The role of macrophages in reproductive-related diseases

Abstract

The study of reproductive immunology includes the role of immunity in reproductive physiology and reproductive-related diseases. Reproductive-related diseases cause low pregnancy rate mainly through ovulation disorders, low-quality sperm production, embryo implantation failure and pregnancy maintenance disorders. Numerous cell types including infiltrating immune cells perform specific functions in the reproductive system. Physiologically macrophages are enriched in the decidua and testes, and macrophages are involved in endometrial receptivity, embryo implantation and spermatogenesis. Pathologically macrophages are associated with alterations of decidual microenvironment in recurrent implantation failure (RIF) and unexplained recurrent miscarriage (uRM), local inflammation in polycystic ovary syndrome (PCOS) and clearance of endometriotic lesions in endometriosis. Although researchers have recently attempted to uncover the pathogenesis and provide effective treatments for the reproductive-related diseases, the specific mechanisms and effective therapies need to be further explored. Here we summarized the latest mechanisms by which macrophages participate in the progression of the reproductive-related diseases, and the promising immune-based treatments. In addition, we discussed decidual macrophage classification and the importance of immune networks in reproduction-related diseases.

Macrophages; Reproduction; Gynecology and obstetrics; Male idiopathic infertility.

1. Introduction

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.

2. The role of macrophages in recurrent implantation failure (RIF)

The definition of RIF varies from center to center, but the cumulative number of embryos transferred and the number of embryo transfer cycles are usually considered [6, 23]. The roles of macrophages in RIF are summarized in Table 1. Abnormal distribution and number of macrophages may be associated with RIF. A study involving 1989 patients with RIF or RM showed that CD163⁺ dMφ accumulated in endometrial glands in nearly 70% cases [24]. Researchers found that abnormal accumulation of macrophages in the endometrial glands caused by adenomyosis may affect embryo implantation in a case report and a retrospective study [25, 26]. In addition, a recent study reported a significant increase in the number of CD163⁺ dMφ in RIF patients compared with normal IVF controls [27]. CCL2 levels were increased in decidual stromal cells in early pregnancy, and CCL2 regulated Treg and monocytes/macrophages migration [28, 29]. The CCL2 levels in patients with RIF were detected to be relatively higher than those in controls in a cross-sectional study, but the authors did not explore the mechanism in detail [30]. The CCL2 signal can only be passed via CCR2 [31]. CCL2-CCR2 axis is closely related to macrophage polarization, and both CD276 and kinesin family member 4A were found to regulate M2 polarization through CCL2-CCR2 axis [32, 33]. Blockade of CCL2-CCR2 axis increased the expression of M1-related HIF1A gene on macrophages [34]; thus, we propose a hypothesis that increased CCL2 may overpolarize macrophages toward the M2 phenotype through CCL2-CCR2 axis influencing polarization-related genes. This M1/M2 imbalance disrupts the pro-inflammatory environment at the maternal-fetal interface in early pregnancy and ultimately leads to failure of embryo implantation [1].

Table 1.

Summary regarding macrophages in recurrent implantation failure (RIF).

Author	Main contributions	Summary
Russell et al. [24]	1. A study involving 1989 patients with RIF or RM showed that decidual CD163+ macrophages accumulated in endometrial glands in nearly 70% cases.	Abnormal distribution and number of macrophages may be associated with RIF.
Tremellen et al. [25]	1.Adenomyosis was associated with aggregation of macrophages in the superficial endometrial glands. 2.This aggregation may interfere with embryo implantation
Tremellen et al. [26]	1.The number of CD163+ macrophages in the gland lumens of the superficial endometrium was increased in adenomyosis patients. 2.Adenomyosis may interfere with embryo implantation through immune mechanisms
Papúchová et al. [27]	1. The number of decidual CD163+ macrophage was greatly increased in RIF patients compared with normal IVF controls
Namlı Kalem et al. [30]	1. The CCL2 levels in patients with RIF were detected to be relatively higher than those in controls.	We propose a hypothesis that increased CCL2 may overpolarize macrophages toward the M2 phenotype through CCL2-CCR2 axis influencing polarization-related genes.
Needham et al. [31]	1. The CCL2 signal can only be passed via CCR2.
Miyamoto et al. [32]	1. CD276 regulated M2 polarization through CCL2-CCR2 axis in ovarian cancer
Zhang et al. [33]	1. Kinesin family member 4A regulated M2 polarization through CCL2-CCR2 axis
Sierra-Filardi et al. [34]	1. Blockade of CCL2-CCR2 axis increased the expression of M1-related HIF1A gene on macrophages.

