Reversible female contraceptives: historical, current, and future perspectives†.

Contraception is a practice with extensive and complicated social and scientific histories. From cycle tracking, to the very first prescription contraceptive pill, to now having over-the-counter contraceptives on demand, family planning is an aspect of healthcare that has undergone and will continue to undergo several transformations through time. This review provides a comprehensive overview of current reversible hormonal and non-hormonal birth control methods as well as their mechanism of action, safety, and effectiveness specifically for individuals who can become pregnant. Additionally, we discuss the latest Food and Drug Administration (FDA)-approved hormonal method containing estetrol and drospirenone that has not yet been used worldwide as well as the first FDA-approved hormonal over-the-counter progestin-only pills. We also review available data on novel hormonal delivery through microchip, microneedle, and the latest FDA-approved non-hormonal methods such as vaginal pH regulators. Finally, this review will assist in advancing female contraceptive method development by underlining constructive directions for future pursuits. Information was gathered from the NCBI and Google Scholars databases using English and included publications from 1900 to present. Search terms included contraceptive names as well as efficacy, safety, and mechanism of action. In summary, we suggest that investigators consider the side effects and acceptability together with the efficacy of contraceptive candidate towards their development.

Blocking serine protease activity prevents semenogelin degradation leading to hyperviscous semen in humans.

Prostate-specific antigen (PSA) is a prostate-specific serine protease enzyme that hydrolyzes gel-forming proteins (semenogelins) and changes the semen from gel-like to watery viscosity, a process called semen liquefaction. Highly viscous semen and abnormal liquefaction reduce sperm motility and contribute to infertility. Previously, we showed that nonspecific serine protease inhibitor (AEBSF) prevented proteolytic degradation of semenogelin in mice. However, it is unclear whether similar effect could be recapitulated in fresh human ejaculates. Therefore, in this study we evaluated the effect of AEBSF on the degradation of semenogelin (SEMG1) and its subsequent impact on semen liquefaction and sperm motility in fresh semen ejaculates collected from healthy men. We found that AEBSF showed a dual contraceptive action where it effectively 1) prevented degradation of SEMG1 resulting in viscous semen and 2) decreased sperm motility in human semen samples. However, the impact of AEBSF on sperm motility and viability could be due to its inhibitory activity toward other serine proteases or simply due to its toxicity. Therefore, to determine whether inhibition of PSA activity alone could disrupt SEMG1 degradation and contribute to hyperviscous semen, a neutralizing PSA antibody was used. We found that PSA antibody effectively prevented SEMG1 degradation with a subtle impact on sperm motility. These findings suggest that the target inhibition of PSA activity can prevent proteolytic degradation of SEMG1 and block liquefaction process, resulting in hyperviscous semen. As it is currently unknown if blocking semen liquefaction alone could prevent pregnancy, it needs further extensive studies before drawing any translational conclusions.

Cell-type specific analysis of physiological action of estrogen in mouse oviducts.

One of the endogenous estrogens, 17ß-estradiol (E2 ) is a female steroid hormone secreted from the ovary. It is well established that E2 causes biochemical and histological changes in the uterus. However, it is not completely understood how E2 regulates the oviductal environment in vivo. In this study, we assessed the effect of E2 on each oviductal cell type, using an ovariectomized-hormone-replacement mouse model, single-cell RNA-sequencing (scRNA-seq), in situ hybridization, and cell-type-specific deletion in mice. We found that each cell type in the oviduct responded to E2 distinctively, especially ciliated and secretory epithelial cells. The treatment of exogenous E2 did not drastically alter the transcriptomic profile from that of endogenous E2 produced during estrus. Moreover, we have identified and validated genes of interest in our datasets that may be used as cell- and region-specific markers in the oviduct. Insulin-like growth factor 1 (Igf1) was characterized as an E2 -target gene in the mouse oviduct and was also expressed in human fallopian tubes. Deletion of Igf1 in progesterone receptor (Pgr)-expressing cells resulted in female subfertility, partially due to an embryo developmental defect and embryo retention within the oviduct. In summary, we have shown that oviductal cell types, including epithelial, stromal, and muscle cells, are differentially regulated by E2 and support gene expression changes, such as growth factors that are required for normal embryo development and transport in mouse models. Furthermore, we have identified cell-specific and region-specific gene markers for targeted studies and functional analysis in vivo.

