Toll-Like Receptor-4 Antagonist (+)-Naltrexone Protects Against Carbamyl-Platelet Activating Factor (cPAF)-Induced Preterm Labor in Mice.

Spontaneous preterm labor is frequently caused by an inflammatory response in the gestational tissues elicited by either infectious or sterile agents. In sterile preterm labor, the key regulators of inflammation are not identified, but platelet-activating factor (PAF) is implicated as a potential rate-limiting effector agent. Since Toll-like receptor (TLR)-4 can amplify PAF signaling, we evaluated whether TLR4 contributes to inflammation and fetal loss in a mouse model of PAF-induced sterile preterm labor, and whether a small-molecule TLR4 inhibitor, (+)-naltrexone, can mitigate adverse PAF-induced effects. The administration of carbamyl (c)-PAF caused preterm labor and fetal loss in wild-type mice but not in TLR4-deficient mice. Treatment with (+)-naltrexone prevented preterm delivery and alleviated fetal demise in utero elicited after cPAF administered by i.p. or intrauterine routes. Pups born after cPAF and (+)-naltrexone treatment exhibited comparable rates of postnatal survival and growth to carrier-treated controls. (+)-Naltrexone suppressed the cPAF-induced expression of inflammatory cytokine genes Il1b, Il6, and Il10 in the decidua; Il6, Il12b, and Il10 in the myometrium; and Il1b and Il6 in the placenta. These data demonstrate that the TLR4 antagonist (+)-naltrexone inhibits the inflammatory cascade induced by cPAF, preventing preterm birth and perinatal death. The inhibition of TLR4 signaling warrants further investigation as a candidate strategy for fetal protection and delay of preterm birth elicited by sterile stimuli.

Immune Cells at the Fetomaternal Interface: How the Microenvironment Modulates Immune Cells To Foster Fetal Development.

Immune cells adapt their phenotypic and functional characteristics in response to the tissue microenvironment within which they traffic and reside. The fetomaternal interface, consisting of placental trophoblasts and the maternal decidua, is a highly specialized tissue with a unique and time-limited function: to nourish and support development of the semiallogeneic fetus and protect it from inflammatory or immune-mediated injury. It is therefore important to understand how immune cells within these tissues are educated and adapt to fulfill their biological functions. This review article focuses on the local regulatory mechanisms ensuring that both innate and adaptive immune cells appropriately support the early events of implantation and placental development through direct involvement in promoting immune tolerance of fetal alloantigens, suppressing inflammation, and remodeling of maternal uterine vessels to facilitate optimal placental function and fetal growth.

Antenatal Suppression of IL-1 Protects against Inflammation-Induced Fetal Injury and Improves Neonatal and Developmental Outcomes in Mice.

Preterm birth (PTB) is commonly accompanied by in utero fetal inflammation, and existing tocolytic drugs do not target fetal inflammatory injury. Of the candidate proinflammatory mediators, IL-1 appears central and is sufficient to trigger fetal loss. Therefore, we elucidated the effects of antenatal IL-1 exposure on postnatal development and investigated two IL-1 receptor antagonists, the competitive inhibitor anakinra (Kineret) and a potent noncompetitive inhibitor 101.10, for efficacy in blocking IL-1 actions. Antenatal exposure to IL-1ß induced Tnfa, Il6, Ccl2, Pghs2, and Mpges1 expression in placenta and fetal membranes, and it elevated amniotic fluid IL-1ß, IL-6, IL-8, and PGF2α, resulting in PTB and marked neonatal mortality. Surviving neonates had increased Il1b, Il6, Il8, Il10, Pghs2, Tnfa, and Crp expression in WBCs, elevated plasma levels of IL-1ß, IL-6, and IL-8, increased IL-1ß, IL-6, and IL-8 in fetal lung, intestine, and brain, and morphological abnormalities: e.g., disrupted lung alveolarization, atrophy of intestinal villus and colon-resident lymphoid follicle, and degeneration and atrophy of brain microvasculature with visual evoked potential anomalies. Late gestation treatment with 101.10 abolished these adverse outcomes, whereas Kineret exerted only modest effects and no benefit for gestation length, neonatal mortality, or placental inflammation. In a LPS-induced model of infection-associated PTB, 101.10 prevented PTB, neonatal mortality, and fetal brain inflammation. There was no substantive deviation in postnatal growth trajectory or adult body morphometry after antenatal 101.10 treatment. The results implicate IL-1 as an important driver of neonatal morbidity in PTB and identify 101.10 as a safe and effective candidate therapeutic.

