Yes Associated Transcriptional Regulator 1 (YAP1) and WW Domain Containing Transcription Regulator (WWTR1) are required for murine pregnancy initiation

Progesterone (Pgr) Cre expressing male mice were crossed to double floxed females ( Yap f/f Wwtr1 f/f ) to generate heterozygous founders. Offspring were then backcrossed to generate Pgr Cre/+ Yap f/f Wwtr1 f/f females for fertility investigation. The resulting mice were genotyped as Pgr Cre/+ Yap f/+ Wwtr1 f/f (pdKO) or Pgr +/+ Yap f/f Wwtr1 f/f but never as Pgr Cre/+ Yap f/f Wwtr1 f/f . Investigation into the Mouse Genome Informatics database revealed that Progesterone and Yap1 are approximately 900kB or 0cM apart, and therefore, it was impossible to generate a full double knockout model utilizing the Pgr Cre . The investigation continued into the role of Yap and Wwtr1 in the murine reproductive tract utilizing the partial double knockout model. Uteri and ovaries were assessed for Cre-mediated recombination of the Yap1 and Wwtr1 alleles ( Figure 1 ). At 3.5dpc, neither Yap1 nor Wwtr1 were differentially expressed in uteri utilizing primers that amplify the floxed exon 3, but the target gene Ccn2 was significantly decreased (unpaired t-test p =0.032, Figure 1A – D ). However, at 5.5dpc, Wwtr1 was significantly decreased in pdKO uteri (unpaired t-test p =0.017, Figure 1B ). Immunohistochemical analysis indicated epithelium and stromal compartment-specific expression of YAP1 and WWTR1 in uteri at 3.5dpc, however expression levels were not significantly different between controls and pdKOs ( Figure 1E – F ). Evidence of Yap1 and Wwtr1 and target gene depletion was not evident in whole ovaries ( Supplementary Figure 1A ). In addition, both YAP1 and WWTR1 protein expression was not significantly different in granulosa cells of 3.5dpc control and pdKO ovaries ( Supplementary Figure 1B ). In addition to these molecular analyses, no structural differences were observed in the ovaries, oviducts, and uteri of virgin females at proestrus or 3.5dpc ( Supplementary Figure 2A – B ). Pgr Cre/+ Yap fl/+ Wwtr1 fl/fl partial double knockouts (pdKO) produced significantly fewer total pups throughout a 6-month breeding trial (n=4, Mixed-effects analysis factor genotype, p =0.006, Figure 2A ). In addition, pdKO females had smaller litter sizes than controls (unpaired t-test p =0.0007, Figure 2B ). The pdKO mice bore approximately 50% fewer pups and litters than the floxed controls throughout the breeding trial ( Supplementary Table 3 ). In addition, the pdKO mice displayed a reduced capacity to produce pups as time progressed (Mantel-Cox test p= 0.007, Figure 2C ). One individual only produced two live-born litters when placed with multiple fertile males ( Figure 2D ). In addition to the subfertility shown throughout the breeding trial, pdKO females in an expanded cohort exhibited significantly reduced first and second litter sizes (Mixed effects analysis factor genotype, p =0.0026, Supplementary Figure 3A ). This was also associated with increased inter-litter timing (Mixed effects analysis factor genotype p <0.0001, factor litter number p =0.0032, Supplementary Figure 3B ). Despite increased inter-litter timing, pdKO body weight measured over an 8-week period matched that of the wild types ( Supplementary Figure 3C ). At the termination of the 6-month breeding trial, n=3 mice per genotype were collected for analyses. These mice did not display any significant differences in body, uterine, or ovarian weight at the time of collection (not shown), and no alterations in reproductive tract structure were observed grossly nor with histological analyses ( Supplementary Figure 2C ). Artificial decidualization was maintained for 5 days, and then uteri were collected for analysis. Representative images show unstimulated (left) and stimulated (right) horns for floxed controls and pdKOs ( Figure 3A ). The uterine wet weight ratio of stimulated to unstimulated horn was not different in the pdKO group compared to controls ( Figure 3B ). However, mRNA expression of decidualization response genes, Wnt4 and Bmp2 , indicated a blunted decidualization response in the stimulated uterine horns of pdKOs compared to controls (Two-way ANOVA p =0.04 Wnt4 , and p =0.006 Bmp2 , Figure 3C ). Pregnancy was assessed in control and