Composition

Abstract

The present invention relates to compositions comprising endometrial stem cells, in particular endometrial mesenchymal stem cells (EnMSCs), for use in methods for the treatment of poor ovarian response, and to methods for banking endometrial stem cells, in particular endometrial mesenchymal stem cells.

Description

[Technical Field]

[0001] The present invention relates to compositions for use in methods for the treatment of poor ovarian response (POR), and methods for making such compositions comprising endometrial stem cells. [Background technology]

[0002] For example, a significant portion of female subjects, including infertile female subjects of all ages, experience infertility as a result of poor ovarian response, which is a disorder characterized by a reduced follicular response resulting in a reduced number of retrieved oocytes.

[0003] According to the Bologna criteria described by Ferraretti et al. [1], to define a poor response in IVF, at least two of the following three characteristics must be present: (i) advanced maternal age or any risk factor for POR, (ii) a previous poor ovarian response (≤3 oocytes with a conventional stimulation protocol), and (iii) an abnormal ovarian reserve test (i.e., an antral follicle count (AFC) of <3–6 follicles or an AMH of <0.5–1.1 ng / ml).

[0004] Two episodes of POR after maximal stimulation are sufficient to define a patient as a poor responder in the absence of an advanced maternal age or an abnormal ovarian reserve test (ORT). By definition, the term POR refers to an ovarian response, and therefore, one stimulation cycle is considered essential for the diagnosis of POR. However, because both advanced age and an abnormal ORT indicate reduced ovarian reserve and can act as surrogates for the outcome of an ovarian stimulation cycle, older patients with an abnormal ORT may be classified as poor responders.

[0005] Many studies have been carried out to investigate the effects of different stimulation protocols (2-4), such as increasing gonadotropin dosage, using dehydroepiandrosterone (DHEA) (5, 6), testosterone (7), and growth hormone (8, 9) on the number of retrieved oocytes, pregnancy rate, and live birth rate of POR through IVF / ICSI cycles. However, due to lack of efficacy, more precise and effective methods need to be followed. Therefore, new strategies should be available to manage infertility in POR and rescue them from oocyte / embryo donation.

[0006] Subjects diagnosed with POR generally have decreased anti-Müllerian hormone (AMH) levels, increased follicle-stimulating hormone (FSH), decreased antral follicle counts (AFC), and poor oocyte quality compared to healthy female subjects of the same age. Furthermore, POR subjects exhibit reduced follicular response and a reduced ovarian capacity to produce oocytes. Oocytes produced by POR female subjects are typically of inferior quality compared to oocytes produced by women with good ovarian reserve. The occurrence of POR is associated with the process of follicle depletion and reduced oocyte quality, and a significant number of POR subjects aspire to have biological offspring. Currently, the only way to achieve pregnancy in these subjects is through assisted reproductive technology (ART), such as in vitro fertilization (IVF), which requires ovarian stimulation with high doses of gonadotropins. Various modalities have been attempted to improve outcomes in POR subjects undergoing assisted reproductive technology. These include the use of high-dose FSH treatment, luteinizing hormone (LH) replacement, gonadotropin-releasing hormone (GnRH) antagonist cycles, and supplemental treatments such as estradiol priming, growth hormone, L-arginine, and dehydroepiandrosterone (DHEA). These gonadotropin treatments and fertility treatments have negative side effects, such as abdominal pain, nausea, vomiting, weight gain, acne, breast pain or tenderness, and mood swings, which may lead to the discontinuation of treatment. Furthermore, gonadotropin treatments are widely used to promote the development of early antral follicles into the preovulatory phase, but many POR subjects do not respond to gonadotropin treatment. Female POR subjects may have a low number of oocytes during oocyte retrieval, which results in fewer embryos for implantation and a lower chance of conception compared to female subjects with normal ovarian reserve. These POR subjects may require mid-cycle cancellation of their IVF cycle due to either lack of follicular development, lack of retrieved oocytes, lack of successful fertilization, or increased pregnancy failure (e.g., high miscarriage rates, which are thought to be due to the initial poor quality of oocytes found in POR subjects).This leaves many POR subjects to turn to either donor oocyte programs or adoption programs.

[0007] Zafardoust et al.

[10] described the use of autologous menstrual blood-derived mesenchymal stromal cells to improve pregnancy rates in POR subjects. However, EnSCs derived from endometrial tissue may have higher telomerase activity, a lower percentage of necrotic cells, less contaminants, and / or higher cell proliferation compared to menstrual stem cells. The present invention provides a method for preparing these cells from endometrial tissue and their advantageous use for treating POR subjects.

[0008] Managing POR subjects presents a challenge for infertility specialists, with most subjects aspireing to have biological offspring. Therefore, there is a need for a POR treatment that has at least one of the following advantages: fewer negative side effects, tolerance of autologous oocytes, new oocyte formation, improved oocyte quality, conception without IVF, tolerance of natural conception, re-establishment of lower FSH serum levels (toward normal serum levels) compared to the POR state, re-establishment of higher AMH serum levels (toward normal serum levels) compared to the POR state, and re-establishment of higher estradiol (E2) serum levels (toward normal serum levels) compared to the POR state. Serum levels are understood to mean blood serum levels. Summary of the Invention

[0009] The present invention provides a composition comprising endometrial stem cells, preferably endometrial mesenchymal stem cells, for use in a method for treating poor ovarian response. The present invention further relates to methods for producing and banking compositions comprising endometrial stem cells, particularly endometrial mesenchymal stem cells.

[0010] It has been surprisingly found that compositions according to the present invention give rise to germ cell-like cells and / or re-establish certain hormone serum levels, such as at least one of the following: AMH, FSH and estradiol, to non-POR serum levels (towards normal serum levels).

