Seminal fluid effects on female reproductive function in laboratory, livestock, and domestic species

Abstract

When seminal fluid is delivered into the female reproductive tract by natural mating or artificial insemination, seminal fluid components elicit biological responses from female tract cells and tissues. This “male to female seminal fluid signaling” affects the chances of conception and pregnancy, depending on the balance of signals in the seminal fluid – which can vary in relation to the genetics and physiological state of individual males. Seminal plasma is instrumental in eliciting female responses, and sperm also contribute. Signaling agents include cytokines and prostaglandins synthesized in the seminal vesicles and other male accessory glands, delivered to the female tract in soluble form, as cargo of seminal extracellular vesicles, or in association with sperm. These agents modulate oviduct and endometrium secretions, and activate immune cell recruitment and gene expression changes in female reproductive tissues, that in turn initiate an adaptive immune response to semen and the conceptus. Collectively these effects can exert either permissive or inhibitory consequences for conception, embryo development, and receptivity to implantation. Extensive studies show seminal fluid effects in porcine, equine, bovine, ovine and canine species, with variations between species due to the site of seminal fluid deposition and female reproductive tract anatomy. Greater insight into the biological function and molecular basis of seminal fluid effects in females has potential applications in novel treatments and management practices to support optimal reproductive outcomes in domestic, livestock and endangered animal species.

Introduction

The biological function of seminal fluid includes effects on the female reproductive tract, with consequences for the chances of conception and reproductive success [1]. Effects in female tissues are mediated largely by seminal plasma, a complex mix of bioactive proteins and other agents produced in response to androgens by male accessory sex organs including the seminal vesicle, prostate and epididymis [2, 3]. Emerging studies show sperm also participate by delivering signaling agents to female reproductive tract cells and tissues [4, 5]. Despite their diverse reproductive tract anatomy and physiology, all species exhibit female tract responses to seminal fluid after coitus, with beneficial or sometimes inhibitory consequences—depending on the composition of seminal fluid signals – that impact reproductive success [1, 6, 7]. Many effects of seminal fluid in the female reproductive tract involve the female immune response, that in turn affect sperm survival and access to oocytes, embryo development and implantation, and quality of placental and fetal development. Developing knowledge of this emerging aspect of reproductive biology is bringing new insight to reproduction science with potential practical applications in many animal species of economic, environmental, and social importance. This review provides a summary of current understanding of the biological effects and physiological significance of male-to-female seminal fluid signaling in a range of mammalian species. In the sections below, we first detail the physiology and immunology of the female response to seminal fluid, and then summarize the experimental evidence for effects on female reproductive events and their consequences in a range of mammalian species.

Effects of seminal fluid in the female reproductive tract

There are substantial differences between species in the anatomy of the female reproductive tract and site of seminal fluid deposition, meaning that different fractions and components of seminal fluid access different female tract compartments and elicit different types of responses (Fig. 1). In the sections below, we provide an overview of the pro-fertility effects of seminal fluid in the cervix, the ovary, the oviduct, and the endometrial lining of the uterus.

Fig. 1.

Summary of current evidence indicating the location in the female reproductive tract of seminal fluid deposition after natural mating (either cranial/anterior vagina (V), or uterus (Ut)), and tissue sites of interaction and physiological responses to seminal fluid, in a range of mammalian species including human, rodent, cattle, pig, sheep, rabbit, horse, camel, dog, cat, and monkey (see text for details on each species). *In artificial insemination (in utero insemination (IUI) in human), washed sperm are usually administered transcervically into the uterus. Created with BioRender.com.

Seminal fluid and microbial clearance

The first and most highly conserved aspect of the response is the acute response to seminal fluid in the cervix within minutes after mating. Upon seminal fluid contact, polymorphonuclear (PMN) cells (predominantly neutrophils) are released into the luminal cavity of the vagina and cervix and engage directly with seminal plasma and sperm. Neutrophil infiltration can extend into the higher reproductive tract, particularly in species such as mice and pigs where whole seminal fluid accesses the uterus. This inflammation-like reaction to semen was first observed in rabbits [8, 9], and has since been described in almost every other mammalian species examined [6, 10]. Rapid leukocytosis also occurs in the cervix of women [11], the uterine lumen of mice [12], and in reproductive tracts of cows [13], dogs [14], horses [15], rabbits [9, 16], sheep [17], and pigs [18], all generally within an hour of mating. Studies in rabbits provided the first insight on post-mating leukocytosis and showed extravasation of leukocytes from peripheral blood within 45 min [16], followed by efflux across the reproductive tract epithelium and accumulation of ~50 million leukocytes within the cervical lumen, and many more in the uterus [9, 16]. The infiltrating neutrophils act to clear excess sperm and seminal debris, and promote recovery of the sterile status of the tract to defend against reproductive tract infection [19]. Potent chemotactic agents in seminal fluid such as interleukin (IL)-8 directly elicit female neutrophilic recruitment across the epithelial barrier, and activate neutrophils to kill and phagocytose microbes [20]. Additionally, seminal fluid contains high levels of diverse anti-microbial peptides such as β-defensins produced in the testis and epididymis that boost anti-microbial activity in the female [21].

Seminal fluid and sperm selection

The second key role for neutrophils recruited into the luminal space after mating or sperm deposition is to selectively remove damaged sperm by phagocytosis or sequestration into neutrophil extracellular traps (NETs). This process entraps a large proportion of ejaculated sperm, retarding their ability to reach the oviduct, access oocytes, and progress to fertilization [22,23,24,25]. Studies in humans suggest neutrophils can selectively remove morphologically abnormal spermatozoa [26], suggesting molecular features determine susceptibility. Repeated mating in rabbits showed specific subsets of sperm can avoid neutrophil capture, even when females mate with more than one male in close succession [27]. The molecules by which neutrophils selectively interact with sperm are not fully defined [28], but one key marker is phosphatidyl serine externalized to the outer leaflet of the surface membrane of sperm during the early stages of apoptotic cell death [29] particularly in sperm exhibiting DNA damage [30]. Components of the diverse repertoire of glycan structures on the sperm glycocalyx acquired during sperm development and maturation likely also contribute to determining sperm fate [31]. Contact with seminal plasma can affect the nature of sperm engagement with neutrophils [22, 24, 25, 32,33,34] in varying ways depending on the species (see details below).

From an evolutionary perspective, there may be female and male advantages for selection and inactivation of particular sperm, such that genetically superior or phenotypically more competent male gametes are favored to promote the likelihood of offspring fitness [35]. It remains to be determined whether neutrophil-mediated mechanisms of sperm selection can respond to environmental factors that differentially favor subsets of sperm in different circumstances.

Seminal fluid and ovulation

In some species, seminal fluid modulates ovarian physiology to promote ovulation and corpus luteum formation (Fig. 1). The effects of seminal fluid in the ovary are thought to occur via unique counter-current exchange mechanisms where prostaglandins and other small molecules can be transferred to the ovarian artery via the uterine vein [36], and through an endocrine action whereby ovulation-inducing factors access the hypothalamic-pituitary axis in the female brain [37].

Effects of seminal fluid on ovulation are most evident in the induced ovulatory species, camels and alpacas, where ovulation is triggered after delivery of seminal plasma by mating or by artificial administration [38, 39]. Rabbit seminal plasma has a similar effect after administration to hormone-primed does [40]. Porcine seminal plasma administered just prior to ovulation reduces the interval between luteinizing hormone surge and ovulation, and elevates plasma progesterone concentration [41, 42]. Similar ovulation-inducing activity can be demonstrated in cattle, horses, and rodents, implying that seminal fluid may also facilitate ovulation in these species [43, 44].

