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Background: MicroRNAs (miRNAs) are presented in the uterine lumen of many mammals, and in vitro experiments have determined that several miRNAs are important for the regulation of endometrial and trophoblast functions. Our aim was to identify and contrast the miRNAs present in extracellular vesicles (EVs) in the uterine lumen fluid (ULF) at the onset of attachment in cattle pregnancies (gestation d 18) initiated by artificial insemination (AI) or by the transfer of an in vitro-produced blastocyst (IVP-ET). A third group had no conceptus after the transfer of an IVP embryo., Results: The abundance of 263 annotated miRNAs was quantified in the EVs collected from ULF. There was an increase in the transcript abundance of 20 miRNAs in the ULF EVs from the AI pregnant group, while 4 miRNAs had a lower abundance relative to the group not containing a conceptus. Additionally, 4 miRNAs were more abundant in ULF EVs in the AI pregnant group relative to IVP-ET group (bta-mir-17, bta-mir-7-3, MIR7-1, MIR18A). Specific miRNAs in the ULF EVs were co-expressed with messenger RNAs expressed in extra-embryonic tissues and endometrium, including genes that are known to be their targets., Conclusions: The results provide biological insights into the participation of miRNAs in the regulation of trophoblast proliferation and differentiation, as well as in endometrium receptivity. The knowledge that in vitro cultured embryos can contribute to the altered abundance of specific miRNAs in the uterine lumen can lead to the development of corrective approaches to reduce conceptus losses during the first month of pregnancy in cattle., (© 2024. The Author(s).)

Cows with metritis (uterine disease) during the first 1 to 2 weeks postpartum have lower pregnancy rates when inseminated later postpartum (typically >10 weeks). We hypothesized that metritis and the disease-associated uterine microbiome have a long-term effect on endometrial gene expression. Changes in gene expression may inform a mechanism through which disease lowers pregnancy rates. A total of 20 cows were enrolled at 1 to 2 weeks postpartum to either metritis (clinical disease; n = 10) or healthy (control; n = 10) groups and randomly assigned to be slaughtered at approximately 80 and 165 dpp (mid-lactation). The microbiome of the reproductive tract was sampled to confirm the presence of pathogens that are typical of metritis. In addition to the original clinical diagnosis, study cows were retrospectively assigned to uterine-disease and control groups based on the composition of their microbiome. There was no effect of early postpartum uterine disease on the uterine microbiome at mid-lactation (time of slaughter). Nonetheless, early postpartum metritis and the disease microbiome were associated with a large number of differentially-expressed genes at mid-lactation primarily in the caruncular compared with the inter-caruncular endometrium. Gene enrichment analysis identified oxidative phosphorylation as the primary pathway increased in caruncular endometrium of diseased cows whereas growth factor signaling pathways were reduced. The current study demonstrated that metritis and a uterine disease microbiome leave a sustained imprint on gene expression in the caruncular endometrium that may explain lower fertility in cows with postpartum uterine disease., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Postnatal development of the uterus involves the specification of undifferentiated epithelium into uterine-type epithelium. That specification is regulated by stromal-epithelial interactions as well as intrinsic cell-specific transcription factors and gene regulatory networks. Here, we present a co-culture system to study the effects of stromal-derived factors on epithelial cell growth and differentiation into organoids. First, we describe epithelial cell isolation and organoid growth characterization. Second, we detail a co-culture system that allows the study of stromal-derived paracrine factors on epithelial development. For complete details on the use and execution of this protocol, please refer to Rizo et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

A central determinant of pregnancy success is proper development of the conceptus (embryo/fetus and associated extraembryonic membranes including the placenta). Although the gross morphology and histology of the bovine placenta have been well studied, the cellular and molecular mechanisms regulating placenta development and trophoblast differentiation and function remain essentially undefined. Here, single-cell transcriptome (scRNA-seq) analysis was performed on the day 17 bovine conceptus and chorion of day 24, 30, and 50 conceptuses (n = 3-4 samples per day) using the 10X Genomics platform. Bioinformatic analyses identified cell types and their ontogeny including trophoblast, mesenchyme, and immune cells. Loss of interferon tau-expressing trophoblast uninucleate cells occurred between days 17 and 30, whereas binucleate cells, identified based on expression of placental lactogen (CSH2) and specific pregnancy-associated glycoprotein genes (PAGs), first appeared on day 24. Several different types of uninucleate cells were present in day 24, 30, and 50 samples, but only one (day 24) or two types of binucleate cells (days 30 and 50). Cell trajectory analyses provided a conceptual framework for uninucleate cell development and binucleate cell differentiation, and bioinformatic analyses identified candidate transcription factors governing differentiation and function of the trophoblasts. The digital atlas of cell types in the developing bovine conceptus reported here serves as a resource to discover key genes and biological pathways regulating its development during the critical periods of implantation and placentation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The uterus is vital for successful reproduction in mammals, and two different types of epithelia (luminal and glandular) are essential for embryo implantation and pregnancy establishment. However, the essential cellular and molecular factors and pathways governing postnatal epithelium maturation, determination, and differentiation in developing uterus are yet to be elucidated. Here, the epithelium of the neonatal mouse uterus was isolated and subjected to single-cell transcriptome (scRNA-seq) analysis. Both the undifferentiated epithelium and determined luminal epithelium were heterogeneous and contained several different cell clusters based on single-cell transcription profiles. Substantial gene expression differences were evident as the epithelium matured and differentiated between postnatal days 1 to 15. Two new glandular epithelium-expressed genes ( Gas6 and Cited4 ) were identified and validated by in situ hybridization. Trajectory analyses provided a framework for understanding epithelium maturation, lineage bifurcation, and differentiation. A candidate set of transcription factors and gene regulatory networks were identified that potentially direct epithelium lineage specification and morphogenesis. This atlas provides a foundation important to discover intrinsic cellular and molecular mechanisms directing uterine epithelium morphogenesis during a critical window of postnatal development., Competing Interests: Competing interests statement:The authors declare no competing interest.

There is a current need for new biomarkers of spermatozoa quality, that consistently and correctly identify spermatozoa that will successfully contribute to subsequent embryo development. This could improve the standardization of semen analysis, decrease early embryo mortality, and use these biomarkers as a selection tool before servicing females. This study utilized imaging techniques to identify potential biomarkers of sperm quality, using sires previously classified as high ( n  = 4) or low ( n  = 4) performing at producing blastocysts in vitro. Spermatozoa were assessed before and following a gradient purification protocol, to understand how populations of cells are impacted by such protocols and may differ between in vivo and in vitro use. Pre-gradient samples from low-performing sires had an increased incidence of DNA damage, although post-gradient samples from high-performing sires were found to have an increased incidence of DNA damage. When evaluating morphology via fluorescent microscopy, the most prevalent defects in pre-gradient samples from high-performing sires were tail defects, which are successfully removed during purification processing. The most prevalent defects in pre-gradient samples from low-performing sires were aggresome defects located in the head, which would be brought into an oocyte upon fertilization and may impair embryo development. Image-based flow cytometry (IBFC) was employed to quantify defect prevalence to evaluate a greater sample size decreasing the variability that exists in manual assessments. Using IBFC, aggresome defects were again identified in the heads of spermatozoa from low-performing sires. Post-gradient samples from low-performing sires had a significantly greater ( p  < 0.05) incidence of aggresome defects than post-gradient samples from high-performing sires. Additionally, IBFC was used to evaluate spermatozoa viability following gradient purification. Distinct populations of sperm cells were identified. High-performing sires had more spermatozoa in the population deemed most viable than low-performing sires. This study demonstrated that spermatozoa defects vary in populations before and following gradient purification, indicating that it may be beneficial to separately evaluate semen for in vivo and in vitro use. Furthermore, a prevalent defect in low-performing sires that could explain a discrepancy between successful fertilization and embryo development was identified. Therefore, elucidating a malfunction regulated by sire, that could potentially affect early embryo development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fallon, Diaz-Miranda, Hamilton, Sutovsky, Zigo, Spencer and Ortega.)