pdKO mice at various time points including implantation (4.5dpc, n=6–8), post-implantation (5.5dpc, n=6–10), early pregnancy (7.5dpc, n=5–7), placentation initiation (9.5dpc, n=5–7), and post-placentation (12.5dpc, n=8–10) ( Figure 4A – B ). The gross morphology of the uteri was normal at all time points except 12.5dpc when pdKO mice exhibited many resorption sites ( Figure 4A ). Quantification of normal implantation sites, when assessed by blue dye reaction, revealed variance in phenotype. At 4.5dpc, the time of implantation, pdKO females were split with 50% of mice with positive implantation sites with blue dye injection and 50% of mice without positive implantation sites ( Figure 4B ). However, by 5.5dpc, most pdKO females had positive implantation sites with numbers comparable to controls ( Figure 4B ). At 7.5dpc and 9.5dpc, the number of mice with positive implantation sites decreased to ~40%, with ~60% not having positive implantation sites at 9.5dpc ( Figure 4B ). At 12.5dpc, embryonic loss was evident with visually identifiable resorption sites ( Figure 4B ). In addition, across all time points measured, pdKO females exhibited a higher likelihood of being not pregnant compared to controls (33% of individuals, Fisher’s exact test p <0.0001, Figure 4C ). Determination of the cause of pregnancy failure prompted the investigation into ovulation and fertilization rates and embryo transport. Post-fertilization at 1.5dpc, when all ovulated oocytes should be in the oviducts, we noted comparable numbers of unfertilized oocytes (UFO) and 2-cell embryos in pdKOs and controls ( Figure 5A ). However, at 3.5dpc, the time of embryo entry into the uterus, we noted the emergence of two mutually exclusive groups: pdKO females with embryos found only within the oviducts and pdKO females with embryos found only within the uterus ( Figure 5B – C ). The total number of visual corpus lutea did not differ between pdKOs and controls, nor did the total number of embryos flushed at 3.5dpc ( Figure 5D ). In addition, visualization of oviducts indicated appropriate folding patterns ( Supplementary Figure 4A )( 13 ). Individual uteri were imaged utilizing tissue clearing to allow visualization of embryos using the whole-mount immunofluorescent protocol. Embryos found within pdKO uteri at 3.5dpc were 50% of the quantity found within control uteri ( Supplementary Figure 4B and 4C ). To disseminate the cause of delayed embryo entry into the maternal endometrium combined with partially delayed implantation and to rationalize delayed embryonic development, we investigated endometrial receptivity at 3.5dpc ( Figure 6 ). The progesterone receptor expression by both mRNA and protein was not altered in pdKO uteri at 3.5dpc ( Figure 6A and B ). However, we noted significantly decreased target gene expression, Ihh, and Nr2f2 , in 3.5dpc uteri (unpaired t-test, p <0.01, Figure 6A ). In addition, luminal epithelial expression of proliferation marker Ki67 was maintained in pdKO uteri ( Figure 6C ). Interestingly, estrogen receptor signaling decreased in pdKOs compared to controls at 3.5dpc (Unpaired t-test, p <0.03, Supplementary Figure 5 ). Implantation chamber morphology was notably normal when visualized with whole uterine imaging at 5.5dpc ( Figure 7A ). However, pdKO uteri contained embryos that morphologically appeared delayed with decreased elongation compared to controls at 5.5dpc ( Figure 7B ). Most embryos visualized (59%) within pdKO uteri were delayed ( Figure 7C ). Overall, implantation chamber length was not affected despite delayed embryos being found in most implantation sites within pdKOs visualized ( Figure 7D ). Delayed embryos with decreased elongated morphology had 3D surface volumes significantly lower compared to normal elongated embryos found within both control and pdKO implantation sites (unpaired t-test, p= 0.01 compared to normal control embryos and p =0.001 compared to normal pdKO embryos Figure 7E ). Additionally, the ratio of embryo volume to implantation chamber length of those embryos that were identified as delayed was significantly decreased (unpaired t-test, p <0.01, Figure 7F ). This finding necessitates further investigation of the factors contributing to delayed embryo development and implantation in our mouse model. Whole uterine