Estrogen and progesterone receptors in human papilloma virus-related cervical neoplasia

Estrogen (ER) and progesterone (PR) receptors in the normal uterine cervix, cervical intraepithelial neoplasia and invasive carcinoma were studied in consecutive samples from Hospital do Câncer, São Paulo, between 1996 and 1997. Tissue was collected by removing a fragment of the tumoral area using a 5-mm diameter biopsy punch, followed by removal of a macroscopically normal area as close as possible from the tumor. Histopathological confirmation was obtained for all specimens analyzed. A total of 24 normal tissues, 17 cases of cervical intraepithelial neoplasia and 7 of invasive carcinomas were studied. The ER/PR ratio was determined by immunohistochemistry using monoclonal antibodies specific for each receptor. Adjacent tissue slides were submitted to generic PCR for human papillomavirus (HPV) DNA detection followed by typing by dot blot hybridization. About half (45.8%) of the tumors were HPV DNA positive while 29.1% of the patients were also HPV positive in their respective normal tissue. ER was negative in the tumoral epithelium of 11 HPV-positive patients (P = 0.04). There was a trend in the ER distribution in normal tissue that was opposite to that from lesions, but it was not statistically significant (P = 0.069). No difference in ER distribution in stromal tissues was observed between HPV-positive and HPV-negative tissues. PR staining was negative in the epithelium of all cases studied. The results obtained from this small number of cases cannot be considered to be conclusive but do suggest that factors related to viral infection affect the expression of these ER/PR cervix receptors.

Effects of metformin treatment on luteal phase progesterone concentration in polycystic ovary syndrome

The causes of luteal phase progesterone deficiency in polycystic ovary syndrome (PCOS) are not known. To determine the possible involvement of hyperinsulinemia in luteal phase progesterone deficiency in women with PCOS, we examined the relationship between progesterone, luteinizing hormone (LH) and insulin during the luteal phase and studied the effect of metformin on luteal progesterone levels in PCOS. Patients with PCOS (19 women aged 18-35 years) were treated with metformin (500 mg three times daily) for 4 weeks prior to the test cycle and throughout the study period, and submitted to ovulation induction with clomiphene citrate. Blood samples were collected from control (N = 5, same age range as PCOS women) and PCOS women during the late follicular (one sample) and luteal (3 samples) phases and LH, insulin and progesterone concentrations were determined. Results were analyzed by one-way analysis of variance (ANOVA), Duncan's test and Karl Pearson's coefficient of correlation (r). The endocrine study showed low progesterone level (4.9 ng/ml) during luteal phase in the PCOS women as compared with control (21.6 ng/ml). A significant negative correlation was observed between insulin and progesterone (r = -0.60; P < 0.01) and between progesterone and LH (r = -0.56; P < 0.05) concentrations, and a positive correlation (r = 0.83; P < 0.001) was observed between LH and insulin. The study further demonstrated a significant enhancement in luteal progesterone concentration (16.97 ng/ml) in PCOS women treated with metformin. The results suggest that hyperinsulinemia/insulin resistance may be responsible for low progesterone levels during the luteal phase in PCOS. The luteal progesterone level may be enhanced in PCOS by decreasing insulin secretion with metformin.

Quantification of fecal estradiol and progesterone metabolites in Syrian hamsters (Mesocricetus auratus)

Alternative methods to the utilization of laboratory animal blood and its by-products are particularly attractive, especially regarding hamsters due to their small size and difficulties in obtaining serial blood samples. Steroid hormone metabolite quantification in feces, widely used in studies of free-ranging or intractable animals, is a non-invasive, non-stressor, economical, and animal saving technique which allows longitudinal studies by permitting frequent sampling of the same individual. The present study was undertaken to determine the suitability of this method for laboratory animals. Estradiol and progesterone metabolites were quantified by radioimmunoassay in feces of intact, sexually mature female Syrian hamsters during the estrous cycle (control) and in feces of superovulated females. Metabolites were extracted by fecal dilution in ethanol and quantified by solid phase radioimmunoassay. Median estrogen and progesterone concentrations were 9.703 and 180.74 ng/g feces in the control group, respectively. Peaks of estrogen (22.44 ± 4.54 ng/g feces) and progesterone (655.95 ± 129.93 ng/g feces) mean fecal concentrations respectively occurred 12 h before and immediately after ovulation, which is easily detected in this species by observation of a characteristic vaginal postovulatory discharge. Median estrogen and progesterone concentrations (28.159 and 586.57 ng/g feces, respectively) were significantly higher in superovulated animal feces (P < 0.0001). The present study demonstrated that it is possible to monitor ovarian activity in Syrian hamsters non-invasively by measuring fecal estradiol and progesterone metabolites. This technique appears to be a quite encouraging method for the development of new endocrinologic studies on laboratory animals.