In Brief: The first week of gestation is a period of major pregnancy loss in cattle, this study reveals that the male plays a key role in regulating embryonic development during this time., Abstract: The impact of sire on preimplantation embryonic development in cattle remains poorly understood. This study evaluated differences in embryos produced in vitro from sires with varying capacities to produce blastocysts. Sires classified as high (HP) and low performing (LP) based on their ability to produce embryos were used to better understand how sire regulates embryonic development. By monitoring development, it was determined that the most common arrest stage was the five- to six-cell stage. Embryos (four to six cells) from HP and LP sires were then analyzed for autophagic activity, where embryos for LP sires exhibited increased autophagy than HP-derived embryos. Transcriptome analysis of four-cell embryos found that embryos from LP sires might have issues in sperm mitochondrial clearance, histone retention, and DNA damage, while HP sires had increased expression of genes involved in transcription, chromosome segregation, and cell division. In conclusion, LP sires had an increased proportion of embryos arresting at the five- to six-cell stage, and these embryos had higher rates of cellular stress due to paternal contributions from the spermatozoon.

Pregnancy loss is a significant problem when embryos produced in vitro are transferred to a synchronized uterus. Currently, mechanisms that underlie losses of in vitro-produced embryos during implantation are largely unknown. We investigated this problem using cattle as a model of conceptus attachment by analyzing transcriptome data of paired extraembryonic membrane and endometrial samples collected on gestation days 18 and 25, which spans the attachment window in cattle. We identified that the transfer of an in vitro-produced embryo caused a significant alteration in transcript abundance of hundreds of genes in extraembryonic and endometrial tissues on gestation days 18 and 25, when compared to pregnancies initiated by artificial insemination. Many of the genes with altered transcript abundance are associated with biological processes that are relevant to the establishment of pregnancy. An integrative analysis of transcriptome data from the conceptus and endometrium identified hundreds of putative ligand-receptor pairs. There was a limited variation of ligand-receptor pairs in pregnancies initiated by in vitro-produced embryos on gestation day 18, and no alteration was observed on gestation day 25. In parallel, we identified that in vitro production of embryos caused an extensive alteration in the coexpression of genes expressed in the extraembryonic membranes and the corresponding endometrium on both gestation days. Both the transcriptional dysregulation that exists in the conceptus or endometrium independently and the rewiring of gene transcription between the conceptus and endometrium are a potential component of the mechanisms that contribute to pregnancy losses caused by in vitro production of embryos., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Postnatal development of the uterus involves specification of undifferentiated epithelium into uterine-type epithelium. That specification is regulated by stromal-epithelial interactions as well as intrinsic cell-specific transcription factors and gene regulatory networks. This study utilized mouse genetic models of Esr1 deletion, endometrial epithelial organoids (EEO), and organoid-stromal co-cultures to decipher the role of Esr1 in uterine epithelial development. Organoids derived from wild-type (WT) mice developed a normal single layer of columnar epithelium. In contrast, EEO from Esr1 null mice developed a multilayered stratified squamous type of epithelium with basal cells. Co-culturing Esr1 null epithelium with WT uterine stromal fibroblasts inhibited basal cell development. Of note, estrogen treatment of EEO-stromal co-cultures and Esr1 conditional knockout mice increased basal epithelial cell markers. Collectively, these findings suggest that Esr1 regulates uterine epithelium lineage plasticity and homeostasis and loss of ESR1 promotes altered luminal-to-basal differentiation driven by ESR1-mediated paracrine factors from the stroma., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

The postpartum uterus involutes to its pre-pregnant and fully functional state within approximately 60 d after calving. Uterine glands are essential for fertility but little is known about their regeneration postpartum. Likewise, the effect of uterine disease (metritis) on gland regeneration is unknown. We hypothesized that uterine glands would be regenerated early postpartum and that metritis would be associated with slower gland regeneration to affect their numbers later postpartum during the breeding period. Postpartum dairy cows were diagnosed as healthy (n = 17 and 9 for experiment [Exp.] 1 and 2) or metritis (n = 17 and 10 for Exp. 1 and 2, respectively) at 7 to 10 d postpartum. Cows were slaughtered at approximately 1 mo (Exp. 1) or approximately 80 or 165 d (Exp. 2) postpartum for the collection of the uterus. Uterine tissue was sectioned and the number of glandular cross-sections per unit area was counted and cross-sectional area measured. Cellular proliferation within the luminal epithelium (LE) and glandular epithelium (GE) was quantified by MKI67 (marker of cellular proliferation) immunohistochemistry. In early postpartum cows (Exp. 1), the greatest amount of MKI67 staining was found in the deep endometrium (cells closest to the myometrium). Cows with purulent material in the uterine lumen at d 30 slaughter (Exp. 1) had fewer endometrial glands per unit area in the deep and middle endometrium when compared with nonpurulent cows. The MKI67 staining was less in the deep endometrial GE and LE for purulent compared with nonpurulent cows. Estrus cyclicity was associated with a greater number of gland cross-sections in the deep and middle endometrium. Later postpartum (80 and 165 d; Exp. 2), there was greater glandular development compared with Exp. 1 and a tendency for a lesser number of gland cross-sections per unit area in diseased cows without an effect on MKI67 staining in the GE or LE. We conclude that uterine disease slows the development of uterine glands early postpartum (by 1 mo) through a mechanism that involves cellular proliferation within the GE. The impact of the early postpartum disease on glandular development later postpartum (Exp. 2) appeared to be less. Additional time, therefore, may allow recovery of the GE in later postpartum cows., (© 2023.)

