Thymus-Derived Regulatory T Cells Exhibit Foxp3 Epigenetic Modification and Phenotype Attenuation after Mating in Mice.

Regulatory T cells (Tregs) are essential for maternal tolerance in allogeneic pregnancy. In preeclampsia, Tregs are fewer and display aberrant phenotypes, particularly in the thymic Treg (tTreg) compartment, potentially because of insufficient priming to male partner alloantigens before conception. To investigate how tTregs as well as peripheral Tregs (pTregs) respond to male partner seminal fluid, Foxp3+CD4+ Tregs were examined in the uterus and uterus-draining lymph nodes in virgin estrus mice and 3.5 d postcoitum. Mating elicited 5-fold increases in uterine Tregs accompanied by extensive Treg proliferation in the uterus-draining lymph nodes, comprising 70% neuropilin 1+ tTregs and 30% neuropilin 1- pTregs. Proliferation marker Ki67 and suppressive competence markers Foxp3 and CTLA4 were induced after mating in both subsets, and Ki67, CTLA4, CD25, and GITR were higher in tTregs than in pTregs. Analysis by t-stochastic neighbor embedding confirmed phenotypically distinct tTreg and pTreg clusters, with the proportion of tTregs but not pTregs among CD4+ T cells expanding in response to seminal fluid. Bisulphite sequencing revealed increased demethylation of the Treg-specific demethylation region in the Foxp3 locus in tTregs but not pTregs after mating. These data show that tTregs and pTregs with distinct phenotypes both respond to seminal fluid priming, but the Foxp3 epigenetic signature is uniquely increased in tTregs. We conclude that reproductive tract tTregs as well as pTregs are sensitive to local regulation by seminal fluid, providing a candidate mechanism warranting evaluation for the potential to influence preeclampsia susceptibility in women.

Prednisolone in early pregnancy inhibits regulatory T cell generation and alters fetal and placental development in mice.

Corticosteroids have been utilised in the assisted reproduction setting with the expectation of suppressing aberrant immune activation and improving fertility in women. However, the effects of corticosteroids on fertility, and on pregnancy and offspring outcomes, are unclear. In this study, mice were administered prednisolone (1 mg/kg) or PBS daily in the pre-implantation phase, and effects on the adaptive immune response, the implantation rate, fetal development and postnatal outcomes were investigated. Prednisolone disrupted the expected expansion of CD4+ T cells in early pregnancy, inhibiting generation of both regulatory T cells (Treg cells) and effector T cells and suppressing IFNG required for T cell functional competence. Prednisolone caused an 8-20% increase in the embryo implantation rate and increased the number of viable pups per litter. In late gestation, fetal and placental weights were reduced in a litter size-dependent manner, and the canonical inverse relationship between litter size and fetal weight was lost. The duration of pregnancy was extended by ~ 0.5 day and birth weight was reduced by ~ 5% after prednisolone treatment. Viability of prednisolone-exposed offspring was comparable to controls, but body weight was altered in adulthood, particularly in male offspring. Thus, while prednisolone given in the pre-implantation phase in mice increases maternal receptivity to implantation and resource investment in fetal growth, there is a trade-off in long-term consequences for fetal development, birth weight and offspring health. These effects are associated with, and likely caused by, prednisolone suppression of the adaptive immune response at the outset of pregnancy.

Recent advances in reproductive research in Australia and New Zealand: highlights from the Annual Meeting of the Society for Reproductive Biology, 2022.

In 2022, the Society for Reproductive Biology came together in Christchurch New Zealand (NZ), for its first face-to-face meeting since the global COVID-19 pandemic. The meeting showcased recent advancements in reproductive research across a diverse range of themes relevant to human health and fertility, exotic species conservation, and agricultural breeding practices. Here, we highlight the key advances presented across the main themes of the meeting, including advances in addressing opportunities and challenges in reproductive health related to First Nations people in Australia and NZ; increasing conservation success of exotic species, including ethical management of invasive species; improvements in our understanding of developmental biology, specifically seminal fluid signalling, ovarian development and effects of environmental impacts such as endocrine-disrupting chemicals; and leveraging scientific breakthroughs in reproductive engineering to drive solutions for fertility, including in assisted reproductive technologies in humans and agricultural industries, and for regenerative medicine.

