Biomedical Chemistry: Research and Methods 2021, 4(1), e00141

CONTENTS

INTRODUCTION

1. AZOOSPERMIA: CAUSES, CLASSIFICATION AND MECHANISMS

2. ADIPOSE DERIVED MESENCHYMAL STEM CELLS IN TREATMENT OF NON OBSTRUCTIVE AZOOSPERMIA

REFERENCES

Table 1Summary of the Relevant Studies

Adipose Derived Mesenchymal Stem Cells Restore Spermatogenesis in Male non Obstructive Azoospermia

A. Tamadon1, M.B. Askarov2, U. Zhanbyrbekuly3, R.A. Zhankina3*, D.T. Saipiyeva3, A.K. Ibragimov4, B.E. Kadirova3, A. Yessenuly4, R.S. Sherkhanov3, M. A. Suleiman3

1Bushehr University of Medical Sciences, Moallem St., Bushehr, Iran
2National Scientifc Medical Center, 42 Abylai Khan Av., Nur-Sultan, 010000, Kazakhstan
3Astana Medical University, 49a Beybitshilik St., Nur-Sultan, 010000, Kazakhstan;
*e-mail: rano_amiko2007@mail.ru
4Ecomed clinic, 1b Saryarka Av., Nur-Sultan, 010000, Kazakhstan

Key words: assisted reproductive technologies; male infertility factor; non obstructive azoospermia; mesenchymal stem cells; adipose tissue; bone marrow

DOI: 10.18097/BMCRM00141

INTRODUCTION

In the recent years, the emerging field of stem cell therapy has opened a new era of regenerative medicine. Being a totally new approach in the treatment of various diseases the diverse potential of stem cells is in a focus of research of many scientists in molecular biology, genetic engineering and general medicine [1].

Stem cells have the ability of self-sustaining throughout the life and are capable of differentiating into cells of various lineages. There are several types of stem cells found in human tissues [2]. Among them, mesenchymal stem cells (MSCs) derived from bone marrow and adipose tissue are considered to be successful in terms of their application for cell therapy. MSCs are multipotent human stromal stem cells able to self-renew. The general properties of MSCs include: high proliferative potential, adhesion to plastic, symmetric and asymmetric division, fibroblast-like morphology, easily induced differentiation, and formation of colonies in a culture [3-5]. 

MSC were first described by Friedenstein et al [6-7] demonstrating the existence of stromal stem cells in bone marrow and lymphoid organs. Their discovery confirmed that the bone marrow contained a distinct population of stem cells capable of forming clones of connective and hematopoietic cell lines. Approximately 30% of the bone marrow aspirate isolated by this team consisted of MSC. These cells showed plastic adhesion capacity and were able to differentiate into chondrocytes, fibroblasts, adipocytes and myoblasts [8-11]. Thus they have been classified as multipotent cells. Their therapeutic effect is based on the ability to secrete a number of signal molecules that modulate functions of various cells in human body [12]. 

MSCs promote growth of hematopoietic progenitors thus creating specific microenvironment (niche) and facilitating migration of hematopoietic stem cells (HSC) [13-15]. MSCs express a number of markers, including: STRO-1, Sca-1, SH-2, SH-3, SH-4, Thy-1, CD29, CD44, CD71, CD120a, CD106, CD124.

MSCs represent a rather dynamic system in the bone marrow, which consists of differentiated fibroblasts, endothelium, cytokines, reticular cells, and components of the extracellular matrix. Cell interactions within the same lineage and with adjacent ones occur through adhesion molecules and specific receptors [16].

MSCs are popular amongst scientists and clinicians due to their differentiation potential, low immunogenicity, and active participation in tissue repair and regeneration after migration to damaged sites [17]. For a long period, MSC isolation was considered as technically difficult due to traumatic specifics of fat removal and bone marrow aspiration [18-20]. 

Bone marrow is the main source of MSCs, and their aspiration still represents the most traumatic link in the chain of the whole MSC isolation procedure. The MSC number, differentiation potential and the viability of bone marrow MSCs (BM MSC) demonstrate the age-related decrease [21]. In this regard, the ongoing search for alternative sources of MSC continues. MSCs derived from adipose tissue (AT MSCs) can be alternative solution for BM MSCs due to their comparable differentiation and therapeutic potential [13]. 

