Progesterone receptor in uterine glands is required for pregnancy establishment in mice.

Expression of the PGR was dynamic in the uterine epithelium throughout the estrous cycle, early pregnancy, and after parturition during the postpartum period ( Fig. S1 ). Immunofluorescence analysis revealed PGR mainly in the GE on gestational day (GD) 1, whereas on GD 2, PGR was detected in both LE and GE. PGR expression was maximal in the LE on GD 3 before declining on GD 4. By GDs 6 and 8, immunoreactive PGR was essentially undetectable in both the LE and GE at the embryo implantation site ( Fig. 1A and Fig. S1 ). To specifically delete Pgr in the GE, Pgr f/f mice ( 24 ) were crossed with Prss29-Cre mice( 25 ). Cre recombinase activity in Prss29-Cre mice is initiated in the GE on GD 4 resulting in complete recombination and deletion of genes by GD 8( 25 ). Immunofluorescence analysis of PGR and FOXA2 confirmed an 85% GE-specific PGR conditional knockout in Prss29 Cre/Cre Pgr f/f ( Pgr gecKO) primiparous females by GD 4 ( Fig. 1B - C and Fig. S2 ). In contrast and as expected, PGR was detected in the GE of GD 4 uteri from nulliparous Pgr gecKO mice that had never experienced pregnancy ( Fig. S3 ). A 6-month breeding trial with wild-type males of proven fertility found that Pgr gecKO females exhibited progressive subfertility compared to control female mice. Control mice had more ( p < 0.01) litters and pups per litter. Notably, Pgr gecKO females exhibited a parity-dependent decline in fertility, producing an average of 1.2 ± 1.82 pups per litter from the second litter onward, compared to 6.55 ± 2.77 pups in controls ( Fig. 1D - F ) with no difference in inter-birth interval (data not shown). After the 6-month breeding trial, uteri were removed at diestrus. No apparent differences in histoarchitecture of the uterus or FOXA2-positive glands were found between the uterus of Pgr gecKO and control females ( Fig. 1G and Fig. S4 ), indicating that PGR expression in the GE is crucial for fertility but does not affect GE cell abundance and morphology. To determine the nature of the fertility defect in mice with GE ablation of PGR, mice were mated to males of proven fertility. Both control and Pgr gecKO mice displayed normal mating behavior, as determined by the presence of postcopulatory vaginal plugs. By the second pregnancy, a reduction in embryo implantation sites was evident by GD 6 in the uterus of Pgr gecKO compared to control mice ( Fig. 2A - B ), consistent with the parity-dependent subfertility observed in the breeding trial. The embryo implantation sites in primiparous Pgr gecKO mice were not different from control mice ( Fig. 2C ). Implanted embryos in Pgr gecKO mice were positioned on the antimesometrial (AM) side of the uterus, and LE removal observed on the lateral sides of implanting embryos with noticeable stromal cell decidualization ( Fig. 2C ). Expression of prostaglandin endoperoxide synthase two (PTGS2), a marker of stromal cell decidualization( 31 ), was present in decidual cells adjacent to implanting embryos in both control and Pgr gecKO uteri ( Fig. 2C ). Likewise, secondary decidual zone (SDZ) formation was evident at the implantation sites based on Ki67 expression. To assess stromal cell decidualization, control and Pgr gecKO mice were subjected to an artificial decidualization protocol. The uterine horn of primiparous control and Pgr gecKO mice exhibited robust deciduoma formation in response to the stimulus ( Fig. 2D - E ). Of note, Bmp2 and Wnt4 , key downstream targets of P4 signaling in the stroma that are critical for stromal cell decidualization( 32 , 33 ), were expressed normally in Pgr gecKO mice at GD 6 implantation sites and in artificially induced decidual stromal cells ( Fig. S5 and Fig. S6 ). These findings suggest that the fertility defect observed in Pgr gecKO female mice is due to defects in embryo implantation. Successful implantation requires both a competent embryo and a receptive uterus, with uterine receptivity primarily regulated by the ovarian steroid hormones E2 and P4 acting through their respective nuclear receptors, ESR1 and PGR( 16 , 18 , 20 , 34 ). Immunostaining of GD 4 first pregnancy uteri found no differences in the spatiotemporal distribution of ESR1 in the LE, GE, and stroma between control and Pgr gecKO mice ( Fig. 3A ). To coordinate uterine receptivity, P4 must inhibit E2 