Bio-modulated mice epithelial endometrial organoids by low-level laser therapy serves as an invitro model for endometrial regeneration.

Endometrial regeneration is a dynamic process that is not well understood. The destruction of the endometrium with the formation of intrauterine adhesions is known as Asherman's syndrome. The lesions range from minor to severe adhesions and their impact on pregnancy is well documented. Operative hysteroscopy is the mainstay of diagnosis and treatment of intrauterine adhesions. Nevertheless, the recurrence rates remain high. It was recorded that low-level laser therapy in low doses has a stimulatory effect on different tissues while the high dose produces a suppressive effect. Organoid is a three-dimensional assembly that displays architectures and functionalities similar to in vivo organs that are being developed from human or animal stem cells or organ-specific progenitors through a self-organization process. Our prospective was to study the effect of Low-Level Laser Therapy (LLLT) on mouse epithelial endometrial organoids regarding cell proliferation and endometrial regeneration as a new modality of treatment. An in vitro clinical trial to generate mouse epithelial organoid model and testing LLLT using He:Ne 632.8 nm device on organoids proliferation, function, and their response to ovarian hormones was performed. Trying endometrial regeneration by culturing organoids with decellularized uterine matrix (DUM) and studying the LLLT effect on the regeneration process. LLLT produced a proliferative effect on the epithelial mouse organoids confirmed by Ki67 and PCNA IHC. The organoids could regenerate the epithelial layer of the endometrium in vitro on DUM and LLLT could help in this process. In conclusion, organoids whether control or bio-stimulated proved a new modality to regenerate the endometrium.

Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion.

Retinoblastoma protein (RB) encoded by Rb1 is a prominent inducer of cell cycle arrest (CCA). The hormone progesterone (P4 ) promotes CCA in the uterine epithelium and previous studies indicated that P4 activates RB by reducing the phosphorylated, inactive form of RB. Here, we show that embryo implantation is impaired in uterine-specific Rb1 knockout mice. We observe persistent cell proliferation of the Rb1-deficient uterine epithelium until embryo attachment, loss of epithelial necroptosis, and trophoblast phagocytosis, which correlates with subsequent embryo invasion failure, indicating that Rb1-induced CCA and necroptosis of uterine epithelium are involved in embryo invasion. Pre-implantation P4 supplementation is sufficient to restore these defects and embryo invasion. In Rb1-deficient uterine epithelial cells, TNFα-primed necroptosis is impaired, which is rescued by the treatment with a CCA inducer thymidine or P4 through the upregulation of TNF receptor type 2. TNFα is expressed in the luminal epithelium and the embryo at the embryo attachment site. These results provide evidence that uterine Rb1-induced CCA is involved in TNFα-primed epithelial necroptosis at the implantation site for successful embryo invasion.

Uterine Epithelial Progesterone Receptor Governs Uterine Receptivity Through Epithelial Cell Differentiation.

Progesterone receptor (PGR) is indispensable for pregnancy in mammals. Uterine PGR responds to the heightened levels of ovarian progesterone (P4) after ovulation and regulates uterine gene transcription for successful embryo implantation. Although epithelial and stromal P4-PGR signaling may interact with each other to form appropriate endometrial milieu for uterine receptivity and the subsequent embryo attachment, it remains unclear what the specific roles of epithelial P4-PGR signaling in the adult uterus are. Here we generated mice with epithelial deletion of Pgr in the adult uterus (Pgrfl/flLtfCre/+ mice) by crossing Pgr-floxed and Ltf-Cre mice. Pgrfl/flLtfCre/+ mice are infertile due to the impairment of embryo attachment. Pgrfl/flLtfCre/+ uteri did not exhibit epithelial growth arrest, suggesting compromised uterine receptivity. Both epithelial and stromal expressions of P4-responsive genes decreased in Pgrfl/flLtfCre/+ mice during the peri-implantation period, indicating that epithelial Pgr deletion affects not only epithelial but stromal P4 responsiveness. In addition, uterine LIF, an inducer of embryo attachment, was decreased in Pgrfl/flLtfCre/+ mice. The RNA-seq analysis using luminal epithelial specimens dissected out by laser capture microdissection revealed that the signaling pathways related to extracellular matrix, cell adhesion, and cell proliferation are altered in Pgr fl/flLtf Cre/+ mice. These findings suggest that epithelial PGR controls both epithelial and stromal P4 responsiveness and epithelial cell differentiation, which provides normal uterine receptivity and subsequent embryo attachment.

Differential roles of uterine epithelial and stromal STAT3 coordinate uterine receptivity and embryo attachment.

