Enhancing breast cancer screening with urinary biomarkers and Random Forest supervised classification: A comprehensive investigation.

OBJECTIVES: Urinary sex hormones are investigated as potential biomarkers for the early detection of breast cancer, aiming to evaluate their relevance and applicability, in combination with supervised machine-learning data analysis, toward the ultimate goal of extensive screening. METHODS: Sex hormones were determined on urine samples collected from 250 post-menopausal women (65 healthy - 185 with breast cancer, recruited among the clinical patients of Candiolo Cancer Institute FPO-IRCCS (Torino, Italy). Two analytical procedures based on UHPLC-MS/HRMS were developed and comprehensively validated to quantify 20 free and conjugated sex hormones from urine samples. The quantitative data were processed by seven machine learning algorithms. The efficiency of the resulting models was compared. RESULTS: Among the tested models aimed to relate urinary estrogen and androgen levels and the occurrence of breast cancer, Random Forest (RF) proved to underscore all the other supervised classification approaches, including Partial Least Squares - Discriminant Analysis (PLS-DA), in terms of effectiveness and robustness. The final optimized model built on only five biomarkers (testosterone-sulphate, alpha-estradiol, 4-methoxyestradiol, DHEA-sulphate, and epitestosterone-sulphate) achieved an approximate 98% diagnostic accuracy on replicated validation sets. To balance the less-represented population of healthy women, a Synthetic Minority Oversampling TEchnique (SMOTE) data oversampling approach was applied. CONCLUSIONS: By means of tunable hyperparameters optimization, the RF algorithm showed great potential for early breast cancer detection, as it provides clear biomarkers ranking and their relative efficiency, allowing to ground the final diagnostic model on a restricted selection five steroid biomarkers only, as desirable for noninvasive tests with wide screening purposes.

Demographic and hormonal evidence for menopause in wild chimpanzees.

Among mammals, post-reproductive life spans are currently documented only in humans and a few species of toothed whales. Here we show that a post-reproductive life span exists among wild chimpanzees in the Ngogo community of Kibale National Park, Uganda. Post-reproductive representation was 0.195, indicating that a female who reached adulthood could expect to live about one-fifth of her adult life in a post-reproductive state, around half as long as human hunter-gatherers. Post-reproductive females exhibited hormonal signatures of menopause, including sharply increasing gonadotropins after age 50. We discuss whether post-reproductive life spans in wild chimpanzees occur only rarely, as a short-term response to favorable ecological conditions, or instead are an evolved species-typical trait as well as the implications of these alternatives for our understanding of the evolution of post-reproductive life spans.

The Use of Morning Urinary Gonadotropins and Sex Hormones in the Management of Early Puberty in Chinese Girls.

CONTEXT: Although gonadotropin-releasing hormone stimulation test (GnRHST) is the gold standard in diagnosing central precocious puberty (CPP), it is invasive, expensive, and time-consuming, requiring multiple blood samples to measure gonadotropin levels. OBJECTIVE: We evaluated whether urinary hormones could be potential biomarkers for prepuberty or postpuberty, aiming to simplify the current diagnosis and prognosis procedure. METHODS: We performed a cross-sectional study of a total of 355 girls with CPP in National Clinical Research Center for Child Health in China, including 258 girls with positive and 97 girls with negative results from GnRHST. Twenty patients received GnRH analogue (GnRHa) treatment and completed a 6-month follow up. We measured luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol, prolactin, progesterone, testosterone, and human chorionic gonadotropin in the first morning voided urine samples. RESULTS: Their urinary LH levels and the ratios of LH to FSH increased significantly with the advancement in Tanner stages. uLH levels were positively associated with basal and peak LH levels in the serum after GnRH stimulation. A cutoff value of 1.74 IU/L for uLH reached a sensitivity of 69.4% and a specificity of 75.3% in predicting a positive GnRHST result. For the combined threshold (uLH ≥ 1.74 + uLH-to-uFSH ratio > 0.4), the specificity reached 86.6%. After 3 months of GnRHa therapy, the uLH and uFSH levels decreased accordingly. CONCLUSION: uLH could be a reliable biomarker for initial CPP diagnosis and screening; uLH could also be an effective marker for evaluating the efficacy of clinical treatment.