Ruminants have a unique placenta in comparison to other mammalian species. Initially, they possess a non-invasive epitheliochorial type of placenta during conceptus elongation. As the conceptus trophectoderm begins to attach to the luminal epithelium (LE) of the endometrium, binucleate cells (BNCs) develop within the trophoblast of the chorion. The BNCs migrate and fuse with the uterine LE to form multinucleate syncytial plaques in sheep and hybrid trinucleate cells in cattle. This area of the ruminant placenta is semi-invasive synepitheliochorial. The BNCs form the foundation of the placental cotyledons and express unique placenta-specific genes including pregnancy-associated glycoproteins and chorionic somatomammotropin hormone 2 or placental lactogen. Attachment and interdigitation of cotyledons into endometrial caruncles form placentomes that are subsequently vascularized to provide essential nutrients for growth of the fetus. This chapter review will discuss historical and current aspects of conceptus implantation and placenta development in ruminant ungulates with a focus on cattle and sheep. Single-cell analysis promises to provide a much more detailed understanding of the different cell populations and insights into pathways mediating trophoblast and placenta. This fundamental is required to understand pregnancy loss and develop strategies to improve pregnancy outcomes in ruminants., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Uterine glands and, by inference, their secretions impact uterine receptivity, blastocyst implantation, stromal cell decidualization, and placental development. Changes in gland function across the menstrual cycle are primarily governed by the steroid hormones estrogen (E2) and progesterone (P4) but can also be influenced by extrinsic factors from the stroma. Using a human endometrial epithelial organoid system, transcriptome and proteome analyses identified distinct responses of the organoids to steroid hormones and prostaglandin E2 (PGE2). Notably, P4 and PGE2 modulated the basolateral secretion of organoid proteins, particularly cystatin C (CST3), serpin family A member 3 (SERPINA3), and stanniocalcin 1 (STC1). CST3, but not SERPINA3 or STC1, attenuated the in vitro stromal decidualization response to steroid hormones and PGE2. These findings provide evidence that uterine gland-derived factors impact stromal cell decidualization, which has implications for pregnancy establishment and fertility in women., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Ruminants have a semi-invasive placenta, which possess highly vascularized placentomes formed by maternal endometrial caruncles and fetal placental cotyledons and required for fetal development to term. The synepitheliochorial placenta of cattle contains at least two trophoblast cell populations, including uninucleate (UNC) and binucleate (BNC) cells that are most abundant in the cotyledonary chorion of the placentomes. The interplacentomal placenta is more epitheliochorial in nature with the chorion developing specialized areolae over the openings of uterine glands. Of note, the cell types in the placenta and cellular and molecular mechanisms governing trophoblast differentiation and function are little understood in ruminants. To fill this knowledge gap, the cotyledonary and intercotyledonary areas of the mature day 195 bovine placenta were analyzed by single nuclei analysis. Single-nuclei RNA-seq analysis found substantial differences in cell type composition and transcriptional profiles between the two distinct regions of the placenta. Based on clustering and cell marker gene expression, five different trophoblast cell types were identified in the chorion, including proliferating and differentiating UNC and two different types of BNC in the cotyledon. Cell trajectory analyses provided a framework for understanding the differentiation of trophoblast UNC into BNC. The upstream transcription factor binding analysis of differentially expressed genes identified a candidate set of regulator factors and genes regulating trophoblast differentiation. This foundational information is useful to discover essential biological pathways underpinning the development and function of the bovine placenta.

The Notch signaling pathway is required for reproductive success. This pathway activates its transcriptional effector, recombination signal binding protein for immunoglobulin kappa J (Rbpj), to induce transcription of its target genes. This signaling pathway is required for successful decidualization, implantation, and uterine repair following parturition. To identify the compartmental specific roles of the Notch signaling pathway in the establishment of pregnancy, we generated epithelial and decidual stromal cell specific knockouts of Rbpj utilizing lactoferrin iCre and Prl8A2 iCre, respectively. Both conditional knockout mouse models were fertile. The Rbpj epithelial knockout mice displayed 27% resorption sites at E15.5, but this did not significantly impact the number of live born pups compared with controls. In addition, the Rbpj epithelial knockout mice displayed increased estrogen signaling in their stromal compartment. Given that both mouse models exhibited fertility comparable to control animals, the epithelial and stromal specific nature of the iCre recombinases utilized, and previously published Rbpj total uterine knockout mouse models, we conclude that Notch effector Rbpj signaling is required at the initiation of pregnancy to support decidualization in stromal cells, but that Rbpj is not required in the epithelial compartment nor is it required for post-implantation pregnancy success., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Endometrial health is affected by molecular processes that underlie estrogen responses. We assessed estrogen regulation of endometrial function by integrating the estrogen receptor α (ESR1) cistromes and transcriptomes of endometrial biopsies taken from the proliferative and mid-secretory phases of the menstrual cycle together with hormonally stimulated endometrial epithelial organoids. The cycle stage-specific ESR1 binding sites were determined by chromatin immunoprecipitation and next-generation sequencing and then integrated with changes in gene expression from RNA sequencing data to infer candidate ESR1 targets in normal endometrium. Genes with ESR1 binding in whole endometrium were enriched for chromatin modification and regulation of cell proliferation. The distribution of ESR1 binding sites in organoids was more distal from gene promoters when compared to primary endometrium and was more similar to the proliferative than the mid-secretory phase ESR1 cistrome. Inferred organoid estrogen/ESR1 candidate target genes affected formation of cellular protrusions and chromatin modification. Comparison of signaling effected by candidate ESR1 target genes in endometrium vs organoids reveals enrichment of both overlapping and distinct responses. Our analysis of the ESR1 cistromes and transcriptomes from endometrium and organoids provides important resources for understanding how estrogen affects endometrial health and function., (Published by Oxford University Press on behalf of the Endocrine Society 2022.)

The aim was to examine the effect of sire fertility status on conceptus-induced changes in the bovine endometrial transcriptome. To generate elongated conceptuses, Day 7 blastocysts produced in vitro using frozen-thawed sperm from Holstein Friesian bulls (3 High fertility, HF and 3 Low fertility, LF) were transferred in groups of 5-10 into synchronized heifers ( n = 7 heifers per bull) and recovered following slaughter on Day 15. Day 15 endometrial explants recovered from the uterine horn ipsilateral to the corpus luteum were recovered from synchronized cyclic heifers ( n = 4). Explants from each heifer were co-cultured for 6 h in RPMI medium alone (Control) or with 100 ng/ml ovine recombinant interferon tau (IFNT) or with a single conceptus from each HF or LF bull. After 6 h, explants were snap frozen and stored at -80°C. Extracted mRNA was subjected to RNA-seq and the resulting data were analyzed with R software. The numbers of differentially expressed genes (DEG; FDR<0.05) were: HF vs. Control: 956; LF vs. Control: 1021; IFNT vs. Control: 1301; HF vs. LF: 2. Unsurprisingly, the majority of DEG (658) were common to all comparisons and were related to IFNT-induced changes in the endometrium. Prior to applying the adjusted p -value, there were 700 DEG between HF and LF, with 191 and 509 genes more expressed in HF or LF, respectively ( p < 0.05). Overrepresentation analysis of KEGG pathways (FDR<0.05), revealed that DEG with higher expression in LF were involved in cell cycle and proteolysis, while those upregulated DEG by HF conceptuses were strongly associated with immune process pathways, such as TNF, NF-kappa B, cytokine-cytokine receptor interaction, and TLR signaling. These pathways were also enriched by DEG upregulated by IFNT compared to the Control. Furthermore, only the HF, and not the LF group, affected the expression of most genes in these pathways ( p < 0.05) according to a negative binomial regression model. Finally, a weighted gene co-expression network analysis revealed two clusters of co-expressed genes associated with the HF conceptuses ( p < 0.05), which were also enriched for the aforementioned pathways. In conclusion, HF conceptuses, similar to IFNT treatment, stimulated multiple pathways involved in immune response, which were apparently not affected by LF conceptuses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 O’Callaghan, Sánchez, Rabaglino, McDonald, Liu, Spencer, Fair, Kenny and Lonergan.)

