CFP1 governs uterine epigenetic landscapes to intervene in progesterone responses for uterine physiology and suppression of endometriosis.

Progesterone (P4) is required for the preparation of the endometrium for a successful pregnancy. P4 resistance is a leading cause of the pathogenesis of endometrial disorders like endometriosis, often leading to infertility; however, the underlying epigenetic cause remains unclear. Here we demonstrate that CFP1, a regulator of H3K4me3, is required for maintaining epigenetic landscapes of P4-progesterone receptor (PGR) signaling networks in the mouse uterus. Cfp1f/f;Pgr-Cre (Cfp1d/d) mice showed impaired P4 responses, leading to complete failure of embryo implantation. mRNA and chromatin immunoprecipitation sequencing analyses showed that CFP1 regulates uterine mRNA profiles not only in H3K4me3-dependent but also in H3K4me3-independent manners. CFP1 directly regulates important P4 response genes, including Gata2, Sox17, and Ihh, which activate smoothened signaling pathway in the uterus. In a mouse model of endometriosis, Cfp1d/d ectopic lesions showed P4 resistance, which was rescued by a smoothened agonist. In human endometriosis, CFP1 was significantly downregulated, and expression levels between CFP1 and these P4 targets are positively related regardless of PGR levels. In brief, our study provides that CFP1 intervenes in the P4-epigenome-transcriptome networks for uterine receptivity for embryo implantation and the pathogenesis of endometriosis.

The Ratio of Mitochondrial DNA to Genomic DNA Copy Number in Cumulus Cell May Serve as a Biomarker of Embryo Quality in IVF Cycles.

Previous studies have reported that the mitochondrial DNA (mtDNA) contents of cumulus cells (CCs) in ovarian follicular fluid are correlated with embryo quality. Quantification of mtDNA CCs has been suggested as a biomarker of embryo viability. The aim of this study was to determine the relationship between mitochondrial DNA (mtDNA)/genomic DNA (gDNA) ratio in CCs and IVF outcomes such as fertilization rates and embryo quality in infertile women. This is an observational study on 144 cumulus-oocyte complexes obtained from 144 patients undergoing IVF-intracytoplasmic sperm injection (ICSI) at a single fertility center. The CCs in ovarian follicular fluid from patients undergoing IVF-ICSI were collected by ovum pick-up. A relative copy number quantification was used to determine mtDNA/gDNA ratio. Quantitative real-time PCR for various markers (ß2M and mtMinArc gene) was used to determine average mtDNA/gDNA ratio of CCs. Investigation of the correlation between mtDNA/gDNA ratio in CCs and IVF outcomes showed no statistically significant correlation between the mtDNA/gDNA ratio in CCs and fertilization rates. However, mtDNA/gDNA ratio and embryo quality showed a statistically significant positive correlation. A significantly higher mtDNA/gDNA ratio was observed in the good quality embryo group compared with the poor quality embryo group (P < 0.05). In addition, the mtDNA/gDNA ratio showed negative correlation with the patient's age (correlation coefficient= -0.228, P < 0.05). Results of this study demonstrate a negative correlation of mtDNA/gDNA ratio in CCs with patient's age, and a low copy number of mtDNA in CCs may have adverse effects on embryo quality in IVF cycles. These results suggest that the ratio of mtDNA/gDNA in CCs may serve as a biomarker in predicting IVF outcomes.

Different Cre systems induce differential microRNA landscapes and abnormalities in the female reproductive tracts of Dgcr8 conditional knockout mice.