Targeted LC-MS/MS analysis of steroid glucuronides in human urine.

Conjugation with glucuronic acid is one of the major metabolic reactions in human steroid hormone catabolism. Recently, increasing interest has been raised concerning the biological roles of steroid glucuronides. We have therefore developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 15 urinary steroid hormone glucuronides in human urine: androsterone glucuronide (An-G), etiocholanolone glucuronide (Etio-G), epiandrosterone glucuronide (epiAn-G), dihydrotestosterone glucuronide (DHT-G), dehydroepiandrosterone glucuronide (DHEA-G), testosterone glucuronide (T-G), epitestosterone glucuronide (epiT-G), estrone glucuronide (E1-3 G), 17ß-estradiol 17-glucuronide (E2-17 G), 17ß-estradiol 3-glucuronide (E2-3 G), estriol 16-glucuronide (E3-16 G), pregnenolone glucuronide (Preg-G), tetrahydro-11-deoxycorticosterone 3-glucuronide (THDOC-3 G), cortisol 21-glucuronide (F-G) and pregnanediol glucuronide (PD-G). Sample workup included protein precipitation and solid phase extraction. Internal standards were used to correct for the loss of analytes during sample preparation and analysis. The method showed good linearity (R2≥0.99) and recovery ranged from 89.6 % to 113.8 %. Limit of quantification ranged from 1.9 nmol/L for F-G to 21.4 nmol/L for An-G. Intra-day and inter-day accuracy and precision were below 15 % for all quality controls. The method was successfully applied to 67 urine samples from children and adolescents in whom total concentrations of free and conjugated steroids had been previously determined by GC-MS after enzymatic hydrolysis. Free and sulfated steroids were also measured by LC-MS/MS. In general, the sums of the respective glucuronidated, sulfated and free forms of an analyte corresponded well with its total amount determined after enzymatic hydrolysis by GC-MS. Regarding the most prominent steroid metabolites, the total mean levels of androsterone and etiocholanolone showed an increase up to 5820.0 nmol/L and 4017.8 nmol/L in the group of 15-20 year-old children, respectively. Glucuronide conjugates (4374.3 nmol/L and 3588.5 nmol/L, respectively) dominated. DHEA was excreted mostly as sulfate (0-1 month of age: 184.5 nmol/L; 15-20 years of age: 1618.4 nmol/L) in all age groups. Cortisol was present predominantly as sulfate (mean: 173.8 nmol/L) in newborns. Levels of sulfated cortisol decreased with age, its glucuronidated form increased. The levels of free cortisol were relatively constant throughout childhood. Sex hormones were preferably excreted as glucuronides. In general, steroid hormone metabolites were conjugated to various extents with glucuronic acid or sulfuric acid and their ratio changed over lifetime.

Content matters: Cyclic effects on women's voices depend on social context.

Women's voices reportedly sound more attractive during the fertile days compared to the non-fertile days of their menstrual cycle. Here we investigated whether the speech content modulates the cyclic changes in women's voices. We asked 72 men and women to rate how interested they were in getting to know the speaker based on her voice. Forty-two naturally cycling women were recorded once during the late follicular phase (high fertility) and once during the luteal phase (low fertility) while speaking sentences of neutral and social content. Listeners were more interested in getting to know the speakers when hearing sentences with social content. Furthermore, raters were more interested in getting to know the speakers when these were recorded in the late follicular than in the luteal phase, but only in sentences with social content. Notably, levels of reproductive hormones (EP ratio) across the cycle phases did not significantly predict the preference for late follicular voices, but echoing the perceptual ratings, there was a significant EP ratio x speech content interaction. Phonetic analyses of mean fundamental frequency (F0) revealed a main effect of menstrual cycle phase and speech content but no interaction. Employing an action-oriented task, the present study extends findings of cycle-dependent voice changes by emphasising that speech content critically modulates fertility effects.