Seminal fluid and embryotrophic cytokines

A further key role for seminal fluid signaling is to influence the cytokine and growth factor environment that modulates development of the preimplantation embryo. As it traverses the oviduct prior to implantation in the uterus, the embryo is exposed to cytokines and growth factors secreted into the luminal compartment [45, 46]. Embryos express cytokine receptors from conception until implantation and several cytokines exert different effects on blastocyst cell number and viability, gene expression and developmental competence [47, 48]. The identity and biological effects of several growth factors and cytokines targeting the pre-implantation embryo have been reviewed previously [45, 46]. Cytokines including granulocytemacrophage colony-stimulating factor (GM-CSF, or CSF-2), CSF-1, and leukemia inhibitory factor (LIF) promote blastocyst development, while others including tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL), and the Type 1 cytokine interferon-γ (IFNG) exert exert potent inhibitory effects [49].

The secretion of these cytokines is regulated at the mRNA level principally by ovarian steroid hormones and, especially in species where seminal fluid reaches the higher tract, by the signaling factors present in seminal fluid. The importance of seminal fluid signaling as a regulator of female tract cytokines is again best indicated by experiments in mice and other rodents. When the effect of seminal plasma is evaluated by comparing expression of oviduct cytokines in females mated to intact males and females mated with males from which the seminal vesicles are surgically removed, there are adverse effects on oviduct cytokine expression. In the absence of seminal plasma stimulation, oviduct expression of embryotrophic LIF, GM-CSF and IL-6 was significantly decreased, while embryotoxic cytokine TRAIL was elevated – resulting in a reduced proportion of females achieving pregnancy [50].

There are now substantial studies showing seminal fluid capacity to induce cytokines from uterine and oviductal epithelial cells is highly conserved across mammalian species. Several in vitro and in vivo studies in livestock species show similar effects of seminal plasma on induction of GM-CSF, IL-6, IL-8 and many other cytokines with embryotrophic effects (see sections below for details).

Effects of seminal fluid on endometrial receptivity for embryo implantation

An important consequence of the female immune response to seminal fluid is to promote immune adaptation to facilitate embryo attachment and implantation. For successful pregnancy, a specialised state of active immune ‘tolerance’ must exist from the very earliest time the embryo first contacts the maternal tissues, which for most species occurs within days of conception. In healthy pregnancy the conceptus does not experience immunological attack at implantation, even though it expresses foreign antigens, including some encoded by paternally-inherited major histocompatibility (MHC) genes. A combination of strategies prevent rejection of the implanting embryo and later the fetal/placental unit - some aspects of the immune response are circumvented, while others are engaged and activated to promote tolerance [51]. Seminal fluid has a direct role in providing immune-regulatory signals, specifically paternal antigens and cytokines, which facilitate induction of pregnancy tolerance [52]. Although implantation of embryos formed ex vivo clearly can occur without contact with seminal fluid, as exemplified in humans and other species where embryo transfer is utilized, there is evidence that endometrial changes induced by seminal plasma improve the likelihood of successful initiation of pregnancy [53].

The benefit of seminal fluid contact is likely to be mediated via immunological effects. Induction of immune tolerance is facilitated by immune cell recruitment and activation in response to seminal fluid. As well as neutrophils, insemination induces macrophages, dendritic cells and T cells to accumulate in the mucosal lining of the cervix, as well as the uterus in mice and some other species [1, 54]. Studies in rodents provide a comprehensive understanding of the molecular and cellular events regulating the immune response to seminal fluid that has informed studies in other animals, all collectively pointing to a conserved mechanism by which seminal fluid affects establishment and progression of pregnancy. These leukocytes are recruited after seminal fluid factors interact with epithelial cells lining the reproductive tissues to cause expression and release of a range of pro-inflammatory cytokines and chemokines that in turn cause leukocytes to extravasate from the blood stream and enter the mucosal surface. Once in the endometrium, these cells take up antigens delivered in seminal fluid, and traffic to draining lymph nodes where they activate an adaptive immune response. The responding T cells are then recruited back into the uterus where they affect several aspects of embryo implantation and placental development.

In species where seminal fluid is largely contained within the cervix such as humans, a similar cascade of immune changes is induced in the mucosal lining of the ectocervix, where dendritic cells, macrophages and T cells are recruited after seminal fluid deposition [55, 56]. There are immune consequences in the endometrial lining of the uterus that promote tolerance and favour embryo implantation, and this may be partly due to indirect effects mediated via draining lymph nodes, and partly due to direct effects of sperm reaching the higher reproductive tract [57].

Specific types of immune cells are required to support the decidualization response in the uterus and to regulate invasion of conceptus cells at implantation [51]. Studies in mice and humans clearly demonstrate that macrophages, dendritic cells, uterine natural killer cells, and T cells are all involved in supporting implantation and allowing a robust placenta to develop [52]. Understanding of the immune response to implantation is less well developed in other species, but emerging studies point to comparable mechanisms across mammalian species [58,59,60].

A key element of this active immune tolerance is engagement of special T lymphocytes known as regulatory T cells (Treg cells) [61]. Treg cells are critical for preventing inappropriate immune activation [62, 63] via suppressing effector functions in T cells, B cells, NK cells, dendritic cell and macrophages. In the context of reproduction, Treg cells are essential for tolerance of male and female gametes, embryos, and the fetal-placental tissues. Uterine Treg cells are induced to proliferate in draining lymph nodes and then recruited into the uterus before embryo implantation. Once in place, they constrain inflammation and permit the immune system to allow the intimate associations between maternal cells and the conceptus-derived placental trophoblasts required for placental development and fetal growth. Genetic mismatch, particularly in the MHC genes, serves to increase the numbers of Treg cells in the implantation site and this stimulates the likelihood of healthy pregnancy and benefits fetal growth. This is clearly evident in mice where congenic lines differing only in MHC genes demonstrate the significance of MHC disparity for litter size and fetal weight, due to the facilitating effects of uterine NK cells and Treg cells on transformation of the uterine vasculature to increase vascular supply to the placenta [64, 65]. A similar requirement for Treg cells is seen in the endometrium of pigs for successful implantation [66], particularly in embryo transfer settings when there is complete MHC disparity between the recipient sow and transferred embryos [67].

Studies in mice show a crucial role for seminal fluid in preparation for embryo implantation is through the provision of antigens and cytokines that stimulate expansion of the endometrial Treg cell population [54]. To ensure enough Treg cells are present in the implantation site, their activation and proliferation during the time between conception and implantation is pivotal, and the quality of response at that time sets the immunological scene for the remainder of pregnancy [65, 68]. The female T cell response to paternal MHC antigens is initiated immediately after immune cells first contact those antigens transmitted within seminal fluid at mating, such that the T cell pool generated during the pre-implantation period is expanded by the time of implantation [69, 70]. Soluble MHC is present in seminal plasma and cell-associated MHC is carried by sperm, extracellular vesicles, seminal leukocytes and/or desquamated genital tract epithelial cells. Dendritic cells recruited into the uterus in response to seminal fluid-induced cytokines are critical for taking up male partner-derived antigens and eliciting Treg cell-mediated tolerance [71,72,73]. Studies in humans implicate a similar mechanism whereby seminal fluid induces a T cell response [56]. A recent study in pigs shows a comparable effect of seminal plasma in selectively inducing uterine Treg cells [66]. Whether similar mechanisms occur in other species remains to be established.

