Transcriptome Analyses of Myometrium from Fibroid Patients Reveals Phenotypic Differences Compared to Non-Diseased Myometrium.

Uterine fibroid tissues are often compared to their matched myometrium in an effort to understand their pathophysiology, but it is not clear whether the myometria of uterine fibroid patients represent truly non-disease control tissues. We analyzed the transcriptomes of myometrial samples from non-fibroid patients (M) and compared them with fibroid (F) and matched myometrial (MF) samples to determine whether there is a phenotypic difference between fibroid and non-fibroid myometria. Multidimensional scaling plots revealed that M samples clustered separately from both MF and F samples. A total of 1169 differentially expressed genes (DEGs) (false discovery rate < 0.05) were observed in the MF comparison with M. Overrepresented Gene Ontology terms showed a high concordance of upregulated gene sets in MF compared to M, particularly extracellular matrix and structure organization. Gene set enrichment analyses showed that the leading-edge genes from the TGFß signaling and inflammatory response gene sets were significantly enriched in MF. Overall comparison of the three tissues by three-dimensional principal component analyses showed that M, MF, and F samples clustered separately from each other and that a total of 732 DEGs from F vs. M were not found in the F vs. MF, which are likely understudied in the pathogenesis of uterine fibroids and could be key genes for future investigation. These results suggest that the transcriptome of fibroid-associated myometrium is different from that of non-diseased myometrium and that fibroid studies should consider using both matched myometrium and non-diseased myometrium as controls.

Placental Transcriptome Adaptations to Maternal Nutrient Restriction in Sheep.

Placental development is modified in response to maternal nutrient restriction (NR), resulting in a spectrum of fetal growth rates. Pregnant sheep carrying singleton fetuses and fed either 100% (n = 8) or 50% (NR; n = 28) of their National Research Council (NRC) recommended intake from days 35-135 of pregnancy were used to elucidate placentome transcriptome alterations at both day 70 and day 135. NR fetuses were further designated into upper (NR NonSGA; n = 7) and lower quartiles (NR SGA; n = 7) based on day 135 fetal weight. At day 70 of pregnancy, there were 22 genes dysregulated between NR SGA and 100% NRC placentomes, 27 genes between NR NonSGA and 100% NRC placentomes, and 22 genes between NR SGA and NR NonSGA placentomes. These genes mediated molecular functions such as MHC class II protein binding, signaling receptor binding, and cytokine activity. Gene set enrichment analysis (GSEA) revealed significant overrepresentation of genes for natural-killer-cell-mediated cytotoxicity in NR SGA compared to 100% NRC placentomes, and alterations in nutrient utilization pathways between NR SGA and NR NonSGA placentomes at day 70. Results identify novel factors associated with impaired function in SGA placentomes and potential for placentomes from NR NonSGA pregnancies to adapt to nutritional hardship.

Characterization and regulation of extracellular vesicles in the lumen of the ovine uterus†.

Secretions of the endometrium are vital for peri-implantation growth and development of the sheep conceptus. Extracellular vesicles (EVs) are present in the uterine lumen, emanate from both the endometrial epithelia of the uterus and trophectoderm of the conceptus, and hypothesized to mediate communication between those cell types during pregnancy establishment in sheep. Size-exclusion chromatography and nanoparticle tracking analysis determined that total EV number in the uterine lumen increased from days 10 to 14 of the cycle but was lower on days 12 and 14 of pregnancy in sheep. Intrauterine infusions of interferon tau (IFNT) did not affect total EV number in the uterine lumen. Quantitative mass spectrometric analyses defined proteins and lipids in EVs isolated from the uterine lumen of day 14 cyclic and pregnant sheep. In vitro analyses found that EVs decreased ovine trophectoderm cell proliferation and increased IFNT production without effects on gene expression as determined by RNA-seq. Collective results support the idea EVs impact conceptus growth during pregnancy establishment via effects on trophectoderm cell growth.

Prostaglandin-endoperoxide synthase 2 is not required for preimplantation ovine conceptus development in sheep.

