Alessandra Gambineri

Type-2 Familial Partial Lipodystrophy (FPLD2), a rare lipodystrophy caused by LMNA mutations, is characterized by a loss of subcutaneous fat from the trunk and limbs and excess accumulation of adipose tissue in the neck and face. Several studies have reported that the mineralocorticoid receptor (MR) plays an essential role in adipose tissue differentiation and functionality. We previously showed that brown preadipocytes isolated from a FPLD2 patient’s neck aberrantly differentiate towards the white lineage. As this condition may be related to MR activation, we suspected altered MR dynamics in FPLD2. Despite cytoplasmic MR localization in control brown adipocytes, retention of MR was observed in FPLD2 brown adipocyte nuclei. Moreover, overexpression of wild-type or mutated prelamin A caused GFP-MR recruitment to the nuclear envelope in HEK293 cells, while drug-induced prelamin A co-localized with endogenous MR in human preadipocytes. Based on in silico analysis and in situ protein ligation assays, we could suggest an interaction between prelamin A and MR, which appears to be inhibited by mineralocorticoid receptor antagonism. Importantly, the MR antagonist spironolactone redirected FPLD2 preadipocyte differentiation towards the brown lineage, avoiding the formation of enlarged and dysmorphic lipid droplets. Finally, beneficial effects on brown adipose tissue activity were observed in an FPLD2 patient undergoing spironolactone treatment. These findings identify MR as a new lamin A interactor and a new player in lamin A-linked lipodystrophies.

An imbalance in sex hormones has an important impact on type 2 diabetes (T2DM) mainly through the involvement of visceral adipose tissue. Androgens have an interesting sex-dimorphic association with T2DM, since hyperandrogenism in females and hypogonadism in males are risk factors for T2DM. Thus, treatments aimed at correcting hyperandrogenism in females and hypogonadism in males may prevent the development of T2DM or help in its treatment.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder among premenopausal women affecting up to 20% of the population. Even though androgen excess is the main diagnostic criterion for PCOS, the evaluation of hyperandrogenemia relies on methods with low accuracy and mainly able to measure total androgen amounts. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) allows high accuracy steroids measurement and if applied to saliva, it also allows the measurement of free or biovalable fractions. We performed seventeen-hours circadian salivary androgens analysis and serum steroids circadian profile in 9 classic PCOS (oligomenorrhea + hirsutism/high testosterone + polycystic ovary morphology) and 7 fertile controls. Subjects woke-up at 0700 h and collected saliva by direct spitting in fasting condition. At 0800 h subjects went to hospital for blood withdrawal, second saliva collection and anthropometric data recording. The blood collection was repeated at 1000 h and 1600 h. Saliva was collected every hour until 2300 h. Daily food intake and menstrual phase were standardized. Steroids were measured by validated LC–MS/MS methods and area under curve (AUCg) was computed. Compared to controls, PCOS girls were younger (P=0.023) and displayed higher BMI (P=0.017) and waist circumference (P=0.011). Both baseline levels and circulating hormones daily exposure were higher in PCOS: testosterone (P=0.001; P=0.006) androstenedione (PP=0.004; P=0.001) and 17OHProgesterone (PP=0.002; P=0.006) and 17OHpregnenolone (P=0.013; P=0.001) levels compared to controls. No differences were found for serum cortisol, cortisone, corticosterone, 11-deoxycortisol, progesterone, estradiol and dihydrotestosterone. PCOS displayed higher salivary androgens both at basal levels and in daily exposure: testosterone (4.41±0.96 vs 13.5±6.9 pg/ml, PPPPP=0.007; 113272.3±47145.4 vs 185457.8±67046.8 AUCg, P=0.018) and 17OHProgesterone (10.5±4.9 vs. 25.1±10.2 pg/ml, P=0.010; below sensitivity limit vs 14643.0±2790.0 AUCg) compared to controls. PCOS subjects displayed high serum testosterone, androstenedione, DHEA, 17OHProgesterone, 17OHpregnenolone and estrone, both at baseline and in daily exposure. Increased serum androgens resulted in high salivary androgens too, showing the salivary androgens profiling as promising tool for non-invasive diagnostic work-up of PCOS patients.

