Marcos Felipe Silva-de-Sá

The Role of Androgen Hormones in Early Follicular Development

Background. Although chronic hyperandrogenism, a typical feature of polycystic ovary syndrome, is often associated with disturbed reproductive performance, androgens have been shown to promote ovarian follicle growth in shorter exposures. Here, we review the main effects of androgens on the regulation of early folliculogenesis and the potential of their application in improving follicular in vitro growth. Review. Androgens may affect folliculogenesis directly via androgen receptors (ARs) or indirectly through aromatization to estrogen. ARs are highly expressed in the granulosa and theca cells of early stage follicles and slightly expressed in mature follicles. Short-term androgen exposure augments FSH receptor expression in the granulosa cells of developing follicles and enhances the FSH-induced cAMP formation necessary for the transcription of genes involved in the control of follicular cell proliferation and differentiation. AR activation also increases insulin-like growth factor (IGF-1) and its receptor gene expression in the granulosa and theca cells of growing follicles and in the oocytes of primordial follicles, thus facilitating IGF-1 actions in both follicular recruitment and subsequent development. Conclusion. During the early and intermediate stages of follicular maturation, locally produced androgens facilitate the transition of follicles from the dormant to the growing pool as well as their further development.

Hysteroscopy as a standard procedure for assessing endometrial lesions among postmenopausal women

CONTEXT AND OBJECTIVES Endometrial cancer is the most prevalent type of malignant neoplasia of the genital tract. The objective of this study was to calculate the sensitivity, specificity, accuracy and positive and negative predictive values for diagnostic hysteroscopy, in comparison with histopathological tests, for all lesions of the endometrial cavity. DESIGN AND SETTING Retrospective descriptive study at the public tertiary-level university hospital of Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo. METHODS Diagnostic hysteroscopy was indicated in the following instances: endometrial thickness > 4 mm in asymptomatic patients; postmenopausal bleeding; and irregular endometrium or endometrium difficult to assess from ultrasound, with or without vaginal bleeding. Ultrasound evaluations were carried out no more than three months prior to hysteroscopy. RESULTS There were 510 patients, with a mean age of 61.1+/-2.0 years and mean time elapsed since the menopause of 12.7+/-2.5 years. Endometrial biopsies were performed on 293 patients (57.5%). Histopathological analysis showed that 18 patients presented endometrial carcinoma or typical or atypical hyperplasia, and none of them presented endometrial thickness of less than 8 mm. No significant differences were found between the median thicknesses of the various benign lesions (p > 0.05). In our data, the sensitivity, specificity, accuracy and positive and negative predictive values for cancer or hyperplasia were 94.4%, 97.0%, 96.8%, 68% and 99.6%, respectively. CONCLUSIONS Our results suggest that hysteroscopy is valuable as a diagnostic tool for malignant/hyperplastic and benign lesions, except for submucous myomas, for which the sensitivity was only 52.6%.

Cryopreservation time does not decrease follicular viability in ovarian tissue frozen for fertility preservation

OBJECTIVE: To determine the effect of storage duration on cryopreserved ovarian tissue using fresh and frozen-thawed samples. METHODS: Seventeen fertile patients underwent an ovarian biopsy during elective laparoscopic tubal ligation. The tissue sample was divided into three parts: one part was processed fresh (FG), and two were slowly frozen, cryopreserved for 30 (G30) or 180 days (G180), thawed and analyzed. Follicular density, follicular viability, and steroidogenic capacity were assessed. RESULTS: We observed no differences between the groups in follicular density, which was assessed in hematoxylin and eosin–stained tissue sections. A heterogeneous follicular distribution was observed in the parenchyma, with a mean density of 361.3±255.4, 454.9±676.3, and 296.8±269.0 follicles/mm3 for FG, G30 and G180, respectively (p = 0.46). Follicular viability was greater in FG (93.4%) when compared with the cryopreserved tissues (70.8% for G30 (p<0.001) and 78.4% for G180 (p<0.001)), with no difference in viability between the frozen samples (p>0.05). The steroidogenic capacity of the tissue was not significantly reduced following cryopreservation. CONCLUSION: The slow freezing procedures used for ovarian cryopreservation are capable of preserving follicular viability and maintaining the steroidogenic capacity of the tissue despite a roughly 30% decrease in follicular viability. Furthermore, short-term storage of ovarian tissue does not appear to compromise follicle integrity.

