The role of macrophages in reproductive-related diseases

Male infertility accounts for approximately 50% of global infertility cases, and 30%–40% of the causes are unclear [ 17 ]. Testicular macrophages are the largest immune cell population in the human testis, and the subpopulation of resident macrophages was CD163 + M2 macrophages [ 124 , 125 ]. The roles of macrophages in male reproduction physiology and male idiopathic infertility are summarized in Table 5 . Testicular macrophages play a pivotal role in spermatogenesis, steroid production and immune privilege. Macrophages were localized near undifferentiated spermatogonia, and these macrophages expressed factors related to spermatogonial growth and differentiation, such as colony stimulating factor 1 (CSF1) [ 126 ]. In the absence of macrophages undifferentiated spermatogonia exhibited defect in differentiation, and the number of proliferative spermatogonia was decreased [ 126 ]. 25-Hydroxycholesterol secreted by testicular macrophages improved the testosterone production by Leydig cells [ 127 ]. Corticosterone produced by testicular macrophages suppressed the secretion of proinflammatory cytokines, maintaining the immune privilege in the testis [ 128 ] Abbreviation and full name cross-reference table was shown in Table 7 . Table 5 Summary regarding macrophages in male idiopathic infertility. Table 5 Author Main contributions Summary DeFalco et al. [ 126 ] 1. Macrophages were localized near undifferentiated spermatogonia. 2. Macrophages expressed factors related to spermatogonial growth and differentiation. 3. In the absence of macrophages undifferentiated spermatogonia exhibited defect in differentiation, and the number of proliferative spermatogonia was decreased. Testicular macrophages play a pivotal role in spermatogenesis, steroid production and immune privilege. Lukyanenko et al. [ 127 ] 1. 25-Hydroxycholesterol secreted by testicular macrophages improved the testosterone production by Leydig cells. Wang et al. [ 128 ] 1. Corticosterone produced by testicular macrophages suppressed the secretion of proinflammatory cytokines, maintaining the immune privilege in the testis. Lin et al. [ 129 ] 1. Ubiquitin-specific protease 2 (USP2) affected spermatogenesis because it is merely expressed in late-elongated sperm. Testicular macrophages participate in the pathogenesis of male idiopathic infertility. Bedard et al. [ 130 ] 2. The USP2−/− male mouse had severe infertility, the infertility was related to defects in sperm motility. Hashimoto et al. [ 131 ] 3. The USP2 in macrophages facilitated sperm movement and hyperactivation, and USP2 maintained GM-CSF expression in testicular macrophages, GM-CSF was also associated with sperm mobility. Frungieri et al. [ 132 ] 1. In the testis of idiopathic infertility, the number of CD68 + macrophages was increased. 2. These macrophages expressed IL-1 and TNF-α. The pro-inflammatory cytokines may damage sperm. Matzkin et al. [ 133 ] 1. The prostaglandin D 2 (PGD 2 ) produced by testicular macrophages was increased via β1-and β2-adrenergic receptors (ADRs). 2. The α1-and β3-ADRs expressed on testicular macrophages were observed in idiopathic infertility men; however, these two subtypes were not expressed on testicular macrophages in fertility men. Yu et al. [ 134 ] 1. The increased estradiol may be a risk factor for infertility. Estrogen promoted the expression of growth arrest–specific 6 (GAS6) on Leydig cells, and GAS6 bound to the phosphatidylserine exposed on the Leydig cell surface. 