[0011] The compositions of the present invention have at least one of the following advantages: fewer negative side effects, tolerance of autologous oocytes, new oocyte formation, increased oocyte quality, conception without IVF, tolerance of natural conception, restoration of ovarian function, oogenesis, re-establishment of lower FSH serum levels compared to the POR state (towards normal serum levels), re-establishment of higher AMH serum levels compared to the POR state (towards normal serum levels), and re-establishment of higher estradiol (E2) serum levels compared to the POR state (towards normal serum levels). DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention provides a composition comprising endometrial stem cells for use in a method for the treatment of poor ovarian response, wherein said endometrial stem cells are derived from an endometrial tissue sample.

[0013] The composition preferably comprises autologous and human endometrial mesenchymal stem cells.

[0014] definition As used herein, the term "marker" or "biomarker" (also known as "biological marker" or "molecular marker" or "genetic marker" or "protein marker," etc.) refers to a measurable indicator of some biological state or condition or disease or disease-related complication. A biomarker can be any molecule (inorganic or organic molecule, protein, or nucleotide sequence) or substance circulating in blood or present in serum, biological fluids, or tissues, or a gene or polynucleotide expressed in a specific tissue or cell. In an embodiment of the invention, the marker is a protein and / or hormone present in or expressed in the serum of a human subject or of the subject's blood, or in endometrial cells or stem cells of a human subject or derived from a subject.

[0015] As used herein, the term "estradiol" (also known as "estrogen" or "estradiol" or "E2" or "17β-estradiol", etc.) refers to a measurable indicator of several biological states.

[0016] As used herein, the term "passage" (also known as "cell passaging" etc.) refers to subculture. The passage number is the number of times a cell culture has been subcultured.

[0017] It will further be appreciated that all numbers referred to in the applicable methods generally have a deviation of about 10%.

[0018] POR A POR subject is a female subject who has a reduced response to ovarian stimulation or reduced fertility compared to a female subject of comparable age.

[0019] POR is distinct from menopause or premature ovarian failure (POF, also known as primary ovarian insufficiency (POI)), as defined by the Practice Committee of the American Society for Reproductive Medicine 2015

[11] . Female subjects diagnosed with POI / POF are under 40 years of age, have postmenopausal FSH serum levels, and have had no menstrual periods for 3-6 months (e.g., secondary amenorrhea or oligomenorrhea). In POI / POF subjects, FSH serum levels are above 40 μL / mL on at least two separate occasions, AMH serum levels are below 1 ng / mL, and AFC is less than 3.

[0020] POR is characterized by a poor response to IVF stimulation, FSH serum levels above 10 mlU / mL, AMH serum levels below 1.1 ng / mL, and an AFC below 3 to 6. POR can be further diagnosed by high FSH and / or high estradiol serum levels measured in the early follicular phase (e.g., days 2-3 after the start of the menstrual cycle) during a clomiphene challenge test, and / or reduced ovarian volume.

[0021] Basal FSH serum levels can be a good predictor of the size of the remaining follicle pool. Elevated basal serum FSH levels indicate POR, and female subjects with elevated basal FSH serum levels frequently have fewer oocytes retrieved in IVF programs. Basal serum levels of FSH and LH on days 2-3 of the menstrual cycle are preferably used to screen for ovarian reserve. Day 1 of the menstrual cycle is understood to be the first day of menstruation. The first half of the menstrual cycle is the follicular phase, followed by the second half, the luteal phase, both of which last approximately 14 days in an average 28-day menstrual cycle. The luteal phase is also referred to as the "secretory phase." FSH serum levels should be measured at various times to rule out discontinuous ovarian activity as a cause of increased gonadotropins. However, as long as the assessment of ovarian reserve is related to the management of infertility, elevated FSH serum levels alone are of limited value.

[0022] Most estradiol in healthy non-POR subjects is produced by the granulosa cells of the ovary by aromatization of androstenedione (produced in theca cells) to estrone, followed by conversion of estrone to estradiol by 17β-hydroxysteroid dehydrogenase.

[0023] Recovery of ovarian dysfunction, such as during POR treatment, can be assessed using serum E2 levels in animals and human subjects over defined time intervals, such as 1 week, 6 weeks, 1 month, 3 months, 6 months, etc., using statistical comparisons between post-treatment and pre-treatment serum E2 levels.

[0024] An increase in basal serum E2 levels after administration of a composition according to the present invention may be considered as a positive sign of treatment effectiveness, in addition to at least one of the following: a decrease in FSH serum levels, an increase in AMH serum levels, and an increase in the number of antral follicles.

[0025] The amount of estradiol (E2) in serum of the blood of animals and human subjects may be measured using the enzyme-linked immunosorbent assay (ELISA) technique.

[0026] AMH is preferably another marker for poor ovarian response. In healthy non-POR subjects, AMH is secreted by cells during the development of the ovarian follicle (ovarian follicle). AMH serum level between 2 and 6 ng / mL is considered normal. A serum AMH level below 1.1 ng / mL may further indicate POR.

[0027] EnSC The endometrium regrows from 1-2 mm thick after menstrual shedding to 14 mm thick during the secretory phase (i.e., luteal phase) of the menstrual cycle and can be completely regenerated after childbirth and in postmenopausal female subjects when exposed to estradiol replacement therapy. Even after extensive iatrogenic destructive procedures such as resection, the endometrium regrows in some female subjects (approximately 25-75% of female subjects) who continue to bleed.

[0028] The endometrium consists of two layers: the functional layer and the basal layer. The functional layer contains the upper two-thirds of the glands surrounded by a loosely vascularized stroma. The basal layer, which serves as the embryonic source for replacement of the functional layer with a new one each menstrual cycle, is composed of the glands, stroma, and the lower one-third of the large blood vessels. The functional layer is shed monthly with menstrual blood resulting from changes in hormone levels and is rapidly remodeled after menstruation.

[0029] This extensive regenerative capacity suggests that the endometrium possesses a stem cell base that supports tissue maintenance / regrowth. Endometrial stem cells (EnSCs) were initially thought to be located only in the stratum basale. New evidence indicates that some stem cells exist in the functional layer of the endometrium.