Mechanism of semen liquefaction and its potential for a novel non-hormonal contraception†.

Semen liquefaction is a proteolytic process where a gel-like ejaculated semen becomes watery due to the enzymatic activity of prostate-derived serine proteases in the female reproductive tract. The liquefaction process is crucial for the sperm to gain their motility and successful transport to the fertilization site in Fallopian tubes (or oviducts in animals). Hyperviscous semen or failure in liquefaction is one of the causes of male infertility. Therefore, the biochemical inhibition of serine proteases in the female reproductive tract after ejaculation is a prime target for novel contraceptive development. Herein, we will discuss protein components in the ejaculates responsible for semen liquefaction and any developments of contraceptive methods in the past that involve the liquefaction process.

Serine protease inhibitor disrupts sperm motility leading to reduced fertility in female mice†.

Inhibition of the sperm transport process in the female reproductive tract could lead to infertility. We previously showed that a pan-serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), blocked semen liquefaction in vivo and resulted in a drastic decrease in the number of sperm in the oviduct of female mice. In this study, we used a mouse model to test the efficacy of AEBSF as a reversible contraceptive, a sperm motility inhibitor, and a spermicide. Additionally, this study evaluated the toxicity of AEBSF on mouse vaginal tissues in vivo and human endocervical cells in vitro. We found that female mice treated with AEBSF had significantly less pups born per litter as well as fertilization rates in vivo compared to the vehicle control. We then showed that AEBSF reduced sperm motility and fertilization capability in vitro in a dose-dependent manner. Furthermore, AEBSF also exhibited spermicidal effects. Lastly, AEBSF treatment in female mice for 10 min or 3 consecutive days did not alter vaginal cell viability in vivo, similar to that of the vehicle and non-treated controls. However, AEBSF decreased cell viability of human ectocervical (ECT) cell line in vitro, suggesting that cells in the lower reproductive tract in mice and humans responded differently to AEBSF. In summary, our study showed that AEBSF can be used as a prototype compound for the further development of novel non-hormonal contraceptives for women by targeting sperm transport in the female reproductive tract.

Roles of steroid hormones in oviductal function

The oviduct (known as the fallopian tube in humans) is the site for fertilization and pre-implantation embryo development. Female steroid hormones, estrogen and progesterone, are known to modulate the morphology and function of cells in the oviduct. In this review, we focus on the actions of estrogen and progesterone on secretory, ciliated, and muscle cell functions and morphologies during fertilization, pre-implantation embryo development, and embryo transport in humans, laboratory rodents and farm animals. We review some aspects of oviductal anatomy and histology and discuss current assisted reproductive technologies (ARTs) that bypass the oviduct and their effects on embryo quality. Lastly, we review the causes of alterations in secretory, ciliated, and muscle cell functions that could result in embryo transport defects.

AMPK is required for uterine receptivity and normal responses to steroid hormones.

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.

Negative elongation factor is essential for endometrial function.

