Patterns of menstrual cycle, menstrual pain and medication usage in young women from high- and middle-income countries.

RESEARCH QUESTION: Do patterns of the menstrual cycle, menstrual pain and the use of medication for menstrual pain differ between young women from high-income countries (HIC) and middle-income countries (MIC)? DESIGN: A multinational, multicentre, cross-sectional study using pen-and-paper questionnaires was conducted between 2016 and 2021 to assess patterns of the menstrual cycle, menstrual pain and the use of medication for menstrual pain. Various parameters were evaluated to identify high-risk factors for severe menstrual pain in women from two HIC (n = 1550) and nine MIC (n = 7139). RESULTS: From a total of 9114 young women, 4920 medical students (HIC n = 696, MIC n = 4224) and 3769 nursing students (HIC n = 854, MIC n = 2915) were included in this study. Compared with those from HIC, a significantly higher proportion of medical and nursing students from MIC reported cyclic pain (83.9% and 86.8%, respectively) and acyclic pain (33.8% and 31.9%, respectively) (both P < 0.001). Multivariate regression analysis revealed that low body mass index and early onset of menarche were independent risk factors for severe cyclic/acyclic pain among women from HIC, and a family history of menstrual pain was a risk factor for severe cyclic/acyclic pain among women from HIC and MIC. CONCLUSIONS: Differential patterns of the menstrual cycle, menstrual pain and use of medication for menstrual pain were found between young women from HIC and MIC. A proper educational programme may be necessary for these women and healthcare providers to understand the consequences of intractable cyclic/acyclic pain, in order to facilitate early detection and timely management of menstrual pain and its negative consequences, such as endometriosis.

Association between Uterine Adenomyosis and Infertility: Role of Axonemal Alteration in Apical Endometria.

ABSTRACT: Uterine adenomyosis is an estrogen-dependent chronic inflammatory condition and may cause painful symptoms, abnormal uterine bleeding, and/or subfertility/infertility. It is characterized by the presence of endometrial glands and stroma within the myometrium causing enlargement of the uterus as a result of reactive hyperplastic and/or hypertrophic change of the surrounding myometrium. Similar to endometriosis, adenomyosis has a negative impact on female fertility. Abnormal uterotubal sperm transport, tissue inflammation, and the toxic effect of chemical mediators have been proposed as contributing factors. Inflammation-induced damage of the mucosal cilia in the fallopian tube has been reported. Besides other proposed mechanisms, our most recent study with transmission electron microscopy analysis indicated that microvilli damage and an axonemal alteration in the apical endometria occur in response to endometrial inflammation. This may be involved in the negative fertility outcome in women with adenomyosis. 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.

The role of innate and adaptive immunity in endometriosis.

The innate and adaptive immune systems are the two key branches that determine host protection at all mucosal surfaces in human body, including the female reproductive tract. The pattern recognition receptors within the host that recognize pathogen-associated molecular patterns are expressed on the cells of the innate immune system. Rapidly reactive, theinnate immune system, responds immediately to the presence of infectious or other non-self agents, thereby launching an inflammatory response to protect the host until the activation of slower adaptive immune system. Macrophages, dendritic cells, and toll-like receptors are integral components of the innate immune system. In contrast, T-helper (Th1/Th2/Th17) cells and regulatory T (Treg) cells are the primary components of adaptive immune system. Studies showed that the growth and progression of endometriosis continue even in unilateral ovariectomized animal suggesting that besides ovarian steroid hormones, the growth of endometriosis could be regulated by innate/adaptive immune systems in pelvic environment. Recent reports demonstrated a potential role of Th1/Th2/Th17/Treg cells either individually or collectively in the initiation, maintenance, and progression of endometriosis. Herewe review the fundamental knowledge of innate and adaptive immunity and elaborate the role of innate and adaptive immunity in endometriosis based on both human and experimental data.

Bacterial infection in endometriosis: a silver-lining for the development of new non-hormonal therapy?

The pathogenesis of endometriosis is a hotly debated topic, yet still cloaked in multiple layers of hypothetical theories. A recent report raises the possibility that bacterial infection, especially those of the genus Fusobacterium, may be the cause of endometriosis, at least in certain women. More importantly, the demonstration that treatment with broad-spectrum antibiotics significantly reduced the size of lesions in a mouse endometriosis model rekindles the hope for new non-hormonal treatments. The development of new therapies has been plagued by strings of unsuccessful clinical trials over the last two decades. Is this antibiotic therapy, a silver lining for the research and development of non-hormonal drugs for endometriosis?

