AMH Concentrations in Peritoneal Fluids of Women With and Without Endometriosis

Background: As its name indicates, anti-Mullerian hormone (AMH) is primarily found as an inhibitor of the Mullerian duct in male fetus. On the other hand, AMH may act as a mediator of Mullerian duct-derived female tissue, such as endometrium in normal and pathological conditions. However, the role of AMH in the functional regulations of endometriosis is not well understood. It can be hypothesized that AMH in peritoneal fluids may affect the activity of peritoneal endometriosis. In this study, we investigated the levels of AMH in peritoneal fluids (PF) in women with and without endometriosis. Methods: PF were collected during laparoscopy from 90 women diagnosed as having advanced endometriosis (rASRM stage III, n = 30; stage IV, n = 60), and 32 women without endometriosis were served as control. Paired serum samples were also collected before the surgery. AMH in PF and serum were measured by ELISA. Individual clinical information was collected. AMH levels were compared according to the presence of endometriosis. The expression of AMHR2 in peritoneal endometriotic lesions obtained during laparoscopy was examined by immunohistochemistry. Results: AMH levels in PF were positively and significantly correlated with serum AMH levels in both women with and without endometriosis (R2 = 0.17, P < 0.0001; R2 = 0.30, P = 0.001, respectively). Serum AMH levels were inversely and significantly correlated with age in women with endometriosis (R2 = 0.092, P = 0.004) and in control women without statistical significance (R2 = 0.078, P = 0.12). AMH levels in PF were also inversely but not significantly correlated with age in women with and without endometriosis (R2 = 0.029, P = 0.11 and R2 = 0.027, P = 0.37, respectively). Mean age and serum AMH levels were not significantly different between two groups. On the other hand, AMH levels in PF were significantly lower in women with endometriosis compared to those of control women [2.15 ± 2.13 (mean ± SD) vs. 4.40 ± 4.77 ng/mL, P = 0.0001]. AMHR2 are localized at glandular epithelium and stromal cells in the ectopic endometrium of peritoneal endometriosis. Conclusions: Women with endometriosis may present lower PF AMH levels even if they retain serum levels similar to women without disease. As peritoneal endometriosis expresses a specific receptor for AMH, lower AMH levels in PF of women with advanced endometriosis may be involved in the pathophysiology of peritoneal endometriosis

Accumulation of fibrosis and altered perifollicular stromal differentiation in vitrified‐thawed human ovarian tissue xenografted to nude mice.

Purpose: Ovarian tissue cryopreservation and its auto-transplantation is promising technique in fertility preservation. Longevity of grafted tissue is limited though mechanism of follicle reduction is not fully understood. We evaluated histological alteration of vitrified-thawed ovarian tissue that grafted to nude mice. Materials and Methods: Human ovarian tissue was cryopreserved by vitrification. After thawing, they were grafted to mesentery of nude mice. Twelve weeks after transplantation, the implants were removed and histologically examined. The presence of follicles, the degree of fibrosis, and TUNEL staining in surrounding cortex were evaluated. The stromal expressions of alpha-smooth muscle actin (aSMA) were determined.Results: Normal ovarian cortex was decreased, and fibrotic area were significantly increased after grafting. The distributions of developmental stage of follicles shifted toward activation of follicular growth. Stromal TUNEL staining was increased in frozen/thawed tissue. The expression of aSMA were found in perifollicular stroma of growing follicles, which were decreased in grafted tissue associated with reduction of cortical stroma.Conclusions: Fibrosis, reduced cortical stroma, and activation of dormant follicleswere concomitantly observed in grafted ovarian cortex, which may relate to limited longevity. Perifollicular aSMA expression can be regarded as a marker of the competence of cortical stroma that regulate follicular development

Comprehensive immune complexome analysis detects disease-specific immune complex antigens in seminal plasma and follicular fluids derived from infertile men and women