Hormonal Profiles of Menstrual Bleeding Patterns During the Luteal-Follicular Transition.

CONTEXT: Menstrual cycle function is determined by a complex endocrine axis that controls the ovaries and endometrium. While the late luteal phase is characterized by declining progesterone and estrogen, how these hormonal profiles relate to menstrual bleeding patterns is not well understood. OBJECTIVE: Characterize associations between luteal phase hormonal profiles and subsequent menstrual bleeding patterns, specifically spotting before bleeding. DESIGN, SETTING, AND PARTICIPANTS: We examined creatinine-adjusted urinary estrone 3-glucuronide (E13G) and pregnanediol 3-glucuronide (Pd3G) levels in relation to spotting in 116 premenopausal women (ages 20-47) who kept daily menstrual diaries and collected first morning urine samples for ≥ 2 consecutive cycles or 1 luteal-follicular transition (n = 283 transitions). We used linear mixed models to estimate associations between luteal phase hormone levels and spotting before bleeding. MAIN OUTCOME MEASURE(S) AND RESULTS: Transitions with ≥ 1 days of spotting before menstrual bleeding (n = 118) had greater luteal phase Pd3G levels vs nonspotting transitions (n = 165). Differences in Pd3G between spotting and nonspotting transitions were largest at menses onset (34.8%, 95% confidence interval, 18.9%, 52.7%). Pd3G levels for spotting transitions dropped to similar levels as nonspotting transitions an average of 1 day later, which aligned with the first day of bleeding for transitions with contiguous spotting. Spotting transitions were preceded by slower rates of Pd3G decline than nonspotting transitions, whereas E13G declines were similar. CONCLUSIONS: Self-reported bleeding patterns may provide insight into luteal phase Pd3G levels. First bleed appears to be the best choice for defining the end of the luteal phase and achieving hormonal consistency across transitions.

Urine metabolomic analysis for monitoring internal load in professional football players.

INTRODUCTION: The design of training programs for football players is not straightforward due to intra- and inter-individual variability that leads to different physiological responses under similar training loads. OBJECTIVE: To study the association between the external load, defined by variables obtained using electronic performance tracking systems (EPTS), and the urinary metabolome as a surrogate of the metabolic adaptation to training. METHODS: Urine metabolic and EPTS data from 80 professional football players collected in an observational longitudinal study were analyzed by ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry and assessed by partial least squares (PLS) regression. RESULTS: PLS models identified steroid hormone metabolites, hypoxanthine metabolites, acetylated amino acids, intermediates in phenylalanine metabolism, tyrosine, tryptophan metabolites, and riboflavin among the most relevant variables associated with external load. Metabolic network analysis identified enriched pathways including steroid hormone biosynthesis and metabolism of tyrosine and tryptophan. The ratio of players showing a deviation from the PLS model of adaptation to exercise was higher among those who suffered a muscular lesion compared to those who did not. CONCLUSIONS: There was a significant association between the external load and the urinary metabolic profile, with alteration of biochemical pathways associated with long-term adaptation to training. Future studies should focus on the validation of these findings and the development of metabolic models to identify professional football players at risk of developing muscular injuries.

Gynaecological diagnosis by ultrasound and the measurement of urinary sex steroid hormones in female orangutans.

Gynaecological diagnoses were carried in three adult female orangutans (Pongo spp.) using ultrasound, and their estrous states were estimated by measuring urinary sex steroid hormone concentrations using enzyme immunoassay. Ultrasound diagnosis revealed that the endometrial thickness and follicle size were correlated with the oestrogen-3-glucuronide concentrations in the follicular phase. In addition, administration of the ovulation inducer human chorionic gonadotropin (hCG) had the strongest effect on the pregnanediol-3-glucuronide (PdG) concentration when the follicle size was 22.3 mm, suggesting that the follicle reaches this size before ovulation. The similarity between this and the maximum ovarian follicle size in humans (approximately 20 mm) indicates that the ancestral reproductive characteristics may have been retained in these species.