induced proliferation in the uterine epithelium( 22 , 35 ). No differences in cell proliferation, based on Ki67 expression, were detected in the LE and stromal cells of Pgr gecKO mice on GD 4 ( Fig. 3B ). Of note, a small number of cells around the GE were Ki67 positive in Pgr gecKO uterus, but not in the GE of control GD 4 uteri ( Fig. 3B ). Next, transcriptome analysis (RNA-seq) was performed using RNA extracted from the entire uterus of control and Pgr gecKO mice on GD 4 during their second pregnancy. The RNA-seq analysis identified (Log2FC > 1 , p < 0.05) only 87 differentially expressed genes (DEGs) in Pgr gecKO as compared to control mice ( Fig. 3C and Table S1 ). The 43 decreased genes were enriched in pathways related to embryo implantation, humoral immune response, and B cell mediated immunity, whereas the 44 increased genes were associated with negative regulation of hormone and protein secretion, acute inflammatory response, and membrane export processes ( Fig. 3C ). The DEGs were then interrogated for PGR binding sites using an existing ChIP-seq data( 18 ). That analysis identified 32 DEGs (e.g., Hp , Lif, Ttr, Lcn2, Clca1, Spink1, Itgam ) which contained PGR binding sites and thus are potentially regulated by activated PGR. Further integration with a uterine epithelial-specific transcriptome dataset from a previous study ( 36 ) found that 19 of the 87 DEGs were expressed in the GE on GD 4 (e.g., Hp, Lif, Ttr, Lipf, Lcn2, Prss29, Prss28, Spink1, Wfdc3 ) ( Fig. 4D -E). Leukemia inhibitory factor (LIF) is essential for blastocyst implantation in mice and transiently induced in the GE on GD 4 in response to E2 from the ovaries( 7 , 9 ). Expression of Lif and several GE-specific PGR responsive genes ( Prss29 , Prss28 , Spink1 ) were also markedly reduced in the Pgr gecKO uterus( 37 - 39 ). In situ hybridization confirmed the decrease in both Lif and Prss29, but not Foxa2 , in the Pgr gecKO uterus ( Fig. 3F ). This data suggests that the decrease in Lif is not due to a reduction in GE cell number or alterations in FOXA2 ( Fig. 3F ). Interestingly, in situ hybridization analysis found reduced Indian hedgehog ( Ihh) mRNA specifically in the GE of Pgr gecKO mice ( Fig. S7 ). Ihh is expressed in the LE and GE on GD 4( 40 ). The reduction in Ihh was not detected in the bulk RNA-seq analysis ( Fig. 3C and Table S1 ), likely because the LE is a much more abundant cell type than the GE in the mouse uterus ( Fig. S7 ). Of note, Ihh is a P4-responsive gene crucial for PGR-mediated communication between uterine epithelia and stroma during early pregnancy( 41 ). To evaluate whether the absence of LIF on GD 4 in Pgr gecKO mice was the underlying cause of embryo implantation failure, two intraperitoneal injections of recombinant mouse LIF were administered to primiparous Pgr gecKO females on GD 4( 12 ). Embryo implantation was initially evaluated on GDs 6 and 8 ( Fig. 4A - B and Fig S9A - B ). The LIF-treated Pgr gecKO mice had ( p > 0.10) comparable numbers of embryo implantation sites to that observed in control mice. In the LIF-injected Pgr gecKO mice, the implantation site was surrounded by PTGS2-positive decidualized stromal cells. No differences were observed in cell proliferation (Ki67) or apoptosis (Cleaved Caspase-3) between control and LIF-treated Pgr gecKO mice ( Fig. 4C and Fig. S8 ). Additionally, there was no difference in ESR1 expression in the stroma ( Fig. S8 ), which is critical for decidualization and embryo implantation( 13 ). To confirm the absence of progesterone signaling in the LIF-treated mice with implantation sites, the progesterone-responsive gene SPINK1 was assessed by IF on GD 8( 39 ). In Pgr gecKO mice, SPINK1 abundance was below the detection limit, whereas controls showed robust SPINK1 expression in the GE. The results confirm the absence of PGR signaling in the GE of LIF-rescued mice ( Fig. 4D ). To further interrogate pregnancy status in the LIF-rescued Pgr gecKO mice ultrasound imaging was conducted on GD 8 and 12 ( Fig. S10A ). The number and appearance of implantation sites detected on GDs 8 and 12 were indistinguishable between control and LIF-treated Pgr gecKO mice. LIF-treated Pgr gecKO mice remained pregnant and delivered the same number of pups as controls at term ( Fig. S10B ). Moreover, LIF supplementation was able to rescue a third pregnancy from those same Pgr gecKO mice ( Fig. S10B ).