Introduction: Clinical and laboratory hyperandrogenisms indistinctly contribute to PCOS diagnosis; however, they differ in hormonal and metabolic correlates and require proper therapeutic strategies. Circulating biomarkers of hirsutism were not identified so far, with elevated testosterone being observed only in half of hirsute patients. Reasons for such inconsistencies possibly relies in previously used immunoassays, nowadays recognized as not adequate because of poor reliability and limited steroid panel. The present study aimed at defining the circulating steroid fingerprint that specifically distinguishes hirsutism by LC-MS/MS profiling of a broad steroid panel. Methods: Sixteen serum steroids were determined in 352 patients (age 14–49 years) in follicular phase. The independent effect of ovarian dysfunction (OD; oligo-amenorrhea and/or PCO morphology), hyperandrogenemia (testosterone≥1.56 nmol/L and/or androstenedione≥5.72 nmol/L) and hirsutism (modified-Ferriman-Gallway score≥8) on circulating steroids was valued. Moreover, hirsute (n=74) vs not-hirsute (n=47) women were compared within patients showing both OD and hyperandrogenemia. Results: OD directly associated with LH (P=0.048) and LH/FSH (PPP=0.002), 17OHprogesterone/progesterone (PP=0.004), androstenedione/dehydroepiandrosterone (PP=0.026), dehydroepiandrosterone (P=0.029), dehydroepiandrosterone-sulfate (P=0.034) and 11deoxycortisol/17OHprogesterone (P=0.003). Hyperandrogenemia positively associated with mFG-score (P=0.012), ACTH, LH and LH/FSH (all PP=0.025) and HOMA-IR (P=0.045). Hyperandrogenemia positively associated with 17OHpregnenolone, progesterone, dehydroepiandrosterone, 17OHprogesterone, estrone (all PP=0.004), dihydrotestosterone (P=0.009), corticosterone, 11deoxycortisol, cortisol (all PP=0.009), 17OHprogesterone/progesterone (PP=0.014), and negatively with dehydroepiandrosterone-sulfate/dehydroepiandrosterone, testosterone/androstenedione, estrone/androstenedione, estradiol/testosterone, dihydrotestosterone/testosterone and cortisol/11deoxycortisol (all PP=0.014) and inversely with HDL (P=0.041). Hirsutism also associated with lowering progesterone (P=0.007) and 17OHprogesterone (P=0.003) and increasing androstenedione/17OHprogesterone (PP=0.021). Compared with not-hirsute, hirsute women with OD and hyperandrogenemia exhibited higher BMI (P=0.002) and dehydroepiandrosterone/17OHpregnenolone (P=0.011), and lower SHBG (P=0.001), 17OHprogesterone (P=0.030), testosterone (P=0.004), androstenedione/dehydroepiandrosterone (P=0.024) and testosterone/androstenedione (P=0.039). Conclusions: Androstenedione excess in OD apparently originates by gonads, as indicated by imbalanced gonadotropin, low progesterone, increased 17hydroxylase activity and reduced adrenal secretion. Hyperandrogenemia is featured by insulin resistance, combined ovarian and adrenal hypersecretion of the overall steroid profile, and reduced precursors’ conversion in downstream active steroids. Hirsutism is characterized by obesity and dyslipidemia, by low 17OHprogesterone and, in the PCOS context, by mild hypertestosteronemia and reduced SHBG, overall suggesting that this phenotype could result by increased free-androgens at the pilosebaceous unit.

