Decreased expression of human heat shock protein 70 in the endometria and pathological lesions of women with adenomyosis and uterine myoma after GnRH agonist therapy

ObjectiveA prominent stress reaction in the pelvis of women with endometriosis and the role of human heat shock protein 70 (HSP70) in inflammation and the growth of endometriosis has been recently demonstrated. We report here expression of HSP70 in tissues derived from GnRH agonist (GnRHa)-untreated and -treated women with adenomyosis and uterine myoma.Study designThis is a case-controlled biological study. Biopsy specimens were collected from pathological lesions and eutopic endometria/autologous myometria of 30 women with adenomyosis, 35 women with uterine myoma and 15 control women during laparoscopy, laparotomy and hysteroscopy. Fourteen women with adenomyosis and 20 women with uterine myoma were treated with GnRHa for a variable period of 3?6 months before surgery. The immunoexpressions of HSP70 and CD68-positive M? in endometria, lesions/myometria were examined by immunohistochemistry. The immunoreactivity of HSP70 in tissues was analyzed by quantitative-histogram (Q-H) scores.ResultsComparing to control women, HSP70 immunoexpression was significantly higher in endometria/myometria and pathological lesions of women with adenomyosis and myoma. A significant positive correlation between Q-H scores of HSP70 and CD68-positive M? numbers was found in the endometria derived from women with adenomyosis (r = 0.388). Treatment with GnRHa significantly decreased Q-H scores of HSP70 in pathological lesions and endometria/myometria of women with adenomyosis and uterine myoma comparing to similar tissues derived from GnRHa-untreated women.ConclusionA variable amount of tissue stress reaction occurred in endometria and pathological lesions of women adenomyosis and uterine myoma that can be effectively suppressed after GnRHa treatment

ENDOCELL-Seud : a Delphi protocol to harmonise methods in endometrial cell culturing

culturing of endometrial cells obtained from the uterine mucosa or ectopic sites is used to study molecular and cellular signalling relevant to physiologic and pathologic reproductive conditions. However, the lack of consensus on standard operating procedures for deriving, characterising and maintaining primary cells in two- or three-dimensional cultures from eutopic or ectopic endometrium may be hindering progress in this area of research. Guidance for unbiased in vitro research methodologies in the field of reproductive science remains essential to increase confidence in the reliability of in vitro models. We present herein the protocol for a Delphi process to develop a consensus on in vitro methodologies using endometrial cells (ENDOCELL-Seud Project). A steering committee composed of leading scientists will select critical methodologies, topics and items that need to be harmonised and that will be included in a survey. An enlarged panel of experts (ENDOCELL-Seud Working Group) will be invited to participate in the survey and provide their ratings to the items to be harmonised. According to Delphi, an iterative investigation method will be adopted. Recommended measures will be finalised by the steering committee. The study received full ethical approval from the Ethical Committee of the Maastricht University (ref. FHML-REC/2021/103). The study findings will be available in both peer-reviewed articles and will also be disseminated to appropriate audiences at relevant conferences

Pathogenesis of Human Adenomyosis: Current Understanding and Its Association with Infertility

The aim of this review article was to summarize our current understanding on the etiologies and pathogenesis of human adenomyosis and to clarify the relative association between adenomyosis and infertility. The exact pathogenesis of adenomyosis is still elusive. Among different reported concepts, direction invagination of gland cells from the basalis endometrium deep into myometrium is the most widely accepted opinion on the development of adenomyosis. According to this concept, endometrial epithelial cells and changed fibroblasts, abnormally found in the myometrium in response to repeated tissue injury and/or disruption at the endometrium-myometrium interface (EMI), elicit hyperplasia and hypertrophy of the surrounding smooth muscle cells. In this review, a comprehensive review was performed with a literature search using PubMed for all publications in English and Japanese (abstract in English), related to adenomyosis and infertility, from inception to April 2021. As an estrogen-regulated factor, hepatocyte growth factor (HGF) exhibits multiple functions in endometriosis, a disease commonly believed to arise from the functionalis endometrium. As a mechanistic basis of gland invagination, we investigated the role of HGF, either alone or in combination with estrogen, in the occurrence of epithelial-mesenchymal transition (EMT) in adenomyosis. Aside from microtrauma at the EMI, metaplasia of displaced Müllerian remnants, differentiation of endometrial stem/progenitor cells within the myometrium and somatic mutation of some target genes have been put forward to explain how adenomyosis develops. In addition, the possible role of microRNAs in adenomyosis is also discussed. Besides our knowledge on the conventional classification (focal and diffuse), two recently proposed classifications (intrinsic and extrinsic) of adenomyosis and the biological differences between them have been described. Although the mechanistic basis is unclear, the influence of adenomyosis on fertility outcome is important, especially considering the recent tendency to delay pregnancy among women. Besides other proposed mechanisms, a recent transmission election microscopic (TEM) study indicated that microvilli damage and an axonemal alteration in the apical endometria of human adenomyosis, in response to endometrial inflammation, may be involved in negative fertility outcomes. We present a critical analysis of the literature data concerning the mechanistic basis of infertility in women with adenomyosis and its impact on fertility outcome