Urinary Sex Steroid and Glucocorticoid Hormones Are Associated With Muscle Mass and Strength in Healthy Adults.

CONTEXT: Sex steroid hormones exhibit anabolic effects whereas a deficiency engenders sarcopenia. Moreover, supraphysiological levels of glucocorticoids promote skeletal muscle atrophy, whereas physiologic levels of glucocorticoids may improve muscle performance. OBJECTIVE: To study the relationship between both groups of steroid hormones at a physiological range with skeletal muscle mass and function in the general population. DESIGN: Cross-sectional analysis of the associations between urinary excreted androgens, estrogens, glucocorticoids, and steroid hormone metabolite ratios with lean mass and handgrip strength in a population-based cohort. SETTING: Three centers in Switzerland including 1128 participants. MEASURES: Urinary steroid hormone metabolite excretion by gas chromatography-mass spectrometry, lean mass by bioimpedance analysis, and isometric handgrip strength by dynamometry. RESULTS: For lean mass a strong positive association was found with 11ß-OH-androsterone and with most glucocorticoids. Androsterone showed a positive association in middle-aged and older adults. Estriol showed a positive association only in men. For handgrip strength, strong positive associations with androgens were found in middle-aged and older adults, whereas positive associations were found with cortisol metabolites in young to middle-aged adults. CONCLUSIONS: Sex steroids and glucocorticoids are strongly positively associated with skeletal muscle mass and strength in the upper limbs. The associations with muscle strength appear to be independent of muscle mass. Steroid hormones exert age-specific anabolic effects on lean mass and handgrip strength. Deficits in physical performance of aged muscles may be attenuated by androgens, whereas glucocorticoids in a physiological range increase skeletal muscle mass at all ages, as well as muscle strength in particular in younger adults.

Sex Hormones in Women With Elevated Breast Cancer Risk Undergoing Weight Loss.

Sedentary lifestyles and obesity are known risk factors for breast cancer. Elevated estrogen levels correlate with obesity and, independently, with increased breast cancer risk. Lifestyle interventions that reduce obesity may mitigate this risk, potentially via estrogen pathways. In a 6-month lifestyle intervention, overweight/obese women with high breast cancer risk were randomized to control (n = 7) or intervention (n = 6) and analyzed for sex hormone levels. Serum and urine hormones were evaluated by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and sex hormone binding globulin (SHBG) by enzyme-linked immunosorbent assay (ELISA). Serum estrone (E1) and estradiol (E2) were reduced by 12.1% and 50.8%, respectively, at 9 months in the intervention group, which differed from controls (p = .043 and .020). This contrasted with a 73.3% increase in urine E1 at 6 months in the intervention group (p = .035). These results suggest that a lifestyle intervention led to a favorable estrogen profile in relation to breast cancer risk.

Effects of night shift on the cognitive load of physicians and urinary steroid hormone profiles - a randomized crossover trial.