Pausing of RNA polymerase II (Pol II) during early transcription, mediated by the negative elongation factor (NELF) complex, allows cells to coordinate and appropriately respond to signals by modulating the rate of transcriptional pause release. Promoter proximal enrichment of Pol II occurs at uterine genes relevant to reproductive biology; thus, we hypothesized that pausing might impact endometrial response by coordinating hormonal signals involved in establishing and maintaining pregnancy. We deleted the NELF-B subunit in the mouse uterus using PgrCre (NELF-B UtcKO). Resulting females were infertile. Uterine response to the initial decidual stimulus of NELF-B UtcKO was similar to that of control mice; however, subsequent full decidual response was not observed. Cultured NELF-B UtcKO stromal cells exhibited perturbances in extracellular matrix components and also expressed elevated levels of the decidual prolactin Prl8a2, as well as altered levels of transcripts encoding enzymes involved in prostaglandin synthesis and metabolism. Because endometrial stromal cell decidualization is also critical to human reproductive health and fertility, we used small interfering to suppress NELF-B or NELF-E subunits in cultured human endometrial stromal cells, which inhibited decidualization, as reflected by the impaired induction of decidual markers PRL and IGFBP1. Overall, our study indicates NELF-mediated pausing is essential to coordinate endometrial responses and that disruption impairs uterine decidual development during pregnancy.-Hewitt, S. C., Li, R., Adams, N., Winuthayanon, W., Hamilton, K. J., Donoghue, L. J., Lierz, S. L., Garcia, M., Lydon, J. P., DeMayo, F. J., Adelman, K., Korach, K. S. Negative elongation factor is essential for endometrial function.

Crucial role of estrogen for the mammalian female in regulating semen coagulation and liquefaction in vivo.

Semen liquefaction changes semen from a gel-like to watery consistency and is required for sperm to gain mobility and swim to the fertilization site in the Fallopian tubes. Kallikrein-related peptidases 3 (KLK3) and other kallikrein-related peptidases from male prostate glands are responsible for semen liquefaction by cleaving gel-forming proteins (semenogelin and collagen). In a physiological context, the liquefaction process occurs within the female reproductive tract. How seminal proteins interact with the female reproductive environment is still largely unexplored. We previously reported that conditional genetic ablation of Esr1 (estrogen receptor α) in the epithelial cells of the female reproductive tract (Wnt7aCre/+;Esr1f/f) causes female infertility, partly due to a drastic reduction in the number of motile sperm entering the oviduct. In this study, we found that post-ejaculated semen from fertile wild-type males was solidified and the sperm were entrapped in Wnt7aCre/+;Esr1f/f uteri, compared to the watery semen (liquefied) found in Esr1f/f controls. In addition, semenogelin and collagen were not degraded in Wnt7aCre/+;Esr1f/f uteri. Amongst multiple gene families aberrantly expressed in the absence of epithelial ESR1, we have identified that a lack of Klks in the uterus is a potential cause for the liquefaction defect. Pharmacological inhibition of KLKs in the uterus replicated the phenotype observed in Wnt7aCre/+;Esr1f/f uteri, suggesting that loss of uterine and seminal KLK function causes this liquefaction defect. In human cervical cell culture, expression of several KLKs and their inhibitors (SPINKs) was regulated by estrogen in an ESR1-dependent manner. Our study demonstrates that estrogen/ESR1 signaling in the female reproductive tract plays an indispensable role in normal semen liquefaction, providing fundamental evidence that exposure of post-ejaculated semen to the suboptimal microenvironment in the female reproductive tract leads to faulty liquefaction and subsequently causes a fertility defect.

Conditional knockout mice for the distal appendage protein CEP164 reveal its essential roles in airway multiciliated cell differentiation.

