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).

Paracrine stimulation of interstitial collagenase (MMP-1) in the human endometrium by interleukin 1α and its dual block by ovarian steroids

In the cycling human endometrium, the expression of interstitial collagenase (MMP-1) and of several related matrix metalloproteinases (MMPs) follows the late-secretory fall in sex steroid plasma concentrations and is thought to be a critical step leading to menstruation. The rapid and extensive lysis of interstitial matrix that precedes menstrual shedding requires a strict control of these proteinases. However, the mechanism by which ovarian steroids regulate endometrial MMPs remains unclear. We report here that, in the absence of ovarian steroids, MMP-1 expression in endometrial fibroblasts is markedly stimulated by medium conditioned by endometrial epithelial cells. This stimulation can be prevented by antibodies directed against interleukin 1α (IL-1α) but not against several other cytokines. Ovarian steroids inhibit the release of IL-1α and repress MMP-1 production by IL-1α-stimulated fibroblasts. In short-term cultures of endometrial explants obtained throughout the menstrual cycle, the release of both IL-1α and MMP-1 is essentially limited to the perimenstrual phase. We conclude that epithelium-derived IL-1α is the key paracrine inducer of MMP-1 in endometrial fibroblasts. However, MMP-1 production in the human endometrium is ultimately blocked by ovarian steroids, which act both upstream and downstream of IL-1α, thereby exerting an effective control via a “double-block” mechanism.

Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity

This study was undertaken to determine the modulation of uterine function by chorionic gonadotrophin (CG) in a nonhuman primate. Infusion of recombinant human CG (hCG) between days 6 and 10 post ovulation initiated the endoreplication of the uterine surface epithelium to form distinct epithelial plaques. These plaque cells stained intensely for cytokeratin and the proliferating cell nuclear antigen. The stromal fibroblasts below the epithelial plaques stained positively for α-smooth muscle actin (αSMA). Expression of αSMA is associated with the initiation of decidualization in the baboon endometrium. Synthesis of the glandular secretory protein glycodelin, as assessed by Western blot analysis, was markedly up-regulated by hCG, and this increase was confirmed by immunocytochemistry, Northern blot analysis, and reverse transcriptase-PCR. To determine whether hCG directly modulated these uterine responses, we treated ovariectomized baboons sequentially with estradiol and progesterone to mimic the hormonal profile of the normal menstrual cycle. Infusion of hCG into the oviduct of steroid-hormone-treated ovariectomized baboons induced the expression of αSMA in the stromal cells and glycodelin in the glandular epithelium. The epithelial plaque reaction, however, was not readily evident. These studies demonstrate a physiological effect of CG on the uterine endometrium in vivo and suggest that the primate blastocyst signal, like the blastocyst signals of other species, modulates the uterine environment prior to implantation.

In vivo responses of the primate corpus luteum to luteinizing hormone and chorionic gonadotropin

Although it is well established that the secretory activity of the corpus luteum absolutely depends on the presence of pituitary-derived luteinizing hormone (LH), it is unknown why the life span of the corpus luteum is extended during early pregnancy by the placental production of chorionic gonadotropin (CG) but regresses in the presence of LH despite the fact that CG and LH have similar actions on the corpus luteum. To compare the responses of the corpus luteum to LH and human CG (hCG), cynomolgus monkeys whose endogenous gonadotropin secretion was blocked during the luteal phase of the menstrual cycle with a gonadotropin-releasing hormone antagonist were i.v. infused with either LH or CG. Infusion of LH at a constant rate overcame the gonadotropin-releasing hormone antagonist-mediated premature luteal regression but failed to prolong the functional life span of the corpus luteum. Continuous infusions of hCG did not effect a pregnancy-like pattern of gonadotropin secretion, but the functional life span of the corpus luteun was extended in two of three animals. Infusion of either LH or hCG in an exponentially increasing manner prolonged the functional life span of the corpus luteum beyond its normal duration. These results indicate that luteal regression at the termination of nonfertile menstrual cycles is caused by a large reduction in the responsiveness of the aging corpus luteum to LH, which can be overcome by elevated concentrations of either LH or CG.

