Sarah A. Robertson

Inflammatory processes in preterm and term parturition

A role for the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) is evident in term and preterm delivery, and this is independent of the presence of infection. All uterine tissues progress through a staged transformation near the end of pregnancy that leads from relative uterine quiescence and maintenance of pregnancy to the activation of the uterus that prepares it for the work of labour and production of stimulatory molecules that trigger the onset of labour and delivery. The uterus is activated by pro-inflammatory cytokines through stimulation of the expression and production of uterine activation proteins (UAPs). One of these actions is the stimulation of prostaglandin (PG) synthesis. Particularly important for labour is PGF(2alpha) and its receptor, PTGFR. In addition, pro-inflammatory cytokines are able to increase the synthesis of matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF) and the progesterone receptor C isoform, which leads to decreased tissue progesterone responsiveness. Some of these effects are replicated by PGF(2alpha), suggesting that it may act via its receptor to amplify the direct actions of cytokines. In turn, VEGF may enhance leukocyte recruitment to the uterus, and MMP-9 may promote activation of inactive pro-form cytokines. Pro-inflammatory cytokines also decrease the activity of 11beta-hydroxysteroid dehydrogenase, which likely increases intrauterine cortisol concentrations. In turn, cortisol may drive PG synthesis. Together these feed-forward mechanisms activate the uterus, trigger the production of uterine contractile stimulants and lead to labour and delivery.

Seminal plasma and male factor signalling in the female reproductive tract

In mammals, insemination results in the transmission of seminal factors that act, in the female reproductive tract, to promote sperm survival, to “condition” the female immune response to tolerate the conceptus and to organise molecular and cellular changes in the endometrium to facilitate embryo development and implantation. These events are initiated when signalling agents, including transforming growth factor-β and other cytokines and prostaglandins secreted by seminal vesicle and prostate glands, interact with epithelial cells in the cervix and uterus to activate cytokine synthesis and to induce cellular and molecular changes resembling a classical inflammatory cascade. The consequences are the recruitment and activation of macrophages, granulocytes and dendritic cells, which have immune-regulatory and tissue-remodelling roles that culminate in improved endometrial receptivity to the implanting embryo. Cytokines elicited by seminal activation have embryotrophic properties and also contribute directly to the optimal development of the early embryo. This review summarises our current understanding of the physiology of responses to seminal plasma in the female reproductive tract and considers the evolutionary significance of seminal plasma in influencing female tissues to promote the success of pregnancy.

Regulatory T-cells and immune tolerance in pregnancy: a new target for infertility treatment?

BACKGROUND Adaptation of the maternal immune response to accommodate the semi-allogeneic fetus is necessary for pregnancy success, and disturbances in maternal tolerance are implicated in infertility and reproductive pathologies. T regulatory (Treg) cells are a recently discovered subset of T-lymphocytes with potent suppressive activity and pivotal roles in curtailing destructive immune responses and preventing autoimmune disease. METHODS A systematic review was undertaken of the published literature on Treg cells in the ovary, testes, uterus and gestational tissues in pregnancy, and their link with infertility, miscarriage and pathologies of pregnancy. An overview of current knowledge on the generation, activation and modes of action of Treg cells in controlling immune responses is provided, and strategies for manipulating regulatory T-cells for potential applications in reproductive medicine are discussed. RESULTS Studies in mouse models show that Treg cells are essential for maternal tolerance of the conceptus, and that expansion of the Treg cell pool through antigen-specific and antigen non-specific pathways allows their suppressive actions to be exerted in the critical peri-implantation phase of pregnancy. In women, Treg cells accumulate in the decidua and are elevated in maternal blood from early in the first trimester. Inadequate numbers of Treg cells or their functional deficiency are linked with infertility, miscarriage and pre-eclampsia. CONCLUSIONS The potency and wide-ranging involvement of Treg cells in immune homeostasis and disease pathology indicates the considerable potential of these cells as therapeutic agents, raising the prospect of their utility in novel treatments for reproductive pathologies.

Seminal Fluid Drives Expansion of the CD4+CD25+ T Regulatory Cell Pool and Induces Tolerance to Paternal Alloantigens in Mice