Considering the involvement of immune cells in maternal-fetal interface in embryo implantation, intrauterine administration of peripheral blood mononuclear cells (PBMC) prior to embryo transfer becomes a promising treatment (shown in Table 6). For fresh embryo transfer, the improvement in implantation, pregnancy and live birth rate via PBMC immunotherapy has been recognized [35]. For frozen/thawed embryo transfer, the improvement of implantation and pregnancy rate is mainly reported in patients who had at least three or four implantation failures [36]; moreover, the promotion of live birth rate is controversial, and cleavage stage transfer may be more beneficial for live birth delivery [36]. It is currently believed that the therapeutic effect is mainly due to the cytokines (eg. TNF-α, IL-1β and IFN-γ) released by PBMC. These cytokines maintain the pro-inflammatory microenvironment required for embryo implantation and promote embryo invasion [37], and hormonal pretreatment for PBMC greatly stimulates cytokine release [38]. Intrauterine administration of PBMC helps RIF patients improve implantation, pregnancy and live birth rate (shown in Table 6), but this treatment mechanism was rarely studied. PBMC is a mixture of peripheral blood cells, and mainly consists of monocytes and lymphocytes. The fate of PBMC after entering the uterine cavity and which immune cells and cytokines play a key role are worth investigating.

Table 6.

Clinical trials in PBMC immunotherapy.

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.

In summary, abnormal distribution and increased number of M2 macrophages are associated with RIF, but the mechanisms have been inadequately studied. Increased M2 macrophages may disrupt the decidual pro-inflammatory microenvironment during implantation, thus leading to RIF. The therapeutic effect of PBMC on RIF is satisfactory, but the specific mechanisms of treatment have also been inadequately studied.

3. The role of macrophages in unexplained recurrent miscarriage (uRM)

WHO defines three or more consecutive miscarriages before the 20 weeks of gestation as recurrent miscarriage (RM) [39]. At least 50% of the causes of recurrent miscarriage are unknown, namely unexplained recurrent miscarriage (uRM) [10]. The roles of macrophages in uRM are summarized in Table 2. In the decidua of uRM patients, researchers have commonly observed an abnormal increase in M1 dMφ or an abnormal decrease in M2 dMφ. The excessively pro-inflammatory environment resulting from the M1/M2 imbalance is responsible for uRM. PD-1 is a co-inhibitory molecule that belongs to the CD28 superfamily, and is expressed on B cells, T cells, NK cells, macrophages, activated monocytes and dendritic cells. PD-L1 is the ligand of PD-1 [40]. PD-1/PD-L1 axis was involved in macrophage polarization and trophoblast invasion. PD-1 expressed on dMφ and PD-L1 expressed on trophoblasts were reduced in uRM patients. PD-1 deficiency induced macrophage polarization into M1 phenotype [41]. PD-L1 deficiency inhibited trophoblast invasion through ERK/MMP pathway [42]. Indoleamine 2, 3-dioxygenase (IDO), an intracellular cytoplasmic enzyme, transfers tryptophan (Trp) to N-formyl kynurenine [43]. IDO⁺ dMφ is significantly lower in uRM patients than in normal control, and IDO⁺ dMφ displayed M2 phenotype [44]. The expression of IDO was also reduced in the decidua of RM patients [45]. In a miscarriage mouse model, IDO supplementation reduced miscarriage rate by suppressing the inflammatory response [46]. Researchers have reported that IDO was a protective factor for early pregnancy, and that IDO and IDO⁺ dMφ was reduced in uRM decidua [44, 45], but researchers have also reported that IDO was elevated in uRM villi, and there is no significant difference in the expression of IDO in control decidua and in RM decidua [47]; thus, more studies are needed to prove the role of IDO in uRM. Cathepsin E is an intracellular aspartic protease belonging to the pepsin family [48]. Reduced dMφ-derived cathepsin E has been observed in uRM patients, and live birth rate was reduced in cathepsin E−/− mouse, cathepsin E disrupted M1/M2 imbalance [49]. dMφ-derived IL-33 was reduced in uRM patients, dMφ-derived IL-33 promoted M2 bias at the maternal-fetal interface [50]. Histone deacetylases (HDACs) are histone modification enzymes, HDAC8 expressed on dMφ was decreased in uRM patients [51]. Decreased HDAC8 resulted in a decrease of CD163 expressed on dMφ, and decreased HDAC8 promoted dMφ apoptosis via ERK signaling pathway [51]. Receptor activator of nuclear factor NK-κB ligand (RANKL), which is secreted by human embryonic trophoblasts and maternal decidual stromal cells, induced dMφ toward the M2 phenotype. Impaired expression of RANKL caused dMφ abnormal polarization in vivo and increased the fetal loss rate in a mouse model [52]. Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor expressed in trophoblasts and dMφ. This receptor activation is associated with M2 polarization. PPARγ⁺ dMφ was significantly reduced in uRM patients, and reduced PPARγ⁺ dMφ may lead to an inflammatory response against the fetus [53]. The role of macrophages in the pathogenesis of uRM is shown in Figure 1.

