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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.

It has been hypothesized that circulating concentrations of estradiol during the preovulatory period, can impact subsequent progesterone concentrations. Ovulation was synchronized in nonlactating beef cows (

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.

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.

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. Summary sentence Through utilization of delayed implantation, SPP1 mRNA and protein are localized in high levels to the luminal epithelium of interimplantation sites, to focal regions of the implantation chamber and to uterine natural killer cells in the decidua, suggesting roles for implantation in mice.

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.

CRISPR-Cas9 gene editing technology provides a method to generate loss-of-function studies to investigate, in vivo, the specific role of specific genes in regulation of reproduction. With proper design and selection of guide RNAs (gRNA) designed to specifically target genes, CRISPR-Cas9 gene editing allows investigation of factors proposed to regulate biological pathways involved with establishment and maintenance of pregnancy. The advantages and disadvantages of using the current gene editing technology in a large farm species is discussed. CRISPR-Cas9 gene editing of porcine conceptuses has generated new perspectives for the regulation of endometrial function during the establishment of pregnancy. The delicate orchestration of conceptus factors facilitates an endometrial proinflammatory response while regulating maternal immune cell migration and expansion at the implantation site is essential for establishment and maintenance of pregnancy. Recent developments and use of endometrial epithelial "organoids" to study endometrial function in vitro provides a future method to screen and target specific endometrial genes as an alternative to generating a gene edited animal model. With continuing improvements in gene editing technology, future researchers will be able to design studies to enhance our knowledge of mechanisms essential for early development and survival of the conceptus.

All mammalian uteri contain glands in their endometrium that develop only or primarily after birth. In mice, those endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantation, stromal cell decidualization, and placental development. Here, we describe a new uterine glandular epithelium (GE) specific Cre recombinase mouse line that is useful for the study of uterine gland function during pregnancy. Utilizing CRISPR-Cas9 genome editing, Cre recombinase was inserted into the endogenous serine protease 29 precursor (Prss29) gene. Both Prss29 mRNA and Cre recombinase activity was specific to the GE of the mouse uterus following implantation, but was absent from other areas of the female reproductive tract. Next, Prss29-Cre mice were crossed with floxed forkhead box A2 (Foxa2) mice to conditionally delete Foxa2 specifically in the endometrial glands. Foxa2 was absent in the glands of the post-implantation uterus, and Foxa2 deleted mice exhibited complete infertility after their first pregnancy. These results establish that Prss29-Cre mice are a valuable resource to elucidate and explore the functions of glands in the adult uterus.

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.Summary sentence: There are dynamic changes in the lipid and metabolite composition of the uterine lumen during early pregnancy in sheep.

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.Summary sentenceConceptus-induced, interferon-tau-dependent genes were identified within bovine endometrial epithelial and stromal fibroblast cells providing critical information regarding conceptus-induced modifications to specific endometrial compartments in cattle.

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

An estimated 75% of unsuccessful pregnancies are due to implantation failure. Investigating the causes of implantation failure is difficult as decidualization and embryo implantation is a dynamic process. Here we describe a new decidua specific iCre recombinase mouse strain. Utilizing CRISPR/Cas9-based genome editing, a mouse strain was developed that expresses iCre recombinase under the control of the endogenous prolactin family 8, subfamily a, member 2 (Prl8a2) promoter. iCre recombinase activity was examined by crossing with mTmG or Sun1-GFP reporter alleles. iCre activity initiated reporter expression at gestational day 5.5 in the primary decidual zone and continued into mid-gestation (gestational day 9.5), with expression highly concentrated in the anti-mesometrial region. No reporter expression was observed in the ovary, oviduct, pituitary, or skeletal muscle, supporting the tissue specificity of the Prl8a2iCre in the primary decidual zone. This novel iCre line will be a valuable tool for in vivo genetic manipulation and lineage tracing to investigate functions of genetic networks and cellular dynamics associated with decidualization and infertility.

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.

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.

We conducted an integrated analysis of gene expression and chromatin structure of mouse uterus to understand the regulation of uterine-expressed genes on gestation day 4 (GD4) during the peri-implantation period. The genes expressed in the uterus showed a significant association (p < .0001) with the presence of the nucleosome-free region (open chromatin) in the 5′-untranslated region of the genes. The majority of these upstream open chromatins harbored a common class of regulatory elements known as upstream open reading frames. We also compared the gene expression profiles between the uterus and brain which showed that specific gene pairs were expressed in a correlated manner, either positively or negatively. In addition, specific ligand/receptor genes showed coordinated patterns of expression between the uterus and brain on GD4, and the level of expression of these ligand/receptors altered significantly in the brain during late pregnancy (GD15) compared with the peri-implantation period (GD4). Collectively, these results suggest that regulation of the uterine genes during the peri-implantation period is likely to have a functional link with the maternal brain in pregnant mice.