Conceptus development and elongation is required for successful pregnancy establishment in ruminants and is coincident with the production of interferon τ (IFNT) and prostaglandins (PGs). In both the conceptus trophectoderm and endometrium, PGs are primarily synthesized through a prostaglandin-endoperoxide synthase 2 (PTGS2) pathway and modify endometrial gene expression and thus histotroph composition in the uterine lumen to promote conceptus growth and survival. Chemical inhibition of PG production by both the endometrium and the conceptus prevented elongation in sheep. However, the contributions of conceptus-derived PGs to preimplantation conceptus development remain unclear. In this study, CRISPR-Cas9 genome editing was used to inactivate PTGS2 in ovine embryos to determine the role of PTGS2-derived PGs in conceptus development and elongation. PTGS2 edited conceptuses produced fewer PGs, but secreted similar amounts of IFNT to their Cas9 control counterparts and elongated normally. Expression of PTGS1 was lower in PTGS2 edited conceptuses, but PPARG expression and IFNT secretion were unaffected. Content of PGs in the uterine lumen was similar as was gene expression in the endometrium of ewes who received either Cas9 control or PTGS2 edited conceptuses. These results support the idea that intrinsic PTGS2-derived PGs are not required for preimplantation embryo or conceptus survival and development in sheep.

Integrative analysis of the forkhead box A2 (FOXA2) cistrome for the human endometrium.

The pioneer forkhead box (FOX)A2 transcription factor is specifically expressed in the glands of the uterus, which are central to endometrial function and fertility. In mice, FOXA2 is a critical regulator of uterine gland development in the neonate and gland function in the adult. An integrative approach was used here to define the FOXA2 cistrome in the human endometrium. Genome-wide mapping of FOXA2 binding intervals by chromatin immunoprecipitation sequencing was performed using proliferative (P)- and midsecretory (MS)-phase endometrium and integrated with the transcriptome determined by RNA sequencing. Distinctive FOXA2 binding intervals, enriched for different transcription factor binding site motifs, were detected in the P and MS endometrium. Pathway analysis revealed different biologic processes regulated by genes with FOXA2 binding intervals in the P and MS endometrium. Thus, FOXA2 is postulated to regulate gene expression in concert with other transcription factors and impact uterine gland development and function in a cycle phase-dependent manner. Analyses also identified potential FOXA2-regulated genes that influence uterine receptivity, blastocyst implantation, and stromal cell decidualization, which are key events in pregnancy establishment.-Kelleher, A. M., Behura, S. K., Burns, G. W., Young, S. L., DeMayo, F. J., Spencer, T. E. Integrative analysis of the forkhead box A2 (FOXA2) cistrome for the human endometrium.

Protein Synthesis by Day 16 Bovine Conceptuses during the Time of Maternal Recognition of Pregnancy.

Interferon Tau (IFNT), the conceptus-derived pregnancy recognition signal in cattle, significantly modifies the transcriptome of the endometrium. However, the endometrium also responds to IFNT-independent conceptus-derived products. The aim of this study was to determine what proteins are produced by the bovine conceptus that may facilitate the pregnancy recognition process in cattle. We analysed by mass spectrometry the proteins present in conceptus-conditioned media (CCM) after 6 h culture of Day 16 bovine conceptuses (n = 8) in SILAC media (arginine- and lysine-depleted media supplemented with heavy isotopes) and the protein content of extracellular vesicles (EVs) isolated from uterine luminal fluid (ULF) of Day 16 pregnant (n = 7) and cyclic (n = 6) cross-bred heifers on day 16. In total, 11,122 proteins were identified in the CCM. Of these, 5.95% (662) had peptides with heavy labelled amino acids, i.e., de novo synthesised by the conceptuses. None of these proteins were detected in the EVs isolated from ULF. Pregnancy-associated glycoprotein 11, Trophoblast Kunitz domain protein 1 and DExD-Box Helicase 39A were de novo produced and present in the CCM from all conceptuses and in previously published CCM data following 6 and 24 h. A total of 463 proteins were present in the CCM from all the conceptuses in the present study, and after 6 and 24 h culture in a previous study, while expression of their transcripts was not detected in endometrium indicating that they are likely conceptus-derived. Of the proteins present in the EVs, 67 were uniquely identified in ULF from pregnant heifers; 35 of these had been previously reported in CCM from Day 16 conceptuses. This study has narrowed a set of conceptus-derived proteins that may be involved in EV-mediated IFNT-independent embryo-maternal communication during pregnancy recognition in cattle.

Identification of loci associated with conception rate in primiparous Holstein cows.