Table 2.

Summary regarding macrophages in unexplained recurrent miscarriage (uRM).

Author	Main contributions	Summary
Zhang et al. [41]	1.PD-1 expressed on dMφ and PD-L1 expressed on trophoblasts were reduced in uRM patients. 2.PD-1 deficiency induced macrophage polarization into M1 phenotype.	The imbalance of macrophage M1/M2 ratio has been observed in abortions	The excessively pro-inflammatory environment resulting from the M1/M2 imbalance and abnormal trophoblast function regulated by macrophages are responsible for uRM.
Chen et al. [42]	1. PD-L1 deficiency inhibited trophoblast invasion through ERK/MMP pathway.
Huang et al. [44]	1.IDO+ dMφ is significantly lower in uRM patients than in normal control, 2.IDO+ dMφ displayed M2 phenotype.
Wei et al. [45]	1. The expression of IDO was also reduced in the decidua of RM patients.
Cheng et al. [46]	1. In a miscarriage mouse model, IDO supplementation reduced miscarriage rate by suppressing the inflammatory response
Goto et al. [49]	1.Reduced dMφ-derived cathepsin E has been observed in uRM patients. 2.Live birth rate was reduced in cathepsin E−/− mouse. 3.Cathepsin E disrupted M1/M2 imbalance.
Sheng et al. [50]	1.dMφ-derived IL-33 was reduced in uRM patients. 2.dMφ-derived IL-33 promoted M2 bias at the maternal-fetal interface
Yao et al. [51]	1.Histone deacetylases (HDACs) are histone modification enzymes. 2.HDAC8 expressed on dMφ was decreased in uRM patients. 3.Decreased HDAC8 resulted in a decrease of CD163 expressed on dMφ. 4.Decreased HDAC8 promoted dMφ apoptosis via ERK signaling pathway.
Meng et al. [52]	1.Receptor activator of nuclear factor NK-κB ligand (RANKL), which is secreted by human embryonic trophoblasts and maternal decidual stromal cells, induced dMφ toward the M2 phenotype. 2.Impaired expression of RANKL caused dMφ abnormal polarization in vivo and increased the fetal loss rate in a mouse model.
Kolben et al. [53]	1.Peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor expressed in trophoblasts and dMφ, is associated with M2 polarization. 2.In RM PPARγ+ dMφ was significantly reduced, reduced PPARγ+ dMφ may lead to an inflammatory response against the fetus.
Ding et al. [54]	1.CD86+ dMφ was increased in uRM patients. 2.Elevated Fas ligand (FasL) expressed on CD86+ dMφ was also observed. 3.The elevated FasL was a potential cause for RM by mediating trophoblast apoptosis.	Macrophages are involved in uRM by affecting trophoblast function.
Wang et al. [55]	1.Ubiquitin-specific protease 2a (USP2a) improved trophoblast migration and invasion via PI3K/Akt/GSK3b signaling pathway. 2.TGF-β secreted by M2 dMφ enhanced the USP2a expression on trophoblasts.
Hao et al. [56]	1.dMφ was associated with decreased nitric oxide (NO) in RM decidua. 2.Protein L-arginine methyltransferase 3 (PRMT3) expressed on dMφ was increased in RM patients. 3.Asymmetrical dimethylarginine (ADMA), a protein degradation product mediated by PRMT3, abnormally accumulated in decidua. 4.ADMA as endogenous nitric oxide synthase inhibitor decreased NO production in decidua. In addition, increased PRMT3 expressed on dMφ promoted trophoblast apoptosis.
Bruno et al. [57]	1. LMWH increased the expression of CD206 and HLA-DR in macrophages, increased CCL20 secretion and reduced CCL2 and CCL22 secretion.		The potential therapeutic strategies via regulating M1/M2 balance bring hope for uRM patients.
Cui et al. [58]	1. M1 dMφ was increased in uRM patients, and Rev-erbα was reduced in M1 dMφ.
Cui et al. [59]	1. SR9009, an agonist of Rev-erbα reduced M1-like polarization via PI3K/Akt signaling pathway.
Li et al. [60]	1.MSCs prevented fetal loss in a mouse model. 2.MSCs inhibited CD4+T cell proliferation and the M2 phenotype in a tumor necrosis factor-stimulating gene 6 dependent manner

Figure 1.