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.

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.

Immune determinants of endometrial receptivity: a biological perspective.

Immune cells are essential for endometrial receptivity to embryo implantation and early placental development. They exert tissue-remodeling and immune regulatory roles-acting to promote epithelial attachment competence, regulate the differentiation of decidual cells, remodel the uterine vasculature, control and resolve inflammatory activation, and suppress destructive immunity to paternally inherited alloantigens. From a biological perspective, the endometrial immune response exerts a form of "quality control"-it promotes implantation success when conditions are favorable but constrains receptivity when physiological circumstances are not ideal. Women with recurrent implantation failure and recurrent miscarriage may exhibit altered numbers or disturbed function of certain uterine immune cell populations-most notably uterine natural killer cells and regulatory T cells. Preclinical and animal studies indicate that deficiencies or aberrant activation states in these cells can be causal in the pathophysiological mechanisms of infertility. Immune cells are, therefore, targets for diagnostic evaluation and therapeutic intervention. However, current diagnostic tests are overly simplistic and have limited clinical utility. To be more informative, they need to account for the full complexity and reflect the range of perturbations that can occur in uterine immune cell phenotypes and networks. Moreover, safe and effective interventions to modulate these cells are in their infancy, and personalized approaches matched to specific diagnostic criteria will be needed. Here we summarize current biological understanding and identify knowledge gaps to be resolved before the promise of therapies to target the uterine immune response can be fully realized.

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.

Endocrine Disruptor Compounds-A Cause of Impaired Immune Tolerance Driving Inflammatory Disorders of Pregnancy?

Endocrine disrupting compounds (EDCs) are prevalent and ubiquitous in our environment and have substantial potential to compromise human and animal health. Amongst the chronic health conditions associated with EDC exposure, dysregulation of reproductive function in both females and males is prominent. Human epidemiological studies demonstrate links between EDC exposure and infertility, as well as gestational disorders including miscarriage, fetal growth restriction, preeclampsia, and preterm birth. Animal experiments show EDCs administered during gestation, or to either parent prior to conception, can interfere with gamete quality, embryo implantation, and placental and fetal development, with consequences for offspring viability and health. It has been presumed that EDCs operate principally through disrupting hormone-regulated events in reproduction and fetal development, but EDC effects on maternal immune receptivity to pregnancy are also implicated. EDCs can modulate both the innate and adaptive arms of the immune system, to alter inflammatory responses, and interfere with generation of regulatory T (Treg) cells that are critical for pregnancy tolerance. Effects of EDCs on immune cells are complex and likely exerted by both steroid hormone-dependent and hormone-independent pathways. Thus, to better understand how EDCs impact reproduction and pregnancy, it is imperative to consider how immune-mediated mechanisms are affected by EDCs. This review will describe evidence that several EDCs modify elements of the immune response relevant to pregnancy, and will discuss the potential for EDCs to disrupt immune tolerance required for robust placentation and optimal fetal development.

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.

Effect of Intralipid infusion on peripheral blood T cells and plasma cytokines in women undergoing assisted reproduction treatment.