Neonatal uterine bleeding: Risk factors and its association with endometriosis-related symptoms later in life.

OBJECTIVE: We examined the prevalence and risk factors in association with neonatal uterine bleeding (NUB) by retrospective search of contemporary and historical medical records and investigated the possible association between the history of NUB at birth and endometriosis-related symptoms later in life who are now young women. STUDY DESIGN: This was a retrospective case-controlled cohort study and prospective evaluation of web-based questionnaire survey on symptoms related to endometriosis among young women born with and without NUB. Multiple regression analysis was performed incorporating various confounding variables that may influence the occurrence of NUB or the reporting of endometriosis symptoms later in life. RESULTS: Among the 1093 female neonates born between 1996 and 2000, 105 of them had NUB, yielding with a prevalence of 9.6 %. Of the 807 female babies born between 2013 and 2017, 25 (3.1 %) had NUB. Multiple Logistic regression analysis indicated that younger age of the mother [odds ratio (OR) = 0.92, 95 % confidence interval (CI) = 0.85-1.00, P = 0.048] and longer gestational age of 39 weeks (OR = 3.04, 95 % CI = 1.43-6.45, P = 0.004) and of ≥ 40 weeks (OR = 4.54, 95 % CI = 2.20-9.39, P < 0.0001) of gestation were significantly associated with the occurrence of NUB. While the possibility of recall bias cannot be ruled out, newborn females who had a history of NUB appeared to complain of various endometriosis-related symptoms later in life during adulthood. CONCLUSIONS: We confirmed the validity of the reported prevalence and risk factors of NUB. NUB indeed occurs with a prevalence of 3-10% during the historical and contemporary period. Longer gestational age and younger maternal age may be considered as high-risk factors for the occurrence of NUB. The clinical relevance of our findings remains to be elucidated. Future prospective studies, preferably with larger sample sizes and the inclusion of NUB cases after discharge from the hospital, may further illuminate some unresolved issues. We also need to confirm the endometriosis-related symptoms in women with and without history of NUB via more definitive diagnosis such as imaging and histology.

Hepcidin as a key regulator of iron homeostasis triggers inflammatory features in the normal endometrium.

Menstrual blood, containing high iron levels, can undergo retrograde transport into the abdominal cavity. Excess iron causes oxidative stress and inflammation. Iron metabolism is regulated by hepcidin, and serum hepcidin levels are increased in patients with endometriosis; however, the functions of hepcidin in normal endometrium remain unclear. We therefore aimed to examine hepcidin concentrations in patients with endometriosis and to determine if iron accumulation and hepcidin increased the production of reactive oxygen species (ROS) and inflammation in normal endometrial cells. We determined hepcidin levels in peritoneal fluid and menstrual blood from patients with and without endometriosis (25/16 and 15/15 patients, respectively). We also examined the effects of hepcidin on ferroportin expression, iron accumulation, and ROS generation in normal endometrial stromal cells (NESCs) from 20 women who underwent surgery for uterine leiomyoma, using immunohistochemistry and immunofluorescence analyses and analyzed its effect on the expression of inflammatory cytokines by real-time polymerase chain reaction. There was no significant difference in iron concentrations in menstrual blood or peritoneal fluid between women with and without endometriosis; however, women with endometriosis had significantly higher hepcidin levels in menstrual blood. Hepcidin reduced the expression of ferroportin in NESCs and promoted the accumulation of ferrous iron. Hepcidin plus ferrous iron increased the production of ROS and inflammatory cytokines compared with ferrous iron alone. These results indicate that women with endometriosis have high hepcidin levels in menstrual blood, leading to increased iron production, oxidative stress, and inflammation, which may, in turn, promote the development of endometriosis.

Is neonatal uterine bleeding responsible for early-onset endometriosis?