BACKGROUND: Subfertility is a major issue facing the dairy industry as the average US Holstein cow conception rate (CCR) is approximately 35%. The genetics underlying the physiological processes responsible for CCR, the proportion of cows able to conceive and maintain a pregnancy at each breeding, are not well characterized. The objectives of this study were to identify loci, positional candidate genes, and transcription factor binding sites (TFBS) associated with CCR and determine if there was a genetic correlation between CCR and milk production in primiparous Holstein cows. Cows were bred via artificial insemination (AI) at either observed estrus or timed AI and pregnancy status was determined at day 35 post-insemination. Additive, dominant, and recessive efficient mixed model association expedited (EMMAX) models were used in two genome-wide association analyses (GWAA). One GWAA focused on CCR at first service (CCR1) comparing cows that conceived and maintained pregnancy to day 35 after the first AI (n = 494) to those that were open after the first AI (n = 538). The second GWAA investigated loci associated with the number of times bred (TBRD) required for conception in cows that either conceived after the first AI (n = 494) or repeated services (n = 472). RESULTS: The CCR1 GWAA identified 123, 198, and 76 loci associated (P < 5 × 10- 08) in additive, dominant, and recessive models, respectively. The TBRD GWAA identified 66, 95, and 33 loci associated (P < 5 × 10- 08) in additive, dominant, and recessive models, respectively. Four of the top five loci were shared in CCR1 and TBRD for each GWAA model. Many of the associated loci harbored positional candidate genes and TFBS with putative functional relevance to fertility. Thirty-six of the loci were validated in previous GWAA studies across multiple breeds. None of the CCR1 or TBRD associated loci were associated with milk production, nor was their significance with phenotypic and genetic correlations to 305-day milk production. CONCLUSIONS: The identification and validation of loci, positional candidate genes, and TFBS associated with CCR1 and TBRD can be utilized to improve, and further characterize the processes involved in cattle fertility.

Validation of 46 loci associated with female fertility traits in cattle.

BACKGROUND: Subfertility is one challenge facing the dairy industry as the average Holstein heifer conception rate (HCR), the proportion of heifers that conceive and maintain a pregnancy per breeding, is estimated at 55-60%. Of the loci associated with HCR, few have been validated in an independent cattle population, limiting their usefulness for selection or furthering our understanding of the mechanisms involved in successful pregnancy. Therefore, the objectives here were to identify loci associated with HCR: 1) to the first artificial insemination (AI) service (HCR1), 2) to repeated AI services required for a heifer to conceive (TBRD) and 3) to validate loci previously associated with fertility. Breeding and health records from 3359 Holstein heifers were obtained after heifers were bred by AI at observed estrus, with pregnancy determined at day 35 via palpation. Heifer DNA was genotyped using the Illumina BovineHD BeadChip, and genome-wide association analyses (GWAA) were performed with additive, dominant and recessive models using the Efficient Mixed Model Association eXpedited (EMMAX) method with a relationship matrix for two phenotypes. The HCR1 GWAA compared heifers that were pregnant after the first AI service (n = 497) to heifers that were open following the first AI service (n = 405), which included those that never conceived. The TBRD GWAA compared only those heifers which did conceive, across variable numbers of AI service (n = 712). Comparison of loci previously associated with fertility, HCR1 or TBRD were considered the same locus for validation when in linkage disequilibrium (D' > 0.7). RESULTS: The HCR1 GWAA identified 116, 187 and 28 loci associated (P < 5 × 10- 8) in additive, dominant and recessive models, respectively. The TBRD GWAA identified 235, 362, and 69 QTL associated (P < 5 × 10- 8) with additive, dominant and recessive models, respectively. Loci previously associated with fertility were in linkage disequilibrium with 22 loci shared with HCR1 and TBRD, 5 HCR1 and 19 TBRD loci. CONCLUSIONS: Loci associated with HCR1 and TBRD that have been identified and validated can be used to improve HCR through genomic selection, and to better understand possible mechanisms associated with subfertility.

Progesterone effects on extracellular vesicles in the sheep uterus.