cross-sections at 7.5dpc from pdKO (n=3) and floxed controls (n=4) were subjected to bulk mRNA sequencing to elucidate the transcriptional repercussions associated with the loss of Yap1 and Wwtr1 in the pregnant uterus. Samples were separated on an MDS plot based on genotype ( Figure 8A ). We identified 16,674 total genes after filtering and 1,785 differentially expressed genes (DEG), with 884 genes upregulated and 901 downregulated in the knockouts compared to controls ( Figure 8B ). Differentially expressed genes (DEG) included eight genes from the Hippo signaling pathway including upstream regulators ( Amotl1 and Amotl2 ), kinases ( Sav1 and Lats2 ), transcription factor ( Tead4 ), transcriptional effectors (Yap1 and Wwtr1 ), and target genes ( Ccn2 and Birc5 ) ( Figure 8C ). Decreased differentially expressed genes also included four decidualization response genes ( Figure 8D ). DEG contributed to Gene Ontology Cellular Component terms like extracellular matrix, collagen trimer, and collagen-containing extracellular matrix, indicating that loss of Yap1 and Wwtr1 affected genes involved in cellular structure ( Figure 8E ). In addition, upregulated genes were enriched for Rat Genome Database (RGD) disease terms like pre-eclampsia, endometriosis, liver cirrhosis, and pulmonary fibrosis, indicating that pdKO uteri were enriched for genes connected with reproductive diseases associated with pregnancy loss and fibrosis, which has classically been associated with Hippo signaling ( Figure 8F ).

Pgr Cre/+ ( 19 ) mice were crossed to Yap fl/fl Wwtr1 fl/fl ( 20 , 21 ) to generate Pgr Cre/+ Yap fl/+ Wwtr1 fl/fl partial double knockouts (pdKO). The Pgr Cre/+ mice are a mixed background of 129Sv × C57BL/6, the Yap fl/fl Wwtr1 fl/fl mice are 129SvEv. Animals were housed and maintained in a designated animal care facility at Michigan State University on a 12-hour light/dark cycle with free access to food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of Michigan State University. Females were co-housed with proven fertile wild-type males for a 6-month breeding trial. Males were rotated in or out of cages if females did not produce a live born litter within one-month from time of set up. For timed mating experiments, proven fertile wildtype males were placed in female cages in the evening. Seminal plugs were checked each morning with day of plug designated at 0.5 days post coitus (dpc). Tail vein injection with Chicago blue dye served as a positive identifier for implantation sites at all time points. Following blue dye injection, female mice were sacrificed for collection at a variety of timepoints including 1.5, 3.5, 4.5, 5.5, 7.5, 9.5, and 12.5dpc. Body weight, uterine wet weight, ovarian wet weight, implantation site number, and gross morphology were catalogued. Uterine, oviductal, and ovarian tissues were divided and flash frozen or stored in RNAlater for downstream RNA and protein analyses or fixed in 4% paraformaldehyde for histological analysis. A subset of tissues were fixed in 10% DMSO in methanol for tissue clearing and advanced light sheet microscopy. Sexually mature (8 weeks or older) female mice were ovariectomized followed by two weeks of rest. Animals were treated with three daily subcutaneous injections of 100ng 17β-estradiol followed by two days of rest then three daily injections of 1mg progesterone plus 6.7ng 17β-estradiol. Six hours following injection on the third day, an intraluminal scratch was performed surgically on the anti-mesometrial side of the endometrium of one uterine horn utilizing a blunted 25G needle. The unscratched uterine horn served as an unstimulated hormonal control. Decidualization reaction was maintained with daily subcutaneous injection of 1mg progesterone plus 6.7ng 17β-estradiol for 5 total days followed by euthanasia (n=5/genotype). Uterine tissues were collected as described above. Decidual reaction was measured by uterine wet weight ratio of stimulated/unstimulated horn and molecular markers Wnt4 and Bmp2 by qPCR of stimulated uterine horn compared to unstimulated horn from the same animal. Sexually mature female mice (8 weeks or older) were mated to proven fertile wild-type males. Oviductal flushes were performed with phosphate buffered saline (PBS) by inserting a 30G needle into the infundibulum