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.

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.

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.

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.

Association between FOXP3+ regulatory T-cells and occurrence of peritoneal lesions in women with ovarian endometrioma and dermoid cysts.

RESEARCH QUESTION: Is there any relationship between numbers of FOXP3+ regulatory T-cells (Treg) and occurrence of peritoneal lesions in women with ovarian endometrioma and dermoid cysts? DESIGN: Retrospective and prospective case-controlled cohort study. Peritoneal lesions were collected from 27 women with ovarian endometrioma and 25 women with dermoid cysts. Peritoneal fluid was collected from 36 women with ovarian endometrioma and 42 women with dermoid cysts. Tissue expression of Forkhead box P3 (FOXP3), one of the transcription factors of Treg cells, and transforming growth factor-beta (TGF-ß) were examined by immunohistochemistry. Interleukin-6 (IL-6) and TGF-ß levels in the peritoneal fluid were measured by enzyme-linked immunosorbent assay. RESULTS: Ovarian endometrioma cases with coexisting peritoneal lesions were significantly more frequent than dermoid cyst cases with coexistent peritoneal lesions (269/350 [76.9%] versus 74/414 [17.9%]; P < 0.001). Numbers of FOXP3+ Treg cells were significantly higher in peritoneal lesions of women coexistent with ovarian endometrioma (F = 21.52, P < 0.001) and dermoid cysts (F = 22.01, P < 0.001) compared with women without peritoneal lesions. Higher FOXP3+ Treg cell numbers in pathological lesions corresponded with significantly higher TGF-ß (P < 0.001) and lower IL-6 (P = 0.020) levels in peritoneal fluid of women with peritoneal lesions compared with women without lesions. CONCLUSIONS: This study confirms current speculation that endometriosis is related to alteration in Treg cells, causing survival and implantation of ectopic endometrial lesions in women with endometrioma and dermoid cysts. The findings may clarify why only 10% of women in the general population develop endometriosis despite cyclic menstruation with retrograde flow occurring in >90% of women.

Biological differences between focal and diffuse adenomyosis and response to hormonal treatment.

RESEARCH QUESTION: Is there any difference in ovarian steroid receptor expression and pattern of fibrosis in focal and diffuse adenomyosis and response to hormonal treatment? DESIGN: Prospective controlled study where biopsy samples were prospectively collected after surgery from 30 women with focal adenomyosis, 21 women with diffuse adenomyosis and 20 women with uterine myoma. Some of these women underwent 3-6 months of treatment with gonadotrophin-releasing hormone agonist (GnRHa) before surgery. Tissue expression of oestrogen receptor (ER) and progesterone receptor (PR) was analysed by immunohistochemistry. Distribution of tissue fibrosis was examined by Masson's trichrome staining with computer-based image analysis of fibrosis in tissues derived from women with and without adenomyosis. RESULTS: There was no difference in ER/PR expression in gland cells/stromal cells of adenomyotic lesions on the ipsilateral side of focal adenomyosis and the anterior/posterior walls of diffuse adenomyosis. Compared to myoma tissues, a relatively decreased expression of ovarian steroid receptors was observed in both focal and diffuse adenomyosis. Image analysis of tissue fibrosis indicated more fibrosis in both focal and diffuse adenomyosis compared to fibrosis in the myometrium derived from women with uterine myoma. The pattern of fibrosis was no different in tissues derived from GnRHa-treated and -untreated women with focal and diffuse adenomyosis. CONCLUSIONS: No difference was found in the expression of ER/PR and entity of fibrosis between women with focal and diffuse adenomyosis regardless of GnRHa treatment. A lower expression of ER/PR compared to myoma tissue potentially clarifies the biological rationale of non-response to hormonal therapies for adenomyosis.

Biological differences between intrinsic and extrinsic adenomyosis with coexisting deep infiltrating endometriosis.

