An Assessment of the Mechanophysical and Hormonal Impact on Human Endometrial Epithelium Mechanics and Receptivity.

The endometrial epithelium and underlying stroma undergo profound changes to support and limit embryo adhesion and invasion, which occur in the secretory phase of the menstrual cycle during the window of implantation. This coincides with a peak in progesterone and estradiol production. We hypothesized that the interplay between hormone-induced changes in the mechanical properties of the endometrial epithelium and stroma supports this process. To study it, we used hormone-responsive endometrial adenocarcinoma-derived Ishikawa cells growing on substrates of different stiffness. We showed that Ishikawa monolayers on soft substrates are more tightly clustered and uniform than on stiff substrates. Probing for mechanical alterations, we found accelerated stress-relaxation after apical nanoindentation in hormone-stimulated monolayers on stiff substrates. Traction force microscopy furthermore revealed an increased number of foci with high traction in the presence of estradiol and progesterone on soft substrates. The detection of single cells and small cell clusters positive for the intermediate filament protein vimentin and the progesterone receptor further underscored monolayer heterogeneity. Finally, adhesion assays with trophoblast-derived AC-1M-88 spheroids were used to examine the effects of substrate stiffness and steroid hormones on endometrial receptivity. We conclude that the extracellular matrix and hormones act together to determine mechanical properties and, ultimately, embryo implantation.

How Mechanical Forces Change the Human Endometrium during the Menstrual Cycle in Preparation for Embryo Implantation.

The human endometrium is characterized by exceptional plasticity, as evidenced by rapid growth and differentiation during the menstrual cycle and fast tissue remodeling during early pregnancy. Past work has rarely addressed the role of cellular mechanics in these processes. It is becoming increasingly clear that sensing and responding to mechanical forces are as significant for cell behavior as biochemical signaling. Here, we provide an overview of experimental evidence and concepts that illustrate how mechanical forces influence endometrial cell behavior during the hormone-driven menstrual cycle and prepare the endometrium for embryo implantation. Given the fundamental species differences during implantation, we restrict the review to the human situation. Novel technologies and devices such as 3D multifrequency magnetic resonance elastography, atomic force microscopy, organ-on-a-chip microfluidic systems, stem-cell-derived organoid formation, and complex 3D co-culture systems have propelled the understanding how endometrial receptivity and blastocyst implantation are regulated in the human uterus. Accumulating evidence has shown that junctional adhesion, cytoskeletal rearrangement, and extracellular matrix stiffness affect the local force balance that regulates endometrial differentiation and blastocyst invasion. A focus of this review is on the hormonal regulation of endometrial epithelial cell mechanics. We discuss potential implications for embryo implantation.

Ultrastructural changes in endometrial desmosomes of desmoglein 2 mutant mice.

The intercellular binding of desmosomal junctions is mediated by cadherins of the desmoglein (Dsg) and desmocollin (Dsc) type. Dsg2 mutant mice with deletion of a substantial segment of the extracellular EC1-EC2 domain, which is believed to participate in homo- and heterophilic desmosomal cadherin interactions, develop cardiac fibrosis and ventricular dilation. Widening of the intercellular cleft and complete intercalated disc ruptures can be observed in the hearts of these mice. Since a reduced litter size of homozygous Dsg2 mutant mice was noted and a functional correlation between desmosomes and embryo implantation has been deduced from animal studies, we looked for an alteration of desmosomes in uterine endometrial epithelium. Shape and number of desmosomes as well as the expression of Dsg2 and the desmosomal plaque protein desmoplakin (Dsp) were investigated by electron microscopy and immunohistochemistry in 12 oestrous-dated mice (7 wild type and 5 homozygous Dsg2 mutant mice) at the age of 9-17 weeks. The immunohistochemical detection of Dsg2 was diminished in the mutants and the number of desmosomes was significantly reduced as revealed by electron microscopy. In addition, the intercellular desmosomal space measured in electron micrographs was considerably widened in the Dsg2 mutants. The increased intercellular spacing can be explained by the partial deletion of the extracellular EC1-EC2 domain of Dsg2. Whether these changes explain the reduced number of offspring of homozygous Dsg2 mutant mice remains to be further investigated.

Insufficient Angiogenesis: Cause of Abnormally Thin Endometrium in Subfertile Patients?

INTRODUCTION: This study investigated subfertile patients with abnormally thin endometrium after infertility treatment. As they had adequate serum concentrations of hormones, an endometrial factor for subfertility was suspected. METHODS: To elucidate the cause of subfertility, endometrial biopsies were taken in each patient in the late proliferative and mid-secretory phases of one menstrual cycle. Endometrial biopsies from women with normal menstrual cycles and confirmed fertility who were undergoing hysterectomy for benign uterine disease were used as positive controls. The tissue samples were investigated for steroid hormone receptor expression and for the proliferation marker Ki-67. Immunohistochemistry was performed with antibodies against the marker molecules for endometrial receptivity - ß 3 integrin, VEGF, LIF, and CD56 (large granular lymphocytes, LGLs). RESULTS: The steroid hormone receptors for estrogen (E2) and progesterone (P) were expressed normally (at the first biopsy) and were down-regulated (at the second biopsy) within the cycle. Strikingly, all of the marker molecules investigated showed negative or weak and inadequate expression in the mid-secretory phase. Numbers of LGLs remained as low as in the proliferative phase. In contrast, fertile patients were found to express these marker molecules distinctly in the mid-secretory phase. CONCLUSIONS: It may be hypothesized that a severe deficiency of these angiogenesis-related marker molecules leads to defective development of the endometrium, which remains thin. Deficient angiogenetic development may thus provide an explanation for the endometrial factor that causes infertility. Further investigations will need to focus on identifying the regulating factors that act between steroid receptor activation and the expression of these marker molecules.

Redistribution of adhering junctions in human endometrial epithelial cells during the implantation window of the menstrual cycle.

The human uterine epithelium is characterised by remarkable plasticity with cyclic changes in differentiation that are controlled by ovarian steroid hormones to optimise conditions for embryo implantation. To understand whether and how cell-cell adhesion is affected, the localisation of junction proteins was studied throughout the menstrual cycle. Expression patterns were examined by immunofluorescence in 36 human endometrial specimens of different cycle stages. Antibodies against the desmosomal proteins desmoplakin 1/2 (Dp 1/2) and desmoglein 2 (Dsg 2), the adherens junction proteins E-cadherin and ß-catenin and also the common junctional linker protein plakoglobin showed a strong subapical staining during the proliferative phase until the early luteal phase (day 20). In the mid- to late luteal phase, however, these junctional proteins redistributed over the entire lateral plasma membranes. In contrast, tight junction proteins (ZO-1, claudin 4) remained at their characteristic subapical position throughout the menstrual cycle. mRNA levels of Dp 1/2, E-cadherin and ZO-1 obtained by real time RT-PCR were not significantly changed during the menstrual cycle. The observed redistribution of desmosomes and adherens junctions coincides with the onset of the so called implantation window of human endometrium. We propose that this change is controlled by ovarian steroids and prepares the endometrium for successful trophoblast invasion.