Fecal estradiol and progesterone metabolite levels in the three-toed sloth (Bradypus variegatus)

The present study was carried out to assess the possibility of measuring fecal steroid hormone metabolites as a noninvasive technique for monitoring reproductive function in the three-toed sloth, Bradypus variegatus. Levels of the estradiol (E2) and progesterone (P4) metabolites were measured by radioimmunoassay in fecal samples collected over 12 weeks from 4 captive female B. variegatus sloths. The validation of the radioimmunoassay for evaluation of fecal steroid metabolites was carried out by collecting 10 blood samples on the same day as defecation. There was a significant direct correlation between the plasma and fecal E2 and P4 levels (P < 0.05, Pearson's test), thereby validating this noninvasive technique for the study of the estrous cycle in these animals. Ovulation was detected in two sloths (SL03 and SL04) whose E2 levels reached 2237.43 and 6713.26 pg/g wet feces weight, respectively, for over four weeks, followed by an increase in P4 metabolites reaching 33.54 and 3242.68 ng/g wet feces weight, respectively. Interestingly, SL04, which presented higher levels of E2 and P4 metabolites, later gave birth to a healthy baby sloth. The results obtained indicate that this is a reliable technique for recording gonadal steroid secretion and thereby reproduction in sloths.

Cortisol and 17-a-hydroxy-progesterone levels in infants with refractory hypotension born at 30 weeks of gestation or less

Refractory hypotension is frequent in very low-birth weight infants, whose hypothalamic-pituitary-adrenal axis has been suggested to be immature. The objective of the present study was to evaluate basal cortisol and 17-a-OH-progesterone in the first 36 h of life in preterm infants with and without refractory hypotension (mean arterial blood pressure below the lower limit for gestational age throughout the study despite aggressive volume expansion and use of vasopressors). Thirty-five infants with £30 weeks of gestation and a birth weight £1250 g, with no postnatal use of corticosteroid or death in the first 48 h were studied. Mean arterial pressure was measured every 4 h during the first 48 h. Cortisol and 17-a-OH-progesterone were determined at 12 and 36 h and patients were divided into refractory hypotensive (N = 15) and control (N = 20) groups. The groups were not different regarding type of delivery, use of prenatal corticosteroid, requirement of mechanical ventilation, use of vasopressor drugs, morphine, fentanyl, prophylactic indomethacin, and mean sample timing. Although refractory hypotensive newborns were more immature, were smaller, suffered more deaths after 48 h of life and had a higher SNAPPE-2 score, their cortisol and 17-a-OH-progesterone levels were not different from controls at 12 h and at 36 h. The increase of cortisol in newborns with refractory hypotension 36 h after birth was significantly higher than in controls. Despite the fact that refractory hypotensive very low-birth weight neonates were submitted to a very stressful condition, their cortisol and 17-a-OH-progesterone levels were similar to controls.

