Immunolocalization of the Anti-Müllerian Hormone (AMH) in Caprine Follicles and the Effects of AMH on In Vitro Culture of Caprine Pre-antral Follicles Enclosed in Ovarian Tissue.

The aims of this study were to evaluate the localization, by immunohistochemistry, of the anti-Müllerian hormone (AMH) in goat ovaries and to investigate its effects on the in vitro survival and development of caprine pre-antral follicles enclosed in fragments of ovarian tissue. Pre-antral follicles were cultured in vitro for 1 or 7 days in α-MEM(+) in the absence or presence of kit ligand (KL; 50 ng/ml, positive control) or AMH (50 or 150 ng/ml). The results showed that AMH was localized in oocytes and granulosa cells from the primordial follicle to antral follicle stages. Addition of AMH maintained the percentage of developing follicles, similar to that in the uncultured control; however, the percentage of developing follicles was significantly lower than that in the cultured control and KL. Nonetheless, addition of AMH to the culture medium did not affect survival rates and follicular growth. In conclusion, this study demonstrated that the expression of AMH varies according to the compartment and stage of follicular development. Furthermore, AMH inhibits the activation of caprine primordial follicles.

Serum markers of oxidative stress and endometriosis.

PURPOSE OF INVESTIGATION: To assess the changes secondary to chronic inflammation in women with and without pelvic endometriosis by the determination of serum thiols and carbonyls. MATERIALS AND METHODS: Sixty-seven women with endometriosis consecutively submitted to laparoscopy and 41 women without endometriosis consecutively submitted to tubal ligation (control group) were selected. Serum levels of total thiols and carbonyls were determined in both groups. RESULTS: Patients with endometriosis had significantly lower thiol levels than controls (342.37 +/- 142.09 microM vs 559.60 +/- 294.05 microM) (p < 0.001), as well as significantly lower carbonyl levels (8.97 +/- 3.76 microM vs 16.40 +/- 9.26 microM) (p < 0.001). Other clinical characteristics were not associated with changes in marker levels. The cutoff point established by the ROC curve was 396.44 microM for the thiols, with 73.1% sensitivity and 80.5% specificity, and 14.9 microM for the carbonyls, with 94% sensitivity and 51.2% specificity. CONCLUSIONS: The serum thiol levels revealed an increase in oxidative stress related to the development of pelvic endometriosis.

Is the mouse follicle culture a good model for the goat with respect to the development of preantral follicles in vitro?

The present study evaluated the efficiency of using 2 culture media developed for mice and for goats in the in vitro preantral follicle culture of each species. Murine and caprine secondary follicles were cultured in vitro with human recombinant follicle-stimulating hormone (murine medium) or with bovine recombinant follicle-stimulating hormone in association with growth hormone (caprine medium). The results showed that murine follicles cultured in caprine medium had lower (P < 0.05) rates of follicular survival and growth, whereas for caprine follicles, these variables were not affected by the type of medium used (P > 0.05). After in vitro maturation, a higher (P < 0.05) number of oocytes that resumed meiosis were observed in the murine medium for both species. In contrast, only in the caprine species estradiol production was significantly superior when the caprine medium was used. Higher progesterone production was observed in the presence of the murine medium only for murine follicles (P < 0.05). In conclusion, murine and caprine preantral follicles cultured under the same in vitro culture medium conditions respond differently; caprine oocytes grown in vitro in the presence of the murine medium show the greatest developmental competence among the tested combinations. Therefore, under the present experimental conditions, the mouse follicle culture has proved be a good model for the development of new culture media for caprine preantral follicles.

Alginate hydrogel matrix stiffness influences the in vitro development of caprine preantral follicles.

This study examined caprine follicular development in different concentrations of alginate matrix to determine the optimal conditions for culture. Caprine preantral follicles were cultured in a two-dimensional system (control) or a three-dimensional encapsulated system in 0.25%, 0.5%, or 1% alginate (ALG 0.25, ALG 0.5, and ALG 1, respectively). A higher percentage of morphologically normal follicles developed in ALG 0.5 and ALG 1 than in ALG 0.25 or the control (P < 0.05). The rate of antrum formation, however, was higher in ALG 0.25 than in ALG 0.5 and ALG 1 conditions (P < 0.05), but similar to the control. Follicles cultured in ALG 0.25 had higher growth rates and meiotic resumption than those cultured in ALG 0.5, ALG 1, or the control (P < 0.05). Moreover, follicles cultured in ALG 0.25 had higher levels of estradiol and progesterone than those cultured in ALG 0.5, ALG 1, or the control, as well as higher levels of CYP19A1 and HSD3B mRNA. In conclusion, a three-dimensional system that uses ALG 0.25 fosters the in vitro development of caprine preantral follicles and increases the rate of meiotic resumption.

Exposing bovine cumulus-oocyte complexes to aromatizable androgen restore serum-induced low estradiol production in vitro.

