Project description:In order to clarify the cause of reproductive failure, we conducted global endometrial gene expression analysis in fertile and subfertile cows. Hierarchical cluster analysis with the expression levels of mitochondrial DNA encoded genes was divided these cows into two clusters. One cluster was composed of fertile cows, the other cluster contained subfertile cows, and the expressions of mitochondrial DNA encoded genes in subfertile cows were higher than those in fertile cows
Project description:The miRNA profiles were measured using small-RNA sequencing in beef heifers sampled at weaning that was retrospectively classified as fertile or subfertile following the breeding protocol. To accomplish this, the miRNA profiles were generated from the blood samples (10 mL) collected from crossbred heifers (Angus-Simmental) at the time of weaning (~238 days after birth). Peripheral white blood cells (PWBC) were extracted from the blood samples and stored at -80°C until further processing. During the breeding season, all the heifers followed the same breeding protocol, estrus synchronization, and fixed-time artificial insemination (AI). Fourteen days following the fixed-time AI, the non-pregnant heifers were exposed to fertile bulls for 60 days. Depending on the presence or absence of conceptus at 75 days following AI, heifers were classified as fertile for those who were pregnant through artificial insemination, pregnant to natural breeding (P-NB), or subfertile for those who were not pregnant. Heifers from fertile (n = 7) and subfertile (n = 7) groups were considered for the study. Total RNA was extracted from the PWBC of 14 samples and was subjected to small RNA library preparation and sequencing. After quality control, adapter trimming, and alignment, mature miRNAs were used for differential expression analysis. The read counts were transformed to counts per million (CPM), and raw counts with CPM < 1 in 50% of the samples were filtered out. The filtered raw counts were analyzed using DESeq2 v 1.26.0 to identify differentially expressed miRNAs (DEMIs). The DEMIs identified with a p-value < 0.05 and absolute (log2 fold change) > 0.5 were considered significant. With the subfertile heifers as the reference group, we identified 16 DEMIs between fertile and subfertile groups. To determine the genes targeting the DEMIs, we downloaded the target genes for each DEMI and retained only those genes expressed in the PWBCs. For the miRNA-gene correlation, we used the partial correlation and information theory (PCIT) approach to identify the significant gene-miRNA correlated pairs. The significant genes correlated with the miRNAs identified pathways including MAPK, ErbB, HIF-1, FoxO, p53, mTOR, T-cell receptor, insulin and GnRH signaling, apoptosis, and pathways regulating pluripotency of stem cells in the fertile group while cell cycle, p53 signaling pathway and apoptosis pathways in the subfertile group.
Project description:Endometrial receptivity is imperative to achieving pregnancy in humans. A disruption in the development of endometrial receptivity is responsible for recurrent implantation failures (RIF) of endometrial origin. To further understand the molecular mechanisms behind the endometrial receptivity process, we used the 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) method to compare and quantify the proteomes from endometrial biopsies of three different endometrial statuses (fertile women, IUD carriers and RIF patients). Overall, iTRAQ allowed to identify 1,889 non-redundant proteins. Of these, 188 were differentially expressed proteins (DEP) (p-value < 0.05) among the three endometrial groups. Pairwise comparisons revealed 133 significant DEP in fertile vs. IUD carriers and 158 DEP in RIF vs. IUD carriers. However, no DEP were identified between fertile and RIF patients. Western blot validation of three DEP involved in endometrial receptivity (Plastin 2, Lactotrasferrin, and Lysozyme) confirmed our iTRAQ results. Moreover, functional KEGG enrichment revealed that complement and coagulation cascades and peroxisome were the two most significant pathways for the RIF vs. IUD comparison and ribosome and spliceosome for the fertile vs. IUD comparison, as possible important pathways involved in the endometrial receptivity acquisition. Our findings confirm that an IUD introduces numerous changes in the endometrial protein profile when compared to fertile and RIF endometria, revealing some key proteins involved in endometrial receptivity. The lack of DEP between fertile and RIF patient endometria suggest either that idiopathic RIF may not have an endometrial origin, with other as-yet-unknown factors involved.
