Gustavo Arantes Rosa Maciel

Triple-negative and luminal A breast tumors: differential expression of miR-18a-5p, miR-17-5p, and miR-20a-5p

New concepts in epigenetics, microRNAs, and gene expression analysis have significantly enhanced knowledge of cancer pathogenesis over the last decade. MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate gene expression by base pairing with target messenger RNAs (mRNAs), resulting in the repression of translation or the degradation of mRNA. To compare the carcinogenic process in tumors with different prognoses, we used real-time RT-PCR to evaluate the miRNA expression profiles of 24 triple-negative breast invasive ductal carcinoma, 20 luminal A breast invasive ductal carcinoma, and 13 normal breast parenchyma controls. We extracted total RNA from tissues fixed in formol and embedded in paraffin (FFPE). Results revealed the upregulation of miR-96-5p (9.35-fold; p = 0.000115), miR-182-5p (7.75-fold; p = 0.000033), miR-7-5p (6.71-fold; p = 0.015626), and miR-21-5p (6.10-fold; p = 0.000000) in tumors group. In addition, the expression of miR-125b-5p (4.49-fold; p = 0.000000) and miR-205-5p (4.36-fold; p = 0.006098) was downregulated. When the expression profiles of triple-negative and luminal A tumors were compared, there was enhanced expression of miR-17-5p (4.27-fold; p = 0.000664), miR-18a-5p (9.68-fold; p = 0.000545), and miR-20a-5 (4.07-fold; p = 0.001487) in the triple-negative tumors compared with luminal A. These data suggest that there is a similar regulation of certain miRNAs in triple-negative and luminal A tumors. However, it is possible that differences in the expression of miR-17-92 cluster will explain the phenotypic differences between these molecular tumor subtypes.

Metformin in normal-weight hirsute women with polycystic ovary syndrome with normal insulin sensitivity

Fifteen normal-weight (body mass index (BMI) 21.50 ± 1.65 kg/m2) hirsute women with polycystic ovary syndrome and normal insulin sensitivity were treated with 850 mg metformin orally, three times daily, for 4 months. Before and at the end of the treatment, clinical data as well as serum concentrations of sex steroid hormones, gonadotropins, fasting plasma glucose and insulin, insulin resistance – homeostasis model assessment (HOMA-IR), carbohydrate tolerance and the area under the curve for insulin (AUCinsulin) were analyzed. Three patients withdrew from the study. Seven of the remaining 12 patients presented menstrual pattern improvement, followed by ovulatory cycles at the end of the treatment period. There were no changes in BMI and hirsutism score. A significant (p < 0.05) decrease in luteinizing hormone (LH) (from 8.18 ± 4.34 to 5.05 ± 1.53 IU/ml), testosterone (from 104.66 ± 27.54 to 82.00 ± 23.05 ng/dl), fasting insulin (from 9.66 ± 4.79 to 7.83 ± 3.06 µIU/ml), AUCinsulin (from 9239 ± 3285 to 7660 ± 2565 µUI/ml × min) and HOMA-IR (from 2.15 ± 1.2 to 1.67 ± 0.74), and a significant increase in follicle-stimulating hormone (FSH) (from 4.05 ± 1.53 to 5.96 ± 2.13 IU/ml), were observed at the end of the treatment period. A higher LH and a lower FSH predicted clinical improvement, while basal insulin and AUCinsulin showed lower predictive value.

Systematic review of cell adhesion molecules and estrogen receptor expression in the endometrium of patients with polycystic ovary syndrome

Infertility associated with polycystic ovary syndrome (PCOS) could be related to many mechanisms including endometrial factors.

Androgen receptor gene polymorphism and polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is characterized by ovulatory dysfunction and hyperandrogenism. Its etiopathology is not well understood but genetic factors seem to have a role. Polymorphism of the androgen receptor (AR) gene has been associated with different androgen pattern diseases.

Differences in neonatal exposure to estradiol or testosterone on ovarian function and hormonal levels

Exposure to an excess of androgen or estrogen can induce changes in reproductive function in adult animals that resemble polycystic ovary syndrome in humans. However, considerable differences exist among several types of animal models. Little is known about the molecular features of steroidogenesis and folliculogenesis in the ovaries of rats exposed to different sex steroids as neonates. Here, we evaluated the impact of androgen and estrogen exposure on the ovaries of adult female rats during their neonatal period in the gene expression of Lhr and Cyp17a1, two key players of steroidogenesis. We also assessed hormone levels, folliculogenesis and the theca-interstitial cell population. The study was performed on the second postnatal day in thirty female Wistar rats that were sorted into the following three intervention groups: testosterone, estradiol and vehicle (control group). The animals were euthanized 90 days after birth. The main outcomes were hormone serum levels, ovary histomorphometry and gene expression of Lhr and Cyp17a1 as analyzed via quantitative real-time PCR. We found that exposure to excess testosterone in early life increased the LH and testosterone serum levels, the LH/FSH ratio, ovarian theca-interstitial area and gene expression of Lhr and Cyp17a1 in adult rats. Estrogen induced an increase in the ovarian theca-interstitial area, the secondary follicle population and gene expression of Lhr and Cyp17a1. All animals exposed to the sex steroids presented with closed vaginas. Our data suggest that testosterone resulted in more pronounced reproductive changes than did estrogen exposure. Our results might provide some insight into the role of different hormones on reproductive development and on the heterogeneity of clinical manifestations of conditions such as polycystic ovary syndrome.