Background: Autoimmune reactions and subsequent inflammation may underlie spermatogenic dysfunction and endometriosis-related infertility.The aim of this study is to identify disease-specific antigens in immune complexes (ICs) in seminal plasma (SP) and in follicular fluid (FF).Methods: Immune complexome analysis, in which nano-liquid chromatography-tandem mass spectrometry is employed to comprehensively identify antigens incorporated into ICs in biological fluids, was performed for specimens collected from infertile couples undergoing assisted reproduction.Forty-two male patients consisting of subjects with oligozoospermia (n=6), asthenozoospermia (n=8), and normal semen analysis (n=28). Fifty-eight female patients consisting of subjects with ovarian endometriosis (n=10) and control women without disease (n=48).Results: Four disease-specific antigens were identified in subjects with oligozoospermia, while five disease-specific antigens were detected in subjects with asthenozoospermia, some of which are involved in sprematogenesis. Eight antigens were detected only in subjects with endometriosis.Conclusion: Functional characteristics of disease-specific antigens were found to correspond to the pathogenesis of male and female infertility. The formation of ICs may contribute to spermatogenic dysfunction and endometriosis-related infertility via loss of function of the related proteins. Immune complexome analysis is expected to be a valuable tool for the investigation of novel diagnostic methods and treatment strategies for infertility

AMH producing purely cystic virilizing adult granulosa cell tumor in 17 years old girl: a case report and review of literatures

Background: Androgen-producing granulosa cell tumor in adolescent girl is rare condition and clinical characteristics are not fully elucidated. Case presentation: Seventeen years old girl complained of secondary amenorrhea was referred to our out-patient consultation. Markedly elevated serum testosterone, LH, and AMH levels were noted. Mild hirsutism and clitoromegaly were presented. Transabdominal ultrasonography and MRI revealed cystic mass occupied pelvic cavity probably originated from left ovary. Right ovary showed polycystic appearance. Laparoscopic left ovarian cystectomy was performed. After the surgery, her menstruation resumed along with normalized hormonal parameters, and clinical hyperandrogenism were improved. Since the scarcity of cellular lining of inner cyst wall, definitive pathological diagnosis was difficult. After the consultation with gynecological pathologist, the tumor was diagnosed as sex cord stromal tumor, highly suspicious for adult granulosa cell tumor. Residual left salpingo-oophorectomy was performed by additional laparoscopic surgery. Her serum testosterone and AMH levels were remained low with regular menstrual cycles and no evidence of recurrence. Conclusions: Androgen-producing cystic granulosa cell tumor is rare gynecological disorders, which need both gynecologic oncological and endocrinological approach. Its clinical manifestations may bring some clues to the pathogenesis of ovulatory dysfunctions, such as polycystic ovary syndrome

Prenatal Vitamin E Treatment Improves Lung Growth in Fetal Rats with Congenital Diaphragmatic Hernia

The aim of this study was to test the effects of vitamin E on fetal lung growth in rats with congenital diaphragmatic hernia (CDH). Experimental congenital diaphragmatic hernia (CDH) was induced in rat fetuses by maternal administration of 100 mg nitrofen by gastric gavage on day 9.5 of gestation. Vitamin E was provided at days 16-20 of gestation, at 30 IU/day. Cesarean section was performed at day 21 of gestation. Immunohistochemistry was performed using anti-surfactant protein A (SP-A) and anti-SP-B polyclonal antibodies. RT-PCR evaluated SP-A and SP-B mRNA expressions. The lung weight/body weight ratio in rats with CDH was lower than the control (p<0.01). The number of type II pneumocytes positive for SP-A in untreated CDH rats (n=20) was lower than the control (n=20). The relative amounts of SP-A and SP-B were significantly higher in vitamin Etreated CDH rats (n=20) than untreated CDH rats (p<0.05). Our results suggest that antenatal vitamin E treatment increases the production of surfactant proteins in hypoplastic lung of rats with the CDH

Significance of p53-Binding Protein 1 Nuclear Foci in Cervical Squamous Intraepithelial Lesions: Association With High-Risk Human Papillomavirus Infection and P16INK4a Expression

