The Journal of Steroid Biochemistry and Molecular Biology

Poor nutrition during pregnancy causes permanent metabolic and/or structural adaptation in offspring. The adrenal gland produces various steroid hormones during pregnancy. Thus, this study aimed to evaluate the influence of diet during pregnancy on the adrenal glands of Wistar rats. For this, 10-week-old pregnant Wistar rats (p, n=15) and non-pregnant rats (np, n=15) were divided into three groups and received a normoproteic control diet (C, 17% casein, n=5), isocaloric low-protein diet (PR, 6% casein, n=5), or 50% calorie restriction (CR, 50% of the diet consumed by group C), over a period of 21 days. On the 21st day of gestation (21dG, p groups) or on the 21st day of diet (np groups), after anesthetic deepening, the right adrenal gland was collected, weighed (total mass), and prepared for inclusion in Paraplast(R) for histomorphometric and immunohistochemical analysis (Ki-67, glucocorticoid receptors (GR), and mineralocorticoid receptor (MR)) in the different areas of the gland. Data, expressed as the mean {+/-} SD, were evaluated by one-way analysis of variance with Tukeys post-test (p < 0.05). CR in pregnancy increased the amount of GR, MR, and Ki-67 receptors in the adrenal gland. The npRC group showed highest GR staining compared to the animals that received a normal diet. Protein restriction in pregnancy decreases adrenal MR. The results allowed us to conclude that even without altering the weight of the adrenal glands, the pRC group suffered the most from stress during the study, suggesting that CR associated with pregnancy can cause morphofunctional changes in the adrenal glands.

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysone receptors (EcRs) and at least one membrane GPCR receptor (DopEcR) and displays numerous pharmacological effects in mammals. However, its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ER{beta} receptor. The goal of our study was to better understand 20E mechanism of action. A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) was used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with Angiotensin-(1-7), the endogenous ligand of Mas. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using small interfering RNA (siRNA) or pharmacological inhibitors. 17{beta}-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin-(1-7) antagonists. A mechanism involving cooperation between Mas receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the Mas receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and indeed this mechanism may explain the close similarity between angiotensin-(1-7) and 20E effects. Our findings open a lot of possible therapeutic developments by stimulating the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.

BackgroundThe increasing amount of data points to the circadian timing system as an essential part of processes regulating androgen homeostasis. However, the relationship between stress response, timekeeping-, and steroidogenesis-related systems is unexplored. ObjectiveThe purpose of the study was to analyze the stress-response of the testosterone-producing Leydig cells depending on the stressful events time. Materials and methodsThe study was designed to follow the effects of 3-hour immobilization (IMO) applied at different periods during the day. The IMO performed once (1xIMO) or repeated in 10 consecutive days (10xIMO). Principal-component-analysis (PCA) followed the expression study of the clock and steroidogenic-related genes in Leydig cells. ResultsBoth types of IMO in all investigated periods increased corticosterone and decreased testosterone blood level. Transcriptional analysis revealed different sensitivity to IMO events depending on the circadian time. The majority of steroidogenesis-related genes (Lhcgr, Cyp11a1, Cyp17a1, Hsd3b1/2) were down-regulated in the inactive but unchanged or even up-regulated in the active phase of the day. Both types of IMO potentiated the expression of clock elements Bmal1/BMAL1, Per1/PER1 regardless of the days stage and reduced Reverba in the inactive phase. The PCA confirmed a major shift, for both IMO-types, in the transcription of steroidogenesis and clock genes across the inactive/active phase. Further, the diurnal pattern of the glucocorticoid receptor (Nr3c1/GR) expression in Leydig cells was increased in the inactive phase due to 10xIMO. The observed time-dependent IMO-response of the Leydig cells correlated with different GR engagements. DiscussionStress- and the circadian-system coordinatively shape Leydig cells physiology, assuming diverse GR engagement as a possible factor in mediating the diurnal effect of stress. ConclusionThe Leydig cells stress-response depends on the time of the stressful situation, emphasizing the importance of circadian activity in supporting androgen homeostasis and male fertility.

