Cytoskeletal organization of bee ovarian follicles during oogenesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of total abdominal hysterectomy on ovarian blood supply in women of reproductive age

Objective. The purpose of this study was to evaluate the effect of total abdominal hysterectomy on ovarian blood supply using transvaginal color Doppler ultrasonography in women of reproductive age. Methods. This prospective study included 61 women aged 40 years or younger who were divided into 2 groups: group 1, comprising 31 patients who underwent total abdominal hysterectomy (TAH), and group 2, comprising 30 women with no abnormalities. Inclusion criteria included normal ovarian function at baseline, with basal follicle-stimulating hormone levels of less than 15 mUI/mL, normal body weight, no tobacco use, and no history of laparotomy or ovarian disease. Ovarian arterial blood supply by determination of the pulsatility index (PI) on Doppler analysis and ovarian volume on transvaginal ultrasonography were assessed at baseline and at 6 and 12 postoperative months. The Student t test, profile analysis, and Friedman and Mann-Whitney tests were used in the statistical analysis of data. Results. Statistical analysis of baseline data revealed that both groups were homogeneous. At months 6 and 12, greater ovarian volumes and lower PI values were observed in patients who underwent TAH (P < .05). By the end of the study, in 8 of the 31 patients who underwent TAH (25.5%), benign ovarian cysts were observed. in the control group, all the parameters studied remained unchanged. Conclusions. The reduced PI values observed on Doppler ultrasonography suggested a decrease in the resistance flow in the ovarian arteries in women of reproductive age who underwent TAH.

Elevated glycodelin-A concentrations in serum and peritoneal fluid of women with ovarian endometriosis

The aim of this study was to evaluate serum and peritoneal fluid (PF) glycodelin-A concentrations in women with ovarian endometriosis. Ninety-nine matched pairs of serum and PF samples were included in our study. The case group comprised 57 women with ovarian endometriosis and the control group 42 healthy women undergoing sterilization or patients with benign ovarian cysts. Glycodelin-A concentrations were measured using ELISA. Endometriosis patients had significantly higher serum and PF glycodelin-A concentrations compared to controls, and this increase was observed in both proliferative and secretory cycle phases. Glycodelin-A concentrations were more than 10-fold higher in PF than in serum and correlated with each other. Intensity and frequency of menstrual pain positively correlated with glycodelin-A concentrations. Sensitivity and specificity of glycodelin-A as a biomarker for ovarian endometriosis were 82.1% and 78.4% in serum, and 79.7% and 77.5% in PF, respectively. These results indicate that Glycodelin-A has a potential role as a biomarker to be used in combination with other, independent marker molecules.

Increased levels of biglycan in endometriomas and peritoneal fluid samples from ovarian endometriosis patients

Abstract In our previous low-density-array gene-expression analysis we found an increased expression of biglycan gene in ovarian endometriosis patients. In the present study we evaluated biglycan expression at the protein level in tissue, serum and peritoneal fluid (PF) from ovarian endometriosis patients, patients with benign ovarian cysts and healthy women. Twenty samples of endometriomas and 27 of control tissues (benign ovarian cysts and eutopic endometrium of healthy women) were obtained laparoscopically or by curettage. Serum and PF samples were collected from 56 ovarian endometriosis patients and 40 controls (patients with benign cysts and healthy women). Tissue biglycan levels and serum and PF biglycan concentrations were determined by Western blotting and ELISA, respectively. Biglycan was detected in endometriomas and in benign cysts tissues but differed in glycosylation levels. The PF biglycan concentrations were significantly increased in ovarian endometriosis patients (mean ± SD = 220.3 ± 190.5 pg/mg protein) compared to the whole control group (101.9 ± 94.7 pg/mg protein, p < 0.001), while serum concentrations did not differ significantly. Biglycan appears to be involved in ovarian pathologies and probably has different roles in benign cysts as compared to ovarian endometriomas.

Ovarian and uterine development and hormonal feedback mechanism in a 46 XX patient with CYP19A1 deficiency under low dose estrogen replacement.

