Quantitative analysis of 34 sex (pro)hormones, conjugates and bioactive oxidation products thereof in human plasma by GC- and LC-MS/MS and systematic investigation of overestimations of analyte concentrations not accounted for by method validation.

When investigating endocrine disorders, it is essential to assess a comprehensive quantitative profile of sex (pro)hormones in plasma including conjugates. Thus, the present study aimed to develop and validate a comprehensive mass spectrometry-based multimethod combining the direct analysis of unconjugated sex (pro)hormones and oxidation products thereof (by GC), as well as their sulfates and glucuronides present in higher concentrations (by LC) with the indirect quantification of glucuronides present in lower concentrations after selective glucuronide hydrolysis (by GC) and its application to plasma derived from ten pre- and postmenopausal women and men each. Even guideline-compliant validation experiments cannot completely reflect overestimation of analyte concentrations due to effects depending on the individual ratio of analytes (i.e. chemical formation of analytes or incomplete removal of interfering analytes). Thus, the extent of processes not accounted for by the calibration strategy were investigated and maximum over- or underestimations of analyte concentrations were assessed for each plasma sample individually. 34 analytes were successfully calibrated, validated (median accuracy 101.1 %, median inter-day precision 8.1 %) and 31 were detected above the detection limit in plasma samples. The sporadic maximum individual over- or underestimation of analyte concentrations amounted to less than 20 %.

Associations among neighborhood walkability, metal exposure, and sex steroid hormone levels: Results from Hangzhou Birth Cohort Study â ¡.

BACKGROUND: Neighborhood walkability may influence maternal-fetal exposure to environmental hazards and maternal-fetal health (e.g., fetal growth restriction, reproductive toxicity). However, few studies have explored the association between neighborhood walkability and hormones in pregnant women. METHODS: We included 533 pregnant women from the Hangzhou Birth Cohort Study II (HBCS-II) with testosterone (TTE) and estradiol (E2) measured for analysis. Neighborhood walkability was evaluated by calculating a walkability index based on geo-coded addresses. Placental metals were measured using inductively coupled plasma mass spectrometry (ICP-MS). TTE and E2 levels in umbilical cord blood were measured using chemiluminescence microparticle immunoassay (CMIA). Linear regression model was used to estimate the relationship between the walkability index, placental metals, and sex steroid hormones. Effect modification was also assessed to estimate the effect of placental metals on the associations of neighborhood walkability with TTE and E2. RESULTS: Neighborhood walkability was significantly linked to increased E2 levels (P trend=0.023). Compared with participants at the first quintile (Q1) of walkability index, those at the third quintiles (Q3) had lower chromium (Cr) levels (ß = -0.212, 95% CI = -0.421 to -0.003). Arsenic (As), cobalt (Co), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), antimony (Sb), selenium (Se), tin (Sn), and vanadium (V) were linked to decreased TTE levels, and cadmium (Cd) was linked to increased TTE levels. No metal was significantly associated with E2 levels in trend analysis. In the analysis of effect modification, the associations of neighborhood walkability with TTE and E2 were significantly modified by Mn (P = 0.005) and Cu (P = 0.049) respectively. CONCLUSION: Neighborhood walkability could be a favorable factor for E2 production during pregnancy, which may be inhibited by maternal exposure to heavy metals.

The profile of steroid hormones in human fetal and adult ovaries.