Menopause Leading to Increased Vaginal Wall Thickness in Women with Genital Prolapse: Impact on Sexual Response

INTRODUCTION Hypoestrogenism causes structural changes in the vaginal wall that can lead to sexual dysfunction. A reduction in vaginal wall thickness has been reported to occur after menopause, although without precise morphometry. AIM To measure vaginal wall thickness in women with genital prolapse in normal and hypoestrogenic conditions and to correlate sexual dysfunction with vaginal wall thickness and estradiol levels. METHODS Surgical vaginal specimens from 18 normoestrogenic and 13 postmenopausal women submitted to surgery for genital prolapse grades I and II were examined. Patients were evaluated for FSH, estradiol, prolactin, glycemia, and serum TSH levels. For histological analysis, samples were stained with Massons trichrome and hematoxylin-eosin. Sexual function was assessed by the Golombok-Rust Inventory of Sexual Satisfaction (GRISS). MAIN OUTCOME MEASURES GRISS questionnaire, histological analysis, morphometric methods, Massons trichrome. RESULTS The vaginal wall was thicker in the postmenopausal than premenopausal group (2.72 +/- 0.72 mm and 2.16 +/- 0.43, P = 0.01, and 2.63 +/- 0.71 mm and 2.07 +/- 0.49 mm, P = 0.01, for the anterior and posterior walls, respectively). These thicknesses seem to be due to the muscular layer, which was also thicker in the postmenopausal group (1.54 +/- 0.44 and 1.09 +/- 0.3 mm, P = 0.02, and 1.45 +/- 0.47 and 1.07 +/- 0.44 mm, P = 0.03, for the anterior and posterior wall, respectively). The vaginal epithelium was thinner in the middle segment than in the proximal one in the posterior wall (0.17 +/- 0.07 mm, 0.15 +/- 0.05 mm, 0.24 +/- 0.09 mm, P = 0.02). There was no correlation between coital pain, vaginal wall thickness, and estradiol levels in either group. CONCLUSION The vaginal wall is thicker after menopause in women with genital prolapse. In this study, vaginal thickness and estrogen levels were not related to sexual dysfunction.

Increased basal FSH levels as predictors of low‐quality follicles in infertile women with endometriosis

To determine whether basal levels of follicle‐stimulating hormone (FSH) and anti‐müllerian hormone (AMH), antral follicle count (AFC), and the numbers of dominant follicles, oocytes, and mature oocytes retrieved after ovarian stimulation differed between infertile women with endometriosis and healthy women undergoing assisted reproduction techniques (ART).

Terapêutica com tibolona em mulheres diabéticas na pós-menopausa: parâmetros clínicos e laboratoriais de segurança

OBJETIVO: determinar o perfil de seguranca clinico-laboratorial da terapia com tibolona em mulheres portadoras de diabetes mellitus nao-insulinodependente (DMNID). METODO: estudo prospectivo, longitudinal, aberto e controlado envolvendo 24 mulheres na pos-menopausa portadoras de DMNID, tratadas sequencialmente com placebo (6 meses) e tibolona 2,5mg/dia (6 meses). Parâmetros clinicos, antropometricos, bioquimicos, hormonais e ultra-sonograficos foram avaliados no periodo basal, apos 6 (tempo 1) e 12 meses de acompanhamento (tempo 2). Analise estatistica foi realizada utilizando-se ANOVA para medidas repetidas, com nivel de significância 5%. RESULTADOS: efeitos colaterais surgiram apenas durante uso da tibolona (cefaleia e mastalgia em 8,3% e sangramento genital em 16,6%). Houve diminuicao significativa dos sintomas climatericos avaliados atraves do indice de Blatt-Kuperman [22,2 ± 7,1 (basal) vs. 13,6 ± 6,7 (tempo 1) vs. 3,1 ± 3,3 (tempo 2); p< 0,0001]. Apos uso da tibolona, evidenciamos reducoes significativas no percentual de gordura corporal, pressao arterial diastolica, niveis de transaminases, triglicerideos e HDL-colesterol. Nao houve variacoes significativas na pressao arterial sistolica, frequencia cardiaca, indice de massa corporal, relacao cintura/quadril, glicemia de jejum, hemoglobina glicosilada, ureia, colesterol total e LDL-colesterol. A avaliacao ultra-sonografica nao revelou variacoes significativas do volume uterino e espessura endometrial. CONCLUSAO: o tratamento com tibolona em curto prazo mostrou bom perfil de seguranca clinico-laboratorial em pacientes na pos-menopausa portadoras de DMNID.

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.