Collectively, there is now compelling evidence to demonstrate that seminal fluid can influence a range of compartments in the female reproductive tract. When the composition of seminal plasma factors is optimal, the result is to ensure that high quality sperm access oocytes, to reduce the transmission of sexually-transmitted infection, to promote ovulation and corpus luteum development, and to maximise the chances of conception, embryo development and implantation. These effects are summarized in Fig. 2.

Fig. 2.

Schematic representation of current understanding of the biological effects by which seminal fluid signaling induces a permissive response to increase the chances of reproductive success. The information in the Figure is drawn from mouse, but there is evidence for similar effects in a range of other mammalian species (see text for details). Permissive seminal fluid component TGFB, working together with immune-regulatory agents CD38 and E-series prostaglandins (PGE), promotes recruitment of neutrophils, dendritic cells, and macrophages in the cervix and uterus. Seminal fluid factors IL-8 and IFNG can attenuate these permissive effects, exerting inhibitory actions when present at high concentrations. Neutrophils traffic into the luminal space to remove microbes and eliminate suboptimal sperm, leaving high quality sperm to access oocytes. Embryotrophic cytokines and growth factors induced by seminal fluid are secreted into the luminal cavity to support preimplantation embryo development. Dendritic cells carry seminal fluid antigens to draining lymph nodes and elicit activation and proliferation of immune-suppressive Treg cells. Treg cells then leave the lymph nodes and circulate via peripheral blood to be recruited into the implantation site where they interact with other immune cells (macrophages and uterine natural killer (uNK) cells) to promote decidualization and generate an endometrial environment conducive to embryo implantation and placental development. Created with BioRender.com.

Male to female signaling factors in seminal fluid

Several studies have investigated the identity of active factors present in seminal fluid. Early experiments in mice using surgical ablation to remove the male accessory glands demonstrated that many active factors in seminal fluid are carried in the seminal plasma fraction [74], either as soluble agents or within the cargo of extracellular vesicles [75]. These agents are derived from seminal vesicles and the prostate, with smaller proportions produced by the testis, epididymis, bulbourethral and periurethral glands [76, 77]. Sperm also carry signaling agents and contribute to mediating effects of seminal fluid on the female reproductive tract immune response [4, 78]. Many sperm-borne signaling factors are likely to be acquired as seminal extracellular vesicles absorbed onto the sperm surface after release from the seminal vesicle, prostate, and epididymis [73, 77, 79].

The seminal plasma factors that signal to female cells and tissues include cytokines, sex hormones, and prostaglandins [57, 80, 81]. Although the specific tissue origins of some key signaling components in seminal plasma are known, others remain to be identified. Using protein chromatography, the cytokine transforming growth factor-β (TGFG) synthesised in the seminal vesicle was identified as the principal active factor in mouse seminal plasma that induces the uterine leukocytic response [82]. TGFβ has also been identified as an important seminal fluid signaling factor in women [83], pigs [84] and sheep [85]. Studies to evaluate TGFβ in seminal plasma of other species are not yet complete.

In addition to TGFG, prostaglandins are powerful signaling agents present in seminal plasma. In particular, prostaglandin E (PGE) has been identified to be present in human seminal plasma, particularly in the 19-hydroxy (19-OH) form [86]. In vitro experiments using human cervical explants has shown that 19-OH PGE induces the expression of IL-8 and suppresses the anti-inflammatory molecule, secretory leukocyte protease inhibitor [87]. While PGE is undetectable in rodent and porcine seminal fluid, prostaglandins are present in horse seminal plasma and may play a role in seminal fluid signaling in this species [88].

Other active factors potentially contribute to seminal fluid signaling. Experiments in humans show effects of IL-8 [89] and soluble HLA-G [90], while the cysteine-rich secretory protein-3 (CRISP3) has been implicated in the equine [32]. Bacterial products including lipopolysaccharide [91] and potentially other ligands for Toll-like receptors (TLRs) appear to be involved. While TLR4 expressed by uterine epithelial cells is required to elicit a female response in mice [92, 93], in cattle and other species, TLR2-mediated signaling is paramount for sperm-mediated induction of cytokines in reproductive tract epithelial cells [94, 95].

Not all agents act to promote gene expression changes in female tract cells; IFNG is a potent inhibitor of seminal fluid signaling mediated by TGFB [96]. There is emerging evidence that metabolic perturbation, infection, and/or exposures to environmental toxins can modulate the composition of seminal plasma, impacting the balance of permissive and inhibitory agents and therefore the quality of the female immune response to seminal fluid [97,98,99,100].

Studies in several species shown that sperm also contributes to the female immune response to seminal fluid. While the molecular basis of direct sperm-mediated signaling remains unclear, in mice TLR4 ligands carried by sperm are essential [4]. Extracellular vesicles derived from the seminal vesicle, prostate and epididymis can be absorbed onto the sperm surface and are likely to be involved in eliciting the female tract response [79]. It seems likely that different species will utilize a range of factors in different proportions, depending on the species and the anatomical site of semen deposition. Substantial research will be required to identify the full range of active factors present in seminal fluid of different species.

In several species, effects of seminal plasma on ovulation are mediated by nerve growth factor-β (NGFB). Originally described in camel seminal plasma, NGFB acts to induce pituitary release of luteinizing hormone to cause ovulation [37, 101], as well as promoting corpus luteum development and steroidogenic function [102, 103]. NGFB is similarly implicated as the ovulation-inducing factor in rabbit and cattle [104]. The ovulation-inducing properties of NGFB in seminal plasma may have effects in a broad range of species [37].

In summary, data to date points to seminal plasma containing a wide array of different molecular signaling agents from various male reproductive gland tissues. Importantly, the composition of seminal fluid cytokines and other components are not static – their abundance and relative balance shift in response to various physiological and pathological states. Many health conditions and environmental exposures can attenuate the levels of permissive cytokines, and elevate pro-inflammatory cytokines in seminal fluid [105]. This includes local and systemic viral and bacterial infection, metabolic disturbances, toxins and drugs, and other inflammatory injuries and exposures [57, 77]. In broad terms, the anti-inflammatory cytokines in seminal fluid appear to exert beneficial or ‘permissive’ effects on fertility. In contrast, pro-inflammatory cytokines in seminal fluid are associated with reduced fertility, particularly in excessive abundance when the balance between anti-inflammatory and pro-inflammatory cytokines tips too low.

Female effects of seminal fluid in different mammalian species

Seminal fluid signaling in mice

Mice are an important model species for defining the effects of seminal fluid on the female tract response, and are the species in which the female response has been studied most extensively (Table 1). In mice, an immune response to seminal fluid is initiated when seminal proteins interact with estrogen-primed cervical and uterine epithelial cells to activate synthesis of pro-inflammatory cytokines including GM-CSF, IL-6, macrophage chemotactic protein-1, IL-8 (KC), and an array of chemokines [93]. The pro-inflammatory cytokines elicit recruitment and accumulation in the subepithelial stromal tissue of leukocytes including macrophages, dendritic cells, and granulocytes [74, 106]. Large populations of neutrophils migrate through the epithelial surface and efflux into the luminal cavity [12, 74, 107]. This leukocytic response is transient and is resolved prior to embryo implantation, in parallel with declining inflammatory cytokine release [108].