ObjectivesGlucocorticoids, mediated by the activation of the HPA axis, affect metabolic responses, insulin resistance, lipolysis and body fat distribution. Dehydroepiandrosterone-sulfate (DHEA-S) is a hormone produced by the adrenal glands and a precursor of sex hormones. The balance and interaction between cortisol and DHEA-S can significantly affect body composition. This study aimed to investigate the relationship between cortisol and DHEA-S levels, cortisol/DHEA-S ratio, and body composition in Korean men and women. MethodsIn total, 802 adults participated in this study between January 2018 and March 2023. Socio-demographic data and lifestyle factors were assessed using questionnaires. Body composition, clinical blood pressure, and metabolic variables, including cortisol and DHEA-S levels, were assessed. Cortisol and DHEA-S scores were analyzed in relation to height, body weight(BW), body mass index(BMI) and waist circumference(WC) according to age and sex. ResultsParticipants had a mean age of 52.6{+/-}11.7 years. Cortisol levels adjusted for age and gender were negatively correlated with BW, WC and BMI. This result was more significant in women than in men. DHEA-S levels were positively correlated with height, BW and WC after adjusting for age. The cortisol/DHEA-S ratio was associated with lower height and BW after adjusting for gender. Logistic regression for cortisol, DHEA-S and the cortisol/DHEA-S ratio in the prediction of central obesity was significant for men after adjusting for age and BMI. ConclusionsElevated cortisol concentrations are associated with lower adiposity. DHEA-S levels were positively correlated with height and body mass. The prediction of central obesity was associated with cortisol and the cortisol/DHEA-S ratio in men and negatively associated with DHEA-S.

PurposeInducing puberty in hypogonadal patients enables achieving normal final adult height, healthy bone mass accrual and improves fertility potential. Reliable availability and access to medicines remain a challenge around the world, particularly in low income countries. We aim to study the availability/access to medications used for inducing and maintaining puberty in centers within the Arab region. Patients and MethodsA cross-sectional survey was conducted using a link to an online questionnaire which was emailed to paediatric endocrinologists in the Arab region. The questionnaire consisted of three questions related to availability of various forms of sex hormones. Results99 physicians from 16 countries participated in the study. The commonest available form of estrogen was conjugated estrogen (29% of centers) followed by ethinylestradiol in 26%. Depot estradiol was available in 11centers while topical estrogen preparations of gel and patches were available in 6 and 10 centers respectively. Medroxy progesterone was available in 26% of the centers followed by noresthisterone (24%). The combined forms of oral and transdermal patches of estrogen/progestorne were available in 35 and 9% of centers. Intramuscular testosterone (Sustanon) was the most commonly available preparation of testosterone followed by the depot injection (Nebido), oral testosterone and testosterone gel and cream. ConclusionsWe report the first availability data of medications used for puberty induction and maintenance in paediatric hypogonadism in the Arab region. Recommended preparations for this purpose are not widely available. Creating essential list of medications used in paediatric endocrinology disorders might improve availability, access and consequently practice.

PRX-3140 is a partial agonist to the 5-hydroxytryptamine receptor 4 (5-HT4) and a ligand for the sigma-1 (S1R) and sigma-2 (S2R) receptors. Although few publications have inferred S1R agonists/antagonists modulate blood glucose, Di et.al (2017) reported S1R deficiency in knockout mice impacted regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis, with a dexamethasone-induced reduction in level of corticosterone markedly attenuated in S1R -/- knockout mice, implicating S1R in feedback response to the HPA axis. The hypothesis that S1R deficiency causes down-regulation of the glucocorticoid receptor (GR) and attenuates GR-mediated feedback inhibition of HPA axis, as well as stress response of HPA axis, suggest that the inverse, the activation of S1R under normal conditions, may modulate glucocorticoid insulin suppression (as a direct S1R-GR effect) as well as cortisol levels (producing HPA axis feedback inhibition). In the present study, coadministration of 10 {micro}M PRX-3140 with 100 nM cortisol significantly increased insulin release (to 74.8 ng/ml, P-value <0.0001). Similar effects were observed when cells were exposed to dexamethasone (Dex), with 10 {micro}M PRX-3140 and 10 nM Dex producing 1.87-fold significantly more insulin than 10 nM Dex alone. Daily glucose concentrations in the 14-day clinical study (NCT00384423) of PRX-3140 demonstrate a reduction for 10 mg once-daily at days 1, 7, 10, and 15. Urine free cortisol levels at 10, 30, 100 and 200 mg dose levels of PRX-3140 demonstrated a larger reduction at 7 and 14 days compared to placebo. As an agonist of S1R that acts as a chaperone of GR, PRX-3140 has demonstrated GR modulating effects in INS-1 cells and in 14-day clinical studies in healthy adults with low incidence of side effects. The results of the present study suggest that S1R activation, with PRX-3140 and NP-18-2 S1R agonists, modulates glucocorticoid insulin suppression and cortisol levels.