RESEARCH QUESTION: Is there a biological difference between intrinsic and extrinsic adenomyosis with coexisting deep infiltrating endometriosis (DIE)? DESIGN: In this prospective controlled study, biopsy specimens were collected after surgery from 23 women with intrinsic adenomyosis and 10 women with extrinsic adenomyosis with coexisting DIE lesions. Histological evaluation was carried out by immunoreaction to Ber-EP4 (epithelial cell marker) and CD10 (stromal cell marker). Tissue expression of oestrogen and progesterone receptors was analysed by immunohistochemistry. Tissue fibrosis was examined by Masson's trichrome staining with computer-based image analysis of fibrosis in respective samples. RESULTS: The detection rate of coexistent DIE was significantly higher in women with extrinsic adenomyosis (9/10 [90.0%]) than in women with intrinsic adenomyosis (3/23 [13.0%]; P < 0.001). The pattern of Ber-EP4-stained glands and CD10-stained stromal cells of extrinsic adenomyosis was similar to that of coexistent DIE lesions. In contrast, the pattern of gland and stromal cells was similar to the endometrium in the cases with intrinsic adenomyosis. Unlike extrinsic adenomyosis, progesterone receptor expression was significantly decreased in both gland cells (P < 0.05) and stromal cells (P < 0.05) of intrinsic adenomyosis. Although relatively more fibrosis was seen in biopsy samples of extrinsic adenomyosis and coexistent DIE than in intrinsic adenomyosis and their coexistent DIE, no significant difference was found. CONCLUSIONS: Extrinsic adenomyosis may be considered as adenomyosis externa based on a close histological and biological relationship between extrinsic adenomyosis and coexistent DIE. Our findings may contribute to the understanding of a possible biological origin of two newly classified intrinsic and extrinsic adenomyosis.

Bacterial contamination hypothesis: a new concept in endometriosis.

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.

Involvement of hepatocyte growth factor-induced epithelial-mesenchymal transition in human adenomyosis.

Adenomyosis is commonly believed to arise from the basalis endometrium. As an estromedin growth factor, hepatocyte growth factor (HGF) exhibits multiple functions in endometriosis, a disease commonly believed to arise from the functionalis endometrium. Here, we investigated the role of HGF in the occurrence of epithelial-mesenchymal transition (EMT) in adenomyosis. Full-thickness-biopsy specimens from endometrium to myometrium were collected after hysterectomy from women with and without adenomyosis. The relationship between HGF and E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell markers) was examined at the gene and protein levels using endometrial epithelial cells (EECs) in culture and tissues by quantitative RT-PCR and immunohistochemistry. The gene and protein expressions of two transcriptional repressors of E-cadherin, SLUG and SNAIL, were examined using Ishikawa cells and in response to HGF and estrogen (E2). HGF down-regulated E-cadherin and up-regulated N-cadherin mRNA expression in EECs, and an inverse relationship in protein expression between HGF and E-cadherin was observed in basalis endometria derived from women with diffuse and focal adenomyosis. HGF induced morphological changes of EECs from a cobblestone-like appearance to spindle-shaped cells and promoted migration of EECs. Ishikawa cells exhibited up-regulation of SLUG/SNAIL gene expression in response to both HGF and E2 with an additive effect between them. HGF- and E2-promoted SLUG/SNAIL gene expression was significantly abrogated after pretreatment of cells with anti-HGF antibody or ICI 182720, an estrogen receptor antagonist. HGF may be involved in gland invagination deep into the myometrium by inducing EMT at the endo-myometrial junction in women with adenomyosis.

Outcome of conservative laparoscopic surgery for adnexal torsion through one-stage or two-stage operation.

AIM: We investigated the outcome on ovarian appearance and occurrence of adhesion after conservative laparoscopic surgery for adnexal torsion during reproductive age. MATERIAL AND METHODS: From April 2009 to September 2012, we treated patients with clinically suspected adnexal torsion who desired future pregnancy. We performed conservative surgery, such as cystectomy or detorsion at one-stage operation, but switched to salpingo-oophorectomy in complicated cases. We evaluated adnexal condition and pattern of adhesion by careful assessment with two-stage laparoscopy or second-look laparoscopy after first surgery. RESULTS: Mean age of patients was 25 ± 8 years. Among 37 patients with suspected adnexal torsion, 18 (49%) had adnexal torsion at first surgery. Conservative treatment was carried out in 14 of 18 cases. We obtained informed consent for second-look laparoscopy or two-stage operation in six of these 14 cases. Among these six patients, two cases were treated with only detorsion by one-stage operation and cystectomy was performed in the other four cases at first operation. At subsequent surgery, the ovary appeared normal in six cases with occurrence of mild to moderate adhesion around the adnexal lesion. Of note, two cases with para-ovarian cyst had torsion that showed complete tubal occlusions and associated severe adhesions. No major complications (peritonitis, thrombotic emboli) were observed after conservative laparoscopic surgery. CONCLUSION: Conservative laparoscopic surgery is a safe procedure to preserve ovarian function in women with adnexal torsion. Careful attention and measures should be considered during follow-up management with the fact in mind that adhesion is a common occurrence and even tubal occlusion may occur in some cases.