Mental and physical stress is common in physicians during night shifts. Neurocognitive effects of sleep deprivation as well as alterations in hormonal and metabolic parameters have previously been described. The aim of this crossover study was to evaluate the effects of night-shift work with partial sleep deprivation on steroid hormone excretion and possible associations with mood, sleep characteristics and cognitive functions in physicians. In total, 34 physicians (mean age 42 ± 8.5 years, 76.5% male) from different departments of the General Hospital of Vienna, Austria, were randomly assigned to two conditions: a regular day shift (8 h on duty, condition 1) and a continuous day-night shift (24 h on duty, condition 2). In both conditions, physicians collected a 24 h urine sample for steroid hormone concentration analysis and further completed psychological tests, including the sleep questionnaire (SF-A), the questionnaire for mental state (MDBF) and the computer-assisted visual memory test (FVW) before and at the end of their shifts, respectively. Although mean sleep deprivation during night shift was relatively small (~1.5 h) the impairment in participants' mental state was high in all three dimensions (mood, vigilance and agitation, p ≤ 0.001). Sleep quality (SQ), feeling of being recovered after sleep and mental balance decreased (p ≤ 0.001), whereas mental exhaustion increased (p < 0.05). Moreover, we could show a nearly linear relationship between most of these self-rating items. Testing visual memory participants made significantly more mistakes after night shift (p = 0.011), however, mostly in incorrectly identified items and not in correctly identified ones (FVW). SQ and false identified items were negatively correlated, whereas SQ and time of reaction were positively associated. It is assumed that after night shift, a tendency exists to make faster wrong decisions. SQ did not influence correctly identified items in FVW. In contrast to previous investigations, we found that only excretion rates for pregnanetriol and androsterone/etiocholanolone ratios (p < 0.05, respectively) were slightly reduced in 24-h urine samples after night shift. A considerable stimulation of the adrenocortical axis could not be affirmed. In general, dehydroepiandrosteron (DHEA) was negatively associated with the sense of recreation after sleep and with the time of reaction and positively correlated with correctly identified items in the FVW test. These results, on the one hand, are in line with previous findings indicating that stress and sleep deprivation suppress gonadal steroids, but, on the other hand, do not imply significant adrenocortical-axis stimulation (e.g. an increase of cortisol) during the day-night shift.

In-house monitoring of steroid hormone metabolites in urine informs breeding management of a giant anteater (Myrmechophaga tridactyla).

Although numbers of giant anteaters within North American facilities have been steadily increasing for the last 15 years, the population now exhibits an unstable age distribution with genetically valuable individuals nearing reproductive senescence. Contributing to this issue is the Association of Zoos and Aquariums (AZA) described lack of standardization of breeding introduction practices and high risk of female injury occurring during such pairings. This report describes the development of a successful breeding protocol at Cleveland Metroparks Zoo based on hormone monitoring and efficient communication between science and animal management teams that minimizes risk of female injury. By training a female giant anteater for urine sample and body weight data collection, staff members accurately predicted estrus, and timed breeding introductions to facilitate positive interactions between the male and female. Such training also allowed for careful monitoring of two pregnancies through parturition (169-184 days from breeding) and post-partum return to estrus (114-129 days from parturition). Urinary hormone monitoring revealed a sharp progestogen increase averaging >five-fold over basal levels (0.52 ± 0.05 ng/mg creatinine) which was sustained throughout the second half of pregnancy. Mean regular estrous cycle length (n = 14 cycles), was calculated as 46.17 ± 1.39 days, measured as days between estrogen peaks of mean concentration 2.27 ± 0.19 ng/mg creatinine. This report summarizes impressive collaborative efforts among multiple zoological departments to achieve extensive hormonal and body weight monitoring from a female giant anteater, adding valuable information on reproductive parameters, and specifics for novel hormone assay techniques.

Associations of Acrylamide Intake With Urinary Sex Hormone Levels Among Preschool-Age Japanese Children.

Acrylamide may affect the sex hormone system in the prepubertal period. This study examined the cross-sectional associations between dietary acrylamide intake and sex hormone levels among preschool-age Japanese children. The study was conducted in 2006 among 230 boys and 198 girls aged 3-6 years in Aichi, Japan. Acrylamide intake was assessed using 3-day diet records. Urinary concentrations of estrone, estradiol, testosterone, and androst-5-ene-3ß, 17ß diol (hereafter referred to as androstenediol) were measured by liquid chromatography-electrospray ionization-tandem mass spectrometry. Sex hormone levels were adjusted for urinary creatinine levels. The estimated acrylamide intake was 1.00 µg/kg of body weight per day. After controlling for age and other covariates, acrylamide intake was significantly positively associated with urinary levels of testosterone and androstenediol in boys. On average, boys in the highest quartile of acrylamide intake had 96.9% higher testosterone (95% confidence interval: -1.8, 295; P for trend = 0.048) and 34.5% higher androstenediol levels (95% confidence interval: -5.9, 92.3; P for trend = 0.04) than boys in the lowest quartile. In girls, there were no significant associations between acrylamide intake and the hormones measured. Acrylamide intake may alter androgen metabolism in preschool-age boys. Because this is a first observation, our findings require confirmation in additional studies.