The steroidal module of the Athlete Biological Passport (ABP) targets the use of exogenous androgenous anabolic steroids (EAAS) in elite sport by monitoring urinary steroid profiles. Urine and blood samples were collected weekly during two consecutive OCP cycles (8 weeks) in 15 physically active women to investigate the low urinary steroid concentrations and putative confounding effect of OCP. In urine, testosterone (T) and/or epitestosterone (E) were below the limit of quantification of 1 ng/mL in 62% of the samples. Biomarkers variability ranged between 31% and 41%, with a significantly lesser variability for ratios (with the exception of T/E (41%)): 20% for androsterone/etiocholanolone (p < 0.001) and 25% for 5-androstane-3,17{beta}-diol/5{beta}-androstane-3,17{beta}-diol (p < 0.001). In serum, variability for testosterone (T; 24%), androstenedione (A4; 23%), dihydrotestosterone (DHT; 19%) and T/A4 (16%) was significantly lower than urinary biomarkers (p < 0.001). Urinary A/Etio increased by > 18% after the first two weeks (p < 0.05) following blood loss. In contrast, T (0.98 nmol/L during the first week), and T/A4 (0.34 the first week) decreased significantly by more than 25% and 17% (p<0.05), respectively in the following weeks. Our results outline steroidal variations during the OCP cycle highlighting exogenous hormonal preparations as confounder for steroid concentrations in blood. Low steroid levels in urine samples have a clear detrimental impact on the subsequent interpretation of steroidal variations for the ABP. With a greater analytical sensitivity and lesser variability for steroids in serum vs. urine in healthy active women, serum represents a complementary matrix to urine in the ABP steroidal module.

BackgroundCongenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is characterized by a broad clinical spectrum, ranging from salt-wasting to nonclassical forms. Genotype-phenotype correlations based on predicted residual enzymatic activity have been widely studied, but data from Latin American populations remain scarce. Additionally, the influence of mutational burden on phenotype prediction has not been fully explored. ObjectiveTo evaluate the genotype-phenotype correlation and the impact of mutational burden on predictive accuracy in a Colombian cohort of patients with CAH. MethodsWe conducted a cross-sectional study of patients with confirmed CAH enrolled in a specialized rare disease program. Genotypic classification was based on predicted residual enzymatic activity (Null, A, B, C), and clinical phenotype was categorized as salt-wasting (SW), simple virilizing (SV), or nonclassical (NC). Genotype-phenotype concordance was defined as exact category agreement. Mutational burden was defined as the total number of pathogenic variants, dichotomized as low ([&le;]2 mutations) or high (>2). Penalized logistic regression (Firth method) was used to evaluate associations between mutational burden, sex, and concordance. ResultsAmong 48 patients with available genetic data, genotype-phenotype concordance was highest in severe genotypes: 100% in Null and 85.7% in Group A. In contrast, concordance declined in Group B (33.3%) and Group C (44.4%). Individuals with high mutational burden had significantly lower odds of concordance (OR = 0.18; 95% CI: 0.03-0.94). No significant interaction between sex and mutational burden was observed. More than one-third of Group C patients exhibited more severe phenotypes than predicted. ConclusionsOur findings support established genotype-phenotype correlations in CAH, particularly for severe genotypes. However, increased mutational burden was associated with reduced predictive accuracy, suggesting the need to consider total mutation load in clinical assessment and genetic counseling.