We aimed in this study to assess whether serum-decreased bovine cumulus-oocyte complex (COC) steroidogenesis during in vitro maturation (IVM) is caused by deficient androgen milieu. For this approach, bovine COCs were cultured in serum-supplemented IVM medium in the presence or absence of 1 µM androstenedione. After 24 h of culture, medium was collected and analyzed for its content of estradiol-17ß (E2) and progesterone (P4). Medium E2 content markedly increased after incubation of COCs with androstenedione (17.52 ± 1.86 ng/ml to the androgen group; 0.32 ± 0.05 ng/ml to the non-androgen group). No significant difference in the P4 content was detected despite the presence of androstenedione (21.83 ± 1.61 ng/ml to the androgen group; 21.73 ± 1.67 ng/ml to the non-androgen group). Our data provide compelling evidence that bovine COCs steroidogenesis remains functional during culture in serum-supplemented medium and suggest that serum-induced decreased COCs estradiol secretion is caused by deficiency of an aromatizable androgen source.

Preventing Microbial Contamination during Long-Term In Vitro Culture of Human Granulosa-Lutein Cells: An Ultrastructural Analysis.

Purpose. To investigate whether the addition of antibiotic/antimycotic during human granulosa-lutein cells (GLCs) isolation and cell-plating procedures prevents microbial contamination after 144 h of culture and also evaluate the effects of contamination on GLCs ultrastructure and steroid secretion. Methods. GLCs obtained from five women submitted to assisted reproductive techniques (ARTs) were isolated with PBS supplemented with antibiotic/antimycotic or PBS nonsupplemented and cultured for 144 h. GLCs were evaluated by transmission electron microscopy (TEM), and estradiol (E2) and progesterone (P4) secretion was assayed by chemiluminescence. Results. Although no contaminating microorganisms were identified by light microscopy, TEM analyses revealed several bacterial colonies in culture dishes of GLCs isolated with only PBS. Bacterial contamination disrupted the adherence of the GLCs to the culture plate interfering with monolayer formation affecting the growth pattern of GLCs. Various cellular debris and bacteria were observed, and no organelles were found in the cytoplasm of infected cells. While bacterial contamination decreased estradiol media levels, it increased progesterone, as compared with noncontaminated group. Conclusion. Taken together, our data showed that the addition of a high dose of antibiotic/antimycotic during the isolation and cell-plating procedures prevents microbial contamination of long-term GLCs culture as its effects on cells growth and function in vitro.

VIP expression in vessels of the vaginal wall: relationship with estrogen status.

OBJECTIVE: Vasoactive intestinal peptide (VIP) is a neuropeptide with elevated expression in regions that control urogenital functions. Estrogen appears to modulate VIP expression in various organs, but this effect has not been demonstrated in the vaginal wall. The aim of this study was to evaluate the influence of estrogen status on VIP expression in vessels of the vaginal wall. METHODS: Surgical specimens were removed from the vaginal walls of 18 premenopausal women and 12 postmenopausal women who were given surgery for genital prolapse grade I or II. Vaginal specimens were stained with estrogen receptor-alpha (ER-α) and VIP antibodies. Levels of follicle stimulating hormone (FSH), estradiol, prolactin, fasting glucose and serum thyroxine stimulating hormone were also measured. Estrogen status was assessed on the basis of FSH and ER-α scores. RESULTS: The vaginal walls of premenopausal women had significantly higher ER-α scores than those of menopausal women (premenopausal group, 3.6 ± 2.2; menopausal group, 1.4 ± 1.8; p = 0.01). Premenopausal women also had significantly higher levels of VIP in the vaginal wall than menopausal women (p = 0.02). Increasing age was associated with lower level of VIP staining (odds ratio 0.88; 95% confidence interval 0.78-0.99). CONCLUSION: Levels of ER-α and VIP expression in the posterior vaginal wall were higher in premenopausal than in menopausal women, but VIP expression was not associated with estrogen status. Age was an independent predictor of VIP staining in vaginal wall biopsies.

Anti-müllerian hormone is the best predictor of poor response in ICSI cycles of patients with endometriosis.

PURPOSE: To correlate ovarian reserve (OR) markers with response in assisted reproduction techniques (ART) and determine their ability to predict poor response among patients with endometriosis (EDT). METHODS: We evaluated ART cycles of 27 women with EDT and 50 with exclusive male factor. Basal follicle stimulating hormone (FSH) and anti-müllerian hormone (AMH) levels were determined. Ovarian response to gonadotropin stimulation was assessed and correlation coefficients calculated between the variables and reserve markers. Areas under the curve (AUC) determined ability of tests to predict poor response. RESULTS: AMH was significantly correlated with response in both groups and it was the only marker with significant discriminative capacity to predict poor response among EDT (AUC = 0.842; 95% CI: 0.651-0.952) and control group (AUC = 0.869; 95% CI: 0.743-0.947). CONCLUSION: Infertile patients with endometriosis can benefit from the pre-therapeutic assessment of OR markers. However, regardless of disease presence, only AMH predicts poor response to stimulus.

High prevalence of polycystic ovary syndrome in women born small for gestational age.

