Significance of mast cells in spermatogenesis, implantation, pregnancy, and abortion: Cross talk and molecular mechanisms

Both subsets of MCs including MCTC (tryptase‐positive, chymase‐positive) and MCT (tryptase‐positive, chymase‐negative) are present in the testis and epididymis. Increased number of MCs, higher levels of MC‐released tryptase in testis and seminal plasma of males with fertility problems, and promoting sperm motility in individuals with oligozoospermia after using MC blockers provide evidence that MCs may play a role in male infertility/subfertility disturbances. MC‐released tryptase and histamine contribute to the fibrosis and may disrupt spermatogenesis. MCs not only influence the process of spermatogenesis but also have effects on the function of other testis‐residing cells. MC‐derived histamine may influence the steroidogenesis of Leydig cells by acting through H1R and H2R receptors. Additionally, the interaction between MC‐released ATP and P2X receptors expressed on the peritubular cells may induce the production of the pro‐inflammatory mediators by peritubular cells. Further investigations showed that MCs may be involved in the pathology of female infertility during implantation, pregnancy, and abortion. In the uterus, MCT subtype is abundant in myometrium and adjacent basal layer while MCTC subtype is distributed in all layers. MCs in response to hormones mainly estradiol and progesterone become activated and release a wide range of mediators including histamine, VEGF, proteases, and metalloproteinases (MMPs) that have a role in different stages of pregnancy. An increasing influx of MCs to the cervix during the pregnancy occurs that helps to the physiologic cervical ripening. While MMPs degrade the extracellular matrix (ECM), VEGF modulates neovascularization and histamine influences the embryo implantation. MC‐derived histamine may have a positive effect during implantation due to its participation in tissue remodeling. MC proteases including tryptase and chymase activate the precursors of MMP2 and MMP9 to mediate ECM degradation during the physiologic menstrual cycle. There is a line of evidence that MCs have a role in abortion by releasing TNF‐α.


| INTRODUC TI ON
Infertility in men is a widespread problem which could be the result of disruption of hypothalamic-pituitary-testicular axis, 1 cryptorchidism, 2 varicocele, 3 testicular tumors, 4

microbial infections
(including Neisseria spp.), 5 and inflammation. 6 Testicular disorders have roots in abnormal development or proliferation of the cells involved in spermatogenesis. 7 There are three phases in the process of spermatogenesis: spermatogonia proliferation, meiosis of spermatocytes, and spermiogenesis to produce haploid spermatids and finally functional sperms. Interestingly, c-KIT/SCF interaction plays a role in spermatogonial proliferation and development 8 ( Figure   1A). Orchestration of immune responses in testis is intricate due to the immune-privileged status of the organ and the function of the blood-testis barrier. Moreover, tight junctions between Sertoli cells are capable of protecting germ cell autoantigens within seminiferous tubules from autoimmune scenarios. 9 Testicular immune privilege guarantees the immunologic protection of germ cells during spermatogenesis from an immune attack. Moreover, the localized cells of innate immunity protect the organ from pathogens that threaten the organ from blood circulation and genitourinary tract. 10 Sertoli cells and Leydig cells secrete activin A, TGF-β, PDL-1, growth arrest-specific gene (Gas6), protein S, and testosterone, which possess immunosuppressive properties. Additionally, macrophages and MCs secrete IL-10 and TGF-β to suppress the immune responses 10 ( Figure   1B). A slight increase in MCs number in the testis and epididymis during infancy followed by decreasing their number during childhood and then increasing during adolescence shows that their number varies in different developmental stages of life. 11 Investigations with immunohistological techniques revealed an increase in the number of testicular MCs in men with fertility disturbances. Interestingly, a shift from MC T -predominant MC subtype in healthy men-to MC TC in patients with fertility problems was reported. 12 Histologically, human testicular MCs can be divided into interstitial and peritubular MCs. The latter group localizes in the lamina propria or close to seminiferous tubules. 13 In contrast to interstitial MCs (also known as globoid MC), peritubular MCs (elongated or fascicular MCs) were found near seminiferous tubules or in the lamina propria itself. 13,14 MCs not only are found in male reproductive organs, but also abound in female reproductive organs including uterus and ovaries. MCs become activated in response to various stimuli in the uterus environment including estrogens, and neurotransmitters after which they release mediators that influence fined tuned biologic processes such as implantation of the blastocyst into the endometrium and tissue remodeling. 15 MCs produce a variety of inflammatory mediators and are believed to have a role in cervical ripening. 16 The number of MCs in cervical biopsies is significantly higher in women at term compared with pregnant females in the first trimester. 16 17 Interestingly, the presence of MCs throughout the endometrium layers and producing pro-inflammatory mediators have been reported to contribute to the recurrent pregnancy losses. 18

