Hypothalamic transcriptional expression of the kisspeptin system and sex steroid receptors differs among polycystic ovary syndrome rat models with different endocrine phenotypes.

OBJECTIVES:: Polycystic ovary syndrome is a heterogeneous endocrine disorder that affects reproductive-age women. The mechanisms underlying the endocrine heterogeneity and neuroendocrinology of polycystic ovary syndrome are still unclear. In this study, we investigated the expression of the kisspeptin system and gonadotropin-releasing hormone pulse regulators in the hypothalamus as well as factors related to luteinizing hormone secretion in the pituitary of polycystic ovary syndrome rat models induced by testosterone or estradiol. METHODS:: A single injection of testosterone propionate (1.25 mg) (n=10) or estradiol benzoate (0.5 mg) (n=10) was administered to female rats at 2 days of age to induce experimental polycystic ovary syndrome. Controls were injected with a vehicle (n=10). Animals were euthanized at 90-94 days of age, and the hypothalamus and pituitary gland were used for gene expression analysis. RESULTS:: Rats exposed to testosterone exhibited increased transcriptional expression of the androgen receptor and estrogen receptor-ß and reduced expression of kisspeptin in the hypothalamus. However, rats exposed to estradiol did not show any significant changes in hormone levels relative to controls but exhibited hypothalamic downregulation of kisspeptin, tachykinin 3 and estrogen receptor-α genes and upregulation of the gene that encodes the kisspeptin receptor. CONCLUSIONS:: Testosterone- and estradiol-exposed rats with different endocrine phenotypes showed differential transcriptional expression of members of the kisspeptin system and sex steroid receptors in the hypothalamus. These differences might account for the different endocrine phenotypes found in testosterone- and estradiol-induced polycystic ovary syndrome rats.

Hypothalamic transcriptional expression of the kisspeptin system and sex steroid receptors differs among polycystic ovary syndrome rat models with different endocrine phenotypes

OBJECTIVES: Polycystic ovary syndrome is a heterogeneous endocrine disorder that affects reproductive-age women. The mechanisms underlying the endocrine heterogeneity and neuroendocrinology of polycystic ovary syndrome are still unclear. In this study, we investigated the expression of the kisspeptin system and gonadotropin-releasing hormone pulse regulators in the hypothalamus as well as factors related to luteinizing hormone secretion in the pituitary of polycystic ovary syndrome rat models induced by testosterone or estradiol. METHODS: A single injection of testosterone propionate (1.25 mg) (n=10) or estradiol benzoate (0.5 mg) (n=10) was administered to female rats at 2 days of age to induce experimental polycystic ovary syndrome. Controls were injected with a vehicle (n=10). Animals were euthanized at 90-94 days of age, and the hypothalamus and pituitary gland were used for gene expression analysis. RESULTS: Rats exposed to testosterone exhibited increased transcriptional expression of the androgen receptor and estrogen receptor-β and reduced expression of kisspeptin in the hypothalamus. However, rats exposed to estradiol did not show any significant changes in hormone levels relative to controls but exhibited hypothalamic downregulation of kisspeptin, tachykinin 3 and estrogen receptor-α genes and upregulation of the gene that encodes the kisspeptin receptor. CONCLUSIONS: Testosterone- and estradiol-exposed rats with different endocrine phenotypes showed differential transcriptional expression of members of the kisspeptin system and sex steroid receptors in the hypothalamus. These differences might account for the different endocrine phenotypes found in testosterone- and estradiol-induced polycystic ovary syndrome rats.

Multiple reaction monitoring (MRM)-profiling for biomarker discovery applied to human polycystic ovarian syndrome.

RATIONALE: We describe multiple reaction monitoring (MRM)-profiling, which provides accelerated discovery of discriminating molecular features, and its application to human polycystic ovary syndrome (PCOS) diagnosis. The discovery phase of the MRM-profiling seeks molecular features based on some prior knowledge of the chemical functional groups likely to be present in the sample. It does this through use of a limited number of pre-chosen and chemically specific neutral loss and/or precursor ion MS/MS scans. The output of the discovery phase is a set of precursor/product transitions. In the screening phase these MRM transitions are used to interrogate multiple samples (hence the name MRM-profiling). METHODS: MRM-profiling was applied to follicular fluid samples of 22 controls and 29 clinically diagnosed PCOS patients. Representative samples were delivered by flow injection to a triple quadrupole mass spectrometer set to perform a number of pre-chosen and chemically specific neutral loss and/or precursor ion MS/MS scans. The output of this discovery phase was a set of 1012 precursor/product transitions. In the screening phase each individual sample was interrogated for these MRM transitions. Principal component analysis (PCA) and receiver operating characteristic (ROC) curves were used for statistical analysis. RESULTS: To evaluate the method's performance, half the samples were used to build a classification model (testing set) and half were blinded (validation set). Twenty transitions were used for the classification of the blind samples, most of them (N = 19) showed lower abundances in the PCOS group and corresponded to phosphatidylethanolamine (PE) and phosphatidylserine (PS) lipids. Agreement of 73% with clinical diagnosis was found when classifying the 26 blind samples. CONCLUSIONS: MRM-profiling is a supervised method characterized by its simplicity, speed and the absence of chromatographic separation. It can be used to rapidly isolate discriminating molecules in healthy/disease conditions by tailored screening of signals associated with hundreds of molecules in complex samples.