Interrelationship among annual cycles of sex steroids, corticosterone and body condition in Nyctibatrachus humayuni.

Synergism between extrinsic and intrinsic factors is crucial for the seasonality of reproduction. Environmental factors such as photoperiod and temperature activate the hypothalamus-pituitary-gonadal axis leading to the secretion of steroid hormones that are crucial for reproduction. Sex steroids are not only essential for the maturation of gonads, but also for development of secondary sexual characters in males and reproductive behaviour of both the sexes. In the present study, we quantified the urinary testosterone (UTM) and corticosterone (UCM) metabolites in males and urinary estradiol metabolites (UEM) and UCM in females of Nyctibatrachus humayuni for two consecutive years to determine annual and seasonal variation in the levels of sex steroids, corticosterone and body condition index (BCI). The results show that sex steroids were highest during the breeding season and lowest during the non-breeding season in both the sexes. An increase in UTM and UEM was observed in males and females respectively during the breeding season. Testicular histology showed the presence of all stages of spermatogenesis throughout the year indicating that spermatogenesis is potentially continuous. Ovarian histology showed the presence of vitellogenic follicles only during the breeding season indicating that oogenesis is strictly seasonal. In males, UCM levels were highest during the breeding season, while in females their levels were highest just prior to the breeding season. In males, BCI was highest during the pre-breeding season, declined during the breeding season to increase again during the post-breeding season. In females, BCI was comparable throughout the year. In males, UTM levels were positively correlated with UCM levels but negatively correlated with BCI. Interestingly, UEM, UCM and BCI were not correlated in females. These results indicate that N. humayuni exhibits an associated pattern of reproduction. Quantification of urinary progesterone metabolites (UPM) during the breeding season showed UPM levels were higher in post-spawning females, suggesting the significance of progesterone in ovulation. Further, non-invasive enzyme immunoassay has been successfully standardized in N. humayuni for the quantification of urinary metabolites of steroid hormones.

Urinary Cadmium Excretion Is Associated With Increased Synthesis of Cortico- and Sex Steroids in a Population Study.

Context: Urinary cadmium (Cd) excretion is associated with cancer and cardiovascular morbidity. A potential mechanism could be disturbance of steroidogenesis in gonads and adrenal glands. Objective: We tested whether urinary excretion of Cd is correlated with that of cortico- and sex steroid metabolites in the general adult population. Setting: The Swiss Kidney Project on Genes in Hypertension is a multicentric, family-based population study. Measures: Urinary excretions of steroid hormone metabolites and Cd were measured with separate day and night collections. Associations were analyzed by mixed linear models. Results: Urinary Cd and testosterone excretions in men were significantly correlated (respective day and night ß values [standard error (SE)], 1.378 [0.242], P < 0.0005; and 1.440 [0.333], P < 0.0005), but not in women [0.333(0.257), P = 0.2; and 0.674 (0.361), P = 0.06]. Urinary Cd and cortisol excretions were positively associated in both sexes [day: ß = 0.475 (SE, 0.157), P = 0.0025, and 0.877 (SE, 0.194), P < 0.0005, respectively; night: ß = 0.875 (SE, 0.253), P < 0.0005 and 1.183 (SE, 0.277), P = 0.00002, respectively]. Cd excretion was correlated with mineralocorticoid metabolites excretion, except tetrahydroaldosterone, in both sexes (P < 0.01). There was an independent effect of Cd on sex hormone and corticosteroid synthesis and an interdependent effect on gluco- and mineralcorticoid production. Conclusion: Our findings provide evidence for a global stimulating effect on steroid synthesis already at low-dose Cd exposure. These findings might explain the association of Cd with diseases such as steroid-sensitive cancers or metabolic disorders.