Occult microscopic endometriosis: undetectable by laparoscopy in normal peritoneum.

STUDY QUESTION: Is there any occurrence of hidden (occult) endometriotic lesions in normal peritoneum of women with and without visible endometriosis? SUMMARY ANSWER: We detected a slightly higher occurrence of occult microscopic endometriosis (OME) in normal peritoneum of women with visible endometriosis than in control women. WHAT IS KNOWN ALREADY: Based on a small number of cases, the concept of invisible microscopic endometriosis in visually normal peritoneum has been reported for more than a decade but there is controversy regarding their tissue activity and clinical significance. STUDY DESIGN, SIZE, DURATION: This case-controlled research study was conducted with prospectively collected normal peritoneal samples from 151 women with and 62 women without visible endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Normal peritoneal biopsy specimens from different pelvic sites of were collected during laparoscopy. A histological search of all peritoneal biopsy specimens for the detection of invisible endometriosis was done by immunoreaction to Ber-EP4 (epithelial cell marker), CD10 (stromal cell marker) and Calretinin (mesothelial cell marker). Tissue expression of estrogen/progesterone receptors (ER/PR) and cell proliferation marker, Ki-67, was performed by immunohistochemistry to identify tissue activity. MAIN RESULTS AND THE ROLE OF CHANCE: Three different patterns of OME were detected based on (I) the presence of typical gland/stroma, (II) reactive hyperplastic change of endometrioid epithelial cells with surrounding stroma and (III) single-layered epithelium-lined cystic lesions with surrounding stroma. A higher tendency toward the occurrence of OME was found in women with visible endometriosis (15.2%, 23/151) compared with control women (6.4%, 4/62) (P = 0.06, χ(2) test). The epithelial cells and/or stromal cells of OME lesions were immunoreactive to Ber-EP4 and CD10 but not reactive to Calretinin. ER and PR expression was observed in all patterns of OME lesions. Ki-67 index was significantly higher in pattern I/II OME lesions than in pattern III OME lesions (P< 0.05 for each). LIMITATIONS, REASONS FOR CAUTION: Bias in the incidence rate of OME lesions in this study cannot be ignored, because we could not analyze biopsy specimens from the Pouch of Douglas of women with revised classification of the American Society of Reproductive Medicine Stage III-IV endometriosis due to the presence of adhesions in the pelvis. WIDER IMPLICATIONS OF THE FINDINGS: We re-confirmed a decade long old concept of invisible (occult) endometriosis in visually normal peritoneum of women with visible endometriosis. The existence of a variable amount of tissue activity in these occult lesions may contribute to the recurrence/occurrence of endometriosis or persistence/recurrence of pain manifestation in women even after successful ablation or excision of visible lesions by laparoscopy. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by Grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science. There is no conflict of interest related to this study. TRIAL REGISTRATION NUMBER: Not applicable.

Intra-uterine microbial colonization and occurrence of endometritis in women with endometriosis†.

