BMSCs

BMSCs are primarily present in the bone marrow microenvironment and are a type of adult stem cell. These stem cells have multipotent differentiation potential with low immunogenicity27. Under certain conditions, BMSCs can differentiate into various tissue cells, such as adipocytes and bone cartilage, and also self-renew28. BMSCs are easily isolated and proliferated and can also migrate to damaged tissue29. Due to their immunomodulatory and paracrine features, BMSCs are believed to have therapeutic potential for infertility30. Studies have shown that cytokines-induced BMSCs can migrate to the damaged tissue and then secrete definite cytokines31. Some of these anti-fibrosis and anti-apoptosis cytokines, such as insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF), help ovarian restoration30. Furthermore, anti-inflammatory and anti-oxidative cytokines such as interleukin (IL)-6 secreted by BMSCs can protect ovarian function31.

studies on ovaries indicated that BMSCs are helpful in treating animal models with premature ovarian failure (POF). In a mouse model with POF, BMSCs reactivated ovarian hormone production and folliculogenesis that had been damaged by chemotherapy32. Another study in rats suggests BMSCs can decrease perimenopause- and cisplatin-induced apoptosis in granulosa cells33. In humans, a clinical trial showed that autologous BMSCs might improve POF conditions in patients with idiopathic POF30.

Animal studies and clinical trials indicate successful treatment of endometrial dysfunction with BMSCs. Animal studies have shown that injection of BMSCs resulted in the secretion of various growth factors into the endometrium that can strongly stimulate cell proliferation and differentiation in the microvascular endothelium34, 35, 36. Moreover, in a mouse model with a thin endometrium, BMSC transplantation upregulated endometrial receptivity markers and improved infertility37. In a rat model, injection of BMSCs into the uterine cavity caused high expression of endometrial cell markers, resulting in increased endometrial thickness38. Several studies have shown that BMSCs migrate to the endometrium after systemic infusion. Studies in a mouse model showed that BMSCs migrate from donor male bone marrow into female recipients’ uterus via systemic infusion. For example, immunofluorescence and fluorescent in situ hybridization were performed on the uterus of female mice receiving BMSC transplantations from males. Then, the migration of BMSCs was confirmed by detecting the Y chromosome and the sex-determining region of Chr Y () gene in the uterus of female mice39. Moreover, a clinical trial on women with bone marrow transplants detected donor-derived cells in the uterine tissue, confirming the migration of BMSCs40.

In AS (IUAs and/or intracervical adhesions), BMSC transplantation effectively repaired endometrium damage by upregulating the expression of the estrogen (ER) and progesterone (PR) receptors41. In a rat model with endometrial cavity fibrosis, BMSC injection improved reproductive function by restoring endometrial receptivity and lining35. Clinical studies on AS suggest that the transplantation of prominin 1 (PROM1/CD133) BMSCs into patients can cause endometrial regeneration42. BMSC infusion improved reproductive function in patients by enhancing endometrial vascular density and refining the intensity and duration of menstruation. Surprisingly, some patients became pregnant after treatment without medical intervention42.

Besides these positive effects and features, there are some limitations to using BMSCs, including the need for an invasive procedure to isolate them, their low proliferation capacity (due to a low number of MSCs in the bone marrow), and their potential to differentiate into undesirable cell types with increasing donor age43, 44 (Table 1).

ADSCs

The general characteristics of MSCs, such as self-renewal, immunomodulation, and differentiation, are also seen in ADSCs, which are isolated from adipose tissue, and due to their ease of extraction through liposuction, have been intensively used in therapy38. Several animal studies have shown that ADSCs could be used to treat infertility45. One animal study showed the positive effect of ADSC injection on the viability of ovarian follicles. ADSCs increased the maintenance of grafts in the ovary and improved graft efficacy46, 47, 48. For example, intraperitoneal injection of ADSCs in mice improved ovarian function in chemotherapy-damaged ovaries47. Similarly, ADSCs improved ovarian dysfunction in rat models, increasing the rate of maturing follicles, oocyte number, and corpora lutea by altering the gene expression and secretion of specific paracrine cytokines46. Furthermore, ADSC treatment decreased apoptosis in granulosa cells. Therefore, ADSCs could be an alternative approach for POF therapy that could be useful in clinical applications and regenerative medicine47.