[0030] EnSCs have been identified based on properties such as cell proliferation, long-term culture ability, multilineage differentiation potential, and expression of stem cell markers. Three types of endometrial stem cells exist in the endometrium: epithelial progenitor cells, endometrial mesenchymal stem cells (EnMSCs), and endothelial stem cells. The epithelial progenitor cell population is located within the residual gland in the basal layer. The subpopulation of endometrial stem cells that express CD146 and CD140b / PDGFRb is preferably endometrial mesenchymal stem cells. They are mainly located near small blood vessels in the functional layer and basal layer.

[0031] Epithelial progenitor cells cannot be obtained from menstrual blood because they are not present in menstrual blood. Epithelial progenitor cells are characterized by their positivity for SSEA-1 and LGR5 markers. Epithelial progenitor cells release growth factors, such as FGF2 and EGF, which influence the proliferation of other cells co-isolated from endometrial tissue samples.

[0032] The endometrial tissue sample preferably comprises at least the basal layer, most preferably the basal layer and the functional layer. The endometrial tissue sample is preferably a fresh tissue sample from the secretory phase of the subject's menstrual cycle. A fresh tissue sample from the secretory phase of the subject's menstrual cycle is believed to significantly improve the composition. The endometrial tissue sample can be obtained under sterile conditions. The endometrial tissue sample and the stem cells derived therefrom preferably have fewer contaminants than menstrual blood and the stem cells derived therefrom, which are contaminated with vaginal microorganisms. The endometrial tissue sample preferably has a lower percentage of necrotic cells than menstrual blood samples. The endometrial tissue sample preferably comprises epithelial progenitor cells, endometrial mesenchymal stem cells (EnMSCs), and endothelial stem cells. Endometrial mesenchymal stem cells have higher cell proliferation than menstrual stem cells. The epithelial progenitor cells have a high proliferation rate and telomerase activity, and preferably support the EnSCs in the endometrial tissue sample and the EnMSCs derived from the endometrial tissue sample. Methods for quantifying proliferation rate and telomerase activity include, for example, real-time PCR for telomere markers such as hTERT, and KI67 staining for proliferation as described in

[12] . EnMSCs derived from endometrial tissue may also show increased hTERT levels and therefore increased telomerase activity. Methods for obtaining endometrial tissue samples are known in the art.

[0033] The EnMSCs and EnSCs used in the present invention are preferably endometrial stem cells derived from endometrial tissue rather than menstrual stem cells derived from menstrual blood.EnSCs derived from endometrial tissue may have higher telomerase activity, lower percentage of necrotic cells, less contaminants, and / or higher cell proliferation than menstrual stem cells.Menstrual stem cells are positive for SOX2 and CD117 markers.Furthermore, EnSCs may be characterized by the absence (negative) of at least SOX2 and CD117 markers, and / or the presence (positive) of at least SUSD2, W5C5, and LGR5 markers.

[0034] EnSCs can be characterized by being positive for the SSEA4 and hTERT markers in addition to the SUSD2, W5C5 and LGR5 markers.

[0035] EnSCs can be further characterized by being positive for CD73, CD105, CD90, CD29, CD146, CD166, STRO1, LGR5(EnSC), SSEA-4(EnSC), h-TERT, SUSD2, N-cadherin and Nanog markers, and negative for SOX2 and CD117 markers.

[0036] The EnMSCs or compositions may be further characterized by being positive for CD146, or CD146 and PDGFRb markers.

[0037] The EnMSC or composition may be further characterized in that said endometrial mesenchymal stem cells express Oct-4, CD146 and STRO-1.

[0038] The EnMSCs or compositions may be positive for the CD90, CD146 and CD105 markers, and negative for the CD34 and CD31 markers.

[0039] EnMSCs may be further characterized by the absence (negative) of at least SOX2 and CD117 markers, and / or by the presence (positive) of at least SUSD2, W5C5 and LGR5 markers.

[0040] EnMSCs can be further characterized by being positive for SSEA4 and hTERT markers.

[0041] EnMSCs are easily accessible, low-cost, have minimal ethical hurdles, low immunogenicity, and / or low tumorigenicity. The differentiation potential of EnMSCs is preferably higher than that of other stem cells. For example, EnMSCs can differentiate in vitro into chondrogenic, adipogenic, and osteogenic lineages better than other mesenchymal stem cells.

[0042] EnMSCs are further characterized by one or more of the following: plastic adherence, fibroblast-likeness, multilineage differentiation potential, expression of classical mesenchymal stem cell surface markers, and a stable karyotype in culture.

[0043] EnMSCs have surprisingly been found to give rise to germ cell-like cells and / or re-establish certain hormone serum levels, such as AMH, FSH and estradiol, to non-POR serum levels (towards normal serum levels).

[0044] The EnMSCs are preferably autologous. Autologous is understood to mean derived from the POR subject. Autologous stem cell transplantation is the transplantation of stem cells removed from a subject and transplanted back into the same subject, with optional banking between removal and transplantation.

[0045] method The present invention further provides a method for producing a composition comprising endometrial stem cells, particularly mesenchymal stem cells, comprising: a. immersing an endometrial tissue sample in a balanced salt solution; b. washing the endometrial tissue sample obtained from step a. with a buffered saline solution; c. mincing the endometrial tissue sample obtained from step b. to produce a minced endometrial tissue sample; d. digesting the minced endometrial tissue sample obtained from step c. to produce a digest containing epithelial cells and stem cells; e. centrifuging the digest obtained from step d.; f. separating the digest obtained from step e. into epithelial cell fraction and stem cell fraction by filtration, and optionally sorting stem cells by using specific mesenchymal stem cell markers such as CD146 and / or CD105 and / or CD90; g. culturing the endometrial mesenchymal stem cells from step f. in a culture medium; h. characterizing the endometrial mesenchymal stem cells by flow cytometry, wherein the endometrial mesenchymal stem cells are positive for CD90, CD146, and CD105 markers, and negative for CD34 and CD31 markers; i. Characterization of endometrial mesenchymal stem cells by their multipotent properties, such as differentiation into osteocytes and adipocytes The present invention relates to a method, including:

[0046] When endometrial mesenchymal cells are prepared, an optional step of sorting the stem cells by using specific mesenchymal stem cell markers such as CD146 and / or CD105 and / or CD90 is applied.