STUDY QUESTION: Is there any risk of intra-uterine bacterial colonization and concurrent occurrence of endometritis in women with endometriosis? SUMMARY ANSWER: An increase in intra-uterine microbial colonization and concurrent endometritis occurred in women with endometriosis that was further increased after GnRH agonist (GnRHa) treatment. WHAT IS KNOWN ALREADY: Higher bacterial contamination of menstrual blood and increased endotoxin level in menstrual and peritoneal fluids have been found in women with endometriosis than in control women. However, information on intra-uterine microbial colonization across the phases of the menstrual cycle and possible occurrence of endometritis in women with endometriosis is still lacking. STUDY DESIGN, SIZE AND DURATION: This is a case-controlled study with prospective collection of vaginal smears/endometrial samples from women with and without endometriosis and retrospective evaluation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Vaginal smears and endometrial smears were collected from 73 women with endometriosis and 55 control women. Twenty of the women with endometriosis and 19 controls had received GnRHa therapy for a period of 4-6 months. Vaginal pH was measured by intra-vaginal insertion of a pH paper strip. The bacterial vaginosis (BV) score was analyzed by Gram-staining of vaginal smears and based on a modified Nugent-BV scoring system. A panel of bacteria was analyzed by culture of endometrial samples from women treated with GnRHa or not treated. Immunohistochemcial analysis was performed using antibody against Syndecan-1 (CD138) and myeloperoxidase in endometrial biopsy specimens from women with and without endometriosis. MAIN RESULTS AND THE ROLE OF CHANCE: A significant shifting of intra-vaginal pH to ≥4.5 was observed in women with endometriosis compared with control women (79.3 versus 58.4%, P < 0.03). Compared with untreated women, use of GnRHa therapy also shifted vaginal pH to ≥4.5 in both control women (P = 0.004) and in women with endometriosis (P = 0.03). A higher risk of increasing intermediate flora (total score, 4-6) (P = 0.05) was observed in women with endometriosis who had GnRHa treatment versus untreated women. The number of colony forming units (CFU/ml) of Gardnerella, α-Streptococcus, Enterococci and Escherichia coli was significantly higher in endometrial samples from women with endometriosis than control women (P < 0.05 for each bacteria). GnRHa-treated women also showed significantly higher colony formation for some of these bacteria in endometrial samples than in untreated women (Gardnerella and E. coli for controls; Gardnerella, Enterococci and E. coli for women with endometriosis, P < 0.05 for all). Although there was no significant difference in the occurrence of acute endometritis between women with and without endometriosis, both GnRHa-treated controls and women with endometriosis had a significantly higher occurrence of acute endometritis (P = 0.003 for controls, P = 0.001 for endometriosis versus untreated women). Multiple analysis of covariance analysis revealed that an intra-vaginal pH of ≥4.5 (P = 0.03) and use of GnRHa (P = 0.04) were potential factors that were significantly and independently associated with intra-uterine microbial colonization and occurrence of endometritis in women with endometriosis. These findings indicated the occurrence of sub-clinical uterine infection and endometritis in women with endometriosis after GnRHa treatment. LIMITATIONS, REASONS FOR CAUTION: We cannot exclude the introduction of bias from unknown previous treatment with immunosuppressing or anti-microbial agents. We have studied a limited range of bacterial species and used only culture-based methods. More sensitive molecular approaches would further delineate the similarities/differences between the vaginal cavity and uterine environment. WIDER IMPLICATIONS OF THE FINDINGS: Our current findings may have epidemiological and biological implications and help in understanding the pathogenesis of endometriosis and related disease burden. The worsening of intra-uterine microbial colonization and higher occurrence of endometritis in women with endometriosis who were treated with GnRHa identifies some future therapeutic avenues for the management, as well as prevention of recurrence, of endometriosis. Further studies are needed to examine intra-uterine colonization of a broad range of common bacteria as well as different viruses and their role in the occurrence of endometritis. STUDY FUNDING/COMPETING INTERESTS: 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: Not applicable.

Uterine preservation surgery for placental polyp.

AIM: To examine clinical and surgical performances of cases with placental polyps in which uterine preservation surgery was conducted. METHODS: During the period September 2002 to April 2009, we examined eight cases (hysteroscopic resection, six cases; laparotomy, one case; dilatation and curettage, one case) diagnosed with placental polyp that had been treated with polyp extraction surgery. Imaging evaluation was done using magnetic resonance imaging and 2-D ultrasound. RESULTS: Three of the eight cases (37.5%) had been first-time pregnancies. Most of our cases experienced minimal surgical manipulation after medical abortion. Among them, six cases (75%) were mid-term medical abortions, one case (12.5%) received no treatment after spontaneous abortion, and one case (12.5%) had postsurgical abortion (dilatation and curettage). All cases showed variable amount of blood flow in the internal mass and myometrium by color Doppler ultrasound. Magnetic resonance imaging angiography showed contrast effects in the intrauterine cavity and myometrium in selected cases. The average duration from diagnosis to surgery was 32 days (range, 11-105). Color Doppler revealed a reduction in blood flow in five cases during the waiting period until surgery with an average blood loss of 10 g (range, 0-20) during surgery. CONCLUSION: Use of color Doppler ultrasound may be useful in diagnosing placental polyp. Although hysteroscopic resection of placental polyp is effective in patients hoping for uterine preservation, delaying timing of surgery may reduce blood loss during operative procedure.

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.

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.

Pelvic pain in women with ovarian endometrioma is mostly associated with coexisting peritoneal lesions.