OBJECTIVES: The female reproductive tract comprises several different cell types. Using three representative Cre systems, we comparatively analysed the phenotypes of Dgcr8 conditional knockout (cKO) mice to understand the function of Dgcr8, involved in canonical microRNA biogenesis, in the female reproductive tract. MATERIALS AND METHODS: Dgcr8f/f mice were crossed with Ltficre/+ , Amhr2cre/+ or PRcre/+ mice to produce mice deficient in Dgcr8 in epithelial (Dgcr8ed/ed ), mesenchymal (Dgcr8md/md ) and all the compartments (Dgcr8td/td ) in the female reproductive tract. Reproductive phenotypes were evaluated in Dgcr8 cKO mice. Uteri and/or oviducts were used for small RNA-seq, mRNA-seq, real-time RT-PCR, and/or morphologic and histological analyses. RESULT: Dgcr8ed/ed mice did not exhibit any distinct defects, whereas Dgcr8md/md mice showed sub-fertility and oviductal smooth muscle deformities. Dgcr8td/td mice were infertile due to anovulation and acute inflammation in the female reproductive tract and suffered from an atrophic uterus with myometrial defects. The microRNAs and mRNAs related to immune modulation and/or smooth muscle growth were systemically altered in the Dgcr8td/td uterus. Expression profiles of dysregulated microRNAs and mRNAs in the Dgcr8td/td uterus were different from those in other genotypes in a Cre-dependent manner. CONCLUSIONS: Dgcr8 deficiency with different Cre systems induces overlapping but distinct phenotypes as well as the profiles of microRNAs and their target mRNAs in the female reproductive tract, suggesting the importance of selecting the appropriate Cre driver to investigate the genes of interest.

An autophagic deficit in the uterine vessel microenvironment provokes hyperpermeability through deregulated VEGFA, NOS1, and CTNNB1.

The uterus undergoes vascular changes during the reproductive cycle and pregnancy. Steroid hormone deprivation induces macroautophagy/autophagy in major uterine cell types. Herein, we explored the functions of uterine autophagy using the Amhr2-Cre-driven atg7 deletion model. Deletion of Atg7 was confirmed by functional deficit of autophagy in uterine stromal, myometrial, and vascular smooth muscle cells, but not in endothelial cells. atg7d/d uteri exhibited enhanced stromal edema accompanied by dilation of blood vessels. Ovariectomized atg7d/d uteri showed decreased expression of endothelial junction-related proteins, such as CTNNB1/beta-catenin, with increased vascular permeability, and increased expression of VEGFA and NOS1. Nitric oxide (NO) was shown to mediate VEGFA-induced vascular permeability by targeting CTNNB1. NO involvement in maintaining endothelial junctional stability in atg7d/d uteri was confirmed by the reduction in extravasation following treatment with a NOS inhibitor. We also showed that atg7d/d uterine phenotype improved the fetal weight:placental weight ratio, which is one of the indicators of assessing the status of preeclampsia. We showed that autophagic deficit in the uterine vessel microenvironment provokes hyperpermeability through the deregulation of VEGFA, NOS1, and CTNNB1.Abbreviations: ACTA2: actin, alpha 2, smooth muscle, aortic; Amhr2: anti-Mullerian hormone type 2 receptor; ANGPT1: angiopoietin 1; ATG: autophagy-related; CDH5: cadherin 5; CLDN5: claudin 5; COL1A1: collagen, type I, alpha 1; CSPG4/NG2: chondroitin sulfate proteoglycan 4; CTNNB1: catenin (cadherin associated protein), beta 1; DES: desmin; EDN1: endothelin 1; EDNRB: endothelin receptor type B; F3: coagulation factor III; KDR/FLK1/VEGFR2: kinase insert domain protein receptor; LYVE1: lymphatic vessel endothelial hyaluronan receptor 1; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MCAM/CD146: melanoma cell adhesion molecule; MYL2: myosin, light polypeptide 2, regulatory, cardiac, slow; MYLK: myosin, light polypeptide kinase; NOS1/nNOS: nitric oxide synthase 1, neuronal; NOS2/iNOS: nitric oxide synthase 2, inducible; NOS3/eNOS: nitric oxide synthase 3, endothelial cell; OVX: ovariectomy; PECAM1/CD31: platelet/endothelial cell adhesion molecule 1; POSTN: periostin, osteoblast specific factor; SQSTM1: sequestosome 1; TEK/Tie2: TEK receptor tyrosine kinase; TJP1/ZO-1: tight junction protein 1; TUBB1, tubulin, beta 1 class VI; USC: uterine stromal cell; VEGFA: vascular endothelial growth factor A; VSMC: vascular smooth muscle cell.

