Emma S. Lucas

Uterine Selection of Human Embryos at Implantation

Human embryos frequently harbor large-scale complex chromosomal errors that impede normal development. Affected embryos may fail to implant although many first breach the endometrial epithelium and embed in the decidualizing stroma before being rejected via mechanisms that are poorly understood. Here we show that developmentally impaired human embryos elicit an endoplasmic stress response in human decidual cells. A stress response was also evident upon in vivo exposure of mouse uteri to culture medium conditioned by low-quality human embryos. By contrast, signals emanating from developmentally competent embryos activated a focused gene network enriched in metabolic enzymes and implantation factors. We further show that trypsin, a serine protease released by pre-implantation embryos, elicits Ca2+ signaling in endometrial epithelial cells. Competent human embryos triggered short-lived oscillatory Ca2+ fluxes whereas low-quality embryos caused a heightened and prolonged Ca2+ response. Thus, distinct positive and negative mechanisms contribute to active selection of human embryos at implantation.

Disordered IL-33/ST2 Activation in Decidualizing Stromal Cells Prolongs Uterine Receptivity in Women with Recurrent Pregnancy Loss

Decidualization renders the endometrium transiently receptive to an implanting blastocyst although the underlying mechanisms remain incompletely understood. Here we show that human endometrial stromal cells (HESCs) rapidly release IL-33, a key regulator of innate immune responses, upon decidualization. In parallel, differentiating HESCs upregulate the IL-33 transmembrane receptor ST2L and other pro-inflammatory mediators before mounting a profound anti-inflammatory response that includes downregulation of ST2L and increased expression of the soluble decoy receptor sST2. We demonstrate that HESCs secrete factors permissive of embryo implantation in mice only during the pro-inflammatory phase of the decidual process. IL-33 knockdown in undifferentiated HESCs was sufficient to abrogate this pro-inflammatory decidual response. Further, sequential activation of the IL-33/ST2L/sST2 axis was disordered in decidualizing HESCs from women with recurrent pregnancy loss. Signals from these cultures prolonged the implantation window but also caused subsequent pregnancy failure in mice. Thus, Il-33/ST2 activation in HESCS drives an autoinflammatory response that controls the temporal expression of receptivity genes. Failure to constrain this response predisposes to miscarriage by allowing out-of-phase implantation in an unsupportive uterine environment.

Maternal low-protein diet during mouse pre-implantation development induces vascular dysfunction and altered renin-angiotensin-system homeostasis in the offspring

Environmental perturbations during early mammalian development can affect aspects of offspring growth and cardiovascular health. We have demonstrated previously that maternal gestational dietary protein restriction in mice significantly elevated adult offspring systolic blood pressure. Therefore, the present study investigates the key mechanisms of blood pressure regulation in these mice. Following mating, female MF-1 mice were assigned to either a normal-protein diet (NPD; 18 % casein) or an isocaloric low-protein diet throughout gestation (LPD; 9 % casein), or fed the LPD exclusively during the pre-implantation period (3.5 d) before returning to the NPD for the remainder of gestation (Emb-LPD). All offspring received standard chow. At 22 weeks, isolated mesenteric arteries from LPD and Emb-LPD males displayed significantly attenuated vasodilatation to isoprenaline (P = 0.04 and P = 0.025, respectively), when compared with NPD arteries. At 28 weeks, stereological analysis of glomerular number in female left kidneys revealed no significant difference between the groups. Real-time RT-PCR analysis of type 1a angiotensin II receptor, Na+/K+ ATPase transporter subunits and glucocorticoid receptor expression in male and female left kidneys revealed no significant differences between the groups. LPD females displayed elevated serum angiotensin-converting enzyme (ACE) activity (P = 0.044), whilst Emb-LPD males had elevated lung ACE activity (P = 0.001), when compared with NPD offspring. These data demonstrate that elevated offspring systolic blood pressure following maternal gestational protein undernutrition is associated with impaired arterial vasodilatation in male offspring, elevated serum and lung ACE activity in female and male offspring, respectively, but kidney glomerular number in females and kidney gene expression in male and female offspring appear unaffected.