Adipose tissue is not only a metabolic reservoir for storage and formation of high-energy substrates; it also participates in hormone metabolism [22]. A more profound study of the adipose tissue structure was performed by M. Rodbell; using techniques of proteolytic cleavage, mechanical grinding and differential centrifugation, 2 fractions of adipose tissue were isolated. These included mature adipocytes and more condensed cellular substance, which was later denominated by Rodbell as a stromal-vascular fraction (SVF). SVF is heterogeneous and contains blood cells, pericytes, endothelial cells, fibroblasts, and pre-adipocytes [23]. Studies have shown that SVF is a huge reservoir of MSC. In 2001 Zuk et al. noted that properties of so-called Adipose Derived Stem Cells (ADSCs) were similar to bone marrow mesenchymal stem cells [24-25]. In an adult bone marrow, the proportion of MSCs is 1:50000 to 1:1000000 cells, whereas in adipose tissue, the ratio of MSCs is 1 per 30–1000 cells [26].

AT MSCs are easier and safer to obtain. The primary acquisition of AT MSC is based on the manually performed procedure including the lipoaspirate (LA) fermentation technique [27]. Adipose tissue suitable for MSC isolation can be obtained either by resection of lipodermal skin flaps or by liposuction (LS) [28-34].

Many scientists chose LS as a surgical intervention preferable for aspirating adipose tissue suitable for isolation of MSCs. Due to the little traumatic impact of this operation, no long-term postoperative rehabilitation of patients is required. Currently, there are various techniques for LS implementation as new state-of-theart equipment continues to emerge (ultrasound, laser) [35]. The most popular option though, is classical tumescent LS, where fat tissue in the donor area of the patient’s body is infiltrated with a mixture of sterile saline with low concentrations of local anesthetic and epinephrine [36]. The LS technique can affect both the viability and quantity of MSCs isolated from fat tissue. With classical LS, the negative pressure in the aspirator is reversely proportional to the number of isolated stem cells [37]. According to Matsumoto et al. [20], when this type of surgical intervention is applied, stem cells should be processed no later than 1 day after the extraction of the fat material from the body, since storage of the fatty substrate at a room temperature decreases number of viable stem cells. Small portions of autologous adipose tissue extracted from the patient's body with a syringe are easily processed for MSC isolation, whereas processing of a large volume of aspirated fat is associated with certain difficulties [38]. Currently, researchers use a number of modified original techniques for adipose stem cells isolation [19, 39-40]. With classical liposuction, the aspirate is separated into 3 layers: (i) the top fatty layer containing homogenized mature adipocytes destroyed during the operation; (ii) the middle layer of intact adipose tissue and (iii) the bottom layer containing residuals of the solution infiltrated into the patient's tissue before surgery with plasma and blood cells. Both the top and bottom layers are removed from the container before aspirated fat processing [41]. The remaining middle layer is washed with sterile phosphate buffer with added antibacterial and antimycotic agents to avoid microbial contamination of the material [18, 28, 42]. Next, the adipose tissue is lysed in sterile collagenase solution to release the components of the stromal vascular fraction (SSF) containing stem cells [43, 44]. Different type enzymes are used, but type IA collagenase is the most effective for MSC isolation [27, 28, 42].

In 2001, the Zuk’s team [27] successfully cultured and studied multipotent cells isolated from human autologous adipose tissue (AAT). Following their success, scientists began to search for ways to safely aspirate fat tissue from patients, isolate and cultivate stem cells [30, 32, 35, 37, 41, 45]. There are many reports suggesting effective application of MSCs both in experimental animals and small groups of patients. Currently, wide use of MSC in clinical practice is limited by safety considerations. Despite large numbers of registered preclinical and clinical studies, the safety of MSC-related therapies remains the major concern for clinicians. The main risks of mesenchymal stem cells are proinflammatory properties, tumorigenicity, and fibrosis. Tumorigenicity is one of the most serious ones. On the one hand, MSCs have the ability to converse into tumors, some studies showed that Ewing's sarcoma cells originate from MSCs [46], on the other hand, MSCs can trigger tumor development. MSCs overproduce cytokines, such as growth factors and chemokines, directly acting on surface receptors of cancer cells, thereby regulating tumor enhancement. Immunosuppressive ability of these cells also promotes growth and metastasis of cancer cells [47, 48]. Another feature of MSCs contributing to tumor development is their pro-angiogenic [49]. MSCs exhibit immunosuppressive effects when exposed to sufficiently high levels of proinflammatory cytokines. However, they promote inflammatory responses in the presence of low levels of IFN-γ and TNF-α [49]. This indicates that MSCs must be triggered by inflammatory cytokines to become immunosuppressive, and the inflammatory environment is a critical factor influencing the immune regulation of MSCs. To improve therapeutic effects of MSCs and reduce potential risks, further studies are needed to investigate immunomodulatory effects of MSC, control excessive cytokines, and establish strict standards for preclinical biosafety tests [50].

