Interplay of hormones and metabolite excretion with fern pattern prove saliva as a potent indicator of male reproductive status in Kangayam breed cattle.

Kangayam cattle are one of the drought breeds in India with distinct attributes. Agricultural transformation has led to a decline in many pure-breed indigenous cattle, including the Kangayam breed. Hence, a study on the reproductive physiology of male Kangayam breed cattle is necessary to disentangle problems in the area of livestock improvement. In this study, we investigated the relationship between serum hormones and bio-constituents and ascertained the potential of saliva as an indicator of the reproductive status of Kangayam cattle (Bos indicus). The present study confirms that cholesterol was higher in intact males and lower in prepubertal and castrated males. Testosterone levels were also higher in intact males than in castrated or prepubertal males. Hence, it can be inferred that high cholesterol levels contribute to active derivatization of testosterone in intact males. In contrast, reduced cholesterol availability leads to decreased testosterone synthesis in castrated and prepubertal males. Furthermore, it is reasonable to speculate that testosterone could have influenced salivary fern patterns in intact males, and thus, fern-like crystallization in the saliva was apparent. The unique salivary compounds identified through GC-MS across various reproductive statuses of Kangayam males may advertise their physiological status to conspecifics. In addition, the presence of odorant-binding protein (OBP) in saliva further supports its role in olfactory communication. This study attested to a posssible interlink between gonadal status and serum biochemical profiles. The salivary fern pattern revealed in this study can be used as a predictive tool, and the presence of putative volatiles and OBP adds evidence to the role of saliva in chemical communication.

Diurnal 11-ketotestosterone and 17-hydroxyprogesterone saliva profiles in paediatric classical congenital adrenal hyperplasia.

OBJECTIVES: The most suitable biochemical markers for therapy adjustment in patients with congenital adrenal hyperplasia are controversial. 11-Oxygenated androgens are a promising new approach. The objective of this study was to investigate the diurnal rhythm of 11-ketotestosterone in children and adolescents in saliva and to correlate it with salivary 17-hydroxyprogesterone. METHODS: Fifty-one samples of steroid day-profiles from 17 patients were additionally analysed for 11-ketotestosterone, retrospectively. All patients were treated in our university outpatient clinic for paediatric endocrinology between 2020 and 2022. Steroid day-profiles of 17 patients could be examined. The cohort showed a balanced sex ratio. The median age was 13 years. The measurements for 17-hydroxyprogesterone were carried out during routine care by immunoassay. The measurements of 11-ketotestosterone were performed from frozen saliva samples using an implemented in-house protocol for liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most important outcome were the absolute values for 11-ketotestosterone, their diurnal rhythmicity and the correlation with 17-hydroxyprogesterone. RESULTS: Both steroids show a circadian diurnal rhythm. 17-hydroxyprogesterone and 11-ketotestosterone correlate significantly. 11-Ketotestosterone showed a positive correlation with BMI at all times of the day. CONCLUSIONS: 11-Ketotestosterone shows circadian rhythmicity in our cohort and correlates with 17-hydroxyprogesterone. These findings serve as an important basis for prospective research into 11-oxygenated androgens as therapeutic markers in paediatrics. However, 11-ketotestosterone appears to be very dependent on BMI.

Recovery in elite youth basketball players: The responsiveness of the psychophysiological measurements and the role of testosterone concentration.

This study investigated the recovery responses to the Total Quality Recovery (TQR), Well-Being questionnaire (WBQ), and Heart Rate (HR) responses to Submaximal Running Test (SRT), and the influence of salivary testosterone concentration (TEST) on these responses in 25 elite youth (U15) male basketball players. TQR, WBQ, and HR measurements were assessed after 48 hours of rest (T1), 24 hours after the 1st day of training (T2) and 24 hours after the 2nd day of training (T3). Salivary sampling was conducted at T1 and T3. A significant decrease was observed for TQR (F = 4.06; p = 0.01) and for WBQ (F = 5.37; p = 0.008) from T1 to T3. No difference among the three-time points was observed for HR and HR Recovery, and the TEST concentration did not influence the results. These results show that TQR and WBQ are sensitive to acute transient alterations in training loads (TL) and may be utilized to monitor recovery in elite youth basketball players. The HR related measurements presented limited responsiveness, and the TEST seems not to influence the recovery of these players who are competing at highest performance level.