2.Macrophage phagocytosis was promoted through GAS6 binding to AXL expressed on macrophages, leading to reduced Leydig cells Eubler et al. [ 135 ] 1. The cation channel TRPV2 was expressed on CD206 + testicular macrophages, and elevated estrogen promoted TRPV2 expression, but whether TRPV2 plays a role in infertility is unknown. Table 6 Clinical trials in PBMC immunotherapy. Table 6 Number of RIF patients Oocyte source Embryo stage PBMC pretreatment Main results Reference 35 Human fresh oocyte blastocyst HCG, 2 days Implantation rate, pregnancy rate and live birth rate were promoted Yoshioka et al. [ 35 ] 253 Human frozen/thawed embryo Cleavage and blastocyst No (freshly isolated PBMC) Implantation and pregnancy rate were promoted in patients who had three or more implantation failures; No effect on live birth rate Okitsu et al. [ 145 ] 45 Human fresh oocyte blastocyst CRH, 2 days Pregnancy rate was promoted Makrigiannakis et al. [ 146 ] 54 Human fresh oocyte cleavage HMG, 3days Implantation and pregnancy rate were promoted Madkour et al. [ 147 ] 633 Human frozen/thawed embryo Cleavage and blastocyst HCG, 2 days Implantation rate and pregnancy rate were promoted in patients with four or more implantation failures; live birth rate was promoted in patients receiving cleavage stage embryo transfer Li et al. [ 36 ] 26 Human fresh oocyte cleavage CRH, 2 days Implantation and pregnancy rate were promoted Makrigiannakis et al. [ 148 ] 250 Human frozen/thawed embryo Cleavage and blastocyst CRH, 2 or 3 days Pregnancy rate was promoted in patients with three or more implantation failures Nobijari et al. [ 149 ] 100 Human frozen/thawed embryo Cleavage and blastocyst HCG, 2 days Pregnancy and live birth rate were promoted; miscarriage rate was lower Pourmoghadam et al. [ 38 ] 207 Human frozen/thawed embryo Cleavage and blastocyst HCG, 4 h No effect on implantation rate and live birth rate Mei et al. [ 150 ] RIF: recurrent implantation failure; PBMC: peripheral blood mononuclear cells; HCG: human chorionic gonadotrophin; CRH: corticotropin releasing hormone; HMG: human menopausal gonadotrophin. Table 7 Abbreviation and full name cross-reference table. Table 7 Abbreviation Full Name ADMA Asymmetrical dimethylarginine ADRs Adrenergic receptors AMPK Adenosine monophosphate-activated protein kinase CCL2 Chemokine (C–C motif) ligand-2 CCL22 Chemokine (C–C motif) ligand-22 CCR2 Chemokine (C–C motif) receptor-2 CMKLR1 Chemokine-like receptor 1 CRP C-reactive protein CSF1 Colony stimulating factor 1 dMφ Decidual macrophages dNK Decidual natural killer EPHA3 Erythropoietin-producing hepatocellular carcinoma A3 ERβ Estrogen receptor β ESCs Endometrial stromal cells EV-LGMNP1 Extracellular vesicular legumain pseudogene 1 FAK Focal adhesion kinase FasL Fas ligand GAS6 Growth arrest–specific 6 GM-CSF Granulocyte-macrophage colony stimulating factor HDACs Histone deacetylases IDO Indoleamine 2, 3-dioxygenase IFN-γ Interferon-gamma IGF-1 Insulin-like growth factor-1 IL-10/1β/33 Interleukin-10/1beta/33 IVF In vitro fertilization LMWH Low molecular weight heparin LPS Lipopolysaccharide MAPK Mitogen-activated protein kinase MIF Macrophage migration inhibitory factor MMPs Matrix metalloproteinases MSCs Mesenchymal stem cells mTOR Mammalian target of rapamycin NF-κB Nuclear factor-kappaB NK Natural killer NO Nitric oxide PAMM Placenta-associated macrophages PBMC Peripheral blood mononuclear cells PCOS Polycystic ovary syndrome PD-1 Programmed cell death-1 PD-L1 Programmed cell death-ligand 1 PGD 2 Prostaglandin D 2 PGE2 Prostaglandin E2 PI3K Phosphatidylinositol-3-kinase PPARγ Peroxisome proliferator-activated receptor γ PRMT3 Protein L-arginine methyltransferase 3 RANKL Receptor activator of nuclear factor NK-κB ligand RIF Recurrent implantation failure RM Recurrent miscarriage S1P Sphingosine1-phosphat SIRPα Signal regulated protein α TGF-β Transforming growth factor-beta Th1 T helper type 1 TNF-α Tumor necrosis factor-alpha TSP1 Thrombospondin-1 uRM Unexplained recurrent miscarriage USP2 Ubiquitin-specific protease 2 USP2a Ubiquitin-specific protease 2a Summary regarding macrophages in male idiopathic infertility. Macrophages were localized near undifferentiated spermatogonia. Macrophages expressed factors related to spermatogonial