Some studies indicate that embryonic stem cells very similar to MSCs have been found in the testes [50]. These cells are located in the basal layer of the testicular seminiferous tubules, they can divide asymmetrically and grow into progenitor cells. These cells survive chemotherapy and can trigger germinative cell differentiation. They, therefore, serve as a reserve storage for stem cell population [51]. It is likely that the interaction between these cells and the transplanted MSC plays a crucial role in the fertility restoration.

1. AZOOSPERMIA: CAUSES, CLASSIFICATION AND MECHANISMS

According to WHO criteria [52], the marriage is considered infertile if no pregnancy occurs within 12 months of unprotected sex. This pathology is an important medico-social issue with up to 15% (one in six) of married couples failing to conceive naturally [53-55]. Amongst them, in 45-50% infertility is caused solely by impaired spermatogenesis [56]. Both for spouses and their doctors, a diagnosis of infertility is a beginning of the long way of tests and possibly therapy. Fertility problem is diagnosed in 5-7% of men; 50-60% of infertility cases are linked to the reduced quantity or quality of ejaculate, which may be due to impaired spermatogenesis, slow maturation of spermatozoa in the epididymis or incomplete patency of vas deferens [56-61]. The main causes of male infertility are genetic disorders, urogenital infections, hypogonadism, cryptorchidism, varicocele, ejaculatory disorders, general and systemic diseases, immunological factors [62]. Despite its multifactorial nature, male infertility is not yet fully understood with about half of cases considered to be idiopathic or unexplained [63]. Investigation of male fertility usually starts with history, physical examination, and spermogram. In about 15% of patients, spermogram shows no obvious abnormalities [64]. However, it has been shown that sperm cells in infertile men have lower DNA integrity than in fertile ones [35, 65-66]. This is very important, because the genetic information passed on to the next generation, depends on the integrity of the sperm DNA [67]. Since several etiological factors contribute to the defective sperm count, the assessment of sperm DNA fragmentation (SDF) may provide an opportunity to better understand and treat such sperm disorders [67]. 

Azoospermia is classified as obstructive and non-obstructive [68]. In most patients with non-obstructive azoospermia, it is possible to distinguish both clinically by thorough diagnostic workup (history, hormone levels, physical examination). These indicators help to determine with high confidence the type of azoospermia [68]. This is important, since obstructive azoospermia is more favorable due to preservation spermatogenesis [69-70].

Non-obstructive azoospermia (NOA) accounts for 5-10% of infertility cases. It manifests as absence of spermatozoa in ejaculates due to spermatogenic deficiency [71]. In the overwhelming majority of cases, azoospermia is associated with a number of irreversible disorders of the testicles, which lead to inhibition of spermatogenesis [71]. Such disorders are most often linked to endocrine, genetic and inflammatory diseases. Also, non-obstructive azoospermia can be idiopathic [84].

Non-obstructive azoospermia should be thoroughly ruled out in all azoospermic patients [72-73]. Palpation and measurement usually reveals small and flabby testicles typical for  non-obstructive azoospermia. About 85% of the testicular parenchyma is involved in spermatogenesis; the smaller the testes, the less sperm is produced [68, 72]. Based on this, such patients should always have an ultrasound scan of scrotum also to rule out varicose veins of the spermatic cord [74].

In all patients with azoospermia, the levels of FSH, LH, prolactin [72], total testosterone, estradiol, inhibin B should be measured. In most patients with nonobstructive azoospermia, FSH will be increased (> 7.6 IU/ml) [72, 75, 76], and LH is elevated or close to normal. Since negative correlation of FSH and LH secretion is determined by the number of Leydig cells and spermatogonia, the levels of FSH and LH may be normal [77].

Hypogonadism is defined by low total testosterone levels (<300 ng/dL) and occurs in the majority of patients with non-obstructive azoospermia, usually reflecting Leydig cell deficiency [78-80].

Obesity can be associated with low total testosterone levels, thereby serum estradiol levels increase due to elevated aromatization of androgens in peripheral tissues [81-83]. Low testosterone in obese patients may also reflect adaptation to altered SHBG, rather than true testosterone deficiency [84]. Therefore, it is necessary to assess both estradiol and SHBG in patients with azoospermia and obesity. Estradiol > 60 pg/ml suppresses LH and FSH secretion and directly inhibits testosterone biosynthesis [81]. These tests can help to decide the treatment strategy before a testicular biopsy is implemented. Due to daily fluctuations in testosterone levels, blood samples are collected before 10:00 am [78, 81].