STUDY QUESTION: Is the occurrence of pelvic pain in women with ovarian endometrioma associated with coexisting peritoneal lesions (PLs)? SUMMARY ANSWER: Pelvic pain in women with ovarian endometrioma is usually associated with coexisting PLs. An increased tissue inflammatory reaction with elevated prostaglandin (PG) production may be responsible for the generation of pain. WHAT IS KNOWN ALREADY: Severe pelvic pain in women with ovarian endometrioma is reported to be associated with deeply infiltrating endometriosis. However, information on pelvic pain in women with ovarian endometriosis with and without coexistent peritoneal superficial lesions is limited. STUDY DESIGN, SIZE AND DURATION: Retrospective clinical study with case-controlled biological research using prospectively collected tissue samples derived from women with and without endometriosis and their retrospective evaluation. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed a retrospective cohort study conducted in 2988 cases who had laparoscopic surgery for indications of ectopic pregnancy, tubal infertility and other benign gynecologic diseases. We analyzed the occurrence of pelvic pain in the cases with ovarian endometrioma according to the distribution of coexisting PLs and pattern of intrapelvic adhesions. Inflammatory reaction of eutopic and ectopic endometria was measured by immunoreaction to macrophage marker, CD68. The tissue expression of cyclooxygenase (COX) 2 was examined by immunohistochemistry and tissue concentrations of PG F2α were measured by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 2988 surgical cases, 350 (11.7%) were found to have ovarian endometrioma at laparoscopy. Coexisting PLs were present in 269 of these women and in this group 85.4% of cases experienced pelvic pain and 14.6% had no pain. In contrast, among the 81 women with ovarian endometrioma only, 38.3% cases experienced pelvic pain and 61.7% cases had no pain and the difference between the groups was statistically significant (P < 0.01). The infiltration of CD68-immunoreactive macrophages was significantly higher in the eutopic and ectopic endometria of women with peritoneal endometriosis than in ovarian endometrioma. The tissue expression of COX2 and levels of PGF2α were significantly higher in both the eutopic and ectopic endometria derived from women with peritoneal endometriosis than in similar tissues derived from women with ovarian endometrioma. LIMITATIONS, REASONS FOR CAUTIONS: Lack of evaluation in the detection of general or disseminated deeply infiltrating endometriosis in the pelvic cavity could be a bias or limitation in this study. Further multicenter prospective studies are needed to strengthen our current findings. WIDER IMPLICATIONS OF THE FINDINGS: Our findings may provide some new insights to understand the physiopathology of pelvic pain in women with ovarian cystic endometriosis and may hint at proper surgical manipulation to prevent the recurrence of pelvic pain in these women. 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: Not applicable.

Additive effects of inflammation and stress reaction on Toll-like receptor 4-mediated growth of endometriotic stromal cells.

STUDY QUESTION: Is there any combined effect between inflammation and stress reaction on Toll-like receptor 4 (TLR4)-mediated growth of endometriotic cells? SUMMARY ANSWER: A combined effect of local inflammation and stress reaction in the pelvic environment may be involved in TLR4-mediated growth of endometriotic stromal cells. WHAT IS KNOWN ALREADY: In endometriosis, higher endotoxin levels in the menstrual fluid (MF) and peritoneal fluid (PF) and higher tissue concentrations of human heat shock protein 70 (HSP70) in the eutopic and ectopic endometria promote TLR4-mediated growth of endometriotic cells. STUDY DESIGN, SIZE AND DURATION: This is a case-controlled research study with prospective collection and retrospective evaluation of sera, MF, PF and endometrial tissues from 43 women with and 20 women without endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: PF was collected from 43 women with endometriosis and 20 control women during laparoscopy. Sera and endometrial biopsy specimens were collected from a proportion of these women. MF was collected from a separate population of 20 women with endometriosis and 15 control women. HSP70 concentrations in sera, MF, PF and in culture media were measured by ELISA. Gene expression of HSP70 by endometrial cells in response to lipopolysaccharide (LPS) was examined by qRT-PCR. The individual and combined effects of LPS and HSP70 on the secretion of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) by PF-derived macrophages (M[Symbol: see text]) were examined by ELISA, while their effects on endometrial cell proliferation were examined by bromodeoxyuridine and [(3)H]-thymidine incorporation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Concentrations of HSP70 were maximal in MF, intermediate in PF and the lowest in sera. In MF and PF, HSP70 levels were higher in women with endometriosis than in controls. LPS stimulated gene expression and secretion of HSP70 by eutopic endometrial stromal cells (ESCs) and this effect was abrogated after pretreatment of cells with an anti-TLR4 antibody. This effect was significantly higher for ESCs derived from women with endometriosis than for ESCs from control women. Exogenous treatment with either HSP70 or LPS significantly stimulated the production of IL-6 and TNFα by M[Symbol: see text] and promoted the proliferation of ESCs, and a significant additive effect between LPS and HSP70 was observed. While individual treatment with either polymyxin B, an LPS antagonist, or anti-HSP70 antibody was unable to suppress the combined effects of LPS and HSP70 on cytokine secretion or ESC proliferation, pretreatment of cells with the anti-TLR4 antibody was able to significantly suppress their combined effects. LIMITATIONS, REASONS FOR CAUTIONS: Further studies are needed to examine the mutual role between other secondary inflammatory mediators and endogenous stress proteins in promoting pelvic inflammation and growth of endometriotic stromal cells. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that endotoxin and HSP70 are mutually involved in a stress reaction and in inflammation. A combined effect between local inflammation and a stress reaction in pelvic environment may be involved in TLR4-mediated growth of endometriotic cells. Since endometriosis is a multi-factorial disease, it is difficult to explain uniformly its growth regulation by a single factor. Our findings may provide some new insights in understanding the physiopathology or pathogenesis of endometriosis and may hold new therapeutic potential. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Grants-in-Aid for Scientific Research (grant no. 16591671 and 18591837) from the Ministry of Education, Sports, Culture, Science and Technology of Japan (to K.N.K.). There is no conflict of interest related to this study. TRIAL REGISTRATION NUMBER: Not applicable.