BACKGROUND: Reproduction in women is at risk due to exposure to chemicals that can disrupt the endocrine system during different windows of sensitivity throughout life. Steroid hormone levels are fundamental for the normal development and function of the human reproductive system, including the ovary. This study aims to elucidate steroidogenesis at different life-stages in human ovaries. METHODS: We have developed a sensitive and specific LC-MS/MS method for 21 important steroid hormones and measured them at different life stages: in media from cultures of human fetal ovaries collected from elective terminations of normally progressing pregnancy and in media from adult ovaries from Caesarean section patients, and follicular fluid from women undergoing infertility treatment. Statistically significant differences in steroid hormone levels and their ratios were calculated with parametric tests. Principal component analysis (PCA) was applied to explore clustering of the ovarian-derived steroidogenic profiles. RESULTS: Comparison of the 21 steroid hormones revealed clear differences between the various ovarian-derived steroid profiles. Interestingly, we found biosynthesis of both canonical and "backdoor" pathway steroid hormones and corticosteroids in first and second trimester fetal and adult ovarian tissue cultures. 17α-estradiol, a less potent naturally occurring isomer of 17ß-estradiol, was detected only in follicular fluid. PCA of the ovarian-derived profiles revealed clusters from: adult ovarian tissue cultures with relatively high levels of androgens; first trimester and second trimester fetal ovarian tissue cultures with relatively low estrogen levels; follicular fluid with the lowest androgens, but highest corticosteroid, progestogen and estradiol levels. Furthermore, ratios of specific steroid hormones showed higher estradiol/ testosterone and estrone/androstenedione (indicating higher CYP19A1 activity, p < 0.01) and higher 17-hydroxyprogesterone/progesterone and dehydroepiandrosterone /androstenedione (indicating higher CYP17A1 activity, p < 0.01) in fetal compared to adult ovarian tissue cultures. CONCLUSIONS: Human ovaries demonstrate de novo synthesis of non-canonical and "backdoor" pathway steroid hormones and corticosteroids. Elucidating the steroid profiles in human ovaries improves our understanding of physiological, life-stage dependent, steroidogenic capacity of ovaries and will inform mechanistic studies to identify endocrine disrupting chemicals that affect female reproduction.

Sex steroid levels and stress-related markers in pregnant and non-pregnant women and the effect of periodontal therapy.

BACKGROUND: Periodontal disease during pregnancy can produce adverse events; in the current study stress was investigated as an exacerbating factors of periodontal disease. The aims of this study were to evaluate the possible associations between stress and pregnancy through scanning for gingivitis and to explore the effect of non-surgical periodontal therapy (NPT) on stress-related markers (CgA, AA, ß-endorphin, DHEA, sIgA and NPY) and sex steroid levels (estrogen and progesterone) in pregnant and non-pregnant women. MATERIAL AND METHODS: A total of 87 subjects; 22 pregnant women with gingivitis, 25 periodontally healthy pregnant women; 22 non-pregnant women with gingivitis and 15 periodontally healthy non-pregnant women, participated in this study. Periodontal clinical measures, stress hormones and sex steroid levels were measured at baseline and following the periodontal therapy. RESULTS: While periodontal therapy showed an improvement in salivary CgA, AA, ß-endorphin, DHEA, and sIgA levels (p<0.05) in non-pregnant women with gingivitis; neuropeptide Y levels were found to be unaffected (p>0.05). There were no significant changes in salivary CgA, AA, DHEA, sIgA, and neuropeptide Y levels in pregnant women with gingivitis (p>0.05); however, a decrease in ß-endorphin levels was observed after therapy (p<0.05). Pregnant women with gingivitis had higher gingival crevicular fluid (GCF) ß-endorphin levels in comparison to non-pregnant women with gingivitis. CONCLUSIONS: Gingival inflammation can be a psychosocial stress inducing factor during pregnancy. Furthermore, periodontal therapy may assist in reducing stress-related hormone levels in GCF during pregnancy.

Salivary Sex Steroid Levels in Infants and the Relation with Infantile Colic

Objective: The hypothalamic-pituitary-gonadal axis is active during minipuberty, the timing of which coincides with infantile colic. To the best of our knowledge, the relationship between these entities has not been previously investigated. Methods: Saliva samples were collected from 15- to 60-day-old term infants (n=139) between 9 am and 5 pm. Group 1 included infants with infantile colic (n=68, 54.4% female) while the remaining healthy infants constituted Group 2 (n=71, 47.9% female). Salivary levels of estradiol (Esal) in females and testosterone (Tsal) in males were measured by ELISA in duplicate. Results: The median (25th-75th centile) age and birth week for all infants were 33 (29-43) days and 39 (38.1-40) weeks, respectively. Levels of Tsal in males [Group 1, 73.35 (59.94-117.82) pg/mL vs Group 2, 77.66 (56.49-110.08) pg/mL, p=0.956] and Esal in females [Group 1, 3.91 (2.76-5.31) pg/mL vs Group 2, 4.03 (1.63-12.1) pg/mL, p=0.683] were similar. However, in subjects with infantile colic (Group 1), Esal and body mass index (BMI) standard deviation scores of females were slightly correlated (Group 1, rs= 0.393, p=0.016 vs. Group 2, rs= 0.308, p=0.076) and there was a significant correlation between the sampling time and Tsal in males (Group 1, rs= 0.469, p=0.009 vs. Group 2, rs= -0.005, p=0.976). Conclusion: Random salivary sex steroid levels were similar in infants with and without infantile colic. However, in subjects with infantile colic, Esal levels in females were positively correlated with BMI and Tsal levels were higher later in the day among males. Thus, sex steroid production may be altered during minipuberty in subjects with infantile colic.

