The physiological role of macrophages in reproductive organs

The uterus provides a complex environment that supports embryo implantation and development. Macrophages play crucial roles in endometrial remodeling during the menstrual cycle and early pregnancy, maintaining immune tolerance, and supporting placental formation. 56 The endometrium undergoes dynamic changes in response to the menstrual cycle, repeating phases of proliferation, secretion, and menstruation. During these processes, macrophages play key roles in tissue repair, regeneration, and immune response regulation. M1 macrophages mainly handle inflammation and tissue clearance, whereas M2 macrophages support tissue repair and regeneration. Factors such as estrogen, progesterone, and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) regulate these processes, ensuring appropriate immune responses and tissue remodeling. 9 , 57 The endometrium thickens under the influence of estrogen. Macrophages promote angiogenesis and assist endometrial regeneration primarily showing the M2 phenotype and secreting anti‐inflammatory cytokines to support tissue repair and regeneration. 9 , 58 , 59 , 60 Progesterone influences the endometrium to mature and prepare for pregnancy. Macrophages become activated, supporting immune regulation and tissue maturation. During this phase, M2 macrophages maintain an anti‐inflammatory environment, stabilizing the tissue. 9 , 58 The endometrium sheds during menstruation. Macrophages increase in number and lead to tissue clearance and repair. During this phase, M1 macrophages secrete proinflammatory cytokines, managing tissue clearance and inflammation control. 9 During menstruation, the endometrium resembles a “wound” and the wound‐healing process is crucial for maintaining reproductive function. 60 The number of uterine macrophages peaks during menstruation as the progesterone levels decrease. 61 The apoptotic endometrial cells that are shed during menstruation are phagocytosed by macrophages. 62 Additionally, macrophages express matrix metalloproteinases, which are upregulated during menstruation and contribute to tissue breakdown during the menstrual process. 63 Estrogen significantly influences the dynamics and functions of macrophages. Estrogen regulates the phenotype of macrophages at each stage of the menstrual cycle, enabling appropriate immune responses and tissue remodeling. GM‐CSF plays a crucial role in the proliferation and activation of macrophages, and its secretion is regulated throughout the menstrual cycle to optimize macrophage function. 59 Estrogen action is mediated by intracellular estrogen receptors (ERs) (namely, ERα and ERβ) and by G protein‐coupled estrogen receptor 1 (a plasma membrane protein). 64 A previous study has reported that endometrial macrophages express estrogen‐related receptor beta suggesting that these cells are regulated in an estrogen‐dependent manner. 65 However, the expression of estrogen receptors in macrophages is a topic of debate. RNA sequencing data from three datasets (two mouse and one human), which was derived from peritoneal macrophages did not detect ERβ, whereas mRNA expression of ERα and G protein‐coupled estrogen receptor 1 were observed. 66 Interestingly, it has been reported that estrogen promotes M2 phenotype during cutaneous repair, which is an alternatively activated type macrophage, through estrogen receptor α. 67 Uterine macrophages do not express progesterone receptors indicating that they are indirectly regulated through factors secreted by progesterone‐responsive endometrial cells. 68 According to the aforementioned databases, peritoneal macrophages derived from mice and humans do not exhibit the expression of progesterone receptors. 66 In the uterus, macrophages are recruited to the endometrium in response to seminal fluid and early pregnancy signals during the peri‐implantation period. 69 , 70 Uterine macrophages are involved in the remodeling process and induce the expression of epithelial glycoproteins necessary for embryo implantation. 71 They also contribute to decidualization and trophoblast invasion of the placenta. 72 , 73 Zhang and colleagues summarized the transition from M1 to M2 macrophages during pregnancy. 73 During the implantation phase, activated M1 macrophages produced proinflammatory cytokines such as IL‐1β, TNF‐α, and nitric oxide (NO) which promote an inflammatory response necessary for embryo implantation. As the trophoblasts invade the uterine stroma, decidual macrophages exhibit both M1 and M2 profiles, a state that persists until the early second trimester. This dual profile helps prevent embryo rejection while promoting trophoblast invasion and vascular remodeling. To support fetal development, an M2‐dominant environment is established in the uterus by the end of pregnancy. 73 In experiments using CD206DTR mice, we proved that the depletion of M2 macrophages during the implantation phase resulted in implantation failure (Figure  3 ). 74 The mechanisms of implantation failure are described in Figure  4 . An absence of M2 macrophages led to an excessive increase in M1 macrophages inducing abnormal TNF‐α expression and activating the WNT‐β‐catenin pathway in the uterine epithelium. This caused sustained proliferation of epithelial cells and the impairment of decidualization resulted in implantation failure (Figure  3 ). 74 Furthermore, the depletion of M2 macrophages suppressed the leukemia inhibitory factor (LIF)‐JAK‐STAT3 pathway which is crucial for implantation, 75 thus preventing the endometrium from becoming receptive to the embryo. 74 Other groups have also reported that M2 macrophages contribute to implantation through various mechanisms. The induction of immune tolerance, including regulatory T cells (Tregs) and IL‐10 is essential for implantation. 76 , 77 M2 macrophages induce Tregs 78 and their depletion reduces IL‐10, disrupting immune tolerance and leading to implantation failure. 76 LIF is an essential molecule involved in implantation, partly by chemotactic recruitment of macrophages. In LIF knockout mice, which exhibit implantation failure, macrophages in the uterus are reported to decrease by more than 50%, highlighting the close relationship between macrophages and LIF. 79 Impact of M2 macrophage depletion on uterine implantation and decidualization. In CD206 DTR mice lacking M2 macrophages, no implantation sites were observed in the uterus (lower panel). Compared to wild‐type mice (upper panel), CD206 DTR mice exhibited impaired decidualization of endometrial stromal cells. The mechanism of impaired implantation in M2 macrophage depleted mouse. Depletion of M2 macrophages suppressed the leukemia inhibitory factor (LIF)‐JAK‐STAT3 pathway. M2 macrophages induced regulatory T cells (Treg), 78 and Treg depletion reduced IL‐10 disrupting immune tolerance. The absence of M2 macrophages led to an excessive increase in M1 macrophages inducing abnormal TNF‐α expression and activating the WNT‐β‐catenin pathway in the uterine epithelium. Our study based on human samples also showed that the proportion of M1 macrophages and TNF‐α expression in the endometrium of patients with implantation failure was significantly higher suggesting that correcting the M1/M2 macrophage balance could be a potential indicator for treating implantation failure. 74 Similarly, recent clinical studies have demonstrated that the proportion of CD68+ (M1 + M2) and CD163+ (M2) macrophages in the endometrium during the midsecretory phase is significantly associated with IVF‐ET outcomes. 80

