Protocol for the establishment and characterization of an endometrial-derived epithelial organoid and stromal cell co-culture system.

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.

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

In cattle, mating to intact, but not vasectomised, bulls has been shown to modify 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. Therefore, 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 (co-)incubated for 6 h alone (control), or with epididymal sperm or ejaculated sperm, following which transcriptomic changes in the endometrium were evaluated. Epididymal sperm elicited a more dramatic endometrial response than ejaculated sperm, in terms of the number of differentially expressed genes (DEGs). Indeed, RNA-sequencing data analysis revealed 1912 DEGs in endometrial explants exposed to epididymal sperm compared with control explants, whereas 115 DEGs were detected between endometrial explants exposed to ejaculated sperm in comparison to control explants. The top pathways associated with genes upregulated by epididymal sperm included T cell regulation and TNF, NF-KB and IL17 signalling. Interestingly, ejaculated sperm induced downregulation of genes associated with T cell immunity and Th17 differentiation, and upregulation of genes involved in NF-KB signalling, in comparison to epididymal sperm. These data indicate that factors of AG origin modulate the interaction between sperm and the endometrium in cattle.

Estrogen receptor alpha regulates uterine epithelial lineage specification and homeostasis.

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.

Basolateral secretions of human endometrial epithelial organoids impact stromal cell decidualization.

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.

Gene editing provides a tool to investigate genes involved in reproduction of pigs.

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.

The Estrogen Receptor α Cistrome in Human Endometrium and Epithelial Organoids.

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.

Prss29 Cre recombinase mice are useful to study adult uterine gland function.

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.

Notch effector recombination signal binding protein for immunoglobulin kappa J signaling is required for the initiation of endometrial stromal cell decidualization†.

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.

Progesterone Signaling in Endometrial Epithelial Organoids.

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.

Impact of preovulatory estradiol concentrations on subsequent luteal function in beef cattle.

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 (n = 53). Cows that exhibited estrus before gonadotrophin-releasing hormone (GnRH)-induced ovulation (d 0) had greater (p<.01) peak concentrations of estradiol compared with cows that did not express estrus (11.5 ± 0.8 vs. 6.2 ± 0.6 pg/mL), respectively, but there was no difference in ovulatory follicle size (p= .80) or interval from GnRH2 to ovulation (p=.23). Circulating concentrations of progesterone during luteal formation (d 3-7; p=.70 and p=.77) or mid-luteal phase (d 8-14; p=.39 and p=.12) were not affected by elevated periovulatory estradiol or an interaction with day. To investigate the direct influence of estradiol on luteal function, ovulation (d 0) was synchronized in nonlactating beef cows and cows were allocated to three groups (control, n = 5; vehicle injection, n = 4; or an estradiol antagonist (Fulvestrant; ICI 182,780), n = 4. Intrafollicular injection of vehicle (100 µL) or an estradiol antagonist (25 µg Fulvestrant in 100 µL) into the largest follicle occurred on d -2. Concentrations of estradiol increased (p<.0001) from d -2 to 0 but did not differ among groups (p>.50). Furthermore, plasma concentrations of progesterone on d 0 through 20 were not affected by treatment (p=.86). These results indicate that elevated preovulatory estradiol before ovulation was not required to prepare granulosa cells for luteinization or subsequent luteal progesterone secretion but did tend to impact luteal lifespan.

Uterine lumen fluid is metabolically semi-autonomous.

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.

Conceptus interferon gamma is essential for establishment of pregnancy in the pig†.

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.

Uterine glands impact embryo survival and stromal cell decidualization in mice.

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.

Generation and analysis of Prss28 and Prss29 deficient mice using CRISPR-Cas9 genome-editing.

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.

Capture and metabolomic analysis of the human endometrial epithelial organoid secretome.

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.

Insights into the lipidome and primary metabolome of the uterus from day 14 cyclic and pregnant sheep†.

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.

Gene editing to investigate the role of conceptus factors in the establishment of pregnancy in the pig.

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.

Conceptus-induced, interferon tau-dependent gene expression in bovine endometrial epithelial and stromal cells†.

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.

In vitro models of the human endometrium: evolution and application for women's health.

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.

Sexually dimorphic effects of forkhead box a2 (FOXA2) and uterine glands on decidualization and fetoplacental development.

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; LtfiCre/+Foxa2f/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.