The autophagy protein, ATG14 safeguards against unscheduled pyroptosis activation to enable embryo transport during early pregnancy.

Recurrent pregnancy loss (RPL), characterized by two or more failed clinical pregnancies, poses a significant challenge to reproductive health. In addition to embryo quality and endometrial function, proper oviduct function is also essential for successful pregnancy establishment. Therefore, structural abnormalities or inflammation resulting from infection in the oviduct may impede the transport of embryos to the endometrium, thereby increasing the risk of miscarriage. However, the precise cellular mechanisms that maintain the structural and functional integrity of the oviduct are not studied yet. Here, we report that autophagy is critical for maintaining the oviduct homeostasis and keeping the inflammation under check to enable embryo transport. Specifically, the loss of the autophagy-related gene, Atg14 in the oviduct causes severe structural abnormalities compromising its cellular plasticity and integrity leading to the retention of embryos. Interestingly, the selective loss of Atg14 in oviduct ciliary epithelial cells did not impact female fertility, highlighting the specificity of ATG14 function in distinct cell types within the oviduct. Mechanistically, loss of Atg14 triggered unscheduled pyroptosis leading to inappropriate embryo retention and impeded embryo transport in the oviduct. Finally, pharmacological activation of pyroptosis in pregnant mice led to an impairment in embryo transport. Together, we found that ATG14 safeguards against unscheduled pyroptosis activation to enable embryo transport from the oviduct to uterus for the successful implantation. Of clinical significance, these findings provide possible insights on the underlying mechanism(s) of early pregnancy loss and might aid in developing novel prevention strategies using autophagy modulators.

A GREB1-steroid receptor feedforward mechanism governs differential GREB1 action in endometrial function and endometriosis.

Cellular responses to the steroid hormones, estrogen (E2), and progesterone (P4) are governed by their cognate receptor's transcriptional output. However, the feed-forward mechanisms that shape cell-type-specific transcriptional fulcrums for steroid receptors are unidentified. Herein, we found that a common feed-forward mechanism between GREB1 and steroid receptors regulates the differential effect of GREB1 on steroid hormones in a physiological or pathological context. In physiological (receptive) endometrium, GREB1 controls P4-responses in uterine stroma, affecting endometrial receptivity and decidualization, while not affecting E2-mediated epithelial proliferation. Of mechanism, progesterone-induced GREB1 physically interacts with the progesterone receptor, acting as a cofactor in a positive feedback mechanism to regulate P4-responsive genes. Conversely, in endometrial pathology (endometriosis), E2-induced GREB1 modulates E2-dependent gene expression to promote the growth of endometriotic lesions in mice. This differential action of GREB1 exerted by a common feed-forward mechanism with steroid receptors advances our understanding of mechanisms that underlie cell- and tissue-specific steroid hormone actions.

Autophagy is required for stem-cell-mediated endometrial programming and the establishment of pregnancy.

Autophagy plays an important role in the normal growth and morphogenesis of a variety of tissues. Its role in uterine maturation, however, is not fully characterized. Recently, we reported that BECN1 (Beclin1)-dependent autophagy, but not apoptosis, is crucial for stem cell-mediated endometrial programming and the establishment of pregnancy in mice. Upon genetic and pharmacological inhibition of BECN1-mediated autophagy, female mice displayed severe endometrial structural and functional defects leading to infertility. Specifically, conditional loss of Becn1 in the uterus induces apoptosis and results in the gradual loss of endometrial progenitor stem cells. Importantly, the restoration of BECN1-driven autophagy, but not apoptosis in Becn1 conditionally ablated mice promoted normal uterine adenogenesis and morphogenesis. Overall, our findings emphasize the critical role of intrinsic autophagy in endometrial homeostasis and on the molecular underpinnings of uterine differentiation.

SF3B1-dependent alternative splicing is critical for maintaining endometrial homeostasis and the establishment of pregnancy.