[0047] The method for producing a composition comprising endometrial stem cells, preferably endometrial mesenchymal stem cells, preferably comprises autologous endometrial stem cells.

[0048] Step a. of the method for making the composition involves immersing the endometrial tissue sample in a balanced salt solution, for example as a migration medium.

[0049] The endometrial tissue sample is preferably a fresh tissue sample from the secretory phase of the subject's menstrual cycle.

[0050] The balanced salt solution is preferably Hank's solution, which contains 0.5-4 wt% penicillin, 0.5-4 wt% streptomycin, and 0.25-1.5 wt% amphotericin, preferably 1.75-2.25 wt% penicillin, 1.75-2.25 wt% streptomycin, and 0.75-1.25 wt% amphotericin. Alternatives to Hank's solution include Dulbecco's Modified Eagle's Medium:Nutrient Mixture F12 (DMEM / F12) (24 hours after obtaining the endometrial tissue sample) or PBS (4-6 hours after obtaining the endometrial tissue sample).

[0051] Step b. of the method for making the composition involves washing the endometrial tissue sample obtained from step a. with a buffered saline solution.

[0052] The buffered saline solution is preferably PBS-buffered phosphate saline, which contains 0.5-4 wt% penicillin, 0.5-4 wt% streptomycin, and 0.25-1.5 wt% amphotericin, preferably 1.75-2.25 wt% penicillin, 1.75-2.25 wt% streptomycin, and 0.75-1.25 wt% amphotericin. Alternatives to PBS-buffered phosphate saline can be sterile saline (e.g., 0.9 wt% NaCl) or cell culture media such as DMEM / F12.

[0053] Step c. of the method for making the composition comprises mincing the endometrial tissue sample obtained from step b to produce a minced endometrial tissue sample.

[0054] The endometrial tissue sample in step c. may be minced using, for example, a scalpel, razor, or scissors to produce a minced endometrial tissue sample.

[0055] Step d. of the method for producing the composition comprises digesting the endometrial tissue sample obtained from step c. to produce a digest comprising epithelial cells and stem cells.

[0056] The digestion in step d. may be carried out using a proteolytic collagenase 1, such as collagenase A. Digestion with collagenase 1, such as collagenase A, may be for 30 to 120 minutes, preferably 35 to 90 minutes, more preferably 45 to 75 minutes, and most preferably 46 to 60 minutes.

[0057] The digestion in step d may be carried out in PBS-buffered phosphate saline, which contains 0.5-4 wt% penicillin, 0.5-4 wt% streptomycin, and 0.25-1.5 wt% amphotericin, preferably 1.75-2.25 wt% penicillin, 1.75-2.25 wt% streptomycin, and 0.75-1.25 wt% amphotericin. An alternative to PBS-buffered phosphate saline is sterile saline (e.g., 0.9 wt% NaCl).

[0058] Collagenase 1, such as collagenase A, can be obtained from cultures free of animal-derived materials. Collagenase preparations contain the activity of several proteases, including collagenase, caseinase, clostripain, and trypsin. Collagenase A contains levels of proteolytic activity similar to type 1 and type 2 collagenases.

[0059] Step e. of the method for producing a composition comprises centrifuging the digest obtained from step d. The digest in step e. may be centrifuged at room temperature (e.g., 20°C) for 1 to 15 minutes, preferably 2 to 10 minutes, and most preferably 4 to 8 minutes, at 500 to 5000 rpm, preferably 800 to 1500 rpm, and most preferably 1000 to 1300 rpm. Any suitable centrifuge, such as a Hettich Universal 320 centrifuge, can be used. The medium used in the centrifugation step may be DMEM / F12 with 10 wt% FBS.

[0060] Step f. of the method for making the composition involves separating the digest into an epithelial cell fraction and a stem cell fraction by filtration.

[0061] Filtration may include filtering the digest through 70 μm and 40 μm cell strainers or by using a FACS (Fluorescence Activated Cell Sorting) system as a digest sorter. Examples of cell strainers used in accordance with the present invention may be the BD Sterile Cell Strainer 40 micron (BD 352340) and the BD Sterile Cell Strainer 70 micron (BD 352350).

[0062] Step f may be followed by sub-step f2, which involves centrifugation of the resulting stem cell fraction, for example, at 1200-1500 rpm at room temperature for 5 minutes.

[0063] A 70 μm first cell strainer may be used to separate cellular debris and undigested tissue, and a 40 μm second cell strainer may be used to separate epithelial cells.

[0064] Step g. of the method for making the composition comprises culturing the endometrial mesenchymal stem cells from step f. in a medium.

[0065] The endometrial mesenchymal stem cells in step g can be cultured in an incubator at 35-38°C, 2-10% CO2, and 95% humidity for two weeks, preferably at 37°C, 5% CO2, and 95% humidity for two weeks, and most preferably at 37°C, 5% CO2, and 95% humidity for two weeks in medium.

[0066] In the method for producing a composition comprising endometrial mesenchymal stem cells, in step g., the medium is preferably changed every three days.

[0067] The culture medium in step g may contain DMEM / F12, 8-12 wt% FBS, 0.25-1.5 wt% penicillin, and 0.25-1.5 wt% streptomycin, preferably 0.75-1.25 wt% penicillin and 0.75-1.25 wt% streptomycin. The culture medium in step g preferably inhibits or is not suitable for the growth of blood and endothelial cells.

[0068] The doubling time for the cell culture can be 49.9 hours. An example of a growth curve is shown in Figure 1.

[0069] Endometrial mesenchymal stem cells may be passaged 1 to 8 times, preferably 2 to 5 times, and most preferably 3 times, before characterization and administration. After a certain number of passages, such as 3, endometrial mesenchymal stem cells may overgrow with side population cells, endothelial cells, or non-proliferating cells. For example, at and after 3 passages, the cell culture is homogenous.

