PSVIII-28 Dietary impacts on rumen, vaginal, and uterine environments in beef heifers

This experiment examined the effects of diet composition on rumen, vaginal, and uterine environments in beef heifers. Fifteen rumen-cannulated, pubertal Angus-influenced heifers were used in a replicated 3 x 3 Latin square design (28-d periods and 21-d washout intervals). Dietary treatments included (as-fed) diets based on 100% grass hay (HF), 61% grass hay + 39% corn-based concentrate (INT), or 25% grass hay + 75% corn-based concentrate (HG). Treatments were offered individually to heifers once daily at 2% of their body weight. Heifers also received 280 g/d of a mineral mix containing melengestrol acetate. Rumen, vaginal, and uterine fluid samples were collected on d 0 and 28 of each period for pH measurement. Data were analyzed using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC) using results from d 0 as independent covariates, and heifer as the experimental unit. Rumen pH on d 0 did not differ (P = 0.97) among treatments (7.197, 7.194, and 7.188 for HF, INT, and HG, respectively; SEM = 0.038). Ruminal pH on d 28 was greater (P ≤ 0.01) in HF compared with INT and HG (6.805, 6.628, and 6.380, respectively; SEM = 0.049), and greater (P < 0.01) in INT compared with HG. Vaginal and uterine pH on d 0 did not differ (P ≥ 0.24) among HF, INT, and HG (6.914, 6.965, 6.780 of vaginal pH, respectively, SEM = 0.082; 6.644, 6.760, 6,592 of uterine pH, respectively, SEM = 0.079). Uterine and vaginal pH on d 28 also did not differ (P ≥ 0.64) among HF, INT, and HG heifers (6.926, 6.937, 6.918 of vaginal pH, respectively, SEM = 0.051; 6.567, 6.507, and 6.457 of uterine pH, respectively, SEM = 0.084). Therefore, dietary composition impacted rumen pH of beef heifers as expected, but without consequences to their vaginal and uterine pH.

Endometrial Glucose Transporters in Health and Disease

Pregnancy loss is a frequent occurrence during the peri-implantation period, when there is high glucose demand for embryonic development and endometrial decidualization. Glucose is among the most essential uterine fluid components required for those processes. Numerous studies associate abnormal glucose metabolism in the endometrium with a higher risk of adverse pregnancy outcomes. The endometrium is incapable of synthesizing glucose, which thus must be delivered into the uterine lumen by glucose transporters (GLUTs) and/or the sodium-dependent glucose transporter 1 (SGLT1). Among the 26 glucose transporters (14 GLUTs and 12 SGLTs) described, 10 (9 GLUTs and SGLT1) are expressed in rodents and 8 (7 GLUTs and SGLT1) in the human uterus. This review summarizes present knowledge on the most studied glucose transporters in the uterine endometrium (GLUT1, GLUT3, GLUT4, and GLUT8), whose data regarding function and regulation are still lacking. We present the recently discovered SGLT1 in the mouse and human endometrium, responsible for controlling glycogen accumulation essential for embryo implantation. Moreover, we describe the epigenetic regulation of endometrial GLUTs, as well as signaling pathways included in uterine GLUT’s expression. Further investigation of the GLUTs function in different endometrial cells is of high importance, as numerous glucose transporters are associated with infertility, polycystic ovary syndrome, and gestational diabetes.

Physicochemical and Functional Characterization of Female Reproductive Fluids: A Report of the First Two Infants Born Following Addition of Their Mother's Fluids to the Embryo Culture Media

Culture media supplemented with reproductive fluids (RF) have been used in livestock species, improving the efficiency and quality of in vitro produced embryos. However, usefulness in humans is still unknown. In this study, we collected human reproductive fluids (HRFs) ex vivo (from 25 patients undergoing abdominal hysterectomy plus bilateral salpingectomy) and in vivo (from 31 oocyte donors). Afterward, protocols to evaluate their osmolality, pH, total protein concentration, endotoxin level, and sterility were optimized, establishing security ranges for their use as natural additives. In addition, a functional assay was developed with bovine embryos grown in vitro in a medium supplemented with 1% of collected HRFs. Finally, a proof of concept was performed with six patients on post ovulation day 2 to evaluate the full-term viability of embryos grown in media supplemented with autologous uterine fluid, collected under in vivo conditions. Two of the embryos resulted in successful pregnancy and delivery of healthy babies. In conclusion, this study establishes a complete quality control sheet of HRFs as additives for embryo culture media and shows first preliminary data on obtaining healthy offspring derived from embryos grown in media supplemented with HRFs.

Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17β signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.

Uterine Metabolomic Analysis for the Regulation of Eggshell Calcification in Chickens

Eggshell quality is economically important for table eggs and functionally indispensable for hatching eggs. During the formation of eggshell in the uterus, organic matrixes in uterine fluid can control and modify the formation of calcified eggshell. At present, there are limited studies focusing on the effect of uterine organic metabolites on eggshell quality. In this study, an LC-MS-based metabolomic technology was performed to identify the crucial uterine metabolites that differently presented in hens producing eggs with divergent eggshell quality (eggshell strength, thickness, and weight). More than 1000 metabolites were identified in uterine fluid, and six putative metabolites, including phosphatidylcholine, diacylglycerol, verapamil, risedronate, coproporphyrinogen III, and biliverdin, were screened to play crucial roles in eggshell calcification. Then, two trials for oral administration and in vitro calcite crystal growth were conducted to verify the effect of potential different metabolites on the eggshell quality. Verapamil has a temporary effect on decreasing eggshell strength and eggshell thickness. Coproporphyrinogen III could induce smaller calcite crystals to improve eggshell strength while biliverdin could modify crystal morphology by forming rougher faces and rounder edges to strengthen the eggshell. The present study gives us new insight to understand the role of uterine fluid matrixes in eggshell calcification.

Blastocyst trophectoderm endocytic activation, a marker of adverse developmental programming

The mouse preimplantation embryo is sensitive to its environment including maternal dietary protein restriction which can alter the developmental programme and affect lifetime health. Previously, we have shown maternal low protein diet (LPD) causes reduction in blastocyst mTORC1 signalling coinciding with reduced availability of branched-chain amino acids (BCAAs) in surrounding uterine fluid. BCAA deficiency leads to increased endocytosis and lysosome biogenesis in blastocyst trophectoderm (TE), a response to promote compensatory histotrophic nutrition. Here, we first investigated the induction mechanism by individual variation in BCAA deficiency in an in vitro quantitative model of TE responsiveness. We found isoleucine (ILE) deficiency as the most effective activator of TE endocytosis and lysosome biogenesis, with less potent roles for other BCAAs and insulin; cell volume was also influential. TE response to low ILE included upregulation of vesicles comprising megalin receptor and cathepsin-B and the response was activated from blastocyst formation. Second, we identified the transcription factor TFEB as mediating the histotrophic response by translocation from cytoplasm to nucleus during ILE deficiency and in response to mTORC1 inhibition. Lastly, we investigated whether a similar mechanism responsive to maternal nutritional status was found in human blastocysts. Blastocysts from women with high body-mass index, but not the method of fertilisation, revealed stimulated lysosome biogenesis and TFEB nuclear migration. We propose TE lysosomal phenotype as an early biomarker of environmental nutrient stress that may associate with long-term health outcome.

O-026 Advanced Glycation Endproducts: A new player in obesity related infertility