Acupuncture does not ameliorate metabolic disturbances in the P450 aromatase inhibitor-induced rat model of polycystic ovary syndrome.

NEW FINDINGS: What is the central question of this study? The effectiveness of low-frequency electroacupuncture in the treatment of metabolic disorders associated with polycystic ovary syndrome (PCOS), an endocrine-metabolic disorder characterized by an imbalance in sex steroid production, is controversial. What is the main finding and its importance? In a rat model of PCOS induced by the inhibition of P450 aromatase, low-frequency electroacupuncture increased low-density lipoprotein-cholesterol but did not improve the insulin resistance or the adipose tissue dysfunction, suggesting that a balance of sex steroids is needed to restore the metabolic function in this rat model of PCOS. Low-frequency electroacupuncture restores sex steroid synthesis and sympathetic activity in women with polycystic ovary syndrome, which may ameliorate its metabolic disturbances, probably by modulating sympathetic nerve activity or sex steroid synthesis. We investigated whether low-frequency electroacupuncture regulates the metabolic function to the same extent as treatment with estradiol or ß-adrenergic blocking in a rat model of polycystic ovary syndrome induced by a P450 aromatase inhibitor (letrozole). Letrozole (200 µg day-1 ) or placebo pellets were implanted in prepubertal Wistar rats. Six weeks thereafter, rats were treated for 5-6 weeks with the following: low-frequency electroacupuncture (5 days per week); a ß-adrenergic blocker (propranolol hydrochloride, 0.1 mg kg-1 , 5 days per week); or 17ß-estradiol (2.0 µg) every fourth day. Body weight development, body composition, locomotor activity, insulin sensitivity, tissue-specific glucose uptake, lipid profile, adipocyte size, serum concentrations of adiponectin and insulin, and gene expression in inguinal fat were measured. All treatments increased circulating levels of low-density lipoprotein-cholesterol. Estradiol treatment restored locomotor activity and increased insulin sensitivity but did not modify the glucose uptake in muscle and fat. An upregulation of genes related to insulin sensitivity and downregulation of genes related to adipogenesis were observed in subcutaneous adipose tissue from rats exposed to letrozole. Only estradiol treatment normalized the expression of these genes. In conclusion, low-frequency electroacupuncture increased low-density lipoprotein-cholesterol without affecting insulin sensitivity or adipose tissue function, which could suggest effects on hepatic lipid regulation, probably mediated by the action of estradiol or the ß-adrenergic pathway.

The promoter -1031(T/C) polymorphism in tumor necrosis factor-alpha associated with polycystic ovary syndrome.

BACKGROUND: A tumor necrosis factor-alpha is a multifunctional pro-inflammation cytokine, which has been considered as one of pathogenic factors for various diseases. The promoter -1031(T/C) polymorphism in the tumor necrosis factor-alpha gene was reported that it plays a part in reproduction-related diseases. Among these, polycystic ovary syndrome (PCOS) is known to be a common gynecological disease of women in reproductive age women. Here, we performed a comparative study of -1031(T/C) polymorphism of TNF-alpha gene with PCOS in a Korean population. METHODS: The -1031(T/C) polymorphism of TNF-alpha gene was analyzed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in a total of 217 PCOS patients and 144 matched female controls of healthy women. And statistical analysis was performed using HapAnalyzer. X2 test and logistic regression were utilized analyze the association between two groups. A p-value under 0.05 was considered statistically significant. RESULTS: The genotype and allelic frequencies were in Hardy-Weinberg equilibrium (HWE). There was strong association between the -1031(T/C) polymorphism in the promoter region of TNF-alpha gene and PCOS (p-value = 0.0003, odd ratio (OR) = 2.53). In addition, the frequency of C allele was significantly higher in PCOS patients compared with controls. Sequence analyses also showed the -1031(T/C) polymorphism of TNF-alpha gene. CONCLUSION: This is the first study on the -1031(T/C) polymorphism of TNF-alpha gene in PCOS. We concluded that the -1031(T/C) polymorphism of TNF-alpha gene is associated with PCOS in a Korean population. Therefore, it is possible that it may be considered as a clinical biomarker to diagnose for PCOS, and is helpful in understanding the etiology for the pathogenesis of PCOS.