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.

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

Establishment of viviparity in mammals evolved through not only the long-term retainment of the fetus within the maternal uterus but differentiation and expansion of cell layers to form functional membranes to exchange O

In comparison to many other mammalian species, ruminant ungulates have a unique form of placentation. Ruminants initially display an epitheliochorial type of placentation; however, during the period of placental attachment, trophoblast giant binucleate cells (BNC) develop within the chorion to migrate and fuse with the uterine surface epithelium to form syncytial plaques. Binucleate cell migration and fusion continues throughout pregnancy but never appears to breach the basal lamina, beneath the uterine surface or luminal epithelium. Therefore, the semi-invasive type of placentation in ruminants is classified as synepitheliochorial. The endometrium of ruminant species also contains unique specialized aglandular structures termed "caruncles" in which the chorioallantois (cotyledons) interdigitates and forms highly vascularized fetal-maternal "placentomes." This chapter will discuss the current knowledge of early conceptus development during the peri-attachment period, establishment of pregnancy, conceptus attachment, and placentation in ruminant ungulates. The features of placentomes, BNCs, fetomaternal hybrid cells, and multinucleated syncytial plaques of the cotyledonary placenta of ruminant species will be reviewed to highlight the unique form of placentation compared to the placentae of other artiodactyls.

The ASAS Public Policy Committee (PPC) provides updates of Grand Challenges (GCs) which clearly articulate research priorities while providing science-based information for shaping public policy and enhancing future funding for research and education programs in animal sciences (AS). Among the GCs is reproduction of domesticated animals (cattle, swine, sheep, goats, poultry, horses, and aquatic species) that 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 identify 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, has 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 (NIH) and USDA National Institute of Food and Agriculture (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. The “Dual Purpose with Dual Benefit” program encouraged One Health approaches for comparative medicine studies that use farm animal models that mimic human developmental, physiological, and etiological processes to promote human and animal health, better understand disease origins, interspecies transmission and mitigation strategies, and improve efficiency of assisted reproduction technologies. This presentation will review the successes of the 9-year Dual Purpose effort and highlight opportunities for tackling GC research in reproduction of domesticated agricultural animals.

Accurate sire fertility evaluation remains a significant challenge in the livestock industry. Sire conception rate (SCR) has been used since 2008 in the dairy industry to rank sire fertility. The objective was to characterize individual sire pregnancy rate, pregnancy loss, and determine the correlation with the sire’s respective SCR. Our main hypothesis is that large variances in fertility parameters by sire cannot be explained by variance of the SCR values. Data from 6,570 timed artificial inseminations (AI) and 25,287 timed embryo transfers (ET) from 55 dairy farms were retrospectively analyzed to assess: pregnancy rate at day 30 (P30), pregnancy rate at day 60 (P60) and pregnancy loss (PL) during this interval. The effect of sire (AI: n = 39; ET: n = 81) for each reproductive parameter was evaluated. SCR records published prior to individual service were obtained to correlate with fertility parameters. For AI mating, pregnancy rate at P30 was 33.13% (2,177/6,570), and 27.6 % for P60 (1,815/6,570) and PL occurred in 16.6% (362/2,177) of pregnancies. Large variance was observed between sires for each parameter but no or negligible correlation (r < 0.2) with published SCR was observed. For ET, pregnancy rate at P30 was 47.8% (12,082/25,287) and 40.5% for P60 (10,246/25,287) with an overall PL of 15.2% (1,836/12,082). Similar to AI, all fertility parameters were highly variable among sires but no or negligible correlation (r < 0.03) with respective SCR was observed. In summary, the current method to evaluate sire fertility using SCR does not truly represent the field fertility status. Large variance in pregnancy loss between days 30 and 60 of gestation were observed among sires and these phenotypes should be considered when evaluating sire fertility to increase the score reliability.

Uterine glands are essential for the establishment of pregnancy and have critical roles in endometrial receptivity to blastocyst implantation, stromal cell decidualization, and placentation. Uterine gland dysfunction is considered a major contributing factor to pregnancy loss, however our understanding of how glands impact embryo survival and stromal cell decidualization is incomplete. Forkhead box A2 (FOXA2) is expressed only in the glandular epithelium and regulates its development and function. Mice with a conditional deletion of FOXA2 in the uterus are infertile due to defective embryo implantation arising from a lack of leukemia inhibitory factor (LIF), a critical factor of uterine gland origin. Here, a glandless FOXA2-deficient mouse model, coupled with LIF repletion to rescue the implantation defect, was used to investigate the roles of uterine glands in embryo survival and decidualization. Studies found that embryo survival and decidualization were compromised in glandless FOXA2-deficient mice on gestational day 6.5, resulting in abrupt pregnancy loss by day 7.5. These findings strongly support the hypothesis that uterine glands secrete factors other than LIF that impact embryo survival and stromal cell decidualization for pregnancy success.