Comprehensive analysis between volatile organic compound (VOC) exposure and female sex hormones: a cross-sectional study from NHANES 2013-2016.

There is growing evidence suggesting that exposure to volatile organic compounds (VOCs) can pose significant health risks, including interference with the function of the reproductive system. However, there has been a lack of research focused on the impact of common environmental VOCs on the levels of sex hormones in the general female population. In this study, we conducted a cross-sectional analysis utilizing the database of the National Health and Nutrition Examination Survey (NHANES, 2013-2016). A total of 2633 participants were included in this study. The Pearson correlation model revealed the potential of co-exposure or co-toxicity between benzene and 2,5-dimethylfuran. According to GLM models, we discovered a significant positive association between blood levels of 2,5-dimethylfuran and benzene with testosterone levels in women. Subgroup analysis further identified that the women with underweight and healthy weight might be the high-risk subgroup. Bayesian kernel machine regression (BKMR) was applied to further assess the univariate and bivariate exposure-response relationships between multiple VOCs. Our research systemically formulated the possible relationship between exposure to VOCs and female sex hormones, indicating the role of VOCs as a risk factor for endocrine disruption, especially benzene and 2,5-dimethylfuran. These findings have important implications for public health and call for further investigation.

Marcadores bioquímicos propuestos para el estudio de la sarcopenia / Proposed biochemical markers for the study of sarcopenia

La sarcopenia asociada a la edad es una condición clínica caracterizada por una disminución en la fuerza, calidad y cantidad de masa muscular así como también en la función muscular. Un biomarcador se define como una característica que es medible objetivamente y evaluable como indicador de un proceso biológico normal, patológico o respuesta terapéutica a una intervención farmacológica. Los marcadores bioquímicos propuestos para el estudio de la sarcopenia pueden ser categorizados en dos grupos. El primero de ellos evalúa el estatus musculoesquelético; este panel de marcadores está formado por miostatina/folistatina, procolágeno aminoterminal tipo III e índice de sarcopenia. El segundo grupo de marcadores bioquímicos evalúa factores causales, para lo cual se sugiere medir el factor de crecimiento insulino-símil tipo 1 (IGF-1), dehidroepiandrosterona (DHEAS), cortisol, facto-res inflamatorios [proteína C reactiva (PCR), interleuquina 6 (IL-6) y factor de necrosis tu-moral (TNF-a)]. Las recomendaciones realiza-das están basadas en la evidencia científica disponible en la actualidad y la disponibilidad de la metodología apropiada para cada uno de los biomarcadores. (AU)

Sarcopenia is a progressive and generalized skeletal muscle disorder defined by decrease in the strength, quality and quantity of muscle mass as well as in muscle function. A biomarker is defined as a feature objectively measured and evaluated as an indicator of a normal biologic process, a pathogenic process or a pharmacologic response to therapeutic intervention. The biochemical markers proposed for the study of sarcopenia may be classified in two groups. The first group evaluates the musculoskeletal status, made up by myostatin/follistatin, N-terminal Type III Procollagen and the sarcopenia index. The second evaluates causal factors, where the measurement of the following is suggested: hormones insulin-like growth factor-1 (IGF-I), dehydroepiandrosterone sulphate (DHEAS), cortisol, inflammatory factors [C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a)]. The recommendations made are based on scientific evidence currently available and the appropriate methodology availability for each biomarker. (AU)

Effects of elevated serum estrogen on dry eye in women undergoing in vitro fertilisation.