| MA S T CELL B I OLOGY: ORI G IN , DE VELOPMENT, AND AC TIVATION
MCs are long-lived granular cells of innate immunity which develop from CD34+/CD117+ pluripotent progenitor cells (MCPs). 19 These progenitors after being released from bone marrow in circulation migrate to the target organs and differentiate to MCs under the influence of growth factors mainly stem cell factor (SCF). 20 Human MCs are classified into two subtypes, namely MC T which express high levels of tryptase and MC TC that express both tryptase and chymase in their cytoplasmic granules. 19 In rodents, MCs are classified in two subtypes: mucosal MCs (MMCs) and connective tissue MCs (CTMCs). 21 MCs become activated upon recognizing IgE bounded allergens and release a wide spectrum of preformed (including chymase, heparin, histamine, and tryptase), newly synthesized mediators mainly lipid mediators such as prostaglandins and leukotrienes and cytokines (eg, TGF-β, TNF-α, and VEGF). 22

| Testicular MCs
Generally, the number of MCs increases in the peritubular walls of infertile men which is likely due to induced migration of MC precursors (MCPs) to the organ, followed by maturation within the testis. 23 Both MC subtypes (MC TC and MC T ) have been reported in human testis and epididymis. 14 MC-derived tryptase and histamine induce fibrosis and may affect normal spermatogenesis in infertile individuals. 14,24 Studies on undescended testis after orchidopexy revealed an increased number of MCs. Trichrome staining of testicular sections showed a wide range of fibrotic regions. 25 Considering that MC-derived tryptase and chymase have mitogenic effects on fibroblasts, MCs may contribute to the development of testicular fibrosis and collagen deposition. 26 Thickening of tubular walls disrupts the exchange of fluids between the tubular and the interstitial regions. 27 Moreover, MC-derived matrix metalloproteinases (MMPs), especially MMP-9, contribute to the development of fibrosis. 26 Interestingly, estradiol (E2) triggers MC degranulation which results in releasing of β-hexaminidase and leukotrienes (LTs).
α estrogen receptors (αER) expressed on human MCs may respond to high levels of estradiol which are notably higher in cryptorchid testes. 25 Adam et al studied myofibroblastic, peritubular cells which along with laminin, type IV collagen, and fibronectin containing ECM form the walls of seminiferous tubules. They reported that MC-derived tryptase may induce the production of decorin by testicular peritubular cells. Decorin regulates the collagen fibrillogenesis and has the ability to bind to TGF-β and PDGF. Decorin may play a role in men infertility through interfering with signaling of growth factor including epidermal growth factor (EGF). 28 In addition to ECM proteins, testicular peritubular cells produce IL-6, MCP-1, NGF, and GDNF. 29 PAR-2 expressing spermatogonia in seminiferous epithelium could be influenced by tryptase-releasing MCs. 30 Investigations on TCam-2 seminoma cells with high expression levels of PAR-2 showed that tryptase prevents apoptosis and supports their proliferation. 30

| Prostatic MCs
Infiltration of MCs into benign prostatic hyperplasia (BPH) tissues has been reported. Further investigations revealed that MC-derived IL-6 may induce the proliferation of BPH-1 cells through activation of STAT3/cyclin D1 signaling. 36 Additionally, MCs play a role in promoting the proliferation of prostate cancer cells and the transition of epithelial mesenchymal. 37 F I G U R E 1 A, Illustration of the testis cell involvement in spermatogenesis. Anatomy of testis and distribution of cells in seminiferous tubules is also depicted. B, Testis has the immune privilege due to its special anatomy and cross talk between the residing cells. Tight junctions between Sertoli cells protect autoantigens from autoimmune responses. Leydig cells release testosterone, Gas6, and protein S to control the function of Sertoli cells. Additionally, the latter cells produce Activin A and TGF-β to control macrophages and DCs. The function of T cells is also controlled by Sertoli cells-derived PDL-1. Macrophage released IL-10 and Tregs

| Seminal fluid MCs
Analyzing seminal specimens from infertile asthenozoospermic males and healthy control group after toluidine blue-pyronin staining showed that these patients had a higher MC number when compared to healthy men. 38 Cincik et al 39

| Ovarian MCs
Histologic distribution of ovarian MCs differs between humans and rodents in which they can be found in all parts of the organ in humans but are limited to the hilum of the ovary in rodents. 44 An increase in the number of MCs has been reported in ovarian endometriomas. High levels of locally produced estrogen (E2) may activate MCs and induce their degranulation. RBL2H3 cells F I G U R E 2 Presence of MCs in testis: MCs promote fibrosis and induce fibroblast proliferation by releasing tryptase and chymase. ATP/P2X interaction is a part of the interplay between MCs and peritubular cells. Additionally, MC-derived chymase supports the production of AngII which acts on peritubular cells via AT1R. The latter cells produce biglycan which is sensed by TLR-2 and results in releasing pro-inflammatory factors. MCs contribute the cycle by releasing TNFα which boosts the expression of TLR-2 on peritubular cells were shown to activate upon incubation with E2 and release nerve growth factor (NGF). Endometriotic cells treated with E2 were reported to play a role in the recruitment of RBL2H3 cells by releasing SCF, TGF-β, and monocyte chemo-attractant protein-1 (MCP-1). 45 In animal models including cats, rats, and cows, it is evident that the number of ovarian MCs varies in different phases of estrous cycle. 46