Clinically Relevant Laboratory Monitoring of Gender-Affirming Hormone Therapy in Transgender People-Experiences from a Teaching Hospital in the Netherlands.

BACKGROUND: Transgender care is shifting from academic to nonacademic settings leading to use of common (immunoassay) compared to sophisticated (mass spectrometry) methods to monitor estradiol and testosterone during gender-affirming hormone therapy (GAHT). The type of assay can influence results and have significant implications for clinical decision making. An evidence gap is present in recommendations regarding the assay needed to monitor GAHT. The present study aimed to summarize current evidence and evaluate immunoassay estradiol and testosterone concentrations in transgender people visiting a nonacademic hospital for GAHT. METHODS: Clinical practice guidelines on GAHT and scientific literature on assay methodologies were screened and summarized. Laboratory and medical data from 252 patients who visited the transgender outpatient clinic of the Maasstad Hospital for GAHT between 2020 and 2022 were retrospectively analyzed. RESULTS: Our research showed that the most used clinical practice guidelines for GAHT provide hormonal target values without recommending a preferred method. A comprehensive literature search on agreement between immunoassay and mass spectrometry showed substantial heterogeneity in results. Retrospective analysis of our immunoassay measured data in transgender people showed hormonal changes during GAHT that are to be expected from the medication used. CONCLUSIONS: We demonstrate that laboratory monitoring of GAHT in a nonacademic hospital can be done safely by immunoassay in most cases. Only in cases where clinical observation is discordant with the hormonal results do more sophisticated methods need to be deployed. A best practice model was proposed for transgender care in nonacademic hospitals.

Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword.

OBJECTIVES: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.

Relationship between the 2D:4D and prenatal testosterone, adult level testosterone, and testosterone change: Meta-analysis of 54 studies.

The ratio between the hands' second to the fourth finger (2D:4D) is commonly hypothesized to result from prenatal testosterone. The 2D:4D has also been hypothesized to relate to adult-level testosterone and, more recently, to the testosterone response to a challenging situation. In the present work, we tested these core assumptions. Drawing from, in total, 54 studies and 8077 participants, we investigated whether the 2D:4D is related to adult level testosterone (44 studies), testosterone change (6 studies), and prenatal testosterone (10 studies). We found no evidence of the relationship between the above testosterone types and digit ratios. Furthermore, there was no relationship between testosterone and the right and left 2D:4D, male and female 2D:4D, and the 2D:4D and testosterone measurement (i.e., measured in blood or saliva). However, we found some evidence that prenatal testosterone measured in amniotic fluid (but not cord blood) might be related to the digit ratios-further studies are necessary to validate this observation. In summary, considering the current state of knowledge, any conclusions drawn from the assumption of the digit ratios as the proxy for testosterone (prenatal, adult level, or testosterone change under a challenging situation) warrant great caution.

Hormonal residues in chicken and cattle meat: A risk threat the present and future consumer health.

The study was designated to determine some hormonal residues (estradiol, progesterone, and testosterone) in chicken and cattle meat as well as assess the risk of these residues for adult and child consumer health. A total of 92 chicken meat samples were collected from four different chicken farm sales outlets, and 56 cattle meat samples from two brands were collected from markets through 2022 in Assiut City, Egypt. An enzyme-linked immunosorbant assay (ELISA) was used to measure the hormonal residues. Results showed that the estimated daily intake (EDI) of estradiol exceeded acceptable daily intakes (ADIs) in all analyzed samples except four chicken farm meat samples for adults only, which were below 0.05 µg/kg bw, and the hazard index (HI) of estradiol and testosterone residues for adults and children exceeded 1 in all examined samples of the study. The U.S. Food and Drug Administration (FDA) set safe limits for these anabolic hormones to ensure high food quality and quantity for humans, but in Egypt, monitoring and assessment of hormonal residues in food became very urgent as no safe limits have been set till now.

Association between Environmental Exposure to Multiclass Organic Pollutants and Sex Steroid Hormone Levels in Women of Reproductive Age.