[0070] Step h. of the method for producing the composition includes characterizing endometrial mesenchymal stem cells from the cultured stem cell fraction by flow cytometry, wherein the endometrial mesenchymal stem cells are positive for CD90, CD146 and CD105 markers and negative for CD34 and CD31 markers.

[0071] Characterization can be performed using a flow cytometer.

[0072] In step h., the cells may be in Hank's Balanced Salt Solution containing 10 wt% fetal bovine serum (FBS), 0.25-1.5 wt% penicillin, 0.25-1.5 wt% streptomycin, and 0.25-1.5 wt% amphotericin, preferably 0.75-1.25 wt% penicillin, 0.75-1.25 wt% streptomycin, and 0.75-1.25 wt% amphotericin.

[0073] Step h. may be followed by substep h2., which involves centrifugation at 1200-1500 rpm at room temperature for 5 minutes.

[0074] Step i. of the method for producing the composition involves characterizing endometrial mesenchymal stem cells by their multipotent properties, such as differentiation into osteocytes and adipocytes.

[0075] Characterization of endometrial mesenchymal stem cells by their multipotent nature, such as differentiation into osteocytes, may be confirmed by Alizarin Red S staining. Alizarin Red S is an anthraquinone dye used to stain for calcium deposits, which are indicative of mature osteocytes.

[0076] The characterization of endometrial mesenchymal stem cells by their multipotentiality, such as differentiation into adipocytes, may be confirmed by staining with Oil Red O. Oil Red O is a dye that strongly stains lipids.

[0077] 1. A method for producing a composition comprising endometrial mesenchymal stem cells, comprising: a. soaking an endometrial tissue sample in Hank's solution; b. washing the endometrial tissue sample obtained from step a. with a PBS-buffered phosphate saline solution containing penicillin, amphotericin, and streptomycin; c. mincing the endometrial tissue sample obtained from step b. to produce a minced endometrial tissue sample; d. digesting the endometrial tissue sample obtained from step b. with proteolytic collagenase to produce a digest containing epithelial cells and stem cells; e. centrifuging the digest obtained from step d.; f. Separating the digest into epithelial and stem cell fractions by filtration using 70 μm and 40 μm cell strainers; or sorting stem cells by using specific mesenchymal stem cell markers such as CD146 and / or CD105 and / or CD90; g. Culturing the endometrial mesenchymal stem cells from step f. in the culture medium in an incubator at 37°C, 5% CO2 and 95% air humidity for 2 weeks; h. characterizing the endometrial mesenchymal stem cells by flow cytometry, wherein the endometrial mesenchymal stem cells are positive for CD90, CD146, and CD105 markers, and negative for CD34 and CD31 markers; i. Characterization of endometrial mesenchymal stem cells by their multipotent properties, such as differentiation into osteocytes and adipocytes Further preferred is a method comprising:

[0078] formulation The composition comprising endometrial mesenchymal stem cells for use in the method for the treatment of poor ovarian response may have a pH between 7.2 and 7.4.

[0079] A composition comprising endometrial mesenchymal stem cells for use in a method for treating poor ovarian response may comprise a physiologically suitable solution selected from the following: PBS solution, autologous serum, sterile saline (e.g., NaCl 0.9 wt%), or cell culture medium, such as DMEM / F12 used in culture, preferably at a pH of 7.2 to 7.4; preferably, the solution is a PBS solution at a pH of 7.2 to 7.4.

[0080] An example of a physiologically relevant PBS solution is an aqueous solution containing 137 mmol / L NaCl, 2.7 mmol / L KCl, 10 mmol / L Na2HPO4, and 1.8 mmol / L KH2PO4 at a pH of 7.4.

[0081] The composition according to the present invention may contain endometrial mesenchymal stem cells at a concentration of 500,000 to 10 million cells / mL, preferably 950,000 to 8 million cells / mL, and most preferably 1.5 million to 6.5 million cells / mL.

[0082] The composition according to the invention for use as a pharmaceutical may be defined by its effect of increasing serum levels of AMH above 1.1 ng / mL, and / or increasing the number of mature follicles in each ovary, and / or increasing serum levels of estradiol (E2), and / or decreasing serum levels of FSH, and / or increasing ovarian volume, and / or increasing the number of antral follicles, up to six months after implantation.

[0083] Administration of the Composition The compositions according to the present invention can be administered fresh or after being frozen. If administration occurs after cryopreservation, re-cultivation after the thawing process may be performed. During re-cultivation, the cells may reach the logarithmic phase of growth and then be administered.

[0084] The compositions according to the invention may be administered arterially, into the ovarian artery, intraovarianly or into at least one ovary. If administered intraovarian, the cells are preferably injected through the vagina, for example using ultrasound guidance.

[0085] The composition according to the present invention is preferably administered in an amount of 500,000 to 2,000,000 cells per dose, preferably 800,000 to 1,300,000 cells per dose, in 1 to 5 doses, preferably 2 to 4 doses.

[0086] banking The present invention further relates to a method for banking endometrial stem cells, in particular endometrial mesenchymal stem cells or compositions according to the invention.

[0087] The present invention provides a method for banking endometrial mesenchymal stem cells or compositions according to the present invention, comprising: a. Obtaining endometrial mesenchymal stem cells from a subject; b. Checking for bacterial, yeast or fungal contamination, e.g., under a microscope; c. Testing the endometrial mesenchymal stem cell sample for mycoplasma, preferably using Gibco's MycoTect kit (catalog number 15672-017); d. After the endometrial mesenchymal stem cells reach the late logarithmic phase, resuspend the endometrial mesenchymal stem cells in freezing medium at 5,000,000-20,000,000 cells / mL; e. Centrifuge the resuspended endometrial mesenchymal stem cells in a 50 mL Falcon tube at 1000 g for 15 minutes; f. Aspirating the supernatant from the centrifuged endometrial mesenchymal stem cells, adding freezing medium, and grinding the cells until homogenous; g. aliquoting 1 mL of the endometrial mesenchymal stem cells obtained from step f. into vials and freezing the vials in a container in a -20°C freezer for 3 hours; h. Transfer the container to a -80°C freezer and store overnight; i. Storing the vials obtained from step f. in a rack in a liquid N2 tank the next day The present invention further relates to a method, including:

[0088] Methods of banking endometrial stem cells in general or compositions according to the invention in an equivalent manner are likewise part of the present invention.