All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Missouri, Columbia, and were conducted according to the NIH Guide for the Care and Use of Laboratory Animals. Floxed Pgr ( Pgr f/f ) mice ( 24 ) were crossed with Prss29 Cre mice ( 25 ) to generate conditional knockout (cKO) animals. Pgr ( Pgr f/f ) were generously provided by Dr. Francesco Demayo (National Institute of Environmental Health Sciences, Durham, North Carolina) and Dr. John Lydon (Baylor College of Medicine, Houston, Texas). Gestational time points were obtained by the mating 8- to 10-week-old females with males of proven fertility (observation of vaginal plug was considered GD 1). At necropsy, uteri were excised, trimmed of fat, and then frozen in liquid nitrogen or fixed with 4% paraformaldehyde. Eight-week-old control and Prss29 Cre/Cre Pgr f/f ( Pgr gecKO) females (n=5 per genotype) were housed individually and continuously with a male of proven fertility. Litter frequency and size was evaluated for 6 months. Primiparous control (n=5) or Pgr gecKO (n=5) female mice were ovariectomized. After two weeks of rest, 100 ng of E2 was injected s.c. for three consecutive days. After two days, mice were injected s.c. daily with 1 mg P4 and 6.7 ng E2 for three days. At 6 h post-last hormone injection, one uterine horn was surgically exposed. A 25-guage needle was inserted into the uterine lumen, and the antimesometrial side of the lumen was scraped using the needle to provide a decidualization stimulus. The contralateral horn was not scraped as a control. Daily hormone treatments (1 mg P4 and 6.7 ng E2 s.c.) were continued for 5 days. At necropsy, gross morphology of uterus was recorded along with the weight of the scraped (stimulated) and unscraped (unstimulated) uterine horn. A portion of each uterine horn was fixed in 4% paraformaldehyde for histology or snap frozen in liquid nitrogen and stored at −80°C for RNA analyses. Briefly, fixed and paraffin-embedded uteri were sectioned at 5 μm, mounted on slides, and baked for 30 min at 60°C. Sections were then deparaffinized in xylene, and rehydrated to water through a graded alcohol series. Antigen retrieval was performed by incubating sections in 10 mM citrate buffer (pH 6.0) at 95°C for 15 min followed by cooling to room temperature. All sections were blocked with 2.5% (v/v) normal goat serum in PBS (pH 7.2) at room temperature for 30 min and incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: rabbit monoclonal anti-FOXA2 (1:800; Abcam; Cat# ab108422), rabbit monoclonal anti-PGR (1:500; Invitrogen; Cat# MA5-14505), rabbit polyclonal anti-ESR1 (1:2000; Abcam; Cat# ab3575), rabbit polyclonal anti-Ki67 (1:500; Abcam; Cat# ab15580), rabbit monoclonal anti-PTGS2 (1:500; Abcam; Cat# ab179800), rat monoclonal anti-cytokeratin 8 (1:100; EMD millipore; Cat# MABT329), rabbit polyclonal anti-CASP3 (1:500; Cell Signaling Technology; Cat# 9661), and mouse anti-E-Cadherin (1:500; Biosciences; Cat# 610182). Visualization by immunofluorescence was performed with Alexa 488, Alexa 594, or Alexa 647-conjugated secondary antibodies (Jackson ImmunoResearch, #112-545-143, #115-585-46, #111-605-144) at a 1:500 dilution in PBS for 60 min at room temperature. Sections were counterstained with Hoechst 33342 (2 μg/mL; Invitrogen, H3570) before affixing coverslips with ProLong ™ Diamond Antifade Mountant (Invitrogen, 36961). Images were acquired with a Leica DM6 B upright microscope and Leica K8 camera using Leica Application Suite X (LAS X). In situ hybridization analysis was performed to visualize the spatial localization of mRNA. Uteri from control and Pgr gecKO were fixed in 4% PFA, embedded in paraffin, sectioned (5 μm), and mounted on positively charged slides (n=3 genotype/day). In