Toll-like receptor system and endometriosis.

Endometriosis is an estrogen-dependent chronic inflammatory condition associated with variable degrees of pelvic pain and infertility. Studies have showed that the growth and progression of endometriosis continue even in ovariectomized animals. This indicates that besides ovarian steroid hormones, the growth of endometriosis can be regulated by the innate immune system in the pelvic environment. As a component of innate immune system, increased infiltration of macrophages has been described in the intact tissue and peritoneal fluid of women with endometriosis. Different immune cells and dendritic cells express Toll-like receptors (TLR) and exhibit functional activity in response to microbial products. In this review article, we discuss the role of the TLR system in endometrium and endometriosis and outline the involvement of cytokines/endotoxin in causing adverse reproductive outcome. In the first part of this review article, the fundamentals of innate immune system, functional characteristics of TLR and signaling pathways of TLR4 are discussed for easy understanding by the readers.

Intraperitoneal Carboplatin for Ovarian Cancer - A Phase 2/3 Trial.

BACKGROUND: Intraperitoneal chemotherapy has been shown to be effective at reducing mortality for patients with advanced epithelial ovarian cancer but is not widely used in practice. METHODS: We performed the Intraperitoneal Therapy for Ovarian Cancer with Carboplatin (iPocc) trial as an open-label, international, multi-institutional, randomized phase 2/3 clinical trial in women with newly diagnosed epithelial ovarian cancer who underwent laparotomy or laparoscopy. All patients received intravenous paclitaxel (80 mg/m2 on days 1, 8, and 15 of a 21-day cycle). In addition, patients in the control group received intravenous carboplatin (dose-dense intravenous paclitaxel plus intravenous carboplatin [dd-TCiv]), whereas patients in the experimental group received dose-dense intravenous paclitaxel plus intraperitoneal carboplatin (dd-TCip). The primary end point was progression-free survival (PFS). Secondary end points included overall survival, tumor response, treatment completion rate, and incidence of adverse events (AEs). RESULTS: Among 655 patients randomized to treatment, median (95% confidence interval [CI]) PFS was 20.7 (18.1 to 22.8) months for dd-TCiv (n=328) and 23.5 (20.5 to 26.9) months for dd-TCip (n=327; hazard ratio, 0.83; 95% CI, 0.69 to 0.99; P=0.04). The PFS benefit with dd-TCip was consistent in patients with different baseline characteristics, stage, size of residual tumor, age, and performance status. The treatment completion rates were 68.3 and 59.9% in the dd-TCiv and dd-TCip groups, respectively. The incidence of intraperitoneal catheter-related AEs in the dd-TCip group was 10.1%; there were no such AEs in the dd-TCiv group. CONCLUSIONS: In the first-line treatment of advanced epithelial ovarian cancer, intraperitoneal carboplatin resulted in a modest prolongation of PFS when given with dose-dense weekly paclitaxel regardless of residual tumor size, with no impact on noncatheter-related toxicities. (Funded by the Japan Agency for Medical Research and Development, and others; Japan Registry of Clinical Trials number, jRCTs031180141.)