BACKGROUND: There is evidence that intrauterine growth restriction, resulting in newborn girls that are small for gestational age (SGA), may be related to the onset of polycystic ovary syndrome (PCOS). Thus, we studied whether women born SGA have a higher prevalence of PCOS than women born appropriate for gestational age (AGA). METHODS: This was a prospective birth cohort study of 384 women born at term between June 1, 1978, and May 31, 1979, in Ribeirão Preto, Brazil. After exclusion, 165 women effectively participated in this study, of whom 43 were SGA and 122 were AGA. The prevalence of PCOS was analysed. At a mean age of 29 years, the women agreed to follow the study protocol, which included: anamnesis, physical examination, serum tests [follicle stimulating hormone, luteinizing hormone, total and free testosterone, dehydroepiandrostenedione sulphate, 17-OH-progesterone, fasting insulin, sex steroid-binding globulin (SHBG) and fasting glucose] and pelvic ultrasound. Data regarding gestational age, birthweight, age at menarche and maternal data were obtained from the files of the cohort. The adjusted relative risk (RR) values of the SGA, insulin resistance, body mass index, maternal smoking and parity variables were analysed using Poisson regression with robust adjustment of variance for the prediction of PCOS. RESULTS: The prevalence of PCOS was higher in the SGA group than in the AGA group [adjusted RR = 2.44, 95% CI (1.39-4.28)]. Hyperandrogenism was more prevalent in the SGA women than in the AGA women (P = 0.011). Circulating SHBG was lower in the SGA women than in the AGA women (P = 0.041), but fasting insulinemia was similar in both groups. CONCLUSIONS: The prevalence of PCOS in SGA women was twice as high as in AGA women in our study population.

Effects of the levonorgestrel-releasing intrauterine system on cell proliferation, Fas expression and steroid receptors in endometriosis lesions and normal endometrium.

BACKGROUND: The objectives of this study were: (i) to evaluate the effects of the levonorgestrel-releasing intrauterine system (LNG-IUS) on both proliferation and apoptosis markers and hormone receptors of the eutopic and ectopic endometrium of women experiencing pain related to endometriosis and (ii) to compare the results with those obtained with GnRH agonist (GnRHa) injections. METHODS: Pre- and post-treatment endometrium and endometriosis specimens were obtained from 22 women experiencing pain related to endometriosis who were treated with LNG-IUS (n = 11) or GnRHa (n = 11) for 6 months. Changes in the expression of proliferating cell nuclear antigen, Fas, progesterone receptor (PRA) and estrogen receptor alpha (ER-alpha) were analyzed by immunohistochemistry. RESULTS: The cell proliferation index was significantly reduced in the epithelium and stroma of both the eutopic and the ectopic endometrium after treatment with the LNG-IUS and GnRHa. Only LNG-IUS users showed an increased H-score for Fas in the epithelium of the eutopic and ectopic endometrium (P < 0.05). Expression of ER-alpha and PRA by the glandular epithelium was lower in the eutopic endometrium after both treatments, but this reduction was noted in the ectopic endometrium only after LNG-IUS treatments (P < 0.05). No difference was detected between groups for any of the markers. CONCLUSIONS: LNG-IUS reduced both cell proliferation and the expression of PRA and ER-alpha and increased Fas expression in the eutopic and ectopic endometrium of patients with endometriosis. Some of these actions were not observed with GnRHa.

Endometriosis in postmenopausal women without previous hormonal therapy: report of three cases.

INTRODUCTION: Endometriosis is a benign, estrogen-dependent, chronic gynecological disorder associated with pelvic pain and infertility. The disease most commonly affects women during the reproductive age, although postmenopausal patients do rarely present it. These rare occurrences are generally associated with hormonal use. MATERIAL AND METHODS: We present three cases of endometriosis in postmenopausal patients who have no history of hormone therapy and no previous history of endometriosis or infertility. CASE REPORTS: In case 1, a 62-year-old woman presented with acyclic pelvic pain and a left ovarian homogeneous cystic mass. After laparoscopic salpingoophorectomy and histological analysis, an ovarian endometriotic cyst was confirmed. In case 2, a 78-year-old woman presented with a painful abdominal wall mass that was confirmed by ultrasound and tomography. Her past medical history included an abdominal hysterectomy 20 years prior to the discovery of this mass. The lesion was surgically excised and histological analysis showed areas of endometrial stroma and glands surrounded by fibrosis, compatible with endometriosis. In case 3, a 54-year-old woman presented with chronic pelvic pain and a nodule in the rectovaginal septum was noted during gynecological examination. Menopause occurred at 48 years of age. She had no previous dysmenorrhea. Ultrasound confirmed the nodule in the rectovaginal septum. The patient was submitted to a diagnostic colonoscopy that revealed a friable lesion, which was subsequently biopsied. The histological diagnosis was endometriosis. CONCLUSIONS: These three cases of postmenopausal endometriosis support the celomic metaplasia theory for the genesis of this disease.