Macrophage involvement in the pathogenesis of unexplained recurrent miscarriage. dMφ are involved in unexplained recurrent miscarriage pathogenesis through polarization imbalance and inducing trophoblast dysfunction. In patients with unexplained recurrent miscarriage, decidual macrophages have a tendency to polarize toward M1, and M2 polarization is suppressed. The increased M1 macrophages promote the release of pro-inflammatory cytokines, creating an excessively pro-inflammatory maternal-fetal microenvironment (A). In addition, decidual macrophages make it difficult to maintain pregnancy by promoting trophoblast apoptosis and inhibiting trophoblast migration and invasion (B).

Macrophages can also be involved in uRM by affecting trophoblast functions. CD86⁺ dMφ was increased in uRM patients, and elevated Fas ligand (FasL) expressed on CD86⁺ dMφ was also observed. The elevated FasL was a potential cause for RM by mediating trophoblast apoptosis [54]. Ubiquitin-specific protease 2a (USP2a) improved trophoblast migration and invasion via PI3K/Akt/GSK3b/β-catenin signaling pathway. TGF-β secreted by M2 dMφ enhanced the USP2a expression on trophoblasts [55]. dMφ was associated with decreased nitric oxide (NO) in RM decidua. Protein l-arginine methyltransferase 3 (PRMT3) expressed on dMφ was increased in RM patients; thus, asymmetrical dimethylarginine (ADMA), a protein degradation product mediated by PRMT3, abnormally accumulated in decidua of RM patients. Eventually ADMA as endogenous nitric oxide synthase inhibitor decreased NO production in decidua. In addition, increased PRMT3 expressed on dMφ promoted trophoblast apoptosis [56].

Based on the M1/M2 imbalance, potential therapeutic strategies are increasingly being investigated. Low molecular weight heparin (LMWH) is often used to treat uRM. LMWH affected macrophage polarization and the cytokine profile. LMWH increased the expression of CD206 and HLA-DR in macrophages, increased CCL20 secretion and reduced CCL2 and CCL22 secretion [57]. M1 dMφ was increased in uRM patients, and Rev-erbα, a significant clock gene, was reduced in M1 dMφ. SR9009, an agonist of Rev-erbα reduced M1-like polarization via PI3K/Akt signaling pathway; thus, SR9009 is a potential therapeutic strategy [58, 59]. Mesenchymal stem cells (MSCs) have been considered as a possibility for the treatment of various immune diseases. MSCs prevented fetal loss in a mouse model, and MSCs inhibited CD4⁺T cell proliferation and reprogrammed M1 phenotype toward the M2 phenotype in a tumor necrosis factor-stimulating gene 6 dependent manner [60]. MSC's ability to balance the decidual immune microenvironment offers hope for the treatment of uRM.

In summary, the excessively pro-inflammatory microenvironment resulting from the M1/M2 imbalance (Figure 1A) and abnormal trophoblast function regulated by dMφ (Figure 1B) are responsible for uRM. Therapeutic strategy to regulate M1/M2 balance may offer new hope for patients.

4. The role of macrophages in polycystic ovary syndrome (PCOS)

The hypothalamus-pituitary gland-ovaries axis is known to control ovulation. Spleen may also be involved in ovulation and may be a direct source of leukocytes for the ovary during the ovulation cycle [61]. Macrophages that differentiated from splenic monocytes are capable of migrating to the ovary, suggesting that the spleen may act as the ovarian immune cell bank [62]. Bilateral superior ovarian nerve sections in rats affected splenic macrophage apoptosis and regulated the ovarian steroid secretion, suggesting that sympathetic nerve may regulate macrophage activity [63].

PCOS is a disease characterized by excessive androgen levels and polycystic ovaries, and the range of serum testosterone is 45–150 ng/dL (2–5 nmol/L) in women with PCOS [64]. The roles of macrophages in PCOS are summarized in Table 3. Ovary and peripheral blood were in a low-grade inflammatory condition in PCOS patients. Serum C-reactive protein (CRP), lymphocytes and monocytes were significantly higher, and the number of macrophages and lymphocytes was also increased in PCOS ovarian tissues [65]. Splenic macrophages were associated with hyperandrogenism in PCOS. Cytokines secreted by splenic macrophages from PCOS mouse stimulated androstenedione production by granulosa cells and inhibited estradiol production, but the exact cytokines are unknown [66]. Excessive androgen inhibited the macrophage viability and increased macrophage apoptosis [67], and promoted pro-inflammatory cytokines expression, such as TNF-α [68]. The increased TNF-α in turn inhibited estradiol production [68]. In addition, TNF-α stimulated proliferation of theca-interstitial cells, possibly contributing to PCOS development [69]. Chemerin is an immunologically active adipokine. Chemerin was elevated systemically in PCOS patients [70, 71]. Chemerin exhibited induction of M1 polarization and inhibition of M2 polarization in other immune-related diseases [72, 73]; however, whether chemerin plays a regulatory role on macrophage polarization in PCOS is unclear. Ovarian M1 macrophages were increased in androgen-treated rats, and chemerin and chemokine-like receptor 1 (CMKLR1) were increased in ovarian tissues and ovarian macrophages respectively, suggesting that increased chemerin induced CMKLR1+ monocytes recruiting to ovary and these CMKLR1+ monocytes are the source of M1 macrophages. Co-culture assay showed that apoptosis of granulosa cells was dependent on these recruited macrophages [74].