For pregnancy to be established, uterine cells respond to the ovarian hormones, estrogen, and progesterone, via their nuclear receptors, the estrogen receptor (ESR1) and progesterone receptor (PGR). ESR1 and PGR regulate genes by binding chromatin at genes and at distal enhancer regions, which interact via dynamic 3-dimensional chromatin structures. Endometrial epithelial cells are the initial site of embryo attachment and invasion, and thus understanding the processes that yield their receptive state is important. Here, we cultured and treated organoids derived from human epithelial cells, isolated from endometrial biopsies, with estrogen and progesterone and evaluated their transcriptional profiles, their PGR cistrome, and their chromatin conformation. Progesterone attenuated estrogen-dependent gene responses but otherwise minimally impacted the organoid transcriptome. PGR ChIPseq peaks were co-localized with previously described organoid ESR1 peaks, and most PGR and ESR1 peaks were in B (inactive) compartment regions of chromatin. Significantly more ESR1 peaks were assigned to estrogen-regulated genes by considering chromatin loops identified using HiC than were identified using ESR1 peak location relative to closest genes. Overall, the organoids model allowed a definition of the chromatin regulatory components governing hormone responsiveness.

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research., (Published by Oxford University Press on behalf of Society for the Study of Reproduction 2022.)

The proper conceptus elongation in ruminants is critical for the successful placentation and establishment of pregnancy. We have previously shown that the trophectoderm-specific knockdown of LIN28A/B in day 9 ovine blastocysts resulted in increased let-7 miRNAs and reduced conceptus elongation at day 16 of gestation. In this current study, by transcriptome analysis of LIN28A knockdown (AKD) or LIN28B knockdown (BKD) trophectoderm (TE), we explored the downstream target genes of the LIN28-let-7 axis and their roles in the placental and fetal development. We identified 449 differentially expressed genes (DEGs) in AKD TE and 1214 DEGs in BKD TE compared to non-targeting control (NTC). Our analysis further revealed that 210 downregulated genes in AKD TE and 562 downregulated genes in BKD TE were the potential targets of let-7 miRNAs. Moreover, 16 downregulated genes in AKD TE and 57 downregulated and 7 upregulated genes in BKD TE were transcription factors. The DEGs in AKD and BKD TE showed enrichment in the biological processes and pathways critical for placental development and function, and fetal development and growth. The results of this study suggest the potential roles of the LIN28-let-7 axis in placental and fetal development beyond its involvement in trophoblast proliferation and conceptus elongation.

Uterine lumen fluid (ULF) is central to successful pregnancy establishment and maintenance, and impacts offspring wellbeing into adulthood. The current dogma is that ULF composition is primarily governed by endometrial glandular epithelial cell secretions and influenced by progesterone. To investigate the hypothesis that ULF is metabolically semi-autonomous, ULF was obtained from cyclic heifers, and aliquots incubated for various durations prior to analysis by untargeted semi-quantitative metabolomic profiling. Metabolite flux was observed in these ULF isolates, supporting the idea that the biochemical makeup of ULF is semi-autonomously dynamic due to enzyme activities. Subsequent integrative analyses of these, and existing, data predict the specific reactions underpinning this phenomenon. These findings enhance our understanding of the mechanisms leading to pregnancy establishment, with implications for improving fertility and pregnancy outcomes in domestic animals as well as women., (© 2022. The Author(s).)

Establishment and maintenance of pregnancy in the pig is a complex process that relies on conceptus regulation of the maternal proinflammatory response to endometrial attachment. Following elongation, pig conceptuses secrete interferon gamma (IFNG) during attachment to the endometrial luminal epithelium. The objective here was to determine if conceptus production of IFNG is important for early development and establishment of pregnancy. CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies were used to create an IFNG loss-of-function study in pigs. Wild-type (IFNG+/+) and null (IFNG-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. IFNG expression was not detected in IFNG-/- conceptuses on either day 15 or day 17 of pregnancy. Ablation of conceptus IFNG production resulted in the reduction of stromal CD3+ and mast cells, which localized to the site of conceptus attachment on day 15. The uteri of recipients with IFNG-/- conceptuses were inflamed, hyperemic and there was an abundance of erythrocytes in the uterine lumen associated with the degenerating conceptuses. The endometrial stromal extracellular matrix was altered in the IFNG-/- embryo pregnancies and there was an increased endometrial mRNA levels for collagen XVII (COL17A1), matrilin 1 (MATN1), secreted phosphoprotein 1 (SPP1), and cysteine-rich secretory protein 3 (CRISP3), which are involved with repair and remodeling of the extracellular matrix. These results indicate conceptus IFNG production is essential in modulating the endometrial proinflammatory response for conceptus attachment and survival in pigs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Secreted phosphoprotein 1 (SPP1, also known as osteopontin) binds integrins to mediate cell-cell and cell-extracellular matrix communication to promote cell adhesion, migration, and differentiation. Considerable evidence links SPP1 to pregnancy in several species. Current evidence suggests that SPP1 is involved in implantation and placentation in mice, but in vivo localization of SPP1 and in vivo mechanistic studies to substantiate these roles are incomplete and contradictory. We localized Spp1 mRNA and protein in the endometrium and placenta of mice throughout gestation, and utilized delayed implantation of mouse blastocysts to link SPP1 expression to the implantation chamber. Spp1 mRNA and protein localized to the endometrial luminal (LE), but not glandular epithelia (GE) in interimplantation regions of the uterus throughout gestation. Spp1 mRNA and protein also localized to uterine naturel killer (uNK) cells of the decidua. Within the implantation chamber, Spp1 mRNA localized only to intermittent LE cells, and to the inner cell mass. SPP1 protein localized to intermittent trophoblast cells, and to the parietal endoderm. These results suggest that SPP1: (1) is secreted by the LE at interimplantation sites for closure of the uterine lumen to form the implantation chamber; (2) is secreted by LE adjacent to the attaching trophoblast cells for attachment and invasion of the blastocyst; and (3) is not a component of histotroph secreted from the GE, but is secreted from uNK cells in the decidua to increase angiogenesis within the decidua to augment hemotrophic support of embryonic/fetal development of the conceptus., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