Targeting specific macrophage subtypes has the potential for identifying new therapeutic strategies aimed at improving outcomes in folliculogenesis and implantation disorders. Nanotechnology‐based drug delivery systems are also gaining attention. This review demonstrated that research an understanding of ovarian physiology has advanced primarily by using clodronate in mouse experiments leading to a reduction of pan‐macrophages. This treatment can is classified under nanotechnology therapeutics. The modulation of macrophages by clodronate is effective not only for physiological studies but also in mouse models of obesity, 90 melanoma, 91 and endometriosis. 92 However, further investigation is needed for human application. Targeted therapy that employs nanoparticles to specific macrophages could enhance treatment efficacy while minimizing side effects. 88 This approach may enable effective therapy with minimal side effects. In this review, we divided macrophages into M1 or M2 macrophages, and assessed reproductive phenomena from the points of deviation to M1 or M2 macrophages. Modulating macrophage subtypes may offer new therapeutic strategies for folliculogenesis and implantation disorders. In cases of excessive M1 macrophages, treatment with G‐CSF to induce M2 macrophages has been evaluated. 93 Conversely, in M2 macrophage‐dominant conditions, GM‐CSF treatment may shift macrophages to the M1 phenotype. 93 In this review, we introduced the subclassification of M2 macrophages into M2a, M2b, M2c, and M2d. It may be necessary to evaluate M2 macrophages further based on this reclassification. Additionally, while we presented data by broadly classifying macrophages into M1 and M2 categories, it has been said that this M1/M2 classification fails to fully capture the characteristics of macrophages. 94 In the future, macrophage‐targeted therapies might be useful in personalized medicine, and a more precise classification of macrophages is essential.