[0089] The composition according to the present invention may further comprise optional components different from the previously mentioned components of the composition, such as additives, the sum of the previously mentioned components and optional components being 100 wt% of the total composition. Thus, the present invention relates to a composition consisting of the previously mentioned components and optional components.

[0090] It should be noted that the present invention relates to all possible combinations of the features described herein, and in particular to combinations of features that are within the scope of the claims. Thus, it will be recognized that all combinations of features relating to the compositions according to the invention, all combinations of features relating to the methods according to the invention, and all combinations of features relating to the compositions according to the invention and the methods according to the invention are described herein.

[0091] It should further be noted that the terms "including," "comprising," "having," "containing," or "involving" do not exclude the presence of other elements. However, it should be understood that a description of a product / composition comprising certain components also discloses a product / composition consisting of these components. A product / composition consisting of these components may be advantageous in that it provides a simpler and more economical method for the preparation of the product / composition. Similarly, a description of a process comprising certain steps also should be understood to disclose a process consisting of these steps. A process consisting of these steps may be advantageous in that it provides a simpler and more economical method.

[0092] When values ​​are stated as lower and upper limits for a parameter, it is understood that the range created by the combination of the lower limit and upper limit value is also disclosed.

[0093] The present invention will now be elucidated by the following examples, without, however, being limited thereto. [Brief explanation of the drawings]

[0094] [Figure 1] Proliferation curves for EnMSCs after 7 days of cell culture. [Figure 2]Figure 2 shows (A) the morphological characteristics and in vitro differentiation of human EnMSCs into mesenchymal lineages. (a) Human EnMSCs (passage 3) differentiate into (b) mineralizing cells stained with Alizarin Red S. (c) Adipocytes stained with Oil Red O (scale bar: 50 μm). (B) Flow cytometry analysis of isolated human EnMSCs for mesenchymal stem cell (CD90, CD105), endometrial stem cell (CD146), hematopoietic (CD34), and endothelial (CD31) markers. The blue line indicates background fluorescence obtained with isotype control IgG1 for CD31 and IgG2a for CD105, CD90, CD146, and CD34. [Figure 3] Figure 3 shows the following: A: Differentiation of human EnMSCs into germ cell-like cells under four different retinoic acid (RA) concentrations: a - human EnMSCs after 3 days with 20 μM RA, b - differentiated cells after 7 days with 15 μM RA, c - differentiated cells after 7 days with 10 μM RA, d - differentiated cells after 7 days with 5 μM RA, e - control cells after 7 days without RA. B: Immunocytochemical analysis of germ cell-expressed markers in 2D medium 7 days after 10 μM RA induction. Cell nuclei were stained with DAPI (scale bar is 100 μm). [Example]

[0095] EnMSC Human endometrial tissue samples from non-POR subjects were collected during the luteal phase. The human endometrial tissue samples, including the functional and basal layers, were immersed in Hank's solution and washed in Dulbecco's phosphate-buffered saline (2 wt% penicillin, 2 wt% streptomycin, and 1 wt% amphotericin), minced, and then digested with collagenase 1, i.e., Hank's balanced salt solution (HBSS) containing collagenase A (1 mg / mL), at 37°C for 30–45 min with stirring. The resulting digest was then centrifuged in a Hettich Universal 320 centrifuge. The resulting pellet was washed in phosphate-buffered saline (PBS). The resulting centrifuged digest, containing epithelial and stem cells, was then passed through 70 μM and 40 μM strainers (BD Biosciences, USA, 93070) or sorted to remove the glandular epithelial component. Endometrial stem cells (EnSCs) were isolated from plastic flasks (25 cm). 2 The cells were cultured in DMEM / F12 medium containing 10 wt% fetal bovine serum (FBS), 1 wt% antibiotic penicillin, and 1 wt% streptomycin, and then incubated at 37 °C in a humidified chamber (5% CO2 and 95% air humidity) until confluence (Figure 1). Flow cytometry analysis was performed on the resulting human EnMSCs for mesenchymal stem cell (CD90, CD105), endometrial stem cell (CD146), hematopoietic (CD34), and endothelial (CD31) markers. To morphologically characterize and demonstrate in vitro differentiation of human EnMSCs (Figure 2A(a)) into mesenchymal lineages, human EnMSCs (after passage 3) were stained with Alizarin Red (Figure 2A(b)), which indicates differentiation into mineralizing cells, and with Oil Red O (Figure 2A(c)), which indicates differentiation into adipocytes.

[0096] Osteogenic differentiation: Endometrial mesenchymal stem cells were expanded and passaged in DMEM containing 10% FBS. Osteogenic differentiation was performed using 1 cm 2 2 x 10 per 4EnMSCs were induced at passage 3 by seeding at 1000 cells per well until confluence was reached and then incubated for an additional 24 hours. The medium was then replaced with differentiation medium containing DMEM / F12 supplemented with 10% FBS, 10 mM β-glycerophosphate, 0.1 μM dexamethasone, and 200 μM ascorbic acid-2-phosphate. The differentiation medium was replaced every 3–4 days for 21 days.

[0097] Alizarin Red S staining: Cells were washed with PBS and fixed in 10% (v / v) formaldehyde. After 15 minutes, 2% Alizarin Red S (pH 4.1) was added to each flask. The flasks were incubated at room temperature for 20 minutes, and then washed four times with dH2O for 5 minutes with shaking. Secretion of mineralized ECM was observed as red nodules by Alizarin Red S staining.