Noninvasive functional optical spectroscopy of human breast tissue

Near infrared diffuse optical spectroscopy and diffuse optical imaging are promising methods that eventually may enhance or replace existing technologies for breast cancer screening and diagnosis. These techniques are based on highly sensitive, quantitative measurements of optical and functional contrast between healthy and diseased tissue. In this study, we examine whether changes in breast physiology caused by exogenous hormones, aging, and fluctuations during the menstrual cycle result in significant alterations in breast tissue optical contrast. A noninvasive quantitative diffuse optical spectroscopy technique, frequency-domain photon migration, was used. Measurements were performed on 14 volunteer subjects by using a hand-held probe. Intrinsic tissue absorption and reduced scattering parameters were calculated from frequency-domain photon migration data. Wavelength-dependent absorption (at 674, 803, 849, and 956 nm) was used to determine tissue concentration of oxyhemoglobin, deoxyhemoglobin, total hemoglobin, tissue hemoglobin oxygen saturation, and bulk water content. Results show significant and dramatic differences in optical properties between menopausal states. Average premenopausal intrinsic tissue absorption and reduced scattering values at each wavelength are 2.5- to 3-fold higher and 16–28% greater, respectively, than absorption and scattering for postmenopausal subjects. Absorption and scattering properties for women using hormone replacement therapy are intermediate between premenopausal and postmenopausal populations. Physiological properties show differences in mean total hemoglobin (7.0 μM, 11.8 μM, and 19.2 μM) and water concentration relative to pure water (10.9%, 15.3%, and 27.3%) for postmenopausal, hormone replacement therapy, and premenopausal subjects, respectively. Because of their unique, quantitative information content, diffuse optical methods may play an important role in breast diagnostics and improving our understanding of breast disease.

A human axillary odorant is carried by apolipoprotein D.

The characterization of the source of the odor in the human axillary region is not only of commercial interest but is also important biologically because axillary extracts can alter the length and timing of the female menstrual cycle. In males, the most abundant odor component is known to be E-3-methyl-2-hexenoic acid (E-3M2H), which is liberated from nonodorous apocrine secretions by axillary microorganisms. Recently, it was found that in the apocrine gland secretions, 3M2H is carried to the skin surface bound to two proteins, apocrine secretion odor-binding proteins 1 and 2 (ASOB1 and ASOB2) with apparent molecular masses of 45 kDa and 26 kDa, respectively. To better understand the formation of axillary odors and the structural relationship between 3M2H and its carrier protein, the amino acid sequence and glycosylation pattern of ASOB2 were determined by mass spectrometry. The ASOB2 protein was identified as apolipoprotein D (apoD), a known member of the alpha2mu-microglobulin superfamily of carrier proteins also known as lipocalins. The pattern of glycosylation for axillary apoD differs from that reported for plasma apoD, suggesting different sites of expression for the two glycoproteins. In situ hybridization of an oligonucleotide probe against apoD mRNA with axillary tissue demonstrates that the message for synthesis of this protein is specific to the apocrine glands. These results suggest a remarkable similarity between human axillary secretions and nonhuman mammalian odor sources, where lipocalins have been shown to carry the odoriferous signals used in pheromonal communication.