Abstract Background: Aromatase deficiency may result in a complete block of estrogen synthesis because of the failure to convert androgens to estrogens. In females, this results in virilisation at birth, ovarian cysts in prepuberty and lack of pubertal development but virilisation, thereafter. Objective and methods: We studied the impact of oral 17β-estradiol treatment on ovarian and uterine development, and on LH/FSH and inhibin B during the long-term follow-up of a girl harboring compound heterozygote point mutations in the CYP19A1 gene. Results: In early childhood, low doses of oral 17β-estradiol were needed. During prepuberty treatment with slowly increasing doses of E2 resulted in normal uterine and almost normal development of ovarian volume, as well as number and size of follicles. Regarding hormonal feedback mechanisms, inhibin B levels were in the upper normal range during childhood and puberty. Low doses of estradiol did not suffice to achieve physiological gonadotropin levels in late prepuberty and puberty. However, when estradiol doses were further increased in late puberty levels of both FSH and LH declined with estradiol levels within normal range. Conclusion: Complete aromatase deficiency provides an excellent model of how ovarian and uterine development in relation to E2, LH, FSH and inhibin B feedback progresses from infancy to adolescence.

Phospholipase A2 group IIA is elevated in endometriomas but not in peritoneal fluid and serum of ovarian endometriosis patients.

Our previous gene expression analysis identified phospholipase A2 group IIA (PLA2G2A) as a potential biomarker of ovarian endometriosis. The aim of this study was to evaluate PLA2G2A mRNA and protein levels in tissue samples (endometriomas and normal endometrium) and in serum and peritoneal fluid of ovarian endometriosis patients and control women. One-hundred and sixteen women were included in this study: the case group included 70 ovarian endometriosis patients, and the control group included 38 healthy women and 8 patients with benign ovarian cysts. We observed 41.6-fold greater PLA2G2A mRNA levels in endometrioma tissue, compared to normal endometrium tissue. Using Western blotting, PLA2G2A was detected in all samples of endometriomas, but not in normal endometrium, and immunohistochemistry showed PLA2G2A-specific staining in epithelial cells of endometrioma paraffin sections. However, there were no significant differences in PLA2G2A levels between cases and controls according to ELISA of peritoneal fluid (6.0 ± 4.4 ng/ml, 6.6 ± 4.3 ng/ml; p = 0.5240) and serum (2.9 ± 2.1 ng/ml, 3.1 ± 2.2 ng/ml; p = 0.7989). Our data indicate that PLA2G2A is implicated in the pathophysiology of ovarian endometriosis, but that it cannot be used as a diagnostic biomarker.

Panels of cytokines and other secretory proteins as potential biomarkers of ovarian endometriosis.

Endometriosis is a gynecologic disease that is characterized by nonspecific symptoms and invasive diagnostics. To date, there is no adequate noninvasive method for the diagnosis of endometriosis. Although more than 100 potential biomarkers have been investigated in blood and/or peritoneal fluid, none of these has proven useful in clinical practice. The aim to find a suitable panel of biomarkers that would allow noninvasive diagnosis thus remains of interest. We evaluated the concentrations of 16 cytokines and other secretory proteins in serum and peritoneal fluid of 58 women with ovarian endometriosis (cases) and 40 healthy women undergoing sterilization or patients with benign ovarian cysts (controls) using multiplexed double fluorescence-based immunometric assay platform and enzyme-linked immunosorbent assay. Significantly higher concentrations of glycodelin-A were shown in serum, and significantly higher levels of glycodelin-A, IL-6, and IL-8, and lower levels of leptin were measured in the peritoneal fluid of cases versus controls. In serum, the best performance was shown by models that included the ratio of leptin/glycodelin-A and the ratio of ficolin 2/glycodelin-A, whereas in the peritoneal fluid the best models included the ratio of biglycan/leptin, regulated on activation normal T-cell expressed and secreted/IL-6 and ficolin-2/glycodelin-A, and IL-8 per milligram of total protein, all in combination with age. The models using serum and peritoneal fluid distinguished between ovarian endometriosis patients and controls regardless of the menstrual cycle phase with relatively high sensitivity (72.5% to 84.2%), specificity (78.4% to 91.2%), and area under the curve (0.85 to 0.90).