PURPOSE: Sex hormones impact inflammatory and immune-mediated diseases. During IVF (in vitro fertilisation) treatment, circulating estrogen levels increase dramatically (10-50x) alongside changes in other hormones. This study examined changes in dry eye with IVF and its relationship with sex hormones. METHODS: A two visit study was conducted on first day of menstruation when estrogen levels are lowest (baseline visit), and on day 9-11 (peak estrogen visit (PO)) of IVF. Symptoms of dry eye and ocular pain and signs of dry eye were examined. Serum hormone levels were assessed using mass spectrometry and immunoassay. Changes in signs and symptoms and associations were explored. Hierarchical multiple regression analysis assessed factors contributing to signs and symptoms. RESULTS: 40 women (36.2 ± 4.0 years) completed the study. Baseline and PO oestradiol (E2) levels were 28.9 pg/ml (20) (median (IQR)); 1360 pg/ml (1276) respectively. Ocular pain and dry eye symptoms worsened (p = 0.02 and p < 0.01) and tear break up and tear secretion values decreased (p = 0.005 and 0.01) at PO. Higher E2 and lower luteinizing hormone (LH) were associated with worsening of dry eye symptoms (ρ = 0.34 p = 0.03, ρ = -0.49 p = 0.001). Reduction in LH and increase in progesterone (P4) were associated with increased ocular pain (ρ = 0.45, p = 0.004 and ρ = 0.39, p = 0.01). Dry eye symptoms were predicted by LH and tear break up (p = 0.02; R2 = 0.18). CONCLUSIONS: IVF treatment resulted in significantly increased ocular symptoms and tear film alterations although these changes were not clinically significant. Dry eye signs and symptoms were poorly predicted by hormone levels.

Exploring the human chorionic gonadotropin induced steroid secretion profile of mouse Leydig tumor cell line 1 by a 20 steroid LC-MS/MS panel.

The canonical androgen synthesis in Leydig cells involves Δ5 and Δ4 steroids. Besides, the backdoor pathway, eompassing 5α and 5α,3α steroids, is gaining interest in fetal and adult pathophysiology. Moreover, the role of androgen epimers and progesterone metabolites is still unknown. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 20 steroids and used it to investigate the steroid secretion induced by human chorionic gonadotropin (hCG) in the mouse Leydig tumor cell line 1 (mLTC1). Steroids were extracted from 500 µL supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 × 3 mm, 3 µm column, with 100 µM NH4F and methanol as mobile phases, and analyzed by positive electrospray ionization and multiple reaction monitoring. Sensitivity ranged within 0.012-38.0 nmol/L. Intra-assay and inter-assay imprecision were < 9.1% and 10.0%, respectively. Trueness, recovery and matrix factor were within 93.4-122.0, 55.6-104.1 and 76.4-106.3%, respectively. Levels of 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, 17OH-progesterone, androstenedione, epitestosterone, dihydrotestosterone, progesterone, androsterone and 17OH-allopregnanolone were effectively measured. Traces of 17OH-dihydroprogesterone, androstanediol and dihydroprogesterone were found, whereas androstenediol, 17OH-pregnenolone, dehydroepiandrosterone, pregnenolone and allopregnanolone showed no peak. hCG induced an increase of 80.2-102.5 folds in 16OH-progesterone, androstenedione and testosterone, 16.6 in dihydrotestosterone, 12.2-27.5 in epitestosterone, progesterone and metabolites, 8.1 in 17OH-allopregnanolone and ≤ 3.3 in 5α and 5α,3α steroids. In conclusion, our LC-MS/MS method allows exploring the Leydig steroidogenesis flow according to multiple pathways. Beside the expected stimulation of the canonical pathway, hCG increased progesterone metabolism and, to a low extent, the backdoor route.

Comprehensive Sex Steroid Profiling in Multiple Tissues Reveals Novel Insights in Sex Steroid Distribution in Male Mice.