Interferon-gamma inhibits seminal plasma induction of colony-stimulating factor 2 in mouse and human reproductive tract epithelial cells.

Seminal fluid interacts with the female reproductive tract to initiate a permissive immune response that facilitates embryo implantation and pregnancy success. The immune-regulatory cytokine interferon-γ (IFNG), which can be elevated in seminal plasma, is associated with reduced fertility. Here, we investigated how IFNG influences the female immune response to seminal fluid. In human Ect1 cervical epithelial cells, IFNG added at physiologically relevant concentrations substantially impaired seminal plasma-induced synthesis of key cytokines colony-stimulating factor 2 (CSF2) and interleukin-6 (IL6). Seminal fluid-induced CSF2 synthesis was also suppressed in the uterus of mice in vivo, when IFNG was delivered transcervically 12 h after mating. Transforming growth factor B1 (TGFB1) is the major seminal fluid signaling factor which elicits CSF2 induction, and IFNG exhibited potent dose-dependent suppression of CSF2 synthesis induced by TGFB1 in murine uterine epithelial cells in vitro. Similarly, IFNG suppressed TGFB1-mediated CSF2 induction in Ect1 cells and human primary cervical epithelial cells; however, IL6 regulation by IFNG was independent of TGFB1. Quantitative PCR confirmed that CSF2 regulation by IFNG in Ect1 cells occurs at the gene transcription level, secondary to IFNG suppression of TGFBR2 encoding TGFB receptor 2. Conversely, TGFB1 suppressed IFNG receptor 1 and 2 genes IFNGR1 and IFNGR2. These data identify IFNG as a potent inhibitor of the TGFB-mediated seminal fluid interaction with relevant reproductive tract epithelia in mice and human. These findings raise the prospect that IFNG in the male partner's seminal fluid impairs immune adaptation for pregnancy following coitus in women.

Seminal plasma pro-inflammatory cytokines interferon-γ (IFNG) and C-X-C motif chemokine ligand 8 (CXCL8) fluctuate over time within men.

STUDY QUESTION: Do seminal plasma pro-inflammatory cytokines interferon-γ (IFNG) and C-X-C motif chemokine ligand 8 (CXCL8) vary within individual men over time? SUMMARY ANSWER: IFNG exhibits substantial variation that is independent of duration of abstinence but correlates with lipopolysaccharide (LPS) content, while CXCL8 varies moderately in association with duration of abstinence. WHAT IS KNOWN ALREADY: Pro-inflammatory cytokines IFNG and CXCL8 in seminal fluid can adversely impact male and female fertility. Other cytokines as well as sperm parameters fluctuate considerably within individuals over time, but whether IFNG and CXCL8 vary similarly, and the determinants of variance, are unknown. STUDY DESIGN, SIZE, DURATION: Between two and seven semen samples were collected from 14 proven fertile donors at 6-10 week intervals over the course of ~12 months, to assess variation over time in cytokines and LPS, and to investigate relationships with sperm parameters and possible regulatory factors. PARTICIPANTS/MATERIALS, SETTING, METHODS: The concentrations and total amounts per ejaculate of IFNG and CXCL8 were determined using commercial ELISA. Sperm parameters were assessed according to World Health Organization (WHO) IV standards and LPS was measured by limulus amebocyte lysate (LAL) assay. Mixed model analysis was utilized to determine the relative contribution of between- and within-individual factors in explaining variance. Relationships between cytokines, LPS and sperm parameters, as well as effect of age and duration of abstinence, were investigated by correlation analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Within-individual variability contributed to total variance particularly for both IFNG, CXCL8 and LPS, and was a stronger determinant than between-individual variability for IFNG and LPS. Normal sperm motility correlated inversely with CXCL8, and sperm concentration correlated inversely with LPS. Duration of abstinence was a determinant of total CXCL8, but not IFNG or LPS. Associations between LPS, IFNG and CXCL8 suggest IFNG and perhaps CXCL8 are influenced by microbial populations. LIMITATIONS, REASONS FOR CAUTION: A limited number of donors from a single clinic were investigated. Clinical information on complete microbiology, BMI, nutrition, smoking and other lifestyle factors was unavailable. Further studies are required to determine whether the findings can be generalized to larger populations and different ethnicities. WIDER IMPLICATIONS OF THE FINDINGS: These data reveal substantial variation over time in pro-inflammatory seminal fluid cytokines and imply existence of microbial or other environmental regulatory factors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the National Health and Medical Research Council of Australia. The authors have no competing interests to disclose.