The remarkable potential of human endometrium to undergo spontaneous remodeling is shaped by controlled spatiotemporal gene expression patterns. Although hormone-driven transcription shown to govern these patterns, the post-transcriptional processing of these mRNA transcripts, including the mRNA splicing in the endometrium is not studied yet. Here, we report that the splicing factor, SF3B1 is central in driving alternative splicing (AS) events that are vital for physiological responses of the endometrium. We show that loss of SF3B1 splicing activity impairs stromal cell decidualization as well as embryo implantation. Transcriptomic analysis revealed that SF3B1 depletion decidualizing stromal cells led to differential mRNA splicing. Specifically, a significant upregulation in mutually exclusive AS events (MXEs) with SF3B1 loss resulted in the generation of aberrant transcripts. Further, we found that some of these candidate genes phenocopy SF3B1 function in decidualization. Importantly, we identify progesterone as a potential upstream regulator of SF3B1-mediated functions in endometrium possibly via maintaining its persistently high levels, in coordination with deubiquitinating enzymes. Collectively, our data suggest that SF3B1-driven alternative splicing plays a critical role in mediating the endometrial-specific transcriptional paradigms. Thus, the identification of novel mRNA variants associated with successful pregnancy establishment may help to develop new strategies to diagnose or prevent early pregnancy loss.

Beclin-1-dependent autophagy, but not apoptosis, is critical for stem-cell-mediated endometrial programming and the establishment of pregnancy.

The human endometrium shows a remarkable regenerative capacity that enables cyclical regeneration and remodeling throughout a woman's reproductive life. Although early postnatal uterine developmental cues direct this regeneration, the vital factors that govern early endometrial programming are largely unknown. We report that Beclin-1, an essential autophagy-associated protein, plays an integral role in uterine morphogenesis during the early postnatal period. We show that conditional depletion of Beclin-1 in the uterus triggers apoptosis and causes progressive loss of Lgr5+/Aldh1a1+ endometrial progenitor stem cells, with concomitant loss of Wnt signaling, which is crucial for stem cell renewal and epithelial gland development. Beclin-1 knockin (Becn1 KI) mice with disabled apoptosis exhibit normal uterine development. Importantly, the restoration of Beclin-1-driven autophagy, but not apoptosis, promotes normal uterine adenogenesis and morphogenesis. Together, the data suggest that Beclin-1-mediated autophagy acts as a molecular switch that governs the early uterine morphogenetic program by maintaining the endometrial progenitor stem cells.

Gut microbiota and microbiota-derived metabolites promotes endometriosis.

Endometriosis is a pathological condition of the female reproductive tract characterized by the existence of endometrium-like tissue at ectopic sites, affecting 10% of women between the age 15 and 49 in the USA. However, currently there is no reliable non-invasive method to detect the presence of endometriosis without surgery and many women find hormonal therapy and surgery as ineffective in avoiding the recurrences. There is a lack of knowledge on the etiology and the factors that contribute to the development of endometriosis. A growing body of recent evidence suggests an association between gut microbiota and endometriosis pathophysiology. However, the direct impact of microbiota and microbiota-derived metabolites on the endometriosis disease progression is largely unknown. To understand the causal role of gut microbiota and endometriosis, we have implemented a novel model using antibiotic-induced microbiota-depleted (MD) mice to investigate the endometriosis disease progression. Interestingly, we found that MD mice showed reduced endometriotic lesion growth and, the transplantation of gut microbiota by oral gavage of feces from mice with endometriosis rescued the endometriotic lesion growth. Additionally, using germ-free donor mice, we indicated that the uterine microbiota is dispensable for endometriotic lesion growth in mice. Furthermore, we showed that gut microbiota modulates immune cell populations in the peritoneum of lesions-bearing mice. Finally, we found a novel signature of microbiota-derived metabolites that were significantly altered in feces of mice with endometriosis. Finally, we found one the altered metabolite, quinic acid promoted the survival of endometriotic epithelial cells in vitro and lesion growth in vivo, suggesting the disease-promoting potential of microbiota-derived metabolites. In summary, these data suggest that gut microbiota and microbiota-derived metabolome contribute to lesion growth in mice, possibly through immune cell adaptations. Of translational significance, these findings will aid in designing non-invasive diagnostics using stool metabolites for endometriosis.

A genome-wide CRISPR-Cas9 knockout screen identifies essential and growth-restricting genes in human trophoblast stem cells.

The recent derivation of human trophoblast stem cells (hTSCs) provides a scalable in vitro model system of human placental development, but the molecular regulators of hTSC identity have not been systematically explored thus far. Here, we utilize a genome-wide CRISPR-Cas9 knockout screen to comprehensively identify essential and growth-restricting genes in hTSCs. By cross-referencing our data to those from similar genetic screens performed in other cell types, as well as gene expression data from early human embryos, we define hTSC-specific and -enriched regulators. These include both well-established and previously uncharacterized trophoblast regulators, such as ARID3A, GATA2, and TEAD1 (essential), and GCM1, PTPN14, and TET2 (growth-restricting). Integrated analysis of chromatin accessibility, gene expression, and genome-wide location data reveals that the transcription factor TEAD1 regulates the expression of many trophoblast regulators in hTSCs. In the absence of TEAD1, hTSCs fail to complete faithful differentiation into extravillous trophoblast (EVT) cells and instead show a bias towards syncytiotrophoblast (STB) differentiation, thus indicating that this transcription factor safeguards the bipotent lineage potential of hTSCs. Overall, our study provides a valuable resource for dissecting the molecular regulation of human placental development and diseases.