BackgroudAdrenal venous sampling (AVS) is a gold standard procedure to determine the dominant side of aldosterone secretion in patients with primary aldosteronism. Unsuccessful cannulation of right adrenal vein (RAV) is a common problem in performing AVS. ObjectiveTo use calculated aldosterone concentration in the RAV (cAldoRAV) for identifying the dominant side of aldosterone secretion. DesignRetrospective study, 2011-2023. MethodsBased on the assumption that cortisol production from both adrenal glands is equal, aldosterone concentration in the RAV was calculated by using the data from left adrenal vein (LAV) and inferior vena cava. The aldosterone concentration in the LAV (AldoLAV) compared to the cAldoRAV (AldoLAV:cAldoRAV ratio) was then used to determine the dominant side of aldosterone secretion in patients with primary aldosteronism. ResultsOf 117 patients with successful AVS, 95 (81.2%) had concordant results between adrenal imaging and AVS study and were used as the gold standard for studying diagnostic performance. The AldoLAV:cAldoRAV ratio with the cutoff values of [&ge;]3 and [&le;]0.33 could identify unilateral diseases (left-sided and right-sided disease, respectively) with 93.8% sensitivity and 100% specificity. In 22 patients who had discordant results between adrenal imaging and standard AVS interpretation, 11 had concordant results when using the AldoLAV:cAldoRAV ratio. ConclusionsThe AldoLAV:cAldoRAV ratio can determine the dominant side of aldosterone secretion with high sensitivity and specificity. It can not only be used for patients with unsuccessful cannulation of RAV but also increase the concordance rate in those who have discordance between adrenal imaging and standard AVS interpretation.

Deficiency of Cytochrome P450 Steroid 21-hydroxylase (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction along with functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in the Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of 21OH deficiency.

Hyperandrogenism is the excessive production of androgenic hormones resulting in infertility in a number of women. While letrozole and clomiphene citrate have been used to increase chances of achieving pregnancy, their effects on the brain has been scarcely studied. This study examined the effects of clomiphene and letrozole alone or in combination on neurobehavioural and neurochemical changes in female rats exposed to testosterone. Weaned rats were assigned into eight groups of ten each. Animals were grouped as normal control administered vehicle (normal saline) orally at 10 ml/kg or subcutaneously at 2 ml/kg, three groups administered clomiphene (CLOM) at 100 {micro}g/kg, letrozole (LETR) at 5 mg/kg and or a combination of clomiphene and letrozole (CLOM/LETR) orally and saline subcutaneously. There were also four groups Testosterone (Test), Test/CLOM, Test/LETR or Test/CLOM+LETR administered testosterone enantate subcutaneously at 1 mg/100 g. Testosterone or saline was administered from day 1-35, while beginning on day 36, clomiphene, letrozole or saline was administered daily for 10 days. At the end of the dosing period, animals were exposed to different behavioural paradigms. After the behavioural tests, animals were sacrificed, the cerebral cortex was homogenised for the assessment of biochemical assays. The result showed an increase in body weight, food intake, locomotor activity, rearing and self grooming with CLOM, LETR and CLOM/LETR in all treated groups. Decreased spatial working memory and anxiolysis was observed with letrozole and/or clomiphene. Increased oxidative stress, decreased total antioxidant capacity, altered inflammatory cytokines and brain neurotransmitter were observed with letrozole and /or clomiphene. In conclusion, the administration of clomiphene and/or letrozole was associated with significant alterations in brain function, oxidative stress, inflammatory markers and brain neurotransmitter levels.