MicroRNA regulation of immune events at conception.

The reproductive tract environment at conception programs the developmental trajectory of the embryo, sets the course of pregnancy, and impacts offspring phenotype and health. Despite the fundamental importance of this stage of reproduction, the rate-limiting regulatory mechanisms operating locally to control fertility and fecundity are incompletely understood. Emerging studies highlight roles for microRNAs (miRNAs) in regulating reproductive and developmental processes and in modulating the quality and strength of the female immune response. Since endometrial receptivity and robust placentation require specific adaptation of the immune response, we hypothesize that miRNAs participate in establishing pregnancy through effects on key gene networks in immune cells. Our recent studies investigated miRNAs that are induced in the peri-conception environment, focusing on miRNAs that have immune-regulatory roles-particularly miR-223, miR-155, and miR-146a. Genetic mouse models deficient in individual miRNAs are proving informative in defining roles for these miRNAs in the generation and stabilization of regulatory T cells (Treg cells) that confer adaptive immune tolerance. Overlapping and redundant functions between miRNAs that target multiple genes, combined with multiple miRNAs targeting individual genes, indicate complex and sensitive regulatory networks. Although to date most data on miRNA regulation of reproductive events are from mice, conserved functions of miRNAs across species imply similar biological pathways operate in all mammals. Understanding the regulation and roles of miRNAs in the peri-conception immune response will advance our knowledge of how environmental determinants act at conception, and could have practical applications for animal breeding as well as human fertility.

Seminal Plasma Promotes Lesion Development in a Xenograft Model of Endometriosis.

The factors that predispose one-tenth of reproductive-aged women to endometriosis are poorly understood. We determined that genetic deficiency in transforming growth factor ß1 impairs endometriosis-like lesion growth in mice. Given that seminal plasma is an abundant source of transforming growth factor ß, we evaluated the effect of exposure to seminal plasma on the growth of endometrial lesions. Human endometrial explants were exposed to seminal plasma or to control medium before transfer to Prkdc(scid)-mutant (severe combined immunodeficient) mice. Xenografts exposed to seminal plasma showed an eightfold increase in volume and a 4.3-fold increase in weight after 14 days. These increases were associated with increased proliferation of endometrial epithelial cells and enhanced survival and proliferation of human stromal cells compared with those in control lesions, in which human stromal cell persistence was negligible. Although the distribution of macrophages was altered, their number and activation status did not change in response to seminal plasma. Seminal plasma stimulated the production of a variety of cytokines in endometrial tissue, including growth-regulated oncogene, granulocyte macrophage colony-stimulating factor, and IL-1ß. These data suggest that seminal plasma enhances the formation of endometriosis-like lesion via a direct effect on endometrial cell survival and proliferation, rather than via macrophage-mediated mechanisms. These findings raise the possibility that endometrial exposure to seminal plasma could contribute to endometriotic disease progression in women.

Seminal plasma transforming growth factor-ß, activin A and follistatin fluctuate within men over time.