Expression of steroidogenic enzymes in porcine polycystic ovaries.

In the present study the expression pattern of the cholesterol side-chain cleavage cytochrome (P450(scc)), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and aromatase (P450(arom)) was analyzed in the health and polycystic ovaries of gilts by means of the Western blot and immunohistochemistry. The polycystic status of ovaries was induced by i.m. dexamethasone (DXM) injections on days 7-21 of the estrous cycle. Macroscopic observation of ovaries of DXM-treated gilts revealed the presence of cysts (1-2 cm in diameter, with a mean number of 7.0+/-1.2 per ovary), a decrease (P<0.05) in number of small follicles (1-3 mm in diameter), as well as the lack of medium-sized follicles (4-6 mm in diameter) and corpora lutea, as compared to the control animals. The expression of P450(scc) (P<0.01), 3beta-HSD (P<0.05) and P450(arom) (P<0.001) proteins in the cysts was higher than in the medium-sized follicles of the control gilts. Moreover, DXM injections resulted also in an enhancement (P<0.05) in the level of P450(scc) protein in the walls of small follicles as compared to the control gilts. Following DXM administration the immunoreactivity (IR) of P450(scc) in the primordial follicles was lower than in the control group. Comparing to the control gilts, the reaction for this enzyme in DXM-treated animals was observed in secondary follicles, while for 3beta-HSD, in primordial, primary, as well as secondary follicles. The immunostaining for P450(scc) (theca cells) and P450(arom) (granulosa cells) in the small follicles of the DXM-treated gilts were more prominent than those found in the gonads of control animals. However, IR for P450(scc) was not found in the granulosa cells of small follicles in the gilts receiving DXM. The intensity of P450(scc) and P450(arom) labelling was distinctly enhanced in the cysts as compared to the medium follicles of the control animals. Furthermore, in contrary to the medium follicles of the control animals, faint IR for 3beta-HSD was found in the granulosa cell layer of cysts. Our data revealed that both the expression of P450(scc), 3beta-HSD and P450(arom) and localization of these enzymes in polycystic ovaries were different from those, found under physiological conditions. These results suggested that above-mentioned enzymes may, by influencing the ovarian steroid synthesis, play an essential role in the creation and/or course of cystic ovarian disease.

Subcutaneous and omental fat expression of adiponectin and leptin in women with polycystic ovary syndrome.

OBJECTIVE: To assess message expression of adiponectin and leptin in visceral and SC fat in women with polycystic ovary syndrome (PCOS) and in control women. DESIGN: Prospective clinical trial. SETTING: Academic medical centers in Mexico City, Mexico and New York, New York. PATIENT(S): Women with PCOS and control women. INTERVENTION(S): Surgical biopsies of visceral (omental) and subcutaneous (SC) adipose tissue, fasting blood samples, and ultrasound measurements of visceral and SC fat. MAIN OUTCOME MEASURE(S): Messenger RNA assessment of adiponectin and leptin in adipose tissue samples; serum measurements of adiponectin, leptin, glucose, insulin, and hormone levels; measurements of fat quantity by ultrasound. Correlative analyses as well as comparisons between women with PCOS and control women were performed. RESULT(S): Confirming previous data, women with PCOS had more insulin resistance, similar serum leptin, but lower serum adiponectin compared with control women. When control women were divided into quartiles by body mass index (BMI), messenger RNA expression of leptin and adiponectin decreased with increasing BMI. Adiponectin and leptin expression was significantly lower in women with PCOS; in weight-matched patients and control women, leptin and adiponectin expression was statistically significantly lower in SC tissue, and adiponectin expression was statistically significantly lower in omental tissue in women with PCOS. In control women, there was greater expression in SC tissue compared with in visceral tissue. There were significant negative correlations between visceral and SC fat mass by both ultrasound as well as adiponectin and leptin expression in women with PCOS. Serum adiponectin correlated statistically significantly with visceral adiponectin expression (r = 0.64) in women with PCOS, and there was a statistically significant correlation between SC adiponectin expression and the Quantitative Insulin-Sensitivity Check Index as a marker of insulin resistance (r = 0.43). CONCLUSION(S): Adipocytokine expression in fat tissue appears to be down-regulated by an increased fat mass; this is particularly evident in the case of adiponectin expression in women with PCOS. It is probable that insulin resistance is a factor that may contribute, in part, to these findings.

