Clinical phenotypes of adults with monogenic and syndromic genetic obesity.

OBJECTIVE: Considering limited evidence on diagnostics of genetic obesity in adults, we evaluated phenotypes of adults with genetic obesity. Additionally, we assessed the applicability of Endocrine Society (ES) recommendations for genetic testing in pediatric obesity. METHODS: We compared clinical features, including age of onset of obesity and appetite, between adults with non-syndromic monogenic obesity (MO), adults with syndromic obesity (SO), and adults with common obesity (CO) as control patients. RESULTS: A total of 79 adults with genetic obesity (32 with MO, 47 with SO) were compared with 186 control patients with CO. Median BMI was similar among the groups: 41.2, 39.5, and 38.7 kg/m2 for patients with MO, SO, and CO, respectively. Median age of onset of obesity was 3 (IQR: 1-6) years in patients with MO, 9 (IQR: 4-13) years in patients with SO, and 21 (IQR: 13-33) years in patients with CO (p < 0.001). Patients with genetic obesity more often reported increased appetite: 65.6%, 68.1%, and 33.9% in patients with MO, SO, and CO, respectively (p < 0.001). Intellectual deficit and autism spectrum disorder were more prevalent in patients with SO (53.2% and 21.3%) compared with those with MO (3.1% and 6.3%) and CO (both 0.0%). The ES recommendations were fulfilled in 56.3%, 29.8%, and 2.7% of patients with MO, SO, and CO, respectively (p < 0.001). CONCLUSIONS: We found distinct phenotypes in adult genetic obesity. Additionally, we demonstrated low sensitivity for detecting genetic obesity in adults using pediatric ES recommendations, necessitating specific genetic testing recommendations in adult obesity care.

The Role of Anti-Müllerian Hormone in Ovarian Function.

Anti-Müllerian hormone (AMH) is a member of the transforming growth factor ß (TGFß) superfamily, whose actions are restricted to the endocrine-reproductive system. Initially known for its role in male sex differentiation, AMH plays a role in the ovary, acting as a gatekeeper in folliculogenesis by regulating the rate of recruitment and growth of follicles. In the ovary, AMH is predominantly expressed by granulosa cells of preantral and antral follicles (i.e., post primordial follicle recruitment and prior to follicle-stimulating hormone (FSH) selection). AMH signals through a BMP-like signaling pathway in a manner distinct from other TGFß family members. In this review, the latest insights in AMH processing, signaling, its regulation of spatial and temporal expression pattern, and functioning in folliculogenesis are summarized. In addition, effects of AMH variants on ovarian function are reviewed.

Clustering Identifies Subtypes With Different Phenotypic Characteristics in Women With Polycystic Ovary Syndrome.

CONTEXT: Hierarchical clustering (HC) identifies subtypes of polycystic ovary syndrome (PCOS). OBJECTIVE: This work aimed to identify clinically significant subtypes in a PCOS cohort diagnosed with the Rotterdam criteria and to further characterize the distinct subtypes. METHODS: Clustering was performed using the variables body mass index (BMI), luteinizing hormone (LH), follicle-stimulating hormone, dehydroepiandrosterone sulfate, sex hormone-binding globulin (SHBG), testosterone, insulin, and glucose. Subtype characterization was performed by analyzing the variables estradiol, androstenedione, dehydroepiandrosterone, cortisol, anti-Müllerian hormone (AMH), total follicle count (TFC), lipid profile, and blood pressure. Study participants were girls and women who attended our university hospital for reproductive endocrinology screening between February 1993 and February 2021. In total, 2502 female participants of European ancestry, aged 13 to 45 years with PCOS (according to the Rotterdam criteria), were included. A subset of these (n = 1067) fulfilled the National Institutes of Health criteria (ovulatory dysfunction and hyperandrogenism). Main outcome measures included the identification of distinct PCOS subtypes using cluster analysis. Additional clinical variables associated with these subtypes were assessed. RESULTS: Metabolic, reproductive, and background PCOS subtypes were identified. In addition to high LH and SHBG levels, the reproductive subtype had the highest TFC and levels of AMH (all P < .001). In addition to high BMI and insulin levels, the metabolic subtype had higher low-density lipoprotein levels and higher systolic and diastolic blood pressure (all P < .001). The background subtype had lower androstenedione levels and features of the other 2 subtypes. CONCLUSION: Reproductive and metabolic traits not used for subtyping differed significantly in the subtypes. These findings suggest that the subtypes capture distinct PCOS causal pathways.

