In Mexico, 17% of women of reproductive age have infertility issues, which results in 1.4 million of couples in need of assisted reproductive techniques 12. From 9 to 25% of these women may show a poor ovarian response, which is defined as a low oocyte collection after an ovarian stimulation 34. This condition might be associated to Premature Ovarian Insufficiency (POI) characterized by an increment in Follicle Stimulant Hormone (FSH) and Luteinizing Hormone (LH) levels, with a reduction in Anti-Müllerian Hormone (AMH) and estrogen levels, including menstrual modifications before the age of 40 (oligomenorrhea or amenorrhea) 567. This condition has a prevalence of 1 out of 100 women before the age of 40; and in 1 out of 1000 under 30, and the risk variation depends on race, which ranges from 0.1% in Japanese to 1% in Caucasians, and 1.4% in Africans and Hispanics 567. Several POI etiopathogeneses have been described; i.e., autoimmune diseases 8, oxidative stress 9, genetical predisposition 10, radiotherapy and chemotherapy in the treatment of cancer 111213. All of these may cause problems in the collection and maintenance of primordial follicles in addition to follicular atresia and apoptosis of granulosa cells 14151617. Only 5 to 15% of these patients are able to get a spontaneous pregnancy 181920.

In addition to the above, currently, women have an important roll in the labor sector, leading them many times to delay motherhood up to an age where their fertility has diminished so much that prevents them to get pregnant. This is the consequence of a reduction in the number of follicles, which are lost every period. As is well known, women are born with a mean of 1 to 2 million of primordial follicles, which decrease to 500,000 in puberty; about 1000 are lost per month after menarche, and after age of 35, this rate get higher 2122232425. This constitutes the beginning of a reduction in ovarian function characterized by an increase in gonadotrophin levels (LH and FSH), and a decrease in AMH and estrogen levels until menstruation finally stops, phenomenon known as menopause, as a consequence of the low number of follicles, which results in an inability to maintain the menstrual cycle 25242627.

In POI (premature aging) as well as in natural Ovarian Aging (OA), there are many instances where the only option that patients have to get pregnant is to accept an ovule donation 2829. This situation is not welcome by some couples and the lack of treatment for restoring ovarian function have lead some specialists to investigate new treatment alternatives; i.e., mesenchymal stem cells (MSCs).

MSCs are found in embryonic and adult tissue. In both of the cases these cells have the ability of auto-renovation, they show a high proliferation and differentiation rate and the ability to differentiate to different cell lines (multipotent) 30313233. MSCs may be obtained from bone marrow tissue (BM-MSCs) 34, peripheral blood (PB-MSCs) 35, umbilical cord (UC.MSCs) 36, adipose tissue (AD-MSCs) 37, menstrual blood (Men-MSCs) 38, etc. They also may be autologous (self cells) or allogenic (donator cells) cells 394041. Mesenchymal stem cells have been observed to be capable to produce and release growth factors, cytokines, chemokines, enzymes, etc., which induce a paracrine regulation of anti-inflammatory processes, the generation of new blood vessels (angiogenesis), inhibition of apoptosis, and tissular fibrosis 33424344454647. In addition, there are observations in vitro that MSCs grown under suitable conditions are capable to differentiate into cells from the three embryonic germ layers (endoderm, mesoderm, and ectoderm) 4748. The term “mesenchymal“ was coined by Caplan in 1991, because of their ability to differentiate into multiple cell types from connective tissue, in addition to their mesodermal origin 49.

All of these features make MSCs very attractive to be used in medicine as shown by studies carried out in laboratory animals and humans, where some recovery in diseases and pathologies as Alzheimer 50, lateral amyotrophic sclerosis 51, Huntington disease 52, Parkinson disease 53, brain and myocardial infarctions 5455, immune conditions 56, arthrosis 57, restoring of ovarian function 258, etc., has been observed.

Considering all of the above, the objective of this study is to describe the effects that autologous AD-MSCs have on the ovarian reserve and the obtention of blastocytes, in couples that have not conceived successfully after several attempts by diverse assisted reproductive techniques; couples to which an AD-MSCs therapy was offered as an aid to improve their fertility.