ADSCs can also improve fertility in animals by increasing endometrial thickness and the number of endometrial glands and microvessels. Furthermore, since ADSCs can differentiate into endometrial cells, their transplantation can repair the endometrial injury. Treating rat models with ADSCs and estrogen restored endometrial tissue, proved via detecting green fluorescent protein (GFP) in endometrial epithelial cells grafted with GFP-labeled ADSCs49, 50. A clinical trial using autologous ADSCs to repair the endometrium recorded the first successful pregnancies and childbirths. Its results showed that endometrium thickness increased in 20 out of 25 patients after sub-endometrial injection of ADSCs. Thirteen women became pregnant, and nine successful childbirths were recorded51.

Regarding male infertility, ADSCs helped restore fertility and sperm production in rats with azoospermia52. In humans, an study showed that the supernatant product of ADSCs could restore sperm motility in infertile male patients. This effect appeared due to growth factors and bioactive molecules positively affecting sperm motility53. Furthermore, intracytoplasmic injection of sperm showed that paracrine factors in the medium isolated from ADSCs could result in oocyte maturation and embryo formation54.

As mentioned above, ADSCs are a promising source for therapeutic applications because of their ease of isolation and availability in high frequency through liposuction. However, ADSCs are embedded in a complex niche and interact with other factors and cells. After their isolation via liposuction and separation from their niche, ADSC features such as proliferation capacity are reduced, possibly due to local anesthesia, which can negatively impact their survival and quantity43, 55, 56, 57 (Table 1).

MenSCs

Since the need for surgery complicates MSC isolation from bone marrow, adipose tissue, or amniotic fluid, it was necessary to find more accessible alternative sources of MSCs58. Recent studies have shown that endometrial basal layer cells have stem cell characteristics such as self-renewal and proliferation59. Endometrial stem cells are new objects in treating infertility (of unclear origin) in women. However, a biopsy or cuvette to access the endometrial stem cells may harm the endometrium60. Interestingly, MenSCs are functionally and morphologically similar to endometrial stem cells. MenSCs match the International Society for Cell and Gene Therapy criteria for MSC characteristics and express dual ESC and MSC markers61. They proliferate twice as fast and regenerate better than BMSCs and MSCs. This proliferation rate is superior for clinical use because when many stem cells can be quickly isolated from the same source, it is a noninvasive, painless procedure with no ethical issues62, 63.

MenSCs can be used as ideal MSCs for treating endometrial infertility. In a nude mouse model with endometrial damage, MenSCs survived in the endometrium after transplantation38. Furthermore, they improved the rate of embryo implantation by upregulating the levels of keratin, vimentin, and VEGF in the endometrium and increasing endometrial thickness by regulating a protein kinase B (PKB/AKT)-related signaling pathway38, 64 because this kinase plays an important role in cell survival, proliferation, and metabolism65.

A clinical trial on patients with severe AS reported promising results after the transplantation of autologous MenSCs. They showed that MenSCs increased endometrial thickness and improved the rate and quality of pregnancy in patients with AS66. Unlike BMSCs, AMSCs, and UCMSCs, MenSCs have shown no limitations in accessibility, collection, proliferation rate , or heterogeneity. However, further basic and clinical studies are needed (Table 1).

UCMSCs

Human UCMSCs (hUCMSCs), also called Wharton jelly MSCs, can be obtained from newborns’ cord tissue. HUCMSCs have stem cell characteristics such as high proliferation, differentiation potency, low immunogenicity, and prolonged survival time after transplantation67.

Since hUCMSCs are safe and effective, their use has been considered to treat infertility. HUCMSCs repaired damaged endometrium in an animal model68. They also increased the rate of implanted embryos by upregulating vascular and downregulating pro-inflammatory factors69. It has also been shown that uterine niches formed in animals after a cesarean could be effectively treated by intramuscular injection of hUCMSCs70. In clinical trials on patients with IUA, hUCMSC-loaded biodegradable collagen scaffolds have been safely and efficiently implanted into the patient’s uterine cavity. After transplantation, the survival of the hUCMSCs was maintained at the site of endometrial damage57. These results indicate a hopeful future for IUA therapy and demonstrate that tissue engineering with hUCMSCs can increase their therapeutic efficiency71.

Besides the positive characteristics of UCMSCs, they have disadvantages such as limited collection at birth (attributed to uncertainty about the baby’s health), low isolation efficiency, and high heterogeneity72, 73, 74 (Table 1).

