In cycling human endometrium, menstruation is followed by rapid estrogen-dependent growth. Upon ovulation, progesterone and rising cellular cAMP levels activate the transcription factor Forkhead box O1 (FOXO1) in endometrial stromal cells (EnSCs), leading to cell cycle exit and differentiation into decidual cells that control embryo implantation. Here we show that FOXO1 also causes acute senescence of a subpopulation of decidualizing EnSCs in an IL-8 dependent manner. Selective depletion or enrichment of this subpopulation revealed that decidual senescence drives the transient inflammatory response associated with endometrial receptivity. Further, senescent cells prevent differentiation of endometrial mesenchymal stem cells in decidualizing cultures. As the cycle progresses, IL-15 activated uterine natural killer (uNK) cells selectively target and clear senescent decidual cells through granule exocytosis. Our findings reveal that acute decidual senescence governs endometrial rejuvenation and remodeling at embryo implantation, and suggest a critical role for uNK cells in maintaining homeostasis in cycling endometrium.

Decidualizing endometrial stromal cells (EnSC) critically determine the maternal response to an implanting conceptus, triggering either menstruation‐like disposal of low‐fitness embryos or creating an environment that promotes further development. However, the mechanism that couples maternal recognition of low‐quality embryos to tissue breakdown remains poorly understood. Recently, we demonstrated that successful transition of the cycling endometrium to a pregnancy state requires selective elimination of pro‐inflammatory senescent decidual cells by activated uterine natural killer (uNK) cells. Here we report that uNK cells express CD44, the canonical hyaluronan (HA) receptor, and demonstrate that high molecular weight HA (HMWHA) inhibits uNK cell‐mediated killing of senescent decidual cells. In contrast, low molecular weight HA (LMWHA) did not attenuate uNK cell activity in co‐culture experiments. Killing of senescent decidual cells by uNK cells was also inhibited upon exposure to medium conditioned by IVF embryos that failed to implant, but not successful embryos. Embryo‐mediated inhibition of uNK cell activity was reversed by recombinant hyaluronidase 2 (HYAL2), which hydrolyses HMWHA. We further report a correlation between the levels of HYAL2 secretion by human blastocysts, morphological scores, and implantation potential. Taken together, the data suggest a pivotal role for uNK cells in embryo biosensing and endometrial fate decisions at implantation.

The human luteinising hormone choriogonadotropin receptor (LHCGR) is a G-protein coupled receptor activated by both human chorionic gonadotropin (hCG) and luteinizing hormone (LH), two structurally related gonadotropins with essential roles in ovulation and maintenance of the corpus luteum. LHCGR expression predominates in ovarian tissues where it elicits functional responses through cyclic adenosine mononucleotide (cAMP), Ca²⁺ and extracellular signal-regulated kinase (ERK) signalling. LHCGR expression has also been localized to the human endometrium, with purported roles in decidualization and implantation. However, these observations are contentious. In this investigation, transcripts encoding LHCGR were undetectable in bulk RNA sequencing datasets from whole cycling endometrial tissue and cultured human endometrial stromal cells (EnSC). However, analysis of single-cell RNA sequencing data revealed cell-to-cell transcriptional heterogeneity, and we identified a small subpopulation of stromal cells with detectable LHCGR transcripts. In HEK-293 cells expressing recombinant LHCGR, both hCG and LH elicited robust cAMP, Ca²⁺ and ERK signals that were absent in wild-type HEK-293 cells. However, none of these responses were recapitulated in primary EnSC cultures. In addition, proliferation, viability and decidual transformation of EnSC were refractory to both hCG and LH, irrespective of treatment to induce differentiation. Although we challenge the assertion that LHCGR is expressed at a functionally active level in the human endometrium, the discovery of a discrete subpopulation of EnSC that express LHCGR transcripts may plausibly account for the conflicting evidence in the literature.

In every menstrual cycle, progesterone acting on estrogen-primed endometrium elicits an inflammatory decidual reaction, rendering it poised for embryo implantation and transformation into the decidua of pregnancy. Here, we show that the sequential functions of the decidual reaction—implantation and decidualization—pivot on the time-sensitive loss of progesterone-resistant DIO2 ⁺ stromal cells that form a specialized implantation niche and reciprocal expansion of progesterone-dependent PLA2G2A ⁺ predecidual cells. Simultaneously, uterine natural killer (uNK) cell proliferation results in the accumulation of immunotolerant subsets. Examination of endometrial biopsies from 924 women revealed that the recurrence risk of miscarriage closely aligns with the incidence of a weakened or stalled decidual reaction, more so than poor uNK cell expansion. Analysis of paired biopsies obtained in different cycles and modeling in assembloids intimated that prior miscarriages disrupt intercycle endometrial homeostasis and calibration of the decidual reaction. Our findings show that erosion of the decidual reaction following a miscarriage drives the recurrence risk irrespective of maternal age.

Decidualization denotes the differentiation of endometrial stromal cells into specialized decidual cells, essential for embryo implantation and pregnancy. The process requires coordination of progesterone and cAMP signaling, which converge on downstream transcription factors. PGE2 and relaxin, acting, respectively, through Gαs-coupled GPCRs EP2 and RXFP1, are putative candidates for generating cAMP in differentiating stromal cells. Here, we show that PGE2 is less efficacious than relaxin in elevating intracellular cAMP levels in primary stromal cells but more effective at driving the expression of decidual genes. PGE2-and relaxin-induced cAMP generation involves receptor internalization, but EP2 is endocytosed into very early endosomes (VEEs). Perturbation of VEE machinery through depletion of key trafficking proteins; APPL1 and GIPC, dysregulates PGE2-dependent cAMP profiles and disrupts key decidual signaling pathways, resulting in a disordered differentiation response. We demonstrate that regulation of EP2 via internalization is essential for coordinated activation of the downstream signaling cascades that govern decidualization.

Context Novel small molecule inhibitors of the oxytocin receptor (OTR) may have distinct pharmacology and mode of action when compared to first generation oxytocin antagonists when used for the prevention of preterm birth. Objective To determine the mechanism of action of small molecule OTR antagonists retosiban and epelsiban in comparison to the currently used peptide-based compound atosiban. Design Human myometrial samples were obtained at cesarean section and subjected to pharmacological manipulations to establish the effect of antagonist binding to OTR on downstream signaling. Results Retosiban antagonism of oxytocin action in human myometrium was potent, rapid and reversible. Inhibition of inositol 1,4,5-trisphosphate (IP3) production followed single site competitive binding kinetics for epelsiban, retosiban and atosiban. Retosiban inhibited basal production of IP3 in the absence of oxytocin. Oxytocin and atosiban, but not retosiban inhibited forskolin and calcitonin stimulated cAMP production. Inhibition of cAMP was reversed by pertussis toxin. Oxytocin and atosiban, but not retosiban and epelsiban, stimulated ERK1/2 activity in a time a concentration dependent manner. Oxytocin and atosiban stimulated cyclo oxygenase 2 (COX2) activity and subsequent production of prostaglandin E₂ and F_2α. Prostaglandin production was inhibited by rofecoxib, pertussin toxin, and ERK inhibitor U0126. Oxytocin but not retosiban or atosiban stimulated coupling of the OTR to Gα_q G-proteins. Oxytocin and atosiban but not retosiban stimulated coupling of the OTR to Gα_i G-proteins. Conclusions Retosiban and epelsiban demonstrate distinct pharmacology when compared to atosiban in human myometrial smooth muscle. Atosiban displays agonist activity at micromolar concentrations leading to stimulation of prostaglandin production.