STUDY QUESTION: Do seminal plasma transforming growth factor-ß (TGFB) cytokines vary within individuals over time, and does this relate to sperm parameters, age or prior abstinence? SUMMARY ANSWER: Activin A and follistatin, and to a lesser extent TGFB1, TGFB2 and TGFB3, vary within individuals over time, in association with duration of abstinence. WHAT IS ALREADY KNOWN: Seminal plasma TGFB cytokines can influence sperm function and reproductive success through interactions with the female reproductive tract after coitus. Over time, individual sperm parameters fluctuate considerably. Whether seminal fluid TGFB cytokines vary similarly, and the determinants of any variance, is unknown. STUDY DESIGN, SIZE, DURATION: Between two and seven semen samples were collected from each of 14 fertile donors at 6-10 week intervals over the course of 12 months, then seminal plasma cytokines and sperm parameters were measured. PARTICIPANTS/MATERIALS, SETTING AND METHOD: The concentrations and total amounts per ejaculate of TGFB1, TGFB2, TGFB3, activin A and follistatin were determined using commercial assays. Sperm parameters were assessed according to WHO IV standards. Mixed model analysis was utilised to determine the relative contribution of between- and within-individual factors to the variance. Relationships between cytokines and sperm parameters, as well as effect of age and duration of abstinence, were investigated by correlation analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Within-individual variability contributed to the total variance for all cytokines and sperm parameters, and was a stronger determinant than between-individual variability for activin A and follistatin as well as for total sperm concentration and sperm motility. Positive correlations between each of the three TGFB isoforms, and activin and follistatin, suggest co-regulation of synthesis. Duration of abstinence influenced total content of TGFB1, TGFB2, activin A and follistatin. TGFB1 correlated inversely with age. LIMITATIONS, REASONS FOR CAUTION: A limited number of donors from a single clinic were investigated. Clinical information on BMI, nutrition, smoking and other lifestyle factors was unavailable. Further studies are required to determine whether the findings can be generalised to larger populations and different ethnicities. WIDER IMPLICATIONS OF THE FINDINGS: These data reveal substantial variation over time in seminal fluid cytokines and indicate that repeated analyses are required to gain precise representative data on an individual's status. Within-individual variation in seminal fluid components should be taken into account when investigating seminal fluid cytokines. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the National Health and Medical Research Council of Australia, ID453556 and APP1041332. The authors have no competing interests to disclose.

TLR4 Signaling Is a Major Mediator of the Female Tract Response to Seminal Fluid in Mice.

Seminal fluid interacts with epithelial cells lining the female reproductive tract to induce expression of proinflammatory cytokines and chemokines, initiating immune tolerance mechanisms to facilitate pregnancy. TGFB cytokines are key signaling agents in seminal plasma but do not fully account for the female response to seminal fluid. We hypothesized that additional molecular pathways are utilized in seminal fluid signaling. Affymetrix microarray was employed to compare gene expression in the endometrium of mice 8 h after mating with either intact males or seminal fluid deficient (SVX/VAS) males. Bioinformatics analysis revealed TLR4 signaling as a strongly predicted upstream regulator activated by the differentially expressed genes and implicated TGFB signaling as a second key pathway. Quantitative PCR and microbead data confirmed that seminal fluid induces endometrial synthesis of several TLR4-regulated cytokines and chemokines, including CSF3, CXCL1, CXCL2, IL1A, IL6, LIF, and TNF. In primary uterine epithelial cells, CSF3, CXCL1, and CXCL2 were strongly induced by the TLR4 ligand LPS but suppressed by TGFB, while IL1A, TNF, and CSF2 were induced by both ligands. TLR4 was confirmed as essential for the full endometrial cytokine response using mice with a null mutation in Tlr4, where seminal fluid failed to induce endometrial Csf3, Cxcl2, Il6, and Tnf expression. This study provides evidence that TLR4 contributes to seminal fluid modulation of the periconception immune environment. Activation of TLR4 signaling by microbial or endogenous components of seminal fluid is thus implicated as a key element of the female tract response to seminal fluid at the outset of pregnancy in mice.

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.

TGF-ß mediates proinflammatory seminal fluid signaling in human cervical epithelial cells.

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-ß1, TGF-ß2, and TGF-ß3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-ß3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-ß isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-ß induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-ß neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-ß isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-ß in the male partner's seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.

Assessing the influence of preconception diet on male fertility: a systematic scoping review.