Polymorphism in CYP17, GSTM1 and the progesterone receptor genes and its relationship with mammographic density

Radiologic breast density is one of the predictive factors for breast cancer and the extent of the density is directly related to postmenopause. However, some patients have dense breasts even during postmenopause. This condition may be explained by the genes that codify for the proteins involved in the biosynthesis, as well as the activity and metabolism of steroid hormones. They are polymorphic, which could explain the variations of individual hormones and, consequently, breast density. The constant need to find markers that may assist in the primary prevention of breast cancer as well as in selecting high risk patients motived this study. We determined the influence of genetic polymorphism of CYP17 (cytochrome P450c17, the gene involved in steroid hormone biosynthesis), GSTM1 (glutathione S-transferase M1, an enzyme involved in estrogen metabolism) and PROGINS (progesterone receptor), for association with high breast density. One hundred and twenty-three postmenopausal patients who were not on hormone therapy and had no clinical or mammographic breast alterations were included in the present study. The results of this study reveal that there was no association between dense breasts and CYP17 or GSTM1. There was a trend, which was not statistically significant (P = 0.084), towards the association between PROGINS polymorphism and dense breasts. However, multivariate logistic regression showed that wild-type PROGINS and mutated CYP17, taken together, resulted in a 4.87 times higher chance of having dense breasts (P = 0.030). In conclusion, in the present study, we were able to identify an association among polymorphisms, involved in estradiol biosyntheses as well as progesterone response, and radiological mammary density.

Influence of progesterone on GAD65 and GAD67 mRNA expression in the dorsolateral striatum and prefrontal cortex of female rats repeatedly treated with cocaine

Female rats are intensely affected by cocaine, with estrogen probably playing an important role in this effect. Progesterone modulates the GABA system and attenuates the effects of cocaine; however, there is no information about its relevance in changing GABA synthesis pathways after cocaine administration to female rats. Our objective was to investigate the influence of progesterone on the effects of repeated cocaine administration on the isoenzymes of glutamic acid decarboxylase (GAD65 and GAD67) mRNA in brain areas involved in the addiction circuitry. Ovariectomized, intact and progesterone replacement-treated female rats received saline or cocaine (30 mg/kg, ip) acutely or repeatedly. GAD isoenzyme mRNA levels were determined in the dorsolateral striatum (dSTR) and prefrontal cortex (PFC) by RT-PCR, showing that repeated, but not acute, cocaine decreased GADs/β-actin mRNA ratio in the dSTR irrespective of the hormonal condition (GAD65: P < 0.001; and GAD67: P = 0.004). In the PFC, repeated cocaine decreased GAD65 and increased GAD67 mRNA ratio (P < 0.05). Progesterone replacement decreased both GAD isoenzymes mRNA ratio after acute cocaine in the PFC (P < 0.001) and repeated cocaine treatment reversed this decrease (P < 0.001). These results suggest that cocaine does not immediately affect GAD mRNA expression, while repeated cocaine decreases both GAD65 and GAD67 mRNA in the dSTR of female rats, independently of their hormonal conditions. In the PFC, repeated cocaine increases the expression of GAD isoenzymes, which were decreased due to progesterone replacement.

Estrous cycle and stress: influence of progesterone on the female brain

The female brain operates in a constantly changing chemical milieu caused by cyclical changes in gonadal hormones during the estrous cycle (menstrual cycle in women). Such hormones are highly lipophilic and pass readily from the plasma to the brain where they can influence neuronal function. It is becoming clear that the rapid reduction in peripheral circulating progesterone, which occurs during the late diestrous phase of the cycle, can trigger a withdrawal-like response, in which changes in GABA A receptor expression render hyper-responsive certain brain areas involved in processing responses to stressful stimuli. The periaqueductal gray matter (PAG) is recognised as an important region for integrating anxiety/defence responses. Withdrawal from progesterone, via actions of its neuroactive metabolite allopregnanolone, triggers up-regulation of extrasynaptic GABA A receptors on GABAergic neurons in the PAG. As a consequence, ongoing GABAergic tone on the output cells decreases, leading to an increase in functional excitability of the circuitry and enhanced responsiveness to stressful stimuli during the late diestrous phase. These changes during late diestrus could be prevented by short-term neurosteroid administration, timed to produce a more gradual fall in the peripheral concentration of allopregnanolone than the rapid decrease that occurs naturally, thus removing the trigger for the central withdrawal response.