This is a prospective and observational study of 17 women that attended to PRONATAL Clinic (Bité Médica Hospital) between 2019 and 2020. Patients were included in protocols of assisted reproductive techniques (ART) and they were proposed to be included in the protocol of ovarian therapy using autologous AD-MSCs as an adjuvant.

Patients were explained about the nature of the proceeding to be carried out and they accepted AD-MSC therapy as an aid after signing a written informed consent. Additionally, the obtention, handling, and transportation of stem cells collected from adipose tissue was made according to the provisions of the Mexican Official Standard NOM-012-SSA3-2012 (that establishes the Criteria for the Realization of Health Research Projects in Human Beings), NOM-260-SSA1-2015 (Mexican Official Standard Project (10/5/17) about disposition of Stem Cells and Progenitor Cells for Therapeutic and Research Purposes); the provisions of Cofepris (Federal Commission for Protection against Sanitary Hazards), UN recommendations, and international regulations.

All cases were evaluated by means of medical history, laboratory studies (general condition, liver, kidneys, thyroid gland, coagulation profiles, ovarian function (AMH) gynecologically, and by ultrasound (Philips, Affiniti 50, with at least 7 MHz)). Inclusion criteria (patients included in the AD-MSCs protocol): Candidates for AD-MSCs procurement, women with normal karyotype, women of reproductive age, infertility caused by POI or OA (menopause), FSH ≥ 20 IU/mL, women with amenorrhea that showed at least one follicle on day 2 or 11 of the menstrual cycle, AMH < 1.2 ng/mL, patients coming back for examination between the second and sixth month after application of AD-MSCs. Additionally, the control group included patients with AMH ≥ 1.2 ng/mL that were not included in this protocol.

Exclusion Criteria (patients excluded from the AD-MSCs protocol): women with autoimmune diseases, lupus erythematosus, multiple sclerosis, coagulation conditions, breast and ovary cancer, contradictions to get pregnant, and lactating women.

In this study, 17 women were included, which were distributed in different groups as described before. In Table 1 may be observed that the patients do not show statistical differences in age, weight, and height between the groups. AMH showed a numerical reduction when “Control” vs “Amenorrhea” were compared (1.9±0.2 vs 0.1±0.1), and a statistically significant reduction when “Control” vs “POI/OA” were compared (1.9±0.2 vs 0.6±0.1, p≤0.05) (Table 1).

In ultrasound results (Graph 1) it may be observed that F2 keep similar values between CONTROL, POI/OA (PRE AD-MSCs), and POI/OA (POST AD-MSCs) (7.3, 7.6, and 7.9), and AMENORRHEA (POST AD-MSCs) shows a little reduction of F2 when compared to AMENORRHEA (PRE AD-MSCs) (2 vs 1.4). With respect to F11, a statistically significant reduction may be observed in POI/OA (PRE AD-MSCs) compared with the CONTROL (9.5 vs 5.2, p≤0.05), in turn, F11 in POI/OA (POST AD-MSCs) showed a statistically significant increase when compared to POI/OA (PRE AD-MSCs) (5.2 vs 9, p≤0.05). Parallelly, AMENORRHEA (POST AD-MSCs) showed a reduction of F11 when compared with AMENORRHEA (PRE AD-MSCs) (3.3 vs 1.6). In the case of FS2, in Graph 2 may be observed a reduction in AMENORRHEA (PRE AD-MSCs) when compared with the CONTROL (2.5 vs 4.3mm). Also, in Graph 2 a statistically significant reduction of F11 in AMENORRHEA (PRE AD-MSCs) when compared to CONTROL (14.8 vs 3.7mm, p≤0.05) may be observed. By contrast, AMENORRHEA (POST AD-MSCs) keeps an increase of FS11 when compared with AMENORRHEA (PRE AD-MSCs) (3.7 vs 9.1mm).