Maternal Periconceptional and Gestational Low Protein Diet Affects Mouse Offspring Growth, Cardiovascular and Adipose Phenotype at 1 Year of Age

Human and animal studies have revealed a strong association between periconceptional environmental factors, such as poor maternal diet, and an increased propensity for cardiovascular and metabolic disease in adult offspring. Previously, we reported cardiovascular and physiological effects of maternal low protein diet (LPD) fed during discrete periods of periconceptional development on 6-month-old mouse offspring. Here, we extend the analysis in 1 year aging offspring, evaluating mechanisms regulating growth and adiposity. Isocaloric LPD (9% casein) or normal protein diet (18% casein; NPD) was fed to female MF-1 mice either exclusively during oocyte maturation (for 3.5 days prior to mating; Egg-LPD, Egg-NPD, respectively), throughout gestation (LPD, NPD) or exclusively during preimplantation development (for 3.5 days post mating; Emb-LPD). LPD and Emb-LPD female offspring were significantly lighter and heavier than NPD females respectively for up to 52 weeks. Egg-LPD, LPD and Emb-LPD offspring displayed significantly elevated systolic blood pressure at 52 weeks compared to respective controls (Egg-NPD, NPD). LPD females had significantly reduced inguinal and retroperitoneal fat pad: body weight ratios compared to NPD females. Expression of the insulin receptor (Insr) and insulin-like growth factor I receptor (Igf1r) in retroperitoneal fat was significantly elevated in Emb-LPD females (P<0.05), whilst Emb-LPD males displayed significantly decreased expression of the mitochondrial uncoupling protein 1 (Ucp1) gene compared to NPD offspring. LPD females displayed significantly increased expression of Ucp1 in interscapular brown adipose tissue when compared to NPD offspring. Our results demonstrate that aging offspring body weight, cardiovascular and adiposity homeostasis can be programmed by maternal periconceptional nutrition. These adverse outcomes further exemplify the criticality of dietary behaviour around the time of conception on long-term offspring health.

Nutrition of females during the peri-conceptional period and effects on foetal programming and health of offspring

The period around the time of conception is one characterised by considerable cytological and molecular restructuring as ovulation occurs, the oocyte is fertilised and the embryonic developmental programme begins. The intrinsic processes regulating peri-conceptional progression are supplemented by environmental factors, which contribute important metabolic information that influences several aspects of the developmental programme. Indeed, there is growing evidence from different mammalian animal models, reviewed here, that the peri-conceptional environment mediated through maternal nutrition can modify development throughout gestation and affect the physiological and metabolic health of adult offspring. The concept that adult disease risk may owe its origin to the quality of peri-conceptional maternal nutrition is one, which merits further research for mechanistic understanding and devising preventive strategies.

Adaptive responses of the embryo to maternal diet and consequences for post-implantation development

Maternal periconceptional (PC) nutrition, coupled with maternal physiological condition, can impact on reproductive performance and potential across mammalian species. Oocyte quality and embryo development are affected adversely by either nutrient restriction or excess. Moreover, the quality of maternal PC nutrition can have lasting effects through fetal development and postnatally into adulthood. Chronic disease, notably cardiovascular and metabolic disease, and abnormal behaviour have been identified in adult offspring in small and large animal models of PC nutrient restriction. These long-term effects associate with compensatory responses that begin from the time of early embryo development. This review assesses the field of PC nutrition in vivo on short- and long-term developmental consequences in rodent and ruminant models and considers the implications for human health.

Decidualization induces a secretome switch in perivascular niche cells of the human endometrium.

The endometrial perivascular microenvironment is rich in mesenchymal stem-like cells that express type 1 integral membrane protein Sushi domain containing 2 (SUSD2) but the role of these cells in the decidual transformation of this tissue in pregnancy is unknown. We used an antibody directed against SUSD2 (W5C5) to isolate perivascular (W5C5(+)) and nonperivascular (W5C5(-)) fibroblasts from mid-luteal biopsies. We show that SUSD2 expression, and hence the ratio of W5C5(+):W5C5(-) cells, changes in culture depending on cell-cell contact and activation of the Notch signaling pathway. RNA sequencing revealed that cultures derived from W5C5(+) progenitor cells remain phenotypically distinct by the enrichment of novel and established endometrial perivascular signature genes. In an undifferentiated state, W5C5(+)-derived cells produced lower levels of various chemokines and inflammatory modulators when compared with their W5C5(-) counterparts. This divergence in secretomes was switched and became more pronounced upon decidualization, which transformed perivascular W5C5(+) cells into the dominant source of a range of chemokines and cytokines, including leukemia inhibitory factor and chemokine (C-C motif) ligand 7. Our findings suggest that the decidual response is spatially organized at the embryo-maternal interface with differentiating perivascular cells establishing distinct cytokine and chemokine profiles that could potentially direct trophoblast toward maternal vessels and govern local immune responses in pregnancy.