Multiciliated cells of the airways, brain ventricles, and female reproductive tract provide the motive force for mucociliary clearance, cerebrospinal fluid circulation, and ovum transport. Despite their clear importance to human biology and health, the molecular mechanisms underlying multiciliated cell differentiation are poorly understood. Prior studies implicate the distal appendage/transition fiber protein CEP164 as a central regulator of primary ciliogenesis; however, its role in multiciliogenesis remains unknown. In this study, we have generated a novel conditional mouse model that lacks CEP164 in multiciliated tissues and the testis. These mice show a profound loss of airway, ependymal, and oviduct multicilia and develop hydrocephalus and male infertility. Using primary cultures of tracheal multiciliated cells as a model system, we found that CEP164 is critical for multiciliogenesis, at least in part, via its regulation of small vesicle recruitment, ciliary vesicle formation, and basal body docking. In addition, CEP164 is necessary for the proper recruitment of another distal appendage/transition fiber protein Chibby1 (Cby1) and its binding partners FAM92A and FAM92B to the ciliary base in multiciliated cells. In contrast to primary ciliogenesis, CEP164 is dispensable for the recruitment of intraflagellar transport (IFT) components to multicilia. Finally, we provide evidence that CEP164 differentially controls the ciliary targeting of membrane-associated proteins, including the small GTPases Rab8, Rab11, and Arl13b, in multiciliated cells. Altogether, our studies unravel unique requirements for CEP164 in primary versus multiciliogenesis and suggest that CEP164 modulates the selective transport of membrane vesicles and their cargoes into the ciliary compartment in multiciliated cells. Furthermore, our mouse model provides a useful tool to gain physiological insight into diseases associated with defective multicilia.

Extracellular Vesicles and the Oviduct Function.

In mammals, the oviduct (or the Fallopian tube in humans) can be divided into the infundibulum (responsible for oocyte pick-up), ampulla (site of fertilization), isthmus (where preimplantation embryos develop), and uterotubal junction (where embryos transit to the uterus). The oviductal fluid, as well as extracellular vesicles produced from the oviduct epithelial cells, referred to as oEVs, have been shown to improve the fertilization process, prevent polyspermy, and aid in embryo development. oEVs contain molecular cargos (such as miRNAs, mRNAs, proteins, and lipids) that can be delivered and fuse to recipient cells. oEVs produced from the ampulla appear to be functionally distinct from those produced from the isthmus. In multiple species including mice, cats, dogs, pigs, and cows, oEVs can be incorporated into the oocytes, sperm, and embryos. In this review, we show the positive impact of oEVs on gamete function as well as blastocyst development and how they may improve embryo quality in in vitro conditions in an assisted reproductive technology setting for rodents, domestic animals, farm animals, and humans.

Estrogen receptor α is required for oviductal transport of embryos.

Newly fertilized embryos spend the first few days within the oviduct and are transported to the uterus, where they implant onto the uterine wall. An implantation of the embryo before reaching the uterus could result in ectopic pregnancy and lead to maternal death. Estrogen is necessary for embryo transport in mammals; however, the mechanism involved in estrogen-mediated cellular function within the oviduct remains unclear. In this study, we show in mouse models that ciliary length and beat frequency of the oviductal epithelial cells are regulated through estrogen receptor α (ESR1) but not estrogen receptor ß (ESR2). Gene profiling indicated that transcripts in the WNT/ß-catenin (WNT/CTNNB1) signaling pathway were regulated by estrogen in mouse oviduct, and inhibition of this pathway in a whole oviduct culture system resulted in a decreased embryo transport distance. However, selective ablation of CTNNB1 from the oviductal ciliated cells did not affect embryo transport, possibly because of a compensatory mechanism via intact CTNNB1 in the adjacent secretory cells. In summary, we demonstrated that disruption of estrogen signaling in oviductal epithelial cells alters ciliary function and impairs embryo transport. Therefore, our findings may provide a better understanding of etiology of the ectopic pregnancy that is associated with alteration of estrogen signals.-Li, S., O'Neill, S. R. S., Zhang, Y., Holtzman, M. J., Takemaru, K.-I., Korach, K. S., Winuthayanon, W. Estrogen receptor α is required for oviductal transport of embryos.

Uterine epithelial cell estrogen receptor alpha-dependent and -independent genomic profiles that underlie estrogen responses in mice.

