The Male Role in Pregnancy Loss and Embryo Implantation Failure

7. Role of Seminal Plasma in Human Female Reproductive Failure: Immunomodulation, Inflammation, and Infections

Deborah J. Anderson1, 2, 3   and Joseph A. Politch1

(1)

Department of Obstetrics and Gynecology, Boston University School of Medicine, 670 Albany Street, Suite 516, Boston, MA 02118, USA

(2)

Department of Microbiology, Boston University School of Medicine, 670 Albany Street, Suite 516, Boston, MA 02118, USA

(3)

Department of Medicine, Boston University School of Medicine, 670 Albany Street, Suite 516, Boston, MA 02118, USA

Deborah J. Anderson

Email: Deborah.Anderson@BMC.org

Abstract

Human seminal plasma contains factors that can regulate the female immune system and potentially promote reproductive fitness. Adverse effects on fertility and pregnancy may occur when seminal plasma provides insufficient, excessive, or altered signals or when the female partner is incapable of receiving these signals.

Keywords

Seminal plasmaProstateSeminal vesiclesVaginal epitheliumEndometriumCytokinesTGF-betaPGE

Seminal plasma can theoretically affect the reproductive fitness of a female sexual partner in a number of ways: (1) in healthy couples, normal components of seminal plasma such as immunomodulatory, proinflammatory, and growth factors may positively impact the female reproductive tract by inducing tolerance and supporting early pregnancy; (2) the immunomodulatory/proinflammatory effects of seminal plasma thought to enhance fertility and early fetal survival may also predispose women to genital infections which could lead to reproductive failure; (3) alterations in the composition of semen in male partners with pathologic conditions such as genital infections could have detrimental effects on female reproductive function by providing insufficient, excessive, or altered signals. Much of the research on beneficial effects of seminal plasma on fertility has been conducted in animal models and is reviewed elsewhere in this issue. This chapter will focus on human studies and will address potential effects of seminal plasma on human female reproduction. Specifically, we will review the origins and concentrations of immunomodulatory/proinflammatory factors in human seminal plasma in health and disease, their effects on immune defense in the female genital tract that may promote fertility and fetal well-being, and mechanisms whereby these factors may directly or indirectly contribute to female reproductive failure.

7.1 Tissue Origins of Seminal Plasma Components

The male genital tract is contained within an epithelial barrier system that normally limits the penetration of components from the peripheral blood and surrounding tissues (Anderson and Pudney 2005). Therefore, most components of the semen are derived from organs that comprise the male genital tract. Recent proteomics studies indicate that there are over 900 proteins in human seminal plasma (Rodriguez-Martinez et al. 2011; Cadavid et al. 2014). Most of the seminal plasma volume is produced in the seminal vesicles (65–75 %) and prostate (25–35 %) (Mann 1954). Several of the principal organs contribute specific immunomodulatory seminal plasma components which will be discussed in more detail below. The epididymis produces soluble HLA-G (Rajagopalan et al. 2006) and CD52g (Norton et al. 2002); the seminal vesicles are a principle source of prostaglandins (Kelley 1981) and TGF-β (Robertson et al. 2002); the prostate also produces TGF-β (Pannek et al. 1999) along with large amounts of exosomes, subcellular microvesicles enriched in bioactive components including cytokines and small RNAs (Burden et al. 2006; Vojtech et al. 2014). Local antibody production in the male genital tract occurs primarily in the penile urethra (Pudney and Anderson 2011). Other important components of semen, such as cytokines, chemokines, and mucins, are produced by epithelial, immune, and stromal cells throughout the genital tract (Anderson and Pudney 2005) (Fig. 7.1).