Secretory phospholipase A2-X (Pla2g10) is a novel progesterone receptor target gene exclusively induced in uterine luminal epithelium for uterine receptivity in mice.

BACKGROUND: Aberration of estrogen (E2) and/or progesterone (P4) signaling pathways affects expression of their target genes, which may lead to failure of embryo implantation and following pregnancy. Although many target genes of progesterone receptors (PRs) have been identified in uterine stroma, only a few PR targets have been reported in the epithelium. Secretory phospholipase A2-(PLA2)-X, a member of the PLA2 family that releases arachidonic acids for the synthesis of prostaglandins that are important for embryo implantation, is dysregulated in the endometrium of patients suffering from repeated implantation failure. However, it is not clear whether sPLA2-X is directly regulated by ovarian steroid hormones for embryo implantation in the uterus. RESULT: P4 induced the Pla2g10 encoding of secretory PLA2-X in the apical region of uterine LE of ovariectomized mice via PR in both time- and dose-dependent manners, whereas E2 significantly inhibited it. This finding is consistent with the higher expression of Pla2g10 at the diestrus stage, when P4 is elevated during the estrous cycle, and at P4-treated delayed implantation. The level of Pla2g10 on day 4 of pregnancy (day 4) was dramatically decreased on day 5, when PRs are absent in the LE. Luciferase assays of mutagenesis in uterine epithelial cells demonstrated that four putative PR response elements in a Pla2g10 promoter region are transcriptionally active for Pla2g10. Intrauterine delivery of small interfering RNA for Pla2g10 on day 3 significantly reduced the number of implantation sites, reinforcing the critical function(s) of Pla2g10 for uterine receptivity in mice. CONCLUSIONS: Pla2g10 is a novel PR target gene whose expression is exclusively localized in the apical region of the uterine LE for uterine receptivity for embryo implantation in mice.

Perivascular Stem Cell-Derived Cyclophilin A Improves Uterine Environment with Asherman's Syndrome via HIF1α-Dependent Angiogenesis.

Asherman's syndrome (AS) is characterized by intrauterine adhesions or fibrosis resulting from scarring inside the endometrium. AS is associated with infertility, recurrent miscarriage, and placental abnormalities. Although mesenchymal stem cells show therapeutic promise for the treatment of AS, the molecular mechanisms underlying its pathophysiology remain unclear. We ascertained that mice with AS, like human patients with AS, suffer from extensive fibrosis, oligo/amenorrhea, and infertility. Human perivascular stem cells (hPVSCs) from umbilical cords repaired uterine damage in mice with AS, regardless of their delivery routes. In mice with AS, embryo implantation is aberrantly deferred, which leads to intrauterine growth restriction followed by no delivery at term. hPVSC administration significantly improved implantation defects and subsequent poor pregnancy outcomes via hypoxia inducible factor 1α (HIF1α)-dependent angiogenesis in a dose-dependent manner. Pharmacologic inhibition of HIF1α activity hindered hPVSC actions on pregnancy outcomes, whereas stabilization of HIF1α activity facilitated such actions. Furthermore, therapeutic effects of hPVSCs were not observed in uterine-specific HIF1α-knockout mice with AS. Secretome analyses of hPVSCs identified cyclophilin-A as the major paracrine factor for hPVSC therapy via HIF1α-dependent angiogenesis. Collectively, we demonstrate that hPVSCs-derived cyclophilin-A facilitates HIF1α-dependent angiogenesis to ameliorate compromised uterine environments in mice with AS, representing the major pathophysiologic features of humans with AS.

Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor.