situ hybridization was performed following ACD Bio’s guide for the RNAscope ® Multiplex Fluorescent V2 Assay kit (ACD Bio, 323270) with probes for Lif (ACD Bio, 475841-C1), Ihh ( ACD Bio, 1259141-C1), Prss29 (ACD Bio, 1110301-C2), and Foxa2 (ACD Bio, 409111-C3). Slides were baked for 1 h at 60°C, deparaffinized in xylene, and rehydrated in 100% ethanol. Deparaffinized sections were subjected to target antigen retrieval by incubating sections in RNAscope ® target antigen retrieval solution (ACD Bio, 322000) at 95°C for 15 min, followed by hydrogen peroxidase (ACD Bio, 322335) incubation at 37°C for 10 min. Sections were treated with protease plus (ACD Bio, 322331) at 40°C for 30 min then probes were hybridized at 40°C for 2 h. Slides were washed with RNAscope ® wash buffer (ACD Bio, 310091) and subjected to a series of incubations with multiple reagents in the RNAscope ® Multiplex Fluorescent Detection Reagent V2 kit (ACD Bio, 323110) and TSA vivids fluorophores (1:1500 dilution) were assigned to desired channels. Sections were stained with DAPI (ACD Bio, 323110) before affixing a coverslip with ProLong ™ Diamond Antifade Mountant (Invitrogen, 36961). Images were acquired with a Leica DM6 B upright microscope and Leica K8 camera using Leica Application Suite X (LAS X). Uteri were collected and pooled at necropsy and flash-frozen in liquid nitrogen (n = 2 biological replicates per genotype). Total RNA was extracted using RNeasy ® Plus Mini Kit protocol (Qiagen 74134). Quantity and purity of total RNA were determined using a Nanodrop spectrophotometer (Fisher, 840274200) and Qubit ® 3.0 Fluorometer (Invitrogen, Q33216 ). RNA integrity, library preparation, and sequencing were conducted at Azenta Life Sciences (South Plainfield, NJ, USA) (n=2 per genotype). RNA integrity was checked using TapeStation (Agilent Technologies). Libraries were prepared using the NEBNext Ultra II RNA Library Prep for Illumina and sequenced (2 x 150 bp paired end) using Illumina instrument (4000 or equivalent). Raw sequence data (.bcl files) generated from Illumina Hiseq was converted into fastq files and de-multiplexed using Illumina’s bcl2fastq 2.17 software. Raw data quality was assessed, and reads were trimmed with Trimmomatic (v0.39) to remove adapters and low-quality bases. Trimmed reads were aligned to the ENSEMBL genome (GRCm39) using STAR (v2.7.11b), and unique exon-aligned gene counts were obtained with featureCounts (v1.5.3). Gene hit counts were analyzed for differential expression using DESeq2( 28 ),with the Wald test generating p-values and Log2 fold changes. Differentially expressed genes (DEGs) were defined as those with adjusted p-values 1. Enriched GO processes were further explored using SRplot( 29 ). DEGs were integrated with publicly available PGR ChIP-seq dataset that was generated with ovariectomized mice treated with P4 for 6 h ( GSM5393542 )( 18 ). Peak-associated genes were identified with GREAT (Genomic Regions Enrichment of Annotations Tool) (v4.0.4)( 30 ) to determine PGR-regulated genes using the threshold of 100kbp extension in both directions to the nearest gene’s TSS. All mouse experiments were performed using at least three biological replicates. For histological analyses, three tissue sections were examined per mouse. Quantification of PGR-positive cells was performed with three biological and three technical replicates. Cell quantification was done manually using Leica DM6 B upright microscope and Leica K8 camera using Leica Application Suite X (LAS X). Litter size, litter number, and number of implantation sites between controls and Prss29 Cre/Cre Pgr f/f were compared using a student t test. A p value less than 0.05 and 0.01 was defined as statistically significant.

