 37 years. Oocytes collected from the Ruber and Pranor clinics were used exclusively for maturation analysis endpoints, while oocytes collected from Spring Fertility were used for embryogenesis endpoints.

[0302] Stimulus properties In preparation for immature oocyte aspiration in Experiment 1, 25 subjects underwent 3-4 days of stimulation with 300-600 IU rFSH and hCG induction. AMH levels were >1 ng / mL (see below). In preparation for immature oocyte aspiration in Experiment 2, 21 subjects received 200 IU rFSH and hCG induction for 3 consecutive days. To increase the number of donors producing more oocytes, an AMH level >1.5 ng / mL was used as the inclusion criterion (see below). In Experiment 2, six subjects received 3-5 doses of clomiphene citrate (100 mg) with hCG induction, along with 1-2 additional doses of 150 IU rFSH, with the goal of subsequent embryogenesis. An AMH level >2.0 ng was used as the inclusion criterion (see below). Gonadotropin injections were initiated on day 2 of the natural cycle or day 5 after discontinuation of oral contraceptives. A complete listing of the donor stimulation regimen for each donor in the study is shown in Table 1 below.

[0303] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0304] ii. Aspiration of small follicles to collect immature cumulus-oocyte complexes Thirty-six hours after induction injection (10,000 IU hCG), aspiration was performed using transvaginal ultrasound with a needle guide attached to a probe to collect oocytes for co-culture experiments. Aspiration was performed using a double-lumen 19-gauge needle (a double-lumen needle was chosen because of the additional rigidity provided by the second channel inside the needle) with ASP medium (Vitrolife®) without flushing the follicles. Follicular contents were collected using a vacuum pump suction (100 mmHg) via the aspiration needle and tubing connection to a 15 mL round-bottom polystyrene centrifuge tube. In conditions where the end result was embryogenesis, aspiration was performed 36 hours after induction injection (10,000 IU hCG) or 48 hours after the last rFSH injection for non-induced cycles. Follicular contents were collected using a single-lumen 19-gauge or single-lumen 20-gauge needle without follicular flushing, using vacuum pump suction (approximately 200 mmHg) through the aspiration needle and tubing connection to a 15 mL round-bottom polystyrene centrifuge tube. In all cases, as the follicle collapsed, the aspiration needle was rapidly rotated around its long axis to create a curettage effect and assist in the release of COCs into the aspirate. Although follicles were not flushed, the aspiration needle was removed from the subject and frequently flushed throughout the oocyte collection procedure to prevent clotting and needle blockage.

[0305] The follicular aspirates were examined in the laboratory using a dissecting microscope. Because the aspirates tended to contain more blood than typical IVF follicular aspirates, they were washed with HEPES medium (G-MOPS Plus, Vitrolife®) to minimize clotting. In many cases, the aspirates were further filtered through a 70-micron cell strainer (Falcon®, Corning) to improve the oocyte examination process. COCs were transferred using a sterile Pasteur pipette to a dish containing LAG medium (Medicult, CooperSurgical®) until use in the IVM procedure. The number of aspirated COCs represented approximately 40% of the antral follicles found in the subjects' ovaries on the starting day.

[0306] iii. Preparation of ovarian supportive cells (OSCs) OSCs were generated from human i...

Claims

1. A method for inducing in vitro maturation of one or more oocytes previously collected from a human subject for use in assisted reproductive technology (ART) procedures, comprising co-culturing the one or more oocytes with a population of ovarian supporting cells.

2. The method according to claim 1, wherein the subject has not been administered a follicle-inducing agent before the collection of one or more oocytes from the subject.

3. The method according to claim 1, wherein, prior to collecting the one or more oocytes from the subject, the subject is administered one or more follicle-inducing agents during the follicle-inducing period.

4. The aforementioned follicle induction period is (a) 8 days or less; (b) 5 days or less; (c) 3 days or less; (d) 1st to 8th; (e) Day 1 to Day 5, (f) 1 to 3 days, or (g) 3 to 5 days The method according to claim 3, having the duration of [the specified duration].

5. The method according to claim 3, wherein the one or more follicle-inducing agents administered to the subject include follicle-stimulating hormone (FSH), clomiphene citrate, and / or human chorionic gonadotropin (hCG).

