Irmgard Classen-Linke

The first step of embryonic trophoblast invasion involves penetration of the endometrial epithelium, which is a unique biological process involving the junction-free apical surfaces of two distinct epithelia. Although cell–cell junctions have been shown to play a role in the establishment of endometrial receptivity, data on their role in the invasion process are lacking. To examine whether human transepithelial trophoblast invasion is linked to heterologous junction formation between trophoblast and endometrial epithelial cells, we used a three-dimensional in vitro implantation model. Hybridoma-derived AC-1M88 extravillous trophoblast spheroids of defined size (∼300 µm) were placed either on endometrial adenocarcinoma-derived Ishikawa monolayers or on biopsy-derived primary endometrial epithelial cell monolayers. Labeling trophoblast and endometrial cells with different fluorescent dyes allowed differentiation of the two cell populations and monitoring of the invasion process for up to 2 days after attachment. Trophoblast invasion was tracked by fluorescence microscopy and quantified with the help of image analysis tools. We observed collective transepithelial invasion of the trophoblast into the endometrial monolayers. Junction formation was then assessed by transmission electron microscopy and immunofluorescence microscopy. We observed that transepithelial trophoblast invasion was accompanied by de novo formation of tight junctions, adherens junctions, and desmosomes between trophoblast and endometrial cells. The relevance of desmosomal adhesion for invasion was furthermore examined by using blocking antibodies against the desmosomal cadherin desmoglein 2. We could demonstrate that the blocking antibodies stalled the invasion process. Together, we conclude that formation of heterologous cell–cell junctions between trophoblast and endometrial epithelial cells is a robust and important mechanism that facilitates implantation. These findings extend previous studies and offer completely new insights into the functional contribution of cell–cell junction rearrangement to embryo implantation.

Tissue remodeling of human endometrium occurs during the menstrual cycle to prepare for embryo adhesion and invasion. The ovarian steroid hormones 17β-estradiol and progesterone control the menstrual cycle to achieve the receptive state during the “window of implantation” (WOI). Here, we focus on the human endometrial epithelium and its changes in polarity, adhesion, cytoskeletal organization and the underlying extracellular matrix enabling embryo implantation. The adhesion and invasion of the trophoblast via the apical plasma membrane of epithelial cells is a unique cell biological process, which is coupled to partial epithelial–mesenchymal transition (EMT). Given the fundamental species differences during implantation, we restrict the review mainly to the human situation and focus on cell culture systems to study the interaction between human trophoblast and endometrial cells. We summarize current knowledge based on the relatively scarce in vivo data and the steadily growing in vitro observations using various cell culture systems.

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.

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.

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.

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 – β₃ 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. Einleitung Diese Studie untersuchte subfertile Patientinnen mit einem abnorm dünnen Endometrium nach einer Infertilitätsbehandlung. Da ausreichende Hormonkonzentrationen im Blut dieser Frauen gemessen wurden, lag die Vermutung nahe, dass ein endometrialer Faktor für die verminderte Fertilität eine Rolle spielen könnte. Methoden Um möglichen Ursachen für die verminderte Fertilität auf den Grund zu gehen, wurden bei jeder Patientin Biopsien des Endometriums in der späten Proliferationsphase sowie in der mittleren Sekretionsphase eines Menstruationszyklus durchgeführt. Endometriale Biopsien von fertilen Frauen mit regulärem Menstruationszyklus, die sich einer Hysterektomie wegen gutartiger Gebärmutterveränderungen unterziehen mussten, dienten als Positivkontrollen. Die Gewebeproben wurden auf die Expression von Steroidhormonrezeptoren sowie mit dem Proliferationsmarker Ki-67untersucht. Mithilfe von Antikörpern gegen Markermoleküle der endometrialen Rezeptivität: β3-Integrin, VEGF, LIF und CD56 (große granuläre Lymphozyten, LGLs), wurden immunhistochemische Untersuchungen durchgeführt. Ergebnisse Die Steroidhormonrezeptoren für Östrogen (E2) und Progesteron (P) waren bei der ersten in einem Zyklus durchgeführten Biopsie normal exprimiert und in der zweiten Biopsie im selben Zyklus herunterreguliert. Auffallend war, dass die Expression aller untersuchten Marker in der mittleren Sekretionsphase negativ oder schwach bzw. ungenügend war. Die Anzahl der LGLs blieb so niedrig wie in der Proliferationsphase. Dagegen wurden diese Markermoleküle bei fertilen Patientinnen in der mittleren Sekretionsphase deutlich exprimiert. Schlussfolgerungen Es wurde die Hypothese aufgestellt, dass die starke Defizienz dieser Markermoleküle, die in Beziehung zur Angiogenese stehen, zu einer ungenügenden Ausbildung des Endometriums führt, das dann dünn bleibt. Eine unzureichende angiogenetische Entwicklung mag daher der Grund für eine endometriale Insuffizienz sein, die zur Infertilität führt. Weitergehende Studien sind nötig, um die Faktoren zu identifizieren, die zwischen der Steroidrezeptor-Aktivierung und der Expression dieser Markermoleküle regulierend wirken.

Establishing a strong link early on between preclinical coursework and the clinical context is necessary for students to be able to recognize the practical relevance of the curriculum during their preclinical anatomical courses and to transfer knowledge more easily. Our objective was to enhance the clinical relevance of a preclinical anatomy course for second-year medical students of dentistry by implementing an interdisciplinary skills training course on “Palpation of the Head and Neck Muscles” and to measure the learning outcomes. For the curricular development of the expanded course module, Kern’s 6-step approach was applied including subjective evaluation. We used a peer-teaching format supported by an e-learning application. A randomized control study measured effects of the two components (skills training, e-module) on learning outcomes. Four learning methods were compared: (1) lecture, (2) lecture + e-module, (3) lecture + skills training, (4) lecture + skills training + e-module. An objective structured clinical examination (OSCE) was used to measure and compare learning outcomes. The two-way variance analysis demonstrated that participation in the skills training had a statistically significant effect on the OSCE results (p = 0.0007). Students who participated in the skills training did better (φ 107.4 ± 14.4 points) than students who only attended the lecture (φ 88.8 ± 26.2 points). Students who used the e-module but did not attend the skills training earned a slightly but not significantly higher average number of points (φ 91.8 ± 31.3 points) than those who only attended the lecture. The learning outcomes of the skills training were again significantly increased when the training was combined with the e-module (φ 121.8 ± 21.8 points), thus making it the ideal method for achieving the learning objectives defined in this study. The “Palpation of the Head and Neck Muscles” interdisciplinary skills training course linking basic anatomical knowledge and clinical skills led to clearly improved learning outcomes for both, anatomical knowledge and clinical skills. The additional use of an e-learning tool (e-module) improved the learning effect.