Table 1. Evidence of seminal fluid impacts on female reproductive physiology and pregnancy outcomes in different mammalian species.

Species	Demonstrated seminal fluid effects
Rodent	- inducing molecular changes in the uterus relevant to immune changes and uterine receptivity [74, 82, 107, 117, 127, 240,241,242,243,244] - recruitment of neutrophils, macrophages, dendritic cells, T cells including Treg cells and gamma/delta+ T cells [4, 68,69,70,71,72, 110, 111, 114, 116, 117, 243, 245] - increasing blood vessel dilation and blood supply in the uterus [64]- improved implantation rate [50, 120, 122, 124, 243] - improved pregnancy rate [119, 121, 245] - improved offspring health and development [50, 241]
Human	- inducing molecular changes in the vagina, cervix and uterus, relevant to immune changes, uterine receptivity, decidualization and angiogenesis [55, 83, 87, 89, 96, 129, 130, 132,133,134, 137, 246,247,248,249,250,251,252,253,254,255,256,257] - recruitment of tolerogenic dendritic cells, macrophages, NK cells and T cells [11, 27, 55, 136, 138, 258] - promoting decidualization [253]- associated with lower risk of pregnancy disorder preeclampsia [139] - lack of exposure associated with increased risk of pregnancy-induced hypertension [139], lower birth weight [259] and altered offspring metabolic phenotype [260, 261]
Porcine	- inducing LH surge and ovulation [41, 161, 162] - inducing molecular changes (including miRNAs) in the cervix and uterus relevant to the inflammatory pathway, immune response, tissue repairing, reduction-oxidation system, sperm transport and binding, potential ion channel and the neuroendocrine response [66, 149, 150, 152,153,154,155,156,157, 164, 262, 263] - recruitment of polymorphonuclear neutrophils in uterine lavage [18, 264] - suppressing polymorphonuclear cell infiltration in the uterine sub-epithelial connective tissues [144]- recruitment of dendritic cells, macrophages [42, 146, 154], FOXP3+ T cells [66], and CD2+ and CD8+ T cells [158] - increasing vascular bed development and uterine vascularisation [159, 160] - increasing farrowing rate [165, 166]
Rabbit	- inducing gene expression changes in the uterus and cervix [172, 173, 175], including proinflammatory and pro-angiogenic genes after exposure [171] - recruitment of leukocytes [8, 9, 16, 169]
Horse	- inducing molecular changes relevant to the immune response in the endometrium [182, 183, 185, 186, 265] - recruitment and activation of polymorphonuclear eosinophils and neutrophils [15, 178, 186, 191, 194] - suppressing sperm-neutrophil binding [22, 32, 179, 181, 192] and increasing clearance of PMNs [195] - decreasing T cell percentage in peripheral blood [188] and increasing helper T cells in the uterus [187]- increasing time-averaged maximum velocity of uterine arteries [189] - increasing pregnancy rate [22, 179, 191, 196]
Cow	- inducing molecular changes relevant to the immune response [5, 200, 205, 206, 213, 215, 266] - recruitment of polymorphonuclear neutrophils [24, 33, 200]- increases sperm-neutrophil binding [199] and neutrophil extracellular trap formation [24, 204] - increasing uterine blood flow volume [207] - increasing EGF expression in repeat breeders [209] and improving conception rate [208, 210]
Sheep	- inducing an inflammatory response in the uterus and cervix after exposure [220, 221] - reducing sperm-neutrophil binding [222] - recruitment of neutrophils and macrophages [219, 220, 267] - improved pregnancy rate [34, 223, 224]
Dog	- storing of sperm in the female tract [225,226,227]- reducing sperm attachment to polymorphonuclear cells [226] - inducing uterine vasodilation [226] - increasing odds of conception [229]
Camel	- inducing LH surge [233, 268]- promoting ovulation [268, 269] - inducing an inflammatory response in the uterus [235] - inducing endometrial vascular changes [234]
Macaque	- recruitment of CD11c+ antigen presenting cells and T cells [230]
Cat	- inducing ovulation [231] - recruitment of Treg cells [232]

Detailed studies in different surgical and genetic models in mice demonstrate how the female immune response to seminal fluid facilitates establishment of pregnancy. As well as clearance from the uterine cavity of microorganisms introduced at mating [10] and induction of Treg cells to confer tolerance at implantation [69, 70], seminal fluid induces the oviduct and uterus to provide trophic factors that promote development of the pre-implantation embryo [50, 109], and induces expression of embryo attachment ligands and angiogenic factors that promote embryo implantation [110]. Macrophage populations in corpora lutea are augmented by exposure of the female tract to seminal plasma constituents [111], and these cells have central functions in development of the luteal vasculature and the progesterone secretion required for endometrial receptivity [112].

The mechanism by which seminal fluid promotes tolerance to paternal alloantigens is well defined in mice. Studies in T cell transgenic mice clearly demonstrate that priming of the female immune response commences upon exposure to paternally-derived transplantation antigens in the seminal fluid [70, 71]. Within 2 days of conception an expanded population of Treg cells can be detected within the uterine lymph nodes [69, 113, 114]. By day 3.5 post coitum, when embryos implant in mice, Treg cells also accumulate in the uterus and are increased > 2-fold compared with estrus numbers [69, 70].

This increase in Treg cells clearly depends on seminal plasma, as when seminal vesicles are excised from male mice before mating, there is no expansion in Treg cell numbers [69, 70]. TGFB is the prime candidate for this seminal plasma factor, since TGFB is promotes Treg cell generation from precursor T cells [115], and exogenous TGFB increased vaginal Treg cell numbers and prevented spontaneous fetal loss in abortion-prone mice [116]. Sperm also provide Treg-inducing and stabilizing factors, as females mated with vasectomized males show reduced expression of the signature Treg cell transcription factor FOXP3 [70] and have fewer Treg cells in draining lymph nodes [4].

CD4^-CD8^- T cells expressing gamma delta T cell receptors (γδ T cells) are particularly responsive to seminal fluid regulation [117]. Mating with intact males caused a 8.3-fold increase in uterine γδ T cell abundance compared to estrous virgin females, and a 22.4-fold increase in the proportion of γδ T cells expressing proliferation marker Ki67. Seminal plasma was critical for γδ T cell accumulation and activation in the endometrium, and similar changes occurred in uterine-draining lymph nodes but not spleen [117]. This shows that γδ T cells are prominent in the immune response to seminal fluid and imply key roles in uterine immune regulation. It is not yet clear how reproductive tract γδ T cells participate in reproductive events – they may assist in quality control of implanting embryos as well as mucosal immune defence [118].

In mice, the success and quality of the outcome are compromised if females are not exposed to seminal plasma [50]. Experiments in which the seminal vesicle, prostate or coagulating glands are surgically removed from mice, rats and hamsters prior to mating each show that seminal vesicle fluid is the most vital non-sperm component of the ejaculate, and support the interpretation that seminal plasma is essential for optimal sperm survival and fertilization potential [119,120,121,122]. Embryo transfer to recipients prepared without contact with seminal fluid causes elevated fetal loss and abnormality, compared to when vasectomized males are used [123]. Confirmation that the effects of seminal plasma are mediated partly through promoting embryotrophic cytokines in the female tract is provided by embryo transfer studies [50]. In other rodent species, implantation rates and fetal growth are similarly impaired unless females are inseminated prior to embryo transfer [124]. While immune-mediated mechanisms to promote expression of embryotrophic cytokines and boost immune tolerance at implantation are major underlying mechanisms, there is also evidence from transcriptional studies in mice of seminal fluid effects on decidualization, vascular changes, and other non-immune mechanisms promoting endometrial receptivity [108, 117, 125].