BACKGROUND: It has been hypothesized that the origin of early-onset endometriosis could be from endometrial mesenchymal stem cells (eMSCs) in neonatal uterine blood (NUB). There is no information on the possible mechanistic basis linking an association between NUB/neonatal endometrium and development of early-onset endometriosis. In this study we performed a series of experiments to clarify the mechanistic link between NUB and/or neonatal endometrium and development of early-onset endometriosis. METHODS: We retrospectively collected postmortem neonatal endometria (n = 15) and prospectively collected NUB (n = 18) of female babies for the analysis of different biological markers including eMSCs. Immunohistochemical analysis of neonatal endometria was performed to examine the expression patterns of ovarian steroid receptors (ER/PGR), decidualization (prolactin, IGFBP1), pre-decidualization (Glycodelin A, α-SMA), proliferation (Ki-67 index), vascularity (CD31 + cells), immunocompetent CD68+, CD45+, CD56 + cells and some putative markers of eMSCs. Cell transfer method and immunocytochemistry were used to investigate the eMSCs and/or endometrial cells in NUB. RESULTS: Immunohistochemical analysis of postmortem neonatal endometria revealed variable staining response to ER/PGR, decidual markers, and substantial proliferative and angiogenic activity. A moderate to strong immunoexpression of Glycodelin-A was found in both neonatal and adult endometria. The tissue infiltration of CD56+, CD45 + and CD68 + immunocompetent cells was significantly low in neonatal endometria than that in adult endometria (p = 0.0003, p < 0.0001, p = 0.034, respectively). No eMSCs or even endometrial cells were detected in NUB. However, a variable expression of some phenotypes of eMSCs (CD90/CD105) was found in neonatal endometria. CONCLUSIONS: Based on our serial experiments we did not find any supporting evidence for the role of NUB in early-onset endometriosis. Neonatal endometria showed variable expression of ovarian steroid receptors, decidualization, and a substantial amount of proliferative and angiogenic activity. As an alternative mechanism, a significantly less tissue accumulation of immunocompetent cells in neonatal endometria may explain the survival of ER + and PGR + cells should they make entry into the pelvis and consequent development of early endometriosis with the onset of ovarian function. Future study with large sample size and application of modified technological tools is warranted to test the NUB hypothesis and to clarify their biological or clinical significance. TRIAL REGISTRATION: not applicable.

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

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.

Low-Nutrient Environment-Induced Changes in Inflammation, Cell Proliferation, and PGC-1α Expression in Stromal Cells with Ovarian Endometriosis.

Although nutrient status plays an important role in cell metabolism, its significance in endometriosis is obscure. Herein, we investigated the effects of a low-nutrient microenvironment on endometriosis. Stromal cells (SCs) from ovarian endometrioma (OESCs) or normal endometrium without endometriosis (NESCs) were isolated and cultured. A low-nutrient microenvironment was replicated by replacing the culture medium with Hank's balanced salt solution. OESC and NESC proliferation under the low-nutrient condition was measured. The expression of exacerbating factors in endometriosis under the low-nutrient condition was examined at the mRNA and protein levels. OESCs showed higher proliferation than NESCs under the low-nutrient condition. In OESCs, the low-nutrient condition upregulated the mRNA expression of vascular endothelial growth factor (VEGF), interleukin-6 and -8, aromatase, Bcl-2, and peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and downregulated that of BAX and induced transcription of PI.3, PII, and exon II. Western blotting revealed elevated VEGF and PGC-1α expression under the low-nutrient condition in OESCs. These changes coincided with the elevated expression of PGC-1α, which was reduced at the mRNA level upon nutrient status rescue. Endometriosis is exacerbated by altered angiogenesis, inflammation, anti-apoptosis, and local estrogen production while trying to survive under a low-nutrient microenvironment; it may be attributed to PGC-1α-mediated metabolic mechanisms.

A targeted educational programme improves fundamental knowledge of menstrual pain and endometriosis in young women: The Endometriosis Awareness Promotion Project.

RESEARCH QUESTION: Would a properly designed educational programme offered to young women improve their awareness and fundamental knowledge of menstrual pain and endometriosis? DESIGN: A multinational cross-sectional study using a pen-and-paper questionnaire among women aged 19-24 years was conducted between 2017 and 2019 to assess fundamental knowledge of menstrual pain and endometriosis. Improvement in knowledge was also analysed using a separate questionnaire completed before, and 1-3 months after, a group discussion, lecture on menstrual pain and endometriosis, or both. RESULTS: Among three groups of students (college [n = 271], medical [n = 877] and nursing [n = 763]), knowledge of menstrual pain and endometriosis was lowest among college students, modest among nursing students and fair among medical students (P < 0.001 for each). The experience of cyclical pain, even when painkillers were taken, was reported by 15.5%, 4.6% and 3.8% of students, respectively. Most students managed their cyclical pain by enduring it or by taking over-the-counter medication. An informative education programme with group discussions, lectures, or both, was successful in improving knowledge and consequences of menstrual pain and endometriosis. Proper education and dissemination of knowledge to college students failed to motivate them to visit gynaecologists; however, medical and nursing students became highly interested in visiting gynaecologists. CONCLUSIONS: An educational programme can improve awareness and knowledge of endometriosis and dysmenorrhoea among young women. The programme motivated nursing and medical students, but not college students, to seek medical attention for early detection and management of endometriosis.