6. The one or more follicle-inducing agents administered to the subject include FSH, (a) The FSH is administered to the subject at least once a day or once a day. (b) The FSH is administered to the subject in an amount of about 100 international units (IU) to about 1,000 IU per day, and / or (c) The method according to claim 5, wherein the duration of FSH administration is equal to or shorter than the duration of the follicle induction period.

7. The method according to claim 6, wherein the period of FSH administration is shorter than the duration of the follicle induction period, and the period of FSH administration is 1, 2, 3, 4, or 5 days during the follicle induction period.

8. The method according to claim 7, wherein the FSH is administered to the subject at a rate of approximately 200 IU per day for one, two, three, four, or five days during the follicle induction period.

9. The method according to claim 8, wherein the FSH is administered to the subject at a rate of approximately 200 IU per day for three days during the follicular induction period.

10. The one or more follicle-inducing agents administered to the subject include clomiphene citrate, (a) The clomiphene citrate is administered to the subject at least once a day or once a day, (b) The clomiphene citrate is administered to the subject in an amount of approximately 50 mg to approximately 100 mg per day, or in an amount of approximately 50 mg per day, and / or (c) The method according to claim 5, wherein the duration of clomiphene citrate administration is equal to or shorter than the duration of the follicle induction period.

11. The method according to claim 10, wherein the period of administration of clomiphene citrate is shorter than the duration of the follicle induction period, and the period of administration of clomiphene citrate is 1, 2, 3, 4, or 5 days during the follicle induction period.

12. The one or more follicle-inducing agents administered to the subject include hCG, (a) The hCG is administered to the subject at least once a day, (b) The hCG is administered to the subject once, twice, or three times during the follicle induction period, and / or (c) The hCG is, (i) Approximately 200 μg to 700 μg per dose (ii) Approximately 500 μg per dose, or (iii) Approximately 2,500 IU to 10,000 IU per dose The method according to claim 5, wherein the amount administered to the subject is greater than the amount described above.

13. The aforementioned subject is, (a) Subjects who have completed or are not receiving oral contraception within 28 days of the start of the follicle induction period, and / or (b) The method according to claim 3, wherein the follicles are determined to be approximately 6 mm to approximately 8 mm in size before the start of the follicle induction period or before the final administration of the follicle induction agent.

14. (a) The follicle induction period shall begin at least five days after the discontinuation of the contraceptive treatment, (b) The follicle induction period begins on the second day of the menstrual cycle of the subject, and / or (c) The method according to claim 13, wherein the contraceptive treatment comprises administering a gonadotropin-releasing hormone (GnRH) agonist to the subject.

15. The biological sample separated from the subject before collecting the one or more oocytes is, (a) Anti-Müllerian hormone (AMH) concentration of approximately 0.1 ng / mL to approximately 1 ng / mL, or approximately 1 ng / mL to approximately 6 ng / mL, (b) AMH concentration of at least 1 ng / mL, (c) AMH concentration of 6 ng / mL or less, (d) AMH concentration of approximately 0.1 ng / mL to approximately 1 ng / mL, (e) AMH concentration of approximately 2.5 ng / mL to approximately 3.0 ng / mL The method according to claim 1, which is determined to have [a certain characteristic].

16. The method according to claim 15, wherein the biological sample is a blood sample.

17. (a) The subjects are between 18 and 48 years old at the time of collection of one or more oocytes. (b) Before collecting one or more oocytes from the subject, the subject is determined to have a follicle size of approximately 6 mm to approximately 14 mm or a follicle size of 14 mm or less. (c) A total of 20 or fewer oocytes are collected from the subject, and / or (d) The method according to claim 1, wherein a plurality of oocytes are collected from the subject.

18. Before collecting one or more oocytes from the subject, (a) The subject was determined to have a follicle size of approximately 8 mm to approximately 12 mm, (b) The subject is determined to have a follicle size of approximately 8 mm to approximately 9 mm, and / or (c) The method according to claim 17, wherein the follicle size is evaluated by ultrasound image analysis.

19. Multiple oocytes were collected from the subject, (a) 10% to 100% of the oocytes collected from the subject are oocytes in the gestational vesicle (GV) stage or meiotic I (MI) stage, (b) 50% to 100% of the oocytes collected from the subject are GV stage or MI stage oocytes, (c) 70% to 100% of the oocytes collected from the subject are GV stage or MI stage oocytes, (d) 90% to 100% of the oocytes collected from the subject are GV stage or MI stage oocytes, or (e) The method according to claim 1, wherein 100% of the oocytes collected from the subject are oocytes in the GV or MI stage.