of both oviducts at 1.5dpc (n=6 per genotype). Flushed products were collected, counted, categorized, and imaged. Uterine and oviductal flushes were performed at 3.5dpc with uterine flushes performed by inserting a 30G needle into the uterotubal junction and flushing toward the cervix utilizing PBS. Flushed products were similarly collected, counted, categorized, and imaged. Tissues were fixed in 4% paraformaldehyde, dehydrated in ethanol and xylene, and embedded in paraffin. Sections (6 μm) were deparaffinized and rehydrated in a graded alcohol series followed by antigen retrieval (Vector Laboratories, Burlingame, CA) and hydrogen peroxide treatment. Next, sections were blocked and incubated with antibodies against YAP, WWTR1, ER-alpha, PGR, Ki67, SUSD2 or ASMA overnight at 4°C (see Supplementary Table 1 for IHC antibody information). On the following day, sections were incubated with biotinylated secondary antibodies followed by incubation with horseradish peroxidase conjugated streptavidin. Immunoreactivity was detected using the DAB substrate kit (Vector Laboratories) and visualized as brown staining by light microscopy. Incubation with secondary antibody only served as a negative control. Alternatively, after dehydration, slides were stained with Masson’s Trichrome followed by rehydration in a graded ethanol series then cover slipped and visualized by light microscopy. ImageJ image analysis software (NIH, v2.14.0), was utilized to determine a digital HSCORE for staining intensity of luminal epithelium, glandular epithelium, and stromal compartments of each uterine section or for granulosa cells in ovarian cross-sections. Total RNA was isolated from frozen mouse tissue using TRIzol reagent (Invitrogen, Waltham, MA). About 1μg of RNA was reverse transcribed to cDNA using a High-Capacity cDNA Reverse Transcription kit according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Quantitative real-time PCR (qPCR) was performed with SYBR Green PCR Master Mix (Applied Biosystems) using the BioRad CFX Opus 384 qPCR system utilizing primers targeting genes of interest ( Supplementary Table 2 ). Expression was normalized to the average of 36b4 and 18s per sample and fold change determined compared to time matched floxed controls ( Pgr +/+ Yap fl/fl Wwtr1 fl/fl ). Total RNA was isolated from flash frozen mouse uteri utilizing TRIzol reagent as described above. Following isolation, RNA was treated with a RNA Clean and Concentrator kit (Zymo) followed by DNAse treatment (TURBO DNA-free kit, Invitrogen) and stored in nuclease-free water at −80°C. Concentration was determined utilizing a Qubit RNA BR Assay Kit (Invitrogen). Samples were sent for RNA integrity analysis then subsequently sent to a sequencing facility for library preparation and sequencing (Michigan State University Genomics Core). Libraries were prepped with Illumina Stranded mRNA Library Prep kit (Illumina) and sequenced (paired end 150bp) on a NovaSeq 6000 Instrument (Illumina) to an average depth of 40 million read pairs per sample. Reads were quality trimmed, adapters were removed using TrimGalore (version 0.6.10), and quality-trimmed reads were assessed with MultiQC (version 1.7). Trimmed reads were mapped to Mus Musculus GRCm39.110 and gene counts quantified using STAR (version 2.6.0c). Model-based differential expression analysis was performed using edgeR-robust method (version 3.42.4) ( 22 ) in R. Genes with low counts per million (CPM) were removed using the filterByExpr function from edgeR. Multidimensional scaling plots, generated with the plotMDS function of edgeR, were used to verify group separation prior to statistical analysis. DEG were identified as FDR P value less than 0.05. Visualization of DEG was performed utilizing EnhancedVolcano (version 1.18.0) to generate a volcano plot. Counts per million of selected DEG were plotted using the box plot function of ggplot2 (version 3.4.4). Gene set enrichment and visualization was completed using the ShinyGO online tool (version 0.80) ( 23 ). Uteri were dissected from 3.5 (n=2) and 5.5dpc (n=8) pdKO and control females. Whole mount immunofluorescent staining was performed as previously described( 24 ). Briefly, following animal euthanasia, uterine samples were subsequently fixed in DMSO:methanol (1:4) and stored