Changes in the proportion of regulatory T cell subpopulations during menstrual cycle and early pregnancy.

PROBLEM: Regulatory T cells (Tregs) play important roles in diseases occurring in women of reproductive age and pregnancy. Tregs are functionally heterogeneous and can be divided into activated Tregs (aTregs), resting Tregs (rTregs), and non-suppressive Tregs (non-Tregs). The purpose of this study is to investigate the change of Treg subpopulations during the menstrual cycle and early pregnancy. METHOD OF STUDY: Two groups of women were enrolled: healthy women aged 20 to 39 years with normal menstrual cycles and patients scheduled to undergo frozen-thawed blastocyst transfer (FT-BT) (subfertile women). Peripheral blood samples were collected at day 5 of the onset of menstruation (follicular phase), 0-2 days after luteinizing hormone (LH) surge (periovulatory phase), and 7-11 days after LH surge (luteal phase) from 20 healthy women. From 23 subfertile women, samples were collected at day 5 (the day of BT) and day 14 (the day of pregnancy testing) of progestogen administration during FT-BT cycle and 9 weeks of gestation if the patient got pregnant. The proportion of total Treg and its subpopulations among CD4+ T cells was analyzed. RESULTS: TTreg and aTreg proportion expanded during periovulatory phase (p < .01) and after pregnancy (p < .05 for tTreg and p < .01 for aTreg). rTreg proportion was significantly high during periovulatory phase (p < .01) and during luteal phase (p < .01). Non-Tregs showed no significant change. CONCLUSIONS: RTregs and aTregs, especially in luteal phase and after getting pregnant, showed significant changes and may play important roles in women of reproductive age.

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

In vitro: 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. Lay summary: Patient-derived cells cultured in the lab are simple and cost-effective methods used to study biological and dysfunctional or disease processes. These tools are frequently used in the field of reproductive medicine. However, the lack of clear recommendations and standardised methodology to guide the laboratory work of researchers can produce results that are not always reproducible and sometimes are incorrect. To remedy this situation, we define here a method to ascertain if researchers who routinely culture cells in the lab agree or disagree on the optimal laboratory techniques. This method will be used to make recommendations for future researchers working in the field of reproductive biology to reproducibly culture endometrial cells in the laboratory.

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.

Occurrence of chronic endometritis in different types of human adenomyosis.

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.

Progesterone Receptor Status of Epithelial Cells as a Predictive Marker for Postoperative Recurrence of Endometriosis.

CONTEXT: Progesterone resistance including progesterone receptor (PR) deficiency contributes to the pathophysiology of endometriosis; however, whether the PR expression levels in ovarian endometrioma (OE) correlate with the postoperative recurrence of endometriosis remains unclear. OBJECTIVE: This study aimed to investigate the association between PR expression levels in OE and the recurrence of endometriosis. METHODS: OE specimens were obtained from 132 patients who underwent conservative surgery for endometriosis. The PR expression levels were evaluated using the H score after immunohistochemical staining. RESULTS: Of the 132 patients, 36 (27.3%) experienced recurrence and 96 (72.7%) did not. No differences were observed in the patient characteristics between the recurrence and nonrecurrence groups except for follow-up period. PR immunoreactivity in the epithelial cells (ECs) was statistically significantly lower in the recurrent group than in the nonrecurrent group (P < .01); however, this change was not observed in the stromal cells. Moreover, multivariable logistic regression analysis revealed that the H score of PR in ECs was an independent factor and was statistically significantly associated with the recurrence of endometriosis (P = .01). Furthermore, we divided the patients into PR-negative or PR-positive groups. The cumulative recurrence rate in the negative PR group was statistically significantly higher than that in the positive PR group (P = .046). CONCLUSION: Low PR expression levels in OE-ECs may predict the recurrence of endometriosis. The PR status in OE-ECs is associated with the pathophysiology of the recurrence of endometriosis, and optimized postoperative management for endometriosis may be provided by referring to the PR status.