growth and differentiation. In the absence of macrophages undifferentiated spermatogonia exhibited defect in differentiation, and the number of proliferative spermatogonia was decreased. In the testis of idiopathic infertility, the number of CD68 + macrophages was increased. These macrophages expressed IL-1 and TNF-α. The pro-inflammatory cytokines may damage sperm. The prostaglandin D 2 (PGD 2 ) produced by testicular macrophages was increased via β1-and β2-adrenergic receptors (ADRs). The α1-and β3-ADRs expressed on testicular macrophages were observed in idiopathic infertility men; however, these two subtypes were not expressed on testicular macrophages in fertility men. Clinical trials in PBMC immunotherapy. RIF: recurrent implantation failure; PBMC: peripheral blood mononuclear cells; HCG: human chorionic gonadotrophin; CRH: corticotropin releasing hormone; HMG: human menopausal gonadotrophin. Abbreviation and full name cross-reference table. Testicular macrophages participate in the pathogenesis of male idiopathic infertility. Ubiquitin-specific protease 2 (USP2) affected spermatogenesis because it is merely expressed in late-elongated sperm [ 129 ]. The USP2−/− male mouse had severe infertility, the infertility was related to defects in sperm motility [ 130 ]. The USP2 in macrophages facilitated sperm movement and hyperactivation, and USP2 maintained GM-CSF expression in testicular macrophages, GM-CSF was also associated with sperm mobility [ 131 ]. In the testis of idiopathic infertility, the number of CD68 + macrophages was increased, and these macrophages expressed IL-1 and TNF-α. These pro-inflammatory cytokines may damage sperm [ 132 ]. Although the number of CD68 + macrophages was increased in the testis of idiopathic infertility [ 132 ], no specific mechanism of abnormal macrophage polarization was found; hence, it is interesting to explore whether abnormal changes in testicular microenvironment resulting from abnormal macrophage polarization cause male infertility. The prostaglandin D 2 (PGD 2 ) produced by testicular macrophages was increased via β1-and β2-adrenergic receptors (ADRs) [ 133 ]. The α1-and β3-ADRs expressed on testicular macrophages were observed in idiopathic infertility men; however, these two subtypes were not expressed on testicular macrophages in fertility men, but the function of α1-and β3-ADRs was unclear [ 133 ]. The increased estradiol may be a risk factor for infertility. Estrogen promoted the expression of growth arrest–specific 6 (GAS6) on Leydig cells, and GAS6 bound to the phosphatidylserine exposed on the Leydig cell surface. Macrophage phagocytosis was promoted through GAS6 binding to AXL expressed on macrophages, leading to reduced Leydig cells [ 134 ]. The cation channel TRPV2 was expressed on CD206 + testicular macrophages, and elevated estrogen promoted TRPV2 expression, but whether TRPV2 plays a role in infertility is unknown [ 135 ]. In a word, macrophages are indispensable in the normal physiological process of male reproduction by regulating spermatogenesis, hormone production and immune tolerance, and are also important in the pathological processes of male infertility.

In summary, macrophages are essential for embryo implantation, pregnancy maintenance and spermatogenesis. Macrophage polarization and phagocytosis are involved in the development of endometriosis; however, current researches have not been able to reveal the precise regulatory mechanisms of macrophages in reproductive-related diseases, and there is still a long way to go in the exploration of macrophages. The immune system is a complex regulatory network. Although macrophages are enriched in the decidua and testis, the regulatory role of other immune cells cannot be ignored. In future, studies on reproductive-related diseases should pay more attention to the interregulation between macrophages and other immune cells.

