Mesenchymal stem cells to treat female infertility; future perspective and challenges: A review

Abstract Infertility negatively impacts the overall health and social life of affected individuals and couples. Female infertility is their inability to perceive pregnancy. To date, polycystic ovary syndrome, primary ovarian insufficiency, fallopian tube obstruction, endometriosis, and intrauterine synechiae have been identified as the primary causes of infertility in women. However, despite the mutual efforts of clinicians and research scientists, the development of an effective treatment modality has met little success in combating female infertility. Intriguingly, significant research has demonstrated mesenchymal stem cells as an optimal source for treating infertility disorders. Therefore, here we attempted to capsulize to date available studies to summarize the therapeutic potential of mesenchymal stem cells in combating infertility in women by focusing on the underlying mechanism through which stem cells can reduce the effects of ovarian disorders. Furthermore, we also discussed the preclinical and clinical application of stem cell therapy, their limitation, and the future perspective to minimize these limitations.

According to the epidemiological categorization, a female is considered infertile if she remains unsuccessful even after several attempts to be pregnant (4,5). An approximate estimation for the prevalence of overall infertility has indicated that 33-41% of infertility could be due to female cause, while male causes contribute 25-39%, and mixed causes are about 9-39% (6). The statistics reveal that women's infertility is among the most significant factors (7) due to its increasing epidemiology, affects millions of females worldwide (7,8). Accordingly, ovulation disorders, i.e., hypothalamic dysfunction, primary ovarian insufficiency, or polycystic ovary (PCOS), are considered the major cause of female infertility.
At the same time, endometriosis and tubal infertility are also among the preeminent cause of female infertility (7). The ovary is a complex and highly regulated organ where minor dysregulation can ultimately lead to infertility (9). While ovulation and hormonal testing are more commonly used methods for diagnosing female infertility.
Moreover, further advent in technology has introduced some assisted reproduction strategies i.e., intrauterine insemination, and in-vitro fertilization to minimize the damaging effects of ovarian dysfunction (1,(10)(11)(12). Other available treatment options include the intervention of supplements or antioxidants, such as zinc, vitamin E, and L-carnitine. Despite their frequent application, these treatment strategies have shown their limitations and side effects (1,13).
Hence, to date, there is a darn lack of an effective treatment strategy for curing female infertility (14). Therefore, researchers and clinician have been investigating more efficient and novel therapeutic measures for infertility. Among these strategies, stem cell therapy holds significant importance, being undifferentiated cells, their self-renewal for longer duration without developing any change (7).
Nonetheless, as compared to other group of stem cell, mesenchymal stem cells (MSCs) have been indicated to possess advantages over other types of stem cells for being free of ethical concerns and teratoma formation (7,15).
Herein we will discuss the published studies about the cause of infertility, to date available therapies, and application of MSCs to treat the infertility in women.

Prevalence and etiology of female infertility
Infertility is defined as primary and secondary infertility; the primary is attributed to a woman who never perceives pregnancy, while secondary infertility indicates a woman who perceives one successful pregnancy but later becomes incapable of perceiving pregnancy (14). The most common prevalence of primary infertility occurs in developed countries, while secondary infertility has been found as a major cause of infertility in developing countries (16). The other reason for infertility could be attributed to the presence of oocytes in a limited defined number (14) which naturally tend to decline with aging and ultimately enhance the risk of miscarriage (17). In addition to the abnormality of reproductive organs, the other major reason for female infertility is a disturbance of the central nervous system that controls the secretion of hormones in the reproductive system (14) further suggest the correlation between infertility and endocrine disorders that is attributed with the hormonal imbalance in the reproductive  (9,19). However, to date, the major factors and comprehensive mechanism with peculiar biomarkers that result in infertility remains unknown. Therefore, not a single efficient or effective treatment is available to inhibit the causes of female infertility disorder. Further, we will brief on the currently available treatment.

Currently available combat tactics for infertility fighting
As reported in the previous section, endocrine system disorder is among the major contributing factor to female infertility. Therefore, to date, hormone replacement therapy has been widely implicated to treat various types of infertility disorders. For instance, Clomid (a widely used gonadotropin) has been reported to trigger an excess generation of luteinizing hormone and follicle-stimulating hormone to be released by the pituitary gland, which induce the ovulation and promotes follicular growth (20) and ultimately results in the production of multiple eggs (1,21). However, this therapeutic strategy carries the risk of breast cancer (22,23). The other treatment option is fertility drugs to treat ovulation disorders (1). However, these drugs have exhibited some setbacks, such as preterm birth, ovarian hyperstimulation syndrome, multiple births, and ovarian tumor (20). Though surgical therapeutics strategies, i.e., assisted reproductive technologies, ovulation induction, hysteroscopic, laparoscopy, and fallopian tube surgeries (1), and superovulation have shown efficiency in optimizing the condition for perceiving pregnancy. However, the efficacy and safety of these strategies have not been completely evaluated (7), and the outcome of these strategies also remained unsatisfactory.
Nonetheless, despite the advancement in treatment strategies for fertility disorders, the overall percentage of infertility remained more than 80% (24). While the development of an effective therapeutic strategy not only requires considerable attention to the physical, psychological, economic, and time-related factors but also needs to apply the novel technology at the cellular level to understand the underlying molecular mechanism completely. Hence, it is essential to establish an alternative and novel therapeutic modality to cure female infertility. While despite being in their infancy, regenerative medicines offers a promising therapeutic candidate, a future set of comprehensive study is required for its clinical application. Accordingly, further, there will be a discussion about the function, applications, and limitations of stem cell therapeutic strategies.