Maternal serum progesterone, estradiol and estriol levels in successful dinoprostone-induced labor

Hormone-mediated quiescence involves the maintenance of a decreased inflammatory responsiveness. However, no study has investigated whether labor induction with prostanoids is associated with changes in the levels of maternal serum hormones. The objective of this study was to determine whether labor induction with dinoprostone is associated with changes in maternal serum progesterone, estradiol, and estriol levels. Blood samples were obtained from 81 pregnant women at term. Sixteen patients had vaginal birth after spontaneous labor, 12 required cesarean section after spontaneous labor and 16 underwent elective cesarean. Thirty-seven patients had labor induction with dinoprostone. Eligible patients received a vaginal insert of dinoprostone (10 mg) and were followed until delivery. Serum progesterone (P4), estradiol (E2) and estriol (E3) levels and changes in P4/E2, P4/E3 and E3/E2 ratios were monitored from admission to immediately before birth, and the association of these measures with the resulting clinical classification outcome (route of delivery and induction responsiveness) was assessed. Progesterone levels decreased from admission to birth in patients who underwent successful labor induction with dinoprostone [vaginal and cesarean birth after induced labor: 23% (P < 0.001) and 18% (P < 0.025) decrease, respectively], but not in those whose induction failed (6.4% decrease, P > 0.05). Estriol and estradiol levels, P4/E2, P4/E3 and E3/E2 ratios did not differ between groups. Successful dinoprostone-induced labor was associated with reduced maternal progesterone levels from induction to birth. While a causal relationship between progesterone decrease and effective dinoprostone-induced labor cannot be established, it is tempting to propose that dinoprostone may contribute to progesterone withdrawal and favor labor induction in humans.

Roles of estrogen and progesterone in modulating renal nerve function in the rat kidney

The maintenance of extracellular Na+ and Cl- concentrations in mammals depends, at least in part, on renal function. It has been shown that neural and endocrine mechanisms regulate extracellular fluid volume and transport of electrolytes along nephrons. Studies of sex hormones and renal nerves suggested that sex hormones modulate renal function, although this relationship is not well understood in the kidney. To better understand the role of these hormones on the effects that renal nerves have on Na+ and Cl- reabsorption, we studied the effects of renal denervation and oophorectomy in female rats. Oophorectomized (OVX) rats received 17β-estradiol benzoate (OVE, 2.0 mg·kg-1·day-1, sc) and progesterone (OVP, 1.7 mg·kg-1·day-1,sc). We assessed Na+ and Cl-fractional excretion (FENa+ and FECl-, respectively) and renal and plasma catecholamine release concentrations. FENa+, FECl-, water intake, urinary flow, and renal and plasma catecholamine release levels increased in OVX vs control rats. These effects were reversed by 17β-estradiol benzoate but not by progesterone. Renal denervation did not alter FENa+, FECl-, water intake, or urinary flow values vs controls. However, the renal catecholamine release level was decreased in the OVP (236.6±36.1 ng/g) and denervated rat groups (D: 102.1±15.7; ODE: 108.7±23.2; ODP: 101.1±22.1 ng/g). Furthermore, combining OVX + D (OD: 111.9±25.4) decreased renal catecholamine release levels compared to either treatment alone. OVE normalized and OVP reduced renal catecholamine release levels, and the effects on plasma catecholamine release levels were reversed by ODE and ODP replacement in OD. These data suggest that progesterone may influence catecholamine release levels by renal innervation and that there are complex interactions among renal nerves, estrogen, and progesterone in the modulation of renal function.

Behavioral effects of endogenous or exogenous estradiol and progesterone on cocaine sensitization in female rats