Organic pollutant exposure may alter sex steroid hormone levels in both animals and humans, but studies on mixture effects have been lacking and mainly limited to persistent organic pollutants, with few hormones being investigated. Moreover, measurements from a single blood or urine sample may not be able to reflect long-term status. Using hair analysis, here, we evaluated the relationship between multiclass organic pollutants and sex steroid hormones in 196 healthy Chinese women aged 25-45 years. Associations with nine sex steroid hormones, including progesterone, androstenedione (AD), testosterone (T), estrone (E1), and 17ß-estradiol (E2), and eight related hormone ratios were explored on 54 pollutants from polychlorinated biphenyl (PCB), pesticide, and bisphenol families using stability-based Lasso regression analysis. Our results showed that each hormone was associated with a mixture of at least 10 examined pollutants. In particular, hair E2 concentration was associated with 19 pollutants, including γ-hexachlorocyclohexane, propoxur, permethrin, fipronil, mecoprop, prochloraz, and carbendazim. There were also associations between pollutants and hormone ratios, with pentachlorophenol, dimethylthiophosphate, 3-phenoxybenzoic acid, and flusilazole being related to both E1/AD and E2/T ratios. Our results suggest that exposure to background levels of pesticides PCB180 and bisphenol S may affect sex steroid hormone homeostasis among women of reproductive age.

Effect of cosmetic hair treatment and natural hair colour on hair testosterone concentrations.

PURPOSE: Testosterone analysis in hair allows for retrospective evaluation of endogenous testosterone concentrations, but studies devoted to investigating confounders in hair testosterone analysis have hitherto been scarce. The current study examined the stability of testosterone concentrations between two hair samples collected three months apart and investigated two potential confounding factors: natural hair colour and cosmetic hair treatments. METHODS: Testosterone was analysed with an in-house radioimmunoassay with a limit of detection adequate for the purpose. RESULTS: The testosterone concentrations from the two samplings, at baseline and three months later, had an intra-individual correlation of moderate strength (rho = 0.378, p<0.001, n = 146). Hair treatment, such as colouring or bleaching, seemed to increase testosterone concentrations (p = 0.051, n = 191, and in a paired analysis in a subset of the cohort p = 0.005, n = 24), while no effect of natural colour in untreated hair (p = 0.133) could be detected. CONCLUSION: The current results suggest that cosmetic hair treatments need to be considered in hair testosterone analyses and demonstrate the utility of a radioimmunoassay to reliably measure testosterone concentrations in small hair samples in women.

Testosterone promotes dominance behaviors in the Ultimatum Game after players' status increases.

Although testosterone is generally considered to promote dominance behaviors, in humans it fosters behaviors appropriate to achieving and maintaining social status, contingent upon the situation. Recent cross-sectional studies, such as Inoue et al. (Sci Rep 7:5335, 2017), have shown that dominance behaviors induced by testosterone are modulated by high status. Yet, it remains ambiguous whether a rise in social status within real-world social groups reshapes the relationship between testosterone and dominance behavior. To investigate this longitudinal question, we added a second wave to Inoue et al.'s study, collecting further data after an interval of 2 years. Members of a university rugby team that adheres to a rigid hierarchical order rooted in seniority played the Ultimatum Game with teammates and provided saliva for assays of testosterone and cortisol. Our analysis reveals that individuals with higher baseline salivary testosterone levels exhibited more dominance as their position in the hierarchy increased according to their seniority.

Idiopathic hypogonadotropic hypogonadism caused by compound heterozygosity for two novel mutations in the GNRH1 gene: a case report.

BACKGROUND: Idiopathic hypogonadotropic hypogonadism (IHH) is a rare congenital or acquired genetic disorder caused by gonadotropin-releasing hormone (GnRH) deficiency. IHH patients are divided into two major groups, hyposmic or anosmic IHH (Kallmann syndrome) and normosmic IHH (nIHH), according to whether their sense of smell is intact. Here we report a case of novel compound heterozygous mutations in the GNRH1 gene in a 15-year-old male with nIHH. CASE PRESENTATION: The patient presented typical clinical symptoms of delayed testicular development, with testosterone < 3.5 mmol/L and reduced gonadotropin (follicle-stimulating hormone, luteinizing hormone) levels. Two heterozygous variants of the GNRH1 gene were detected, nonsense variant 1: c.85G > T:p.G29* and variant 2: c.1A > G:p.M1V, which disrupted the start codon. CONCLUSIONS: Two GNRH1 mutations responsible for nIHH are identified in this study. Our findings extend the mutational spectrum of GNRH1 by revealing novel causative mutations of nIHH.