Fallopian tubes are essentials component of the female reproductive system and connect the ovaries to the uterus. They play a pivotal role in fertilization and the subsequent implantation of the embryo by facilitating the movement of gametes. 81 Fallopian tubes are known to be affected by various pathological conditions such as infections and endometriosis. Consequently, the development of tubal disease has a relationship with infertility. There are very few animal studies conducted using mice that have investigated the role of macrophages in the fallopian tubes. The below presented studies have primarily focused on humans. Macrophages are primarily located in the epithelium and lamina propria, 82 where they contribute to tissue repair and immune responses. Flow cytometric analysis of immune cells from fimbria specimens showed that the predominant cell type was M1 macrophages, followed by Tregs, CD8 cytotoxic T cells and M2 macrophages. 83 Others found the presence of macrophage in the human tubal epithelium to be minimal. 84 Studies have reported variations in the number of macrophages during the menstrual cycle, with some reporting an increase in the number of macrophages during the secretory phase. 82 , 85 Ovulation is an inflammatory response, following which, the fallopian tubes are exposed to the follicular fluid which is enriched with inflammatory mediators. 28 , 29 , 30 Therefore, these inflammatory mediators increase the number of macrophages in the tubal walls. 86 In conditions such as salpingitis and hydrosalpinx, an increase in the number of macrophages has been reported. 87 , 88 In particular, CD68‐positive M1 macrophages were predominant in salpingitis and were shown to produce proinflammatory cytokines (IL‐6 and IL‐8). 88 Several studies have confirmed that the number of macrophages significantly increases in ectopic pregnancies when compared to normal fallopian tubes. 88 , 89 Proinflammatory cytokines (IL‐6 and IL‐8), derived from macrophages, activate the expression of implantation‐associated molecules and Wnt signaling pathway predisposing the tubal epithelium to an adhesive and receptive state for embryo implantation. 88

Macrophages are critical cells of the immune system. These cells also significantly play roles in reproductive physiology. This review aimed to detail the biological characteristics of macrophages, primarily in the ovaries and uterus. Understanding the diverse functions of macrophages in various stages of the reproductive process is critical to the development of therapeutic strategies. While this review primarily focused on animal studies (mouse models), studies conducted on human participants have been referenced when available. As limitations due to ethical considerations often restrict extensive research in human samples, most of our understanding of macrophages in reproductive organs comes from animal models. Macrophages originate from bone marrow‐derived precursors and are involved in the phagocytosis of foreign substances, antigen presentation, and regulation of inflammation. These cells are found in the ovaries, uterus, fallopian tubes, and mammary glands. 1 Macrophages are also involved in the regulation of the hypothalamic–pituitary–gonadal axis. Macrophages polarize into different functional states in response to environmental signals. 2 , 3 Macrophages exhibit functional plasticity, polarizing into classically activated (M1) macrophages and alternatively activated (M2) macrophages. M2 macrophages are further categorized into subtypes (M2a, M2b, M2c, and M2d), each characterized by distinct surface markers, the type of cytokines secreted, and the functions. 4 Macrophage polarization dynamically shifts according to environmental stimuli, and has an essential role in regulating immune responses and maintaining tissue homeostasis. 5 During the initial stages of infection, M1 macrophages are dominant for eliminating pathogens and promoting inflammation. By contrast, M2 macrophages are dominant for tissue repair phases, for suppressing inflammation, and promoting tissue regeneration. 3 , 6 Chronic inflammation may occur when M1 macrophages are overly activated, leading to tissue damage. In contrast, M2 macrophages promote tumor growth and metastasis in a tumor microenvironment. 7 , 8 M1 macrophages are activated by lipopolysaccharides (LPS) and interferon‐gamma (IFN‐γ) and secrete proinflammatory cytokines necessary for controlling infections. On the other hand, M2 macrophages are activated by IL‐4, IL‐13, and IL‐10 and regulate inflammation and promote tissue repair. M2 macrophages can be further subdivided into M2a, M2b, M2c, and M2d, each induced by different stimuli and having specific functions. 9 , 10 M2a macrophages are induced by IL‐4 and IL‐13, and promote tissue repair and angiogenesis. These cells produce anti‐inflammatory cytokines such as IL‐10. M2b macrophages are induced by immune complexes and toll‐like receptor ligands, secrete increased levels of IL‐10, creating an immunosuppressive environment. M2c macrophages are induced by IL‐10 and TGF‐βcells, and are essential for apoptotic cell clearance and maintaining an anti‐inflammatory environment. M2d macrophages are induced by IL‐6 and LIF, and promote angiogenesis and immune evasion. 9 , 10 However, very few studies have evaluated M2 macrophages subsets in female reproductive organs physiology.

We have no conflicts of interest for this article.