Table 3.

Summary regarding macrophages in polycystic ovary syndrome (PCOS).

Author	Main contributions	Summary
Xiong et al. [65]	1.Serum C-reactive protein (CRP), lymphocytes and monocytes were significantly higher in PCOS patients. 2.The number of macrophages and lymphocytes was also increased in PCOS ovarian tissues.		The inflammatory response involving macrophages and macrophage-related cytokines are associated with the progression of PCOS.
Figueroa et al. [66]	1. In PCOS mouse model, cytokines secreted by splenic macrophages stimulated androstenedione production by granulosa cells and inhibited estradiol production.
Li et al. [67]	1. Excessive androgen inhibited the macrophage viability and increased macrophage apoptosis
Huang et al. [71]	1. Serum chemerin was elevated systemically in PCOS patients.	Chemerin is associated with PCOS, but whether chemerin plays a regulatory role on macrophage polarization in PCOS is unclear.
Ji et al. [72]	1. Chemerin promotes the pathogenesis of preeclampsia by inducing M1 macrophage polarization
Lin et al. [73]	1. Chemerin aggravates colitis by suppressing M2 macrophage polarization.
McCartney et al. [74]	1.Ovarian M1 macrophages were increased in PCOS rat model. 2.Chemerin was increased in ovarian tissues. 3.Chemokine-like receptor 1 (CMKLR1) was increased in ovarian macrophages. 4.Apoptosis of granulosa cells was dependent on these CMKLR1+ macrophages
Zhou et al. [77]	1.MIF was increased in ovarian tissues in PCOS rat model. 2.MIF may participate in PCOS via MAPK signaling pathway.	MIF is involved in PCOS via different signaling pathways, and it is not clear whether MIF affects macrophage migration in PCOS
He et al. [78]	1.MIF was increased in ovarian tissues in PCOS rat model. 2.MIF may participate in PCOS via NF-κB pathway
González et al. [80]	1. Plasma MIF levels were significantly higher in PCOS patients.
Bennett et al. [75]	1. MIF inhibited the ability of macrophages random migration

Macrophage migration inhibitory factor (MIF) was the first pro-inflammatory cytokine, and plays an indispensable role in acute and chronic inflammatory conditions and inhibited the ability of macrophages random migration [75, 76]. MIF was increased in ovarian tissues in PCOS rat model, and MIF may participate in PCOS via MAPK signaling pathway [77]. The increased MIF may also participate in PCOS via NF-κB pathway leading to inflammation [78]. Plasma MIF levels were significantly higher in PCOS patients than healthy women [79, 80]; thus, plasma MIF levels may be a potential predictor of PCOS. MIF is involved in pathogenesis of PCOS, and in vitro experiments have demonstrated that MIF inhibited macrophage migration [75, 76], thus; it is worth investigating whether MIF is involved in PCOS by inhibiting macrophage migration. Researchers found that MIF was positively correlated with obesity [79], but it has also been found that MIF was not associated with obesity in PCOS [80]; thus, more researches are needed to prove the relationship between MIF and obesity in PCOS.

Overall, the inflammatory response involving macrophages and macrophage-related cytokines are associated with the progression of PCOS. Rodent models of PCOS have been widely used [81], but genomic differences between humans and animals are objective. The use of human-derived cells and biocompatible biomaterials to construct ovarian organoid may provide a new model for the study of the mechanisms of PCOS [82, 83].