Effects of seminal plasma on ovulation may also contribute. In mice, seminal plasma effects in the ovary are mediated via recruitment and activation of ovarian macrophages, with consequences for luteal development and elevated progesterone synthesis [111, 112]. Seminal plasma relaxin is also implicated in promoting ovulation through direct effects on connective tissue remodeling in the follicle wall [126, 127].

Seminal fluid signaling in human

The cervix is the site for seminal fluid deposition following coitus in women and is a major effector site for immune responses in the human female genital tract [128]. Early studies documented that artificial insemination (AI) of human sperm into the cervix results in an influx of leukocytes, predominantly neutrophils into the surrounding tissue [11, 27]. The seminal plasma fraction is demonstrated to be important in this process, as in vitro and in vivo experiments show that addition of seminal plasma results in the induction of GM-CSF, IL-6, IL-8, macrophage chemotactic protein (MCP)-1, macrophage inflammatory protein-3A (MIP-3a and IL01A [55, 129]. This transcriptional response is accompanied by leukocyte recruitment of predominantly macrophages and dendritic cells, but also memory T cells [55].

In vitro experiments clearly demonstrate that seminal plasma induces dose-dependent changes in primary endometrial epithelial cell expression of genes encoding IL-1B, IL-6, LIF and TGFB, particularly in cells recovered during the mid-secretory phase [89, 130]. Endometrial tissue fragments respond similarly, with 10% seminal plasma inducing synthesis of MIP-1B, GM-CSF, IL-1B, macrophage-derived chemokine (MDC), IL-10, and growth-regulated oncogene (GRO), linked with enhanced survival of stromal cells and proliferation of endometrial tissue [131]. A recent RNAseq study using endometrial epithelial organoids showed consistent effects of seminal plasma on gene expression. Enrichment analysis revealed pathways linked to immune response, hormone signaling and epithelial-mesenchymal transition, all processes associated with endometrial receptivity [132].

Seminal plasma has been demonstrated to enhance and accelerate the decidualization response in human endometrial stromal cells in vitro [133]. Experiments using primary endometrial epithelial cells show contact with seminal plasma elicits gene expression pathways associated with leukocyte recruitment and endothelial cell migration, as well as cell proliferation and viability in both endometrial epithelial cells and stromal fibroblasts [134]. This was accompanied by robust changes in pro-inflammatory cytokine and chemokine proteins (CCL2, CSF-2, CSF-3, IL-6, CXCL8 [IL-8], vascular endothelial growth factor-A [VEGF-A]) and chemotactic activity for T cells and macrophages [134], indicating that seminal fluid exposure in the endometrium could induce leukocyte recruitment similar to that observed in the cervix post-coitus.

Whether sufficient seminal fluid reaches the endometrial surface to have any impact on human uterine receptivity in vivo has been a contentious point. The cervical mucus selectively releases a proportion of sperm that ascend to the uterus and fallopian tube to fertilize the oocyte. Studies using albumin-coated microbeads demonstrate that substantial peristaltic contractile activity in the uterus acts to propel sperm and particulate material into the higher tract, with microbeads reaching the fallopian tubes within one minute of deposition in the cervix [135]. Furthermore, seminal plasma TGFB is absorbed onto the surface of sperm, showing carriage by sperm reasonably could transport seminal plasma factors to access the endometrium [78].

A limited number of studies point to effects on the uterine immune response in vivo in humans. Using endometrial biopsies, marked increases in CD16^-/CD56^bright uterine natural killer (uNK) cells were found after seminal fluid contact prior to ovulation [136]. In a recent Danish study that utilized RNAseq in endometrial biopsies to evaluate effects of seminal fluid transmission in vivo, consistent effects on endometrial transcriptional response were reported [137]. The gene set enrichment analysis showed a positive enrichment of pathways associated with immune response, cell viability, proliferation, and cellular movement. Moreover, pathways involved in implantation, embryo development, oocyte maturation, and angiogenesis were positively enriched [137].

There is some evidence consistent with a role for seminal plasma in induction of Treg cells in women, although definitive experiments have not yet been conducted. In in vitro experiments, seminal plasma induces the differentiation of tolerogenic dendritic cells, potentially through effects mediated by E-series prostaglandins [138]. TGFB within seminal plasma appears to contribute to this response [83]. Further studies are required to increase understanding of seminal fluid signaling in humans.

Clinical studies support an effect on seminal fluid exposure on the outcomes of pregnancy. Immune disorders of pregnancy such as preeclampsia are more common in the first conception, after a short period of co-habitation, after a long period of co-habitation when barrier contraceptives are used, or when pregnancy is conceived with a new male partner [139, 140]. Data from IVF and related reproductive technologies support a benefit of seminal fluid signaling at conception, with the absence of seminal plasma in the in vitro setting potentially contributing to higher rates of implantation failure and decreased embryo quality [53]. The evidence for a benefit of seminal fluid contact is now compelling, as demonstrated by a meta-analysis of 2,204 patients in seven random controlled trials (RCTs) showing a 23% improvement in clinical pregnancy rate when seminal plasma is delivered to the reproductive tract artificially or by sexual intercourse (relative risk [RR], 1.23; 95% confidence interval [CI], 1.06–1.42; P < 0.006) [53], and subsequent meta-analyses draw similar conclusions [141].

Seminal fluid signaling in pigs

In pigs, seminal fluid induces a well-described inflammatory cascade regulated by similar processes to mouse and humans. Like mice, pigs are intra-uterine inseminators resulting in direct contact between seminal fluid and epithelial cells lining the endometrium. Neutrophils are rapidly recruited from the endometrial stroma into the uterine lumen after insemination [18, 142,143,144], where they appear to sequester a subset of viable spermatozoa, but not non-viable spermatozoa [145]. This neutrophil influx is accompanied by accumulation of macrophages and dendritic cells, granulocytes and lymphocytes in the endometrial stroma [146,147,148]. Experiments with vasectomized boars show that seminal plasma constituents mediate the response [146]. Studies in gilts have shown that specific factors present in seminal plasma interact with uterine cells to induce expression of GM-CSF, IL-6 and MCP-1 and IL-10 and that neuroendocrine stimulation resulting from the physical act of mating is not necessary for this response. Similar to humans and mice, seminal fluid exposure upregulates cytokines IL-6, IL-10 and TGFB1, TGFB2, and TGFB3 in endometrial epithelial cells, as well as genes relevant to tissue repairing and cell adhesion pathways [145, 149]. Transcriptional changes seen in cell culture models are even more pronounced in uterine tissue in vivo, where a total of 1,600 genes were differentially expressed after seminal plasma infusion, with enrichment analysis showing overrepresentation of genes and pathways associated with the immune response, cytokine signaling, cell cycle, cell adhesion, and hormone response [150]. The initial transcriptional response involves sperm and seminal plasma – as well as neuroendocrine effects of the mating event – and extends to the oviduct where genes impacting sperm survival are regulated [151,152,153]. In the endometrium, its impact extends through the pre-implantation phase and may contribute to a more pronounced anti-inflammatory environment as receptivity to attachment is acquired [154, 155]. Seminal plasma-mediated miRNA regulation in the female tract may contribute to these gene expression changes [156].