MC4R variants modulate α-MSH and setmelanotide induced cellular signaling at multiple levels.

CONTEXT: Melanocortin-4 receptor (MC4R) plays an important role in body weight regulation. Pathogenic MC4R variants are the most common cause of monogenic obesity. OBJECTIVE: We have identified 17 MC4R variants in adult and pediatric patients with obesity. Here, we aimed to functionally characterize these variants by analyzing four different aspects of MC4R signaling. In addition, we aimed to analyze the effect of setmelanotide, a potent MC4R agonist, on these MC4R variants. MATERIALS AND METHODS: Cell surface expression and α-MSH- or setmelanotide-induced cAMP response, ß-arrestin-2 recruitment, and ERK activation were measured in cells expressing either wild type (WT) or variant MC4R. RESULTS: We found a large heterogeneity in the function of these variants. We identified variants with a loss of response for all studied MC4R signaling, variants with no cAMP accumulation or ERK activation but normal ß-arrestin-2 recruitment, and variants with normal cAMP accumulation and ERK activation but decreased ß-arrestin-2 recruitment, indicating disrupted desensitization and signaling mechanisms. Setmelanotide displayed a greater potency and similar efficacy as α-MSH, and induced significantly increased maximal cAMP responses of several variants compared to α-MSH. Despite the heterogeneity in functional response, there was no apparent difference in the obesity phenotype in our patients. DISCUSSION: We show that these obesity-associated MC4R variants affect MC4R signaling differently, yet leading to a comparable clinical phenotype. Our results demonstrate the clinical importance of assessing the effect of MC4R variants on a range of molecular signaling mechanisms to determine their association with obesity, which may aid in improving personalized treatment.

GNB1 and obesity: Evidence for a correlation between haploinsufficiency and syndromic obesity.

Most patients with GNB1 encephalopathy have developmental delay and/or intellectual disability, brain anomalies and seizures. Recently, two cases with GNB1 encephalopathy caused by haploinsufficiency have been reported that also show a Prader-Willi-like phenotype of childhood hypotonia and severe obesity. Here we present three new cases from our expert centre for genetic obesity in which GNB1 truncating and splice variants, probably leading to haploinsufficiency, were identified. They all have obesity, hyperphagia and intellectual deficit. The clinical cases and their weight courses are presented, together with a review of all 68 published cases with GNB1 encephalopathy. Information on weight was not mentioned in most of these articles, so we contacted authors for additional clinical information on weight status and hyperphagia. Of the 42 patients whose weight status we could determine, obesity was present in 8 patients (19%). Obesity is significantly over-represented in the group with truncating and splicing variants. In this group, we see an obesity prevalence of 75%. Since GNB1 has been linked to several key genes in the hypothalamic leptin-melanocortin pathway, which regulates satiety and energy expenditure, our data support the potential association between GNB1 haploinsufficiency and genetic obesity. We also suggest GNB1 is a candidate gene for the known obesity phenotype of the 1p36 microdeletion syndrome given this chromosomal region includes the GNB1 gene. Knowledge of an additional obesity phenotype is important for prognosis, early interventions against obesity and awareness when prescribing weight-inducing medication.

Long-term glucocorticoid exposure and incident cardiovascular diseases - the Lifelines cohort.

CONTEXT: Long-term glucocorticoid levels in scalp hair (HairGCs), including cortisol and the inactive form cortisone, represent the cumulative systemic exposure to glucocorticoids over months. HairGCs have repeatedly shown associations with cardiometabolic and immune parameters, but longitudinal data are lacking. DESIGN: We investigated 6341 hair samples of participants from the Lifelines cohort study for cortisol and cortisone levels, and associated these to incident cardiovascular diseases (CVD) during 5-7 years of follow-up. We computed the odds ratio (OR) of HairGC levels for incident CVD via logistic regression, adjusting for classical cardiovascular risk factors, and performed a sensitivity analysis in subcohorts of participants <60 years and >= 60 years. Also, we associated HairGC levels to immune parameters (total leukocytes and subtypes). RESULTS: Hair cortisone levels (available in n = 4701) were independently associated with incident CVD (p < 0.001), particularly in younger individuals (multivariate-adjusted OR 4.21, 95% confidence interval (CI) 1.91-9.07 per point increase in 10-log cortisone concentration (pg/mg), p < 0.001). All immune parameters except eosinophils were associated with hair cortisone (all multivariate-adjusted p < 0.05). CONCLUSIONS: In this large, prospective cohort study, we found that long-term cortisone levels, measured in scalp hair, represent a relevant and significant predictor for future cardiovascular diseases in younger individuals. These results highlight glucocorticoid action as possible treatment target for CVD prevention, where hair glucocorticoid measurements could help identify individuals that may benefit from such treatments.