5. The role of macrophages in endometriosis

Endometriosis is defined as the implantation of endometrial-like tissue (“lesions”) outside the uterine cavity, usually in the pelvic cavity or ovaries. Endometriosis correlates with chronic pelvic pain, menstrual disorders and infertility [13]. Infertility may be due to an inflammatory microenvironment that is not conducive to conception or due to ovulation disorders caused by ovarian endometriosis [84]. The roles of macrophages in endometriosis are summarized in Table 4. Macrophages are associated with pain caused by endometriosis. The concentration of macrophage-derived insulin-like growth factor-1 (IGF-1) was elevated in peritoneal fluid from endometriosis patients and there was a positive correlation between IGF-1 and pain score, possibly because IGF-1 enhanced nerve sensitization and neurogenesis [85]. CT/CC genotype of miR-146b rs1536309 correlated with the risk of pain in endometriosis, the patients carrying the CT/CC genotype of miR-146brs1536309 had reduced expression of miR-146b in macrophages, and in this condition inflammation was enhanced [86].

Table 4.

Summary regarding macrophages in endometriosis.

Author	Main contributions	Summary
Forster et al. [85]	1.The macrophage-derived insulin-like growth factor-1 (IGF-1) was elevated in peritoneal fluid from endometriosis patients 2.There was a positive correlation between IGF-1 and pain score 3.IGF-1 enhanced nerve sensitization and neurogenesis		Macrophages are associated with pain caused by endometriosis.
Zhang et al. [86]	1.CT/CC genotype of miR-146b rs1536309 correlated with the risk of pain in endometriosis. 2.The patients carrying the CT/CC genotype of miR-146brs1536309 had reduced expression of miR-146b in macrophages.
Wu et al. [90]	1. The concentration of prostaglandin (PG) E2 (PGE2) was elevated in peritoneal fluid from patients with endometriosis.	The phagocytic ability of peritoneal macrophages was weakened in patients with endometriosis	The weakened clearance and promoted growth of ESCs eventually cause the development of endometriosis.
Wu et al. [91]	1.PGE2 inhibited the expression of annexin A2 on peritoneal macrophages via the EP2/EP4 receptor-dependent signaling pathway 2.Decreased annexin A2 was associated with weakened macrophage phagocytosis.
Chuang et al. [92]	1.PGE2 inhibited the expression of CD36 on peritoneal macrophages. 2.Decreased CD36 was associated with weakened macrophage phagocytosis.
Liu et al. [94]	1.The expression of CD47 on ectopic endometrial stromal cells (ESCs) was significantly elevated in endometriosis patients 2.As CD47 ligands, thrombospondin-1 (TSP1) and signal regulated protein α (SIRPα) were also increased 3.The elevated CD47 binding to elevated SIRPα expressed on peritoneal macrophages exhausted macrophage phagocytic ability and improved M2 polarization
Liu et al. [96]	1.Heme abnormally accumulated in the peritoneal fluid from patients with endometriosis, 2.High concentration of heme treatment exacerbated the damage to phagocytic ability
Clark et al. [97].	1.The expression of CD200 was upregulated in endometrial-like tissues 2.Soluble CD200 (sCD200) was also increased in the small veins of endometrial-like tissues
Weng et al. [98]	1.The expression of CD200 receptor in peritoneal macrophages was also elevated. 2.The phagocytic ability of macrophages in vitro gradually decreased with the increasing CD200 levels.
Nie et al. [100]	1. Ectopic endometrial homogenates promoted M1 to M2 macrophage polarization.	M2 macrophages were increased in patients with endometriosis, and the increased M2 macrophages are associated with ectopic ESCs proliferation, angiogenesis and lymphangiogenesis
Sun et al. [101]	1. After treatment with Exosomes from endometriosis, the macrophages were polarized towards an M2 phenotype.
Gou et al. [102]	1. The ectopic ESCs-derived CCL2 recruiting macrophages through estrogen receptor β (ERβ) and NF-κB signaling.
Huang et al. [103]	1. The ectopic ESCs-derived exosomal miR-301a-3p enhanced M2 polarization via PTEN-PI3K pathway.
Sun et al. [104]	1.The ectopic ESCs-derived extracellular vesicular legumain pseudogene 1 (EV-LGMNP1) was increased in endometriosis patients. 2.The elevated EV-LGMNP1 induced M2 polarization, and serum increased EV-LGMNP1.
Miller et al. [105]	1.IL-17A could induce macrophage recruitment and M2 polarization
Ono et al. [106]	1.The sphingosine 1-phosphate concentration was greatly increased in peritoneal fluid from patients with endometriosis 2.Sphingosine 1-phosphate induced M2 polarization
Itoh et al. [107]	1. M2 macrophages accelerated ectopic ESCs proliferation through Stat3 activation.
Ono et al. [108]	1. M2 macrophages contributed to the angiogenesis in the endometriosis mouse model.
Chan et al. [109]	1. Macrophages facilitated the invasion of endometrial stromal cells.
Hattori et al. [110]	1. Macrophages-derived VEGF-C and VEGF-D promotes lymphangiogenesis in a VEGFR1-dependent manner.
Nagai et al. [111]	1.MCP-1 is an inducer of macrophage recruitment. 2.Focal adhesion kinase (FAK) inhibitors significantly inhibited the secretion of MCP-1 by ectopic ESCs.		New targets based on the regulation of macrophage function may provide new ideas for the treatment of endometriosis.
Sekulovski et al. [113]	1.M2 macrophages promoted ectopic ESCs viability. 2.This promotion was also inhibited by niclosamide.
Xu et al. [114]	1.EPHA3 promoted macrophage apoptosis and autophagy by inhibiting the mTOR signaling pathway in endometriosis mouse model. 2.EPHA3 and mTOR may be new therapeutic targets
Mattos et al. [120]	1.vGalectin-3 promoted the development of endometriosis. 2.Galectin-3 inhibitors reduced endometriosis by inhibiting angiogenesis and the enrichment of M2 macrophages.