BackgroundBicalutamide is a potential anti-androgen for transgender individuals with feminizing embodiment goals, but use has been limited because of hepatotoxicity in cisgender men with prostate cancer. This study compared transaminase changes in transfeminine adolescents and young adults (AYA) using low-dose bicalutamide with individuals using other methods of androgen blockade. MethodsA retrospective analysis was conducted using electronic health record data for patients starting gender affirming hormone therapy with at least 10 months of follow-up data between 2015 and 2023. Linear mixed models compared change in ALT and AST from baseline and maximum ALT and AST values in bicalutamide and comparison groups. Secondary outcomes included % individuals with ALT and AST elevation more than 1, 2, or 3 times the upper limit of normal (ULN) (Fishers exact test), standardized mean estradiol dose by group (t test), and Tanner staging of breast tissue by group (Fishers exact test). ResultsEighty-four transfeminine AYA (median age 18) taking bicalutamide were compared to 69 transfeminine AYA (median age 19) taking GnRH agonists, spironolactone or no agent in addition to estradiol. In linear mixed models adjusted for baseline age, BMI, baseline ALT or AST, and alcohol use, there was no difference in delta or maximum ALT or AST in bicalutamide and comparison groups. No individuals had an AST or ALT level > 3x ULN. Estradiol doses and Tanner stages were similar between groups in a subgroup analysis of individuals receiving pediatric care. ConclusionBicalutamide was not associated with significant change in transaminases as compared with other anti-androgen regimens over one year. Bicalutamide appears to be a safe anti-androgen for transfeminine individuals at low dose with close monitoring and deserves further study.

Recently, the glutathione S-transferase A3-3 (GST A3-3) homodimeric enzyme was identified as the most efficient enzyme that catalyzes isomerization of the precursors of testosterone, estradiol, and progesterone in the gonads of humans and horses. However, the presence of GST A3-3 orthologs with equally high ketosteroid isomerase activity has not been verified in other mammalian species, even though pig and cattle homologs have been cloned and studied. Identifying GSTA3 genes is a challenge because of multiple GSTA gene duplications (12 in the human genome), so few genomes have a corresponding GSTA3 gene annotated. To improve our understanding of GSTA3 gene products and their functions across diverse mammalian species, we cloned homologs of the horse and human GSTA3 mRNAs from the testes of a dog, goat, and gray short-tailed opossum, with those current genomes lacking GSTA3 gene annotations. The resultant novel GSTA3 mRNA and inferred protein sequences had a high level of conservation with human GSTA3 mRNA and protein sequences ([&ge;] 70% and [&ge;] 64% identities, respectively). Sequence conservation was also apparent for the 13 residues of the "H-site" in the 222 amino acid GSTA3 protein that is known to interact with the steroid substrates. Modeling predicted that the dog GSTA3-3 is a more active ketosteroid isomerase than the goat or opossum enzymes. Our results help us understand the active sites of mammalian GST A3-3 enzymes, and their inhibitors may be useful for reducing steroidogenesis for medical purposes, such as fertility control or treatment of steroid-dependent diseases.

While the human mineralocorticoid receptor (MR) regulates electrolyte homeostasis through aldosterone activation of the kidney MR, the MR also is highly expressed in the brain, where the MR is activated by cortisol. Here, we report the half-maximal response (EC50) and fold-activation by cortisol, aldosterone and other corticosteroids of human MR rs5522, a haplotype containing valine at codon 180 instead of isoleucine found in wild-type MR (Ile-180). MR rs5522 (Val-180) has been studied for actions in the human brain involving coping with stress and depression. We compared the EC50 and fold-activation by corticosteroids of MR rs5522 and wild-type MR transfected into HEK293 cells with either the TAT3 promoter or the MMTV promoter. Parallel studies investigated the binding of MR antagonists, spironolactone and progesterone, to MR rs5522 to investigate their use as antagonists of MR rs5522. In HEK293 cells with the MMTV promoter, MR rs5522 had a slightly higher EC50 compared to wild-type MR and a similar fold-activation for all corticosteroids. In contrast, in HEK293 cells with the TAT3 promoter, MR rs5522 had a higher EC50 (lower affinity) and fold-activation for cortisol compared to wild-type MR (Ile-180), while compared to wild-type MR, the EC50s of MR rs5522 for aldosterone and corticosterone were slightly lower and fold-activation was higher. Spironolactone and progesterone had similar antagonist activity for MR rs5522 and MR (Ile-180) in the presence of MMTV and TAT3 promoters in HEK293 cells indicating these antagonists are potential regulators of brain MR rs5522 to treat hyperactivity that contributes to Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

LH and FSH are two important hormones in the regulation of granulosa cells. Their effects are mediated mainly by cAMP/PKA signaling, bit the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated as well. We studied the involvement of the ERK cascade in LH and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production, downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased-expression of the steroidogenic acute regulatory (StAR) protein, a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is downregulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis, but also activates downregulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents, may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.