A322546_1_En_7_Fig1_HTML.gif

Fig. 7.1

Origin of seminal plasma components

7.2 Beneficial Effects of Immunomodulatory and Proinflammatory Factors in Human Seminal Plasma

Seminal plasma contains very high concentrations of two potent immunomodulatory factors, TGF-β and PGE, which may promote female fertility by suppressing natural immune responses to sperm and the semi-allogeneic fetus. Humoral and cellular immunity to sperm and embryos have been associated with infertility and miscarriage (Kokcu et al. 2012; Hill 1995; Raghupathy 1997). The immunomodulatory roles of seminal TGF-β and PGE were recently reviewed in detail by Robertson (2013) and Doncel (2014). TGF-β is a ubiquitous pleiotropic immunomodulatory factor that has been implicated as a key factor in the modulation of host defense (Wahl et al. 2006; Gorelik and Flavell 2002). Its potent immunoregulatory activity became evident from studies in Tgfb1 and Tgfbr1 knockout mice (mice without TGF-β or the TGF-β receptor), which develop lethal inflammatory disorders early in life (Kulkarni et al. 1993; Marie et al. 2006; Shull et al. 1992). Three isoforms of TGF-β are present in human seminal plasma (TGF-β1, TGF-β2, and TGF-β3); they exist primarily in latent form until activation in the female genital tract by proteases and acid pH (Robertson et al. 2002). TGF-β induces stimulatory or inhibitory effects in human T cells depending on the T cell differentiation status and the stimulatory conditions (Oh and Li 2013). The first evidence that TGF-β plays a critical role at mucosal sites was provided by early studies on TGF-β signaling in the intestine showing that induction of regulatory T cells (Tregs) by TGF-β promoted immunological tolerance to food antigens by active control of innate and adaptive immune responses (Harrison and Powrie 2013). TGF-β has been suggested to be one of the major factors inducing immune tolerance in the female genital tract by inducing differentiation of Treg cells and suppressing the activity of natural killer cells (Robertson et al. 2002), thereby suppressing immunity to antigens expressed on sperm and the implanting embryo. TGF-β also upregulates the expression of proinflammatory cytokines and chemokines in epithelial cells from the female reproductive tract which could stimulate cell growth and angiogenesis to support embryo implantation (Sharkey et al. 2012a) and thus be beneficial to early events in pregnancy. PGE is another potent immunomodulatory factor in semen, capable of modulating immune functions on multiple levels (Quayle et al. 1989). The concentration of PGE in seminal plasma is several orders of magnitude higher than that in blood plasma, although there is a high degree of interindividual variation (Templeton et al. 1978). PGE suppresses macrophage and neutrophil function and the cytotoxic activity of T lymphocytes and natural killer cells and upregulates the inflammatory mediator Cox 2 in vaginal epithelial cells (Templeton et al. 1978). Kelley et al. showed that PGE exposure results in upregulated IL-10 and downregulated IL-12 production, shifting the T cell response from a Th1 (cell-mediated immunity (CMI) dominant) to Th2 (humoral immunity dominant) immune response (Kelley 1981). Soluble HLA-G is also present in semen and has been proposed to suppress adverse NK cell activity directed against invading cytotrophoblast (Rajagopalan et al. 2006). CD52g, a sperm-coating glycoprotein derived from the epididymis, may also play an important role in preventing antisperm immunity and infertility, although it can itself be a target of antisperm antibodies in some infertility patients (Norton et al. 2002). Seminal plasma also contains high concentrations of proinflammatory cytokines and chemokines that could affect fertility by recruiting and activating immune cells in the reproductive tissues and stimulating the production of factors that stimulate cell growth and angiogenesis (Politch et al. 2007). Specifically, seminal plasma has very high concentrations of IL-7, a hematopoietic growth factor that promotes the proliferation of lymphoid progenitors, B cell maturation, and T and NK cell survival (Fry and Mackall 2005), and three chemokines, SDF-1, MCP-1, and IL-8, which may recruit leukocytes to the insemination site to participate in immune defense and scavenger functions. The concentrations and ranges of principal immunomodulatory and proinflammatory factors in semen are provided in Table 7.1. Unpublished data from our laboratory indicate that TGF-β levels are decreased and IL-8 levels are significantly increased in semen from men with leukocytospermia (male genital inflammation) (Politch J, personal communication).