Abstract T regulatory (Treg) cells are implicated in maternal immune tolerance of the conceptus at implantation; however, the antigenic and regulatory signals controlling Treg cells in early pregnancy are undefined. To examine the role of male seminal fluid in tolerance induction, the effect of exposure to seminal fluid at mating on responsiveness to paternal alloantigens was examined using paternal tumor cell grafts and by delayed-type hypersensitivity (DTH) challenge on Day 3.5 postcoitum. Exposure to seminal fluid inhibited rejection of paternal tumor cells, independently of fertilization and embryo development, while seminal fluid from major histocompatability complex (MHC)-dissimilar males was less effective. Similarly, mating with intact males suppressed the DTH response to paternal alloantigens in an MHC-specific fashion. Excision of the seminal vesicle glands diminished the tolerance-inducing activity of seminal fluid. Mating with intact males caused an increase in CD4+CD25+ cells expressing FOXP3 in the para-aortic lymph nodes draining the uterus, beyond the estrus-associated peak in cycling mice. The increase in CD4+CD25+ cells was abrogated when males were vasectomized or seminal vesicles were excised. Collectively, these data provide evidence that exposure to seminal fluid at mating promotes a state of functional tolerance to paternal alloantigens that may facilitate maternal acceptance of the conceptus at implantation, and the effects of seminal fluid are likely to be mediated by expansion of the Treg cell pool. Both seminal plasma and sperm components of the seminal fluid are necessary to confer full tolerance and elicit the Treg cell response, potentially through provision of immune-deviating cytokines and antigens, respectively.

Transforming growth factor β—a mediator of immune deviation in seminal plasma

TGFβ is a potent immune deviating agent, driving active forms of immune tolerance in peripheral tissues through effects on the induction and resolution of inflammatory responses and phenotype skewing in antigen-presenting cells and lymphocytes. The TGFβ content of seminal plasma from human, rodent and livestock species is amongst the highest measured in biological fluids. The seminal vesicle gland is the principal source of TGFβ in the semen of mice, where its synthesis is regulated by testosterone. At insemination, seminal TGFβ is deposited in the female tract and is activated by acidic vaginal pH, enzymes of male or female tract origin, or through cleavage-independent processes involving conformational change after interaction with epithelial cell docking proteins. Seminal TGFβ has been shown to be a principal stimulating agent in the post-coital inflammatory response, and is likely to be essential for induction of immune tolerance to seminal antigens. As well as preventing aberrant immunity to spermatozoa, these events are implicated in priming an appropriate female immune response to embryo implantation, since many seminal antigens are shared by the conceptus. The cascade of immunological events elicited by seminal TGFβ may therefore explain epidemiological observations linking acute and cumulative exposure to semen with successful placental development and pregnancy outcome. Depending on whether the female tract has evolved mechanisms to discriminate seminal antigens from opportunistic pathogens, there may be a detrimental cost of seminal TGFβ in inhibiting protective immunity to agents of sexually transmitted disease including HIV. A better understanding of the significance and role of TGFβ in semen will facilitate development of novel therapies for immune-based infertility disorders.

MicroRNA-Regulated Pathways Associated with Endometriosis

Endometriosis is a prevalent gynecological disease characterized by growth of endometriotic tissue outside the uterine cavity. MicroRNAs (miRNAs) are naturally occurring posttranscriptional regulatory molecules that potentially play a role in endometriotic lesion development. We assessed miRNA expression by microarray analysis in paired ectopic and eutopic endometrial tissues and identified 14 up-regulated (miR-145, miR-143, miR-99a, miR-99b, miR-126, miR-100, miR-125b, miR-150, miR-125a, miR-223, miR-194, miR-365, miR-29c and miR-1) and eight down-regulated (miR-200a, miR-141, miR-200b, miR-142-3p, miR-424, miR-34c, miR-20a and miR-196b) miRNAs. The differential expression of six miRNAs was confirmed by quantitative RT-PCR. An in silico analysis identified 3851 mRNA transcripts as putative targets of the 22 miRNAs. Of these predicted targets, 673 were also differentially expressed in ectopic vs. eutopic endometrial tissue, as determined by microarray. Functional analysis suggested that the 673 miRNA targets constitute molecular pathways previously associated with endometriosis, including c-Jun, CREB-binding protein, protein kinase B (Akt), and cyclin D1 (CCND1) signaling. These pathways appeared to be regulated both transcriptionally as well as by miRNAs at posttranscriptional level. These data are a rich and novel resource for endometriosis and miRNA research and suggest that the 22 miRNAs and their cognate mRNA target sequences constitute pathways that promote endometriosis. Accordingly, miRNAs are potential therapeutic targets for treating this disease.

Seminal Fluid Induces Leukocyte Recruitment and Cytokine and Chemokine mRNA Expression in the Human Cervix after Coitus