With respect to the results of the assisted reproduction lab, it can be observed that NO, MII, and OF showed values larger in CONTROL when compared to POI/OA (Pre AD-MSCs) (6.8, 4.8, and 4 vs 3.5, 2.6, and 1.3) and AMENORRHEA (Pre AD-MSCs) (6.8, 4.8 and 4 vs 0, 0, and 0). On the other hand, POI/OA (POST AD-MSCs) showed an increase of NO, MII, when compared to POI/OA (Pre AD-MSCs) (6.2, 4.2 y 2.5 vs 3.5, 2.6, and 1.3), likewise, NO, MII and OF in AMENORRHEA (POST AD-MSCs) showed a small increase compared to AMENORRHEA (PRE AD-MSCs) (1, 1 and 1 vs 0, 0 and 0). With respect to NB, POI/OA (PRE AD-MSCs) and AMENORRHEA (PRE AD-MSCs) showed a reduction when compared to CONTROL (0, 0 vs 2.5). POI/OA (POST AD-MSCs) showed an increase in NB when compared to POI/OA (PRE AD-MSCs) (3 vs 0), and AMENORRHEA (PRE and POST AD-MSCs) did not show NB (0 vs 0) (Graph 1).

In turn, the findings in patients are resumed in Table 2. It may be observed that AMENORRHEA (Pre AD-MSCs) showed less patients with follicle on day 2 (100 vs 66.6%) than the CONTROL group. In addition, AMENORRHEA (Pre and POST AD-MSCs) showed less patients with follicles on Day 11 compared to the CONTROL group (100 vs 66.6 and 66.6%). On the other hand, there were less couples that showed oocytes and MII in POI/OA (Post AD-MSCs), AMENORRHEA (Pre AD-MSCs), and AMENORRHEA (POST AD-MSCs), when compared to the CONTROL group (87.5, 0, 33.3%vs 100%).

Oocytes (MII, MI, and germinal vesicle), PET (patients with embryo transfer), PTET (Pregnancy rate of patients with embryo transfer), PAP (pregnancy rate for all patients) y MCR (menstrual cycle recovery).

(Table 2) shows less patients with oocytes successfully fertilized in POI/OA (POST AD-MSCs), AMENORRHEA (PRE AD-MSCs), and AMENORRHEA (POST AD-MSCs) when compared to the CONTROL group, and POI/OA (PRE AD-MSCs) (42.8, 0 y 0% vs 66.6 and 66.6) (Table 2). After oocyte fertilization, 100% of the embryos turned to blastocytes in the CONTROL and POI/OA (Post AD-MSCs) groups, whereas the rest of couples ^{POI/OA (PRE AD} did not succeed to turn to blastocytes. In addition to the above, POI/OA (Post AD-MSCs) group showed a higher rate of PTET than CONTROL (75 vs 100%) group, and CONTROL group showed a higher rate of PAP than POI/OA (Post AD-MSCs). In addition, 100% of the patients in the AMENORRHEA (POST AD-MSCS) group showed temporary menstruation.

Finally, when endometrial thickness was analyzed on Day 2, it was observed that CONTROL, AMENORRHEA (PRE AD-MSCs), and AMENORRHEA (POST AD-MSCs) groups showed a reduction when compared to POI/OA (PRE AD-MSCs), and POI/OA (POST AD-MSCs) groups (2.5, 2 and 2.3 vs 3.4 and 3.7mm) (Graph 3). Later, on Day 11, endometrial thickness showed a reduction in POI/OA (PRE AD-MSCs), and POI/OA (POST AD-MSCs); AMENORRHEA (PRE AD-MSCs), and AMENORRHEA (POST AD-MSCs), when compared to the CONTROL group (8.6, 9.4, 3.5 and 6.5 vs 10.3mm). Only the AMENORRHEA (PRE AD-MSCs) group showed a statistically significant difference when compared to the CONTROL group control (10.3 vs 3.5, p≤0.05). POI/OA (POST AD-MSCs) group showed a numerical increase when compared to POI/OA (PRE AD-MSCs) group (9.4 vs 8.6mm). Similarly, the AMENORRHEA (POST AD-MSCs) group showed an increase when compared to the AMENORRHEA (PRE AD-MSCs) group (6.5 vs 3.5mm) (Graph 3).