Body mass index stratified meta-analysis of genome-wide association studies of polycystic ovary syndrome in women of European ancestry.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex multifactorial disorder with a substantial genetic component. However, the clinical manifestations of PCOS are heterogeneous with notable differences between lean and obese women, implying a different pathophysiology manifesting in differential body mass index (BMI). We performed a meta-analysis of genome-wide association study (GWAS) data from six well-characterised cohorts, using a case-control study design stratified by BMI, aiming to identify genetic variants associated with lean and overweight/obese PCOS subtypes. RESULTS: The study comprised 254,588 women (5,937 cases and 248,651 controls) from individual studies performed in Australia, Estonia, Finland, the Netherlands and United States of America, and separated according to three BMI stratifications (lean, overweight and obese). Genome-wide association analyses were performed for each stratification within each cohort, with the data for each BMI group meta-analysed using METAL software. Almost half of the total study population (47%, n = 119,584) were of lean BMI (≤ 25 kg/m2). Two genome-wide significant loci were identified for lean PCOS, led by rs12000707 within DENND1A (P = 1.55 × 10-12) and rs2228260 within XBP1 (P = 3.68 × 10-8). One additional locus, LINC02905, was highlighted as significantly associated with lean PCOS through gene-based analyses (P = 1.76 × 10-6). There were no significant loci observed for the overweight or obese sub-strata when analysed separately, however, when these strata were combined, an association signal led by rs569675099 within DENND1A reached genome-wide significance (P = 3.22 × 10-9) and a gene-based association was identified with ERBB4 (P = 1.59 × 10-6). Nineteen of 28 signals identified in previous GWAS, were replicated with consistent allelic effect in the lean stratum. There were less replicated signals in the overweight and obese groups, and only 4 SNPs were replicated in each of the three BMI strata. CONCLUSIONS: Genetic variation at the XBP1, LINC02905 and ERBB4 loci were associated with PCOS within unique BMI strata, while DENND1A demonstrated associations across multiple strata, providing evidence of both distinct and shared genetic features between lean and overweight/obese PCOS-affected women. This study demonstrated that PCOS-affected women with contrasting body weight are not only phenotypically distinct but also show variation in genetic architecture; lean PCOS women typically display elevated gonadotrophin ratios, lower insulin resistance, higher androgen levels, including adrenal androgens, and more favourable lipid profiles. Overall, these findings add to the growing body of evidence supporting a genetic basis for PCOS as well as differences in genetic patterns relevant to PCOS BMI-subtype.

Longitudinal changes in acylated versus unacylated ghrelin levels may be involved in the underlying mechanisms of the switch in nutritional phases in Prader-Willi syndrome.

INTRODUCTION: Prader-Willi syndrome (PWS) is characterized by a switch from failure to thrive to excessive weight gain and hyperphagia in early childhood. An elevated, more unfavorable ratio between acylated and unacylated ghrelin (AG/UAG ratio) might play a role in the underlying mechanisms of this switch. We aimed to assess the evolution of the appetite regulating hormones acylated ghrelin (AG) and unacylated ghrelin (UAG) and the AG/UAG ratio and their association with the change in eating behavior in children with PWS, compared to healthy age-matched controls. METHODS: Longitudinal study in 134 children with PWS and 157 healthy controls, from The Netherlands, France and Belgium. Levels of AG and UAG and the AG/UAG ratio were measured and nutritional phases as reported for PWS were scored. RESULTS: The AG/UAG ratio was in the first years of life lower in PWS than in controls and started to increase from the age of 3 years, resulting in a high-normal AG/UAG ratio compared to controls. The AG levels remained stable during the different nutritional phases (p=0.114), while the UAG levels decreased from 290 pg/ml in phase 1a to 137 pg/ml in phase 2b (p<0.001). The AG/UAG ratio increased significantly from 0.81 in phase 2a to 1.24 in phase 2b (p= 0.012). CONCLUSIONS: The change from failure to thrive to excessive weight gain and hyperphagia in infants and children with PWS coincides with an increase in AG/UAG ratio. The increase in AG/UAG ratio occurred during phase 2a, thus before the onset of hyperphagia.