Bacterial contamination hypothesis: a new concept in endometriosis

Abstract Background Endometriosis is a multifactorial disease that mainly affects women of reproductive age. The exact pathogenesis of this disease is still debatable. The role of bacterial endotoxin (lipopolysaccharide, LPS) and Toll‐like receptor 4 (TLR4) in endometriosis were investigated and the possible source of endotoxin in the pelvic environment was examined. Methods The limulus amoebocyte lysate test was used to measure the endotoxin levels in the menstrual fluid and peritoneal fluid and their potential role in the growth of endometriosis was investigated. Menstrual blood and endometrial samples were cultured for the presence of microbes. The effect of gonadotrophin‐releasing hormone agonist (GnRHa) treatment on intrauterine microbial colonization (IUMC) and the occurrence of endometritis was investigated. Main findings (Results) Lipopolysaccharide regulates the pro‐inflammatory response in the pelvis and growth of endometriosis via the LPS/TLR4 cascade. The menstrual blood was highly contaminated with Escherichea coli and the endometrial samples were colonized with other microbes. A cross‐talk between inflammation and ovarian steroids or the stress reaction also was observed in the pelvis. Treatment with GnRHa further worsens intrauterine microbial colonization, with the consequent occurrence of endometritis in women with endometriosis. Conclusion For the first time, a new concept called the “bacterial contamination hypothesis” is proposed in endometriosis. This study's findings of IUMC in women with endometriosis could hold new therapeutic potential in addition to the conventional estrogen‐suppressing agent

Occurrence of chronic endometritis in different types of human adenomyosis

Abstract Purpose Human adenomyosis has an adverse effect on female fertility. Exact mechanistic basis is still unclear. We investigated the occurrence of chronic endometritis (CE) in different types of human adenomyosis. Methods This is a prospective non‐randomized observational study enrolling patients with focal (n = 30), diffuse (n = 26), intrinsic (n = 23), and extrinsic (n = 10) adenomyosis. Endometrial biopsy samples were collected from hysterectomy specimens. Immunohistochemical analysis was performed using antibody against CD68 (Mφ marker) with biopsy samples of intrinsic/extrinsic adenomyosis and CD138 (Syndecan‐1), a marker of plasma cells, in all biopsy samples. Results In GnRHa‐untreated groups, a higher trend in the occurrence of CE, as characterized by infiltration of ≥1 plasma cells in endometrial stroma, was found in women with focal (58.8%, p = 0.0849) and diffuse adenomyosis (60.0%, p = 0.0841) comparing to control women (10.0%). In women with focal adenomyosis, ipsilateral side showed a significantly higher occurrence of CE (58.8%) than on the contralateral side (11.7%) (p = 0.043). Tissue infiltration of macrophages in endometria was significantly higher in intrinsic than in extrinsic adenomyosis (p = 0.03) without showing any significant difference in the occurrence of CE between these two variants of adenomyosis. Conclusion A variable occurrence of CE in different types of adenomyosis may be involved in adverse reproductive outcome

Expression profiles of E/P receptors and fibrosis in GnRHa-treated and -untreated women with different uterine leiomyomas.