Rats with steroid-induced polycystic ovaries develop hypertension and increased sympathetic nervous system activity.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder associated with ovulatory dysfunction, abdominal obesity, hyperandrogenism, hypertension, and insulin resistance. METHODS: Our objectives in this study were (1) to estimate sympathetic-adrenal medullary (SAM) activity by measuring mean systolic blood pressure (MSAP) in rats with estradiol valerate (EV)-induced PCO; (2) to estimate alpha1a and alpha2a adrenoceptor expression in a brain area thought to mediate central effects on MSAP regulation and in the adrenal medulla; (3) to assess hypothalamic-pituitary-adrenal (HPA) axis regulation by measuring adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels in response to novel-environment stress; and (4) to measure abdominal obesity, sex steroids, and insulin sensitivity. RESULTS: The PCO rats had significantly higher MSAP than controls, higher levels of alpha1a adrenoceptor mRNA in the hypothalamic paraventricular nucleus (PVN), and lower levels of alpha2a adrenoceptor mRNA in the PVN and adrenal medulla. After exposure to stress, PCO rats had higher ACTH and CORT levels. Plasma testosterone concentrations were lower in PCO rats, and no differences in insulin sensitivity or in the weight of intraabdominal fat depots were found. CONCLUSION: Thus, rats with EV-induced PCO develop hypertension and increased sympathetic and HPA-axis activity without reduced insulin sensitivity, obesity, or hyperandrogenism. These findings may have implications for mechanisms underlying hypertension in PCOS.

Avaliaçäo do uso da metformina em pacientes portadoras da síndrome dos ovários policísticos (SOP): estudo duplo cego randomizado / Evaluation of the treatment of polycystic ovary syndrome (PCOS) with metformin: a double blinded study

Avaliar os efeitos da utilizaçäo da metformina em pacientes portadoras da SOP, sob o ponto de vista clínico e laboratorial. Estudo clínico randomizados duplo cego. Vinte e duas pacientes com diagnóstico de síndrome dos ovários policísticos foram randomizadas em dois grupos: Grupo A, controle (placebo) e Grupo B, que utilizou metformina 850 mg de 12/12 h. O estudo teve duraçäo de 3 meses. Foram realizados exames laboratoriais, ultra-sonográficos e cuidadoso questionário antes e após o tratamento. Houve reduçäo significativa dos níveis de insulina sérica e da resistência insulínica no grupo que utilizou metformina, além disso, 90 por cento das pacientes deste grupo tiveram seu ciclo menstrual regularizado. Também se observou reduçäo, porém näo significativa dos níveis de androgênios, triglicérides e colesterol total com elevaçäo do HDL. A metformina se mostrou uma droga bem tolerada. A metformina é uma opçäo para o tratamento da SOP, sendo uma droga bem tolerada e eficaz em restabelecer a ciclicidade menstrual e reduzir os níveis de insulina destas pacientes.

[The proportion of molecular forms of gonadotropins changes in patients with polycystic ovaries]. / La proporción de formas moleculares de gonadotropinas se altera en pacientes con síndrome de ovarios poliquísticos.

It is known that protein hormones circulate as different molecular forms and the relative proportion of these isoforms changes according to endocrine milieu. In particular gonadotropins, both LH and FSH, isoforms suffer variations related to the estrogen levels; thus sera obstained from menopausal women show a predominance of larger molecular forms which are considered as having lesser biological activity and the administration of estrogen replacement therapy is followed by the appearance of intermediate molecular forms possessing higher biological activity. This chormatographic pattern with predominance of intermediate isoforms is typical at midcycle in sera from normal women at the periovulatory stage. Present study showed that sera obtained from anovulatory women, such as patients with polycystic ovaries a predominance of larger and smaller molecular weight isoforms, exhibiting a chromatographic pattern different from that observed in normal women. It is speculate that there is some imbalance between the ovarian steroid synthesis and gonadotropin production in the stage of tertiary structural conformation.

Expression of vascular permeability factor/vascular endothelial growth factor by human granulosa and theca lutein cells. Role in corpus luteum development.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a cytokine that is overexpressed in many tumors, in healing wounds, and in rheumatoid arthritis. VPF/VEGF is thought to induce angiogenesis and accompanying connective tissue stroma in two ways: 1), by increasing microvascular permeability, thereby modifying the extracellular matrix and 2), as an endothelial cell mitogen. VPF/VEGF has been reported in animal corpora lutea and we investigated the possibility that it might be present in human ovaries and have a role in corpus luteum formation. We here report that VPF/VEGF mRNA and protein are expressed by human ovarian granulosa and theca cells late in follicle development and, subsequent to ovulation, by granulosa and theca lutein cells. Therefore, VPF/VEGF is ideally positioned to provoke the increased permeability of thecal blood vessels that occurs shortly before ovulation. VPF/VEGF likely also contributes to the angiogenesis and connective tissue stroma generation that accompany corpus luteum/corpus albicans formation. Finally, VPF/VEGF was overexpressed in the hyperthecotic ovarian stroma of Stein-Leventhal syndrome in which it may also have a pathophysiological role.