Estrogens exert their activity through estrogen receptor alpha (ERalpha) to stimulate hypertrophy and hyperplasia in the uterus. A uterine epithelial ERalpha conditional knockout mouse model (Wnt7a(Cre+);Esr1(f/f) or cKO) demonstrated that ERalpha in the epithelial cells was dispensable for an initial uterine proliferative response to 17beta-estradiol (E2) but required for subsequent uterine biological responses. This study aimed to characterize the differential gene expression patterns induced by E2 in the presence or absence of epithelial ERalpha. RNA microarray analysis revealed that approximately 20% of the genes differentially expressed at 2 h were epithelial ERalpha independent, as they were preserved in the cKO uteri. This indicates that early uterine transcripts mediated by stromal ERalpha are sufficient to promote initial proliferative responses. However, more than 90% of the differentially expressed transcripts at 24 h were not regulated in the cKO, indicating that the majority of later transcriptional regulation required epithelial ERalpha, especially those involved in mitosis. This shows that loss of regulation of these later transcripts results in blunted subsequent uterine growth after 3 days of E2 treatment. Additionally, progesterone's ability to inhibit E2-induced epithelial cell proliferation was impaired, consistent with a uterine receptivity defect that contributes to cKO infertility. These transcriptional profiles correlate with our previously observed biological responses, in which the initial proliferative response is independent of epithelial ERalpha and thus dependent on stromal ERalpha, yet epithelial ERalpha is essential for subsequent tissue responsiveness.

Hormonal regulation of cilia in the female reproductive tract.

This review intends to bridge the gap between our knowledge of steroid hormone regulation of motile cilia and the potential involvement of the primary cilium focusing on the female reproductive tract functions. The review emphasizes hormonal regulation of the motile and primary cilia in the oviduct and uterus. Steroid hormones including estrogen, progesterone, and testosterone act through their cognate receptors to regulate the development and biological function of the reproductive tracts. These hormones modulate motile ciliary beating and, in some cases, primary cilia function. Dysfunction of motile or primary cilia due to genetic anomalies, hormone imbalances, or loss of steroid hormone receptors impairs mammalian fertility. However, further research on hormone modulation of ciliary function, especially in the primary cilium, and its signaling cascades will provide insights into the pathogenesis of mammalian infertility and the development of contraceptives or infertility treatments targeting primary and/or motile cilia.

Deletion of Kallikrein-related peptidases ( Klks ) has no effect on fertility in mice.

Kallikreins (KLKs) are serine peptidases. It was established that Klks are estrogen-target genes in mouse uteri. However, the functional requirement of KLK family in the uterine function during reproduction is unknown. Here we generated a compound deletion of Klk1b3, Klk1b4, Klk1b5, and Klk1 in a mouse model using CRISPR/Cas9 strategy with four single guide RNAs (sgRNAs) to target the second exon of these four genes that are aligned back-to-back in a single locus spanning 32.95 kb on chromosome 7. We found that both male and female knockout mice are fertile with no apparent health defect compared to wild-type controls. Our data suggest that Klk1b3, Klk1b4, Klk1b5, and Klk1 are not necessary for male and female reproductive function in mice.

Multi-omics analyses and machine learning prediction of oviductal responses in the presence of gametes and embryos.

The oviduct is the site of fertilization and preimplantation embryo development in mammals. Evidence suggests that gametes alter oviductal gene expression. To delineate the adaptive interactions between the oviduct and gamete/embryo, we performed a multi-omics characterization of oviductal tissues utilizing bulk RNA-sequencing (RNA-seq), single-cell RNA-sequencing (scRNA-seq), and proteomics collected from distal and proximal at various stages after mating in mice. We observed robust region-specific transcriptional signatures. Specifically, the presence of sperm induces genes involved in pro-inflammatory responses in the proximal region at 0.5 days post-coitus (dpc). Genes involved in inflammatory responses were produced specifically by secretory epithelial cells in the oviduct. At 1.5 and 2.5 dpc, genes involved in pyruvate and glycolysis were enriched in the proximal region, potentially providing metabolic support for developing embryos. Abundant proteins in the oviductal fluid were differentially observed between naturally fertilized and superovulated samples. RNA-seq data were used to identify transcription factors predicted to influence protein abundance in the proteomic data via a novel machine learning model based on transformers of integrating transcriptomics and proteomics data. The transformers identified influential transcription factors and correlated predictive protein expressions in alignment with the in vivo-derived data. In conclusion, our multi-omics characterization and subsequent in vivo confirmation of proteins/RNAs indicate that the oviduct is adaptive and responsive to the presence of sperm and embryos in a spatiotemporal manner.