The M1/M2 classification of macrophages was referenced to the Th1/Th2 classification of helper T cells [ 136 ]. IFN-γ, LPS, TNFα and GM-CSF act as stimuli for M1 polarization ( Figure 3 A). M1 macrophages highly express CD80 and CD86, and secrete pro-inflammatory cytokines ( Figure 3 A). IL-4, IL-10, IL-13and TGF-β act as stimuli for M2 polarization, and CD206 and CD163 have been suggested as M2 markers [ 5 , 137 ] ( Figure 3 A); however, this simple M1/M2 classification is not perfect. CD163 + macrophages are elevated in tumor microenvironments with Th1 signature, implying that CD163 alone may not be suitable as a marker for M2 [ 138 ]. Combination of CD163 and the transcription factor CMAF more accurately identifies M2 macrophages [ 139 ]. There is no information that CD169 + macrophages in lymph node and spleen can be classified into M1 or M2 macrophages [ 137 ]. The M1/M2 classification is the most commonly used in the study of reproductive-related diseases, but recently researchers have made efforts to break this classification ( Figure 3 B). Strominger et al. divided dMφ into CD11c high and CD11c low macrophages ( Figure 3 B). CD11c high macrophages were associated with lipid metabolism and inflammation, CD11c low macrophages were associated with extracellular matrix formation and tissue growth. Each of these populations secrete both pro- or anti-inflammatory cytokines; thus, these populations cannot be simply classified into M1 or M2 macrophages [ 140 ]. In 2018, Vento-Tormo et al. divided decidual macrophages into ITGAX high dM1 and ITGAX low dM2 using single-cell reconstitution at the maternal-fetal interface, and found the gene expression of IL-10, IL-6, IL-1β was significantly higher in ITGAX high dM1 than in ITGAX low dM2 [ 141 ] ( Figure 3 B). In 2021, Thomas et al. identified placenta-associated macrophages (PAMM) using single-cell sequencing. PAMM was subdivided into 3 populations: PAMM1a (FOLR2 − CD9 hi CCR2 lo/int ), PAMM1b (FOLR2 − CD9 -/int CCR2 + ) and PAMM2 (HLA-DR hi FOLR2 hi ) ( Figure 3 B). PAMM1a adhered to the surface of the placental villi and had a repairing effect, PAMM1b was monocyte and PAMM2 was similar to dM2 that was described by Vento-Tormo et al. [ 142 ]. Whether these new classifications can better categorize macrophage heterogeneity and functions requires further study. Figure 3 Classifications of decidual macrophages. Decidual macrophages can be classified into M1 and M2 macrophages according to pro-inflammatory and anti-inflammatory states (A). In addition, new classifications are gradually being explored (B). Figure 3 Classifications of decidual macrophages. Decidual macrophages can be classified into M1 and M2 macrophages according to pro-inflammatory and anti-inflammatory states (A). In addition, new classifications are gradually being explored (B). One immune cell is not an island, one immune cell usually needs the help of other immune cells and cytokines to function, and the role of immune network consisting of immune cells and cytokines in disease cannot be ignored. The complex immune network has been studied in infection and cancer. For example, in infection, TGF-β secreted by Treg and other immune cells, binds to Treg and builds a bridge between Treg and effector T cells, ultimately leading to the suppression of effector T cell functions [ 143 ]. In cancer, IFN-γ secreted by T helper type 1 (Th1) recruits M1 macrophages, and IL-12 secreted by M1 macrophages recruits and activates Th1 cells, and also promotes the maturation of dendritic cells [ 144 ]. Although researchers have attempted to explore immune networks in reproductive-related diseases, these studies only revealed the relationship of immune cells in numbers and distribution, and did not investigate the molecular interactions between immune cells or the cytokine communication. For example, the number of decidual CD163 + macrophages in RIF patients was greatly higher than those in normal controls, and CD163 + macrophages were positively correlated with CD56 + dNK [ 27 ]. The distribution of CD56 + dNK cells is similar to that of CD8+T cells in the endometrium of RIF patients [ 24 ]; therefore, in future researchers should pay more attention to the role of immune networks and delve deeper into the interaction mechanisms in reproductive-related diseases.

All authors listed have significantly contributed to the development and the writing of this article. Tao duan and Yongsheng yu was supported by 10.13039/501100001809 National Natural Science Foundation of China , China [82071663 & 81902622]. No data was used for the research described in the article. The authors declare no conflict of interest. No additional information is available for this paper.