A comprehensive atlas of sex steroid distribution in multiple tissues is currently lacking, and how circulating and tissue sex steroid levels correlate remains unknown. Here, we adapted and validated a gas chromatography tandem mass spectrometry method for simultaneous measurement of testosterone (T), dihydrotestosterone (DHT), androstenedione, progesterone (Prog), estradiol, and estrone in mouse tissues. We then mapped the sex steroid pattern in 10 different endocrine, reproductive, and major body compartment tissues and serum of gonadal intact and orchiectomized (ORX) male mice. In gonadal intact males, high levels of DHT were observed in reproductive tissues, but also in white adipose tissue (WAT). A major part of the total body reservoir of androgens (T and DHT) and Prog was found in WAT. Serum levels of androgens and Prog were strongly correlated with corresponding levels in the brain while only modestly correlated with corresponding levels in WAT. After orchiectomy, the levels of the active androgens T and DHT decreased markedly while Prog levels in male reproductive tissues increased slightly. In ORX mice, Prog was by far the most abundant sex steroid, and, again, WAT constituted the major reservoir of Prog in the body. In conclusion, we present a comprehensive atlas of tissue and serum concentrations of sex hormones in male mice, revealing novel insights in sex steroid distribution. Brain sex steroid levels are well reflected by serum levels and WAT constitutes a large reservoir of sex steroids in male mice. In addition, Prog is the most abundant sex hormone in ORX mice.

Impact of hypogonadism on bone mineral density and vertebral fractures in HIV-infected men.

PURPOSE: Hypogonadism and osteoporosis are frequently reported in HIV-infected men and, besides multifactorial pathogenesis, they might be directly linked because of testicular involvement in bone health. We evaluated the prevalence of osteoporosis and vertebral fractures (VFs) in HIV-infected men, and assessed their relationship with gonadal function. METHODS: We enrolled 168 HIV-infected men (median age 53). Osteoporosis and osteopenia were defined with T-score ≤ - 2.5SD and T-score between - 1 and - 2.5SD, respectively. VFs were assessed by quantitative morphometric analysis. Total testosterone (TT), calculated free testosterone (cFT), Sex Hormone Binding Globulin (SHBG), Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) were obtained; overt hypogonadism was defined on symptoms and low TT or cFT, and classified into primary and secondary according to gonadotropins; compensated hypogonadism was defined as normal TT and cFT with high LH levels. RESULTS: Overall, osteoporosis and osteopenia were found in 87.5% of patients, and VFs were detected in 25% of them; hypogonadism was identified in 26.2% of cases. Osteoporotic patients had higher SHBG vs those with normal bone mineral density (BMD). Fractured patients were more frequently hypogonadal and with higher SHBG. SHBG showed negative correlation with both spine and femoral BMD, and positive correlation with VFs. In multivariate models, FSH showed negative impact only on femoral BMD, whereas older age and higher SHBG predicted VFs. CONCLUSION: We found a high burden of bone disease and hypogonadism in HIV-infected men, and we showed that the impact of gonadal function on bone health is more evident on VFs than on BMD.

Evaluation of acetamiprid and azoxystrobin residues and their hormonal disrupting effects on male rats using liquid chromatography-tandem mass spectrometry.

Endocrine-disrupting compounds as pesticides affect the hormonal balance, and this can result in several diseases. Therefore, the analysis of representative hormones with acetamiprid (AC) and azoxystrobin (AZ) was a good strategy for the investigation of the endocrine-disrupting activity of pesticides. Hence, a sensitive and rapid analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The method was validated for the analysis of AC, AZ, estriol, estrone, progesterone, and testosterone in the serum, testis, and liver of rats. The correlation between the residues of pesticides and the disturbance of the endocrine system was evaluated. The different mass parameters, mobile phase types, analytical columns, injection volumes, and extraction solvents were compared to get the lowest limit of detection of the studied compounds. The detection limits of AC, AZ, estriol, estrone, progesterone, and testosterone were 0.05, 0.05, 1.0, 10, and 1.0 ng/ml, respectively. The method developed was applied to evaluate the changes in these hormones induced by the duration of exposure to AC and AZ in rat testis and serum. The hormones level in rat serum and testis had a significant decrease as they were oral gavage treated with different high concentrations of studied pesticides. Both pesticides were distributed in the body of rats by the multi-compartment model (liver, testis, and serum).

Sex steroid hormones are associated with mortality in COVID-19 patients: Level of sex hormones in severe COVID-19.

