Generation of porcine endometrial organoids and their use as a model for enhancing embryonic attachment and elongation.

In brief: Porcine endometrial organoids (EOs) were isolated and characterized, revealing distinctive features such as unique extracellular matrix formation, fusion into uterine bud-like structures, and facilitation of embryo elongation. The yield of EOs was significantly enhanced by cryopreservation medium supplemented with the rock inhibitor (Y-27632), resulting in reduced expression of apoptotic mRNAs and microRNAs. Abstract: Endometrial organoids (EOs) are acceptable models for understanding maternal-embryonic cross talk. This study was conducted to generate EOs and optimize their cryopreservation and provide coculture modeling with embryos. The endometrial tissues were used for culturing the organoids inside domes of Matrigel®. To improve the long-term storage of EOs, 10 µM ROCK inhibitor (RI) was added to the cryopreservation medium. Day 7 parthenogenetically activated embryos were cocultured with EOs or EO outgrowths, and embryonic cell numbers and embryo attachment were monitored. Spherical EOs 100-300 µm in size can be retrieved on day 7 of culture, and larger EOs, approximately 1.5 mm in diameter, can be maintained in the Matrigel® dome for 21 days. The nuclear expression of Ki67 indicates that more than 80% of EOs nuclei were proliferative. EOs exhibit unique novel characters such as formation of extracellular matrix and ability for fusion. RI increased the yield and quality of organoids after freezing or thawing. The cell number of cocultured embryos increased five-fold, and the proportion of trophoblast outgrowths increased seven-fold compared with those of control embryos. The embryos cultured with EO-conditioned medium showed a better attachment rate than the other models, and - for the first time - we report embryonic elongation. Immunofluorescence staining of the attached embryos showed CDX2 in the periphery of EOs outgrowths. The 3D assembly and cryopreservation of EOs was optimized, and EO coculture supported embryo attachment, trophoblast outgrowth, and elongation, which would provide a valuable tool for studying the intricate processes involved in porcine embryo implantation.

Melatonin and resveratrol alleviate molecular and metabolic toxicity induced by Bisphenol A in endometrial organoids.

Bisphenol A (BPA), a widespread environmental contaminant, poses concerns due to its disruptive effects on physiological functions of the uterine endometrium. In contrast, melatonin (MT) and Resveratrol (RSV) are under scrutiny for their potential protective roles against BPA-induced damage. For the efficacy and ethical concerns in the animal test, endometrial organoids, three-dimensional models mimicking endometrium, serve as crucial tools for unraveling the impact of environmental factors on reproductive health. This study aimed to comprehensively characterize the morphological, molecular and metabolic responses of porcine endometrial organoids to BPA and assess the potential protective effects of MT and RSV. Porcine uteri were prepared, digested with collagenase, mixed with Matrigel, and incubated at 38°C with 5 % CO2. Passaging involved dissociation through trypsin-EDTA treatment and subculturing. The culture medium was refreshed every 2-3 days. To investigate the environmental impact on reproductive health, endometrial organoids were treated with BPA (0.5 µM), MT (with/without BPA at 0.1 µM), and/or RSV (10 µM). Various molecular screening using gene expression, western blotting, immunofluorescence staining, and metabolites profiling were assessed the effects of BPA, MT, and RSV in terms of cell viability, morphology, reproductivity, and metabolism alteration in the endometrial organoids. As expected, BPA induced structural and molecular disruptions in organoids, affecting cytoskeletal proteins, Wnt/ß-catenin signaling, and epithelial/mesenchymal markers. It triggered oxidative stress and apoptotic pathways, altered miRNA expression, and disrupted the endocannabinoid system. The level of glucose, galactose, and essential amino acids were increased or decreased by approximately 1.5-3 times in BPA-treated groups compared to the control groups (p-value < 0.05), indicating metabolic changes. Moreover, MT and RSV treated groups exhibited protective effects, mitigating BPA-induced disruptions across multiple pathways. For the first time, our study models endometrial organoids, advancing understanding of environmental impacts on reproductive health.

Effects of extracellular vesicles derived from steroids-primed oviductal epithelial cells on porcine in vitro embryonic development.

Extracellular vesicles (EVs) play an active role in regulating different physiological events, however, endocrine control of EVs cargo contents remain poorly understood. In this study, we aimed to isolate EVs from the porcine oviductal epithelial cells (POECs) that were primed with steroid hormones including estradiol (E2) and progesterone (P4), mimicking the in vivo conditions of the reproductive cycle and studied their effects on in vitro produced embryonic development. For this purpose, POECs were treated either with 0 concentration (control) or two different combinations of E2 and P4 including 50 pg/mL E2 + 0.5 ng/mL P4 (group H1), and 10 pg/mL E2 + 35 ng/mL P4 (group H2). Embryos were prepared after in vitro maturation either by parthenogenetic activation or somatic cell nuclear transfer (SCNT) technique. Treating parthenogenetic embryo with EVs, led a significantly higher rate of the blastocyst formation in the group supplemented with each EVs, compared to the control group. In addition, TUNEL assay and gene expression level analysis revealed that apoptosis was significantly reduced in the H2 EVs group. Furthermore, EVs from hormone-primed POECs improved the formation rate of porcine SCNT embryos compared to the control group. While in each EVs supplemented group (control EVs, H1 EVs, H2 EVs), the expression of cell reprogramming-related genes in cloned embryos showed a tendency of increase, the effect was stronger in H1 EVs and H2 EVs. In conclusion, EVs derived from POECs cultured in hormonal conditions simulating the in vivo environment had a positive effect on porcine blastocysts formation, which will likely facilitate in the production of cloned embryos.