Role of ovarian cancer antigen CA125/MUC16 in development and cancer

Cancer antigen 125 (CA125) is a tumor antigen that is routinely used to monitor the disease progress and the outcome of treatment in ovarian cancer patients. Elevated serum levels of CA125 are detected in over 80% of epithelial ovarian cancer patients. CA125 is a high molecular weight (>1M Dalton) mucin-type glycoprotein encoded by the MUC16 gene on human chromosome 19. Although MUC16 has served as the best serum marker for monitoring growth of ovarian cancer, roles for MUC16 in normal physiology and ovarian cancer are largely unknown. To understand the biological functions of MUC16, I characterized a mouse Muc16 homolog on chromosome 9 by means of expression pattern profiling, phenotype analysis of Muc16 knockout mice, and in vitro and in vivo studies of Muc16 null transformed ovarian surface epithelial (OSE) cells. ^ The mouse Muc16 homolog shares a conserved genomic structure with human MUC16. In addition to being expressed in mouse ovarian cancer, mouse Muc16 mRNA and protein were expressed in the mesothelia covering the heart, lung, ovary, oviduct, spleen, testis, and uterus. The conserved genomic structure and expression pattern of mouse Muc16 to human MUC16 suggests that mouse Muc16 is the ortholog of human MUC16. To understand the biological functions of Muc16, I generated Muc16 knockout mice. Muc16 knockout mice were viable, fertile and normal by one year of age. However, between 18 and 24 months of age, Muc16 knockout mice developed various tissue abnormalities such as ovarian cysts and tumors of the liver and other peritoneal organs. To determine the role of MUC16 in ovarian cancer progression, I established Muc16 null transformed ovarian surface epithelial (OSE) cell lines, following the same method to develop mouse model of epithelial ovarian cancer (Orsulic et al., 2002). Loss of Muc16 did not affect cell morphology, cell proliferation rate, or tumorigenic potential. However, Muc16-null OSE cells showed decreased attachment to extracellular matrix proteins as well as to primary mouse peritoneal mesothelial cells. Peritoneal mesothelia are the most frequent implantation sites of ovarian cancer. Furthermore, a pilot transplantation assay suggests that Muc16 null transformed OSE cells formed less disseminated tumors in the peritoneal cavity compared to wild-type OSE cells. ^ In conclusion, these results demonstrate that MUC16 is not required for normal mouse development or reproduction, but plays important roles in tissue homeostasis, ovarian cancer cell adhesion and dissemination. This study provides the first in vivo evidence of the roles of MUC16 in development, as well as ovarian cancer progression and dissemination. These studies offer valuable insights into possible mechanisms of ovarian cancer development and potential molecular targets for ovarian cancer treatment. ^

Expression pattern and developmental behaviour of cellular nucleic acid-binding protein (CNBP) during folliculogenesis and oogenesis in fish