Fifty percent of human peritoneal leukocytes are macrophages [87], and peritoneal macrophages are involved in inflammatory and infectious diseases through various immunomodulatory mechanisms [88]. The role of macrophages in the pathogenesis of endometriosis is shown in Figure 2. The phagocytic ability of peritoneal macrophages was weakened in patients with endometriosis; thus, these peritoneal macrophages lacked the ability to remove endometrial-like tissues in the peritoneal cavity [89]. The concentration of prostaglandin (PG) E₂ (PGE₂) was elevated in peritoneal fluid from patients with endometriosis [90]. PGE₂ inhibited the expression of annexin A2 on peritoneal macrophages via the EP₂/EP₄ receptor-dependent signaling pathway [91], and inhibited the expression of CD36 on peritoneal macrophages [92]. The decreased annexin A2 and CD36 were associated with weakened macrophage phagocytosis. TGF-β inhibitor increased the expression of CD36 in vitro; thus, TGF-β inhibitor may be a potential treatment [93]. The TSP1-CD47-SIRPα axis promoted the development of endometriosis through several ways. CD47 is a phagocytic check point protein, the expression of CD47 on ectopic endometrial stromal cells (ESCs) was significantly elevated in endometriosis patients [94]; furthermore, as CD47 ligands, thrombospondin-1 (TSP1) and signal regulated protein α (SIRPα) were also increased [93, 94]. The elevated CD47 binding to elevated SIRPα expressed on peritoneal macrophages exhausted macrophage phagocytic ability and improved M2 polarization [94], and elevated CD47 reduced the apoptosis of ectopic ESCs [95]. The increased TSP-1 in peritoneal fluid inhibited apoptosis of vascular endothelial cells [94]. Heme abnormally accumulated in the peritoneal fluid from patients with endometriosis, and the high concentration of heme (30 μmol/L) treatment exacerbated the damage to phagocytic ability [96]. The expression of CD200 was upregulated in endometrial-like tissues and soluble CD200 (sCD200) was also increased in the small veins of endometrial-like tissues [97]. The expression of CD200 receptor in peritoneal macrophages was also elevated. The phagocytic ability of macrophages in vitro gradually decreased with the increasing CD200 levels, indicating that CD200 damaged the macrophage phagocytosis and may facilitate the immune escape of ectopic lesions [98]. The expression of CD200 in the plasma was also elevated; thus, plasma CD200 may be may be a new diagnostic strategy for endometriosis [98].

Figure 2.

Macrophage involvement in the pathogenesis of endometriosis. The phagocytosis to ESCs is weakened, resulting in the inability to clear proliferating ectopic ESCs (A). In addition, abnormal components in ectopic ESCs and peritoneal fluid promote M2 polarization. M2 macrophages promote angiogenesis and lymphangiogenesis, as well as promote the proliferation and invasion of ectopic ESCs (B). The weakened clearance and promoted growth of ESCs eventually cause the development of endometriosis.

M2 macrophages were dominant in endometriotic lesions and peritoneal fluid [99]. The polarization of macrophages toward M2 phenotype was upregulated upon exposure to exosomes and homogenates from ectopic ESCs in vitro, implying that ectopic ESCs were responsible for M2 polarization [100, 101]. The ectopic ESCs-derived CCL2 recruiting macrophages through estrogen receptor β (ERβ) and NF-κB signaling [102]. The ectopic ESCs-derived exosomal miR-301a-3p enhanced M2 polarization via PTEN-PI3K pathway [103]. The ectopic ESCs-derived extracellular vesicular legumain pseudogene 1 (EV-LGMNP1) was increased in endometriosis patients. The elevated EV-LGMNP1 induced M2 polarization, and serum increased EV-LGMNP1 suggested that EV-LGMNP1 may have clinical implications [104]. Abnormally elevated components in peritoneal fluid may also be inducers of M2 polarization, such as IL-17A and sphingosine1-phosphate (S1P) [105, 106]. M2 macrophages accelerated ectopic ESCs proliferation through stat3 activation [107], and contributed to the angiogenesis through producing VEGF-A and TGFβ1 [108]. Macrophages facilitated the invasion of endometrial stromal cells [109]. Macrophages-derived VEGF-C and VEGF-D promotes lymphangiogenesis in a VEGFR1-dependent manner [110].