| Uterus-residing MCs
MCs are found in the endometrium as round or ovoid granular cells more often, whereas elongated shapes of them abound in the basal layer and endometrium/myometrium junction. 18

| Placental and decidual MCs
Investigations of placental samples obtained after abortion or inflammation revealed that the number of placental MCs especially chymase+ subtype rises in such pathologic disorders. 52 Chymase-mediated angiotensin II activation may play a role in fetal malformation. 52 Interaction of immune cells including NKCs, macrophages, Tregs, regulatory Bregs, and T cells in the decidua contributes to the formation of the suppressive environment to tolerate fetus as a semi-allograft. 53 Matsuno et al using antihuman tryptase mAb investigated the localization of tryptase+ cells and reported that these cells abound on the maternal side. They compared the primary decidual MCs from the early stages of pregnancy with decidua-derived MCs in terms of protease content and reported them as tryptase + chymase+ MC TC type. 53 One interesting interplay between Tregs and MCs in graft tolerance has been well investigated in which Treg-released IL-9 attracts MCs to graft site and OX40/OX40L interaction stabilizes MCs. The latter cells released TGF-β and IL-10 contribute to the formation of an immunosuppressive microenvironment to protect tolerance. 54 Because the fetus behaves as a semi-allograft, MCs residing in uterus, placenta, and decidua may play a role in the induction and maintaining tolerance. 54

| MA S T CELL S IN IMPL ANTATI ON , PREG NAN C Y, AND DELIVERY
MC-derived histamine is believed to have a positive effect during implantation due to its participation in tissue remodeling. 55 Investigation of the kinetic of implantation in MC-deficient C57BL/6J-Kit W−sh/W−sh (W-sh) revealed impaired implantation when compared with the control group with normal function MCs which could be compensated by local transfer of wild-type (WT) bone marrow-derived MCs (BMMCs). 56 Additionally, MCs support trophoblast survival, placentation, and growth of fetus by secreting galectin-1 (a glycan-binding protein). 56 Rodent studies using disodium cromoglycate to prevent MC degranulation revealed a role for MCderived VEGF which acts as a necessary pro-angiogenic factor to regulate implantation. Releasing VEGF upon MC degranulation was reported to support endothelial cell proliferation and angiogenesis in the uterus of the pregnant rats. 57,58 MC-derived leptin and tryptase like VEGF have angiogenic activity, and the extent of angiogenesis in leiomyomas has been reported to correlate with the expression of these cytokines in MCs granules. 59 Interestingly, a significant rise in pre-term deliveries in women suffering from asthma provides a line of evidence that MCs may play a role in delivery. 60 63 MC-derived MMPs may also play a role in implantation by mediating the tissue remodeling needed for the process. 64,65 MMPs are involved in the histologic remodeling of the endometrium during the menstrual cycle. Their expression during the cycle varies in which they show a higher expression during the menstrual and tissue proliferative phase while a decreased expression levels during the secretory phase. 66 Chymase secreted by uterus-residing MCs supports the pregnancy by mediating the remodeling of spiral arteries to maintain steady blood supplies for fetus 67 (Figure 3).

| MA S T CELL S AND PREG NAN C Y LOSS E S
Accumulation of MCs throughout all layers of the endometrium has been reported in women with a history of pregnancy loss. MC activation in these women resulted in higher levels of SCF, tryptase, heparin sulfate, and MMP-2. 18