PurposeInfants and toddlers with classical congenital adrenal hyperplasia (CAH) are at high risk for adrenal crisis and associated sequelae. To better understand acute illness at this early age, we determined the frequency and severity of acute illness and hospitalizations between 0-4 years of age, both within CAH and compared to controls. We also evaluated the impact of pre-hospital stress-dose hydrocortisone on Emergency Department (ED) visits and hospitalizations. MethodsWe performed a retrospective study of 40 CAH youth and 27 age-matched controls at a tertiary center. Characteristics of acute illnesses during the first 4 years of life were recorded, including fever, vomiting, diarrhea, ED visits, hospitalizations, abnormal electrolytes, and stress-dose hydrocortisone usage. ResultsCAH youth had more frequent illnesses requiring stress-dosing when they were younger than 2 years old [4.0 (1.0-6.0)] compared to when they were 2-4 years old [3.0 (1.0-4.0), P < 0.05], with the most illnesses during their first year of life. As well, CAH infants and toddlers had more hospitalizations younger than 2 years old compared to 2-4 years old (36 vs 2). 25% (3/12) of CAH youth with abnormal electrolytes in the ED did not receive any stress-dosing (oral/IM) prior to the ED, and only 25% (3/12) had received intramuscular hydrocortisone at home. CAH youth had more frequent ED visits (7.4 times as many) and hospitalizations (38 to 0) compared to controls. ConclusionsVery young children with classical CAH are at high risk for acute illness and hospitalizations during their first 2 years of life, and do not receive adequate stress-dosing prior to the ED despite appropriate education. Our findings underscore the need for earlier recognition of acute illness in this vulnerable population and improved education regarding administration of stress-dose hydrocortisone to prevent morbidity.

Infertility, defined as the inability to achieve pregnancy after 12 months of unprotected intercourse, is a major reproductive health concern. In females, hyperandrogenism often contributes to polycystic ovarian syndrome (PCOS), a leading cause of infertility. Although clomiphene and letrozole are widely used as ovulation inducers, their individual efficacy is limited, and the potential benefit of combined therapy remains unclear. This study investigated the effects of clomiphene-letrozole co-administration on gonadotrophic hormones, inflammatory cytokines, antioxidant status, and ovarian histomorphology in a rat model of hyperandrogenism. Thirty female Wistar rats were randomised into five groups (n=6). Group A received saline, while groups B-E were administered testosterone enanthate (10 mg/kg, subcutaneous injection) for 35 days to induce PCOS. Group B served as PCOS control, while groups C, D, and E were additionally treated with clomiphene (100 {micro}g/kg), letrozole (5 mg/kg), or their combination, respectively, for 10 days from day 36. Hormonal assays, cytokine profiling, antioxidant measurements, and ovarian histology were performed. Results showed that the co-administration significantly reduced body and ovary weights, lowered glucose levels, and improved oestradiol and follicle-stimulating hormone profiles compared with PCOS controls. Combination therapy also enhanced antioxidant capacity, reduced lipid peroxidation, and modulated inflammatory cytokines by lowering IL-1{beta} and TNF- while elevating IL-10. Histological evaluation revealed cystic follicles with basement membrane thickening in the PCOS control, consistent with ovarian hyperstimulation; and a reversal with clomiphene and or letrozole treatment. In conclusion, clomiphene-letrozole co-administration demonstrated superior benefits over monotherapy in modulating endocrine, oxidative, and inflammatory parameters, suggesting a potential therapeutic advantage in ovulation induction for PCOS-related infertility.