[0098] Adipogenic differentiation: Cells derived from whole endometrial isolates were expanded and passaged in DMEM with 10% FBS. 2 2 x 10 per 4 EnMSCs were induced at passage 3 by plating single cells at 100 μg / ml, allowing the cells to reach confluence, and then incubated for an additional 24 hours. The medium was then replaced with differentiation medium containing DMEM / F12 supplemented with 10% FBS, insulin (10 μg / ml), dexamethasone (1 mM), indomethacin (200 μg), and isobutylmethylxanthine (0.5 mM). The differentiation medium was changed every 3–4 days for 21 days.

[0099] Oil Red O staining: Oil Red O staining was used to confirm the presence of lipids in differentiated cells. Cells were washed with PBS, fixed in 2% paraformaldehyde, 0.2% glutaraldehyde in PBS for 15 minutes, and then rinsed with PBS. They were then stained with Oil Red O (reconstituted in isopropanol) for 10 minutes, rinsed in 60% isopropanol, and then PBS. Lipid droplets were visualized in red under a light microscope.

[0100] In vitro model For example, differentiation of human EnMSCs into germ cell-like cells for oogenesis / ovarian function regeneration (e.g., oocyte development and / or hormone secretion such as estradiol and AMH). Human endometrial mesenchymal stem cells were induced to differentiate by incubation in culture medium at 37°C, 95% humidity, and 5% CO2 for 7 days. The culture medium was DMEM (Invitrogen, USA) supplemented with 10 wt% FBS, 1 wt% penicillin, 1 wt% streptomycin, and retinoic acid (RA) (Invitrogen, USA) at four different concentrations: 5, 10, 15, and 20 μM (Figure B3b-e). A control group was prepared in which human EnMSCs were cultured in DMEM without RA as a differentiation inducer for the same time as the other four samples (Figure B3f). During culture, the DMEM medium was changed every other day. After 7 days, the cultured human EnMSCs were shown to differentiate into germ cell-like cells by immunofluorescence staining for DAPI, DAZL, and DDX4 (Figure 3A).

[0101] In vivo model The compositions according to the present invention were used in a method for treating POR in a POR rat model. In addition, blank (i.e., + or positive) and negative controls were run.

[0102] The rats were randomly assigned to the following three groups: POR rat group (referred to as stem cell transplantation group in Table 1) (n = 10). The POR rat model was established by intraperitoneal injection of 200 mg / kg cyclophosphamide (CTX) into rats on the first day, followed by intraperitoneal injection of 8 mg / kg / day for 15 consecutive days. Two weeks after the establishment of the POR rat model, the POR rats were injected with EnMSCs (100 μL, 1 × 10 6 A composition containing human EnSCs (at a concentration of 1000 / mL) was injected into the tail vein using a microinjector. Blank control group (referred to as Control+ in Table 1) (n=10). These were non-POR rats, i.e., normal rats that were not given any treatment for 2 weeks after the establishment of the POR rat model in the POR rat group. A negative control group (referred to as "control" in Table 1) (n=10) of the POR rat model was established by intraperitoneal injection of 200 mg / kg of cyclophosphamide (CTX) into rats on the first day, followed by intraperitoneal injection of 8 mg / kg / day for 15 consecutive days. The POR rat group was not treated for two weeks after the establishment of the POR rat model.

[0103] After 1 and 6 weeks of treatment, serum FSH, estradiol (E2), and AMH serum levels were measured. MSC treatment resulted in the re-establishment of normal serum levels of FSH, estradiol (E2), and AMH in non-POR rats.

[0104] Treatment of POR rats with a composition containing EnMSCs resulted in a decrease in serum FSH levels compared to untreated POR rats. Treatment of POR rats with MSCs resulted in an increase in serum AMH and estradiol levels compared to untreated POR rats.

[0105] Clinical Data Female human subjects (hereinafter referred to as subjects) who meet the following inclusion criteria will be recruited: diagnosed with poor ovarian response, serum FSH levels greater than 20 IU / L or AMH serum levels less than 1 ng / mL, no evidence of male infertility, normal karyotype, history of IVF failure or previous poor response, and no more than three oocytes after a conventional ovarian stimulation protocol. Furthermore, subjects will not have the following exclusion criteria: primary amenorrhea, abnormal karyotype (e.g., Turner syndrome, Fragile X syndrome), thyroid dysfunction, severe endometriosis, contraindications to pregnancy, previous personal history of ovarian cancer, previous personal history of breast cancer, history of severe drug allergy or allergic diathesis, autoimmune disease, history of severe familial genetic disease, HIV+, hepatitis B+, hepatitis C+, psychiatric illness, signal transduction disorder, and alcohol or other drug abuse. A composition comprising autologous EnMSCs according to the present invention will be administered intraovarianly to the subject.

[0106] Subjects demonstrate at least one of the following improvements: an increase in serum levels of AMH of more than 1 ng / mL (preferably up to 2.5 ng / mL) for up to 6 months after administration (implantation) of the composition according to the invention; · Increased number of mature follicles in each ovary for up to 6 months after transplantation; · Increased estradiol (E2) serum levels above 30 pg / mL for up to 6 months after transplantation; ·Decreased follicle-stimulating hormone (FSH) serum levels for up to 6 months after implantation; Increased ovarian volume for up to 6 months after transplantation; Increase in antral follicle count from 3 to 6 up to 6 months after implantation.

[0107] All of the data mentioned will be collected during clinical trials, but improvement in one of the mentioned criteria may indicate a positive ovarian response to this medication.

[0108] More specifically, clinical data from two POR patients undergoing autologous endometrial stem cell therapy are presented below.