Project description:We investigated differential gene expression profiles of endometrium during the mid-luteal phase of the estrous cycle between repeat breeding (RB) and normally fertilized cows using microarray analysis. Caruncular (CAR) and intercaruncular (ICAR) endometrium of both ipsilateral and contralateral uterine horns to the corpus luteum were collected from RB (inseminated at least three times but not pregnant) and normally fertile cows on Day 15 of the estrous cycle. Global gene expression profiles of these endometrial samples were analyzed with a 15K custom-made oligo-microarray in cattle. Microarray analysis revealed that 405 and 397 genes were differentially expressed in CAR and ICAR of the ipsilateral uterine horn of RB, respectively when compared with normal cows. In contralateral uterine horn, 443 and 257 differentially expressed genes were identified in the CAR and ICAR of RB, respectively when compared with normal cows. In the analysis of whole uterine (combining the above four portions), RB cows showed up-regulation of 37 genes including PRSS2, GSTA3, PIPOX, CNGA1 and IGG1C and down-regulation of 39 genes including CHGA, KRT35, THBS4, CPXM2 and PRF1 compared with normal cows. Our results may suggest that local regulation of molecular mechanisms in each uterine portion contributes to normal uterine physiology.
Project description:The objective of this study was to evaluate the association between progesterone concentration (P4) at day 4 of the estrous cycle (beginning of diestrus) and endometrial global gene expression at day 9 (middle diestrus) in lactating grazing dairy cows. Blood samples were obtained on days 0, 4 and 9 for P4 measurement by chemiluminescence. Cows were asignated to one of the following groups (n=3/group): cows with low physiological P4 at day 4 (PLP4), cows in anestrous (ANE), cows with high physiological P4 at day 4 (HPP4) and superovulated cows (SO). Endometrial biopsy samples were obtained on day 9 for RNA sequencing. Quality control and determination of differentially expressed genes (DEG; FDR<0.05) were determined using the edgeR package for R. The number of DEG between groups were: HPP4 vs LPP4: 453; HPP4 vs ANE: 623; SO vs LPP4: 603; SO vs ANE: 1102; LPP4 vs ANE: 261 and SO vs HPP4: 0. Functional analysis using the DAVID database revealed that endometrial genes upregulated by low P4 are enriched in the immune system and inflammatory response. Conversely, genes upregulated by high P4 are enriched inmodulation of uterine relaxation-contraction, GnRH signaling pathway, focal adhesion and EGF-like related terms. In conclusion, our results support the concept of changes in P4 at the beginning of the luteal phase could be associated with the endometrial expression of critical genes involved in the preparation of the uterine environment for embryo implantation.
Project description:In order to identify pre-conceptional endometrial dysregulations, we compared the endometrial expression between fertile and IF and RM patients
Project description:We report the transcriptomic profile and PGR cistromic profile for endometrial biopsies obtained from fertile women during the proliferative and mid-secretory phase of the menstrual cycle
Project description:We report the transcriptomic profile and PGR cistromic profile for endometrial biopsies obtained from fertile women during the proliferative and mid-secretory phase of the menstrual cycle
Project description:Pregnancy induces changes in the transcriptome of the bovine endometrium from 15 days after insemination. However, pregnancy is less likely to occur if cows had a postpartum bacterial infection of the uterus. We hypothesized that uterine bacterial infection alters the endometrial transcriptomic signature of pregnancy. To examine the endometrial transcriptomic signature of pregnancy, cows were inseminated 130 days after intrauterine infusion of pathogenic bacteria and endometrium was collected 16 days later for RNA sequencing. We found 171 pregnancy regulated genes in cows 146 days after bacterial infection. When comparing our findings with three previous studies that described the endometrial transcriptomic signature of pregnancy in healthy cows, 24 genes were consistently differentially expressed in pregnancy, including MX1, MX2 and STAT1. However, 12 pregnancy regulated genes were only found in the endometrium of healthy cows, including ISG15 and TRANK1. Furthermore, 28 pregnancy regulated genes were only found in the endometrium of cows following bacterial infection and these were associated with altered iNOS, TLR, and IL-7 canonical signaling pathways. Although 94 predicted upstream regulators were conserved amongst the studies, 14 were found only in the endometrium of pregnant healthy cows, and 5 were found only in cows following bacterial infection, including AIRE, NFKBIA, and DUSP1. In conclusion, there were both consistent and discordant features of the endometrial transcriptomic signature of pregnancy 146 days after intrauterine bacterial infusion. These findings imply that there is an essential transcriptomic signature of pregnancy, but that infection induces long term changes in the endometrium that affect the transcriptomic response to pregnancy.
Project description:In order to identify pre-conceptional endometrial dysregulations, we compared the endometrial expression between fertile and IF and RM patients Total RNA were extracted and used for hybridizing Affymetrix chips (GeneChip Human Genome U133 Plus2.0 Array). Data were normalised by gcRMA method and raw p-values adjusted by Bonferroni procedure (1%). Endometrial biopsy was performed in non conceptional cycle in the middle luteal phase-(5 women were selected in each group).