AMSCs

The amnion is usually discarded as medical waste after delivery. The amniotic tissue, a postpartum membrane, is a rich resource of human MSCs called AMSCs (hAMSCs)75. HAMSCs have some advantages over MSCs. Besides MSC properties, they also have some ESC phenotypic properties. In addition, the methods used to isolate hAMSCs are noninvasive, safe, and without ethical issues. These superior properties make hAMSCs a good target for regenerative medicine76, 69.

In the chemotherapy-induced POI rat model, hAMSC transplantation lessened ovarian injury and improved ovarian function77. In an IUA rat model, hAMSC transplantation downregulated pro-inflammatory cytokines such as IL-1β and tumor necrosis factor-α (TNF-α) and upregulated anti-inflammatory cytokines such as IL-6 and basic fibroblast growth factor (bFGF)78. These results show that hAMSC transplantation could improve endometrial restoration, likely due to their immunomodulatory characteristics79.

In the mouse model of age-related diminished ovarian reserve, hAMSC transplantation improved ovarian function and oocyte maturation by modifying the ovarian microenvironment through the protein kinase, AMP-activated, alpha 2 catalytic subunit (PRKAA2/AMPK)/forkhead box O3 (FOXO3/FOXO3A) signaling pathway80.

Interestingly, the epithelial cells derived from amniotic tissue, called human amniotic epithelial cells (hAECs), also exhibit stem cell features and have the potential for cell-based therapy. Like hAMSCs and all MSCs, hAECs have the potential for multipotential differentiation, immune regulation, and low tumorigenicity81. HAEC transplantation improved endometrial morphology in IUA mouse models, possibly by stimulating endometrial stromal cells’ proliferation and angiogenesis and increasing endometrium thickness58. Moreover, the transplanted hAECs triggered endometrium autophagy in the IUA mouse models, reducing the fibrotic region of the endometrium and thereby improving infertility.

Finally, the most important limitations of AMSCs include restricted pluripotency, reduced numbers with aging, and limited expansion72, 82 (Table 1).

PMSCs

The placenta is a temporary organ made with the participation of fetal and maternal tissues that is excreted after delivery. A population of MSCs can be isolated from the human placenta, called PMSCs83. This tissue is easily accessible (noninvasive) and abundant. It has immunoregulating, self-renewing, and differentiating properties. PMSCs are not donor-age-dependent and express common BMSC markers. Due to their properties, PMSCs are an attractive source of MSCs for stem cell-based therapy84.

PMSCs have been shown to secrete several cytokines, including colony-stimulating factor 3 (CSF3/G‐CSF), IL‐6/‐8/‐10, and C-C motif chemokine ligand 5 (CCL5/RANTES)85. The secretion of KIT ligand (KITLG/SCF) after PMSC transplantation promoted oocyte survival, elevating the expression levels of lin-28 homolog A (), LIM homeobox protein 8 (), NOBOX oogenesis homeobox (), and nanos C2HC-type zinc finger 3 (), thereby improving ovarian function67. These findings suggest that PMSC therapy could be used to treat individuals with infertility and ovarian dysfunction, such as PCOS67.

Two clinical trials by Levy . evaluated the efficacy and safety of PMSCs in treating men with Peyronie’s disease86 and erectile dysfunction (ED)87, reporting beneficial results as a nonsurgical treatment. These two diseases do not inherently cause infertility. However, reports suggest that since these diseases may affect the strength and completeness of ejaculation, they can affect a man’s ability to conceive88.

However, most clinical trials on PMSCs are just at the beginning of the path, do not yet have published results, and require further research89.

Extracellular vesicles derived from MSCs

Stem cells are widely used in reproductive medicine and have direct and indirect (paracrine, such as cytokines) therapeutic effects90. However, due to the limitations of injecting living cells, it is likely better to use their paracrine elements, such as extracellular vesicles and microRNAs (miRNAs). Exosomes are a well-characterized subset of extracellular vesicles that are secreted by MSCs. They encompass various cellular compounds, including lipids, proteins, coding and non-coding RNAs (mRNAs, tRNAs, and miRNAs), DNA fragments, and cell surface proteins91. The compounds present in various exosomes differ based on the origin of the MSCs92. Since exosomes are important carrier organelles for intercellular cross-talk, they play an important role in many physiological functions93.