Effects of follicular versus luteal phase-based strength training in young women

Hormonal variations during the menstrual cycle (MC) may influence trainability of strength. We investigated the effects of a follicular phase-based strength training (FT) on muscle strength, muscle volume and microscopic parameters, comparing it to a luteal phase-based training (LT). Eumenorrheic women without oral contraception (OC) (N = 20, age: 25.9 ± 4.5 yr, height: 164.2 ± 5.5 cm, weight: 60.6 ± 7.8 kg) completed strength training on a leg press for three MC, and 9 of them participated in muscle biopsies. One leg had eight training sessions in the follicular phases (FP) and only two sessions in the luteal phases (LP) for follicular phase-based training (FT), while the other leg had eight training sessions in LP and only two sessions in FP for luteal phase-based training (LT). Estradiol (E2), progesterone (P4), total testosterone (T), free testosterone (free T) and DHEA-s were analysed once during FP (around day 11) and once during LP (around day 25). Maximum isometric force (Fmax), muscle diameter (Mdm), muscle fibre composition (No), fibre diameter (Fdm) and cell nuclei-to-fibre ratio (N/F) were analysed before and after the training intervention. T and free T were higher in FP compared to LP prior to the training intervention (P < 0.05). The increase in Fmax after FT was higher compared to LT (P <0.05). FT also showed a higher increase in Mdm than LT (P < 0.05). Moreover, we found significant increases in Fdm of fibre type ΙΙ and in N/F only after FT; however, there was no significant difference from LT. With regard to change in fibre composition, no differences were observed between FT and LT. FT showed a higher gain in muscle strength and muscle diameter than LT. As a result, we recommend that eumenorrheic females without OC should base the periodization of their strength training on their individual MC.

Factores de prognóstico na gravidez precoce

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

A relação entre as hormonas sexuais e a voz no sexo feminino

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Ovulation and risk of epithelial ovarian cancer

Incessant ovulation is thought to be one of the primary causes of epithelial ovarian cancer. However, the effects of ovulation at different ages and of the various exposures or events that suppress ovulation have not been established. We used data from an Australian case-control study of 791 ovarian cancer cases and 853 controls to examine the effect of ovulation on ovarian cancer risk. The total number of lifetime ovulations was calculated using information provided in a monthly contraceptive/reproductive calendar, as well as incorporating other information such as average menstrual cycle length. An increase of I year's worth of ovulation was associated with a 6% increase in risk of ovarian cancer (95% confidence interval [CI] = 4-8%). Ovulations in the 20-29-year age group were associated with the greatest risk, with a 20% increase in risk associated with each year of ovulation during this age period (95% Cl = 13-26%). When the effects of different exposures that suppress ovulation were compared, there was an indication that some factors may have a greater effect than others. These findings support the theory that incessant ovulation is a major contributor to the occurrence of ovarian cancer and suggest that ovulations during the 20s may be those most associated with disease risk. (C) 2003 Wiley-Liss, Inc.

The effect of hormone replacement therapy and/or exercise on skeletal muscle attenuation in postmenopausal women: a yearlong intervention

Hormone replacement therapy (HRT) has been reported to exert a positive effect on preserving muscle strength following the menopause, however, the mechanism of action remains unclear. We examined whether the mechanism involved preservation of muscle composition as determined by skeletal muscle attenuation. Eighty women aged 50-57 years were randomly assigned to either: HRT, exercise (Ex), HRT + exercise (ExHRT), and control (Co) for 1 year. The study was double-blinded with subjects receiving oestradiol and norethisterone acetate (Kliogest) or placebo. Exercise included progressive high-impact training for the lower limbs. Skeletal muscle attenuation in Hounsfield units (HU) was determined by computed tomography of the mid-thigh. Areas examined were the quadriceps compartment (includes intermuscular adipose tissue), quadriceps muscles, the posterior compartment and posterior muscles. Muscle performance was determined by knee extensor strength, vertical jump height, and running speed over 20 m. Fifty-one women completed the intervention. Vertical jump height and running speed improved in the HRT and ExHRT groups compared with Co (interaction, P < 0.01). For both the quadriceps compartment and quadriceps muscles, HU significantly increased (interaction, P <= 0.005) for HRT, Ex, and ExHRT compared with Co. For the posterior compartment, HU for the HRT and ExHRT were significantly increased compared with Co, while for posterior muscles, ExHRT was significantly greater than Co. Although the effects were modest, the results indicate that HRT, either alone or combined with exercise, may play a role in preserving/improving skeletal muscle attenuation in early postmenopausal women and thereby exert a positive effect on muscle performance.