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.

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.

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.

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.

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.

All mammalian uteri contain glands in the endometrium that develop only or primarily after birth. Gland development or adenogenesis in the postnatal uterus is intrinsically regulated by proliferation, cell–cell interactions, growth factors and their inhibitors, as well as transcription factors, including forkhead box A2 (FOXA2) and estrogen receptor α (ESR1). Extrinsic factors regulating adenogenesis originate from other organs, including the ovary, pituitary, and mammary gland. The infertility and recurrent pregnancy loss observed in uterine gland knockout sheep and mouse models support a primary role for secretions and products of the glands in pregnancy success. Recent studies in mice revealed that uterine glandular epithelia govern postimplantation pregnancy establishment through effects on stromal cell decidualization and placental development. In humans, uterine glands and, by inference, their secretions and products are hypothesized to be critical for blastocyst survival and implantation as well as embryo and placental development during the first trimester before the onset of fetal–maternal circulation. A variety of hormones and other factors from the ovary, placenta, and stromal cells impact secretory function of the uterine glands during pregnancy. This review summarizes new information related to the developmental biology of uterine glands and discusses novel perspectives on their functional roles in pregnancy establishment and success.

The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1−/−) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1−/− embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1−/− embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1−/− conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1−/− embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1−/− gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1−/− embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .

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.

All mammalian uteri contain glands that synthesize or transport and secrete substances into the uterine lumen. Uterine gland development, or adenogenesis, is uniquely a postnatal event in sheep and pigs and involves differentiation of glandular epithelium from luminal epithelium, followed by invagination and coiling morphogenesis throughout the stroma. Intrinsic transcription factors and extrinsic factors from the ovary and pituitary as well as the mammary gland (lactocrine) regulate uterine adenogenesis. Recurrent pregnancy loss is observed in the ovine uterine gland knockout sheep, providing unequivocal evidence that glands and their products are essential for fertility. Uterine gland hyperplasia and hypertrophy during pregnancy are controlled by sequential actions of hormones from the ovary and/or pituitary as well as the placenta. Gland-derived histotroph is transported by placental areolae for fetal growth. Increased knowledge of uterine gland biology is expected to improve pregnancy outcomes, as well as the health and productivity of mothers and their offspring.

The placenta plays a pivotal role in the development of the fetal brain and also influences maternal brain function, but our understanding of communication between the placenta and brain remains limited. Using a gene expression and network analysis approach, we provide evidence that the placenta transcriptome is tightly interconnected with the maternal brain and fetal brain in d 15 pregnant C57BL/6J mice. Activation of serotonergic synapse signaling and inhibition of neurotrophin signaling were identified as potential mediators of crosstalk between the placenta and maternal brain and fetal brain, respectively. Genes encoding specific receptors and ligands were predicted to affect functional interactions between the placenta and brain. Paralogous genes, such as sex comb on midleg homolog 1/scm-like with 4 mbt domains 2 and polycomb group ring finger (Pcgf) 2/ Pcgf5, displayed antagonistic regulation between the placenta and brain. Additionally, conditional ablation of forkhead box a2 ( Foxa2) in the glands of the uterus altered the transcriptome of the d 15 placenta, which provides novel evidence of crosstalk between the uterine glands and placenta. Furthermore, expression of cathepsin 6 and monocyte to macrophage differentiation associated 2 was significantly different in the fetal brain of Foxa2 conditional knockout mice compared with control mice. These findings provide a better understanding of the intricacies of uterus-placenta-brain interactions during pregnancy and provide a foundation and model system for their exploration.-Behura, S. K., Kelleher, A. M., Spencer, T. E. Evidence for functional interactions between the placenta and brain in pregnant mice.

Glands of the uterus are essential for the establishment of pregnancy in mice and their products regulate embryo implantation and stromal cell decidualization critical for pregnancy establishment. Forkhead box A2 (FOXA2) is expressed specifically in the glands and a critical regulator of their differentiation, development and function. Progesterone and FOXA2 regulate members of a serine proteinase gene family (Prss28 and Prss29). Here, CRISPR-Cas9 genome-editing was used to create mice with a heterozygous or homozygous deletion of Prss28 or/and Prss29 to determine their biological roles in uterine function. Female mice lacking Prss28 and Prss29 or both developed normally and were fertile without alterations in uterine histoarchitecture, uterine gland number, or and gene expression. Thus, Prss28 and Prss29 are dispensable for female fertility and do not impact endometrial gland development or uterine function mice.