Although it has been reported that uterine signal transducer and activator of transcription 3 (STAT3) is essential for embryo implantation, the exact roles of uterine epithelial and stromal STAT3 on embryo implantation have not been elucidated. To address this issue, we generated Stat3-floxed/Ltf-iCre (Stat3-eKO), Stat3-floxed/Amhr2-Cre (Stat3-sKO), and Stat3-floxed/Pgr-Cre (Stat3-uKO) mice to delete Stat3 in uterine epithelium, uterine stroma, and whole uterine layers, respectively. We found that both epithelial and stromal STAT3 have critical roles in embryo attachment because all the Stat3-eKO and Stat3-sKO female mice were infertile due to implantation failure without any embryo attachment sites. Stat3-eKO uteri showed indented structure of uterine lumen, indicating the role of epithelial STAT3 in slit-like lumen formation in the peri-implantation uterus. Stat3-sKO uteri exhibited hyper-estrogenic responses and persistent cell proliferation of the epithelium in the peri-implantation uterus, suggesting the role of stromal STAT3 in uterine receptivity. In addition, Stat3-uKO female mice possessed not only the characteristic of persistent epithelial proliferation but also that of indented structure of uterine lumen. These findings indicate that epithelial STAT3 controls the formation of slit-like structure in uterine lumen and stromal STAT3 suppresses epithelial estrogenic responses and cell proliferation. Thus, epithelial and stromal STAT3 cooperatively controls uterine receptivity and embryo attachment through their different pathways.

Levonorgestrel Inhibits Embryo Attachment by Eliminating Uterine Induction of Leukemia Inhibitory Factor.

Progestogens including progesterone (P4) and levonorgestrel (LNG) are clinically used for multiple purposes such as contraception and infertility treatment. The effects of progestogens on the uterus remains to be elucidated. Here we examine the effect of excessive progestogen administration on embryo implantation focusing on the function of uterine leukemia inhibitory factor (LIF), a cytokine that is induced by estrogen and essential for embryo attachment. Treatment of wild-type (WT) female mice with vehicle (control), LNG at the dose of 300 µg/kg/day and P4 at the dose of 10 mg/day from day 1 to day 4 of pregnancy was conducted. LNG-treated and P4-treated mice showed embryo attachment failure on day 5 of pregnancy (The rate of mice with embryo attachment sites [%MAS], 11% and 13%, respectively), while all the control mice had normal attachment sites. Uterine LIF expression was significantly reduced in LNG-treated and P4-treated mice on day 4 evening. Administration of recombinant LIF (rLIF) at the dose of 24 µg/day on day 4 significantly rescued embryo attachment failure in LNG-treated and P4-treated mice (%MAS, 80% and 75%, respectively). Estradiol (E2) administration also rescued embryo attachment failure in LNG-treated mice (%MAS, 83%). Furthermore, excess P4 treatment before implantation decreased decidual P4 receptor (PGR) expression and induced decidualization defect apart from LIF downregulation. These findings indicate that progestogens cause embryo attachment inhibition through downregulation of uterine LIF expression and compromised decidualization through downregulation of PGR independently of LIF reduction. This study may contribute to a better understanding of contraceptive action of progestogens.

Uterine receptivity, embryo attachment, and embryo invasion: Multistep processes in embryo implantation.

BACKGROUND: Recurrent implantation failure is a critical issue in IVF-ET treatment. Successful embryo implantation needs appropriate molecular and cellular communications between embryo and uterus. Rodent models have been used intensively to understand these mechanisms. METHODS: The molecular and cellular mechanisms of embryo implantation were described by referring to the previous literature investigated by us and others. The studies using mouse models of embryo implantation were mainly cited. RESULTS: Progesterone (P4) produced by ovarian corpus luteum provides the uterus with receptivity to the embryo, and uterine epithelial growth arrest and stromal proliferation, what we call uterine proliferation-differentiation switching (PDS), take place in the peri-implantation period before embryo attachment. Uterine PDS is a hallmark of uterine receptivity, and several genes such as HAND2 and BMI1, control uterine PDS by modulating P4-PR signaling. As the next implantation process, embryo attachment onto the luminal epithelium occurs. This process is regulated by FOXA2-LIF pathway and planar cell polarity signaling. Then, the luminal epithelium at the embryo attachment site detaches from the stroma, which enables trophoblast invasion. This process of embryo invasion is regulated by HIF2α in the stroma. CONCLUSION: These findings indicate that embryo implantation contains multistep processes regulated by specific molecular pathways.