Levels of hormones regulating appetite and energy homeostasis in response to a 1.5-Year combined lifestyle intervention for obesity.

Background: Weight loss can induce changes in appetite-regulating hormone levels, possibly linked to increases in appetite and weight regain. However, hormonal changes vary across interventions. Here, we studied levels of appetite-regulating hormones during a combined lifestyle intervention (CLI: healthy diet, exercise and cognitive behavioral therapy). Methods: We measured levels of long-term adiposity-related hormones (leptin, insulin, high-molecular-weight (HMW) adiponectin) and short-term appetite hormones (PYY, cholecystokinin, gastric-inhibitory polypeptide, pancreatic polypeptide, FGF21, AgRP) in overnight-fasted serum of 39 patients with obesity. Hormone levels were compared between T0 (baseline), T1 (after 10 weeks) and T2 (end of treatment, 1.5 years). T0-T1 hormone changes were correlated with T1-T2 anthropometric changes. Results: Initial weight loss at T1 was maintained at T2 (-5.0%, p < 0.001), and accompanied by decreased leptin and insulin levels at T1 and T2 (all p < 0.05) compared to T0. Most short-term signals were not affected. Only PP levels were decreased at T2 compared to T0 (p < 0.05). Most changes in hormone levels during initial weight loss did not predict subsequent changes in anthropometrics, except for T0-T1 decreases in FGF21 levels and T0-T1 increases in HMW adiponectin levels tended to be associated with larger T1-T2 increases in BMI (p < 0.05 and p = 0.05, respectively). Conclusion: CLI-induced weight loss was associated with changes in levels of long-term adiposity-related hormones towards healthy levels, but not with orexigenic changes in most short-term appetite signals. Our data indicates that the clinical impact of alterations in appetite-regulating hormones during modest weight loss remains questionable. Future studies should investigate potential associations of weight-loss-induced changes in FGF21 and adiponectin levels with weight regain.

Glucocorticoids, stress and eating: The mediating role of appetite-regulating hormones.

Disrupted hormonal appetite signaling plays a crucial role in obesity as it may lead to uncontrolled reward-related eating. Such disturbances can be induced not only by weight gain itself but also by glucocorticoid overexposure, for example, due to chronic stress, disease, or medication use. However, the exact pathways are just starting to be understood. Here, we present a conceptual framework of how glucocorticoid excess may impair hormonal appetite signaling and, consequently, eating control in the context of obesity. The evidence we present suggests that counteracting glucocorticoid excess can lead to improvements in appetite signaling and may therefore pose a crucial target for obesity prevention and treatment. In turn, targeting hormonal appetite signals may not only improve weight management and eating behavior but may also decrease detrimental effects of glucocorticoid excess on cardio-metabolic outcomes and mood. We conclude that gaining a better understanding of the relationship between glucocorticoid excess and circulating appetite signals will contribute greatly to improvements in personalized obesity prevention and treatment.

Androgen receptor mutations modulate activation by 11-oxygenated androgens and glucocorticoids.

BACKGROUND: Androgen receptor (AR) ligand-binding domain (LBD) mutations occur in ~20% of all castration-resistant prostate cancer (CRPC) patients. These mutations confer ligand promiscuity, but the affinity for many steroid hormone pathway intermediates is unknown. In this study, we investigated the stimulation of clinically relevant AR-LBD mutants by endogenous and exogenous steroid hormones present in CRPC patients to unravel their potential contribution to AR pathway reactivation. METHODS: A meta-analysis of studies reporting untargeted analysis of AR mutants was performed to identify clinically relevant AR-LBD mutations. Using luciferase reporter and quantitative fluorescent microscopy, these AR mutants were screened for sensitivity for various endogenous steroids and synthetic glucocorticoids used in the treatment of CRPC. RESULTS: The meta-analysis revealed that ARL702H (3.4%), ARH875Y (4.9%), and ART878A (4.4%) were the most prevalent AR-LBD mutations across 1614 CRPC patients from 21 unique studies. Testosterone (EC50: 0.22 nmol/L) and 11-ketotestosterone (11KT, EC50: 0.74 nmol/L) displayed subnanomolar affinity for ARWT. The p.H875Y mutation selectively increased sensitivity of the AR for 11KT (EC50: 0.15 nmol/L, p < 0.05 vs ARWT), whereas p.L702H decreased sensitivity for 11KT by almost 50-fold. While cortisol and prednisolone both stimulate ARL702H, dexamethasone importantly does not. CONCLUSION: Both testosterone and 11KT effectively contribute to ARWT activation, while selective sensitization positions 11KT as a more prominent activator of ARH875Y. Dexamethasone may be a suitable alternative to prednisolone and should be explored in patients bearing the ARL702H.

