Cell Growth Factor and Estrogen Inducing Menstrual Blood-Derived Stem Cells (MenSC) Differentiate into Endometrial Epithelial Cells

Extracted MenSC (Menstrual blood-derived stem cells) from female menstrual blood. Added various exogenous factors in-vitro and simulated the female uterine environment to observe how to make MenSC differentiation into Endometrial epithelial cells by artificial induction. MenSCs were divided into 4 groups: 2.5×10−5 mol/L E group, 1.613 nmol/L EGF group, 2.5×10−5 mol/L E+1.613 nmol/L EGF group, control Group (only MenSCs); the relevant indicators of the experiment includes cell staining and Western Blot to detect CK and VIM protein content; RT-PCR to detect CK-19 mRNA and VIM mRNA. The cell staining results showed that E+EGF group had significant differentiation in 7 days and 14 days. CK-19mRNA of E+EGF group was significantly higher than other groups, and the EGF group expression was obviously higher than that of E group, and VIMmRNA expression is opposite to that. The protein expression had the similar performance. MenSC can differentiate into endometrial epithelial cells after induced by E and EFG; and the co-culture of E and EFG can achieve better differentiation, which proves their work together in MenSC differentiate towards endometrial epithelial cells.

Expression of genes associated with fertility in the uterus and oviduct of heifers challenged with lipopolysaccharide

Summary Lipopolysaccharide (LPS) endotoxemia has been negatively associated with fertility. This study aimed to investigate the effect of LPS-induced inflammation on gene expression associated with bovine fertility in the uterus and oviduct. Sixteen healthy heifers were divided into two groups. The LPS group (n = 8) received two intravenous (i.v.) injections of 0.5 µg/kg of body weight of LPS with a 24-h interval, and the control group (n = 8) received two i.v. injections of saline solution with the same interval of time. All the animals had the follicular wave synchronized. Three days after the second injection of LPS, all animals were slaughtered and uterine and oviduct samples were collected. Gene expression associated with inflammatory response, thermal and oxidative stresses, oviduct environment quality, and uterine environment quality was evaluated. Body temperature and leucogram demonstrated that LPS induced an acute systemic inflammatory response. In the uterus, the expression of PTGS2 and NANOG genes was downregulated by the LPS challenge. However, no change in expression was observed in the other evaluated genes in the uterus, nor those evaluated in the oviduct. In conclusion, the inflammatory process triggered by LPS did not persist in the uterus and oviduct 3 days after challenge with LPS. Nonetheless, reduction in PTGS2 and NANOG expression in the uterus suggested that, indirectly, LPS may have a prolonged effect, which may affect corpus luteum and endometrial functions.

Sperm exposure to accessory gland secretions alters the transcriptomic response of the endometrium in cattle

In a recent study from our group, mating to intact, but not vasectomised, bulls modified the endometrial transcriptome, suggesting an important role of sperm in the modulation of the uterine environment in this species. However, it is not clear whether these changes are driven by intrinsic sperm factors, or by factors of accessory gland (AG) origin that bind to sperm at ejaculation. Thus, the aim of the present study was to determine whether ejaculated sperm, which are suspended in the secretions of the AGs, elicit a different endometrial transcriptomic response than epididymal sperm, which have never been exposed to AG factors. To this end, bovine endometrial explants collected from heifers in oestrus were incubated alone (control), or with epididymal or ejaculated sperm. RNA-sequencing revealed 1912 differentially expressed genes (DEGs) between in endometrial explants exposed to epididymal sperm compared with control explants, whereas 115 DEGs genes detected between endometrial explants exposed to ejaculated sperm in comparison to control explants. In both cases, the top pathways associated with these genes included T cell regulation and NF-KB and IL17 signalling. To confirm whether AG factors were directly responsible for the dampening of the endometrial response elicited by ejaculated sperm, endometrial explants were incubated with epididymal sperm previously exposed, or not, to seminal plasma (SP). Exposure to SP abrogated the downregulation of SQSTM1 by epididymal sperm, and partially inhibited the upregulation of MYL4 and CHRM3 and downregulation of SCRIB. These data indicate that factors of AG origin modulate the interaction between sperm and the endometrium in cattle.

Epigenetics in the Uterine Environment: How Maternal Diet and ART May Influence the Epigenome in the Offspring with Long-Term Health Consequences

The societal burden of non-communicable disease is closely linked with environmental exposures and lifestyle behaviours, including the adherence to a poor maternal diet from the earliest preimplantation period of the life course onwards. Epigenetic variations caused by a compromised maternal nutritional status can affect embryonic development. This review summarises the main epigenetic modifications in mammals, especially DNA methylation, histone modifications, and ncRNA. These epigenetic changes can compromise the health of the offspring later in life. We discuss different types of nutritional stressors in human and animal models, such as maternal undernutrition, seasonal diets, low-protein diet, high-fat diet, and synthetic folic acid supplement use, and how these nutritional exposures epigenetically affect target genes and their outcomes. In addition, we review the concept of thrifty genes during the preimplantation period, and some examples that relate to epigenetic change and diet. Finally, we discuss different examples of maternal diets, their effect on outcomes, and their relationship with assisted reproductive technology (ART), including their implications on epigenetic modifications.