Decreased occurrence of endometriosis in women with Chlamydia trachomatis infection.

PROBLEM: Despite abundant reports on the risk role of uterine outflow tract obstruction in endometriosis, information on the occurrence of endometriosis in women with Chlamydia trachomatis infection causing fallopian tube obstruction is unknown. We investigated the role of Chlamydia trachomatis infection with or without fallopian tubal patency in the occurrence of endometriosis. METHODS: This is a retrospective case-controlled cohort study with 539 women who had laparoscopic surgery for several indications during the period between January, 2003 and June, 2010. Women with ectopic pregnancy, uterine anomaly, chromosomal abnormality, primary amenorrhea, and perimenopausal women were excluded. Endometriosis was diagnosed by laparoscopic inspection and confirmed by histopathology. Tubal patency was diagnosed by HSG or laparoscopic chromopertubation test. Presence of chlamydia infection was examined by RT-PCR and serological test. RESULTS: Two-hundred and seven women were enrolled. Eighty-six (41.5%) women had chlamydia infection. Tubal patency and occurrence of endometriosis were significantly decreased among women with chlamydia infection comparing to women without it (P = .005 and P = .0008, respectively). Even among women with patent tube, laparoscopic detection of endometriosis was significantly decreased in chlamydia infected comparing to non-infected women (P = .02). Multiple logistic regression model revealed that previous history of chlamydia infection significantly decreased the occurrence of endometriosis, and was independent of age, menstrual status, parity and tubal patency (odds ratio .44; 95% confidence interval .24-.80; P = .007). CONCLUSION: A decreased occurrence of peritoneal endometriosis was observed in women with Chlamydia trachomatis infection. The possible impairment of retrograde menstrual flow by chlamydia-infected tubal damage may decrease the risk of endometriosis.

Levofloxacin or gonadotropin releasing hormone agonist treatment decreases intrauterine microbial colonization in human endometriosis.

OBJECTIVE: We examined the hypothesis that antibiotic treatment with or without gonadotropin releasing hormone agonist (GnRHa) may decrease intrauterine infection with consequent decrease in tissue inflammation, cell proliferation and angiogenesis in human endometriosis. STUDY DESIGN: This is a prospective non-randomized observational study. Endometrial/endometriotic samples were collected during surgery from 53 women with endometriosis and 47 control women who were treated with levofloxacin (LVFX, 500 mg, once per os) or GnRHa (1.88 mg/IM for 3 months) before surgery. Endometrial samples were analyzed by broad-range polymerase-chain reaction (PCR) amplification of bacteria targeting V5-V6 region of 16S rRNA gene. Immunohistochemical analysis was performed using antibodies against CD138 (Syndecan-1, a marker of plasma cells), CD68 (marker of macrophages), Ki-67 (cell proliferation marker), and CD31 (vascular cells marker). RESULTS: 16S rDNA metagenome assay indicated that treatment with either of LVFX or GnRHa + LVFX significantly decreased some components of major bacterial genera comparing to untreated group. In women with endometriosis, treatment with either of LVFX or GnRHa + LVFX significantly decreased Gardnerella, Prevotella, Acidibactor, Atopobium, Megasphaera, and Bradyrhizobium (p < 0.05 for each) comparing to untreated group. Cochran-Mantel-Haenszel test indicated that occurrence rate of chronic endometritis was significantly decreased after GnRHa + LVFX treatment comparing to GnRHa treatment group (p = 0.041). These findings were coincided with significantly decreased CD68-stained macrophage infiltration, Ki-67- stained cell proliferation and CD31-stained micro-vessel density in endometria and endometriotic lesions with histology proven improvement in the morphological appearance of ovarian endometrioma. CONCLUSIONS: These findings suggest that clinical administration of a broad-spectrum antibiotic with or without GnRHa may be effective in improving uterine infection with decrease of tissue inflammation, cell proliferation, and angiogenesis in human endometriosis.

An axonemal alteration in apical endometria of human adenomyosis.