at −20°C. Then, samples were rehydrated in 1:1, Methanol:PBST (PBS + 1% Triton X-100) for 15 minutes then washed for 15 minutes in 100% PBST. Tissues were then incubated in a blocking solution of PBS, 1% Triton X-100, and 2% powdered milk for 2 hours at room temperature. Samples were stained with primary antibodies for rat anti-CDH1 (M108, Takara Biosciences), rabbit anti-cytokeratin 8 (MA5–14476, Invitrogen) and rabbit anti-FOXA2 (Abcam, ab108422) diluted at 1:500 in blocking solution for 7 nights at 4°C. Uterine samples were then washed in PBST for 15 minutes twice and 45 minutes four times then incubated with fluorescently conjugated Alexa Fluor 555 Donkey anti-Rabbit IgG secondary antibody (A31572, Invitrogen), and 647 Goat anti-Rat secondary antibody (A21247, Invitrogen) and Hoechst (B2261, Sigma Aldrich) at 1:500 for 2 nights at 4°C. Samples were then washed in PBST for 15 minutes twice and 45 minutes four times, dehydrated in methanol then incubated overnight in 3% H2O2 diluted in methanol. Tissues were then washed in 100% methanol for 15 minutes twice then for 60 minutes and cleared overnight using BABB (benzyl alcohol:benzyl benzoate, 1:2). Stained tissue samples were imaged with a Leica TCS SP8 X Confocal Laser Scanning Microscope System (Leica Microsystems) with a white-light laser, using a 10x air objective. For each uterine horn, z-stacks were generated with a 7.0μm increment, and image analysis was carried out using Imaris v9.2.1 (Bitplane, Zurich, Switzerland). Briefly, confocal LIF files were imported into the Surpass mode of Imaris, and Surface module 3D renderings were used to create structures for the oviductal–uterine junctions, embryos, implantation chambers, and horns as described previously( 25 ). We used the Contour modules for Hoechst and FOXA2 fluorescent signal (embryo), and CDH1 signal (oviduct and implantation chamber). Embryo 3D volume was assessed using the surface statistics function, while embryo implantation chamber length was calculated using the measurement points module of Imaris software. Data were tested for normality then log transformed in the case of non-normal data and analyzed by unpaired t-tests, one-way ANOVA, Fisher’s exact test, or mixed effects analysis as indicated. Data that were not normal were analyzed utilizing Kruskal-Wallis nonparametric tests. Statistical analyses were performed utilizing GraphPad Prism 10 (GraphPad Software) and values were considered significant if p <0.05.

The deficient uterine receptivity and dysregulation of hormonal signals within the pdKO females is interesting. However, the interplay between the Hippo signaling pathway and the hormonal response has been observed before. The functional kinases of the Hippo kinase cascade, serine/threonine kinase 3 and 4 (Stk3/4), have been shown to be differentially expressed throughout the murine estrous cycle primarily under the control of estrogen signaling( 14 ). Indeed, YAP1 expression is also differentially regulated throughout the murine estrous cycle, with its level being highest during estrous and diestrus( 8 ). Importantly, estrus in female mice is when ovulation occurs, and 3 days later is when the window of receptivity begins suggesting that YAP1 is present and may play a role in hormone response during this period. In addition to the cyclical presence of YAP1 as well as activation of the Hippo signaling pathway, the function of this pathway has also been associated with hormonal action. In disease states like breast cancer and endometriosis, YAP1 bound to TEAD transcription factors can induce transcription of ESR1, or alternatively, in endometriosis, YAP1 can regulate upstream factors that influence PGR expression ( 15 , 16 ). These reports support an indirect method of hormonal control and interpretation of the signal, but it is possible within the canonical function of YAP1/WWTR1 as transcriptional coactivators that they could play a direct role in hormone receptor function. In one study, YAP/TEAD complexes bound to ESR1 enhancer regions and estrogen response elements ( 17 ). We propose that within our model, it is possible that the loss of Yap1/Wwtr1 and subsequent binding to TEADs, prevents the acquisition and activation of distal estrogen and progesterone receptor response elements and enhancer recruiters. This