GM-CSF, IL-6 and MCP-1 have chemotactic properties and are implicated in regulating macrophages and dendritic cell recruitment into the endometrial stroma. Interestingly, exposure to sperm in addition to seminal plasma alters the seminal plasma response [154, 157]. While the luminal neutrophil response is resolved within 24 hours [18], the inflammatory cells infiltrating the endometrium appear to persist and undergo differentiation in the tissue for several days, expanding the endometrial MHC class II⁺ dendritic cell pool for the duration of the pre-implantation period [146, 154]. This transition in leukocyte phenotype is likely to be driven by the more rapid termination of inflammatory cytokine synthesis that occurs in gilts exposed to seminal plasma [154]. Treg cells likely contribute to resolution of inflammation and potentially improved receptivity for implantation, as seminal plasma increased endometrial FOXP3⁺ Treg cells on day 6 post-treatment and overall T cell number on day 13 post-treatment, accompanied by upregulated IL-10 expression [66]. Seminal plasma components heparin-binding spermadhesins and PSP-I/PSP-II heterodimers are implicated in mediating CD4⁺ and CD8⁺ T cell recruitment [158]. The leukocytic response to seminal plasma is accompanied by an elevated endometrial vascular response, which may in turn facilitate embryo attachment and development [159, 160].

Two studies have linked elevated expression of embryotrophic cytokines in the endometrium with seminal plasma-induced changes in embryo development. While one study demonstrated advanced development of blastocysts 6 days after infusion [150]. In contrast, another study showed an increase in the number of blastocysts and a lower degree of variance of developmental stage within the embryo population, with potential benefits for implantation success [154].

Studies in pigs have been instrumental for exploring the effects of seminal fluid on ovarian function. In estrous pigs, there is a reduction in the interval between the LH surge and ovulation in response to natural mating [161] or seminal plasma administered transcervically [41, 162]. The effects appear to be mediated by local transport of seminal constituents or semen-induced effector molecules originating in proximal parts of the tract, because the effect is seen only in ovaries ipsilateral to uterine horns receiving the seminal stimulus. Seminal plasma also promotes development of the corpus luteum and steroidogenic capacity [42], with infiltrating macrophages implicated as mediating cells in this process.

The identity of seminal plasma components eliciting the female response in pigs is not well defined. Porcine seminal plasma contains a wide array of cytokines with potential to interact with female tissues including high levels of the neutrophil chemokine IL-8 [163]. TGFB is abundant in porcine seminal plasma, but it is unlikely to be the sole relevant agent as no correlation between TGFB content and boar reproductive performance by AI with diluted semen was found [84]. Seminal extracellular vesicles may play a role in the upregulation of immune and inflammatory genes in the endometrium, as isolated seminal extracellular vesicles elicited gene expression changes in porcine uterine epithelial cells in vitro, with induction of transcripts related to immune response, inflammatory response, and their associated signaling pathways [164]. The effects of seminal extracellular vesicles on female tissues are implicated in pro-fertility effects, with oxytocin levels in seminal extracellular vesicles associated with improved fertility outcomes including higher farrowing rate [165]. In a large on-farm study in Serbia, intrauterine infusion of seminal plasma produced significantly higher farrowing rate and more live-born piglets per litter compared with the control sows, showing that intrauterine infusion of seminal plasma is effective in increasing the fertility rate in artificially inseminated sows, under the conditions of practical intensive pig production [166]. There are also beneficial effects of uterine treatments with seminal or leukocyte antigens at previous estrous events [167, 168], supporting a biological mechanism by which seminal plasma primes the adaptive immune response to seminal antigens.

Seminal fluid signaling in rabbit

The first reported evidence of an inflammatory reaction to semen was observed in rabbits in 1952 [8], when pseudopregnant or estrus rabbits were inseminated with semen and the resulting leukocyte influx was examined. The study demonstrated that a leukocytic influx in response to seminal fluid was evident under both hormonal conditions, but that a stronger response occurred in the uterus of pseudopregnant rabbits. Interestingly, administration of sterile epididymal sperm failed to induce a significant reaction, leading the authors to suggest that bacterial contamination was the reason for the leukocytic influx [8]. Subsequent studies showed that both seminal plasma and sperm contribute to the initiation of the leukocytic response in rabbits [9, 16, 169]. In the presence of seminal plasma, rabbit sperm show similar viability and potentially increased motility, but fertility was not improved [170]. Transmission of seminal fluid by natural mating induced upregulation of TGFB1, TGFB2, TGFB3, VEGFA, CTEN and NGF in the female tract [171, 172], while vaginal infusion of seminal plasma increased NR3C1 expression in the infundibulum and TGFB3 and VEGFA localised to the oviduct [171, 173]. Rabbits have also been utilized to provide evidence for rapid transport of seminal fluid components to distal parts of the female reproductive tract. Using bucks immunized with fd-tet, male-derived Ig could be detected in the uterus within 20 min of intromission [174]. The identity of seminal fluid signaling molecules is unknown, but IL-1B has been identified to be abundantly expressed by the prostate and seminal vesicles, and is suggested to upregulate NGF synthesis in the uterus [175]. Factors that drive decapacitation of sperm in the female tract have also been investigated [176].

Seminal fluid signaling in horse and donkey

In equine species, seminal fluid accesses the uterus after coitus and induces recruitment of neutrophils across the uterine epithelium. Elevated numbers of PMNs are seen in vaginal lavage collected 6 h after sperm or seminal plasma treatment [15, 177, 178]. These neutrophils bind to and then phagocytose sperm [22, 179], through formation of neutrophil extracellular traps (NETs) [33]. The presence of seminal plasma inhibits this process, protecting viable but not non-viable sperm [180]. A role CRISP3 in seminal plasma has been indicated in mediating the interaction between sperm and neutrophils [32], and has been shown to suppress blood PMN binding to live sperm but not dead sperm or bacteria [181].

Seminal plasma treatment induced the expression of proinflammatory cytokines IL-1B and IL-8, and downregulated TNF in equine endometrial biopsies [182]. In cultured endometrial cells, compared to sperm exposure only, seminal plasma elicited differentially expressed genes associated with enriched functional terms including antigen processing and regulation, immune/inflammatory response, and embryo growth and development [183]. Similar effects in vivo were seen after seminal plasma infusion into the uterus – with upregulated PGF2A at 24 h post-treatment [184], and upregulated IL1B, IL8, IL10, TGFB and COX2 at 7 days after infusion in mares [185]. These gene expression changes are linked with altered uterine immune cell populations, specifically increased eosinophil number in uterine biopsies [186]. Insemination with semen increased the number of CD4⁺ helper T cells in endometrial tissues within 48 h after treatment [187], while seminal plasma exposure decreased the percentage of Treg cells in peripheral blood, but had no detectable impact on endometrial Treg number 24 h after treatment [188]. Lasting impacts have been reported, with seminal fluid treatment leading to increased time-averaged maximum velocity of uterine arteries and uterine inflammation [189].