In ruminants, conceptus elongation requires the endometrium and its secretions. The amino acid, carbohydrate, and protein composition of the uterine lumen during early pregnancy has been defined in sheep; however, a comprehensive understanding of metabolomic changes in the uterine lumen is lacking, particularly with respect to lipids. Here, the lipidome and primary metabolome of the uterine lumen, endometrium, and/or conceptus was determined on day 14 of the estrous cycle and pregnancy. Lipid droplets and select triglycerides were depleted in the endometrium of pregnant ewes. In contrast, select ceramides, diglycerides, and non-esterified fatty acids as well as several phospholipid classes (phosphatidylcholine, phosphatidylinositol, phosphatidylglycerols, and diacylglycerols) were elevated in the uterine lumen of pregnant ewes. Lipidomic analysis of the conceptus revealed that triglycerides are particularly abundant within the conceptus. Primary metabolite analyses found elevated amino acids, carbohydrates, and energy substrates, among others, in the uterine lumen of pregnant ewes. Collectively, this study supports the hypothesis that lipids are important components of the uterine lumen that govern conceptus elongation and growth during early pregnancy., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Suboptimal uterine fluid (UF) composition can lead to pregnancy loss and likely contributes to offspring susceptibility to chronic adult-onset disorders. However, our understanding of the biochemical composition and mechanisms underpinning UF formation and regulation remain elusive, particularly in humans. To address this challenge, we developed a high-throughput method for intraorganoid fluid (IOF) isolation from human endometrial epithelial organoids. The IOF is biochemically distinct to the extraorganoid fluid (EOF) and cell culture medium as evidenced by the exclusive presence of 17 metabolites in IOF. Similarly, 69 metabolites were unique to EOF, showing asymmetrical apical and basolateral secretion by the in vitro endometrial epithelium, in a manner resembling that observed in vivo. Contrasting the quantitative metabolomic profiles of IOF and EOF revealed donor-specific biochemical signatures of organoids. Subsequent RNA sequencing of these organoids from which IOF and EOF were derived established the capacity to readily perform organoid multiomics in tandem, and suggests that transcriptomic regulation underpins the observed secretory asymmetry. In summary, these data provided by modeling uterine luminal and basolateral fluid formation in vitro offer scope to better understand UF composition and regulation with potential impacts on female fertility and offspring well-being., Competing Interests: The authors declare no competing interest.

Development of viviparity in mammals requires that the placenta evolves as an intermediate interface between the fetus and maternal uterus. In addition to the retention of the fetus and secretion of nutrients to support growth and development to term, it is essential that viviparous species modify or inhibit the maternal immune system from recognizing the semi-allogeneic fetus. Following blastocyst hatching from its zona pellucida, trophoblast differentiation provides the initial communication to the maternal endometrium to regulate maintenance of progesterone production from the corpus luteum and biological pathways in uterine and conceptus development necessary in the establishment and maintenance of pregnancy. Many conceptus factors have been proposed to serve in the establishment and maintenance of pregnancy. CRISPR-Cas9 gene-editing technology provides a specific and efficient method to generate animal models to perform loss-of-function studies to investigate the role of specific conceptus factors. The utilization of CRISPR-Cas9 gene editing has provided a direct approach to investigate the specific role of conceptus factors in the development and establishment of pregnancy in the pig. This technology has helped address a number of questions concerning peri-implantation development and has altered our understanding of maternal recognition and maintenance of pregnancy in the pig.

Bovine endometrium consists of epithelial and stromal cells that respond to conceptus interferon tau (IFNT), the maternal recognition of pregnancy (MRP) signal, by increasing expression of IFN-stimulated genes (ISGs). Endometrial epithelial and stromal-cell-specific ISGs are largely unknown but hypothesized to have essential functions during pregnancy establishment. Bovine endometrial epithelial cells were cultured in inserts above stromal fibroblast (SF) cells for 6 h in medium alone or with IFNT. The epithelial and SF transcriptomic response was analyzed separately using RNA sequencing and compared to a list of 369 DEGs recently identified in intact bovine endometrium in response to elongating bovine conceptuses and IFNT. Bovine endometrial epithelial and SF shared 223 and 70 DEGs in common with the list of 369 endometrial DEGs. Well-known ISGs identified in the epithelial and SF were ISG15, MX1, MX2, and OAS2. DEGs identified in the epithelial but not SF included a number of IRF molecules (IRF1, IRF2, IRF3, and IRF8), mitochondria SLC transporters (SLC25A19, SLC25A28, and SLC25A30), and a ghrelin receptor. Expression of ZC3HAV1, an anti-retroviral gene, increased specifically within the SF. Gene ontology analysis identified the type I IFN signaling pathway and activation of nuclear factor kappa B transcription factors as biological processes associated with the epithelial cell DEGs. This study has identified biologically relevant IFNT-stimulated genes within specific endometrial cell types. The findings provide critical information regarding the effects of conceptus IFNT on specific endometrial compartments during early developmental processes in cattle., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The endometrium is the inner lining of the uterus that undergoes complex regeneration and differentiation during the human menstrual cycle. The process of endometrial shedding, regeneration, and differentiation is driven by ovarian steroid hormones and prepares the endometrium and intrauterine environment for embryo implantation and pregnancy establishment. Endometrial glands and their secretions are essential for pregnancy establishment, and cross talk between the glandular epithelium and stromal cells appears vital for decidualization and placental development. Despite being crucial, the biology of the human endometrium during pregnancy establishment and most of pregnancy is incomplete, given the ethical and practical limitations of obtaining and studying endometrium from pregnant women. As such, in vitro models of the human endometrium are required to fill significant gaps in understanding endometrial biology. This review is focused on the evolution and development of in vitro three-dimensional models of the human endometrium and provides insight into the challenges and promises of those models to improve women's reproductive health., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The surface epithelium of the bovine endometrium comprises at least 2 cell types (ciliated cells and secretory cells with microvilli), but their distribution and morphological changes over the estrous cycle are poorly understood. The objective was to quantify the number of ciliated cells and assess morphological changes in secretory cells on the uterine surface epithelium during the estrous cycle. Caruncular endometrium (CAR) and intercaruncular endometrium (ICAR) samples were collected from the uterine body, the horn ipsilateral to the corpus luteum or dominant follicle (H-CL/DF), and the horn contralateral to the corpus luteum or dominant follicle (H-NCL/NDF) from heifers following slaughter on d 0 (estrus; n = 5) or d 14 (mid-luteal phase; n = 5) of the estrous cycle. Samples were prepared for scanning electron microscopy at 1,000× magnification. Four to 10 fields (256 × 225 µm) for each sample were examined (n = 567 images). The number of ciliated cells was counted and the surface was scored for the morphology of the secretory cells (0 = absence of microvilli on surface; 3 = 100% of surface covered with microvilli). Ciliated cells were present in both the CAR and ICAR regions. The number of ciliated cells per field increased from d 0 to 14 in CAR and decreased from d 0 to14 in ICAR. The scanning electron microscopy revealed a general lack of uniformity in the lawn of microvilli on the surface of the endometrium. Based on the scores, approximately 25% of the fields had a surface that was <50% covered by microvilli. Depletion of microvilli may be explained by a normal process where apical protrusions are formed and either regress back into the cell surface or break to release their contents into the uterine lumen. These studies support the hypothesis that the surface of the luminal epithelium changes during the estrous cycle through a process that involves remodeling of the apical surface. The morphology of the apical surface may have a key role in governing pregnancy establishment., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Glands of the uterus are essential for pregnancy establishment. Forkhead box A2 (FOXA2) is expressed specifically in the glands of the uterus and a critical regulator of glandular epithelium (GE) differentiation, development, and function. Mice with a conditional deletion of FOXA2 in the adult uterus, created using the lactotransferrin iCre (Ltf-iCre) model, have a morphologically normal uterus with glands, but lack FOXA2-dependent GE-expressed genes, such as leukemia inhibitory factor (LIF). Adult FOXA2 conditional knockout (cKO; Ltf iCre/+ Foxa2 f/f ) mice are infertile due to defective embryo implantation arising from a lack of LIF, a critical implantation factor of uterine gland origin. However, intraperitoneal injections of LIF can initiate embryo implantation in the uterus of adult FOXA2 cKO mice with pregnancies maintained to term. Here, we tested the hypothesis that FOXA2-regulated genes in the uterine glands impact development of the decidua, placenta, and fetus. On gestational day 8.5, the antimesometrial and mesometrial decidua transcriptome was noticeably altered in LIF-replaced FOXA2 cKO mice. Viable fetuses were reduced in FOXA2 cKO mice on gestational days 12.5 and 17.5. Sex-dependent differences in fetal weight, placenta histoarchitecture, and the placenta and metrial gland transcriptome were observed between control and FOXA2 cKO mice. The transcriptome of the placenta with a female fetus was considerably more altered than the placenta with a male fetus in FOXA2 cKO dams. These studies reveal previously unrecognized sexually dimorphic effects of FOXA2 and uterine glands on fetoplacental development with potential impacts on offspring health into adulthood., Competing Interests: The authors declare no competing interest.