The Multifaceted Role of Autophagy in Endometrium Homeostasis and Disease.

Autophagy is a conserved fundamental cellular process with a primary function of catabolizing harmful or surplus cellular contents such as protein aggregates, dysfunctional/long-lived organelles, intracellular pathogens, and storage nutrients. An increasing body of evidence reveals that basal autophagy is essential for maintaining endometrial homeostasis and mediating endometrial-specific functions, including menstrual cycle, embryo implantation, and decidualization. However, perturbed levels of autophagy can lead to severe endometrial pathologies, including endometriosis, endometrial hyperplasia, endometrial cancer, adenomyosis, and leiomyoma. This review highlights the most recent findings on the activity, regulation, and function of autophagy in endometrium physiology and pathology. Understanding the mechanistic roles of autophagy in endometrium homeostasis and disease is key to developing novel therapeutic strategies for endometrium-related infertility and malignancies.

Peroxiredoxin 6 Plays Essential Role in Mediating Fertilization and Early Embryonic Development in Rabbit Oviduct.

The oviduct is a site for early reproductive events including gamete maturation, fertilization, and early embryo development. Secretory cells lining the oviduct lumen synthesize and secrete proteins that interact with gametes and developing embryos. Although previous studies have identified some of the secretory proteins in the oviduct, however, knowledge and their precise specific functions in the oviduct are poorly understood. In this study, by using proteomic approach, we identified a secretory protein, Peroxiredoxin 6 (PRDX6), and evaluated its role in mediating early pregnancy events, fertilization, and embryo development in rabbit oviduct. The expression of PRDX6 was significantly higher in ampulla and isthmus sections of the oviduct in mated animal groups compared to non-mated controls. Furthermore, significant reduction in number of embryos recovered from PRDX6 siRNA-transfected oviductal horn was observed compared to the control contralateral horn. Moreover, in animals receiving PRDX6 siRNA in their oviductal horn, the number of implanted blastocysts was significantly less in the uterus as observed on day 9 post-coital (p.c.). Further, during embryo-rabbit oviduct epithelial cell (ROEC) co-culture, siRNA-mediated PRDX6 silencing attenuated the early embryonic development. Mechanistically, increased levels of ROS and expression of oxidative stress- and inflammation-related proteins were found in PRDX6 siRNA-treated ROEC cells as compared to control cells, implicating that ablation of PRDX6 in the oviduct creates a stress-induced micro-environment detrimental to early embryonic development in oviduct. Taken together, our data suggest that PRDX6 maintains an optimal micro-environment conducive to successful embryo development and can be considered as a candidate to evaluate its therapeutic potential in IVF strategies.

Gut microbiota-derived short-chain fatty acids protect against the progression of endometriosis.

Worldwide, ∼196 million are afflicted with endometriosis, a painful disease in which endometrial tissue implants and proliferates on abdominal peritoneal surfaces. Theories on the origin of endometriosis remained inconclusive. Whereas up to 90% of women experience retrograde menstruation, only 10% develop endometriosis, suggesting that factors that alter peritoneal environment might contribute to endometriosis. Herein, we report that whereas some gut bacteria promote endometriosis, others protect against endometriosis by fermenting fiber to produce short-chain fatty acids. Specifically, we found that altered gut microbiota drives endometriotic lesion growth and feces from mice with endometriosis contained less of short-chain fatty acid and n-butyrate than feces from mice without endometriosis. Treatment with n-butyrate reduced growth of both mouse endometriotic lesions and human endometriotic lesions in a pre-clinical mouse model. Mechanistic studies revealed that n-butyrate inhibited human endometriotic cell survival and lesion growth through G-protein-coupled receptors, histone deacetylases, and a GTPase activating protein, RAP1GAP. Our findings will enable future studies aimed at developing diagnostic tests, gut bacteria metabolites and treatment strategies, dietary supplements, n-butyrate analogs, or probiotics for endometriosis.