IntroductionPolycystic ovary syndrome (PCOS) is a common disorder that is not fully understood. Multiple hormonal and metabolic factors impact on disease pathophysiology resulting in various phenotypic characteristics among the PCOS population. Luteinizing hormone beta subunit (LHB) (protein ID P01229) is mapped on (chr19p13.3) and consists of three exons. Luteinizing hormone (LH) has a central role in stimulation ovarian steroidogenesis, in particular androgen production, and the promotion of ovulation. ObjectivesTo determine if genetic variations of LHB are associated with PCOS among Sudanese families. MethodsA prospective laboratory based cross-sectional study to examine genetic mutations in LHB that associate with PCOS in families (cases; n=35 families, 90 females and controls; n=11 families, 30 females) in Khartoum State, Sudan. Quantitative enzyme linked immuno-sorbent assay (ELISA) and polymerase chain reaction (PCR) with Sanger sequencing were used to analyze biochemical parameters and detect polymorphisms. Protein structure and function bioinformatics analysis was conducted using standard software. ResultsPCOS cases had significantly different biochemical parameters from the controls (LH: p<0.001; testosterone: p<0.001; fasting glucose: p=0.02; insulin: p=0.01; triglycerides: p=0.03; total cholesterol: p<0.001; high density lipoprotein (HDL): p=0.012;low density lipoprotein (LDL): p<0.001). There were no differences in follicle stimulating hormone (FSH) (p=0.984) or prolactin (p=0.068). Sanger sequencing revealed 5 single nucleotide polymorphisms (rs5030775, A18T; rs746167425, R22K; rs1800447, W28R; rs35270001, H30R; and rs34349826, I35T) located on (exon 2) of LHB gene that were statistically correlated with serum LH, Testosterone and insulin levels among PCOS families. ConclusionThis is the first molecular family-based study in Sudan exploring the genetics of the LHB gene in women manifesting PCOS. These novel mutations give further information about the role of genetic inheritance and may explain some of the altered ovarian function and responses in women with PCOS.

Androgen excess in women is associated with the development of PCOS and its abnormalities. The Hypothalamus Pituitary Ovarian axis signaling is altered with excessed androgens, leading to anovulation and infertility. Previous studies in the lab have shown that AR signaling in the pituitary alters gonadotrophin release. Hence, the present pioneering study was an approach to determine the transcriptomic changes responsible to the phenotype seen with DHT excess. RNA seq data showed that 583 genes were differentially regulated by DHT in pituitary, of which 344 were upregulated and 239 downregulated. Pathways involved for these genes included endoplasmic reticulum, Golgi apparatus, calcium signaling and vesicles. Meanwhile, Transcriptional factor analysis showed that majority of the genes changed had Androgen responsive elements.

Androgen excess is one of the most common endocrine disorders of reproductive-aged women, affecting up to 20% of this population. Women with elevated androgens often exhibit hyperinsulinemia and insulin resistance. The mechanisms of how elevated androgens affect metabolic function are not clear. Hyperandrogenemia in a dihydrotestosterone (DHT)-treated female mouse model induces whole body insulin resistance possibly through activation of the hepatic androgen receptor (AR). We investigated the role of hepatocyte AR in hyperandrogenemia-induced metabolic dysfunction by using several approaches to delete hepatic AR via animal-, cell-, and clinical-based methodologies. We conditionally disrupted hepatocyte AR in female mice developmentally (LivARKO) or acutely by tail vein injection of an adeno-associated virus with a liver-specific promoter for Cre expression in ARfl/fl mice (adLivARKO). We observed normal metabolic function in littermate female Control (ARfl/fl) and LivARKO (ARfl/fl; Cre+/-) mice. Following chronic DHT treatment, female Control mice treated with DHT (Con-DHT) developed impaired glucose tolerance, pyruvate tolerance, and insulin tolerance, not observed in LivARKO mice treated with DHT (LivARKO-DHT). Further, during an euglycemic hyperinsulinemic clamp, the glucose infusion rate was improved in LivARKO-DHT mice compared to Con-DHT mice. Liver from LivARKO, and primary hepatocytes derived from LivARKO, and adLivARKO mice were protected from DHT-induced insulin resistance and increased gluconeogenesis. These data support a paradigm in which elevated androgens in females disrupt metabolic function via hepatic AR and insulin sensitivity was restored by deletion of hepatic AR.