Maternal nutrient restriction impairs placental growth and development, but available evidence suggests that adaptive mechanisms exist, in a subset of nutrient restricted (NR) ewes, that support normal fetal growth and do not result in intrauterine growth restriction (IUGR). This study utilized Affymetrix GeneChip Bovine and Ovine Genome 1.0 ST Arrays to identify novel placental genes associated with differential fetal growth rates within NR ewes. Singleton pregnancies were generated by embryo transfer and, beginning on Day 35 of pregnancy, ewes received either a 100% National Research Council (NRC) (control-fed group; n = 7) or 50% NRC (NR group; n = 24) diet until necropsy on Day 125. Fetuses from NR ewes were separated into NR non-IUGR ( n = 6) and NR IUGR ( n = 6) groups based on Day 125 fetal weight for microarray analysis. Of the 103 differentially expressed genes identified, 15 were upregulated and 88 were downregulated in NR non-IUGR compared to IUGR placentomes. Bioinformatics analysis revealed that upregulated gene clusters in NR non-IUGR placentomes associated with cell membranes, receptors, and signaling. Downregulated gene clusters associated with immune response, nutrient transport, and metabolism. Results illustrate that placentomal gene expression in late gestation is indicative of an altered placental immune response, which is associated with enhanced fetal growth, in a subpopulation of NR ewes.

An appropriate female reproductive environment is essential for pregnancy success. In several species, including mice, pigs and horses, seminal plasma (SP) components have been shown to modulate this environment, leading to increased embryo viability and implantation. Due to the characteristics of mating in the aforementioned species, SP comes into direct contact with the uterus. However, it is questionable whether any SP reaches the uterus in species that ejaculate inside the vagina, such as humans and cattle. Hence, we hypothesized that sperm, perhaps acting as a vehicle for SP factors, play a more important role in the modulation of the maternal uterine environment in these species. In addition, changes elicited by SP and/or sperm may originate in the vagina and propagate to more distal regions of the female reproductive tract. To test these hypotheses, a bovine model in which heifers were mated to intact or vasectomized bulls or were left unmated was used. RNA-sequencing of endometrial samples collected 24 h after mating with a vasectomized bull did not reveal any differentially expressed genes (DEGs) in comparison with control samples. However, the endometrium of heifers mated with intact bulls exhibited 24 DEGs when compared to heifers mated with vasectomized bulls, and 22 DEGs when compared to unmated control heifers. The expression of a set of cytokines ( IL6 , IL1A , IL8 , and TNFA ) and candidate genes identified in the endometrial RNA-sequencing ( PLA2G10 , CX3CL1 , C4BPA , PRSS2 , BLA-DQB , and CEBPD ) were assessed by RT-qPCR in the vagina and oviductal ampulla. No differences in expression of these genes were observed between treatments in any region. However, mating to both intact and vasectomized bulls induced an increase in IL1A and TNFA expression in the vagina compared to the oviduct. These data indicate that sperm, but not secretions from the accessory glands alone, induce modest changes in endometrial gene expression after natural mating in cattle. However, it is not clear whether this effect is triggered by inherent sperm proteins or SP proteins bound to sperm surface at the time of ejaculation., (Copyright © 2020 Recuero, Sánchez, Mateo-Otero, Bagés-Arnal, McDonald, Behura, Spencer, Kenny, Yeste, Lonergan and Fernandez-Fuertes.)

Heifer conception rate (HCR) is defined as the percentage of inseminated heifers that become pregnant at each service. The genome-wide association analyses in this study focused on identifying the loci associated with Holstein heifer ( n = 2013) conception rate at first service (HCR1) and the number of times bred (TBRD) to achieve a pregnancy. There were 348 unique loci associated ( p < 5 × 10 -8 ) with HCR1 and 615 unique loci associated ( p < 5 × 10 -8 ) with TBRD. The two phenotypes shared 302 loci, and 56 loci were validated in independent cattle populations. There were 52 transcription factor binding sites (TFBS) and 552 positional candidate genes identified in the HCR1- and TBRD-associated loci. The positional candidate genes and the TFBS associated with HCR1 and TBRD were used in the ingenuity pathway analysis (IPA). In the IPA, 11 pathways, 207 master regulators and 11 upstream regulators were associated ( p < 1.23 × 10 -5 ) with HCR1 and TBRD. The validated loci associated with both HCR1 and TBRD make good candidates for genomic selection and further investigations to elucidate the mechanisms associated with subfertility and infertility.