[0109] 1-Method Patients with a final diagnosis of POR (poor ovarian response) were selected according to the exclusion and inclusion criteria (Table 1) (Table 2). [Table 1] [Table 2]

[0110] The patient received an intraovarian injection of autologous endometrial mesenchymal stem cells into the right ovary (larger ovary) according to Table 2. [Table 3]

[0111] 2-Results After 1 month, patients were followed up according to Tables 4 and 5. [Table 4] [Table 5]

[0112] Six months after cell transplantation, the final results were compared with the initial values ​​for this patient according to Table 6. [Table 6]

[0113] References 1- Ferraretti, A., La Marca, A., Fauser, B., Tarlatzis, B., Nargund, G., Gianaroli, L., & ESHRE working group on Poor Ovarian Response Definition. (2011). ESHRE consensus on the definition of ‘poor response' to ovarian stimulation for in vitro fertilization: The Bologna criteria. Human Reproduction, 26(7), 1616-1624. 2- Alsbjerg, B., Haahr, T., Elbaek, H. O., Laursen, R., Povlsen, B. B., & Humaidan, P. (2019). Dual stimulation using corifollitropin alfa in Bologna criteria poor ovarian responders-a case series. Reproductive Biomedicine Online, 38(5), 677-682. 3- Lin, L.-T., Vitale, S. G., Chen, S.-N., Wen, Z.-H., Tsai, H.-W., Chern, C.-U., & Tsui, K.-H. (2018). Luteal phase ovarian stimulation may improve oocyte retrieval and oocyte quality in poor ovarian responders undergoing in vitro fertilization: Preliminary results from a single-center prospective pilot study. Advances in Therapy, 35(6), 847-856. 4- Kuroda, K., Kitade, M., Kumakiri, J., Jinushi, M., Shinjo, A., Ozaki, R., Ikemoto, Y., Katoh, N., & Takeda, S. (2016). Minimum ovarian stimulation involving combined clomiphene citrate and estradiol treatment for in vitro fertilization of Bolognacriteria poor ovarian responders. Journal of Obstetrics and Gynaecology Research, 42(2), 178-183. 5- Chern, C. U., Tsui, K. H., Vitale, S. G., Chen, S. N., Wang, P. H., Cianci, A., Tsai, H. W., Wen, Z. H., & Lin, L. T. (2018). Dehydroepiandrosterone (DHEA) supplementation improves in vitro fertilization outcomes of poor ovarian responders, especially in women with low serum concentration of DHEA-S: A retrospective cohort study. Reproductive Biology and Endocrinology, 16(1), 90. 6- Zhang,M., Niu,W.,Wang, Y., Xu, J., Bao, X.,Wang, L., Du, L., & Sun, Y. (2016). Dehydroepiandrosterone treatment in women with poor ovarian response undergoing IVF or ICSI: A systematic review and meta-analysis. Journal of Assisted Reproduction and Genetics, 33(8), 981-991. 7- Noventa, M., Vitagliano, A., Andrisani, A., Blaganje, M., Viganò, P., Papaelo, E., Scioscia, M., Cavallin, F., Ambrosini, G., & Cozzolino, M. (2019). Testosterone therapy for women with poor ovarian response undergoing IVF: A meta-analysis of randomized controlled trials. Journal of Assisted Reproduction and Genetics, 36(4), 673-683. 8- Li, X. L., Wang, L., Lv, F., Huang, X. M., Wang, L. P., Pan, Y., & Zhang, X. M. (2017). The influence of different growth hormone addition protocols to poor ovarian responders on clinical outcomes in controlled ovary stimulation cycles: A systematic review and meta-analysis. Medicine (Baltimore), 96(12), e6443. 9- Cai, M. H., Liang, X. Y.,Wu, Y. Q., Huang, R., & Yang, X. (2019).Six-week pretreatment with growth hormone improves clinical outcomes of poor ovarian responders undergoing in vitro fertilization treatment: A self-controlled clinical study. Journal of Obstetrics and Gynaecology Research, 45(2), 376-381. 10. Zafardoust, S., Kazemnejad, S.,Darzi,S., Fahti-Kazerooni, M., Rastegai, H, Mohammadzadeh, A. Improvement of Pregnancy Rate and Live Birth Rate in Poor Ovarian Responders by Intraovarian Administration of Autologous Mentrual Blood Derived-Mesenchymal Stromal Cells: Phse I / II Clinical Trial; Stem Cell Reviews and Reports (2020) 16:755 - 763, https: / / doi.org / 10.1007 / s12015-020-09969-6 [11 Testing and interpreting measures of ovarian reserve: a committee opinion. Practice Committee of the American Society for Reproductive Medicine. Fertil Steril. 2020 ;114:1151-7

[12] Valentijn AJ1, Palial K, Al-Lamee H, Tempest N, Drury J, Von Zglinicki T, Saretzki G, Murray P, Gargett CE, Hapangama DK. SSEA-1 isolates human endometrial basal glandular epithelial cells: phenotypic and functional characterization and implications in the pathogenesis of endometriosis.Hum Reprod. 2013; 28(10):2695-708. doi: 10.1093 / humrep / det285.

Claims

1. A composition comprising endometrial mesenchymal stem cells (EnMSCs) derived from an endometrial tissue sample for use in a method for treating poor ovarian response, wherein the endometrial mesenchymal stem cells are positive for the markers CD90, CD146, and CD105, and negative for the markers CD34 and CD31, and express Oct-4 and STRO-1.

2. The composition of claim 1 , wherein the endometrial mesenchymal stem cells are human autologous endometrial mesenchymal stem cells.

3. 3. The composition of claim 1 or 2, wherein the composition is administered intraovarianly or to at least one ovary.

4. 4. The composition of claim 1, wherein the composition further comprises a physiologically suitable solution selected from the following: PBS solution, autologous serum, sterile saline, or cell culture medium, at a pH of 7.2 to 7.

4.

5. The composition according to any one of claims 1 to 4, wherein the endometrial mesenchymal stem cells are at a concentration of 500,000 to 10 million cells / mL, or 950,000 to 8 million cells / mL, or 1.5 million to 6.5 million cells / mL.

6. 6. The composition of any one of claims 1 to 5, wherein the composition is administered in an amount of 0.5 million to 2 million cells per dose or 0.8 million to 1.3 million cells per dose, in 1 to 5 doses or 2 to 4 doses.

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