Sex Hormone Metabolism and Threatened Abortion.

BACKGROUND The aim of this study was to evaluate changes in sex hormone metabolism in patients with threatened miscarriage. MATERIAL AND METHODS We recruited 73 women in early pregnancy (6-8 weeks of gestation) and divided them into the following 2 groups based on whether they had vaginal bleeding: group A (n=34), the threatened abortion group; and group B (n=39), the normal pregnancy group. Human chorionic gonadotrophin (hCG), estradiol (E2), progesterone (P4), and testosterone (T) serum levels were tested and sex hormone metabolites in the urine were detected using gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS). As the control, data for sex hormones and their metabolites were obtained in normal women of childbearing age without pregnancy (group C: n=23). RESULTS E2 and T serum levels were lower in women with threatened miscarriage (group A). Estrone (E1), E2, estriol (E3), 16α-hydroxyestrone (16α-OHE1), 4-methoxyestrone (4-MeOE1), 2-hydroxyestradiol (2-OHE2), and 4-methoxyestradiol (4-MeOE2) levels were significantly lower in group A (P=0.001, 0.003, 0.009, 0.001, 0.012, 0.032, and 0.047, respectively.). Urine levels of dehydroepiandrosterone (DHEA), androstenedione (A2), and the metabolite of (A2) were also significantly lower in group A (P=0.007, 0.009, and 0.011, respectively). The 2-OHE1/E1, 4-OHE1/E1, 2-MeOE1/E1, and 2-MeOE2/E2 ratios were lower in group B, whereas the 2-OHE2/E2, 4-OHE2/E2, and 4-MeOE2/E2 ratios were dramatically lower in all pregnant women (groups A and B) than in group C. CONCLUSIONS Deficiency in DHEA and abnormal levels of sex hormone metabolites may cause a reduction in the activity of estrogens in women with threatened abortion. These alterations may result in bleeding during the first trimester of pregnancy.

Sex Hormones, Gonadotropins, and Sex Hormone-binding Globulin in Infants Fed Breast Milk, Cow Milk Formula, or Soy Formula.

Measurement of endogenous hormones in early life is important to investigate the effects of hormonally active environmental compounds. To assess the possible hormonal effects of different feeding regimens in different sample matrices of infants, 166 infants were enrolled from two U.S hospitals between 2006 and 2009. The children were classified into exclusive soy formula, cow milk formula or breast milk regimens. Urine, saliva and blood samples were collected over the first 12 months of life. Estradiol, estrone, testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and sex hormone-binding globulin (SHBG) levels were measured in the three matrices. Lower estradiol and LH levels were found in urine and saliva samples of soy formula-fed boys compared to cow formula-fed boys. Higher LH level was found in urine samples of soy formula-fed girls compared to cow formula-fed girls. However, we found neither a neonatal testosterone rise in the boys nor a gender-specific difference in testosterone levels, which suggests that urinary testosterone levels may not accurately reflect blood levels during mini-puberty. Nevertheless, our study shows that blood, urine and saliva samples are readily collectible and suitable for multi-hormone analyses in children and allow examination of hypotheses concerning endocrine effects from dietary compounds.

Evaluation of various approaches to the isolation of steroid hormones from urine samples prior to FASS-MEKC analysis.

The goal of this study was to assess various analytical approaches for the simultaneous and efficient extraction of steroid hormones (cortisone, cortisol, prednisolone, corticosterone, testosterone, 17α-methyltestosterone, epitestosterone, progesterone) from urine samples prior to separation based on field-amplified sample stacking MEKC (FASS-MEKC). FASS-MEKC successfully allowed the compounds to be separated within 12 min using a BGE composed of 5 mM sodium tetraborate, 150 mM boric acid, 50 mM SDS, and 15% methanol. Therefore, many procedures such as solid-phase microextraction, SPE, and dispersive liquid-liquid microextraction (DLLME) were tested and compared using a multivariate tool, namely, cluster analysis. Finally, DLLME-FASS-MEKC was validated and proved a good linearity of calibration curves (R2 above 0.9948) in a concentration range from 50 to 1000 ng/mL for all analytes. The LOD was established at 15 ng/mL, whereas the LOQ was 50 ng/mL. The intra- and interday precision, expressed as RSD%, did not exceed 9.97%. The DLLME-FASS-MEKC method was successfully applied to the analysis of urine samples from healthy volunteers and sportsmen. This methodology could prove to be useful in clinical studies and/or doping control depending on the steroid concentrations required in biomedical applications.