Reproductive-related diseases interrupt the process of successful pregnancy. The implantation of the fertilized egg in the endometrium in a receptive state is the basis for pregnancy. Infertility due to recurrent implantation failure (RIF) and recurrent miscarriage (RM) is often due to an unfavorable endometrial environment that is not conducive to implantation. In addition, the combination of high-quality sperm and mature egg is a prerequisite for the formation of a fertilized egg. Polycystic ovary syndrome (PCOS) and endometriosis interrupt the production of mature eggs, and male infertility hinders sperm production. The key role of macrophage in these diseases has been gradually revealed, and understanding the pathogenesis of these diseases is beneficial for the diagnosis and treatment. Researchers suggested that pregnancy can be divided into three immune stages corresponding to fetal development: the first stage is the pro-inflammatory stage of embryo implantation and placenta formation; the second stage is the anti-inflammatory stage related to fetal development; the third stage is the second pro-inflammatory stage associated with parturition [ 1 ]. This classification emphasizes the importance of immunity in maintaining normal pregnancy. Abundant immune cells infiltrate the decidua in early pregnancy, of which 70% are decidual natural killers (dNK) and 25% are decidual macrophages (dMφ) [ 1 , 2 ]. Decidual macrophages derived from monocytes are known as uterine tissue-resident macrophages, and multiple cytokines contribute to the development of monocytes to macrophages [ 3 ]. Macrophages can be divided into M1 and M2 phenotype according to their activation status. CD86 and CD80 are known M1 markers, CD163 and CD206 are known M2 markers [ 4 ]. M1 macrophages are characterized by pro-inflammatory and antibacterial effects; however, M2 macrophages are characterized by anti-inflammatory effects and promote trophoblast proliferation [ 5 ]. Macrophages participate in the preparation of a receptive endometrium during embryo implantation and the decidualization [ 6 , 7 ]. The live birth rate is approximately 30% per in vitro fertilization (IVF) cycle, but some patients still fail to achieve embryo implantation after three and more high-quality embryo transfer [ 8 ], and suffer from recurrent implantation failure (RIF). The RIF may be due to decreased endometrial receptivity [ 9 ]. Recurrent miscarriage (RM) is also due to abnormality in the decidual microenvironment. The main known causes of RM include genetic factors, anatomical abnormalities, thrombophilic disorders and endocrine disorders, but at least 50% of the causes are unknown, namely unexplained recurrent miscarriage (uRM) [ 10 ]. Polycystic ovary syndrome (PCOS) is a common endocrinopathy in women of reproductive age, accompanied by hyperandrogenism, insulin resistance, polycystic ovaries and infertility [ 11 ]. The activation status of macrophages fluctuated regularly during the different phases of the menstrual cycle in healthy women, but this fluctuation was weakened in PCOS [ 12 ], implying that macrophages are potentially related to PCOS. Endometriosis is defined as the implantation of endometrial-like tissue outside the uterine cavity [ 13 ]. One study reported that nearly 50% of infertility women suffered endometriosis [ 14 ]. In the normal endometrium, a large inflow of macrophages is accompanied by an increase in macrophage-derived cytokines during the secretory and menstrual periods [ 15 ]. In addition, macrophages play an important role in the beginning of endometrial shedding by secreting matrix metalloproteinases (MMPs) during menstruation [ 16 ], implying macrophages may be involved in the pathogenesis of endometriosis. Infertility affects about 15% of couples, male infertility accounts for 50% of all infertility cases, and at least 30% of male infertility cases remain unknown, namely male idiopathic infertility [ 17 ]. Macrophages are the dominant leukocyte type in the testis [ 18 ]. Testicular macrophages are involved in vascularization and morphogenesis during the embryonic period. Macrophages are also associated with the vasculature in the adult testicular interstitium [ 19 , 20 ], and in mouse models, testicular macrophages were reported to support Leydig cell development [ 21 , 22 ]. Therefore, this present paper will review the latest mechanisms by which macrophages participate in the occurrence and progression of the reproductive-related diseases, and will review the promising immune-based treatments. In addition, decidual macrophage classification and the importance of immune networks in reproduction-related diseases will be discussed in this paper.