Proper counseling and management of patients with non-obstructive azoospermia presents a challenge for andrologists, urologists, and reproductive medicine specialists. Despite this, advances in molecular biology, hormone replacement therapy, and microsurgical sperm retrieval, together with modern techniques of in vitro fertilization (IVF), give hope for natural paternity [71]. Due to irreversible nature of spermatogenesis damage in patients with non-obstructive azoospermia, testicular biopsy and assisted reproductive technologies are the only ways to obtain biological off-springs.

Non-obstructive azoospermia is considered to be a condition not responding to drug therapy [71]. Patients with non-obstructive azoospermia are unable to have children of their own and have options of either adoption or using donated sperm [85]. Despite the marked changes in spermatogenesis, these patients still have a chance to conceive. In such situations, the preservation of spermatogenesis may be focal and present in 10% –50% of testicular tissues [86, 87]. For men with non-obstructive azoospermia, testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI) remains the only choice to conceive [88]. However, TESE-ICSI has limited success in patients with non-obstructive azoospermia, as during the first TESE cycle, sperm is found only in 56% of cases, and the subsequent probability of egg fertilization with ICSI is only 41%. As a result, the successful fertilization probability with this technique is only 23% [88]. Advances in assisted reproductive technologies such as intracytoplasmic sperm injections and in vitro fertilization have changed the treatment strategies of non-obstructive azoospermia management [89]. Obtaining spermatozoa is possible only through testicle biopsy, with the subsequent intracytoplasmic injection of sperm into an egg (ICSI) [73, 86, 87, 90]. Treatment of patients with this form of infertility is the most difficult from a psychological and clinical point of view [54, 55, 91].

2. ADIPOSE DERIVED MESENCHYMAL STEM CELLS IN TREATMENT OF NON OBSTRUCTIVE AZOOSPERMIA

In recent years, a significant progress has been achieved  in the treatment of assisted reproductive diseases, and now more than 80% of infertile couples can have children [92]. Due to their unlimited source and high differentiation potential, stem cells are considered as potential new therapeutic agents for the treatment of infertility.

MSC transplantation is a relatively new therapy proposed to induce spermatogenesis and treat male infertility [93]. Since MSC are involved in processes such as cell survival, proliferation, migration, angiogenesis, and immune modulation, these cells are considered as an ideal material 

Germinal cells are the conserved embryonic stem cells, these cells provide spermatogenesis [94]. It is likely that the interaction between these cells and the transplanted MSCs plays a role in the restoration of fertility. A certain combination of growth factors can be used to induce the differentiation of MSCs into cells of the germ cell epithelium [95-96]. Nayernia et al. demonstrated for the first time that rat bone marrow MSCs could differentiate into male germ cells [97].