As p53-binding protein 1 (53BP1) localizes to the sites of DNA double-strand breaks and rapidly forms nuclear foci (NF), and itspresence may be an indicator of endogenous genomic instability (GIN). We previously showed that 53BP1 NF in cervical cellsincrease with neoplastic progression, indicating the significance of 53BP1 expression for the estimation of malignant potential during cervical carcinogenesis. This study aimed to further elucidate the impact of 53BP1 expression as a biomarker for cervical squamous intraepithelial lesion (SIL). A total of 81 tissue samples, including 17 of normal cervical epithelium, 22 of cervical intraepithelial neoplasia (CIN) 1, 21 of CIN2, and 21 of CIN3, from patients positive for high-risk human papillomavirus (HR-HPV)were used for double-label immunofluorescence of 53BP1 and Ki-67/p16INK4a expression and HR-HPV in situ hybridization. We analyzed associations between 53BP1 expression type with parameters such as CIN grade, HR-HPV infection status, p16INK4a expression, and CIN prognosis. Expression type of 53BP1 was significantly associated with histological grade of CIN and HR-HPV in situ hybridization signal pattern (P < .0001). There was a significant correlation between 53BP1 and p16INK4a expression levels(r ? .73, P < .0001). However, there was no association between 53BP1 expression type and CIN prognosis. We propose that 53BP1 expression type is a valuable biomarker for SIL, which can help estimate the grade and GIN of cervical lesions reflecting replication stress caused by the integration of HR-HPV to the host genome

Changes in serum anti-Müllerian hormone levels may predict damage to residual normal ovarian tissue after laparoscopic surgery for women with ovarian endometrioma.

We measured serum anti-Müllerian hormone levels before and after surgery in women undergoing unilateral and monolocular cystectomy for benign ovarian diseases. Comparing to control benign cysts, we found a significant decline in serum anti-Müllerian hormone levels with consequent depletion of follicles in tissue specimens after surgery for women with ovarian endometrioma

Estrogen and progesterone receptor expression in macrophages and regulation of hepatocyte growth factor by ovarian steroids in women with endometriosis

Background: Information regarding macrophage-mediated regulation of hepatocyte growth factor (HGF) by ovarian steroid hormones in women with endometriosis is limited. Therefore, we investigated the regulation of HGF by steroid hormones in isolated macrophages and stromal cells derived from women with or without endometriosis. Methods: We isolated CD68 immunoreactive adherent macrophages in vitro from 46 women with endometriosis and 30 women without endometriosis. Estrogen receptor (ER) and progesterone receptor (PR) expression in macrophages was demonstrated by immunohistochemistry and RT-PCR. Production of HGF in the culture media of basal and ovarian steroid-stimulated macrophages was examined by enzyme-linked immunosorbent assay. Expression of mRNA for HGF and its receptor, c-Met in macrophages and stromal cells in response to ovarian steroid was investigated by RT-PCR. The single and combined effect of HGF and estrogen on the growth of macrophages and stromal cells was analysed by bromodeoxyuridine (BrdU) incorporation. Results: ER and PR were expressed in isolated macrophages and intact tissue at the protein and mRNA levels. Macrophages derived from women with endometriosis produced significantly higher concentration of HGF (352.2 ± 4.9 pg/ml) in conditioned media after treatment with estradiol (10-8 mol/l) than that of basal macrophages (221.5 ± 32.8 pg/ml, P < 0.05) or women without endometriosis (170.6 ± 2.6 pg/ml, P < 0.05). These effects were less evident after treatment with progesterone. Treatment with tamoxifen (10-6 mol/l) reversed the production of HGF and other macromolecules. Secretion of HGF in response to ovarian steroids was further enhanced after activation with lipopolysaccharide. The mRNA expressions of HGF and its receptor, c-Met, were also detected in macrophages and stroma in response to estrogen, suggesting an autocrine regulation. HGF mRNA expression was higher in cells of women with endometriosis than non-endometriosis women. Bromodeoxyuridine incorporation indicated that exogenous stimulation with HGF and estrogen, either alone or in combination, significantly increased the cell proliferation of both endometrial stroma and macrophages compared to that of non-endometriosis or non-treated cells. Conclusion: These results suggest that besides other inflammatory mediators, ovarian steroids also participate in the production of HGF by peritoneal macrophages which may be involved in the growth of endometriosis either alone or in combination with estrogen