OBJECTIVES: Intravenous infusion of Intralipid is an adjunct therapy in assisted reproduction treatment (ART) when immune-associated infertility is suspected. Here, we evaluated the effect of Intralipid infusion on regulatory T cells (Treg cells), effector T cells and plasma cytokines in peripheral blood of women undertaking IVF. METHODS: This prospective, observational pilot study assessed Intralipid infusion in 14 women exhibiting recurrent implantation failure, a clinical sign of immune-associated infertility. Peripheral blood was collected immediately prior to and 7 days after intravenous administration of Intralipid. Plasma cytokines were measured by Luminex, and T-cell subsets were analysed by flow cytometry. RESULTS: A small increase in conventional CD8+ T cells occurred after Intralipid infusion, but no change was seen in CD4+ Treg cells, or naïve, memory or effector memory T cells. Proliferation marker Ki67, transcription factors Tbet and RORγt, and markers of suppressive capacity CTLA4 and HLA-DR were unchanged. Dimensionality-reduction analysis using the tSNE algorithm confirmed no phenotype shift within Treg cells or other T cells. Intralipid infusion increased plasma CCL2, CCL3, CXCL8, GM-CSF, G-CSF, IL-6, IL-21, TNF and VEGF. CONCLUSION: Intralipid infusion elicited elevated pro-inflammatory cytokines, and a minor increase in CD8+ T cells, but no change in pro-tolerogenic Treg cells. Notwithstanding the limitation of no placebo control, the results do not support Intralipid as a candidate intervention to attenuate the Treg cell response in women undergoing ART. Future placebo-controlled studies are needed to confirm the potential efficacy and clinical significance of Intralipid in attenuating cytokine induction and circulating CD8+ T cells.

Therapeutic Potential of Regulatory T Cells in Preeclampsia-Opportunities and Challenges.

Inflammation is a central feature and is implicated as a causal factor in preeclampsia and other hypertensive disorders of pregnancy. Inflammatory mediators and leukocytes, which are elevated in peripheral blood and gestational tissues, contribute to the uterine vascular anomalies and compromised placental function that characterize particularly the severe, early onset form of disease. Regulatory T (Treg) cells are central mediators of pregnancy tolerance and direct other immune cells to counteract inflammation and promote robust placentation. Treg cells are commonly perturbed in preeclampsia, and there is evidence Treg cell insufficiency predates onset of symptoms. A causal role is implied by mouse studies showing sufficient numbers of functionally competent Treg cells must be present in the uterus from conception, to support maternal vascular adaptation and prevent later placental inflammatory pathology. Treg cells may therefore provide a tractable target for both preventative strategies and treatment interventions in preeclampsia. Steps to boost Treg cell activity require investigation and could be incorporated into pregnancy planning and preconception care. Pharmacological interventions developed to target Treg cells in autoimmune conditions warrant consideration for evaluation, utilizing rigorous clinical trial methodology, and ensuring safety is paramount. Emerging cell therapy tools involving in vitro Treg cell generation and/or expansion may in time become relevant. The success of preventative and therapeutic approaches will depend on resolving several challenges including developing informative diagnostic tests for Treg cell activity applicable before conception or during early pregnancy, selection of relevant patient subgroups, and identification of appropriate windows of gestation for intervention.

Periconception onset diabetes is associated with embryopathy and fetal growth retardation, reproductive tract hyperglycosylation and impaired immune adaptation to pregnancy.

Diabetes has been linked with impaired fertility but the underlying mechanisms are not well defined. Here we use a streptozotocin-induced diabetes mouse model to investigate the cellular and biochemical changes in conceptus and maternal tissues that accompany hyperglycaemia. We report that streptozotocin treatment before conception induces profound intra-cellular protein ß-O-glycosylation (O-GlcNAc) in the oviduct and uterine epithelium, prominent in early pregnancy. Diabetic mice have impaired blastocyst development and reduced embryo implantation rates, and delayed mid-gestation growth and development. Peri-conception changes are accompanied by increased expression of pro-inflammatory cytokine Trail, and a trend towards increased Il1a, Tnf and Ifng in the uterus, and changes in local T-cell dynamics that skew the adaptive immune response to pregnancy, resulting in 60% fewer anti-inflammatory regulatory T-cells within the uterus-draining lymph nodes. Activation of the heat shock chaperones, a mechanism for stress deflection, was evident in the reproductive tract. Additionally, we show that the embryo exhibits elevated hyper-O-GlcNAcylation of both cytoplasmic and nuclear proteins, associated with activation of DNA damage (É£H2AX) pathways. These results advance understanding of the impact of peri-conception diabetes, and provide a foundation for designing interventions to support healthy conception without propagation of disease legacy to offspring.