Cocaine sensitization is a marker for some facets of addiction, is greater in female rats, and may be influenced by their sex hormones. We compared the modulatory effects of endogenous or exogenous estradiol and progesterone on cocaine-induced behavioral sensitization in 106 female rats. Ovariectomized female rats received progesterone (0.5 mg/mL), estradiol (0.05 mg/mL), progesterone plus estradiol, or the oil vehicle. Sham-operated control females received oil. Control and acute subgroups received injections of saline, while the repeated group received cocaine (15 mg/kg, ip) for 8 days. After 10 days, the acute and repeated groups received a challenge dose of cocaine, after which locomotion and stereotypy were monitored. The estrous cycle phase was evaluated and blood was collected to verify hormone levels. Repeated cocaine treatment induced overall behavioral sensitization in female rats, with increased locomotion and stereotypies. In detailed analysis, ovariectomized rats showed no locomotor sensitization; however, the sensitization of stereotypies was maintained. Only females with endogenous estradiol and progesterone demonstrated increased locomotor activity after cocaine challenge. Estradiol replacement enhanced stereotyped behaviors after repeated cocaine administration. Cocaine sensitization of stereotyped behaviors in female rats was reduced after progesterone replacement, either alone or concomitant with estradiol. The behavioral responses (locomotion and stereotypy) to cocaine were affected differently, depending on whether the female hormones were of an endogenous or exogenous origin. Therefore, hormonal cycling appears to be an important factor in the sensitization of females. Although estradiol increases the risk of cocaine sensitization, progesterone warrants further study as a pharmacological treatment in the prevention of psychostimulant abuse.

Activation of Progesterone Receptor by ATP

Progesterone-receptor complex from freshly prepared hen oviduct cytosol acquired the ability to bind to isolated nuclei, DNA-cellulose and ATP-Sepharose when incubated with 5-10 mM ATP at 4°C. The extent of this ATP-dependent activation was higher when compared with heat-activation achieved by warming the progesterone- receptor complex at 23 °C. The transformation of progesterone-receptor complex which occurred in a time-dependent manner was only partially dependent on hormone presence. The ATP effect was selective in causing this transformation whereas ADP, AMP and cAMP failed to show any such effect. The non-hydrolizable analogs of ATP, adenosine 5'-[a,/3-methylene]triphosphate and adenosine 5-[/l,y-imido]triphosphate were also found to be ineffective. Presence of 10 mM sodium molybdate blocked both the ATP and the heat-activation of progesterone-receptor complex. Mn" or Mg` had no detectable effect on the receptor activation but the presence of Ca" increased the extent of ATP-activation slightly. EDTA presence (> 5 mM) decreased the extent of receptor activation by about 40 % and was, therefore, not included in the buffers used for activation studies. Divalent cations were also ineffective when tested in the presence of 1- 5 mM EDTA. The properties of progesterone-receptor complex remained intact under the above conditions when analyzed for steroid-binding specificity and Scatchard analysis. However, the ATP-activated progesterone-receptor complex lost the ability to aggregate when tested on low-salt sucrose gradients. ATP was equally effective in activating the rat-uterine estradiol-receptor complex at 4 "C and influenced the transformation of 4-S receptor form into a 5-S form when analyzed on sucrose gradients containing 0.3 M KCI. The presence of ATP also increased the rate of activation of progesterone-receptor complex at 23 °C. These findings suggest a role for ATP in receptor function and offer a convenient method of studying the process of receptor activation at low temperature and mild assay conditions.

Metabolic profiles and progesterone cycles in first lactation dairy cows

This study investigated the ovarian function, metabolic profiles and fertility in first lactation Holstein-Friesian dairy cows (mean 305 day milk yield: 7417 +/- 191 kg, n = 37). Reproductive profiles obtained from milk progesterone analysis were categorized into normal (n = 17) and four abnormal profiles (delayed ovulation, DOV1, n = 9; DOV2, n = 2; persistent corpus luteum, PCL1, n = 6; PCL2, n = 4; 1: immediately post-calving, 2: subsequent cycles). Fifty-five percent of cows had abnormal profiles with half of these being categorized as DOV1. Fertility of DOV1 and DOV2 cows was reduced whereas PCL1 and PCL2 cows had similar reproductive competence to normal profile cows. DOV1 animals had higher milk energy values, lower energy balances, lower dry matter intakes (DMI) and greater body weight and body condition score (BCS) losses post-calving than normal profile animals. DOV1 animals also had lower insulin-like growth factor-I (IGF-I) and higher betahydroxybutyrate (BHB) concentrations and tended to have the lower insulin and glucose concentrations in the pre-service period than normal profile cows. All PCL animals had vulval discharges postpartum. Despite this, the DMI, body weight and BCS changes, IGF-I concentrations and fertility of PCL1 animals was similar to normal profile cows. In conclusion, the high prevalence of delayed ovulation post-calving (DOV1) in primiparous high yielding cows lasted long enough (71 +/- 8.3 days) to have a detrimental impact on fertility and was associated with significant physiological changes. This study did not establish any detrimental effects of PCL profiles on fertility or production parameters. (C) 2002 Elsevier Science Inc. All rights reserved.