Morfologia das células intersticiais de ovários policísticos de ratas: um estudo experimental

PURPOSES: To evaluate the histomorphometry of ovarian interstitial cells, as well as the blood sex steroid concentrations of female rats with polycystic ovaries induced by continuous light. METHODS: Twenty female rats were divided into two groups: Control Group - in the estrous phase (CtrlG), and a group of rats with polycystic ovaries induced by continuous illumination (POG). CtrlG animals were maintained on a light period from 07:00 a.m. to 07:00 p.m., and POG animals with continuous illumination (400 Lux) for 60 days. After this period all animals were anesthetized and blood was collected for the determination of serum estradiol (E2), progesterone (P4), and testosterone (T), followed by removal of the ovaries that were fixed in 10% formalin and processed for paraffin embedding. Five-µm histological sections were stained with hematoxylin and eosin and used for histomorphometric analysis. Morphological analyses, cyst count, determination of concentration and of the nuclear volume of interstitial cells were performed with the aid of a light microscope adapted to a high resolution camera (AxioCam), whose images were transmitted to and analyzed by the computer using AxioVision Rel 4.8 software (Carl Zeiss). Data were analyzed statistically by the Students t-test (p CtrlG=73.2±6.5, p CtrlG=80.6±3.9, p POG=4.2±1.5, p CtrlG=63.6±16.5, p CtrlG=6.9±3.2, p<0.05) compared to CtrlG animals. CONCLUSION: The interstitial cells of the rat polycystic ovary probably originate from ovarian cysts due to the degeneration of granulosa cells and differentiation of the internal theca cells. The elevations of serum testosterone and estradiol were probably due to the significant increase in cell activity and in the area occupied by interstitial cells.

Trp28Arg/Ile35Thr LHB gene variants are associated with elevated testosterone levels in women with polycystic ovary syndrome.

INTRODUCTION Polycystic Ovary Syndrome (PCOS) is a complex endocrine disorder, of multifactorial etiology, which affects 6-10% of women of reproductive age. It is considered the leading cause of anovulatory infertility, menstrual disorders and hyperandrogenism in this population. The genetic basis of PCOS is still largely unknown despite significant family clustering; determining its mode of inheritance is particularly difficult given the heterogenic presentation of the disease. MATERIALS AND METHODS 130 Brazilian women, aged 14-42 years, who met the 2003 Rotterdam criteria for PCOS diagnosis, were included, and 96 healthy women constituted the control group. Presence of hirsutism was classified using the modified Ferriman-Gallwey score (F-G score) as absent (≤7), mild (8-14), and severe (≥15). Blood levels of luteinizing hormone (LH), total testosterone (TT), dehydroepiandrosterone sulfate (DHEA-S) and androstenedione were determined. The coding region of the luteinizing hormone beta-subunit (LHB) gene was amplified and sequenced. Differences in allelic and genotypic frequency distribution of each polymorphism across controls and cases were estimated by the Mantel-Haenszel chi-square or Fishers exact test (p<0.05), and the probability of an association between the detection of a polymorphism and presence of a diagnosis of PCOS, by logistic regression. RESULT(S) Sequencing detected 8 polymorphisms in the LHB gene coding region. Two polymorphisms in linkage disequilibrium were significantly more prevalent in the presence of hyperandrogenemia: rs1800447/rs34349826 (Trp28Arg/Ile35Thr) (p=0.02). CONCLUSION(S) In this series, a modulatory effect of LHB polymorphisms on hyperandrogenemia phenotype of PCOS was observed; however, this finding needs to be replicated in other populations.