We previously demonstrated that 5'-AMP-activated protein kinase (AMPK) is essential for normal reproductive functions in female mice. Conditional ablation of Prkaa1 and Prkaa2, genes that encode the α1 and α2 catalytic domains of AMPK, resulted in early reproductive senescence, faulty artificial decidualization, uterine inflammation and fibrotic postparturient endometrial regeneration. We also noted a delay in the timing of embryo implantation in Prkaa1/2d/d female mice, suggesting a role for AMPK in establishing uterine receptivity. As outlined in new studies here, conditional uterine ablation of Prkaa1/2 led to an increase in ESR1 in the uteri of Prkaa1/2d/d mice, resulting in prolonged epithelial cell proliferation and retention of E2-induced gene expression (e.g. Msx1, Muc1, Ltf) through the implantation window. Within the stromal compartment, stromal cell proliferation was reduced by five-fold in Prkaa1/2d/d mice, and this was accompanied by a significant decrease in cell cycle regulatory genes and aberrant expression of decidualization marker genes such as Hand2, Bmp2, Fst and Inhbb. This phenotype is consistent with our prior study, demonstrating a failure of the Prkaa1/2d/d uterus to undergo decidualization. Despite these uterine defects, ovarian function seemed to be normal following ablation of Prkaa1/2 from peri-ovulatory follicles in which ovulation, luteinization and serum progesterone levels were not different on day 5 of pregnancy or pseudopregnancy between Prkaa1/2fl/fl and Prkaa1/2d/d mice. These cumulative findings demonstrate that AMPK activity plays a prominent role in mediating several steroid hormone-dependent events such as epithelial cell proliferation, uterine receptivity and decidualization as pregnancy is established.

In cattle, embryo transfer into the uterine horn contralateral to the corpus luteum results in a higher incidence of pregnancy loss compared to transfer into the ipsilateral horn. We have previously reported temporal changes in the endometrial transcriptome during the estrous cycle which differ between uterine horns. The objective of this study was to compare the transcriptomic response of endometrium from the ipsilateral and contralateral horns to an elongating conceptus. Cross-bred beef heifers (n = 16) were synchronized and either used to generate day 14 conceptuses following the transfer of in vitro-produced blastocysts or to obtain day 14 endometrial explants. Conceptuses were recovered on day 14 by post-mortem uterine flushing, placed individually on top of explants collected from the ipsilateral (IPSI-D14) or the contralateral (CONTRA-D14) uterine horn of cyclic heifers, and co-cultured for 6 h. The response to a conceptus was markedly different between uterine horns, with 61 and 239 differentially expressed genes (DEGs; false discovery rate <0.05) in the ipsilateral and contralateral horns, respectively, compared to their controls. Direct comparison between IPSI-D1 and CONTRA-D14 revealed 32 DEGs, including CXCL11, CXCL10, IFIT2, RSAD2 and SAMD9. Gene Ontology analysis of these 32 genes revealed ten enriched biological processes, mainly related to immune response and response to an external stimulus. These data indicate that the endometrial response to the presence of a conceptus varies between uterine horns in the same uterus and may contribute to the higher incidence of pregnancy loss following embryo transfer to the contralateral horn.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

The inability of beef cattle to maintain full term pregnancies has become an economic concern for the beef industry. Herd management and nutritional improvements have alleviated environmental impacts on embryonic and fetal loss, yet additional gains can be made through genomic selection. The objectives of this study were to identify loci and gene-sets in crossbred beef heifers associated with the number of services required to become pregnant (TBRD) and heifer conception rate at first service (HCR1). Heifers (n = 709) from a commercial beef operation underwent one round of artificial insemination, before exposure to bulls for natural service for 50 days. Pregnancy and time of conception was determined by ultrasound 35 days after the breeding season. Heifers were genotyped using the GeneSeek (Lincoln, NE) Bovine GGP50K BeadChip prior to genome-wide association analyses (GWAA) conducted using an EIGENSTRAT-like model to identify loci associated (P < 1 × 10-5) with TBRD and HCR1. One locus was associated (P = 8.97 × 10-6) with TBRD on BTA19 and included the positional candidate gene ASIC2, which is differentially expressed in the endometrium of fertility classified heifers, and the positional candidate gene, SPACA3. Gene-set enrichment analyses using SNP (GSEA-SNP) data, was performed and identified one gene-set, oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen as enriched (NES = 3.15) with TBRD and contained nine leading edge genes that contributed to the enrichment of the gene set. The enriched gene-set is involved in catalyzing oxidation-reduction reactions, which have been associated with oxidative stressors impacting pregnancy success. No loci were associated nor gene-sets enriched with HCR1. Identification of loci, positional candidate genes, gene-sets and leading edge genes enriched for fertility facilitate genomic selection that allows producers to select for reproductively superior cattle, reduce costs associated with infertility, and increase percent calf crop., Competing Interests: The authors have declared that no competing interests exist.

LIN28 inhibits let-7 miRNA maturation which prevents cell differentiation and promotes proliferation. We hypothesized that the LIN28- let-7 axis regulates proliferation-associated genes in sheep trophectoderm in vivo. Day 9-hatched sheep blastocysts were incubated with lentiviral particles to deliver shRNA targeting LIN28 specifically to trophectoderm cells. At day 16, conceptus elongation was significantly reduced in LIN28A and LIN28B knockdowns. Let-7 miRNAs were significantly increased and IGF2BP1-3, HMGA1, ARID3B, and c-MYC were decreased in trophectoderm from knockdown conceptuses. Ovine trophoblast (OTR) cells derived from day 16 trophectoderm are a useful tool for in vitro experiments. Surprisingly, LIN28 was significantly reduced and let-7 miRNAs increased after only a few passages of OTR cells, suggesting these passaged cells represent a more differentiated phenotype. To create an OTR cell line more similar to day 16 trophectoderm we overexpressed LIN28A and LIN28B, which significantly decreased let-7 miRNAs and increased IGF2BP1-3, HMGA1, ARID3B, and c-MYC compared to control. This is the first study showing the role of the LIN28- let-7 axis in trophoblast proliferation and conceptus elongation in vivo. These results suggest that reduced LIN28 during early placental development can lead to reduced trophoblast proliferation and sheep conceptus elongation at a critical period for successful establishment of pregnancy.

Context: Poor uterine receptivity is one major factor leading to pregnancy loss and infertility. Understanding the molecular events governing successful implantation is hence critical in combating infertility., Objective: To define Progesterone Receptor (PGR)-regulated molecular mechanisms and epithelial roles in receptivity., Design: RNA-sequencing and PGR-ChIP-seq were conducted in parallel to identify PGR-regulated pathways during the Window of implantation (WOI) in endometrium of fertile women., Setting: Endometrial biopsies from the proliferative and mid-secretory phases were analyzed., Patients or Other Participants: Participants were fertile, reproductive aged (18-37 years) women with normal cycle length, and without any history of dysmenorrhea, infertility, or irregular cycles. In total, 42 endometrial biopsies obtained from 42 women were analyzed in this study., Interventions: There were no interventions during this study., Main Outcome Measures: Here we measured the alterations in gene expression and PGR occupancy in the genome during the WOI, based on the hypothesis that PGR binds uterine chromatin cycle dependently to regulate genes involved in uterine cell differentiation and function., Results: 653 genes were identified with regulated PGR binding and differential expression during the WOI. These were involved in regulating inflammatory response, xenobiotic metabolism, epithelial mesenchymal transition, cell death, interleukin/Signal Transducer And Activator Of Transcription (STAT) signaling, estrogen response, and Mammalian target of rapamycin complex 1 (MTORC1) response. Transcriptome of the epithelium identified 3052 differentially expressed genes, of which 658 were uniquely regulated. Transcription factors Interferon Regulatory Factor 8 (IRF8) and Myocyte Enhancer Factor 2C (MEF2C) were found to be regulated in the epithelium during the WOI at the protein level, suggesting potentially important functions that are previously unrecognized., Conclusion: PGR binds the genomic regions of genes regulating critical processes in uterine receptivity and function., (Published by Oxford University Press on behalf of the Endocrine Society 2019.)