Collectively, the studies described here establish that PGR in the GE is essential for embryo implantation, pregnancy establishment and fertility in mice. A major biological role of PGR in the GE is to regulate nidatory Lif expression in the endometrium during the peri-implantation period. This finding aligns with earlier reports that conditional knockout of PGR in the entire uterine epithelium using Pgr-Cre , Wnt7a-Cre or Ltf - Cre mice, as well as overexpression of PGR, caused infertility due to defects in embryo implantation and/or stromal cell decidualization( 18 , 21 , 22 , 42 ). Uterine glands coordinate essential events required for pregnancy establishment( 4 , 43 ). Indeed, mice that lack uterine glands [conditional deletion of Foxa2 , Ctnbb1 , Lef1 , Wnt4 , Wnt5a , or Wnt7a , or progesterone-induced uterine gland ablation (PUGKO)] uniformly exhibit impaired or markedly reduced nidatory Lif expression in the GE accompanied by defects in embryo implantation and/or stromal cell decidualization( 11 , 44 - 48 ). PGR signaling in the uterus has long been recognized as a key regulator of epithelial-stromal crosstalk necessary for establishing uterine receptivity for embryo implantation( 14 , 20 , 23 , 49 ). Conditional ablation of PGR in uterine epithelial cells using Ltf-Cre and Wnt7a-Cre revealed that loss of PGR alters the expression of genes ( Ihh, Ptch, Nr2f2 ) involved in epithelium-stromal communication and the expression of Lif during the acquisition of endometrial receptivity( 14 , 18 , 20 - 24 ). In the present study, PGR loss specifically in the GE did not disrupt epithelial-stromal communication, allowing for the inhibition of LE cell proliferation and showing no detectable changes in ESR1 or PGR expression in the stroma, LE or GE. Moreover, Bmp2 and Wnt4 , essential downstream effectors of PGR-IHH-NR2F2 signaling in decidualization( 32 , 33 , 50 ), were normally induced in Pgr gecKO mice. These findings underscore cell type-specific functions of PGR in the GE during the establishment of pregnancy. Of note, uterine histoarchitecture was not altered in GE Pgr gecKO mice, thus PGR function in the GE is not essential for epithelial homeostasis after puberty, but rather essential for the uterus to support pregnancy establishment. Integration of our transcriptomic data with publicly available datasets ( 18 , 36 ) revealed PGR-regulated, GE-enriched genes ( Lif , Prss28 , Prss29 , Spink1 ) downregulated in Pgr gecKO uteri. Mice lacking Prss28 and Prss29 are fertile with no obvious uterine defects, whereas the biological role of Spink1 has not yet been determined( 39 , 51 ). LIF was the first GE-derived factor shown to be essential for murine pregnancy and is transiently induced by the nidatory estrogen surge in the GE on GD 4( 7 - 9 ). Mice lacking LIF are infertile due to defective uterine receptivity, blastocyst implantation, and stromal cell decidualization. Notably, the epithelial PGR target Ihh was not differentially expressed in the dataset, likely owing to its continued expression in the LE despite the reduction in the GE population( 52 , 53 ). This observation highlights the need for single-cell RNA sequencing to determine the biological impact of targeted cell type-specific knockouts in low abundant cell population. Conditional ablation of Ihh in the uterus also leads to infertility and unopposed E2 signaling in the uterine epithelium( 52 ). The absence of increased epithelial proliferation in the GE-specific PGR deletion supports the idea that gland-derived IHH is dispensable for regulation of GE and LE proliferation. Both ESR1 and FOXA2 have known roles in regulating Lif expression in the glands of the uterus in response to nidatory E2 from the ovaries( 7 , 12 , 13 , 54 ); however expression of those transcription factors were not altered in the GE of Pgr gecKO mice in the present study. This observation suggests a novel role for the PGR in E2 induction of Lif expression in the GE during early pregnancy. Interestingly, constitutive PGR expression in the uterine epithelium reduced Lif expression in the GE by blocking ESR1 occupancy at the Lif promoter( 18 ). In that same study, while exogenous LIF rescued early implantation, multiple perturbations were noted in later pregnancy( 18 ). Here, in Pgr gecKO mice, exogenous LIF administration rescued successive pregnancies, indicating that PGR, together with ESR1 and FOXA2 signaling, plays a critical role in the positive regulation of Lif . Because Pgr is also expressed in the glands of postpartum uteri, repeated rescue of pregnancy in Pgr gecKO mice suggests that postpartum regeneration and involution are not affected in the absence of glandular Pgr . In addition to mediating endometrial receptivity, LIF is critical for stromal decidualization, and studies have demonstrated that LIF regulates uterine decidualization by inducing a paracrine mechanism involving the production of IHH from the LE( 34 ). Unlike other LIF-deficient models, Pgr gecKO mice here did not exhibit defects in stromal cell decidualization. It is possible that a small number of GE cells retaining PGR may express an amount of Lif sufficient to sustain the Ihh expression in the LE, thereby supporting post-attachment decidualization. Collectively, these observations suggest that the primary biological role of PGR in the GE is to enable E2 induction of Lif expression in the GE during the peri-implantation period. Indeed, other pregnancy process appear to be independent of GE PGR function. Taken together, our results provide evidence that PGR in the glands is essential for establishing uterine receptivity for embryo implantation, which is primarily mediated by effects on nidatory Lif expression in the GE. The broader implications of these findings may extend to reproductive pathologies, where reduced responsiveness to progesterone leads to impaired decidualization and glandular dysfunction. In this regard, both the GE-specific transcription factor, FOXA2, as well as LIF are reduced in the endometrium of women with endometriosis, which may result from gland dysfunction and defects in PGR signaling in endometriosis( 55 - 57 ). Endometrial cancer arises from the uterine glands and loss of PGR expression correlates with increased proliferation, invasion, and poor prognosis( 58 , 59 ). Thus, understanding the mechanisms controlled by PGR in the glands not only provides insights into pregnancy biology but may also uncover potential therapeutic targets for improving fertility outcomes and treating progesterone-associated reproductive diseases.