Progesterone (P4) acts via the endometrium to promote conceptus growth and implantation for pregnancy establishment. Many cells release extracellular vesicles (EVs) that are membrane-bound vesicles of endosomal and plasma membrane origin. In sheep, endometrial-derived EVs were found to traffic to the conceptus trophectoderm. Thus, EVs are hypothesized to be an important mode of intercellular communication by transferring select RNAs, proteins, and lipids between the endometrium and conceptus. Electron microscopy analysis found that the endometrial luminal and glandular epithelia were the primary source of EVs in the uterus of cyclic sheep. Size exclusion chromatography and nanoparticle tracking analysis (NTA) found that total EV number in the uterine lumen increased from day 10 to 14 in cyclic sheep. Next, ewes were ovariectomized and hormone replaced to determine effects of P4 on the endometrium and EVs in the uterine lumen. Transcriptome analyses found that P4 regulated 1611 genes and nine miRNAs in the endometrium. Total EV number in the uterine lumen was increased by P4 treatment. Small RNA sequencing of EVs detected expression of 768 miRNAs and determined that P4 regulated seven of those miRNAs. These studies provide fundamental new information on how P4 influences endometrial function to regulate conceptus growth for pregnancy establishment in sheep.

Extracellular Vesicles Originate from the Conceptus and Uterus During Early Pregnancy in Sheep.

Cells release diverse types of membrane-bound vesicles of endosomal and plasma membrane origin, termed exosomes and microvesicles, respectively. Extracellular vesicles (EVs) represent an important mode of intercellular communication by transferring select RNAs, proteins, and lipids between cells. The present studies tested the hypothesis that the elongating ovine conceptus and uterus produces EVs that mediate conceptus-maternal interactions during early pregnancy. In Study 1, EVs were purified from uterine luminal fluid of Day 14 cyclic sheep. The EVs were fluorescently labeled with PKH67 dye and infused into the uterine lumen of pregnant sheep for 6 days using an osmotic pump. On Day 14, labeled EVs were observed in the conceptus trophectoderm and uterine epithelia, but not in the uterine stroma or myometrium. In Study 2, Day 14 conceptuses were cultured ex vivo for 24 h and found to release EVs into the culture medium. Proteomics analysis of the Day 14 conceptus-derived EVs identified 231 proteins that were enriched for extracellular space and several protein classes, including proteases, protease inhibitors, chaperones and chaperonins. RNA sequencing of Day 14 conceptus-derived EVs detected expression of 512 mRNAs. The top-expressed genes were overrepresented in ribosomal functions and components. Isolated EVs from conceptuses were fluorescently labeled with PKH67 and infused into the uterine lumen of cyclic sheep for 6 days using an osmotic pump. On Day 14, labeled EVs were observed in the uterine epithelia, but not in the uterine stroma or myometrium. Labeled EVs were not observed in the ovary or in other maternal tissues. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia, and are involved in intercellular communication between those cells during the establishment of pregnancy in sheep.

Analysis of the Uterine Epithelial and Conceptus Transcriptome and Luminal Fluid Proteome During the Peri-Implantation Period of Pregnancy in Sheep.

Studies support the idea that uterine epithelia and their secretions have important biological roles in conceptus survival, elongation, and implantation in sheep. The present study evaluated the transcriptome of the uterine luminal epithelium (LE) and glandular epithelium (GE) and the conceptus and proteome of uterine luminal fluid (ULF) during the peri-implantation period of pregnancy. Transcriptome (RNA-sequencing) analysis was conducted in LE and GE isolated from uteri of Day 10, 12, 14, 16, and 20 pregnant sheep by laser capture microdissection. In the LE, the total number of expressed genes increased between Days 10 and 20, whereas expressed genes in the GE increased from Days 10 to 14 and then decreased to Day 20. Most of the expressed genes in LE and GE from Days 10 to 14 are involved in cell survival and growth, whereas genes involved in cell organization and protein synthesis were most abundant on Days 16 and 20. Total expressed genes in the conceptus was greatest on Day 12, decreased to Day 16, and then increased to Day 20. Genes abundantly expressed in the elongating conceptus included IFNT, PTGS2, MGST1, FADS1, and FADS2, whereas SERPINA1, CSH1, and PLET1 were most abundant in the Day 20 conceptus. Proteins, identified by mass spectrometry, increased in the ULF from Days 10 to 16 and are involved in cellular reorganization or are proteases or chaperone proteins. These results support the idea that conceptus elongation and implantation is regulated by both extrinsic and intrinsic factors. This study provides critical information that serves as a foundation to discover new regulatory pathways governing uterine receptivity, conceptus elongation, trophectoderm differentiation, conceptus-endometrial interactions, and pregnancy establishment in ruminants.

Identification of Beef Heifers with Superior Uterine Capacity for Pregnancy.

Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In study one, predominantly Angus heifers were classified based on fertility using serial embryo transfer to select animals with intrinsic differences in pregnancy loss. In each of the four rounds, a single in vitro-produced, high-quality embryo was transferred into heifers on Day 7 postestrus and pregnancy was determined on Days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In study two, fertility-classified heifers were resynchronized and bred with semen from a single high-fertility bull. Blood samples were collected every other day from Days 0 to 36 postmating. Pregnancy rate was determined on Day 28 by ultrasound and was higher in HF (70.4%) than in heifers with low fertility (36.8%; SF and IF). Progesterone concentrations in serum during the first 20 days postestrus were not different in nonpregnant heifers and also not different in pregnant heifers among fertility groups. In study three, a single in vivo-produced embryo was transferred into fertility-classified heifers on Day 7 postestrus. The uteri were flushed on Day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the Day 14 endometrial biopsies of pregnant HF, SF, and IF heifers (n = 5 per group) and analyzed by edgeR-robust analysis. There were 26 differentially expressed genes (DEGs) in the HF compared to SF endometrium, 12 DEGs for SF compared to IF endometrium, and three DEGs between the HF and IF endometrium. Several of the DEG-encoded proteins are involved in immune responses and are expressed in B cells. Results indicate that preimplantation conceptus survival and growth to Day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between Days 14 and 28 when pregnancy recognition signaling and conceptus elongation and implantation must occur for the establishment of pregnancy.

Spatial differences in gene expression in the bovine oviduct.

The aim of this study was to compare the transcriptome of the oviductal isthmus of pregnant heifers with that of cyclic heifers as well as to investigate spatial differences between the transcriptome of the isthmus and ampulla of the oviduct in pregnant heifers. After synchronizing crossbred beef heifers, those in standing oestrus (=Day 0) were randomly assigned to cyclic (non-bred, n=6) or pregnant (artificially inseminated, n=11) groups. They were slaughtered on Day 3 and both oviducts from each animal were isolated and cut in half to separate ampulla and isthmus. Each portion was flushed to confirm the presence of an oocyte/embryo and was then opened longitudinally and scraped to obtain epithelial cells which were snap-frozen. Oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum Microarray analysis of oviductal cells revealed that proximity to the corpus luteum did not affect the transcriptome of the isthmus, irrespective of pregnancy status. However, 2287 genes were differentially expressed (P<0.01) between the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum in pregnant animals. Gene ontology revealed that the main biological processes overrepresented in the isthmus were synthesis of nitrogen, lipids, nucleotides, steroids and cholesterol as well as vesicle-mediated transport, cell cycle, apoptosis, endocytosis and exocytosis, whereas cell motion, motility and migration, DNA repair, calcium ion homeostasis, carbohydrate biosynthesis, and regulation of cilium movement and beat frequency were overrepresented in the ampulla. In conclusion, large differences in gene expression were observed between the isthmus and ampulla of pregnant animals at Day 3 after oestrus.

Biological Roles of Hydroxysteroid (11-Beta) Dehydrogenase 1 (HSD11B1), HSD11B2, and Glucocorticoid Receptor (NR3C1) in Sheep Conceptus Elongation.

In sheep, the elongating conceptus synthesizes and secretes interferon tau (IFNT) as well as prostaglandins (PGs) and cortisol. The enzymes, hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) and HSD11B2 interconvert cortisone and cortisol. In sheep, HSD11B1 is expressed and active in the conceptus trophectoderm as well as in the endometrial luminal epithelia; in contrast, HSD11B2 expression is most abundant in conceptus trophectoderm. Cortisol is a biologically active glucocorticoid and ligand for the glucocorticoid receptor (NR3C1 or GR) and mineralocorticoid receptor (NR3C2 or MR). Expression of MR is not detectable in either the ovine endometrium or conceptus during early pregnancy. In tissues that do not express MR, HSD11B2 protects cells from the growth-inhibiting and/or proapoptotic effects of cortisol, particularly during embryonic development. In study one, an in utero loss-of-function analysis of HSD11B1 and HSD11B2 was conducted in the conceptus trophectoderm using morpholino antisense oligonucleotides (MAOs) that inhibit mRNA translation. Elongating, filamentous conceptuses were recovered on Day 14 from ewes infused with control morpholino or HSD11B2 MAO. In contrast, HSD11B1 MAO resulted in severely growth-retarded conceptuses or conceptus fragments with apoptotic trophectoderm. In study two, clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing was used to determine the role of GR in conceptus elongation and development. Elongating, filamentous-type conceptuses (12-14 cm in length) were recovered from ewes gestating control embryos (n = 7/7) and gestating GR-edited embryos (n = 6/7). These results support the idea that the effects of HSD11B1-derived cortisol on conceptus elongation are indirectly mediated by the endometrium and are not directly mediated through GR in the trophectoderm.