Assessment of exercise-induced stress via automated measurement of salivary cortisol concentrations and the testosterone-to-cortisol ratio: a preliminary study.

In this study, our aim was to validate whether the automated measurement of salivary testosterone and cortisol concentrations and the testosterone-to-cortisol (T/C) ratio, considering their individual circadian rhythms can be used to assess the stress response of male athletes to different exercise intensities accurately and effectively. We measured the salivary testosterone and cortisol concentrations and their respective serum concentrations that were collected from 20 male long-distance runners via passive drooling in the morning and evening for two consecutive days involving different exercise intensities. An electrochemiluminescence immunoassay was performed to evaluate the salivary testosterone and cortisol concentrations. The results showed a positive correlation between the salivary testosterone and cortisol concentrations and their respective serum concentrations. The participants were divided into two groups: with and without interval training. The interval training group showed a significantly higher rate of change in the salivary cortisol concentration and a significantly lower rate of change in the T/C ratio in the evening interval training on day 1 than lower-intensity running on day 2. Our results indicated that the salivary cortisol concentrations and the T/C ratio could distinguish between exercises at different intensities, which may be beneficial for detecting differences in stress responses among athletes.

Differential susceptibility to parenting influences on reactive and proactive aggression: The role of testosterone and cortisol in children.

This study explored whether cortisol and testosterone moderate the effect of parenting style on children's aggressive behavior, in accordance with the diathesis-stress or differential susceptibility theory; i.e., whether children are vulnerable to negative parenting styles (diathesis-stress) or, in addition to this vulnerability, also benefit more from positive parenting (differential susceptibility). The sample group comprised 279 eight-year-old children (154 boys and 125 girls) from Spain. Aggressive behavior was assessed using the "Reactive and Proactive Questionnaire" (RPQ) and parenting styles were measured using the "Parenting Styles and Dimensions Questionnaire" (PSDQ). Additionally, three saliva samples were collected to measure testosterone and cortisol levels, which were analyzed using ELISA. The results revealed that girls' high testosterone levels moderated the association between mothers' authoritarian parenting style and reactive aggression. This result can be explained by the differential susceptibility model. None of the interactions were statistically significant in boys' proactive aggression. The study emphasizes the importance of investigating biological and social factors that may influence aggressive behavior.

Hormonal responses to brief social interactions: The role of psychosocial stress and relationship status.

This study investigated the effects of psychosocial stress on hormonal responses to a social interaction with an opposite-sex individual to test the hypothesis that stress may interfere with or suppress adaptive neuroendocrine responses to courtship opportunities. Heterosexual men and women were randomly assigned prior to arrival to either a control or psychosocial stress condition (Trier Social Stress Test) and subsequently went through a social interaction test with an opposite-sex individual. Expected increases of testosterone for control participants who interacted with opposite-sex individuals were not observed, and changes in testosterone were not observed for those in the psychosocial stress condition either. However, exploratory analyses in control participants showed main and interaction effects of relationship status were significant for both cortisol and testosterone. Specifically, single individuals showed higher levels of cortisol compared to those in a relationship, and single individuals showed significantly higher concentrations of cortisol after a social interaction when compared to individuals who were in a relationship. For testosterone, only individuals in a relationship decreased in testosterone following the social interaction. This study suggests that relationship status and psychosocial stress may be important variables moderating the relationship between an ecological cue of a potential courtship opportunity and subsequent adaptive physiological responses.

Testosterone histories from tusks reveal woolly mammoth musth episodes.