ABSTRACT: In patients with coronavirus disease 2019 (COVID-19), men are more severely affected than women. Multiple studies suggest that androgens might play a role in this difference in disease severity. Our objective was to assess the association between sex hormone levels and mortality in patients with severe COVID-19.We selected patients from the Amsterdam University Medical Centers COVID-19 Biobank, in which patients admitted to hospital in March and April 2020, with reverse transcription-polymerase chain reaction proven severe acute respiratory syndrome-coronavirus-2 infection, were prospectively included. Specifically, we included postmenopausal women (>55 years) and age-matched men, with a mortality of 50% in each group. Residual plasma samples were used to measure testosterone, estradiol, sex hormone binding globulin (SHBG), and albumin. We investigated the association of the levels of these hormones with mortality in men and women.We included 16 women and 24 men in March and April 2020 of whom 7 (44%) and 13 (54%), respectively, died. Median age was 69 years (interquartile range [IQR] 64-75). In men, both total and free testosterone was significantly lower in deceased patients (median testosterone 0.8 nmol/L [IQR 0.4-1.9] in deceased patients vs 3.2 nmol/L [IQR 2.1-7.5] in survivors; P < .001, and median free testosterone 33.2 pmol/L [IQR 15.3-52.2] in deceased patients vs 90.3 pmol/L [IQR 49.1-209.7] in survivors; P = .002). SHBG levels were significantly lower in both men and women who died (18.5 nmol/L [IQR 11.3-24.3] in deceased patients vs 34.0 nmol/L [IQR 25.0-48.0] in survivors; P < .001). No difference in estradiol levels was found between deceased and surviving patients.Low SHBG levels were associated with mortality rate in patients with COVID-19, and low total and free testosterone levels were associated with mortality in men. The role of testosterone and SHBG and potential of hormone replacement therapy needs further exploration in COVID-19.

Environmentally relevant perinatal exposure to DBP disturbs testicular development and puberty onset in male mice.

Di-n-butyl phthalate (DBP) is considered as a potential modifier of puberty. However, different results indicate that DBP plays an accelerated, delayed, or neutral role in the initiation of puberty. Furthermore, whether the effect of DBP on puberty will disrupt the function of reproductive system in the adults is still ambiguous. Therefore, we aimed to investigate the effect of maternal exposure to DBP on the onset of puberty in male offspring mice and the subsequent changes in the development of reproductive system. Here, pregnant mice were treated with 0 (control), 50, 250, or 500 mg/kg/day DBP in 1 mL/kg corn oil administered daily by oral gavage from gestation day (GD) 12.5 to parturition. Compared with the control group, the 50 mg/kg/day DBP group accelerated puberty onset and testicular development were quite remarkable in male offspring mice during early puberty. Furthermore, in 22-day male offspring mice, 50 mg/kg/day DBP induced increased levels of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone in serum, and promoted the expression of steroidogenesis-related genes in the testes. Testicular Leydig cells (LCs) were isolated from the testes of 3-week-old mice and treated with 0 (control), 0.1, 1 mM monobutyl phthalate (MBP, the active metabolite of DBP) for 24 h. Consistent with the in vivo results, the expression of steroidogenesis-related genes and testosterone production were increased in LCs following exposure to 0.1 mM MBP. In adulthood, testes of the male offspring mice exposed to all doses of DBP exhibited adverse morphology compared with the control group. These results demonstrated that maternal exposure to 50 mg/kg/day DBP induced earlier puberty and precocious development of the testis, and eventually damaged the reproductive system in the later life.

Characterization of Benign Breast Diseases and Association With Age, Hormonal Factors, and Family History of Breast Cancer Among Women in Sweden.