Table 7.1

Concentrations of selected components in normal semen

Component

Concentration

References

Immunomodulatory factors

TGF-β1

Sharkey et al. (2012a)

Total

219.3 ± 13.4 ng/mla

Bioactive (% total)

2.3 ± 0.4 ng/mla (1.2 %)

TGF-β2

Total

5.3 ± 0.7 ng/mla

Bioactive (% total)

0.25 ± 0.04 ng/mla (5.3 %)

TGF-β3

Total

172.2 ± 32.8 ng/mla

Bioactive (% total)

3.5 ± 1.2 ng/mla (1.8 %)

PGE-1

7.0 ± 6.0 μg/mla

Gerozissis et al. (1982)

PGE-2

14.0 ± 11.0 μg/mla

PGF-1a

1.0 ± 0.7 μg/mla

PGF-2a

2.0 ± 2.0 μg/mla

IL-7

2,365.8 (1,109.5–3,985.5) pg/mlb

Politch et al. (2007)

HLA-G

82 (29–1,161) U/mlc

Dahl et al. (2014)

Exosomes

~1 Trillion/ejaculate

Vojtech et al. (2014)

Chemokines

MCP-1

3.3 (0.3–81.5) ng/mlb

Politch et al. (2007)

IL-8

1.6 (0.4–14.7) ng/mlb

SDF-1α

5.1 (ND–18.0) ng/mlb

Proinflammatory cytokines

TNF-α

1.5 (ND–40.3) pg/mlb

Politch et al. (2007)

GM-CSF

1.5 (ND–1,190.6) pg/mlb

aMean ± SE

bGeometric mean (range)

cMedian (range)

An exciting new area of research is the study of seminal exosomes. These highly abundant subcellular microvesicles, produced primarily by the prostate but also in the epididymis and seminal vesicles, are enriched in bioactive components including cytokines and small RNAs (miRNA, YRNA, and tRNA) and may play an important role in the fertilization and intercellular communication in the genital tract (Burden et al. 2006; Vojtech et al. 2014; Li et al. 2013). It is estimated that approximately one trillion exosomes are present in a human ejaculate. These small vesicles readily fuse with the plasma membrane of sperm and other cell types to deliver important signaling molecules. A number of immune-related mRNAs are targeted by miRNAs in seminal exosomes; whether miRNAs can be delivered by seminal exosomes in sufficient quantity to target genes and change cellular functions in the vaginal immune cell population is unknown (Vojtech et al. 2014). They have been shown to play a direct role in antiviral immune defense (Madison et al. 2014).

7.3 Evidence for Effects of Seminal Plasma on the Human Female Reproductive Tract

Recently, a meta-analysis was conducted on the role of seminal plasma for improved outcomes during in vitro fertilization. The outcome of IVF treatment in patients exposed to seminal plasma near the time of oocyte pickup or embryo transfer was compared to that of controls with no exposure to seminal plasma (a total of 2,204 patients in seven randomized control trials). They found a statistically significant improvement in the clinical pregnancy rate after seminal plasma exposure (RR 1.24, p = 0.003), but no improvement in the ongoing pregnancy/live birth rate (Crawford et al. 2015). However, this topic is a matter of debate. Michael Bedford has pointed out that virgin animals are perfectly good embryo transfer recipients and that many human IVF programs do not use priming with seminal plasma in conjunction with IVF cycles and obtain good fertilization and pregnancy outcomes. He concludes that whereas a nuanced effect of seminal plasma on fertility outcome in humans cannot be dismissed, many experimental and clinical results demonstrate that the presence of seminal plasma is not essential for the transport and survival of spermatozoa in the female tract, for fertilization, or for implantation and embryonic development (Bedford 2015). We review below evidence from in vitro and clinical studies of the effect of seminal plasma on various regions of the human female genital tract.