Bone morphogenetic protein-6 enhances gonadotrophin-dependent progesterone and inhibin secretion and expression of rnRNA transcripts encoding gonadotrophin receptors and inhibin/activin subunits in chicken granulosa cells

The aims were to examine ovarian expression of bone morphogenetic protein (BMP) ligands/receptor mRNAs in the chicken and to test the hypothesis that theca-derived BMP(s) modulates granulosa cell function in a paracrine manner. RT-PCR revealed expression of multiple BMPs in granulosa and theca cells from prehierarchical and preovulatory follicles with greater expression in theca cells; both cell types expressed BMP receptors-1A, -1B and -II consistent with tissue responsiveness. Preovulatory granulosa cells F1, F2 and F3/4) were cultured with BMP-6 (expressed by theca but not granulosa) in the presence/absence of LH, FSH or 8-Br-cAMP. RMP-6 increased 'basal' and gonadotrophin-induced inhibin-A and progesterone secretion by each cell type but did not enhance the effect of 8-Br-cAMP. This indicates that the observed synergism between BMP-6 and gonadotrophin might involve BMP-induced up-regulation of gonadotrophin receptors. In support of this, BMP-6 alone increased LH-receptor (LHR) mRNA in F1 cells and FSH-receptor (FSHR) mRNA in F1, F2 and F3/4 cells. RMP-6 also enhanced LH/FSH-induced LHR transcript amount in each cell type but did not raise FSHR transcript amounts above those induced by BMP-6 alone. To further explore BMP6 action on inhibin-A secretion, we quantified inhibin/activin subunits (alpha, beta(A), beta(B)) mRNAs. Consistent with its effect on inhibin-A secretion, BMP-6 enhanced 'basal' expression of alpha- and beta(A)-Subunit mRNA in F1, F2 and F3/4 cells, and beta(B)-subunit mRNA in F3/4 cells. BMP-6 markedly enhanced FSH/LH-induced expression of alpha-subunit in all follicles and FSH-induced beta(A)-subunit in F2 and F3/4 follicles but not in F1 follicles. Neither BMP-6 alone, nor FSH/LH alone, affected 'basal' OB mRNA abundance. However, co-treatment with gonadotrophin and BMP-6 greatly increased beta(B)-subunit expression, the response being lowest in F1 follicles and greatest in F3/4 follicles. Collectively, these results support the hypothesis that intra-ovarian OMPs of thecal origin have a paracrine role in modulating granulosa cell function in the chicken in a preovulatory stage-dependent manner.

Bile salt composition is secondary to bile salt concentration in determining hydrocortisone and progesterone solubility in intestinal mimetic fluids.

Simulated intestinal fluids (SIFs) used to assay the solubility of orally administered drugs are typically based on a single bile salt; sodium taurocholate (STC). The aim of this study was to develop mimetic intestinal fluids with a closer similarity to physiological fluids than those reported to date by developing a mixed bile salt (MBS) system (STC, sodium glycodeoxycholate, sodium deoxycholate; 60:39:1) with different concentrations of lecithin, the preponderant intestinal phospholipid. Hydrocortisone and progesterone were used as model drugs to evaluate systematically the influence of SIF composition on solubility. Increasing total bile salt concentration from 0 to 30 mM increased hydrocortisone and progesterone solubility by 2- and ∼25-fold, respectively. Accordingly, higher solubilities were measured in the fed-state compared to the fasted-state SIFs. Progesterone showed the greatest increases in solubility in STC and MBS systems (2-7-fold) compared to hydrocortisone (no significant change; P>0.05) as lecithin concentration was increased. Overall, MBS systems gave similar solubility profiles to STC. In conclusion, the addenda of MBS and lecithin were found to be secondary to the influence of BS concentration. These data provide a foundation for the design of more bio-similar media for pivotal decision-guiding assays in drug development and quality control settings.