Importance: Benign breast diseases (BBDs) are common and associated with breast cancer risk, yet the etiology and risk of BBDs have not been extensively studied. Objective: To investigate the risk of BBDs by age, hormonal factors, and family history of breast cancer. Design, Setting, and Participants: This retrospective cohort study assessed 70 877 women from the population-based Karolinska Mammography Project for Risk Prediction of Breast Cancer (KARMA) who attended mammographic screening or underwent clinical mammography from January 1, 2011, to March 31, 2013, at 4 Swedish hospitals. Participants took part in a comprehensive questionnaire on recruitment. All participants had complete follow-up through high-quality Swedish national registers until December 31, 2015. Pathology medical records on breast biopsies were obtained for the participants, and BBD subtypes were classified according to the latest European guidelines. Analyses were conducted from January 1 to July 31, 2020. Exposures: Hormonal risk factors and family history of breast cancer. Main Outcomes and Measures: For each BBD subtype, incidence rates (events per 100 000 person-years) and multivariable Cox proportional hazards ratios (HRs) with time-varying covariates were estimated between the ages of 25 and 69 years. Results: A total of 61 617 women within the mammographic screening age of 40 to 69 years (median age, 53 years) at recruitment with available questionnaire data were included in the study. Incidence rates and risk estimates varied by age and BBD subtype. At premenopausal ages, nulliparity (compared with parity ≥3) was associated with reduced risk of epithelial proliferation without atypia (EP; HR, 0.62; 95% CI, 0.46-0.85) but increased risk of cysts (HR, 1.38; 95% CI, 1.03-1.85). Current and long (≥8 years) oral contraceptive use was associated with reduced premenopausal risk of fibroadenoma (HR, 0.65; 95% CI, 0.47-0.90), whereas hormone replacement therapy was associated with increased postmenopausal risks of epithelial proliferation with atypia (EPA; HR, 1.81; 95% CI, 1.07-3.07), fibrocystic changes (HR, 1.60; 95% CI, 1.03-2.48), and cysts (HR, 1.98; 95% CI, 1.40-2.81). Furthermore, predominantly at premenopausal ages, obesity was associated with reduced risk of several BBDs (eg, EPA: HR, 0.31; 95% CI, 0.17-0.56), whereas family history of breast cancer was associated with increased risk (eg, EPA: HR, 2.11; 95% CI, 1.48-3.00). Conclusions and Relevance: These results suggest that the risk of BBDs varies by subtype, hormonal factors, and family history of breast cancer and is influenced by age. Better understanding of BBDs is important to improve the understanding of benign and malignant breast diseases.

Effect of aging on clinical features and metabolic complications of women with polycystic ovary syndrome.

PURPOSE: To assess the distribution of clinical features and metabolic abnormalities of polycystic ovary syndrome (PCOS) women according to their age. METHODS: Retrospective study on 602 women (mean age 23.9 ± 6.2 years), diagnosed according to International PCOS Network Guidelines criteria as having PCOS in a University-based Hospital. Anthropometric features, hormonal and metabolic parameters were measured and compared between the different age groups (group A ≤ 20 years; group B 21-30 years; group C > 30 years). RESULTS: Patients in group A were more often hyperandrogenic, while in group C hypertension, dyslipidemia, obesity, impaired fasting glucose, and insulin resistance (IR) were more prevalent. After adjusting for BMI, age correlated positively with sex hormone-binding globulin (SHBG), IR, total- and LDL-cholesterol, and negatively with DHEAS, insulin, and free androgen index (FAI). SHBG was significantly associated with IR and atherogenic dyslipidemia, while FAI levels were linked to hypertension, independently of other factors considered. Furthermore, the regression analysis showed a stronger relationship between BMI and metabolic outcomes, regardless of age. CONCLUSION: Polycystic ovarian syndrome (PCOS) phenotype changes with age. Clinical and biochemical hyperandrogenism are a major concern in young PCOS women, while metabolic burden tends to increase with aging. Some of the cardiovascular risk factors are dependent on FAI and SHBG levels, whereas BMI confirms its key role in the genesis of most of the metabolic sequelae in PCOS, independently of age.

Capillary electrophoresis and liquid chromatography for determining steroids in concentrates of purified water from Päijänne Lake.

The research was done with partial filling micellar electrokinetic chromatography, microemulsion electrokinetic chromatography, and ultra-high performance liquid chromatography. The study focuses on determination of male and female steroids from cold and hot tap water of households in Helsinki City. The district´s raw water is made run from Päijänne Lake through a water tunnel to the purification plants in Helsinki area. The effluents delivered from the plants to households as tap water were sampled and used for the study. They were concentrated with solid phase extraction to exceed the detection limits of the three methods. With partial filling method the limits were 0.50, 0.48, 0.33, and 0.50 mg/L for androsterone, testosterone, progesterone, and testosterone-glucuronide, respectively. In microemulsion method the limit values were 1.33, 1.11, and 0.40 mg/L for androsterone, testosterone, and progesterone, respectively, and 0.83, 0.45, and 0.50 mg/L for hydrocortisone, 17-α-hydroxyprogesterone, and 17-α-methyltestosterone, respectively. In the tap water samples, progesterone concentrations represented the highest values being 0.22 and 1.18 ng/L in cold and hot water, respectively. They also contained testosterone (in all samples), its glucuronide metabolite (in 25% of the samples), and androstenedione (in 75% of the samples). The ultra-high liquid chromatographic method with mass spectrometric detection was used for identification of the steroids at µg/L level.