7.3.1 Vagina and External Genitalia

In human reproduction, semen is deposited into the vagina, and it is this site and the external genitalia that have maximum exposure to semen components. Semen concentrations in vaginal fluid decline after intercourse to approximately 50 % after 1 h and reach baseline after 24 h (Macaluso et al. 1999; Graves et al. 1985). The external genitalia (labia majora and minora) are covered with keratinized skin, and the vagina and ectocervix are lined with specialized nonkeratinizing stratified squamous epithelia (Anderson 2007). These multilayered epithelia normally afford a barrier to external signaling by presenting a wall of cornified enucleated cells on the apical surface which lack most membrane receptors and signaling pathways (Anderson et al. 2014). Lipophilic molecules from semen could be absorbed through the vaginal epithelium to achieve local or systemic effects (Muranishi et al. 1993). In women with certain lower genital infections (e.g., HSV-2, HPV, GC) or epithelial lesions, living functional basal epithelial cells or leukocytic infiltrates in the vaginal epithelium could be exposed and react to seminal components. Vaginal and ectocervical cells grown as monolayers in vitro respond to semen challenge by producing GM-CSF, IL-6, IL-8, and MCP-1 (Sharkey et al. 2007); these cultures are not fully differentiated and represent the basal epithelial layer of the stratified epithelium that is exposed by vaginal lesions or infections. A clinical study that monitored the infiltration of lymphocytes and other WBC population into the ectocervical mucosa following intercourse reported increased numbers of T lymphocytes, macrophages, and dendritic cells (Sharkey et al. 2012b), but whether the signal was transmitted across the stratified squamous epithelium of the ectocervix or via the neighboring endocervix has yet to be determined.

7.3.2 Endocervix

The human endocervix is lined with a single layer of viable columnar epithelial cells that are highly responsive to external signals (Fichorova and Anderson 1999), including seminal plasma (Sharkey et al. 2012a). The opening of the endocervix (cervical os) is protected from bacteria in the vaginal compartment by a thick layer of secreted mucins (Gipson et al. 1997), but seminal components may diffuse through mucus (Cone 2009) or directly contact endocervical epithelial cells after intercourse due to disruption of the mucus barrier or other means of exposure (e.g., cervical ectopy). Studies have documented leukocytic exudates in the cervical canal following intercourse (Pandya and Cohen 1985; Thompson et al. 1992) and infiltrates of macrophages, dendritic cells, and T lymphocytes in the cervical epithelium and stroma (Sharkey et al. 2012b) indicating that the cervix is a region that commonly responds to seminal signaling. Leukocytic infiltrates could play a scavenger role in the clearance of sperm and other seminal factors after intercourse. Less well understood are the potential effects of endocervical epithelial factors produced in response to seminal plasma on other aspects of reproductive function such as sperm capacitation and effects registered in the upper tract pertaining to implantation.

7.3.3 Uterus/Endometrium

Seminal plasma effects on various sites in the female reproductive tract depend on the concentration of seminal factors reaching the tissue. In mice, semen rapidly enters the uterus after coitus (Zamboni 1972), and a number of studies have documented effects of semen on endometrial receptivity and implantation in mice (Robertson et al. 2013). However, there is debate on whether seminal plasma ascends beyond the cervix to enter the upper genital tract (uterus, fallopian tubes) in women. A series of magnetic resonance imaging studies using 99mTc-labeled human albumin microspheres showed radiolabel dispersion into the uterus and fallopian tubes following deposition into the vagina (Kunz et al. 1996; Venter and Iturralde 1979; Zervomanolakis et al. 2007). However, a number of other studies using semen surrogates or vaginal gels have failed to document this effect (Barnhart et al. 20042005; Brown et al. 1997; Chatterton et al. 2004; Louissaint et al. 2012; Mauck et al. 2008). Even if appreciable amounts of soluble vaginal contents do not ascend into the uterus through the endocervix, seminal plasma components could signal cells in the upper reproductive tract if small amounts ascend into this region after intercourse through peristalsis as proposed by some studies or through absorption through the vaginal epithelium. A small percentage (1–15 %) of TGF-beta in semen is associated with the sperm fraction (Sharkey et al. 2012a), therefore making it possible for sperm to provide TGF-β signaling as they ascend into the upper genital tract.