Hormones in biological media reveal endocrine activity related to development, reproduction, disease and stress on different timescales1. Serum provides immediate circulating concentrations2, whereas various tissues record steroid hormones accumulated over time3,4. Hormones have been studied in keratin, bones and teeth in modern5-8 and ancient contexts9-12; however, the biological significance of such records is subject to ongoing debate10,13-16, and the utility of tooth-associated hormones has not previously been demonstrated. Here we use liquid chromatography with tandem mass spectrometry paired with fine-scale serial sampling to measure steroid hormone concentrations in modern and fossil tusk dentin. An adult male African elephant (Loxodonta africana) tusk shows periodic increases in testosterone that reveal episodes of musth17-19, an annually recurring period of behavioural and physiological changes that enhance mating success20-23. Parallel assessments of a male woolly mammoth (Mammuthus primigenius) tusk show that mammoths also experienced musth. These results set the stage for wide-ranging studies using steroids preserved in dentin to investigate development, reproduction and stress in modern and extinct mammals. Because dentin grows by apposition, resists degradation, and often contains growth lines, teeth have advantages over other tissues that are used as records of endocrine data. Given the low mass of dentin powder required for analytical precision, we anticipate dentin-hormone studies to extend to smaller animals. Thus, in addition to broad applications in zoology and palaeontology, tooth hormone records could support medical, forensic, veterinary and archaeological studies.

The association of prenatal amniotic sex hormones and digit ratio (2D:4D) in children aged 5 to 70 months: A longitudinal study.

The sex difference of the 2D:4D digit ratio (female > male)-a proposed marker for prenatal testosterone exposure-is well established. Studies suggest it already exists in utero and is of moderate effect size in adulthood. However, evidence for the claim that 2D:4D reflects prenatal androgen action is limited, and the sex difference may exhibit lability during childhood. In the present study, 244 mothers were recruited in the course of an amniocentesis examination (performed between gestational weeks 14 and 18). Prenatal testosterone (T) and estradiol (E) levels were determined from amniotic fluid for boys and girls. The majority (97.4%, n = 114) of available female T levels (n = 117) were found below the level of quantification. Therefore, only male amniotic fluid data (n = 117) could be included for the analysis of associations between amniotic sex hormones (T levels and T to E ratio (T/E)) and 2D:4D. The families were then invited to each of the five consecutive follow-ups (ages: 5, 9, 20, 40, and 70 months) where children's 2D:4D was measured for both hands. The alternative marker D[r-l] reflects the directional asymmetry of 2D:4D (right subtracted by left 2D:4D) and was subsequently calculated as an additional measure for prenatal T exposure. No significant correlations between amniotic T or the T/E ratio (measured between week 14 and 18 of gestation) with 2D:4D respectively D[r-l] were observed for any time point. There was a significant sex difference (females > males) and a significant age effect with moderate correlations of 2D:4D between time points. 2D:4D increased between 20 and 40 months and between 40 and 70 months of age. The findings raise questions regarding the applicability of 2D:4D as a marker for prenatal androgen action and are discussed in terms of the reliability of obtained digit ratio data as well as in terms of the developmental timing of amniocentesis.

Day-to-day coordination of the stress and reproductive axes: A continuous-time analysis of within-person testosterone and cortisol relationships in athletic and healthy men.

Day-to-day coordination of the stress (i.e., hypothalamic-pituitary-adrenal [HPA]) and reproductive (i.e., hypothalamic-pituitary-gonadal [HPG]) axes is central to allostatic regulation, reproductive success, and survival. Reports of positive, within-person testosterone and cortisol relationships (or coupling) suggest cross-talk of a facilitative nature, but longitudinal evidence is scarce and has methodological and analytical limitations. To address this, we used a continuous-time (CT) model to investigate day-to-day, within-person coupling of testosterone and cortisol in two male cohorts. Salivary testosterone and cortisol fluctuations were monitored in 35 athletic men across two international tournaments (M = 19.3 tests) and in 41 healthy men during normal daily living (M = 27.9 tests). Bayesian CT analysis revealed a diminishing effect of each hormone on itself as time-interval length or lag increased. In both groups, cortisol had a negative lagged effect on testosterone that persisted for around three days. The cortisol effect on testosterone peaked after 0.71 and 0.51 days in athletic (standardized estimate = -0.13) and healthy men (standardized estimate = -0.11), respectively. Further estimates of non-lagged, contemporaneous correlations revealed positive testosterone and cortisol relationships (athlete r = 0.04, healthy r = 0.46). In summary, complex within-person HPA and HPG interplay emerged in two independent male cohorts. Specifically, a rising cortisol concentration was linked to a fall in testosterone concentration at later time points, but concurrently these hormones tended to rise and fall together. Our results suggest that inhibitory and facilitatory hormonal actions coexist on varying timescales, thereby expanding knowledge of HPG and HPA cross-talk in everyday life.