Stem cell therapy in treating female infertility and significance of using MSCs
Based on the preclinical study data, stem cell therapy is the most promising candidate for treating infertility disorder. At the same time, further insight into its differentiation potential characterized stem International Journal of Reproductive BioMedicine Besides, the underlying mechanism indicates that stem cells can be used therapeutically for a variety of ailments that is attributed to the secretion of various mediators particularly cytokines that could effectively modulate the inflammatory and immune activation processes that significantly reduce inflammation-associated tissue damage (38)(39)(40). Hence, MSCs aid in the reduction of ovarian damage by targeting inflammation.
Nonetheless, it is also important to note that most of the above-mentioned research has been carried out using the rodent's model, which significantly differs from humans, particularly regarding the female reproductive system. Collectively, the above-mentioned studies suggest that MSCs therapy can provide longer reproductive life in larger animals, peculiarly, cattle that are more identical to humans, and present an economically admissible model (30). Accordingly, some latest preclinical trial studies and their outcome have been summarized (Table I)  For in vitro experiment, conditioned media from BM-hMSC was exposed to androgen-producing H293R cells and analyzed androgen-producing gene expression.

UC-MSCs and their underlying mechanism for fertility disorders
The  to support these findings. Thus, speculating that regarding female infertility disorder, it is necessary to seek out alternative novel sources of stem cells (more supportive to female reproductive system) rather than traditional applied common sources for stem cells therapy.

EnMSCs
Since EnMSCs are a promising candidate for treating infertility disorders due to their ability of self-renewal, high proliferation, and differentiation (60). It has been found that basal layer of the endometrium is an ample source EnMSCs; however, menstrual blood has also been indicated to contain EnMSCs, known as menstrual  Moreover, granulosa CCM has also been shown to improve follicle development in primordial mice.

Role of cell-free therapy and female infertility disorders
As a result, this provides evidence that human cumulus cells condition medium can support the IVM of mouse GV oocytes when derived from cultures of adherent cumulus cells (77). The effects testicular CCM has also been evaluated in vitro from embryonic stem cells on the development of female germ cells (78)(79)(80). For instance, testicular CCM was found to increase oocyte maturation in both mice and buffalo by secreting a variety of growth factors which enhance in vitro oogenesis (80). Intriguingly, humans also exhibit these factors (81).

Constraints in the clinical application of MSCs therapy for infertility disorders
Although

Challenges and future perspective for application of stem cell therapy infertility disorder
The fact that engineered MSCs combined with the scaffold technique are considered a promising candidate to cure infertility-related disorders in females (43). However, therapeutic application of stem cell therapy remained restricted to few countries due to several technical and ethical limitations (89). Thus, this section will determine the challenges and some potential novel strategies to overcome these challenges. Besides, optimizing extraction and transplantation method of stem cells also remained the major concern for its therapeutic application (92). Besides, the route for administration of stem cell injection is also an important factors effecting the outcome of stem cell therapy (93). For instance, direct administration of stem cells into the ovaries is applied when the target is to restore ovarian function, while intravenous (i.v.) or intraperitoneal (i.p.) that allows fair distribution of stem cells through blood strea, can be used to evaluate the effects of stem cell transplantation for multiple organ system (89).
To overcome the limitations of stem cell exposure, recently, cell-free therapy has been developed (43) and reported to hold several advantages in cell therapy (88). For this purpose, the most practical approach is using exosomes (secreted by MSC), as discussed in the previous section. In addition to cellular therapy, the therapeutic sequel of stem cell treatment can also be improved by combined therapeutic strategies. For instance, biomaterials are increasingly being integrated for fertility disorders, not only to reduce the shear stress caused by stem cell injection but also enhance the probability of cell survival after administration (93). It has been demonstrated that the combined use of collagen scaffolds and stem cells aids in the rapid spread of stem cells to the targeted tissues or organs and enhances the probability of transplanted cells survival at the initial phase of transplantation in-vivo (43). Consistently, its has been shown in an experimental study that combined application of collagen scaffold with UC-MSCs in patients suffering from POF exhibited the successful activation of follicles in dormant ovaries (43). Although integrated therapeutic strategies are considered a potential candidate for developing a safe and efficient therapeutic strategy to overcome the clinical challenges associated with infertility disorders, precise understanding of the signaling mechanism remained elusive. Nonetheless, stem cells-derived exosomes and their vital content, such as miRNAs have also been suggested as the promising therapeutic candidate in the treatment of various ovarian dysfunction, particularly via folliculogenesis and genetic stability and vascular formation (91). However, further studies are required to unveil the exact mechanism. Taken together, despite of these novel molecular therapeutic modalities, further advent in the research and comprehensive knowledge of the underlying molecular mechanism is prerequisite to treat female fertility disorders.

Conclusions
Developing an efficient therapeutic strategy based on the regenerative properties of stem cells holds the confidence and hope of scientists, clinicians, and patients suffering from fertility disorders. However, to obtain significant breakthroughs for the cure of infertility in women and for successful clinical translation of precise and well-designed study plan (from the isolation of stem cells or stem cell-derived molecules to informed, voluntary consent and model of cell delivery including) is essentially required for the success of initial clinical trials in the light of previously reported studies to overcome the limitations. Taken together, the present study attempted to present an overview of previous continued attempts and research studies to sort out the complex web of stem cell-derived therapeutic strategies and their role in treating female fertility disorders.