Currently, a great deal of studies have shown how MSCs help to reduce the symptoms and alterations observed in different pathologies through animal models and clinical assays. All of this, in turn, has allowed the development of better obtention techniques of MSCs from different tissues. In the case of POI, studies of transplants have shown the therapeutic potential by restoring the ovarian structure and function 59. All of the above has offered the opportunity to carry out studies like this one, which pretends to disclose our experience with the use of AD-MSCs as a coadjuvant in the treatment of women with low ovarian response.

Turning to substance, on Day 2 of menstrual cycle it was observed that the application of AD-MSCs did not increase the number of follicles in patients with infertility caused by POI or OA (7.6 vs 7.9). In addition, patients with amenorrhea, even after the application of AD-MSCs, showed a reduction in the number of follicles (2 vs 1.4). On the other hand, the application of AD-MSCs increased the number of follicles on Day 11 in patients with infertility by POI and OA (5.2 vs 9) (Graph 1). Similarly, Herraiz, 2018 60 in a group of 17 patients with low response, observed that after applying 50x106 BM-MSCs to the ovary by catheterization from Day 2 to Day 43, the number of antral follicles (3 vs 8) was increased. Volkova, 2017 61, observed an increase of the number of follicles on Day 21 in 18-week old female mice with chronic oophoritis after an intravenous infusion of BM-MSCs compared to what was observed on Day 10 (9.4 vs 15.3). Similarly, Herraiz,2018 62, reported an increase in the number of follicles from Day 7 to Day 14 (0.24 vs 0.8 follicles/mm3, in histological cuts) after the application of bone-marrow MSCs to immunodeficient mice with ovarian damage induced by chemotherapy; and Fouad, 2015 63, found a higher number of follicles after application of AD-MSCs (4 vs 13) and placental MSCs (4 vs 18) (by intravenous route) to 48 female rats with POI induced by cyclophosphamide. The effect of AD-MSCs on the increment of the number of follicles may also be reflected in the increase of follicles size on Day 11 of the menstrual cycle (POI and OA (13.5 vs 15.7mm) and amenorrhea (3.7 vs 9.1)) (Graph 2). Similarly, ABD-ALLAH, 2013 64 observed a higher number of follicles >70μm after the application of BM-MSCs (X̅:53.9 vs 16) (by intravenous route), in histological cuts from 35 female rabbits with POI induced by cyclophosphamide.

With respect to the results obtained in the laboratory of assisted reproduction, an increase of Total Oocytes (3.5 vs 6.2) was found in patients with POI and OA, which allowed to have a greater number of oocytes in “MII” (2.6 vs 4.2) after the application of AD-MSCs. In addition, patients with amenorrhea increased the possibility to show at least one mature oocyte (0 vs 1) (Graph 1). Similarly, Luján 2020 2, in a case report of a woman (39 years old) diagnosed with poor ovarian response, the application of AD-MSCs (intraovarian) increased the number of MII oocytes obtained (3 to 14). Also, Herraiz,2018 62, reported an increment of the number of MII oocytes (15 vs 30) in immunodeficient mice with ovarian damage induced by chemotherapy after the application of BM-MSCs, and Li, 2018 65 showed a higher number of follicles (11 vs 39) in a group of female mice with POI induced by cyclophosphamide, six weeks after the application of MSCs obtained from a chorionic plate. On the other hand, Herraiz, 2018 60, found that the application of 50x106 BM-MSCs by catheterization in ovary, in a group of patients diagnosed as poor responders, did not increase the amount of MII oocytes (2 vs 2). So far, it can be observed that the benefit of AD-MSCs is higher in patients with POI and OA than in patients that do have menstrual cycle anymore. This might be improved by using a method with a longer administration of MSCs (trying several different doses) as it was observed that ovaries responded very shortly.