BACKGROUND: The last decade has seen increased research on the relationship between diet and male fertility, but there are no clearly defined nutritional recommendations for men in the preconception period to support clinical fertility outcomes. OBJECTIVE AND RATIONALE: The purpose of this scoping review is to examine the extent and range of research undertaken to evaluate the effect(s) of diet in the preconception period on male clinical fertility and reproductive outcomes. SEARCH METHODS: Four electronic databases (MEDLINE and EMBASE via Ovid, CAB Direct, and CINAHL via EBSCO) were searched from inception to July 2023 for randomized controlled trials (RCTs) and observational studies (prospective/retrospective, case-control, and cross-sectional). Intervention studies in male participants or couples aiming to achieve dietary or nutritional change, or non-intervention studies examining dietary or nutritional components (whole diets, dietary patterns, food groups or individual foods) in the preconception period were included. Controls were defined as any comparison group for RCTs, and any/no comparison for observational studies. Primary outcomes of interest included the effect(s) of male preconception diet on clinical outcomes such as conception (natural or via ART), pregnancy rates and live birth rates. Secondary outcomes included time to conception and sperm parameters. OUTCOMES: A total of 37 studies were eligible, including one RCT and 36 observational studies (prospective, cross-sectional, and case-control studies; four studies in non-ART populations) published between 2008 and 2023. Eight reported clinical outcomes, 26 reported on secondary outcomes, and three reported on both. The RCT did not assess clinical outcomes but found that tomato juice may benefit sperm motility. In observational studies, some evidence suggested that increasing fish or reducing sugar-sweetened beverages, processed meat or total fat may improve fecundability. Evidence for other clinical outcomes, such as pregnancy rates or live birth rates, showed no relationship with cereals, soy and dairy, and inconsistent relationships with consuming red meat or a 'healthy diet' pattern. For improved sperm parameters, limited evidence supported increasing fish, fats/fatty acids, carbohydrates and dairy, and reducing processed meat, while the evidence for fruits, vegetables, cereals, legumes, eggs, red meat and protein was inconsistent. Healthy diet patterns in general were shown to improve sperm health. WIDER IMPLICATIONS: Specific dietary recommendations for improving male fertility are precluded by the lack of reporting on clinical pregnancy outcomes, heterogeneity of the available literature and the paucity of RCTs to determine causation or to rule out reverse causation. There may be some benefit from increasing fish, adopting a healthy dietary pattern, and reducing consumption of sugar-sweetened beverages and processed meat, but it is unclear whether these benefits extend beyond sperm parameters to improve clinical fertility. More studies exploring whole diets rather than singular foods or nutritional components in the context of male fertility are encouraged, particularly by means of RCTs where feasible. Further assessment of core fertility outcomes is warranted and requires careful planning in high-quality prospective studies and RCTs. These studies can lay the groundwork for targeted dietary guidelines and enhance the prospects of successful fertility outcomes for men in the preconception period. Systematic search of preconception diet suggests that increasing fish and reducing sugary drinks, processed meats and total fat may improve male fertility, while consuming healthy diets, fish, fats/fatty acids, carbohydrates and dairy and reducing processed meat can improve sperm health.

A disrupted FOXP3 transcriptional signature underpins systemic regulatory T cell insufficiency in early pregnancy failure.

Regulatory T (Treg) cell defects are implicated in disorders of embryo implantation and placental development, but the origins of Treg cell dysfunction are unknown. Here, we comprehensively analyzed the phenotypes and transcriptional profile of peripheral blood Treg cells in individuals with early pregnancy failure (EPF). Compared to fertile subjects, EPF subjects had 32% fewer total Treg cells and 54% fewer CD45RA+CCR7+ naive Treg cells among CD4+ T cells, an altered Treg cell phenotype with reduced transcription factor FOXP3 and suppressive marker CTLA4 expression, and lower Treg:Th1 and Treg:Th17 ratios. RNA sequencing demonstrated an aberrant gene expression profile, with upregulation of pro-inflammatory genes including CSF2, IL4, IL17A, IL21, and IFNG in EPF Treg cells. In silico analysis revealed 25% of the Treg cell dysregulated genes are targets of FOXP3. We conclude that EPF is associated with systemic Treg cell defects arising due to disrupted FOXP3 transcriptional control and loss of lineage fidelity.

Immune regulatory cytokines in seminal plasma of healthy men: A scoping review and analysis of variance.