7.4 Potential Adverse Effects of Seminal Plasma

7.4.1 Insufficient Seminal Plasma Immunoregulation Could Permit Immunity Against Sperm and the Conceptus

In some couples, the man’s seminal plasma may be deficient in some of the immunoregulatory compounds described above, or the female partner could be nonresponsive to seminal plasma signaling (e.g., deficient in receptors or proteases that activate TGF-β) due to genetic abnormalities, genital infections, or other circumstances. Lack of immune tolerance/immunosuppression in the genital tract could promote the synthesis of antibodies associated with female reproductive failure including antisperm and antiphospholipid antibodies or the generation of a T cell response to the conceptus that can lead to miscarriage (Kokcu et al. 2012; Hill 1995; Raghupathy 1997).

7.4.2 Regulation of Cervicovaginal Immune Defense by Seminal Plasma: Increased Susceptibility to Infections?

Memory T cells, including antigen-specific cytotoxic CD8+ T cells, reside throughout the female genital tract and are thought to play a major role in host defense at this site. They are concentrated in the transformation zone of the cervix, where seminal plasma exerts its maximum effect (Pudney et al. 2005). Chemokines in semen attract T cells, along with PMNs and macrophages, to the endocervix after intercourse and may promote immune defense during this vulnerable window of infection. But TGF-β and PGE may also exert a tolerizing or immunosuppressive effect on these critical immune defense cells and impede their ability to clear an infection. Viral and bacterial genital infections have been associated with infertility and miscarriage in women (Novy et al. 2008).

7.4.3 Proinflammatory Factors in Seminal Plasma Could Promote HIV Infection

Genital inflammation is a risk factor for HIV transmission due to the recruitment of HIV target cells into the mucosal epithelium and their activation which makes cells more vulnerable to infection and enables cells to produce more HIV once infected (Mayer and Venkatesh 2011). TGF-beta and PGE act on cervical epithelial cells to upregulate the synthesis of proinflammatory cytokines and chemokines which recruit leukocytes and regulate their immune function (Sharkey et al. 2012a). Suppression of T cell immunoactivity and promotion of inflammation in the genital tract by seminal plasma could promote the sexual transmission of HIV-1. This topic has recently been reviewed by Doncel et al. (2014) and Rametse et al. (2014).

7.5 Conclusions

Seminal fluid improves the pregnancy rate in laboratory and farm animals, and a number of components in seminal plasma have been identified that have beneficial effects on reproduction. The extent to which seminal plasma affects fertility in humans is not fully understood, but there is considerable evidence that seminal plasma affects immune cell populations in the lower genital tract. Several studies have documented leukocytic infiltration into the endocervical canal following intercourse, possibly due to chemokines in semen, and these cells may play a scavenger role and/or secrete factors that can positively or negatively affect fertility. In vitro studies have demonstrated that seminal plasma components are immunosuppressive and induce the differentiation of Treg cells that can mediate tolerance in the genital tract to foreign antigens on sperm and trophoblast cells. This effect of seminal plasma must be tightly controlled so as not to weaken immune defense to sexually transmitted pathogens. If there is too little immunomodulation by semen, cellular and humoral immune responses could develop against antigens expressed on sperm or the conceptus and negatively affect female reproductive function. However, too much immunosuppression or the induction of inflammation by seminal plasma could predispose women to genital infections which also can adversely affect reproductive function.

Acknowledgment

Supported by NIH grant U19AI096398 (DJA).

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