would explain why the loss of Yap1/Wwtr1 during the receptive window leads to a failure of PGR and ESR1-regulated genes to be activated despite appropriate expression of receptor levels during this time ( Summary Figure ). The mechanistic aspect of this hypothesis remains to be tested but opens a path for novel roles of YAP1/WWTR1 function within the murine uterus. We showed that depletion of Yap1 and Wwtr1 led to a lack of decidualization response and maternal remodeling. YAP1 and WWTR1 are classically noted to respond to extracellular changes in stiffness transduced through the Hippo signaling pathway and decidualization and pregnancy processes, which rely on appropriate extracellular changes to alter the maternal endometrial environment( 7 ). They also classically regulate the transcription of extracellular and structural component genes in addition to cell cycle regulatory genes( 7 ). Unsurprisingly, the loss of Yap1/Wwtr1 led to the enrichment of DEG in GO Cellular Component terms like plasma membrane, collagen-containing matrix, and extracellular matrix ( Supplementary Table 6 ). Within the upregulated and downregulated DEG of the bulk mRNA sequencing, specific classes of genes included extracellular matrix genes such as collagens, CCNs, laminins, and extracellular remodeling genes like matrix metalloproteases ( Supplementary Tables 6 and 7 ). However, differential gene expression analysis of whole tissue makes it difficult to determine whether altered wound healing or fibrosis occurs in the pdKO females. Given the phenotypic data that show a progressive loss of fertility in the pdKO females, it is likely that either aberrant wound repair mechanisms or increased scarring and associated fibrosis are occurring. Both wound healing and fibrosis-associated markers were found within the DEG at 7.5dpc, including the decrease of angiogenic markers Vegfc , Vegfb , increased Pdgfrb , and increased expression of Il13ra1 and Il4ra1 . Angiogenesis is associated with wound healing but is negatively associated with fibrosis( 18 ). These results suggest that loss of Yap1 / Wwtr1 leads to decreased wound healing during pregnancy through the downregulation of proangiogenic factors. However, vasculature responds to a lack of angiogenic signal by upregulating receptor expression on the vascular smooth muscle( 18 ). In addition, IL13RA1 forms a heteromeric complex with IL4RA and activation of this receptor complex is widely considered pro-fibrotic, supporting that the pdKO uteri may be exhibiting increased fibrosis( 18 ). Indeed, pathway enrichment for upregulated DEG in Disease RGD included disease terms for fibrotic diseases like pulmonary fibrosis and liver cirrhosis ( Supplementary Table ). It is likely that loss of Yap1/Wwtr1 contributes to both a lack of wound healing and increased fibrosis within the murine uterus during pregnancy and this contributes to the repetitive pregnancy loss seen within the pdKO females. The incomplete penetrance of phenotype in the pdKO females can be attributed to a lack of recombination of one Yap1 allele. Both Progesterone receptor and Yap1 are located on chromosome 9 in mice with Pgr being located at 8899834–8968612 bp on the sense strand and Yap1 being located at 7932000–8004597 bp on the antisense strand (Mgi). Both Pgr and Yap1 are located at 2.46cM and due to the basics of recombination efficiency, the likelihood of recombination for these two genes is nearly 0%. Unfortunately, this contributes to the only possible genotype being Pgr Cre on the sense strand and one deleted allele of Yap1 on the antisense strand within any given Pgr expressing cell within the murine uterus. The proximity of these genes led to the partiality of the double knockout and the lack of complete phenotype. Despite the incomplete penetrance of phenotype, we observed significant differences in pdKO females compared to controls, which compromised fertility. It is likely that complete conditional uterine knockout of Yap1 combined with Wwtr1 would lead to a much more severe effect, including fully compromised decidualization and fertility due to a lack of maternal remodeling. Currently, we are working to generate cell type-specific knockouts to determine the endometrial compartmental contributions of total Yap1 and Wwtr1 knockout.