20. (a) The population of ovarian supporting cells includes ovarian granulosa cells and / or ovarian stromal cells, wherein the ovarian granulosa cells are forkheadbox protein L2 (FOXL2) positive and / or the ovarian stromal cells are nuclear receptor subfamily 2 group F member 2 (NR2F2) positive and / or (b) The method according to claim 1, wherein the ovarian supporting cells are obtained by differentiation of a population of induced pluripotent stem cells (iPSCs).

21. The aforementioned population of ovarian supporting cells is (a) Approximately 50,000 to 500,000 ovarian supporting cells, or (b) Approximately 50,000 ovarian supporting cells, approximately 55,000 ovarian supporting cells, approximately 60,000 ovarian supporting cells, approximately 65,000 ovarian supporting cells, approximately 70,000 ovarian supporting cells, approximately 75,000 ovarian supporting cells, approximately 80,000 ovarian supporting cells, approximately 85,000 ovarian supporting cells, approximately 90,000 ovarian supporting cells, approximately 95,000 ovarian supporting cells, approximately 100,000 ovarian Supporting cells, approximately 105,000 ovarian supporting cells, approximately 110,000 ovarian supporting cells, approximately 115,000 ovarian supporting cells, approximately 120,000 ovarian supporting cells, approximately 125,000 ovarian supporting cells, approximately 130,000 ovarian supporting cells, approximately 135,000 ovarian supporting cells, approximately 140,000 ovarian supporting cells, approximately 145,000 ovarian supporting cells, or approximately 150,000 ovarian supporting cells The method according to claim 1, including the method described in claim 1.

22. The method according to claim 20, wherein the ovarian supporting cells include steroid-producing granulosa cells and / or the ovarian supporting cells include estradiol-producing steroid-producing granulosa cells.

23. The ovarian supporting cells mentioned above were obtained by differentiation of a population of induced pluripotent stem cells (iPSCs), and these iPSCs are (a) One or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, (b) Two or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, (c) Three or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, (d) Four or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, or (e) All five transcription factors: FOXL2, NR5A1, GATA4, RUNX1, and RUNX2 The method according to any one of claims 1 to 22, modified to express the following:

24. (a) The ovarian supporting cells are cryopreserved and thawed before the co-culture with the one or more oocytes. (b) The one or more oocytes are co-cultured with the population of ovarian supporting cells for about 12 to about 120 hours, and / or (c) The method according to claim 1, wherein the co-culture is carried out in an adherent co-culture system or a suspension co-culture system.

25. (a) Before and / or after the co-culture, the one or more oocytes are evaluated with respect to parameters selected from the group consisting of total oocyte score, oocyte maturation rate from GV to MII stage, oocyte maturation rate from GV to MI stage, oocyte maturation rate from MI to MII stage, average oocyte shape, average oocyte size, average cytoplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality. (b) The one or more oocytes are exposed after the co-culture and / or (c) The method according to claim 1, further comprising isolating one or more meiotic (MII) oocytes from a mixture produced by co-culturing one or more oocytes collected from the subject with the population of ovarian supporting cells.

26. The method according to claim 25, wherein one or more meiotic (MII) oocytes produced by co-culturing one or more oocytes collected from the subject with the population of ovarian supporting cells are separated from the mixture, the subject has undergone autologous ART treatment, and the method further comprises contacting each of the one or more MII oocytes with a mature sperm cell.

27. (a) The one or more MII-stage oocytes are cryopreserved and thawed before contact, or the one or more MII-stage oocytes are not cryopreserved and thawed before contact. (b) The contact includes, and / or, in vitro fertilization (IVF) of one or more MII-stage oocytes or intracytoplasmic sperm injection (ICSI) into one or more MII-stage oocytes. (c) The method of claim 25, wherein the contact results in embryo formation.

28. The aforementioned contact leads to embryo formation, (a) The embryo is transferred to the uterus of the subject, (b) The embryo is transferred to the uterus of the subject approximately three or five days after contact between the one or more MII-stage oocytes and mature spermatids, or (c) The method according to claim 26 or 27, wherein the embryo is transferred to the target uterus, and the embryo transferred to the target uterus is a blastocyst-stage embryo.