Differential expressions of estrogen/progesterone receptors (ER/PR) and individual component of extracellular matrices derived from fibroid are reported. Information on the pattern of change in ER/PR expression and amount of tissue fibrosis after hormonal treatment is unclear. We investigated pattern of change in ER/PR expression and percentage of tissue fibrosis in different uterine leiomyomas after gonadotropin-releasing hormone agonist (GnRHa) treatment. Biopsy specimens from fibroids and adjacent myometria were collected after surgery from women with submucosal myoma (SMM, n = 18), intramural myoma (IMM, n = 16) and subserosal myoma (SSM, n = 17). A proportion of women in each group of fibroid underwent treatment with GnRHa for a variable period of 3-6 months. Tissue expression of ER and PR was analyzed by immunohistochemistry. In vitro cell proliferation effect of GnRHa on human umbilical vein endothelial cells (HUVECs) was examined. Distribution of tissue fibrosis was examined by Masson's trichrome staining with computer-captured image analysis of fibrosis derived from different types of fibroid. PR content was significantly higher than ER in tissues derived from GnRHa-untreated women with SMM and SSM (p = 0.04 for both). Comparing to untreated group, GnRHa-treatment significantly decreased either ER or PR expression in different fibroids. Exogenous treatment with GnRHa dose-dependently decreased proliferation of HUVECs. No significant difference was observed in the percentage of fibrosis in tissues collected from GnRHa-treated and -untreated women with fibroids. The distribution of fibrosis in myoma/myometria and occurrence of fibrosis in perivascular area showed an increasing trend with higher age of the women and with larger size of fibroids. Our findings suggest that despite estrogen dependency, higher PR content in GnRHa-untreated group may indicate a potential role of progesterone in leiomyoma growth. Although GnRHa therapy may shrink fibroids and reduce risk of bleeding during surgery, the occurrence of diffuse tissue fibrosis may impair effective reduction of fibroid size after hormonal treatment

Association between tissue stress reaction and ACE2/TMPRSS2 expression in endometria of reproductive aged women before and during Covid-19 pandemic

Abstract Background A potential concern has been raised regarding fertility and reproductive outcome during the Covid-19 pandemic with growing stress and anxiety. However, information on the association between tissue stress reaction and expression profiles of SARS-CoV-2 viral entry proteins, ACE2 and TMPRSS2, in endometria collected from women before (pre-pandemic) and during the Covid-19 pandemic (in-pandemic) is unknown. We aim to investigate the relationship between the expression of stress-reactive proteins and of ACE2 and TMPRSS2 in endometria collected from women during these two different time frames. Methods We retrospectively retrieved tissue blocks of endometrial samples from 25 women in 2019 (pre-pandemic) and 25 women in 2020 (in-pandemic) who underwent hysterectomy for different gynecological indications. Immunohistochemical analysis was performed with endometrial tissue samples that were collected before and during the pandemic, using respective antibodies targeting ACE2/TMPRSS2, ADRB2 and NK1R (stress and anxiety receptor markers, respectively). The quantification of immunoreactive cells for each marker was calculated by the immunoreactive score (IRS) analysis. This retrospective cohort study was limited to small sample size. Results No significant differences in the IRS of ACE2 and TMPRSS2 were found between the endometria that were collected before and during the pandemic with a lack of correlation between ACE2 and TMPRSS2 expression in respective endometria (r = 0.11, pre-pandemic; r = 0.04, in-pandemic). The immunostaining levels of stress marker, ADRB2 were significantly higher in the endometria of in-pandemic group (p = 0.015) comparing to that of pre-pandemic group. Pearson’s correlation coefficient analysis showed a significant correlation in the expression between ADRB2 and TMPRSS2 (r = 0.41, p = 0.042) in the endometria of in-pandemic group but not in the pre-pandemic group. Conclusion The rise in stress and anxiety among women during current pandemic may elicit substantial amount of tissue stress reaction with consequent increase in the expression of SARS-CoV-2 viral entry proteins in their endometria. A lack of correlation between ACE2 and TMPRSS2 expression in endometria may reassure women in their reproductive age that they are not more susceptible to infection by SARS-CoV-2 and suggest that stressful women during this pandemic can safely decide to conceive naturally or by artificial reproductive technology