Urinary sex steroid hormone and placental leucine aminopeptidase concentration differences between live births and stillbirth of Bornean orangutans (Pongo pygmaeus).

BACKGROUND: Under the environment of pregnancy, the placenta assumes an important steroidogenic role in the maintenance of pregnancy. METHODS: Urinary placental leucine aminopeptidase (PLAP), estrone-3-glucuronide (E1 G), and pregnanediol-3-glucuronide (PdG) concentrations were compared among five pregnancies (four live births and one stillbirth) in four orangutans. RESULTS: The gestation period of the stillbirth (223 days) was shorter than that of the live births (239-254 days). In females who gave a live birth, average PLAP and E1 G concentrations increased until the delivery. Conversely, in the female who gave a stillbirth, PLAP concentration failed to increase, and E1 G concentration was significantly low in late pregnancy period. Regarding PdG concentrations, there was no significant difference among all pregnancies. CONCLUSIONS: This is the first study reporting a change in urinary PLAP, E1 G, and PdG concentrations during orangutan stillbirth and live birth pregnancies. The findings will assist in developing pregnancy screening tests.

Biological sex identification in the endangered dusky gopher frog (Lithobates sevosa): a comparison of body size measurements, secondary sex characteristics, ultrasound imaging, and urinary hormone analysis methods.

BACKGROUND: Accurate sex identification techniques are important for wildlife demographic studies and for genetic management of captive breeding colonies. Various non-invasive methods for identification of biological sex in the weakly dimorphic endangered dusky gopher frog (DGF; Lithobates sevosa) were explored to support planned recovery efforts for this species including breeding and augmentation of wild populations. METHODS: Body size (snout-vent length and body weight) measurements, observation of nuptial pads, ultrasound imaging, and urinary hormone analysis for testosterone and estrone were performed on 27 male and 19 female DGFs. For each method, the mean and range of measurement values were determined for male and female DGFs housed in a captive breeding population. The ability of these methods to accurately predict the true biological sex of the individuals was assessed retrospectively. RESULTS: Body size measurements were of limited use for sex identification purposes, as males and females demonstrated overlapping body lengths and weights. Observation of the presence/absence of nuptial pads in males and females, respectively, proved to be accurate and easy to perform in most cases. Ultrasound imaging was useful for predicting the sex of female frogs, particularly when females were gravid. Commercial enzyme immunoassay kits were validated to measure urinary hormones in the DGF. Mean urinary testosterone (males: 2.22 ± 0.38 ng/ml; females: 0.92 ± 0.11 ng/ml) and estrone (males: 0.08 ± 0.01 ng/ml; females: 1.50 ± 0.39 ng/ml) concentrations were significantly (p < 0.05) different between the sexes. However, there was some overlap in hormone concentrations between the sexes. When a ratio of testosterone (T) to estrone (E) concentrations was calculated for each individual, males demonstrated significantly greater T/E ratios compared to females (p < 0.05). Use of this ratio showed greater accuracy in predicting the sex of the animal compared to using testosterone or estrone concentrations alone. CONCLUSIONS: Monitoring for presence/absence of nuptial pads and using urinary testosterone to estrone hormone ratios were the most accurate methods for identifying the biological sex of adult DGFs. Urinary hormone measurements for sex identification may be useful in other weakly dimorphic and monomorphic amphibian species in both ex situ and in situ settings.