Metoclopramide-induced hyperprolactinemia effects on the pituitary and uterine prolactin receptor expression

UNLABELLED In this work we have evaluated the gene expression profile of prolactin and prolactin receptor in the pituitary and the uterus of female mice with metoclopramide-induced hyperprolactinemia treated with estrogen and/or progesterone. For this purpose, 49 Swiss female mice were allocated to seven groups. INTERVENTIONS 50-day treatment with metoclopramide, progesterone and estrogen. Our results showed that in the pituitary, metoclopramide-induced hyperprolactinemia increased prolactin expression. In the castrated animals, progesterone, with or without estrogen, produced an increase in prolactin. Pituitary prolactin receptor and the estrogen and progesterone treatment were responsible for the rise in PRLR-S2. In the uterus, no differences in prolactin expression were found between the different study groups. PRLR-S1 had its expression reduced in all castrated animals as against the castrated group treated with vehicle. In the noncastrated animals, PRLR-S2 rose in the metoclopramide-treated group, and, in the castrated animals, its expression diminished in all groups in relation to the vehicle-treated castrated controls. An increase in PRLR-S3 was found in the oophorectomized animals treated with a combination of estrogen and progesterone. PRLR-L rose in the oophorectomized animals treated with progesterone in isolation or in association with estrogen. These findings suggest that metoclopramide associated to progesterone or estrogen may determine an increase in pituitary prolactin and PRLR-S2 expression. The estrogen-progesterone may enhance the expression of PRLR-S3 and PRLR-L isoform of prolactin receptor.

Heterotopic ovarian transplantation results in less apoptosis than orthotopic transplantation in a minipig model.

BackgroundOvarian autotransplantation has shown increasing promise as a clinical method for the preservation of fertility and hormonal function. However, information regarding the success rate of this type of transplantation is limited. We hypothesized that results vary according to the site of the ovarian transplantation. To test this hypothesis, fresh or cryopreserved ovarian strips were autotransplanted to orthotopic or heterotopic sites. The strips were later collected, and the morphology and expression of selected markers of apoptosis were evaluated. We compared the Bax, Bcl-2 and cleaved caspase-3 staining levels and the morphometric aspects of autotransplanted fresh and cryopreserved ovarian strips placed at orthotopic and heterotopic sites in minipigs.MethodsForty female minipigs were allocated to the following five groups: group 1 (control), ovarian tissue removed during oophorectomy; group 2, transplantation of fresh ovarian strips to a heterotopic site; group 3, transplantation of fresh ovarian strips to an orthotopic site; group 4, transplantation of cryopreserved ovarian strips to a heterotopic site; and group 5, transplantation of ovarian trips to an orthotopic site. On day 7 after transplantation, ovarian strips were collected, and the morphology and expression of apoptosis markers were evaluated.ResultsIn all groups, follicles across all stages of development were detected. The numbers of primordial, primary and secondary follicles were similar in all groups, but the numbers of antral follicles were lower in the cryopreserved groups in comparison with freshly derived ovarian tissue, with no significant differences observed between fresh and cryopreserved transplants. In all transplanted groups, Bcl-2 expression was lower and Bax expression was higher than in the control group. Furthermore, increased expression of apoptosis markers was detected in fresh intraperitoneal transplants. Lastly, the expression of cleaved caspase-3 was higher in the cryopreserved orthotopic group compared with the heterotopic group.ConclusionsOrthotopic and heterotopic ovarian strip transplantations are feasible options using these techniques. Importantly, we found that heterotopic transplantation preserves ovarian follicle integrity to a greater degree (i.e., lower expression of apoptosis markers) than orthotopic transplantation, and cryopreservation does not exacerbate expression of apoptosis’s markers. These findings have major clinical applications and enhance the discussion regarding the heterotopic transplantation of ovarian tissue.

The progesterone and estrogen modify the uterine prolactin and prolactin receptor expression of hyperprolactinemic mice

Abstract The aim of this study was to evaluate the effects of metoclopramide-induced hyperprolactinemia on the prolactin (PRL) and PRL receptor’s expression in the uterus of mice. For this purpose, 49 Swiss mice were divided into the following groups: GrSS (non-ovariectomized mice given vehicle); GrMET (non-ovariectomized mice treated with metoclopramide); OvSS (ovariectomized mice given vehicle); OvMET (ovariectomized mice treated with metoclopramide); OvMET+17βE (ovariectomized mice treated with metoclopramide and 17β estradiol); OvMET+MP (ovariectomized mice treated with metoclopramide and micronized progesterone); OvMET+17βE+MP (ovariectomized mice treated with metoclopramide and a solution of 17β estradiol and micronized progesterone). Immunohistochemical analyzes were evaluated semi-quantitatively. Our results showed that GrMET, OvMET+MP, and OvMET+17βE+MP presented strong PRL expression. OvMET and OvMET+17βE presented mild reaction, while GrSS and OvSS presented weak reaction. Concerning PRL receptor, OvMET+MP and OvMET+17βE+MP showed strong reaction; GrMET, OvSS, and OvMET+17βE showed mild reaction; and GrSS and OvMET showed weak reaction. These findings suggest that progesterone alone or in combination with estrogen may increase the expression of uterine PRL and PRL receptor.