STUDY QUESTION: Is there any change in the distribution of microvilli and microtubules in the apical endometria of women with adenomyosis? SUMMARY ANSWER: We observed microvilli damage in the apical endometria and an axonemal alteration characterized by abnormal distribution of longitudinal bundles of microtubules within microvilli in women with adenomyosis. WHAT IS KNOWN ALREADY: Human adenomyosis has a negative impact on female fertility. Abnormal utero-tubal sperm transport, tissue inflammation and toxic effect of chemical mediators have been proposed as contributing factors. Inflammation-induced damage of mucosal cilia in the Fallopian tube has been reported. However, information on inflammation-induced damage of microvilli on the apical endometrial cells and its core bundles of microtubules in adenomyosis remains unknown. STUDY DESIGN, SIZE, DURATION: This is a prospective cohort study with subjects undergoing laparoscopic surgery or hysterectomy for clinical indication and evaluations of endometrial biopsy samples in two academic university hospitals. During the period between March 2015 and December 2018, endometrial biopsy samples were prospectively collected from 15 control women and 45 women with adenomyosis for immunohistochemical analysis and a separate cohort of 10 control women with cervical intraepithelial neoplasia Grade 3 (CIN3) and 20 women with adenomyosis for analysis by immunohistochemistry and transmission electron microscopy (TEM). PARTICIPANTS/MATERIALS, SETTING, METHODS: For immunohistochemical study, endometrial biopsy samples were prospectively collected from 15 control women with fibroids, 25 women with focal adenomyosis and 20 women with diffuse adenomyosis after surgery. The diagnosis of fibroid and adenomyosis was made clinically by transvaginal ultrasonography and magnetic resonance imaging and confirmed by histology. Immunohistochemical analysis was performed retrospectively using antibody against CD68 (marker of macrophages) in endometrial biopsy specimens of women with and without adenomyosis. TEM was performed with the apical endometria collected from a separate cohort of 10 control women with CIN3 and 20 women with focal and diffuse adenomyosis for the identification of any change in the distribution of microvilli and longitudinal bundles of microtubules within microvilli. MAIN RESULTS AND ROLE OF CHANCE: Comparing to control endometria and contralateral side, tissue infiltration of macrophages (Mφ) in the endometria was significantly higher on the ipsilateral side of focal adenomyosis (P = 0.02 and P = 0.03, respectively) and anterior/posterior walls of diffuse adenomyosis (P = 0.01 for both). In a subgroup analysis of patients with focal adenomyosis with and without symptoms, the endometria of symptomatic women displayed a tendency of higher Mφ infiltration on the ipsilateral side than in asymptomatic women (P = 0.07). Comparing to contralateral side endometria of symptomatic women, Mφ infiltration was significantly higher in the endometria of symptomatic women collected from the ipsilateral side of focal adenomyosis (P = 0.03). We found a significantly less tissue infiltration of Mφ in the endometria of women with CIN3 than that in endometria of women with focal adenomyosis. TEM analysis showed that number of microvilli in the endometria was significantly decreased on the ipsilateral side (P = 0.003) comparing to that on the contralateral side of focal adenomyosis. The Chi-squared test indicated that cases with abnormal (disruption in the normal arrangement of 9 peripheral pairs + 1 central pair) microtubules (MT) were significantly higher in women with adenomyosis than in cases with normal patterns (P = 0.0016). While contralateral side displayed significantly less abnormal MT (P = 0.0002), ipsilateral side of focal adenomyosis showed significantly higher abnormal MT (P = 0.0164) comparing to normal patterns. Cases with symptomatic adenomyosis showed significantly higher abnormal MT than normal MT (P = 0.0004). An axonemal alteration characterized by abnormal structural distribution of microtubules within microvilli in the apical endometria in response to endometrial inflammation may be involved in adverse reproductive outcome in women with adenomyosis. LIMITATIONS, REASONS FOR CAUTION: The average age of women in this study was high that may be associated with overall decline in fertility regardless of the presence or absence of adenomyosis or endometriosis. We collected endometrial biopsy samples from two completely separate cohorts of women for analysis by immunohiostochemistry and TEM. We need future follow-up study with increased sample size and from the same patients to precisely clarify the mechanistic link between axonemal alteration and negative fertility outcome. WIDER IMPLICATIONS OF THE FINDINGS: Our current findings may have some biological implication to better understand the endometrial epithelial biology and pathology in women with adenomyosis and may open the avenue for future study in other reproductive diseases. The ultra-structural abnormalities of microvilli and microtubules in the apical endometria in response to tissue inflammatory reaction may clarify the possible association between negative fertility outcome and adenomyosis. Our findings may be clinically useful during counseling with symptomatic patients with adenomyosis desiring pregnancy. STUDY FUNDING/COMPETING INTEREST (S): This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology of Japan. There is no conflict of interest related to this study. TRIAL REGISTRATION NUMBER: N/A.

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.