Progesterone-dependent and -independent modulation of luminal epithelial transcription to support pregnancy in cattle

In cattle, starting 4-5 days after estrus, pre-implantation embryonic development occurs in the confinement of the uterine lumen. Cells in the endometrial epithelial layer control the molecular traffic to and from the lumen and, thereby determine luminal composition. Starting early post-estrus, endometrial function is regulated by sex-steroids, but the effects of progesterone on luminal cells transcription have not been measured in vivo. First objective was to determine the extent to which progesterone controls transcription in luminal epithelial cells 4 d (D4) after estrus. Second objective was to discover luminal transcripts that predict pregnancy outcomes, when the effect of progesterone is controlled. Endometrial luminal epithelial cells were collected from embryo transfer recipients on D4 using a cytological brush and their transcriptome determined by RNASeq. Pregnancy by embryo transfer was measured on D30 (25 pregnant and 18 non-pregnant). Progesterone concentration on D4 was associated positively (n= 182) and negatively (n= 58) with gene expression. Progesterone-modulated transcription indicated an increase in oxidative phosphorylation, biosynthetic activity and proliferation of epithelial cells. When these effects of progesterone were controlled, different genes affected positively (n= 22) and negatively (n= 292) odds of pregnancy. These set of genes indicated that a receptive uterine environment was characterized by the inhibition of phosphoinositide signaling and innate immune system responses. A panel of 25 genes predicted the pregnancy outcome with sensitivity and specificity ranging from 64-96% and 44-83%, respectively. In conclusion, in the early diestrus, both progesterone-dependent and -independent mechanisms regulate luminal epithelial transcription associated with pregnancy outcomes in cattle.

Preparing for implantation

A new laboratory model helps to understand the role of senescent cells in fostering a uterine environment that can support an embryo.

PSIII-9 Supplementation of beta-carotene or fatty acids alters production of prostaglandins in bovine endometrial epithelial cells

Differential prostaglandin secretion from the bovine endometrium can be used as a marker for an embryotropic or embryotoxic uterine environment. Beta-carotene has antioxidant properties and is the precursor for retinol, which has been shown to improve early embryonic development in vivo and in vitro. Furthermore, dietary fatty acid supplementation, specifically eicosapentaenoic acid (EPA) and decosahexaenoic acid (DHA) has been shown to alter prostaglandin production. The objective of this study was to determine prostaglandin production of endometrial cells following treatment with beta-carotene, EPA, or DHA. Bovine endometrial epithelial cells were treated for 24 hours with serum-free media supplemented with either 10 µM beta-carotene, 10 µM EPA, 10 µM DHA or ethanol (>1% volume/volume) vehicle control. After treatment, concentrations of PGE2 and PGF2a were analyzed in media via commercially available ELISA kits. Concentrations and ratios of prostaglandins were analyzed via ANOVA using the mixed procedure in SAS version 9.4. Beta-carotene treatment decreased PGE2 (P < 0.0001) and PGF2a (P = 0.0003) concentrations in media compared to controls. However, the ratio of PGE2:PGF2a was not different (P = 0.1203) between beta-carotene and controls. DHA treatment decreased PGE2 (P < 0.0001) concentrations in media but did not alter (P = 0.1079) PGF2a concentrations in media compared to controls. The ratio of PGE2:PGF2a was not different (P = 0.6343) between DHA and controls. EPA treatment did not alter (P = 0.1503) PGE2 concentrations in media compared to controls. Conversely, PGF2a concentrations were decreased (P = 0.0088) in media treated with EPA compared to controls. Therefore, the ratio of PGE2:PGF2a was increased (P = 0.0116) between EPA versus controls. These studies demonstrate that in vitro supplementation of EPA may alter the endometrial synthesis of prostaglandins to be more embryotropic. Therefore, EPA may be therapeutic for in vivo trials to influence the early uterine environment.

Dietary polyphenols in the intervention of gestational diabetes

Background: The impact of gestational diabetes mellitus GDM is severe since it is directly associated with the detrimental intra-uterine environment, which may lead to enhanced risk of development of fetal metabolic disorders and affects mother and offspring both. Plant derived polyphenols are reported for their protective roles against a variety of human diseases; however, the impact of adherence to polyphenols on the risk of GDM has not been exhaustively reviewed. Objective: Aim of this review is to summarize relevant studies linking polyphenols and polyphenol rich dietary patterns by carefully considering the mechanisms of their action to the risk of GDM and its management. Method: Data from PubMed, ScienceDirect, and Springer online databases and updates from the Indian Council of Medical Research (ICMR) were studied. Conclusion: Present review provides a comprehensive report on the association between plant polyphenols and lower risk of GDM, suggesting that polyphenol-rich dietary patterns may be important in preventing the development of GDM and its healthy management.

PAEP and cilia gene splicing changes in endometrial glands during the implantation window in women with recurrent pregnancy loss

ABSTRACTEndometrial glands are essential for fertility, consisting of ciliated and secretory cells that facilitate a suitable uterine environment for embryo implantation. This study sought to determine whether an endometrial gland specific transcriptome and splicing profile are altered in women with recurrent pregnancy loss. Our data provide a comprehensive catalogue of cilia and PAEP gene isoforms and relative exon usage in endometrial glands. We report a previously unannotated endometrial gland cilia transcript GALNT11 and its susceptibility to exon skipping. Key endometrial receptivity gene transcripts are also reported to change in endometrial glands of women with recurrent pregnancy loss. The endometrial gland cilia and PAEP targets identified in this study could be used to identify a perturbed endometrium, isolate causes of recurrent pregnancy loss and develop targeted therapies in personalised medicine.