Characterization of the Endometrium of Women with Reproductive Failure

Differentiation of human endometrial stromal cells (HESCs) into decidual cells represents a highly coordinated process essential for embryo implantation. Following the post-ovulatory rise in progesterone levels HESCs undergo biochemical and morphological transformation in a process known as decidualization. It heralds the end of the mid-secretory phase implantation window, defined as the limited period during which progesterone-driven changes in the luminal epithelium allow apposition, attachment and invasion of a developmentally competent blastocyst. Failure of the endometrium to acquire a receptive phenotype is widely viewed as a major cause of infertility and IVF treatment failure. Conversely, recurrent pregnancy loss (RPL) is associated with impaired decidualization. Firstly, analysis of mid-secretory endometrial biopsies from RPL patients demonstrated that there is a decreased expression of the decidual marker, Prolactin but increased levels of pro-inflammatory cytokine, Prokineticin-1. Secondly, HESCs were then identified to mount a highly coordinated but transient inflammatory response that renders the endometrium receptive, by rapidly releasing Interleukin-33, up-regulating its cognate transmembrane receptor-ST2L and other pro-inflammatory mediators before mounting an anti-inflammatory response that includes down-regulation of ST2L and increased secretion of the soluble decoy receptor sST2. In agreement, only during the transient pro-inflammatory phase of the decidual process did HESCs secrete factors conducive to implantation in mice. Failure of HESCs to constrain this pro-inflammatory response appeared to prolong the implantation window and was associated with RPL. Furthermore, deregulated Serum and Glucocorticoid Kinase 1 (SGK1) activity in the endometrium during the window of implantation either interfered with embryo implantation, leading to infertility, or compromised the integrity of the decidual-placental interface, resulting in pregnancy loss. A constitutively active SGK1 mutant was expressed in luminal epithelial cells of the mouse uterus; perturbing uterine fluid handling and abolished embryo implantation. However, implantation was not impaired in Sgk1-deficient mice, although there was evidence of fetal demise. RPL was also associated with lower SGK1 induction in decidualizing HESCs and impaired expression of oxidative stress defence genes. In summary, impaired decidualization or unfettered endometrial receptivity carries an obvious risk of implantation of developmentally delayed or compromised embryos thus triggering a spectrum of pathological events, leading to miscarriage or predisposing for obstetrical complications. Together, these findings provide fundamental insights of uterine receptivity and describe a novel paradigm of reproductive failure with potentially far-reaching clinical implications

β-Klotho as a Negative Regulator of the Peptide Transporters PEPT1 and PEPT2

Background/Aims: β-Klotho, a transmembrane protein expressed in several tissues including the brain and the kidney, is critically important for inhibition of 1,25(OH)2D3 formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. Soluble klotho is a β-glucuronidase participating in the regulation of several ion channels and carriers. The present study explored the effect of β-Klotho protein on the peptide transporters PEPT1 and PEPT2. Methods: cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes and glycine-glycine (2 mM)-induced inward current (IGly) taken as measure of glycine-glycine transport. Measurements were made without or with prior 24 h treatment with soluble β-Klotho protein (30 ng/ml) in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Ussing chamber experiments were employed to determine electrogenic peptide transport across intestinal epithelia of klotho deficient (kl-/-) and corresponding wild type (kl+/+) mice. Results: IGly was observed in PEPT1 and in PEPT2 expressing oocytes but not in water injected oocytes. In both, PEPT1 and PEPT2 expressing oocytes IGly was significantly decreased by treatment with soluble β-Klotho protein. As shown for PEPT1, β-klotho protein decreased significantly the maximal transport rate without significantly modifying the affinity of the carrier. The effect of β-Klotho on PEPT1 was reversed by DSAL. Intestinal IGly was significantly larger in kl-/- than in kl+/+ mice. Conclusion: β-Klotho participates in the regulation of the peptide transporters PEPT1 and PEPT2

LEFTY2 Controls Migration of Human Endometrial Cancer Cells via Focal Adhesion Kinase Activity (FAK) and miRNA-200a

Background: LEFTY2, a suppressor of cell proliferation, tumor growth, regulator of stemness and embryonic differentiation, is a negative regulator of cancer cell reprogramming. Malignant transformation may lead to migration requiring loss of adhesion and gain of migratory activity. Signaling involved in the orchestration of migration, proliferation and spreading of cells include focal adhesion kinase (FAK) and adhesion molecule E-cadherin. Aims: The present study explored whether LEFTY2 influences the proliferation marker MKi67, FAK activity, E-cadherin abundance and migration of Ishikawa human endometrial carcinoma cells. Moreover, the study explored the involvement of microRNA-200a (miR-200a), which is known to regulate cellular adhesion by targeting E-Cadherin. Methods: FAK activity was estimated from FAK phosphorylation quantified by Western blotting, migration utilizing a wound healing assay, miR-200a and MKi67 expression levels utilizing qRT-PCR, cell proliferation and apoptosis using BrdU and Annexin V staining, respectively, and E-Cadherin (E-Cad) abundance, using confocal microscopy. Results: LEFTY2 (25 ng/ml, 48 hours) treatment was followed by decrease of MKi67 expression, FAK activity and migration. LEFTY2 upregulated miRNA-200a and E-Cad protein level in Ishikawa cells. The effect of LEFTY2 on migration was mimicked by FAK inhibitor PF 573228 (50 µM). Addition of LEFTY2 in the presence of PF-573228 did not result in a further significant decline of migration. Conclusion: In conclusion, LEFTY2 down-regulates MKi67 expression and FAK activity, up-regulates miR-200a and E-cadherin, and is thus a powerful negative regulator of endometrial cell proliferation and migration