Yazawa et al. proved that spermatogonial stem cells were able to differentiate into steroidogenic cells, such as Leydig cells, both in vivo and in vitro [98]. BM MSCs transplanted into testes of busulfan-induced azoospermic rats, promoted differentiation into Sertoli and Leydig cells [99]. In one study [100], MSCs derived from perivascular cells of the human umbilical cord were able to differentiate into germ cell-like cells exposed to a cocktail of growth factors such as leukemia inhibiting factor (LIF), glial neurotrophic factor (GDNF), retinoid acid, testosterone, and follicle-stimulating hormone. Fluorescence in situ hybridization (FISH) also showed that 10% –30% of cells gave rise to haploid cells. The results obtained in the expression analysis evidenced that these cells could produce Sertoli-like cells under the same conditions [101]. These observations suggest that the functioning of similar signaling pathways promotes the development of Sertoli cells and germ cells. Asgari et al. found that factors secreted by Sertoli cells could lead to the differentiation of MSCs into primary germ cells (PGCs) [101]. This model of morphology and expression of MSC, co-cultured with Sertoli cells, was similar to germ cells, confirming their differentiation into male cells [102]. Results of preclinical trials have demonstrated that fertility can be restored through MSC transplantation. In 2012, Sabbaghi et al.  transplanted BM MSCs into the testes of rats with azoospermia modeled by testicular torsion. However, the markers expression of germ cell epithelium showed differentiation into germ cells [103]. Transplantation of MSCs into convoluted tubules of rats with busulfan-induced azoospermia helped to restore spermatogenesis [104-106]. BM-MSC transplantation improves expression of germ cell markers in the testes and can be proposed as a suitable method for the treatment of infertility. According to many researches, the increased expression of testicular germ cell markers after BM-MSC transplantation enables to suggest this method for treatment of male infertility [105, 107]. Moreover, MSCs may be involved in the suppression of antisperm antibodies (ASA) [108] and can reduce factors that lead to infertility caused by testicular torsion through reduction of apoptosis and oxidative stress and stimulation of testosterone production [109]. Ghasemzadeh-Hasankolaei et al. transplanted BM MSCs into testes of infertile rats and observed their differentiation in spermatogonia [110]. This study demonstrated improved differentiation capacity of MSCs, potentially leading to more remarkable restoration of testes ability to spermatogenesis compared to hematopoietic stem cells [110]. They found a substantial elevation of mRNA in three meiosis genes 6 weeks after injection of umbilical cord blood stem cells. The injection method was attributed to the ability of MSCs to differentiate in spermatogonia and other supporting spermatogenesis cells by increasing regulation of gene expression in spermatogenesis [111]. Fertility in male rats with busulfan-induced azoospermia was restored by transplantation of adipose tissue (AT) MSCs [106]. Cells with a green-fluorescent protein as a surface marker have been found on both sides: outside the basement membrane and inside the convoluted tubules. This confirms the idea that MSCs can participate in spermatogenesis in two ways: by supporting spermatogonial stem cells (SSCs) and differentiating into spermatozoa. Cord blood MSC transplantation is an effective method for increasing expression of germ cell stem cells in busulfaninduced azoospermic models [112]. The differentiation of AT MSCs into testicular germ cells suggests that cell therapy can help reverse pathological changes in the testicular convoluted tubules. AT MSCs recreate the microenvironment of the convoluted tubules through production of germ cells in the recipient’s seminiferous tubules [106]. Monsefi et al. showed that transplanted AT MSCs can differentiate into germ cells in the convoluted tubules of Wistar rats. It was found that both AT MSC and BM MSCs are effective in treatment of azoospermia in animals. Successful spermatogenesis was achieved in guinea pigs with busulfan-induced azoospermia following injection of BM MSCs [113]. Allogenic AT MSCs could differentiate into cells similar to spermatogenic epithelium in vitro, providing theoretical and experimental background for clinical use of AT MSCs in treatment of infertility in animals such as guinea pigs with azoospermia [114]. In rats, BM MSCs mitigate the toxic effect of cisplatin on testes at both genetic and molecular levels [113]. Currently, 3 possible mechanisms of testicular function restoration during MSC-induced tissue regeneration are investigated: 1) MSCs can differentiate into target cells [115]; 2) the transplanted cells secrete growth factors, stimulating restoration of the recipient's cellular function [116] 3) MSCs connect with endogenous cells, restoring the function of damaged cells [117]. Sertoli cells are immunotolerant [117] and this, possibly, contributes to the protection of transplanted allogeneic cells from a post-transplant inflammatory or immune response, and the survival of donor BM MSCs. H. Chen et al. showed that sperm differentiation is possible after transplantation of cord blood MSCs into the testicular convoluted tubes in immunodeficient rats [118].

In 2016, scientists from Jordan presented observational data of patients who were given intratesticular injections of CD34/CD133 cells.

CD34 - the absolute number of hematopoietic stem cells

CD133 - prominin-1 is a glycoprotein encoded in humans by the PROM gene.

These patients were followed up 5 years post-transplantation. No complications were recorded. 27 histological changes were found in 9 patients (33%). In 7%, growth of spermatids in the testes was confirmed, in 11% mature sperm was detected on spermograms. Spermatocytes and spermatozoa after stem cell transplantation appeared in 26%. Long-term observations recorded 6 natural conceptions, 2 births and 1 successfully performed IVF in three married couples [119]. In another study, 6 patients with nonobstructive azoospermia with negative results of microsurgical sperm extractionand levels of FSH > 25 mlU/ml (norm 1.4-13.6 mU/L) and inhibin B <16 (norm 148365 ng/ml) received intra-testicular injections of autologous MSCs. All patients showed a positive hormonal response. During treatment, the concentration of FSH reduced to 16.3 ± 5.6 mlU/ml, and inhibin B increased to 14.5 ng/ml. MicroTESE detected Germinogenic cells in 3 patients. Pregnancy was reported in two couples, one terminated at week 12 and another had 7-month normal development [120].

The presented data prove the great potential of MSCs in restoration of fertility in patients with non-obstructive azoospermia. Mastering and successfully applying this technique in clinical practice can help a vast group of patients to revive spermatogenesis and enjoy fatherhood.

Table 1 summarizes results of relevant studies.

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Table 1. Summary of the Relevant Studies

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