Generation of Oviductal Glycoprotein 1 Cre Mouse Model for the Study of Secretory Epithelial Cells of the Oviduct.

The epithelial cell lining of the oviduct plays an important role in oocyte pickup, sperm migration, preimplantation embryo development, and embryo transport. The oviduct epithelial cell layer comprises ciliated and nonciliated secretory cells. The ciliary function has been shown to support gamete and embryo movement in the oviduct, yet secretory cell function has not been well characterized. Therefore, our goal was to generate a secretory cell-specific Cre recombinase mouse model to study the role of the oviductal secretory cells. A knock-in mouse model, Ovgp1Cre:eGFP, was created by expressing Cre from the endogenous Ovgp1 (oviductal glycoprotein 1) locus, with enhanced green fluorescent protein (eGFP) as a reporter. EGFP signals were strongly detected in the secretory epithelial cells of the oviducts at estrus in adult Ovgp1Cre:eGFP mice. Signals were also detected in the ovarian stroma, uterine stroma, vaginal epithelial cells, epididymal epithelial cells, and elongated spermatids. To validate recombinase activity, progesterone receptor (PGR) expression was ablated using the Ovgp1Cre:eGFP; Pgrf/f mouse model. Surprisingly, the deletion was restricted to the epithelial cells of the uterotubal junction (UTJ) region of Ovgp1Cre:eGFP; Pgrf/f oviducts. Deletion of Pgr in the epithelial cells of the UTJ region had no effect on female fecundity. In summary, we found that eGFP signals were likely specific to secretory epithelial cells in all regions of the oviduct. However, due to a potential target-specific Cre activity, validation of appropriate recombination and expression of the gene(s) of interest is absolutely required to confirm efficient deletion when generating conditional knockout mice using the Ovgp1Cre:eGFP line.

Uterine epithelial estrogen receptor α is dispensable for proliferation but essential for complete biological and biochemical responses.

Female fertility requires estrogen to specifically stimulate estrogen receptor α (ERα)-dependent growth of the uterine epithelium in adult mice, while immature females show proliferation in both stroma and epithelium. To address the relative roles of ERα in mediating estrogen action in uterine epithelium versus stroma, a uterine epithelial-specific ERα knockout (UtEpiαERKO) mouse line was generated by crossing Esr mice with Wnt7a-Cre mice. Expression of Wnt7a directed Cre activity generated selective deletion of ERα in uterine epithelium, and female UtEpiαERKO are infertile. Herein, we demonstrate that 17ß-estradiol (E(2))-induced uterine epithelial proliferation was independent of uterine epithelial ERα because DNA synthesis and up-regulation of mitogenic mediators were sustained in UtEpiαERKO uteri after E(2) treatment. IGF-1 treatment resulted in ligand-independent ER activation in both wild-type (WT) and UtEpiαERKO and mimicked the E(2) stimulatory effect on DNA synthesis in uterine epithelium. Uterine epithelial ERα was necessary to induce lactoferrin, an E(2)-regulated secretory protein selectively synthesized in the uterine epithelium. However, loss of uterine epithelial ERα did not alter the E(2)-dependent progesterone receptor (PR) down-regulation in epithelium. Strikingly, the uterine epithelium of UtEpiαERKO had robust evidence of apoptosis after 3 d of E(2) treatment. Therefore, we surmise that estrogen induced uterine hyperplasia involves a dispensable role for uterine epithelial ERα in the proliferative response, but ERα is required subsequent to proliferation to prevent uterine epithelial apoptosis assuring the full uterine epithelial response, illustrating the differential cellular roles for ERα in uterine tissue and its contribution during pregnancy.

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.