| CLINIC AL IMPLI C ATI ON OF M C INVOLVEMENT IN FERTILIT Y AND PREG NAN C Y
Considering the reviewed role of MCs in human infertility, they can potentially be a key cell type to target. Among the targeting approaches is the application of MC blockers. Intraperitoneally, injection of ketotifen fumarate in rats with experimental autoimmune orchitis and testicular cord torsion was reported to have beneficial effects in the prevention of MC infiltration and reducing inflammation. 73 Prevention of MC activation and applying stabilizing agents have been also investigated in human models. Hibi et al reported the benefits of tranilast administration in the treatment of F I G U R E 3 Role of MCs in implantation and pregnancy: MCs protect the surveillance of blastocyst and trophoblast by releasing a variety of mediators (In blue). The mechanism of action of each mediator is mentioned in black. TNFα, unlike other mediators, acts as an abortogenic mediator. MCs-derived chymase may act on spiral arteries and promote their remodeling oligoasthenozoospermia and severe idiopathic oligozoospermia which improved semen parameters including total sperm count. 74,75 Yamamoto et al after administration of tranilast in a placebo-controlled study reported a pregnancy rate of 28.6% in the test group compared with 0% in the control group. 76 The beneficial effects of ketotifen, another MC blocker on men fertility, were reported by Saharkhiz et al This group of researchers included 40 infertile couples with asthenospermic infertility, and men included in the test group received oral ketotifen. A comparison of sperm parameters prior and after taking ketotifen showed an improvement in the test group. Sperm motility improved ranging from 16.7% to 21.4%. The rate of pregnancy was reported 12.5% in infertile couples. 77 The benefits of Ketotifen have also been reported in increasing the rate of pregnancy in post-varicocelectomy. 78 Additionally, improvement of sperm morphology in male patients with leukocytospermia was reported after using ketotifen. Decreasing the number of leukocytes and significant production of sperms with normal morphology were observed in 4 and 8 weeks post-treatment, respectively. 79

Aspects of MC involvement in male fertility which require further investigations
Ref.
It is not clear whether the increase in the number of the MCs within testis is due to induced migration of MCPs or MC infiltration 23 The role of MC mediators in seminal fluid needs to be studied. Tryptase is the main mediator investigated 41,42 The effects of MC mediators on a variety of sperm parameters including morphology and motility need further investigations 11 Our understanding of the histopathologic effects of mastocytosis on male fertility and testis is quite poor 85 SCF/c-KIT interaction plays a pivotal role in spermatogenesis, and sperm precursors express c-KIT. The molecular mechanism by which SCF/c-KIT interaction contributes to the development of testis germ cells, and the possible sources of SCF needs further study 86 Testicular MCs express melatonin receptors, and melatonin has anti-proliferative and anti-inflammatory effects on MCs. The possibility of controlling testicular MCs by targeting melatonin receptors in vivo and in vitro needs to be studied 87 One interesting theme for investigating the role of MCs in maintaining the function of the testis is their capability of expressing a wide variety of TLRs and MHC class II molecules, therefore the ability to participate in the host-pathogen defense 10 The major feature of orchitis is the infiltration of leukocytes into the testis which results in damaging its anatomical structures especially the seminiferous epithelium. In many settings, MC mediators orchestrate the infiltration of inflammatory cells into tissues. The role of MC mediators in the recruitment of inflammatory cells into testis remains unclear 10 β-endorphin released during stress stimulates MC degranulation. The possibility of MC involvement in the impact of stress on infertility is poorly understood 88 Testis benefits from an immunosuppressive microenvironment to protect the spermatogenesis. The immunosuppressive role of MCs in terms of mediators and cross talk with immunoregulatory cells is unclear 10 The presence of other potential testis-residing cells rather than MCs with the ability of histamine production needs more investigations 32 The interaction between oxidative stress and prostate-residing MCs in prostatitis and tissue damage is unclear 89 Recruitment of MCs through CXCL12/CXCR4 and other chemokine-based pathways into prostate tissue needs further investigation 90 The selective expansion and shifting MC T to MC TC in men with infertility disorders and the possible effect of testis microenvironment on driving the shift could be considered for investigation 12 Selective targeting of tryptase as the main MC-released mediator involved in the thickening of testis tubular walls instead of blocking MC degranulation may be promising 27

Aspects of MC involvement in female fertility which require further investigations
The role of MCs in implantation and placental angiogenesis in women with recurrent pregnancy loss needs to be elucidated 48 MC releases mediators with immunosuppressive property; however, the immunosuppressive role of MCs in decidua and placenta has not been elucidated 53 Hormonal changes throughout the menstrual cycle may affect the biologic function of MCs. In this regard, for example, estradiol is capable of inducing MC degranulation 55 Considering that MCs play a role in transplantation biology, and that from the immunologic point of view fetus is a semi-allograft, the role of MC mediators and cross talk with other immune cells residing in the uterus, decidua, and placenta in establishing a state of tolerance during pregnancy need to be studied 54 Trafficking of MCPs from circulation to target tissues is controlled by a molecular mechanism including integrin-based interactions. The exact molecular mechanism of trafficking of MCPs in genital organs of females and also involved adhesion molecules, integrin, and ligands could be considered for further investigation. Targeting the trafficking of MCPs into genital organs may be a promising approach to clarify the effects of MC presence during pregnancy 91 Monitoring the pregnancy in women with mastocytosis may elucidate further aspects of MC involvement in female fertility/infertility MCs and NKCs counterbalance their effects to support a normal spiral artery remodeling and placentation. 83 Table 1

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.