The uterus is comprised of two tissue compartments, the endometrium and the myometrium. The endometrium contains diverse cell types, luminal epithelium (LE), glandular epithelium (GE), stromal, immune, and endothelial cells( 1 , 2 ). Coordinated, cell type specific interactions among these endometrial cell types are essential for establishing uterine receptivity and pregnancy. The GE of the uterus have recognized biological roles in pregnancy establishment in both mice and humans( 3 - 6 ). Leukemia inhibitory factor ( Lif ) is expressed during the window of uterine receptivity on gestational day (GD) 4 specifically in the GE in response to estrogen produced by the ovaries, and LIF was the first cytokine found to be required for embryo implantation in mice( 7 - 9 ). The infertility phenotype of Lif null mice, as well as studies of mice and sheep lacking uterine glands, supports a principal role for uterine gland-derived factors in pregnancy establishment and maintenance( 10 - 13 ). Establishment of a receptive uterus for embryo implantation and subsequent pregnancy maintenance is governed by the cell type specific actions of the ovarian steroid hormones estrogen (E2) and progesterone (P4)( 14 - 16 ). Estrogen, acting through estrogen receptor alpha (ESR1), stimulates uterine epithelial cell proliferation during the early pre-implantation period and induces LIF production and secretion from the GE, which is essential for embryo implantation and thus establishment of pregnancy in mice( 8 , 9 , 17 ). Progesterone, acting through the progesterone receptor (PGR), prepares the uterine epithelium for embryo implantation by inhibiting epithelial proliferation prior to embryo attachment, and directing epithelial-stromal crosstalk for stromal cell decidualization and post-implantation embryo development( 18 - 22 ). Indeed, uterine epithelial ablation of PGR using Ltf-Cre and Wnt7a-Cre mouse models altered the expression of genes involved in pregnancy establishment, including Lif ( 14 , 18 , 20 - 24 ). Although conditional knockout (cKO) mouse models have demonstrated essential roles for P4-PGR signaling in both the epithelium and stroma, a cell type specific role for PGR in the GE remains undefined. In the present study, we conditionally deleted Pgr in the GE of the uterus using the Prss29-Cre mouse model( 25 ). Prss29, also known as implantation serine proteinase 2, is expressed specifically and abundantly in GE of the mouse uterus particularly after embryo implantation ensues( 26 , 27 ). This genetic approach effectively ablates PGR in the GE after embryo implantation, avoiding any effects of PGR loss during development of the uterus before puberty. Ablation of PGR in the GE of the adult uterus did not impact success of the first pregnancy; however, a decline in fertility was observed in subsequent pregnancies. Transcriptomic and histologic analyses of early pregnant uteri revealed that loss of PGR signaling in the GE reduced expression of classical PGR target genes, as well as peri-implantation Lif expression on GD 4. Intraperitoneal injections of recombinant LIF on GD 4 fully restored fertility in mice lacking PGR in the GE by eliciting embryo implantation and pregnancy success to term. Taken together, these findings provide direct in vivo evidence that PGR within the GE is required for peri-implantation Lif expression, but PGR does not have an essential role in GE function after embryo implantation and during the rest of pregnancy.