Glucocorticoid Receptor Antagonism Improves Glucose Metabolism in a Mouse Model of Polycystic Ovary Syndrome.

Context: Polycystic ovary syndrome (PCOS) is a complex metabolic disorder associated with obesity, insulin resistance, and dyslipidemia. Hyperandrogenism is a major characteristic of PCOS. Increased androgen exposure is believed to deregulate metabolic processes in various tissues as part of the PCOS pathogenesis, predominantly through the androgen receptor (AR). Notably, various metabolic features in PCOS are similar to those observed after excess glucocorticoid exposure. Objective: We hypothesized that glucocorticoid receptor (GR) signaling is involved in the metabolic symptoms of PCOS. Methods: In a PCOS model of chronic dihydrotestosterone (DHT) exposure in female mice, we investigated whether GR signaling machinery was (de)regulated, and if treatment with a selective GR antagonist alleviated the metabolic symptoms. Results: We observed an upregulation of GR messenger RNA expression in the liver after DHT exposure. In white adipose tissues and liver we found that DHT upregulated Hsd11b1, which encodes for the enzyme that converts inactive into active glucocorticoids. We found that preventive but not therapeutic administration of a GR antagonist alleviated DHT-induced hyperglycemia and restored glucose tolerance. We did not observe strong effects of GR antagonism in DHT-exposed mice on other features like total fat mass and lipid accumulation in various tissues. Conclusion: We conclude that GR activation may play a role in glucose metabolism in DHT-exposed mice.

Cohort profile: a prospective Australian cohort study of women's reproductive characteristics and risk of chronic disease from menarche to premenopause (M-PreM).

PURPOSE: Previous studies have identified associations between individual reproductive factors and chronic disease risk among postmenopausal women. However, few have investigated the association of different markers of reproductive function, their interactions and risk factors of chronic disease among women approaching menopause. The Menarche-to-PreMenopause (M-PreM) Study aims to examine the relationship between reproductive factors across the reproductive lifespan and risk indicators for chronic disease among women in their early-to-mid-40s. The purpose of this cohort profile paper is to describe the rationale, study design and participant characteristics of the M-PreM Study. PARTICIPANTS: Women born in 1973-1978 who participated in the Australian Longitudinal Study on Women's Health (ALSWH) were invited to undertake a clinical or self-administered assessment. A total of 1278 women were recruited from June 2019 to June 2021. FINDINGS TO DATE: The study measures included functional, cognitive and cardiometabolic tests, anthropometry, spirometry, respiratory health questionnaires, physical activity, sleep patterns, sex hormones, and cardiovascular and metabolic markers; whereas blood and saliva samples were used for the analysis of genetic variants of genes associated with reproductive characteristics and chronic disease. The mean age of the clinic and self-assessed participants was 44.6 and 45.3 years, respectively. The menopausal status of participants was similar between the two arms of the study: 38%-41% premenopausal, 20% perimenopausal, and 36% took oral contraception or hormone replacement therapy. Approximately 80% of women had at least one child and participants reported experiencing pregnancy complications: preterm birth (8%-13% of pregnancies), gestational diabetes (10%) and gestational hypertension (10%-15%). FUTURE PLANS: The biomedical data collected in the M-PreM Study will be linked to existing ALSWH survey data on sociodemographic factors, health behaviour, reproductive function, and early life factors collected over the past 20 years and health administrative data. The association between reproductive factors and risk indicators of chronic disease will be analysed.

Loss of Anti-Müllerian Hormone Signaling in Mice Affects Trabecular Bone Mass in a Sex- and Age-Dependent Manner.