Macrophages are pivotal in the development of endometriosis, and the drugs based on the regulation of macrophage function are promising therapeutic approaches. MCP-1 is an inducer of macrophage recruitment. Focal adhesion kinase (FAK) inhibitors significantly inhibited the secretion of MCP-1 by ectopic ESCs [111]. The growth of endometriosis-like lesions was reduced by niclosamide via STAT3 and NF-ĸB signaling [112], M2 Macrophages promoted ectopic ESCs viability, this promotion was also inhibited by niclosamide [113]. The expression of erythropoietin-producing hepatocellular carcinoma A3 (EPHA3) was reduced, but the expression of mammalian target of rapamycin (mTOR) was increased in mouse with endometriosis [114]. Xu et al. found that EPHA3 promoted macrophage apoptosis and autophagy by inhibiting the mTOR signaling pathway in mouse model of endometriosis [114]; thus, EPHA3 and mTOR hold potential as therapeutic targets for endometriosis. The authors attempted to use this mechanism to explain the pathogenesis of endometriosis, but the authors only studied the changes in EPHA3 and mTOR protein levels and did not examine in detail which specific molecules in the mTOR signaling pathway play a role. Previous studies have found that macrophage autophagy can be inhibited via PI3K/Akt/mTOR pathway [115], AMPK/mTOR/TFEB pathway [116] and TREM2/mTOR axis [117]; moreover, macrophage apoptosis can be inhibited via Akt/mTOR [118] and AMPK/mTOR pathway [119]. Based on the above discussion, it is worthwhile to investigate which molecules that cause mTOR inhibition are driven by EPHA3 ultimately leading to increased apoptosis and autophagy of macrophages in endometriosis. Galectin-3 promoted the development of endometriosis, galectin-3 inhibitors reduced endometriosis by inhibiting angiogenesis and the enrichment of M2 macrophages [120]. M2 macrophage are dominant in endometriosis patients in the absence of disruption for immune system, In the presence of infection, the number of M1 macrophages increased and replaced the dominance of M2 macrophages [121]. M1 macrophage-derived secretions were beneficial for lesion clearance via polarizing M2 to M1 [122]. LPS-induced macrophages reduced endometriosis-like lesion growth in an IL-10-dependent manner [123]; thus, whether the development of endometriosis can be inhibited by inducing a mild inflammatory response is an interesting question.

Overall, in terms of clinical symptom, macrophages are involved in pain caused by endometriosis. In terms of the mechanism of endometriosis, macrophages play a key role in the weakened clearance (Figure 2A) and promoted growth of ESCs (Figure 2B), and both processes promoted development of endometriosis.

6. The role of macrophages in male idiopathic infertility

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.

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 D2 (PGD2) 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 7.

Abbreviation and full name cross-reference table.

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
PGD2	Prostaglandin D2
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

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 D₂ (PGD₂) 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.

7. Discussion

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 3A). M1 macrophages highly express CD80 and CD86, and secrete pro-inflammatory cytokines (Figure 3A). 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 3A); 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 3B). Strominger et al. divided dMφ into CD11c^high and CD11c^low macrophages (Figure 3B). 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^highdM1 and ITGAX^lowdM2 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^highdM1 than in ITGAX^lowdM2 [141] (Figure 3B). In 2021, Thomas et al. identified placenta-associated macrophages (PAMM) using single-cell sequencing. PAMM was subdivided into 3 populations: PAMM1a (FOLR2⁻CD9^hiCCR2^lo/int), PAMM1b (FOLR2⁻CD9^-/intCCR2⁺) and PAMM2 (HLA-DR^hiFOLR2^hi) (Figure 3B). 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).

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.

8. Conclusion

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.

Declarations

Author contribution statement

All authors listed have significantly contributed to the development and the writing of this article.

Funding statement

Tao duan and Yongsheng yu was supported by National Natural Science Foundation of China, China [82071663 & 81902622].

Data availability statement

No data was used for the research described in the article.

Declaration of interest's statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.