Peroxisome proliferator activator receptor gamma (PPARG) regulates conceptus elongation in sheep.

The ovine blastocyst hatches from the zona pellucida by Day 8 and develops into an ovoid or tubular conceptus (embryo and associated extraembryonic membranes) that grows and elongates into a filamentous form between Days 12 and 16. The trophectoderm of the elongating conceptus synthesizes and secretes interferon tau (IFNT) as well as prostaglandins (PGs) via prostaglandin synthase two (PTGS2). Intrauterine infusion of a PTGS2 inhibitor prevents conceptus elongation in sheep. Although many PGs are secreted, PGI2 and PGJ2 can activate nuclear peroxisome proliferator activator receptors (PPARs) that heterodimerize with retinoic X receptors (RXRs) to regulate gene expression and cellular function. Expression of PPARD, PPARG, RXRA, RXRB, and RXRG is detected in the elongating ovine conceptus, and nuclear PPARD and PPARG are present in the trophectoderm. Consequently, PPARD and PPARG are hypothesized to have essential roles in conceptus elongation in ruminants. In utero loss-of-function studies of PPARD and PPARG in the ovine conceptus trophectoderm were conducted using morpholino antisense oligonucleotides (MAOs) that inhibit mRNA translation. Elongating, filamentous-type conceptuses were recovered from ewes infused with a control morpholino or PPARD MAO. In contrast, PPARG MAO resulted in severely growth-retarded conceptuses or conceptus fragments with apoptotic trophectoderm. In order to identify PPARG-regulated genes, PPARG chromatin immunoprecipitation sequencing and RNA sequencing were conducted using Day 14 ovine conceptuses. These analyses revealed candidate PPARG-regulated genes involved in biological pathways, including lipid and glucose uptake, transport, and metabolism. Collectively, results support the hypothesis that PTGS2-derived PGs and PPARG are essential regulators of conceptus elongation, with specific roles in trophectoderm survival and proliferation.

Oviduct-Embryo Interactions in Cattle: Two-Way Traffic or a One-Way Street?

This study examined the effect of the presence of single or multiple embryos on the transcriptome of the bovine oviduct. In experiment 1, cyclic (nonbred, n = 6) and pregnant (artificially inseminated, n = 11) heifers were slaughtered on Day 3 after estrus, and the ampulla and isthmic regions of the oviduct ipsilateral to the corpus luteum were separately flushed. Oviductal epithelial cells from the isthmus region, in which all oocytes/embryos were located, were snap-frozen for microarray analysis. In experiment 2, heifers were divided into cyclic (nonbred, n = 6) or pregnant (multiple embryo transfer, n = 10) groups. In vitro-produced presumptive zygotes were transferred endoscopically to the ipsilateral oviduct on Day 1.5 postestrus (n = 50 zygotes/heifer). Heifers were slaughtered on Day 3, and oviductal isthmus epithelial cells were recovered for RNA sequencing. Microarray analysis in experiment 1 failed to detect any difference in the transcriptome of the oviductal isthmus induced by the presence of a single embryo. In experiment 2, following multiple embryo transfer, RNA sequencing revealed 278 differentially expressed genes, of which 123 were up-regulated and 155 were down-regulated in pregnant heifers. Most of the down-regulated genes were related to immune function. In conclusion, the presence of multiple embryos in the oviduct resulted in the detection of differentially expressed genes in the oviductal isthmus; failure to detect changes in the oviduct transcriptome in the presence of a single embryo may be due to the effect being local and undetectable under the conditions of this study.

Yes Associated Transcriptional Regulator 1 (YAP1) and WW Domain Containing Transcription Regulator (WWTR1) are required for murine pregnancy initiation.