In vertebrates, folliculogeneis establishes an intricate system for somatic cell-oocyte interaction, and ultimately leads to the acquisition of their respective competences. Although the formation process and corresponding interactions are strikingly similar in diverse organisms, knowledge of genes and signaling pathways involved in follicle formation is very incomplete and the underlying molecular mechanisms remain enigmatic. CNBP has been identified for more than ten years, and the highest level of CNBP transcripts has been observed in adult zebrafish ovary, but little is known about its functional significance during folliculogeneis and oogenesis. In this study, we clone CNBP cDNA from gibel carp (Carassius auratus gibelio), and demonstrate its predominant expression in gibel carp ovary and testis not only by RTPCR but also by Western blot. Its full-length cDNA is 1402 bp, and has an ORF of 489 nt for encoding a peptide of 163 aa. And its complete amino acid sequence shared 68.5%-96.8% identity with CNBPs from other vertebrates. Based on the expression characterization, we further analyze its expression pattern and developmental behaviour during folliculogeneis and oogenesis. Following these studies, we reveal an unexpected discovery that the CagCNBP is associated with follicular cells and oocytes, and significant distribution changes have occurred in degenerating and regenerating follicles. More interestingly, the CagCNBP is more highly expressed in some clusters of interconnected cells within ovarian cysts, no matter whether the cell clusters are formed from the original primordial germ cells or from the newly formed cells from follicular cells that invaded into the atretic oocytes. It is the first time to reveal CNBP relevance to folliculogeneis and oogenesis. Moreover, a similar stage-specific and cell-specific expression pattern has also been observed in the gibel carp testis. Therefore, further studies on CNBP expression pattern and developmental behaviour will be of significance for understanding functional roles of CNBP during gametogenests. (c) 2005 Elsevier B.V. All rights reserved.

EARLY LIFE EXPOSURE TO GENISTEIN AND DAIDZEIN DISRUPTS STRUCTURAL DEVELOPMENT OF REPRODUCTIVE ORGANS IN FEMALE MICE

In mice, exposure to isoflavones (ISO), abundant in soy infant formula, during the first 5 d of life alters structural and functional development of reproductive organs. Effects of longer exposures are unknown. The study objective was to evaluate whether exposure to a combination of daidzein and genistein in the first 10 compared to 5 d of life results in greater adverse effects on ovarian and uterine structure in adult mice. Thirteen litters of 8–12 pups were cross-fostered and randomized to corn oil or ISO (2 mg daidzein + 5 mg genistein/kg body weight/d) for the first 5 or 10 d of life. The 10-d protocol mimicked the period when infants are fed soy protein formula (SPF) but avoids the time when suckling pups can consume the mother’s diet. Body and organ weights and histology of ovaries and uteri were analyzed. There were no differences in the ovary or uterus weight, number of ovarian follicles, number of multiple oocyte follicles, or percent of ovarian cysts with 5 or 10 d of ISO intervention compared to respective controls. The 10-d ISO group had higher body weights from 6 d to 4 mo. of age and a higher percent of hyperplasia in the oviduct than the respective control. Lower numbers of ovarian corpus lutea and a higher incidence of abnormal changes were reported in the uteri of both ISO groups compared to their respective controls. Five- and 10-d exposure to ISO had similar long-lasting adverse effects on the structures of ovaries and uterus in adult mice. Only the 10-d ISO exposure resulted in greater body weight gain at adulthood.

Efeitos da Histerectomia Total Abdominal sobre o Fluxo Sangüíneo Ovariano

Objetivo: avaliar os efeitos da histerectomia total abdominal (HTA) sobre o fluxo sangüíneo ovariano, em mulheres no menacme, por meio da dopplervelocimetria e ultra-sonografia transvaginal. Métodos: estudo prospectivo no qual foram incluídas 61 mulheres, com idade igual ou inferior a 40 anos. As pacientes foram divididas em dois grupos: G1, com 31 pacientes submetidas à HTA, e G2, com 30 mulheres normais não submetidas à cirurgia. Somente foram incluídas pacientes eumenorréicas, ovulatórias, não-obesas ou fumantes, sem cirurgias ou doenças ovarianas prévias. Avaliou-se o fluxo sangüíneo das artérias ovarianas, inicialmente e aos 6 e 12 meses, pelo índice de pulsatilidade (IP) na dopplervelocimetria, e o volume ovariano pela ultra-sonografia transvaginal (US). Para análise estatística empregou-se teste t pareado, análise de perfil, teste de Friedman e teste de Mann-Whitney. Resultados: na comparação estatística inicial os grupos foram homogêneos quanto às características epidemiológicas e quanto aos demais parâmetros avaliados neste estudo. Nas pacientes submetidas à histerectomia, observaram-se aos 6 e 12 meses aumento do volume ovariano ao US e diminuição do IP avaliado pela dopplervelocimetria (p<0,05), quando confrontadas ao controle. Aos 12 meses, em 8 das 31 pacientes pós-HTA (25,5%) verificou-se ocorrência de cistos ovarianos de aspecto benigno. No grupo controle não houve alteração de nenhum desses parâmetros. Conclusão: a redução do IP na dopplervelocimetria das artérias ovarianas sugere aumento do fluxo sangüíneo ovariano pós-histerectomia total abdominal em mulheres no menacme.