29. An in vitro composition comprising a population of ovarian supporting cells, one or more GV or MI stage oocytes, and one or more diluents or additives.

30. The composition according to claim 29, wherein the population comprises about 10,000 to about 100,000 ovarian supporting cells.

31. The aforementioned population of ovarian supporting cells is (a) Approximately 50,000 to 100,000 ovarian supporting cells, or (b) Approximately 50,000 ovarian supporting cells, approximately 55,000 ovarian supporting cells, approximately 60,000 ovarian supporting cells, approximately 65,000 ovarian supporting cells, approximately 70,000 ovarian supporting cells, approximately 75,000 ovarian supporting cells, approximately 80,000 ovarian supporting cells, approximately 85,000 ovarian supporting cells, approximately 90,000 ovarian supporting cells, approximately 95,000 ovarian supporting cells, or approximately 100,000 ovarian supporting cells The composition according to claim 30, comprising:

32. The aforementioned ovarian supporting cells are (a) containing steroid-producing granulosa cells, (b) comprising estradiol-producing steroid-producing granulosa cells, and / or (c) Obtained through the differentiation of iPSC populations. The composition according to claim 29.

33. The aforementioned ovarian supporting cells were obtained by differentiation of a population of iPSCs. The iPSC mentioned above is (a) One or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, (b) Two or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, (c) Three or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, (d) Four or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, or (e) All five transcription factors: FOXL2, NR5A1, GATA4, RUNX1, and RUNX2 The composition according to any one of claims 29 to 32, which has been modified to express the following:

34. The composition according to claim 29, wherein the ovarian supporting cells are cryopreserved.

35. A cell culture medium containing a population of ovarian supporting cells and one or more GV or MI stage oocytes.

36. The cell culture medium according to claim 35, wherein the population comprises approximately 10,000 to approximately 150,000 ovarian supporting cells.

37. The aforementioned population of ovarian supporting cells is (a) Approximately 50,000 to 150,000 ovarian supporting cells, or (b) Approximately 50,000 ovarian supporting cells, approximately 55,000 ovarian supporting cells, approximately 60,000 ovarian supporting cells, approximately 65,000 ovarian supporting cells, approximately 70,000 ovarian supporting cells, approximately 75,000 ovarian supporting cells, approximately 80,000 ovarian supporting cells, approximately 85,000 ovarian supporting cells, approximately 90,000 ovarian supporting cells, approximately 95,000 ovarian supporting cells, approximately 100,000 ovarian Supporting cells, approximately 105,000 ovarian supporting cells, approximately 110,000 ovarian supporting cells, approximately 115,000 ovarian supporting cells, approximately 120,000 ovarian supporting cells, approximately 125,000 ovarian supporting cells, approximately 130,000 ovarian supporting cells, approximately 135,000 ovarian supporting cells, approximately 140,000 ovarian supporting cells, approximately 145,000 ovarian supporting cells, or approximately 150,000 ovarian supporting cells The cell culture medium according to claim 36, comprising:

38. The aforementioned ovarian supporting cells are (a) containing steroid-producing granulosa cells, (b) comprising estradiol-producing steroid-producing granulosa cells, and / or (c) Obtained through the differentiation of iPSC populations. The cell culture medium according to claim 35.

39. The aforementioned ovarian supporting cells were obtained by differentiation of a population of iPSCs. The iPSC mentioned above is (a) One or more transcription factors selected from FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, (b) Two or more transcription factors from among FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, (c) Three or more transcription factors from among FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, (d) Four or more transcription factors from among FOXL2, NR5A1, GATA4, RUNX1, and RUNX2, or (e) All five transcription factors: FOXL2, NR5A1, GATA4, RUNX1, and RUNX2 A cell culture medium according to any one of claims 35 to 37, which is modified to express a specific compound.

40. The cell culture medium according to claim 35, wherein the cell culture medium is cryopreserved.

41. A kit comprising a composition according to any one of claims 29 to 32 and 34 or a cell culture medium according to any one of claims 35 to 38 and 40, and an accompanying document, wherein the accompanying document instructs the user of the kit to co-culture the population of ovarian supporting cells with one or more oocytes according to the method described in any one of claims 1 to 22 and 24 to 27.

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