Objective: This study was aimed to evaluate the prevalence of non classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-NCAH) in hyperandrogenic women in the reproductive age attending our Endocrinology Unit and to identify specific phenotypic traits among clinical, biochemical and hormonal features. Setting: Outpatient Unit of Endocrinology, S. Orsola-Malpighi University Hospital of Bologna, Italy. Patients and methods: Among a population of 1079 women who attended our Unit from 2003 to 2017 for hyperandrogenic complaints, we selected 70 subjects having basal 17 hydroxyprogesterone (17OHP) levels in the follicular phase of the menstrual cycle ≥200 ng/dl measured by immunoassay. All these 70 patients performed CYP21A2 gene analysis by direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). They also performed an ovarian ultrasonography, a 1-24ACTH test, a complete basal steroid profiling measured both by immunoassay and by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), all in the follicular phase of the menstrual cycle. Results: Twenty-three patients resulted affected by 21-NCAH, 15 were found to be heterozygotes for the 21-hydroxylase deficiency (21-HTZ) and 32 had no mutations for CYP21A2gene(C). As expected, these three groups resulted significantly different for basal 17OHP levels measured by immunoassay even though there was an overlap among them [21-NCAH:1971±2550 ng/dl (from 228 to 12,420); 21 HTZ: 287±85 ng/dl (from 196 to 452); C: 305±100 ng/dl (from 201 to 624)]. At variance, basal 17OHP measured by LC-MS/MS discriminated the 21-NCAH group from the others without any overlap [21-NCAH: 16.9±23.1 ng/ml (from 3.5 to 51.5); 21 HTZ: 1.7±0.9 ng/ml (from1.1 to 2.9); C: 1.4±0.3 ng/ml (from 1.1 to 1.8)]. Furthermore, in 21-NCAH patients 21-deoxycortisol levels were found significantly higher than in the other groups (0.58±0.32 ng/ml vs. 0.14±0.08 ng/ml in 21 HTZ and 0.11±0.06 ng/ml in C, P<0.05). In addition, cortisol and corticosterone measured by LC-MS/MS were significantly lower in 21-NCAH group (cortisol: 61.6±46.9 ng/ml; corticosterone 1.1±0.9 ng/ml) than in 21-HTZ (cortisol: 182±27 ng/ml; corticosterone: 12.1±9.7 ng/ml) and C (cortisol: 169±26 ng/ml; corticosterone 14.4±5.2 ng/ml) groups. Conclusion: This study confirms that the prevalence of 21-NCAH of new diagnosis among hyperandrogenic women in the reproductive age is high accounting for 2.2%. In addition, it demonstrates that a basal blood steroid profiling measured by LC-MS/MS in the follicular phase of the menstrual cycle and composed by 17OHP, 21-deoxycortisol, cortisol and corticosterone is a valid diagnostic tool in 21-NCAH patients.

Though LC-MS/MS is replacing immunoassays for major glucocorticoids and androgens routine assessment, the measurement of other sex steroids and precursors is still challenging and available only in experienced research laboratories. Our aim was to develop a simple-prep and rapid 2D-LC-MS/MS method for the simultaneous measurement of estrone (E1), estradiol (E2), dihydrotestosterone (DHT) and 17OHpregnenolone. Serum volumes of 300ul were spiked with internal standards and processed by single-step liquid-liquid-extraction. Extracts were injected onto Prominence HPLC - LCMS-8050 triple-quadrupole platform (Shimadzu). Analytes were purified by perfusion column, separated on a Gemini C6-phenyl (100×2 mm, 5 um) column (Phenomenex), ionized by electrospray and detected by specific multiple reaction monitoring transition in 11min run. The method showed high functional sensitivity (E2 and DHT: 9.8 pg/ml, E1: 4.9 pg/ml, 17OHpregnenolone: 39.1 pg/ml), proper accuracy (86.4–102.1%) and intra- (0.9–4.9%) and inter-assay (2.1–11.6%) imprecision. Sex and age specific reference intervals were estimated. The LC-MS/MS method was compared with routine E1 and E2 immunoassays. The Passing&Bablok analysis revealed high correlation between Roche-Modular III ECLIA and LC-MS/MS for E2 measurement both at all values (n=82; r:0.991) and at E2 levels n=28; r:0.960), with respectively slight or absent proportional (slope (90CI): 1.06 (1.02–1.09) and 096 (0.85–1.11)) and systematic (intercept (90CI): −5.50 (−10.4–−1.68) and −0.78 (−6.94–4.96)) bias. DSL8700 direct RIA for E1 measurement showed poor correlation with LC-MS/MS (n=42; r:0.872), with relevant proportional (slope (90CI): 076 (063–0.94)) and systematic (intercept (90CI): 18.4 (9.4–22.9)) bias. The LC-MS/MS assay showed optimal performance to sensitively and accurately determine routinely-assayed estrogens, confirming the reliability of new-generation ECLIA for E2, and highlighting direct RIA unreliability in determining E1. The assay further allowed the determination of important though not-routinely assayed DHT and 17OHpregnenolone. The bench- and run-time required by the LC-MS/MS method allow the integration of steroid profiling capability both in research and clinical settings.