Insemination with fertile sperm suspended in seminal plasma was reported in one study to yield a significantly improved pregnancy rate compared to suspension in semen extender (77% vs. 5%) [22], but not all studies show a similar benefit [178]. The inflammatory response to seminal fluid likely contributes to a major issue in equine species of persistent endometritis [190]. It may reflect an uncontrolled manifestation of the physiological inflammation normally elicited by seminal fluid, as a persistent pro-inflammatory shift in uterine cytokines was induced by repeatedly frozen-and-thawed semen in mares susceptible to endometritis [185].

Studies into the effectiveness of AI in the female donkey have compared various semen extenders with seminal plasma in glycine, or ethylene glycol cryoprotected sperm [191]. While the use of semen extenders improved total and progressive motility of sperm, there was an improved pregnancy rate when seminal plasma was present (8/13 pregnant with use of seminal plasma, 3/13 pregnant with use of extender). The degree of neutrophil-mediated clearance of sperm was suppressed by seminal plasma in jennies due to reduced sperm-neutrophil attachment [191, 192]. This may reflect effects of seminal plasma on neutrophil activation state [193], as seminal plasma facilitates the neutrophil response to sperm [194], and promotes faster clearance after insemination [195]. There may be benefits for fertility as faster neutrophil clearance was associated with improved fertility [195], and jennies inseminated with thawed sperm with added seminal plasma showed a trend towards improved fertility that was significant when uterine luminal fluid was also supplemented [196]. Together, these observations are consistent with a regulatory role for seminal plasma during the peri-conceptional period in the donkey.

Seminal fluid signaling in cows

Bovine semen, unlike equine semen, is deposited into the vagina and sperm migrate through the cervix into the uterus leaving the bulk of seminal plasma behind in the lower tract [24]. However, ejaculated sperm are coated in seminal plasma proteins to allow seminal plasma to access the bovine uterus [197, 198]. The potential effects of seminal plasma in the uterus are relevant, given that intra-uterine AI is commonly used to maximize reproductive efficiency. In studies utilizing intra-uterine AI, there is evidence for neutrophil migration following AI, even when seminal plasma proteins are extensively diluted [24, 199, 200]. A greater proportion of sperm are sequestered by neutrophils when sperm are from low quality, as opposed to high quality bulls, suggesting differential susceptibility to neutrophil-mediated killing depending on bull factors [199].

Several studies show sperm-neutrophil binding and NET formation are elevated by components of seminal plasma [24]. Others report that addition of bovine seminal plasma to diluted or washed sperm acts to increase sperm viability, motility and protection [197, 201, 202], with DNase activity present in seminal plasma potentially acting to free entangled sperm [33]. Seminal plasma causes bovine neutrophils to have more cell membrane damage, to produce less reactive oxygen species (ROS) following mitogenic stimulation, and suppresses transmigration in response to IL-8 [203]. Bull factors may impact the nature of this interaction – it has been reported that seminal plasma from younger bulls generates increased NET formation compared to old bulls [204].

In bovine endometrial cell culture models, seminal plasma treatment elicits a pro-inflammatory response. Similar to humans and mice, this is characterised by upregulation of cytokines including IL1B, IL6, CSF2 and TGFB1 [205, 206]. Intrauterine delivery of either raw semen or seminal plasma, like natural mating, caused increased uterine blood flow volume compared to the placebo group [207]. Intravaginal seminal plasma deposition into repeat breeder females acted to normalize low EGF concentrations in the endometrium and blood [208,209,210]. Seminal plasma from low fertility bulls generated higher production of TGFB1-3, IL-6 and IL-8 in bovine endometrial epithelial cells compared to high fertility bulls [211], but another study showed similar effects of seminal plasma on the transcriptome of bovine endometrial epithelial cells regardless of nutritional plane [212]. Consistent with other species studied, TGFB is implicated as an active factor in seminal plasma. TGFB1 and TGFB2 isoforms both induced IL6 expression in bovine endometrial cells [213]. There are particular technical challenges of working with bull seminal plasma in this context – it contains RNase and may lower endometrial RNA integrity and compromise RNA quality in gene expression studies [214]. This may also explain why seminal plasma at 4% negatively affects endometrial epithelial cell number and viability in vitro, especially in semen from low fertility bulls [211].

Epididymal sperm or ejaculated bovine sperm also caused transcriptomic changes in endometrial explants, including IL1A, IL1B, IL6, TNFA and CXCL8 [199], and other genes associated with T cell and NF-kB signaling [215]. The fertility status of bulls did not have an overt impact on the inflammatory response induced by frozen-thawed sperm in follicular phase uterine explants [199]. Pointing to a key role for TLR2 ligands associated with sperm in mediating a sperm-epithelial interaction, sperm pre-incubation with TLR2 agonists upregulated transcription of pro-inflammatory cytokines TNFA, IL1B, IL8 and PTGES, while TLR2 inhibition led to a weaker inflammatory response [5].

Overall, these studies suggest that bovine SP has a regulatory effect on inflammatory processes in the uterus following insemination. Whether there are significant impacts on fertility is not clear – there are contradicting findings regarding effects on pregnancy rate. An initial study showed no effect on pregnancy rates in dairy cows or beef cows after delivery of 0.5 ml of seminal plasma at the time of AI [216]. A follow-up study reported supplementation of pooled seminal plasma at the time of AI in dairy cows did not impact pregnancy rate, but increased heifer calf birth weight after AI with X chromosome-sorted sperm [206]. A positive effect was seen when vaginal infusion of seminal plasma into repeat breeder cows acted to double the conception rate compared to control treatment [210]. In another study in beef cows, seminal plasma was reported to alter expression of immune response genes in the endometrium and to increase uterine artery resistance at mid-gestation, but no changes in offspring development were detected [217]. The reasons for discrepancies between studies are not clear but may include technical challenges of preparing and storing seminal plasma to maintain optimal biological activity. There may be potential for addition of exogenous seminal plasma components such as TGFB to leverage biological mechanisms to improve fertility, however no effect on pregnancy rates in beef cows and only a non-statistically significant marginal benefit in dairy cows was observed in trials when 40 ng TGFB was delivered with sperm at the time of insemination [216]. Another study using a lower 10 ng dose of TGFB1 also reported no effect [218]. Given that current management practise for cattle AI is to extensively dilute seminal plasma, any change to this practise to include seminal plasma or its components would require research evidence to validate the efficacy of the approach and ensure no risks exist.

Seminal fluid signaling in sheep

As observed in other species, mating in sheep induces a transient inflammatory response that draws macrophages and neutrophils into the cervical and uterine tissues within hours of insemination [219, 220]. In the sheep, like the human, semen is deposited into the cranial vagina and around the cervical os, rather than directly into the uterus. Ex vivo studies using cervical explant tissue showed that frozen-thawed epididymal spermatozoa together with seminal plasma elicited major changes in gene expression in cervical explants with differentially expressed genes enriched in pathways relevant to inflammation [221], including induction of many pro-inflammatory cytokine genes induced by seminal fluid in humans and rodents. Studies of the uterine response show that both sperm and seminal plasma contribute to eliciting macrophage and neutrophil infiltration as well as increased IL-8 secretion. Exposure to whole semen or washed sperm but not seminal plasma alone induced secretion of GM-CSF. A limitation of this study was the possible contribution of bacterial contamination, as antibiotic addition partially reduced the neutrophil response to seminal fluid [220].