Successful reproduction is an impeccably complex process. The coordination of intricate molecular signals with large-scale physiological changes imparts many opportunities for error. An unfortunate statistic is that 20% of couples attempting to reproduce are infertile from either male or female contributions (NICHD). Within mammals, the fertilization of ovulated oocyte(s) is just the beginning, assuming that ovulation is not compromised. Following fertilization and early embryonic development, an embryo implants into a receptive maternal endometrium that is significantly remodeled throughout pregnancy to support gestation( 1 ). In humans and mice, successful reproduction requires the coordinated efforts of many molecular and cellular signals and the appropriate interplay between systems, including the neuroendocrine, immune, vascular, and female reproductive tract. Humans undergo spontaneous endometrial stromal cell decidualization, a terminal differentiation of the underlying stroma within the endometrium, which is induced by increasing levels of progesterone during the secretory phase of the menstrual cycle( 1 ). This process is critical to control trophoblast invasion and allow appropriate embryo invasion and occurs spontaneously each cycle in the absence of a conceptus( 2 , 3 ). In mice, decidualization only occurs when an embryo physically attaches to the maternal endometrial epithelium that communicates with the stroma and initiates decidualization( 4 ). In humans, decidualization prepares the maternal endometrium for implantation while in mice implantation initiates decidualization. In both mice and humans, decidualization controls the level of trophoblast invasion and is integral to pregnancy establishment. Therefore, understanding the molecular mechanisms that underlie these complex coordinated events is essential to identifying mechanisms that may go awry and contribute to infertility. Many molecular pathways have been implicated in decidualization and implantation, including WNT, NOTCH, and HIPPO signaling( 5 ). Of particular interest is the mechanosensing HIPPO pathway. This pathway was first identified in Drosophila and so named due to mutations in this pathway leading to increased body size( 6 ). The Hippo signaling pathway in mammals controls organ size, growth, and proliferation and does so by sensing and responding to changes in the extracellular environment, like the presence and tension of nearby cells, as well as growth factor availability( 7 ). The Hippo pathway is a kinase cascade whereby external signals like those mentioned above induce the phosphorylation of MST1/2 through a variety of upstream signals. MST1/2 phosphorylates LATS1/2, which then phosphorylates YAP1 and WWTR1, two transcriptional cofactors. YAP1 and WWTR1 are either bound or degraded in the cytoplasm and therefore inactive when the HIPPO signaling pathway is active. Conversely, when the HIPPO signaling pathway is deactivated, YAP1 and WWTR1 translocate to the nucleus and bind their canonical partner transcription factors, the TEADs, where they regulate directly gene transcription of extracellular and cytoplasmic matrix components, cell cycle genes, and indirectly by acting as distal enhancer recruiters( 7 ). This pathway is well conserved among mammals and has been implicated to play a role in female reproductive function. In the murine uterus, YAP1 exhibits dynamic expression with the highest levels of mRNA and protein expression at estrus, an estrogen-dominated stage( 8 ). In addition, YAP1 is significantly increased and phosphorylated with 17β-estradiol treatment of ovariectomized mice, suggesting YAP1’s importance in specific phases of the estrus cycle( 8 ). Beyond the estrus cycle, YAP1 is expressed throughout the murine endometrium in early pregnancy prior to embryo implantation at embryonic day 0.5 and beyond( 9 ). Post-implantation Yap1 and its targets, Ctgf and Ankrd1 , are significantly increased. A similar response is observed in oil-induced decidualization, suggesting a potential role for YAP1 in maternal preparation of pregnancy( 9 ). In addition, conditional deletion of Yap1 and its homolog, Wwtr1 (formerly known as Taz ), under Anti-Mullerian hormone receptor type 2 driven Cre expression, results in degradation of the oviductal isthmus myosalpinx complicating embryo transport, suggesting an essential role for YAP1 and WWTR1 in structural integrity( 10 ). Beyond what is known in the mouse, YAP1 and WWTR1 have been independently identified as critical for endometrial stromal cell decidualization in vitro . YAP1 expression increases in the first 48 hours of in vitro endometrial stromal cell decidualization, and knockdown by shYAP prior to induction of in vitro decidualization results in a compromised decidualization response( 11 ). In addition, WWTR1 increases during in vitro decidualization at day 6, and the knockdown of WWTR1 also compromises the expression of decidualization markers IGFBP1 and dPRL (unpublished)( 12 ). These studies highlight the roles of YAP1 and WWTR1 in in vitro decidualization, but this potential has yet to be explored in vivo . In this study, we generated conditional knockout of YAP1 and WWTR1 under the Progesterone receptor Cre to explore the potential roles and regulation of these Hippo homologs in pregnancy.