Sustained release of the CCR5 inhibitors CMPD167 and maraviroc from vaginal rings in rhesus macaques

Antiretroviral entry inhibitors are now being considered as vaginally administered microbicide candidates for the prevention of the sexual transmission of human immunodeficiency virus. Previous studies testing the entry inhibitors maraviroc and CMPD167 in aqueous gel formulations showed efficacy in the macaque challenge model, although protection was highly dependent on the time period between initial gel application and subsequent challenge. In this paper, we describe the sustained release of maraviroc and CMPD167 from matrix-type silicone elastomer vaginal rings both in vitro and in vivo. Both inhibitors were released continuously during 28 days from rings in vitro at rates of 100 to 2,500 µg/day. In 28-day pharmacokinetic studies in rhesus macaques, the compounds were measured in the vaginal fluid and vaginal tissue; steady-state fluid concentrations were ~10(6)-fold greater than the 50% inhibitory concentrations (IC(50)s) for simian human immunodeficiency virus 162P3 inhibition in macaque lymphocytes in vitro. Plasma concentrations for both compounds were very low. The pretreatment of macaques with Depo-Provera (DP), which is commonly used in macaque challenge studies, was shown to significantly modify the biodistribution of the inhibitors but not the overall amount released. Vaginal fluid and tissue concentrations were significantly decreased while plasma levels increased with DP pretreatment. These observations have implications for designing macaque challenge experiments and also for ring performance during the human female menstrual cycle.

A study of some constituents in human female saliva

Changes in the concentration of some constituents in women's saliva during the menstrual cycle were studied. Saliva was used because it is easier to collect than other body fluids and is continuously available for analysis. Glucose, the enzyme 17-Acetyl-D-glucosaminidase (NAG) and Calcium which are saliva constituents and belong to three different chemical groups were selected for the study. Several analytical techniques were investigated. The fluorometric assay procedure was found to be the best because of its specificity and sensitivity for the estimation of these constituents. resides the fluorametric method a spectrophotometric method was used in the NAG determination and an atomic absorption method in the calcium estimation. Glucose was estimated by an enzymatic method. This is based on the reaction of glucose with the enzymes glucose oxidase and peroxidase to yield hydrogen peroxide, which in turn oxidises a non-fluorescent substrate, p-hydroxyphenylacetic acid, to a highly fluorescent product. The saliva samples in this determination had to be centrifuged at high speed, heated in a boiling water bath, centrifuged again and then treated with a mixture of cation and anion resins to remove the substances that inhibited the enzyme system. In the determination of the NAG activity the saliva samples were diluted with citric acid/phosphate buffer, and then centrifuged at high speed. The assay was based on the enzymic hydrolysis of the non-fluorescent substrate 4-Methyl-umbelli1eryl-p-D-glucosaminide to the highly fluorescent 4-Methyl-umbelliferone• Calcium was estimated by a fluorometric procedure based upon the measurement of the fluorescence produced by the complex formed between calcein blue and calcium, at pH 9 - 13. From the results obtained from the analysis of saliva samples of several women it was found that glucose showed a significant increase in its level around the expected time of ovulation. This was found in seven cycles out of ten. Similar results were found with the enzyme NAG. No significant change in the calcium levels was observe& at any particular time of the cycle. The levels of the glucose, the activity of the enzyme NAG and the concentration of the calcium were found to change daily, and to differ from one subject to another and in the same subject from cycle to cycle. The increase observed it salivary glucose levels and the enzyme NAG activity could be monitored to predict the time of ovulation.