Leuprolide acetate reduces both in vivo and in vitro ovarian steroidogenesis in infertile women undergoing assisted reproduction

Despite the probable inhibitory effects of GnRH analogues on ovarian steroidogenesis in vitro, their association with assisted reproduction protocols shows favorable results. This suggests that there are important differences in the behaviors of these drugs when administered in vivo versus in vitro. To clarify these differences, this study was designed to analyze the effect of leuprolide acetate (LA) on ovarian steroidogenesis in women undergoing In Vitro Fertilization (IVF). A prospective, randomized open label study was conducted on 14 women (26-35 years): seven receiving only gonadotrophins (Group 1) and seven receiving gonadotrophin plus LA at 1mg/day (Group 2). The LA in vivo effect was determined with serum and follicular fluid (FF) samples and via luteinized granulosa cell cultivation (GCC), where cells were obtained during oocyte retrieval after ovarian hyperstimulation. In vitro analysis was performed via addition of LA to GCC only for Group 1 (without LA) at progressively higher concentrations (0, 10(-12), 10(-9) and 10(-6)M). In vivo, the main observation was a reduction in androgen production in Group 2, represented by lower androstenedione production in FF (G1=6479+/-3458; G2=3021+/-1119 ng/ml; p=0.04) and a lower testosterone peak in GC at 96h (G1=0.64+/-0.12 ng/ml; G2=0.50+/-0.19 ng/ml; P=0.02), but a higher fertilization rate (G1=67%; G2=83%; p=0.009). In vitro, testosterone, estradiol and progesterone were also reduced by LA, even though this reduction occurred for progesterone only at the highest LA dosage (10(-6)M; 606.0+/-114.3 ng/ml versus 1524.0+/-246.5 ng/ml; p=0.02). Results show that LA reduces ovarian steroidogenesis in vivo by essentially inhibiting androgen synthesis; whereas, in vitro, ovarian steroidogenesis is reduced overall.

MALDI mass spectrometry reveals that cumulus cells modulate the lipid profile of in vitro-matured bovine oocytes

ABSTRACT The influence of cumulus cells (CC) on the lipid profile of bovine oocytes matured in two different lipid sources was investigated. Cumulus-oocyte complexes (COC) or denuded oocytes (DO) were matured in tissue culture medium (TCM) supplemented with fetal bovine serum (FBS) or serum substitute supplement (SSS). Lipid profiles of TCM, serum supplements, immature CC and oocyte (IO), and in vitro-matured oocytes from COC and DO were then analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and submitted to partial least squares-discriminant analysis (PLS-DA). The developmental competence of such oocytes was also assessed. Differences in lipid composition were observed between two types of sera and distinctly influenced the lipid profile of CC. As revealed by PLS-DA, the abundance of specific ions corresponding to triacylglycerols (TAG) or phospholipids (PL) were higher in COC compared to DO both supplemented with FBS or SSS and to some extent affected the subsequent DO in vitro embryo development. DO exposed to SSS had however a marked diminished ability to develop to the blastocyst stage. These results indicate a modulation by CC of the oocyte TAG and PL profiles associated with a specific cell response to the serum supplement used for in vitro maturation.

Effect of soybean phosphatidylcholine on lipid profile of bovine oocytes matured in vitro

The phospholipid (PL) composition of embryo and oocyte membranes affects thermal phase behavior and several physicochemical properties such as fluidity and permeability. The characterization of PL profiles and the development of suitable in vitro maturation (IVM) protocols, that are able to modify membranes composition, may result in significant improvements in oocyte developmental potential and cryotolerance. Using soybean phosphatidylcholine (PC) as a model supplement, we evaluated the effect of PL supplementation during IVM on bovine cumulus-oocyte-complex (COC). Substantial changes in the lipid profiles of oocyte membrane were observed and associated with pre-implantation data. The propensity of the PC supplement to become soluble in the maturation medium and/or diffuse into mineral oil was also assessed. Oocytes were matured in TCM without supplementation, i.e. control, (n=922) or supplemented with 50 or 100μM PC (n=994). The maturation media and mineral oil pre- and post- IVM, along with control and PC-treated oocytes were then analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and the lipid profiles were compared via principal component analysis (PCA). Soybean PCs are bioavailable and stable in IVM medium; further, PCs did not diffuse to the mineral oil, which also remained unaltered by the metabolism of treated oocytes. PC supplementation at 100μM resulted in substantially greater relative abundances of polyunsatured PL, namely PC (32:1), PC (34:2), PC (36:6), PC (36:4), and PC (38:6), in oocyte membrane. These differences indicated that short-term exposure to the PC supplement could indeed modify the lipid composition of IVM-oocytes in a dose-dependent manner. Membrane incorporation of polyunsaturated molecular species of PC was favored, and does so without compromising the viability of the subsequent embryo in regards to cleavage, blastocyst development and hatching rate. The reported approach will allow for the development of novel strategies to modulate oocyte membrane dynamics and structure.