After the intracytoplasmic spermatozoid injection (ICSI) to the oocytes collected, an increase of the number of “fertilized oocytes” was observed after the application of AD-MSCs in patients with POI, OA (50 to 59.5%) and amenorrhea (0 vs 100% corresponding to 1 patient out of 1) (Graph 1), which agrees with Zafardous, 2020 66, who found an increase of the number of “fertilized oocytes” after applying MSCs derived from menstrual blood (men-MSCs) (76 vs 92%) in 15 patients with POI. This allowed patients with POI and OA to show a higher number of blastocysts after the application of AD-MSCs, which coincides with Zafardous, 2020 66, who report an increase of 0 to 3 blastocysts in patients with POI. Patients with amenorrhea did not show any blastocysts.

In Table 2 it is clear how patients with POI and OA get benefits from the administration of AD-MSCs as an adjuvant for low ovarian response as evidenced by a higher number of patients showing MII oocytes, blastocysts, and that got pregnant (0 vs 3). The number of patients that restore temporary their menstrual cycle increases and so their possibility to obtain at least one mature oocyte, which is concordant with Edessy M, 2015 67, who report two menstruation recoveries and only one pregnancy in a group of 10 patients with POI after the application of BM-MSCs. In turn, Gupta S, 2018 68, in a case report of a 45 year old patient with infrequent menstruation for at least three years, found that the application of BM-MSCs lead to obtain three oocytes, which allowed a successful pregnancy and the birth of a baby girl.

In addition, when endometrial thickness was measured, an increase was found on Day 11 of menstrual cycle in patients with POI, OA (8.6 to 9.4mm) and with amenorrhea (3.5 to 6.5 mm) after the administration of AD-MSCs, which might be associated to the recovery of the ovarian function and an endocrine communication existing between ovaries and uterus 67697071. Tersoglio, 2020 72, found similar effects of MSCs when administered directly in uterus in a study where an increase in endometrial thickness (5.2 a 9.9mm) was reported in 29 patients with thin endometrium to which endometrial MSCs (en-MSCs) were administered. On the other hand, Singh, 2020 73, found that the application of BM-MSCs in endometrium increases the thickness thereof in women with Asherman's syndrome (2.6 to 4.2) and endometrial atrophy (3.6 to 5.9).

Theoretically, the results obtained in this study might reflect disturbances related to FSH such as polymorphisms of FSH receptors (FSHr), low sensitivity to FSHr induced by damage caused by free radicals, environmental contaminants, life style, radiotherapy, autoimmune response, chemotherapy, and diet, in addition to LH variants that could turned it less reactive 74757677; therefore, the application of AD-MSCs might help to reduce these disturbances by generating new blood vessels 78, downregulating the immune response 79, providing a more suitable microenvironment 8081, and avoiding the apoptosis of several cell types derived from possible niches of the ovary 8283. In addition, these niches might also be responsible of generating a microenvironment in response to possible internal and external factors that may be benefic for the quiescence or differentiation (to epithelial and follicular ovarian cells) of AD-MSCs, which favors the restauration of the ovarian function 81.

Finally, notwithstanding that there is a great deal of evidence showing the benefits of MSCs as a therapy for a large number of pathologies, in the specific case of ovarian conditions, said benefits are poorly known, in addition to the scarce knowledge of the action mechanisms associated to each one of the pathologies studied so far. Therefore, it will be of great importance to carry out more studies like this that provide data to expand the knowledge about this matter.

The results of this study suggest that the application of AD-MSCs directly to the ovary improve the ovarian function, stimulate endometrial growth, and induce the collection of a larger number of follicles, which makes it possible to obtain more MII oocytes which, in turn, increase the possibility to obtain blastocysts of a higher quality and therefore, a pregnancy, in patients with POI or OA.

In patients with amenorrhea, the application of AD-MSCs restores menstruation temporary, and increases the possibility to collect at least one oocyte per menstrual cycle.

Additionally, the performance of more studies with the aim to find out the exact mechanisms by which AD-MSCs restore the ovarian function is suggested. This way, we will be able to observe whether there is a tendency in favor of using AD-MSCs as a therapy or treatment for POI and OA.