Pharmacological chaperones for the oxytocin receptor increase oxytocin responsiveness in myometrial cells

Oxytocin is a potent uterotonic agent administered to nearly all patients during childbirth in the United States. Inadequate oxytocin response can necessitate Cesarean delivery or lead to uterine atony and postpartum hemorrhage. Thus, it may be clinically useful to identify patients at risk for poor oxytocin response and develop strategies to sensitize the uterus to oxytocin. Previously, we showed that the V281M variant in the oxytocin receptor (OXTR) gene impairs OXTR trafficking to the cell surface, leading to a decreased oxytocin response in cells. Here, we sought to identify pharmacological chaperones that increased oxytocin response in cells expressing WT or V281M OXTR. We screened nine small-molecule agonists and antagonists of the oxytocin/vasopressin receptor family and identified two, SR49059 and L371,257, that restored both OXTR trafficking and oxytocin response in HEK293T cells transfected with V281M OXTR. In hTERT-immortalized human myometrial cells, which endogenously express WT OXTR, treatment with SR49059 and L371,257 increased the amount of OXTR on the cell surface by two- to fourfold. Furthermore, SR49059 and L371,257 increased the endogenous oxytocin response in hTERT-immortalized human myometrial cells by 35% and induced robust oxytocin responses in primary myometrial cells obtained from patients at the time of Cesarean section. If future studies demonstrate that these pharmacological chaperones or related compounds function similarly in vivo, we propose that they could potentially be used to enhance clinical response to oxytocin

Obesity, Second Stage Duration, and Labor Outcomes in Nulliparous Women

Objective: This study aimed to estimate second stage duration and its effects on labor outcomes in obese versus nonobese nulliparous women. Study design: This was a secondary analysis of a cohort of nulliparous women who presented for labor at term and reached complete cervical dilation. Adjusted relative risks (aRR) were used to estimate the association between obesity and second stage characteristics, composite neonatal morbidity, and composite maternal morbidity. Effect modification of prolonged second stage on the association between obesity and morbidity was assessed by including an interaction term in the regression model. Results: Compared with nonobese, obese women were more likely to have a prolonged second stage (aRR: 1.48, 95% CI: 1.18-1.85 for ≥3 hours; aRR: 1.65, 95% CI: 1.18-2.30 for ≥4 hours). Obesity was associated with a higher rate of second stage cesarean (aRR: 1.78, 95% CI: 1.34-2.34) and cesarean delivery for fetal distress (aRR: 2.67, 95% CI: 1.18-3.58). Obesity was also associated with increased rates of neonatal (aRR: 1.38, 95% CI: 1.05-1.80), but not maternal morbidity (aRR: 1.06, 95% CI: 0.90-1.25). Neonatal morbidity risk was not modified by prolonged second stage. Conclusion: Obesity is associated with increased risk of neonatal morbidity, which is not modified by prolonged second stage of labor

Naturally occurring genetic variants in the oxytocin receptor alter receptor signaling profiles

The hormone oxytocin is commonly administered during childbirth to initiate and strengthen uterine contractions and prevent postpartum hemorrhage. However, patients have wide variation in the oxytocin dose required for a clinical response. To begin to uncover the mechanisms underlying this variability, we screened the 11 most prevalent missense genetic variants in the oxytocin receptor

Mitochondrial Dysfunction and Apoptosis in Cumulus Cells of Type I Diabetic Mice

Impaired oocyte quality has been demonstrated in diabetic mice; however, the potential pathways by which maternal diabetes exerts its effects on the oocyte are poorly understood. Cumulus cells are in direct contact with the oocyte via gap junctions and provide essential nutrients to support oocyte development. In this study, we investigated the effects of maternal diabetes on the mitochondrial status in cumulus cells. We found an increased frequency of fragmented mitochondria, a decreased transmembrane potential and an aggregated distribution of mitochondria in cumulus cells from diabetic mice. Furthermore, while mitochondrial biogenesis in cumulus cells was induced by maternal diabetes, their metabolic function was disrupted as evidenced by lower ATP and citrate levels. Moreover, we present evidence suggesting that the mitochondrial impairments induced by maternal diabetes, at least in part, lead to cumulus cell apoptosis through the release of cytochrome c. Together the deleterious effects on cumulus cells may disrupt trophic and signaling interactions with the oocyte, contributing to oocyte incompetence and thus poor pregnancy outcomes in diabetic females

Early Pregnancy Loss: A Role For Glucose Utilization In The Endometrial Stroma

Recurrent pregnancy loss affects a large number of women, and the causes of this disorder remain largely unknown. One of the key steps in establishing a successful pregnancy is embryo implantation. While embryo abnormalities may certainly lead to pregnancy loss, it is equally important for the uterine endometrium to adequately progress to, what is termed, a receptive state. The endometrium is composed of several cell types, including the epithelial, stromal, endothelial and a number of resident immune cells. A hallmark of endometrial receptivity is the differentiation of the endometrial stromal cell (ESC) compartment into the decidua. During this process, termed decidualization, the ESCs undergo a number of morphological and functional changes, which results in their ability to support early embryo growth and development prior to full placental function. Decidual cells show evidence of drastic changes in glucose metabolism, which is evidenced by their high stores of glycogen granules. We, therefore, hypothesized that increased glucose uptake by ESCs is necessary for decidualization and that this excess glucose is differentially metabolized in the decidual cells versus undifferentiated stroma. We first explored the abundance of GLUT mRNAs in primary ESCs isolated from mice and humans. We found that GLUT1 mRNA is the most abundant among glucose transporter family members in the ESCs of both species studied, but several other transporters also show moderately high mRNA levels. We also determined that GLUT1 is required for proper decidualization of human ESCs since in vitro knockdown of this gene significantly lowered expression of the known decidualization markers, PRL and IGFBP1. Lastly, we also demonstrated that metabolism of glucose through the pentose phosphate pathway is a requirement for proliferation and decidualization of endometrial stroma. Inhibition of the pentose phosphate pathway led to a significant decrease in the decidual reaction both in vitro and in vivo. Taken together, the outcomes of this study highlight two distinct steps of glucose utilization, uptake via Glut1 and flux through the pentose phosphate pathway, which are both necessary for proper ESC decidualization. Abnormal glucose utilization by decidualizing ESCs may provide a mechanistic explanation for idiopathic recurrent pregnancy loss

Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells

Oxytocin acts through the oxytocin receptor (OXTR) to modulate uterine contractility. We previously identified OXTR genetic variants and showed that, in HEK293T cells, two of the OXTR protein variants localized to the cell surface less than wild-type OXTR. Here, we sought to measure OXTR in the more native human myometrial smooth muscle cell (HMSMC) line on both the cell-surface and across the whole cell, and used CRISPR editing to add an HA tag to the endogenous OXTR gene for anti-HA measurement. Quantitative flow cytometry revealed that these cells possessed 55,000 ± 3200 total OXTRs and 4900 ± 390 cell-surface OXTRs per cell. To identify any differential wild-type versus variant localization, we transiently transfected HMSMCs to exogenously express wild-type or variant OXTR with HA and green fluorescent protein tags. Total protein expression of wild-type OXTR and all tested variants were similar. However, the two variants with lower surface localization in HEK293T cells also presented lower surface localization in HMSMCs. Overall, we confirm the differential surface localization of variant OXTR in a more native cell type, and further demonstrate that the quantitative flow cytometry technique is adaptable to whole-cell measurements