OBJECTIVE: Seminal plasma cytokines are associated with fertility and reproductive health, but progressing their clinical utility is hampered by absence of reference data on concentration ranges of relevant cytokines in healthy men. We employed a systematic approach to assemble current evidence on the concentrations of immune regulatory cytokines present in seminal plasma (SP) of normozoospermic and/or fertile men and evaluated the impact of different platform methodologies for cytokine quantification. EVIDENCE REVIEW: A systematic literature search was performed utilising PubMed, Web of Science and Scopus. Databases were searched from inception until 30th June 2022 inclusive, using combinations of keywords pertaining to seminal fluid and cytokines, and was restricted to human participants. Original data with values reported as concentration of specific cytokines in SP of men clearly defined as fertile or normozoospermic were extracted from studies written in English. RESULTS: A total of 3769 publications were initially identified, of which 118 fulfilled the eligibility criteria for inclusion. A total of 51 individual cytokines are detectable in SP of healthy men. The number of studies reporting on each cytokine range from 1 to >20. The reported concentrations for many cytokines linked with fertility status, including IL6, CXCL8/IL8, and TNFA, are highly variable between published studies. This is associated with the different immunoassay methodologies utilised and may be exacerbated by a lack of validation of assays to ensure suitability for SP assessment. Due to the large variation between studies, accurate reference ranges for healthy men cannot be determined from the published data. CONCLUSIONS: The concentrations of cytokines and chemokines detected in SP is inconsistent and highly variable between studies and cohorts, limiting current capacity to define reference ranges for cytokine concentrations in fertile men. The lack of standardisation in methods used to process and store SP, and variation in platforms used to evaluate cytokine abundance, are factors contributing to the observed heterogeneity. To progress the clinical utility of SP cytokine analysis will require standardisation and validation of methodologies so that reference ranges for healthy fertile men can be defined.

Regulatory T cells are paramount effectors in progesterone regulation of embryo implantation and fetal growth.

Progesterone (P4) is essential for embryo implantation, but the extent to which the pro-gestational effects of P4 depend on the maternal immune compartment is unknown. Here, we investigate whether regulatory T cells (Treg cells) act to mediate luteal phase P4 effects on uterine receptivity in mice. P4 antagonist RU486 administered to mice on days 0.5 and 2.5 postcoitum to model luteal phase P4 deficiency caused fewer CD4+Foxp3+ Treg cells and impaired Treg functional competence, along with dysfunctional uterine vascular remodeling and perturbed placental development in midgestation. These effects were linked with fetal loss and fetal growth restriction, accompanied by a Th1/CD8-skewed T cell profile. Adoptive transfer at implantation of Treg cells - but not conventional T cells - alleviated fetal loss and fetal growth restriction by mitigating adverse effects of reduced P4 signaling on uterine blood vessel remodeling and placental structure and by restoring maternal T cell imbalance. These findings demonstrate an essential role for Treg cells in mediating P4 effects at implantation and indicate that Treg cells are a sensitive and critical effector mechanism through which P4 drives uterine receptivity to support robust placental development and fetal growth.

Molecular filtration properties of the mouse expanded cumulus matrix: controlled supply of metabolites and extracellular signals to cumulus cells and the oocyte.

While formation of the expanded cumulus matrix and its importance for oocyte maturation and ovulation are well described, its function in these processes remains unknown. The degree of expansion and expression of cumulus matrix genes are positively correlated with oocyte quality, suggesting that this matrix plays a key role in oocyte maturation. Based on recognized filtration properties of analogous matrices, we investigated whether the cumulus matrix acts as a molecular filter by assessing diffusion of fluorescently labeled dextrans (neutral and negatively charged) and hydrophilic (glucose) and hydrophobic (cholesterol) metabolites in cumulus oocyte complexes (COCs). Expanded in vivo-matured COCs resisted absorption of glucose and cholesterol compared to unexpanded COCs. In vitro-matured (IVM) COCs have a pronounced deficiency in cumulus matrix proteins and have poor oocyte quality. Here we demonstrate that IVM cumulus matrix has deficient filtration properties, with dextran and glucose and cholesterol molecules diffusing more readily into IVM than in vivo-matured COCs. Taking the inverse approach, we found that prostaglandin E2 (PGE2), synthesized by cumulus cells, is retained within the matrix of in vivo-matured COCs but IVM COCs have reduced capacity to retain PGE2, secreting significantly more into the medium. This is the first demonstration of a biophysical property of the cumulus matrix. The ability to regulate metabolite supply from the surrounding environment while sequestering vital signaling factors, such as PGE2, is likely to impact oocyte maturation. Thus, IVM may reduce oocyte quality due to dysregulated control of metabolites and signaling molecules.

Measuring Reactive Oxygen Species in Semen for Male Preconception Care: A Scientist Perspective.