As observed in humans and mice, ovine seminal plasma contains the active signaling factor TGFB [85]. Other biologically active factors in seminal plasma attenuate the interaction between sperm and neutrophils, and heat-labile seminal plasma proteins are implicated [222]. This activity may contribute to the increase in pregnancy rate seen when epididymal sperm is exposed to seminal plasma before insemination, through effects on the surface glycocalyx of sperm that confer greater capacity to avoid neutrophil binding, aiding cervical transit [25, 34]. That this treatment yields a greater benefit after cervical insemination than after intrauterine insemination [223] suggests that seminal plasma contact with the cervix is necessary to deliver the benefit of contact. However, another study contradicts these findings and suggests direct effects of sperm in the endometrium, as incubation of frozen-thawed sperm with seminal plasma did not affect pregnancy rate after either cervical or intrauterine insemination, but did yield improvement after laparoscopic uterine insemination [224].

Seminal fluid signaling in dogs

There are a limited number of studies examining the role of seminal fluid in the immune response to coitus in bitches. Following coitus, canine sperm are deposited in the cranial vagina and distributed throughout the female reproductive tract via vaginal and uterine contractions. Due to the potential length between coitus and fertilization in canine (up to 9 days), studies have focused on sperm storage in the reproductive tract and it has been established that epithelial cells of the uterine gland and uterine tube junction tether sperm and act to provide a sperm reservoir [225,226,227]. This interaction is thought to promote sperm survival and fertility and reduce polyspermy [228]. In the first study to document the canine response to seminal fluid, seminal fluid delivered by AI was demonstrated to induce uterine vasodilation [226]. Further, sperm attachment to uterine epithelium was shown to be hindered without the presence of seminal plasma, presumably due to sperm interaction with neutrophils. The possibility that seminal plasma components attenuate sperm interactions with neutrophils in the female tract after insemination was demonstrated in a study where insemination using frozen-thawed spermatozoa with prostatic fluid increased the odds of conception [229].

Seminal fluid signaling in other species

In a number of other species, there is evidence for seminal fluid effects on the female reproductive tract and potential consequences for female reproductive investment. In macaques, an inflammatory response in the cervix is seen after seminal plasma exposure, accompanied by cervical recruitment of CD11c⁺ antigen presenting cells and local CD4⁺ T cell response [230]. In cats, subcutaneous administration of seminal plasma increased serum progesterone level and induced ovulation [231]. Assessment of Treg cell-associated genes Foxp3, Ctla4 and Cd25 supports the conclusion that Treg cells are higher in early pregnancy compared to late pregnancy, suggesting that seminal fluid may induce uterine Treg cells in cats as it does in other species [232]. Studies in camels have focussed primarily on the effects of seminal plasma on induction of ovulation mediated by seminal plasma NGFB [37] and its endocrine effects to promote the LH surge preceding ovulation [233]. Interestingly studies in llamas suggest seminal plasma acts to promote uterine receptivity through effects on endometrial vascularization [234] and uterine cytokine response [235], in part mediated by seminal plasma NGFB. Although the focus of this review is mammals, effects of seminal fluid on the female tract likely extend to birds – for example, there is evidence that sperm within the fowl oviduct are phagocytized by recruited immune cells, likely to be macrophages [236].

Conclusions

In all species examined so far, seminal fluid induces a biological response in the female reproductive tract that involves a range of immunological changes with consequences for reproductive success (Fig. 2). Evaluation of responses in rodents and large animal species has allowed considerable insight into the molecular and cellular basis for this response and its role in the physiology of embryo implantation and establishing successful pregnancy. There are remarkable similarities in the female response across species, although variation exists between species in the site of seminal fluid deposition and manifestation of semen-induced effects, potentially aligning with differences in reproductive strategy between species.

A key underlying mechanism in the female response to seminal fluid across all species involves immune cells and mediators. In all species this commences when sperm cause recruitment and activation of neutrophils in the initial aspect of the female tract response, in order to maintain tract sterility and select and remove suboptimal and surplus sperm. Another common feature is the capacity of seminal plasma to modulate this sperm-mediated response, and future studies will determine whether female sperm selection is responsive to male physiological status and environmental exposures. In species where seminal fluid reaches the uterus without extensive dilution, such as mice and pigs, factors in seminal plasma are the key driver of responses in the higher reproductive tract, and sperm also contribute by interacting with seminal plasma factors to modulate effects on endometrial cytokine expression. It is clear in these species that the consequent immune response influences female receptivity to embryo implantation and placental development, such that immune tolerance may or may not be induced, depending on the balance of seminal plasma signals. In both species, exposure to seminal fluid alters the dynamics of pre-implantation embryo development with an increase in the number of fertilized oocytes attaining viable blastocyst stage. Both also provide evidence that seminal factors promote corpus luteum development and progesterone synthesis. In species where the cervix is the site of seminal fluid deposition and only a small proportion of sperm reach the higher tract, the most dramatic effects of seminal fluid are seen locally in the cervix and changes in the uterus are less pronounced and more difficult to detect. Nevertheless emerging studies consistently show effects in humans, as well as sheep and cows, of signaling factors derived from seminal plasma and associated with sperm on a female immune response in the higher tract.

The broad commonality in female responses between species is consistent with an evolutionarily conserved pathway that has substantial influence on the reproductive process. This raises the question of the biological and evolutionary value of female responses to seminal fluid in mammals, a key area that requires further investigation. A compelling interpretation is that females utilize male seminal fluid signals as cues in a form of cryptic female choice, by which the female immune response selectively discerns the quality and compatibility of mating partners and invests reproductive resources accordingly, to maximize fitness of future populations [1, 237]. Studies in animal models, and a limited number of in vitro studies in human, suggest this may be associated with direct inhibitory effects on female reproductive tract receptivity. Elevated pro-inflammatory cytokines in seminal fluid are likely to be key mediators of an inhibitory response, through suppressing generation of female immune tolerance, as well as exerting adverse effects on sperm [57]. There may also be male partner-specific effects afforded by MHC genes that contribute to the degree and quality of response elicited by different males, perhaps mediated through immunological memory with consequences for future reproductive opportunities [238].

Clearly there is still much to learn about how and why seminal fluid signaling varies between species, the evolutionary value of this event in reproductive biology, and the potential benefit that seminal fluid may or may not impart for reproductive success in different animals. The utility of interventions where beneficial effects exist would be particularly relevant in species commonly bred using AI, where purified sperm is combined with artificial semen extenders for fertilization and the normal process of seminal fluid signaling is averted. Future studies should focus on defining the effects of seminal fluid exposure on conception and implantation, as well as fetal development and postnatal growth trajectory, viability and health of offspring, especially in pigs, sheep, cattle and other economically important species, or species where managed breeding is challenging. To harness the benefit of this knowledge, it will be important to understand the complexity of permissive as well as inhibitory factors in seminal plasma, and to understand how they are differentially regulated in the male reproductive tract. It will also be critical to develop consistent, proven approaches to recover various components of seminal fluid and to store and utilize them appropriately, so that bioactivity is not compromised and between-laboratory variation in experimental materials and results is avoided [239]. A better understanding of seminal fluid signaling in a range of animal species is expected to lead to a better understanding of this important area of biology, and has considerable potential to reveal strategies to improve breeding management and ensure maximal reproductive efficiency.

Conflict of interests

The authors have nothing to declare.

Funding Statement

The publication fee of this article was covered by JSPS KAKENHI Grant Number 22HP2009.