Loss of endometrial plasticity in recurrent pregnancy loss

Menstruation drives cyclic activation of endometrial progenitor cells, tissue regeneration, and maturation of stromal cells, which differentiate into specialized decidual cells prior to and during pregnancy. Aberrant responsiveness of human endometrial stromal cells (HESCs) to deciduogenic cues is strongly associated with recurrent pregnancy loss (RPL), suggesting a defect in cellular maturation. MeDIP‐seq analysis of HESCs did not reveal gross perturbations in CpG methylation in RPL cultures, although quantitative differences were observed in or near genes that are frequently deregulated in vivo. However, RPL was associated with a marked reduction in methylation of defined CA‐rich motifs located throughout the genome but enriched near telomeres. Non‐CpG methylation is a hallmark of cellular multipotency. Congruently, we demonstrate that RPL is associated with a deficiency in endometrial clonogenic cell populations. Loss of epigenetic stemness features also correlated with intragenic CpG hypomethylation and reduced expression of HMGB2, coding high mobility group protein 2. We show that knockdown of this sequence‐independent chromatin protein in HESCs promotes senescence and impairs decidualization, exemplified by blunted time‐dependent secretome changes. Our findings indicate that stem cell deficiency and accelerated stromal senescence limit the differentiation capacity of the endometrium and predispose for pregnancy failure. Stem Cells 2016;34:346–356

Induction of 11β-HSD 1 and Activation of Distinct Mineralocorticoid Receptor- and Glucocorticoid Receptor-Dependent Gene Networks in Decidualizing Human Endometrial Stromal Cells

The actions of glucocorticoids at the feto-maternal interface are not well understood. Here, we show that decidualization of human endometrial stromal cells (HESCs) in response to progesterone and cAMP signaling is associated with a strong induction of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) expression and enzyme activity. Decidualization also triggered a gradual decrease in glucocorticoid receptor (GR) expression and reciprocal increase in mineralocorticoid receptor (MR) levels. Gene expression profiling of differentiating HESCs after small interfering RNA (siRNA)-mediated knockdown of either GR or MR identified 239 and 167 significantly regulated genes, respectively. Interestingly, GR-repressed genes were enriched for Krüppel-associated box domain containing zinc-finger proteins, transcriptional repressors involved in heterochromatin formation. In agreement, GR knockdown was sufficient to enhance trimethylated H3K9 levels in decidualizing cells. Conversely, we identified several MR-dependent genes implicated in lipid droplet biogenesis and retinoid metabolism. For example, the induction in differentiating HESCs of DHRS3, encoding a highly conserved enzyme that catalyzes the oxidation/reduction of retinoids and steroids, was enhanced by aldosterone, attenuated in response to MR knockdown, and abolished upon treatment with the MR antagonist RU26752. Furthermore, we demonstrate that decidualization is associated with dynamic changes in the abundance and distribution of cytoplasmic lipid droplets, the formation of which was blocked by RU26752. In summary, progesterone drives local cortisol biosynthesis by decidual cells through induction of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), leading to transcriptional regulation of distinct GR and MR gene networks involved in epigenetic programming and lipid and retinoid metabolism, respectively.

Elevated Periimplantation Uterine Natural Killer Cell Density in Human Endometrium Is Associated With Impaired Corticosteroid Signaling in Decidualizing Stromal Cells

Background: Decidualizing human endometrial stromal cells (HESCs) profoundly up-regulate 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), the enzyme that converts inert cortisone to active cortisol. We postulated that the induction of a cortisol gradient upon decidualization of the periimplantation endometrium may impact on the uterine natural killer (uNK) cell population and on local expression of corticosteroid-dependent target genes. Methods: Midluteal endometrial biopsies (n = 55) were processed for uNK cell (CD56) analysis and primary HESC cultures. The cultures remained either untreated or were decidualized for 4 or 8 days. A tissue microarray was constructed from endometria with normal (n = 18) and elevated uNK cell (n = 18) scores. An abnormal uNK cell test was defined as greater than 5% CD56+ cells in the subluminal stroma. Results: Increased uNK cell density was associated with lower endometrial expression of 11βHSD1 and mineralocorticoid receptor (MR) but not glucocorticoid receptor in vivo. Elevated uNK cell density also corresponded to impaired induction of key decidual markers (11βHSD1, prolactin, and insulin-like growth factor binding protein-1) and MR-dependent enzymes (dehydrogenase/reductase member 3 and retinol saturase) in differentiating HESC cultures. Increased uNK cell density in vivo was not associated with increased in vitro expression of either IL-15 or IL-11, two cytokines implicated in uNK cell regulation. Conclusions: Elevated levels of uNK cells in the stroma underlying the surface epithelium are associated with inadequate cortisol biosynthesis by resident decidualizing cells and suboptimal induction of key MR-dependent enzymes involved in lipid biogenesis and the retinoid transport pathway. Our observations suggest that uNK cell testing identifies those women at risk of reproductive failure due to relative uterine cortisol deficiency.