text Globally, 39% of the adult population is overweight or obese, with the prevalence of obesity following an upward trajectory over the recent decades (WHO). Up to 30% of women of reproductive age in Western countries are obese before conception, and obese women experience higher rates of infertility and pregnancy complications than lean women; however, the mechanisms underpinning obesity-related infertility are poorly understood. Advanced Glycation Endproducts (AGEs) are a proinflammatory modification of proteins exposed to sugars, formed through the Maillard reaction. AGEs are elevated four-fold in the uterine fluid of obese, infertile women, compared to lean. AGEs equimolar to those in the obese microenvironment negatively impact the functions of endometrial epithelial and stromal cells, and adhesion and invasion of trophoblast cells, reducing the potential for successful maternal-fetal interactions (Antoniotti et al., 2018). This research further investigated preimplantation embryo development and endometrial cell functions in the presence of AGEs equimolar to those in obese uterine fluid. Altered local environments in very early life can set offspring up for a lifetime of health or disease (DoHAD); thus, uterine AGEs may contribute to the prevalence of non-communicable disease in children of obese parents. Preimplantation mouse embryos were cultured in vitro with AGEs equimolar with uterine fluid concentrations from lean and obese women, and their development and implantation potential assessed. “Obese” AGEs did not impact the proportion of embryos reaching blastocyst stage by day 4, but significantly reduced the proportion of blastocysts hatching by day 5 (P < 0.01). AGEs equimolar with the obese uterine environment detrimentally impacted trophectoderm formation and function: reduced trophectoderm cell number (P < 0.01), reduced outgrowth on fibronectin (indicative of reduced implantation potential, (P < 0.01), but did not increase cell apoptosis (TUNEL assay). RAGE antagonism, but neither metformin nor antioxidants, improved trophectoderm cell number. Thus, obesity-associated AGEs link obesity and reduced fertility through poor placentation potential of embryos (Hutchison et al, 2020). Endometrial epithelial cell function was examined in the presence of lean and obese concentrations of AGEs. Obese AGEs significantly reduced the rate of proliferation (xCelligence real time cell analysis) of the endometrial epithelial cell line ECC-1 versus lean AGEs (P = 0.04). Antioxidants successfully restored the rate of proliferation in the presence of obese AGEs (P = 0.7 versus lean AGEs). Subsequently, human endometrial epithelial organoid culture was utilised as a more physiologically relevant experimental paradigm. When cultured as organoids, primary endometrial epithelial cells were functionally responsive to obesity-associated AGEs, expressing both RAGE and TLR4. The morphology of organoids in culture was not impacted by the presence of obese AGEs versus lean; however, the proliferation of epithelial cells retrieved from organoid culture was altered by obese AGEs versus lean. Obese AGEs also increased the secretion of proinflammatory CXCL16 versus vehicle control (P = 0.04) while increased secretion of other proinflammatory cytokines and chemokines including TNFa approached significance in the presence of obese AGEs. As the inflammatory milieu is altered in the uterine fluid of infertile women, elevated AGEs may promote an infertile endometrial inflammatory environment. AGEs link obesity and reduced fertility, being detrimental to preimplantation embryo development and endometrial cell function when present at concentrations equal to those in obese uterine fluid. Antioxidants and RAGE antagonism provide beneficial effects to cell function in the presence of obesity-associated AGEs. This research provides evidence supporting AGEs as a factor contributing to obesity related infertility, and as an emerging frontier for reproductive health. Clinically, reduction of uterine AGEs may improve fertility for obese couples wishing to conceive. Antoniotti et al (2018). Hum Rep. 33(4), 654-665. PMID: 29471449 Hutchison et al (2020). RBMO. 41(5), 757-766. PMID: 32972872

GLUT4 in Mouse Endometrial Epithelium: Roles in Embryonic Development and Implantation

GLUT4 is involved in rapid glucose uptake among various kinds of cells to contribute to glucose homeostasis. Prior data have reported that aberrant glucose metabolism by GLUT4 dysfunction in the uterus could be responsible for infertility and increased miscarriage. However, the expression and precise functions of GLUT4 in the endometrium under physiological conditions remain unknown or controversial. In this study, we observed that GLUT4 exhibits a spatiotemporal expression in mouse uterus on pregnant days 1–4; its expression especially increased on pregnant day 4 during the window of implantation. We also determined that estrogen, in conjunction with progesterone, promotes the expression of GLUT4 in the endometrial epithelium in vivo or in vitro. GLUT4 is an important transporter that mediates glucose transport in endometrial epithelial cells (EECs) in vitro or in vivo. In vitro, glucose uptake decreased in mouse EECs when the cells were treated with GLUT4 small interfering RNA (siRNA). In vivo, the injection of GLUT4-siRNA into one side of the mouse uterine horns resulted in an increased glucose concentration in the uterine fluid on pregnant day 4, although it was still lower than in blood, and impaired endometrial receptivity by inhibiting pinopode formation and the expressions of leukemia inhibitory factor (LIF) and integrin ανβ3, finally affecting embryonic development and implantation. Overall, the obtained results indicate that GLUT4 in the endometrial epithelium affects embryo development by altering glucose concentration in the uterine fluid. It can also affect implantation by impairing endometrial receptivity due to dysfunction of GLUT4.