In mice, seminal fluid elicits an inflammation-like response in the female genital tract that activates immune adaptations to advance the likelihood of conception and pregnancy. In this study, we examined whether similar changes in leukocyte and cytokine parameters occur in the human cervix in response to the male partner’s seminal fluid. After a period of abstinence in proven-fertile women, duplicate sets of biopsies were taken from the ectocervix in the periovulatory period and again 48 h later, 12 h after unprotected vaginal coitus, vaginal coitus with use of a condom, or no coitus. A substantial influx of CD45+ cells mainly comprising CD14+ macrophages and CD1a+ dendritic cells expressing CD11a and MHC class II was evident in both the stratified epithelium and deeper stromal tissue after coitus. CD3+CD8+CD45RO+ T cells were also abundant and increased after coitus. Leukocyte recruitment did not occur without coitus or with condom-protected coitus. An accompanying increase in CSF2, IL6, IL8, and IL1A expression was detected by quantitative RT-PCR, and microarray analysis showed genes linked with inflammation, immune response, and related pathways are induced by seminal fluid in cervical tissues. We conclude that seminal fluid introduced at intercourse elicits expression of proinflammatory cytokines and chemokines, and a robust recruitment of macrophages, dendritic cells, and memory T cells. The leukocyte and cytokine environment induced in the cervix by seminal fluid appears competent to initiate adaptations in the female immune response that promote fertility. This response is also relevant to transmission of sexually transmitted pathogens and potentially, susceptibility to cervical metaplasia.

Maternal tract factors contribute to paternal seminal fluid impact on metabolic phenotype in offspring

Significance Events at conception shape the future growth and health of offspring, to impact life course potential and disease susceptibility. The environment and experiences of both parents contribute to programming offspring phenotype through epigenetic modifications imparted before embryo implantation. How the father transmits this information remains elusive. Possible pathways include the sperm genome and epigenome, postejaculatory effects of seminal fluid on sperm, and indirect actions of seminal fluid on various female factors regulating embryo development. In this study, we provide evidence that seminal fluid acts to influence both sperm integrity and the balance of embryotrophic and embryotoxic signals in the female reproductive tract, in turn affecting embryo development and programming of future adiposity and metabolic phenotype in male offspring. Paternal characteristics and exposures influence physiology and disease risks in progeny, but the mechanisms are mostly unknown. Seminal fluid, which affects female reproductive tract gene expression as well as sperm survival and integrity, provides one potential pathway. We evaluated in mice the consequences for offspring of ablating the plasma fraction of seminal fluid by surgical excision of the seminal vesicle gland. Conception was substantially impaired and, when pregnancy did occur, placental hypertrophy was evident in late gestation. After birth, the growth trajectory and metabolic parameters of progeny were altered, most profoundly in males, which exhibited obesity, distorted metabolic hormones, reduced glucose tolerance, and hypertension. Altered offspring phenotype was partly attributable to sperm damage and partly to an effect of seminal fluid deficiency on the female tract, because increased adiposity was also evident in adult male progeny when normal two-cell embryos were transferred to females mated with seminal vesicle-excised males. Moreover, embryos developed in female tracts not exposed to seminal plasma were abnormal from the early cleavage stages, but culture in vitro partly alleviated this. Absence of seminal plasma was accompanied by down-regulation of the embryotrophic factors Lif, Csf2, Il6, and Egf and up-regulation of the apoptosis-inducing factor Trail in the oviduct. These findings show that paternal seminal fluid composition affects the growth and health of male offspring, and reveal that its impact on the periconception environment involves not only sperm protection but also indirect effects on preimplantation embryos via oviduct expression of embryotrophic cytokines.

Reactive Oxygen Species and Sperm Function—In Sickness and In Health

The ability of spermatozoa to generate reactive oxygen species (ROS) has been appreciated since the 1940s. It is a universal property of mature spermatozoa from all mammalian species and a major contributor to the oxidative stress responsible for defective sperm function. The mechanisms by which oxidative stress limits the functional competence of mammalian spermatozoa involve the peroxidation of lipids, the induction of oxidative DNA damage, and the formation of protein adducts. ROS production in these cells involves electron leakage from the sperm mitochondria, triggered by a multitude of factors that impede electron flow along the electron transport chain. The net result of mitochondrial ROS generation is to damage these organelles and initiate an intrinsic apoptotic cascade, as a consequence of which spermatozoa lose their motility, DNA integrity, and vitality. This pathway of programmed senescence also results in the exteriorization of phosphatidylserine, which may facilitate the silent phagocytosis of these cells in the aftermath of insemination, in turn influencing the female tract immune response to sperm antigens and future fertility. Despite the vulnerability of sperm to oxidative stress, it is also clear that normal sperm function depends on low levels of ROS generation in order to promote the signal transduction pathways associated with capacitation. Modulators of ROS generation by spermatozoa may therefore have clinical utility in regulating the fertilizing capacity of these cells and preventing the development of antisperm immunity. Achievement of these objectives will require a systematic evaluation of pro- and antioxidant strategies in vivo and in vitro.

Cytokine-Leukocyte Networks and the Establishment of Pregnancy

PROBLEM: Factors in seminal plasma stimulate an intense but transient inflammatory response in the murine endometrium at mating. The aim of our current studies is to delineate the cytokine‐leukocyte interactions comprising this response and to elucidate the significance of these events in changes in the maternal immune system and as determinants of pregnancy outcome.