Specific surface molecules on exosomes facilitate their interactions with their target cells. They include MHC I and II molecules, galectin, integrin, intercellular adhesion molecule 1 (ICAM1), and collagen94. Exosomes can trigger cell migration, proliferation, viability, angiogenesis, oogenesis, spermatogenesis, and acrosome reaction. They can alter different signaling pathways and are involved in gene expression, immune system downregulation, and embryo implantation induction95. Exosomes have an important effect on the reproductive mechanism and can be used to treat reproductive diseases such as POI and ED95. In POI models, transplantation of hAMSC-derived exosomes decreased apoptosis in tissue grafts by upregulating SMAD family member 5 () expression to downregulate caspase-3 (CASP3), caspase-8 (CASP8), and Fas cell surface death receptor (FAS)/Fas ligand (FASLG/FasL)96. HAMSC-derived exosomes also upregulate SMAD family members 2 () and 3 (), enhancing granulosa cell (GC) proliferation, activating ovulation, and producing corpus luteum.

It has been suggested that an exosome combination from several sources (MSCs, hUCMSCs, and bovine granulosa) may be effective for treating asthenozoospermia because it could reduce the reactive oxygen rate97. In addition, combining exosomes from retinal astroglial, cardiomyocytes, and MSCs was suitable for treating endometriosis in male rats with type 2 diabetes. Combining these exosomes likely inhibited induced cell death, macrophage infiltration, and angiogenesis in the ectopic endometrium98, 99, 100.

Regarding mouse mating, tetraspanin proteins (., CD81 and CD9) vital for oocyte fertilization are produced by oocyte-derived exosomes, and oocytes produced by mice lacking these proteins cannot fuse with sperm101, 102. Placenta-derived exosomes are important in decreasing T regulatory cells and downregulating the maternal immune system during gestation103, 104, 105 by secretion of MHC-I, MHC-II, UL16 binding protein 1–5 (ULBP1–5), and FASLG105.

Overall, using exosomes is less risky and more feasible than stem cells because they protect their contents from damaging enzymes, do not induce inflammation, and do not generate teratomas105.

The role of microRNAs in driving MSC therapeutic outcomes

Most of the therapeutic effects of MSCs are due to paracrine producers, especially through the release of soluble factors such as miRNAs and exosomal miRNAs. In other words, miRNAs mediate the effects of MSCs and are therefore considered new therapeutic factors106.

Studies have shown that miRNAs inhibit target mRNA translation and thereby have a critical effect on regulating stem cell differentiation and regeneration107. The miRNAs may show similar effects in regulating stem cells’ pathological and physiological features related to ovarian function. In female rats with cyclophosphamide-induced POF, GC apoptosis was associated with the upregulation of in BMSCs108. GCs are somatic steroidogenic cells surrounding the oocytes and are essential for developing oocytes since they generate nutrients and growth factors109. It has been shown that a high miR-21 level in BMSCs reduces their expression of programmed cell death protein 4 (), inducing GC apoptosis and leading to abnormal oocyte development110. In addition, overexpression in BMSCs may increase estradiol and decrease follicle-stimulating hormone levels108.

Besides cytoplasmic miRNAs, exosomal miRNAs can modulate intercellular signal transduction and regulate molecular mechanisms in various diseases111. Two studies have shown the role of some BMSC-derived exosomal miRNAs in activating the restoration of ovarian function in an animal model of POF4. BMSC-derived exosomal miR-644-5p regulated tumor protein p53 (TP53) signaling, inhibiting GC death112. BMSC-derived exosomal miR-144-5p regulated phosphatase and tensin homolog (PTEN) in rats with chemotherapy-induced POF, restoring ovarian function. These findings show that gene expression regulation by miRNAs could be a subset of BMSC-based therapy113.

Co-transplantation of MSCs with other elements

The effectiveness of transplantation protocols combined with other factors was examined in a rat model. Treating IUA with MenSCs combined with platelet-rich plasma (PRP) improved proliferation and angiogenesis and morphologically restored the endometrium108. In addition, combining estrogen with ADSCs induced endometrial tissue regeneration in a rat model38. Co-transplantation of PRP and MSCs into an animal model of POI increased IGF-1 and transforming growth factor (TGF)-β levels and C-X-C motif chemokine ligand 12 () expression (an anti-inflammatory chemokine). Indeed, PRP reduced inflammatory responses and the extent and number of follicular atresia114. Applying a collagen scaffold with ADSCs in preclinical experiments on rats with POF led to long-term ADSC maintenance in their ovaries. This long duration improved ovarian function and fertility restoration in rats115. Co-transplanting spermatogonial stem cells with TGF-ß1-treated MSCs in mice improved fertility efficiency because TGF-ß1 treatment preferentially conducts MSCs to the testis where their secretion elements could recover the testicular niche116.