Development of extraction separation technology based on deep eutectic solvent and magnetic nanoparticles for determination of three sex hormones in milk.

A rapid, reliable and eco-friendly method for the determination of three sex hormones in five kinds of milk was developed and validated by combining vortex-assisted liquid-liquid microextraction (VALLME) and magnetic solid-phase extraction (MSPE). Deep eutectic solvents (DESs) such as choline chloride/urea were considered as the extraction solvent in VALLME and multi-walled carbon nanotubes (MMWCNTs) were used as the adsorbent which could adsorb DESs on the surface. The optimum experimental conditions were as follows: amount of MMWCNTs for 10 mg, volume of acetone for 4 mL, no sodium chloride and extraction pH at 7. After the optimization of several main variables, satisfactory sensitivity levels were achieved as low as 1.0-1.3 ng mL-1 and 2.5-4.5 ng mL-1 for the limit of method detections and the limit of method quantitation, respectively. The recoveries of the three hormones in different milk samples were in the range of 80.1%-116.4%. Consequently, this method is suitable for monitoring the trace amount of sex hormones in milk matrices.

Steroid reference intervals in women: influence of menopause, age and metabolism.

OBJECTIVE: To investigate the impact of age, obesity and metabolic parameters on 13 circulating steroids in reproductive and menopausal age. To define reference intervals (RIs). DESIGN: Cross-sectional. METHODS: Three hundred and twenty five drug-free, healthy and eumenorrheic women were selected from the general population. Independent relationships of LC-MS/MS-determined steroid levels with age, BMI and metabolic parameters were estimated. Reference sub-cohorts were defined for calculating upper and lower limits in reproductive age, menstrual phases and menopause, and these were compared with limits in dysmetabolic sub-cohorts. RESULTS: Lower androgens, pro-androgens and estrogens, but higher cortisol and metabolites were found in menopausal compared to reproductive age women. Androgens and precursors decreased during reproductive age (P < 0.001-P = 0.002) but not after menopause. 17OH-progesterone decreased with BMI (P = 0.006) and glucocorticoids with waist circumference (P < 0.001P = 0.002) in reproductive age, but increased with triglycerides (P=0.011P=0.038) after menopause. Inverse associations of dihydrotestosterone with BMI (P=0.004) and HDL-cholesterol (P=0.010), estrone with total cholesterol (P=0.033) and estradiol with triglycerides (P=0.011) were found in reproductive age. After menopause, estrone increased with waist circumference (P<0.001) and decreased with insulin resistance (P=0.012). Ovarian steroid RIs were estimated in menstrual phases and menopause. Age- and reproductive status-specific RIs were generated for androgens, precursors and corticosteroids. Lower limits for reproductive age cortisol (P=0.020) and menopausal 11-deoxycortisol (P=0.003) in dysmetabolic sub-cohorts were reduced and increased, respectively, compared to reference limits. CONCLUSIONS: Obesity and dysmetabolism differently influence circulating steroids in reproductive and menopausal status. Age, menstrual and menopausal status-specific RIs were provided by LC-MS/MS for a broad steroid panel.

Supercritical fluid chromatography hyphenated to mass spectrometry for metabolomics applications.

While supercritical fluid chromatography was developed over 50 years ago, it is only over the past 15 to 20 years that it has become routinely utilized. Along with the commercialization of a new generation of instruments, during the last 20 years supercritical fluid chromatography has improved performance, reliability, and robustness. Supercritical fluid chromatography is fully compatible with mass spectrometric techniques. This review compiles the application of supercritical fluid chromatography separations coupled to mass spectrometry instrumentation for the exploration, profiling, and quantitation of metabolites during the last two decades. The selection of metabolites chosen for this article have direct applications in preclinical models of disease and clinical applications as potential biomarkers of disease including lipids, steroid hormones, bile acids, polar metabolites, peptides, and proteins.