Preditores Clínicos e Histopatológicos de Tumor Trofoblástico Gestacional pós-Mola Hidatiforme Completa

Objetivo: definir os preditores clínicos e histopatológicos mais eficientes da evolução da mola hidatiforme completa (MHC) para tumor trofoblástico gestacional (TTG). Métodos: estudo prospectivo clínico e histopatológico de todas as portadoras de MHC, atendidas entre 1990 e 1998 no Hospital das Clínicas de Botucatu -- UNESP. A avaliação clínica pré-esvaziamento molar classificou a gravidez molar em: MHC de alto risco e MHC de baixo risco. Foram analisados os preditores clínicos para TTG, estabelecidos por Goldstein et al.¹ e por outros autores2--10. A avaliação histopatológica incluiu a determinação do diagnóstico de MHC, segundo os critérios de Szulman e Surti11, e o reconhecimento dos fatores de risco para TTG, de Ayhan et al.8. Os preditores clínicos e histopatológicos foram correlacionados com o desenvolvimento de TTG pós-molar. Resultados: em 65 portadoras de MHC, cistos do ovário maiores que 6 cm e tamanho uterino maior que 16 cm foram os preditores clínicos mais eficientes de TTG. A proliferação trofoblástica, a atipia nuclear, a necrose/hemorragia, a maturação trofoblástica e a relação cito/sinciciotrofoblasto não foram preditores significativos para TTG. A correlação entre preditor clínico e histopatológico para o desenvolvimento de TTG não foi possível porque nenhum parâmetro histopatológico foi significativo. Conclusões: mais estudos são necessários para avaliar possíveis preditores de persistência (TTG) e sua aplicação no contexto clínico das MHC. Enquanto isso, a determinação seriada de hCG sérico permanece o único indicador prognóstico seguro para TTG pós-MHC.

Cisto ovariano em vacas de leite: incidência, resposta à aplicação de GnRH e desempenho reprodutivo

A incidência de cistos ovarianos, a resposta ao tratamento com GnRH e os efeitos da ocorrência de cisto no desempenho reprodutivo e na taxa de descarte foram determinados em vacas lactantes da raça Holandesa. Vacas lactantes (n=333) foram avaliadas semanalmente por ultrassonografia a partir da quarta semana pós-parto, visando à detecção de corpos lúteos (CL) e de folículos ovarianos maiores que 10mm. Na sétima semana pós-parto, as vacas foram classificadas: em ciclando (n=248; presença de CL em um dos exames ultrassonográficos); em anestro (n=54; ausência de CL e de folículos >25mm) e com cisto (n=31; ausência de CL e presença de estruturas >25mm), quando foram distribuídas em: grupo-controle (n=16; sem tratamento) e grupo-tratamento (n=15; vacas que receberam uma aplicação de GnRH). A taxa de cura foi de 60,0% no grupo das vacas tratadas e de 87,5% no grupo-controle. As vacas com cistos apresentaram maior intervalo parto-primeira inseminação artificial (P<0,05; 91,4±8,3 vs. 77,8±2,5), maior número de serviços por concepção (P<0,05; 4,4±1,2 vs. 3,6±1,5), maior intervalo parto-concepção (P<0,05; 214,8±25,9 vs. 174,9±7,7) e maior taxa de descarte (P<0,05; 41,2 vs. 21,8%) do que as vacas ciclando. Vacas diagnosticadas com cisto na sétima semana pós-parto apresentaram recuperação espontânea, embora o intervalo parto-concepção e a taxa de descarte tenham sido maiores para essas vacas.