Endometrial stromal cell decidualization is required for pregnancy success. Although this process is integral to fertility, many of the intricate molecular mechanisms contributing to decidualization remain undefined. One pathway that has been implicated in endometrial stromal cell decidualization in humans in vitro is the Hippo signaling pathway. Two previously conducted studies showed that the effectors of the Hippo signaling pathway, YAP1 and WWTR1, were required for decidualization of primary stromal cells in culture. To investigate the in vivo role of YAP1 and WWTR1 in decidualization and pregnancy initiation, we generated a Progesterone Cre mediated partial double knockout (pdKO) of Yap1 and Wwtr1. Female pdKOs exhibited subfertility, a compromised decidualization response, partial interruption in embryo transport, blunted endometrial receptivity, delayed implantation and subsequent embryonic development, and a unique transcriptional profile. Bulk mRNA sequencing revealed aberrant maternal remodeling evidenced by significant alterations in extracellular matrix proteins at 7.5 days post-coitus in pdKO dams and enrichment for terms associated with fertility-compromising diseases like pre-eclampsia and endometriosis. Our results indicate a required role for YAP1 and WWTR1 for successful mammalian uterine function and pregnancy success.

Spatial Transcriptomic Analysis Identifies Epithelium-Macrophage Crosstalk in Endometriotic Lesions.

The mechanisms underlying the pathophysiology of endometriosis, characterized by the presence of endometrium-like tissue outside the uterus, remain poorly understood. This study aimed to identify cell type-specific gene expression changes in superficial peritoneal endometriotic lesions and elucidate the crosstalk among the stroma, epithelium, and macrophages compared to patient-matched eutopic endometrium. Surprisingly, comparison between lesions and eutopic endometrium revealed transcriptional similarities, indicating minimal alterations in the sub-epithelial stroma and epithelium of lesions. Spatial transcriptomics highlighted increased signaling between the lesion epithelium and macrophages, emphasizing the role of the epithelium in driving lesion inflammation. We propose that the superficial endometriotic lesion epithelium orchestrates inflammatory signaling and promotes a pro-repair phenotype in macrophages, providing a new role for Complement 3 in lesion pathobiology. This study underscores the significance of considering spatial context and cellular interactions in uncovering mechanisms governing disease in endometriotic lesions.

Spheroids as a model for endometriotic lesions.

The development and progression of endometriotic lesions are poorly understood, but immune cell dysfunction and inflammation are closely associated with the pathophysiology of endometriosis. There is a need for 3D in vitro models to permit the study of interactions between cell types and the microenvironment. To address this, we developed endometriotic spheroids (ES) to explore the role of epithelial-stromal interactions and model peritoneal invasion associated with lesion development. Using a nonadherent microwell culture system, spheroids were generated with immortalized endometriotic epithelial cells (12Z) combined with endometriotic stromal (iEc-ESC) or uterine stromal (iHUF) cell lines. Transcriptomic analysis found 4,522 differentially expressed genes in ES compared with spheroids containing uterine stromal cells. The top increased gene sets were inflammation-related pathways, and an overlap with baboon endometriotic lesions was highly significant. Finally, to mimic invasion of endometrial tissue into the peritoneum, a model was developed with human peritoneal mesothelial cells in an extracellular matrix. Invasion was increased in the presence of estradiol or pro-inflammatory macrophages and suppressed by a progestin. Taken together, our results strongly support the concept that ES are an appropriate model for dissecting mechanisms that contribute to endometriotic lesion development.

Development of a core outcome set for ventilation trials in neurocritical care patients with acute brain injury: protocol for a Delphi consensus study of international stakeholders.

INTRODUCTION: There is little consensus and high heterogeneity on the optimal set of relevant clinical outcomes in research studies regarding extubation in neurocritical care patients with brain injury undergoing mechanical ventilation. The aims of this study are to: (1) develop a core outcome set (COS) and (2) reach consensus on a hierarchical composite endpoint for such studies. METHODS AND ANALYSIS: The study will include a broadly representative, international panel of stakeholders with research and clinical expertise in this field and will involve four stages: (1) a scoping review to generate an initial list of outcomes represented in the literature, (2) an investigator meeting to review the outcomes for inclusion in the Delphi surveys, (3) four rounds of online Delphi consensus-building surveys and (4) online consensus meetings to finalise the COS and hierarchical composite endpoint. ETHICS AND DISSEMINATION: This study received ethical approval from the French Society of Anesthesia and Critical Care Medicine Institutional Review Board (SFAR CERAR-IRB 00010254-2023-029). The study results will be disseminated through communication to stakeholders, publication in a peer-reviewed journal, and presentations at conferences. TRIAL REGISTRATION NUMBER: This study is registered with the Core Outcome Measures in Effectiveness Trials (COMET) Initiative.