BACKROUND: CRISPR/Cpf1 is a class II, type V RNA-guided endonuclease that is distinct from the type II CRISPR/Cas9 nuclease, widely used for genome editing. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR/Cas9 system. The applications of CRISPR to rodent embryos for the production of knock-out (KO) mice have been achieved mainly by microinjection, which requires heavily-equipped instruments with skillful hands. Here, we evaluated the genome editing efficiency between Cpf1/mRNA and Cpf1/ribonuclear protein (RNP) in mouse embryos, and established an easy, fast, and technically less demanding method to produce KO mice using electroporation of the Cfp1/RNP system. METHODS: The efficiency of electroporation-based delivery of AsCpf1/mRNA and AsCpf1/RNP to target exon 3 of leukemia inhibitory factor (Lif) into mouse zygotes was evaluated. Embryos that developed to the two-cell stage after zygote electroporation were transferred into the oviducts of surrogate mothers to produce AsCpf1-mediated LIF KO mice. The genome editing efficiency of blastocysts and pups was tested using the T7E1 assay and/or DNA sequencing. Congenital abnormalities and reproductive phenotypes in LIF KO mice produced by electroporation with AsCpf1/RNP were examined. RESULTS: Survival and two-cell development of electroporated zygotes were comparable between the AsCpf1/mRNA and AsCpf1/RNP groups, whereas genome editing efficiency was relatively higher in the AsCpf1/RNP group (13.3% vs 18.1% at blastocyst and 33.3% vs 45.5% at offspring), respectively. Two mouse lines with a frameshift mutation in exon 3 of the Lif gene were established from the AsCpf1/RNP group. All congenital abnormalities of LIF KO mice produced by AsCpf1/RNP electroporation were observed. AsCpf1-mediated LIF KO mice showed postnatal growth retardation and implantation failure, both of which are major phenotypes of LIF KO mice generated by conventional gene targeting. CONCLUSION: Electroporation of AsCpf1/RNP at the zygote stage is an efficient genome editing method to produce KO mice.

Oestrogen-induced expression of decay accelerating factor is spatiotemporally antagonised by progesterone-progesterone receptor signalling in mouse uterus.

Decay accelerating factor (DAF) is upregulated in the fetoplacental trophoblast, which protects the fetus from maternal complement injury. DAF was found to be downregulated in the endometrium of patients with repeated implantation failure. Thus, we examined the molecular mechanisms of DAF expression regulation by ovarian steroid hormones in the mouse uterus. Immunofluorescence staining demonstrated its exclusive localisation in the apical region of the epithelium in the uterus. Oestrogen (E2) significantly induced Daf mRNA in a time-dependent manner. Progesterone (P4) did not have any significant effect on Daf expression; however, it negatively modulated E2-induced DAF expression and RU486 effectively interfered with the inhibitory action of P4 in the uterus. During early pregnancy DAF was higher on Day 1 of pregnancy, but significantly decreased from Day 3, which is consistent with its E2-dependent regulation. Interestingly, DAF expression seemed to be influenced by the implanting blastocyst on Day 5 and it was gradually increased during preimplantation embryo development with peak levels at blastocyst stages. We demonstrated that E2-dependent DAF expression is antagonised by P4-progesterone receptor signalling in the uterine epithelium. Spatiotemporal regulation of DAF in the uterus and preimplantation embryos suggest that DAF functions as an immune modulator for embryo implantation and early pregnancy in mice.

Estrogen-induced transcription factor EGR1 regulates c-Kit transcription in the mouse uterus to maintain uterine receptivity for embryo implantation.

Early growth response 1 (Egr1) is a key transcription factor that mediates the action of estrogen (E2) to establish uterine receptivity for embryo implantation. However, few direct target genes of EGR1 have been identified in the uterus. Here, we demonstrated that E2 induced EGR1-regulated transcription of c-Kit, which plays a crucial role in cell fate decisions. Spatiotemporal expression of c-Kit followed that of EGR1 in uteri of ovariectomized mice at various time points after E2 treatment. E2 activated ERK1/2 and p38 to induce EGR1, which then activated c-Kit expression in the uterus. EGR1 transfection produced rapid and transient induction of c-KIT in a time- and dose-dependent manner. Furthermore, luciferase assays to measure c-Kit promoter activity confirmed that a functional EGR1 binding site(s) (EBS) was located within -1 kb of the c-Kit promoter. Site-directed mutagenesis and chromatin immunoprecipitation-PCR for three putative EBS within -1 kb demonstrated that the EBS at -818/-805 was critical for EGR1-dependent c-Kit transcription. c-Kit expression was significantly increased in the uterus on day 4 and administration of Masitinib, a c-Kit inhibitor, effectively interfered with embryo implantation. Collectively, our results showed that estrogen induces transcription factor EGR1 to regulate c-Kit transcription for uterine receptivity for embryo implantation in the mouse uterus.

Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

The harmonized actions of ovarian E2 and progesterone (P4) regulate the proliferation and differentiation of uterine cells in a spatiotemporal manner. Imbalances between these hormones often lead to infertility and gynecologic diseases. Whereas numerous factors that are involved in P4 signaling have been identified, few local factors that mediate E2 actions in the uterus have been revealed. Here, we demonstrate that estrogen induces the transcription factor, early growth response 1 ( Egr1), to fine-tune its actions in uterine epithelial cells (ECs) that are responsible for uterine receptivity for embryo implantation. In the presence of exogenous gonadotrophins, ovulation, fertilization, and embryonic development normally occur in Egr1-/- mice, but these animals experience the complete failure of embryo implantation with reduced artificial decidualization. Although serum levels of E2 and P4 were comparable between Egr1+/+ and Egr1-/- mice on d 4 of pregnancy, aberrantly reduced levels of progesterone receptor in Egr1-/- uterine ECs caused enhanced E2 activity and impaired P4 response. Ultrastructural analyses revealed that Egr1-/- ECs are not fully able to provide proper uterine receptivity. Uterine mRNA landscapes in Egr1-/- mice revealed that EGR1 controls the expression of a subset of E2-regulated genes. In addition, P4 signaling was unable to modulate estrogen actions, including those that are involved in cell-cycle progression, in ECs that were deficient in EGR1. Furthermore, primary coculture of Egr1-/- ECs with Egr1+/+ stromal cells, and vice versa, supported the notion that Egr1 is required to modulate E2 actions on ECs to prepare the uterine environment for embryo implantation. In contrast to its role in ECs, loss of Egr1 in stroma significantly reduced stromal cell proliferation. Collectively, our results demonstrate that E2 induces EGR1 to streamline its actions for the preparation of uterine receptivity for embryo implantation in mice.-Kim, H.-R., Kim, Y. S., Yoon, J. A., Yang, S. C., Park, M., Seol, D.-W., Lyu, S. W., Jun, J. H., Lim, H. J., Lee, D. R., Song, H. Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

Deficiency in DGCR8-dependent canonical microRNAs causes infertility due to multiple abnormalities during uterine development in mice.

DGCR8 is an RNA-binding protein that interacts with DROSHA to produce pre-microRNA in the nucleus, while DICER generates not only mature microRNA, but also endogenous small interfering RNAs in the cytoplasm. Here, we produced Dgcr8 conditional knock-out mice using progesterone receptor (PR)-Cre (Dgcr8(d/d)) and demonstrated that canonical microRNAs dependent on the DROSHA-DGCR8 complex are required for uterine development as well as female fertility in mice. Adult Dgcr8(d/d) females neither underwent regular reproductive cycles nor produced pups, whereas administration of exogenous gonadotropins induced normal ovulation in these mice. Interestingly, immune cells associated with acute inflammation aberrantly infiltrated into reproductive organs of pregnant Dgcr8(d/d) mice. Regarding uterine development, multiple uterine abnormalities were noticeable at 4 weeks of age when PR is significantly increased, and the severity of these deformities increased over time. Gland formation and myometrial layers were significantly reduced, and the stromal cell compartment did not expand and became atrophic during uterine development in these mice. These results were consistent with aberrantly reduced stromal cell proliferation and completely failed decidualization. Collectively, we suggest that DGCR8-dependent canonical microRNAs are essential for uterine development and physiological processes such as proper immune modulation, reproductive cycle, and steroid hormone responsiveness in mice.