Survival and growth of the bovine conceptus is dependent on endometrial secretions or histotroph. Previously, serial blastocyst transfer was used to classify heifers as high fertile (HF), subfertile (SF), or infertile (IF). Here, we investigated specific histotroph components (proteins and metabolites) in the uterine lumen of day 17 fertility-classified heifers. Interferon tau (IFNT) was more abundant in uterine lumenal fluid (ULF) of pregnant HF than SF animals as the conceptus was longer in HF heifers. However, no differences in endometrial expression of selected classical and nonclassical interferon-stimulated genes (ISGs) were observed, suggesting that IFNT signaling in the endometrium of pregnant HF and SF heifers was similar. Pregnancy significantly increased the abundance of several proteins in ULF. Based on functional annotation, the abundance of a number of proteins involved in energy metabolism, oxidative stress, amino acid metabolism, and cell proliferation and differentiation were greater in the ULF of pregnant HF than SF heifers. Metabolomics analysis found that pregnancy only changed the metabolome composition of ULF from HF heifers. The majority of the metabolites that increased in the ULF of pregnant HF as compared to SF heifers were associated with energy and amino acid metabolism. The observed differences in ULF proteome and metabolome are hypothesized to influence uterine receptivity with consequences on conceptus development and survival in fertility-classified heifers., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pig conceptuses secrete estrogens (E2), interleukin 1 beta 2 (IL1B2), and prostaglandins (PGs) during the period of rapid trophoblast elongation and establishment of pregnancy. Previous studies established that IL1B2 is essential for rapid conceptus elongation, whereas E2 is not essential for conceptus elongation or early maintenance of the corpora lutea. The objective of the present study was to determine if conceptus expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and release of PG are important for early development and establishment of pregnancy. To understand the role of PTGS2 in conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing PTGS2 using CRISPR/Cas9 technology. Wild-type (PTGS2+/+) and null (PTGS2-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. Immunolocalization of PTGS2 and PG production was absent in cultured PTGS2-/- blastocysts on day 7. PTGS2+/+ and PTGS2-/- blastocysts were transferred into surrogate gilts, and the reproductive tracts were collected on either days 14, 17, or 35 of pregnancy. After flushing the uterus on days 14 and 17, filamentous conceptuses were cultured for 3 h to determine PG production. Conceptus release of total PG, prostaglandin F2⍺ (PGF2α), and PGE in culture media was lower with PTGS2-/- conceptuses compared to PTGS2+/+ conceptuses. However, the total PG, PGF2α, and PGE content in the uterine flushings was not different. PTGS2-/- conceptus surrogates allowed to continue pregnancy were maintained beyond 30 days of gestation. These results indicate that pig conceptus PTGS2 is not essential for early development and establishment of pregnancy in the pig., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Survival and growth of the bovine conceptus (embryo and associated extraembryonic membranes) are dependent on endometrial secretions or histotroph found in the uterine lumen. Previously, serial embryo transfer was used to classify heifers as high fertile (HF), subfertile (SF), or infertile (IF). Here, we investigated specific histotroph components [glucose, prostaglandins (PGs), and lipids] in the uterine lumen of day 17 pregnant and open fertility-classified heifers. Concentrations of glucose in the uterine lumen were increased by pregnancy but did not differ among fertility-classified heifers. Differences in expression of genes encoding glucose transporters and involved with glycolysis and gluconeogenesis were observed between conceptuses collected from HF and SF heifers. In the uterine lumen, PGE2 and PGF2α were increased by pregnancy, and HF heifers had higher concentrations of PGE2, PGF2α, and 6-keto-PFG1α than SF heifers. Differences were found in expression of genes regulating PG signaling, arachidonic acid metabolism, and peroxisome proliferator-activated receptor signaling among conceptuses and endometrium from fertility-classified heifers. Lipidomics was conducted exclusively in samples from HF heifers, and phosphatidylcholine was the main lipid class that increased in the uterine lumen by pregnancy. Expression of several lipid metabolism genes differed between HF and SF conceptuses, and a number of fatty acids were differentially abundant in the uterine lumen of pregnant HF and SF heifers. These results support the ideas that uterine luminal histotroph impacts conceptus survival and programs its development and is a facet of dysregulated conceptus-endometrial interactions that result in loss of the conceptus in SF cattle during the implantation period of pregnancy establishment., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The objectives of this study were to identify loci, positional candidate genes, gene-sets, and pathways associated with spontaneous abortion (SA) in cattle and compare these results with previous human SA studies to determine if cattle are a good SA model for humans. Pregnancy was determined at gestation day 35 for Holstein heifers and cows. Genotypes from 43,984 SNPs of 499 pregnant heifers and 498 pregnant cows that calved at full term (FT) were compared to 62 heifers and 28 cows experiencing SA. A genome-wide association analysis, gene-set enrichment analysis-single nucleotide polymorphism, and ingenuity pathway analysis were used to identify regions, pathways, and master regulators associated with SA in heifers, cows, and a combined population., Results: Twenty-three loci and 21 positional candidate genes were associated ( p < 1 × 10 -5 ) with SA and one of these (KIR3DS1) has been associated with SA in humans. Eight gene-sets (NES > 3.0) were enriched in SA and one was previously reported as enriched in human SA. Four master regulators ( p < 0.01) were associated with SA within two populations., Conclusions: One locus associated with SA was validated and 39 positional candidate and leading-edge genes and 2 gene-sets were enriched in SA in cattle and in humans.

The human endometrium is essential in providing the site for implantation and maintaining the growth and survival of the conceptus. An unreceptive endometrium and disrupted maternal-conceptus interactions can cause infertility due to pregnancy loss or later pregnancy complications. Despite this, the role of uterine glands in first trimester human pregnancy is little understood. An established organoid protocol was used to generate and comprehensively analyze 3-dimensional endometrial epithelial organoid (EEO) cultures from human endometrial biopsies. The derived EEO expand long-term, are genetically stable, and can be cryopreserved. Using endometrium from 2 different donors, EEO were derived and then treated with estrogen (E2) for 2 d or E2 and medroxyprogesterone acetate (MPA) for 6 d. EEO cells were positive for the gland marker, FOXA2, and exhibited appropriate hormonal regulation of steroid hormone receptor expression. Real-time qPCR and bulk RNA-sequencing analysis revealed effects of hormone treatment on gene expression that recapitulated changes in proliferative and secretory phase endometrium. Single-cell RNA sequencing analysis revealed that several different epithelial cell types are present in the EEO whose proportion and gene expression changed with hormone treatment. The EEO model serves as an important platform for studying the physiology and pathology of the human endometrium., Competing Interests: The authors declare no competing interest.

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.