Oxidative stress and elevated levels of seminal and sperm reactive oxygen species (ROS) may contribute to up to 80% of male infertility diagnosis, with sperm ROS concentrations at fertilization important in the development of a healthy fetus and child. The evaluation of ROS in semen seems promising as a potential diagnostic tool for male infertility and male preconception care with a number of clinically available tests on the market (MiOXSYS, luminol chemiluminescence and OxiSperm). While some of these tests show promise for clinical use, discrepancies in documented decision limits and lack of cohort studies/clinical trials assessing their benefits on fertilization rates, embryo development, pregnancy and live birth rates limit their current clinical utility. In this review, we provide an update on the current techniques used for analyzing semen ROS concentrations clinically, the potential to use of ROS research tools for improving clinical ROS detection in sperm and describe why we believe we are likely still a long way away before semen ROS concentrations might become a mainstream preconception diagnostic test in men.

Regulatory T Cell Proportion and Phenotype Are Altered in Women Using Oral Contraception.

Regulatory T (Treg) cells are a specialized CD4+ T cell subpopulation that are essential for immune homeostasis, immune tolerance, and protection against autoimmunity. There is evidence that sex-steroid hormones estrogen and progesterone modulate Treg cell abundance and phenotype in women. Since natural oscillations in these hormones are modified by hormonal contraceptives, we examined whether oral contraception (OC) use impacts Treg cells and related T cell populations. T cells were analyzed by multiparameter flow cytometry in peripheral blood collected across the menstrual cycle from healthy women either using OC or without hormonal contraception and from age-matched men. Compared to naturally cycling women, women using OC had fewer Treg cells and an altered Treg cell phenotype. Notably, Treg cells exhibiting a strongly suppressive phenotype, defined by high FOXP3, CD25, Helios, HLADR, CTLA4, and Ki67, comprised a lower proportion of total Treg cells, particularly in the early- and mid-cycle phases. The changes were moderate compared to more substantial differences in Treg cells between women and men, wherein women had fewer Treg cells-especially of the effector memory Treg cell subset-associated with more T helper type 1 (Th1) cells and CD8+ T cells and lower Treg:Th1 cell and Treg:CD8+ T cell ratios than men. These findings imply that OC can modulate the number and phenotype of peripheral blood Treg cells and raise the possibility that Treg cells contribute to the physiological changes and altered disease susceptibility linked with OC use.

Sperm modulate uterine immune parameters relevant to embryo implantation and reproductive success in mice.

Seminal fluid factors modulate the female immune response at conception to facilitate embryo implantation and reproductive success. Whether sperm affect this response has not been clear. We evaluated global gene expression by microarray in the mouse uterus after mating with intact or vasectomized males. Intact males induced greater changes in gene transcription, prominently affecting pro-inflammatory cytokine and immune regulatory genes, with TLR4 signaling identified as a top-ranked upstream driver. Recruitment of neutrophils and expansion of peripheral regulatory T cells were elevated by seminal fluid of intact males. In vitro, epididymal sperm induced IL6, CXCL2, and CSF3 in uterine epithelial cells of wild-type, but not Tlr4 null females. Collectively these experiments show that sperm assist in promoting female immune tolerance by eliciting uterine cytokine expression through TLR4-dependent signaling. The findings indicate a biological role for sperm beyond oocyte fertilization, in modulating immune mechanisms involved in female control of reproductive investment.

Roles of male reproductive tract extracellular vesicles in reproduction.

Extracellular vesicles (EVs) are secreted cell-derived membrane structures present in all organisms across animal, bacterial, and plant phyla. These vesicles play important roles in cell-cell communication in many processes integral to health and disease. Recent studies demonstrate that EVs and their cargo have influential and conserved roles in male reproduction. While EVs have been isolated from virtually all specialized tissues comprising the male reproductive tract, they are best characterized in the epididymis (epididymosomes) and seminal fluid (seminal fluid extracellular vesicles or prostasomes). Broadly speaking, EVs promote reproductive success through supporting sperm development and function, as well as influencing the physiology of female reproductive tract cells after mating. In this review, we present current knowledge on the composition and function of male reproductive tract EV populations in both normal physiology and pathology, and argue that their functions identify them as critical regulators of fertility and fecundity.