Changes in tissue inflammation, angiogenesis and apoptosis in endometriosis, adenomyosis and uterine myoma after GnRH agonist therapy

Background: Information is limited regarding multifunctional role of GnRH agonist (GnRHa) therapy in reproductive diseases. We investigated pattern of changes in inflammatory reaction, micro-vessel density and apoptosis in the tissues collected from women with endometriosis, adenomyosis and uterine myoma who were treated with or without GnRHa therapy. Methods: Biopsy specimens were collected from lesions, myometria and corresponding endometria of 45 women with ovarian endometrioma, 35 women with adenomyosis, and 56 women with uterine myoma. A fraction of these women were treated with GnRHa therapy for a variable period of 3-6 months before surgery. We performed immunohistochemical analysis of CD68, a macrophage (Mφ) marker, and von Willebrand factor (VWF), a vessel marker, using respective antibodies. The changes in apoptosis were examined using TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay and by the immunoexpression of activated caspase-3 in tissues after GnRHa therapy. Results: The infiltration of CD68-positive M φ and VWF-positive micro-vessel density were significantly decreased in the endometria of women with endometriosis, adenomyosis and uterine myoma in the GnRHa-treated group when compared with that in the non-treated group. A marked decrease in inflammatory and angiogenic responses were observed in lesions and myometria of these diseases. When compared with non-treated group, a significantly increase in apoptotic index (apoptotic cells per 10 mm2 area) and quantitative-histogram (Q-H) scores of activated caspase-3 after GnRHa therapy were observed in the eutopic endometria, lesions and myometria of these diseases. Conclusion: GnRH agonist was able to markedly reduce the inflammatory reaction and angiogenesis and significantly induce apoptosis in tissues derived from women with endometriosis, adenomyosis and uterine myoma. These multiple biological effects at the tissue level may be involved in the regression of these reproductive diseases

Effect of human seminal fluid on the growth of endometrial cells of women with endometriosis.

OBJECTIVES: We investigated the effect of human seminal fluid on the growth of endometrial cells derived from women with and without endometriosis. STUDY DESIGN: Seminal plasma (SP) was collected from 18 healthy fertile men. Serum, peritoneal fluid (PF) and tissue specimens of eutopic and ectopic endometrium were collected from 45 women with endometriosis and 20 women without endometriosis during laparoscopic surgery. Prostaglandin (PG) E2, hepatocyte growth factor (HGF), and estradiol (E2) levels in each sample of SP, serum and PF were measured by enzyme-linked immunosorbent assay. The growth pattern of cells derived from eutopic and ectopic endometria in response to SP was examined by 5-bromo-2-deoxyuridine (BrdU) incorporation assay. RESULTS: Seminal plasma was able to significantly stimulate the growth of epithelial cells and stromal cells derived from the eutopic and ectopic endometria of women with endometriosis (2-3-fold) when compared with control media. The SP-promoted proliferation of both gland cells and stromal cells derived from eutopic endometria was also remarkably higher in women with endometriosis than that of women without endometriosis. Although levels of PGE2, HGF and E2 in SP were variable when compared with other body fluids, the levels of PGE2 and HGF in SP were significantly higher than those in either peritoneal fluid or serum of women with or without endometriosis. Pretreatment of cells with individual anti-PGE2 antibody, anti-HGF antibody and two selective estrogen receptor modulators, tamoxifen and raloxifene was unable to suppress SP-mediated growth of endometrial cells. However, pretreatment of cells with combined anti-PGE2 antibody plus anti-HGF antibody or combined anti-PGE2 antibody plus anti-HGF antibody plus tamoxifen or raloxifene was able to significantly suppress SP-promoted growth of eutopic and ectopic endometrial cells. CONCLUSION: Human seminal fluid enriched with different macromolecules may promote the growth of endometrial cells derived from women with endometriosis. Our findings may suggest some detrimental effect of unprotected sexual intercourse in women with endometriosis