Gonadal Function in Male Patients With Metastatic Renal Cell Cancer Treated With Sunitinib.

BACKGROUND/AIM: Single-agent tyrosine kinase inhibitors are still prescribed as first-line treatment to a relevant subgroup of patients with metastatic renal cell carcinoma (mRCC). These agents are known to cause disfunction of many endocrine glands (e.g., thyroid). In this two-step trial, we aimed to assess gonadal function among male patients with mRCC treated with sunitinib. PATIENTS AND METHODS: We enrolled a first cross-sectional cohort of pre-treated (>6 months) patients and a subsequent cohort of treatment-naïve patients who were prospectively followed-up. All patients were screened for hypogonadism and received a Functional Assessment of Cancer Therapy - General (FACT-G) questionnaire at study entry and after 6 months of therapy. Patients who were candidates for testosterone replacement therapy (TRT) also received a FACT-G questionnaire at baseline and 3 months after supplementation. RESULTS: Among the 30 enrolled patients, the prevalence of hypogonadism was found to be higher in those receiving sunitinib for a longer period (27.3% at baseline, 41.7% in the first 6 months, and 68.4% after 9 months of therapy). The testosterone level of patients correlated with quality of life (R=0.32). A total of six patients received TRT, with a significant improvement in their global quality of life after the first 3 months of treatment. CONCLUSION: An increasing prevalence of hypogonadism was seen among male patients who received long-term treatment with sunitinib. TRT was associated with relevant improvements in quality of life. These findings corroborate similar published observations and encourage the assessment of gonadal function in male patients with mRCC under treatment with sunitinib.

Steroid Profiling in the Amniotic Fluid: Reference Range for 12 Steroids and Interest in 21-Hydroxylase Deficiency.

CONTEXT: Determination of steroid levels in the amniotic fluid gives some insight on fetal adrenal and gonadal functions. OBJECTIVE: Our objectives were to establish reference ranges of 12 steroid levels throughout pregnancy and to compare them with steroid levels from pregnancies with fetuses presenting with 21-hydroxylase deficiency (21OHD). METHODS: Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was applied to 145 "control" amniotic fluid samples from gynecology activity (12 + 6 to 32 + 4 gestational weeks, GW). The following steroids were analyzed according to gestational age and compared to 23 amniotic fluid samples from fetuses with classic 21OHD confirmed by molecular studies: delta-4-androstenedione (D4), dehydroepiandrosterone (DHEA), 17-hydroxyprogesterone (17OHP), 11-deoxycortisol (11OH), 21-deoxycortisol (21OH), corticosterone, deoxycorticosterone (DOC), testosterone, pregnenolone, 17-hydroxypregnenolone (17Pregn), cortisol, and cortisone. Chromosomal sex was determined by karyotype and gestational age by biometric measurements. RESULTS: Analysis of control samples showed a statistically significant difference for D4 and testosterone levels according to fetal sex. Cortisol, corticosterone, and DOC had lower concentrations before 20 GW than after 20 GW, whereas 17Pregn and pregnenolone had higher concentrations before 20 GW. This allowed us to establish age- and sex-dependent reference values. We observed higher 21OH, 17Pregn, D4, and testosterone levels in females with 21OHD than female controls. The ratios 17OHP/17Pregn, D4/DHEA, and 11OH/17OHP appeared discriminant for the diagnosis of 21OHD. CONCLUSION: Our study provides information on fetal steroidogenesis and suggests reference values for 12 steroids during pregnancy. This allows a prenatal diagnosis of 21OHD within 24 hours and might be useful in the diagnosis of other variations of sex development.