Down-regulation of angiopoietin-1 expression in menorrhagia

Angiogenesis is an essential component of endometrial repair and regeneration following menses. Perturbation of this process is associated with menorrhagia, a common gynecological disorder that results in excessive menstrual bleeding. Angiopoietin-1 (Ang-1) promotes vascular maturation via the Tie-2 receptor, while angiopoietin-2 (Ang-2) is its natural antagonist that destabilizes vessels and initiates neovascularization in the presence of vascular endothelial growth factor. To test the hypothesis that menorrhagia arises as a result of poor signal for vascular maturation, we have examined the expression of Ang-1, Ang-2, and Tie-2 in endometrium throughout the menstrual cycle from 30 normal women and 28 patients with menorrhagia. Ribonuclease protection assay and Western blot analysis showed Ang-2 expression was consistently higher than Ang-1 in normal endometrium throughout the cycle. However, with menorrhagia Ang-1 mRNA and protein were not detected or down-regulated, while Ang-2 was observed at similar levels in both normal and menorrhagic endometrium resulting in a greater than a 50% decrease in the ratio of Ang-1 to Ang-2 protein. In situ hybridization and immunohistochemical studies supported these findings and revealed cyclical changes in the expression of Ang-1 and Ang-2. These results suggest that the angiopoietin/Tie-2 system promotes vascular remodeling in endometrium and loss of normal Ang-1 expression may contribute to the excessive blood loss observed in menorrhagia.

Parâmetros bioquímicos e níveis de IgE total e específicas para aeroalérgenos em mulheres obesas com síndrome dos ovários policísticos

Introduction: Polycystic ovary syndrome (PCOS) whose classic features (menstrual irregularity of oligo/ amenorrhea type, chronic anovulation, infertility and hyperandrogenism clinical and/ or biochemical), is associated with aspects of metabolic syndrome (MS), as obesity and insulin resistance. The level of obesity determines different levels of inflammation, increasing cytokines participants of metabolic and endocrine functions, beyond modulate the immune response. Metabolic changes, added to the imbalance of sex hormones underlying irregular menstruation observed in (PCOS) can trigger allergic processes and elevation of total and specific IgE antibodies indicate that a sensitization process was started. Objective: To evaluate the influence of PCOS on biochemical parameters and levels of total and specific IgE to aeroallergens in obese women. Methods: After approval by the Committee of Ethics in Research, were recruited 80 volunteers with BMI ≥ 30 kg/m2 and age between 18 and 45 years. Among these, 40 with PCOS according to the Rotterdam criteria and 40 women without PCOS (control group). All participants were analysed with regard to anthropometric, clinical, gynecological parameters, interviewed using a questionnaire, and underwent blood sampling for realization of laboratory tests of clinical biochemistry: Total cholesterol, LDL-cholesterol, HDL- cholesterol, Triglycerides, Fasting glucose, Urea, Creatinine, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and immunological: total and specific IgE to Dermatophagoides pteronyssinus, Blomia tropicalis, Dermatophagoides farinae and Dermatophagoides microceras.Statistical analysis was performed using SPSS 15.0 software through the chi-square tests, Fisher, Student t test and binary logistic regression, with significance level (p <0.05). Results: It was observed in the group of obese women with PCOS that 29 (72.5%) had menstrual cycle variable and 27 (67.5%) had difficulty getting pregnant. According to waist-hip ratio, higher average was also observed in obese PCOS (0.87). Blood level of HDL (36.9 mg/dL) and ALT (29.3 U/L) were above normal levels in obese women with PCOS, with statistically significant relationship. In the analysis of total and specific IgE to D. pteronyssinus high results were also prevalent in obese PCOS, with blood level (365,22 IU/mL) and (6.83 kU/L), respectively, also statistically significant. Conclusions: Observed predominance of cases with high levels of total IgE in the group of obese women with PCOS, 28 (70%) of the participants, whose mean blood concentration of the group was 365.22 IU/mL. In the analysis of Specific IgE between the groups, the allergen Dermatophagoides pteronyssinus showed greater dispersion and average the results of sensitization in the group of obese PCOS, whose mean blood concentration was 6.83 kU/l. Keywords: Obesity, Allergens and Polycystic Ovary Syndrome