Ovariectomy-induced osteoporosis in mice results from an abrupt loss of ovarian sex steroids. Anti-Müllerian hormone knockout (AMHKO) mice show a gradual but accelerated ovarian aging, and therefore may better resemble osteoporosis following natural menopause. To study the impact of AMH signaling deficiency on bone, we compared trabecular and cortical bone parameters in 2-, 4-, 10-, and 16-month-old male and female wild-type (WT), AMHKO, and AMH type II receptor knockout (MRKI) mice using micro computed tomography (microCT). Goldner's staining was performed to confirm the observed bone phenotype. Both male and female AMHKO and MRKI mice showed age-dependent loss of trabecular bone (P < 0.001). However, reproductive-aged female AMHKO and MRKI mice had higher BV/TV compared with WT (P < 0.001), coinciding with increased growing follicle numbers (P < 0.05) and increased estrus inhibin B levels (AMHKO: P < 0.001; MRKI: P < 0.05) but normal inhibin A, estrogen, and progesterone levels. In aged female AMHKO and MRKI mice BV/TV did not differ from WT mice due to greater trabecular bone loss between 10 and 16 months compared with WT mice. At these ages, AMHKO and MRKI mice had reduced growing follicle numbers (P < 0.05) and reduced inhibin B levels (P < 0.001). At age 10 months, female MRKI mice had increased cortical bone parameters compared with WT mice (P < 0.01). Bone parameters of male AMHKO and MRKI mice did not differ from male WT mice. In conclusion, AMH signaling deficiency results in a sex- and age-dependent effect on predominantly trabecular bone. Our results further suggest that reproductive hormones beyond estrogen may contribute to bone homeostasis.

Sexual Dimorphism in Transcriptional and Functional Glucocorticoid Effects on Mouse Skeletal Muscle.

Muscle atrophy is common in patients with increased glucocorticoid exposure. Glucocorticoid effects are often sex-specific, and while different glucocorticoid responses between male and female subjects are reported, it is unclear why this is. In this study, we evaluated the effects of corticosterone and synthetic glucocorticoid treatment on muscle atrophy in male and female mice. We found that corticosterone treatment reduced grip strength in female mice only, whereas muscle mass was reduced in both sexes. Skeletal muscle transcriptional responses to corticosterone treatment were more pronounced and widespread in male mice. Synthetic glucocorticoid treatment reduced grip strength in both sexes, while female mice were more sensitive to muscle atrophy than male mice. To evaluate the role of androgens, chemically-castrated male mice were treated with synthetic glucocorticoids. We observed additively reduced muscle mass, but did not observe any interaction effects. Although sex differences in glucocorticoid responses in skeletal muscle are partly influenced by androgen signaling, further studies are warranted to fully delineate the underlying mechanisms.

Obesity and Hyperphagia With Increased Defective ACTH: A Novel POMC Variant.

OBJECTIVE: Patients with pro-opiomelanocortin (POMC) defects generally present with early-onset obesity, hyperphagia, hypopigmentation and adrenocorticotropin (ACTH) deficiency. Rodent models suggest that adequate cleavage of ACTH to α-melanocortin-stimulating hormone (α-MSH) and desacetyl-α-melanocortin-stimulating hormone (d-α-MSH) by prohormone convertase 2 at the KKRR region is required for regulating food intake and energy balance. METHODS: We present 2 sisters with a novel POMC gene variant, leading to an ACTH defect at the prohormone convertase 2 cleavage site, and performed functional studies of this variant. RESULTS: The patients had obesity, hyperphagia and hypocortisolism, with markerly raised levels of ACTH but unaffected pigmentation. Their ACTH has reduced potency to stimulate the melanocortin (MC) 2 receptor, explaining their hypocortisolism. CONCLUSION: The hyperphagia and obesity support evidence that adequate cleavage of ACTH to α-MSH and d-α-MSH is also required in humans for feeding control.

Comparison of 3 Different AMH Assays With AMH Levels and Follicle Count in Women With Polycystic Ovary Syndrome.

CONTEXT: Anti-Müllerian hormone (AMH) levels strongly correlate with the number of antral follicles (total follicle count, TFC) in the ovary. In women with polycystic ovary syndrome (PCOS), this is reflected by significantly increased serum AMH levels. Different assays have been developed to measure AMH. However, little is known about the interassay correlation in women with increased AMH levels. OBJECTIVE: To investigate the correlation of AMH values between different AMH assays and with TFC in PCOS patients. METHODS: AMH levels were measured in 1660 PCOS patients, using 3 different AMH assays: Gen II (Beckman Coulter); picoAMH (Ansh Labs); and Elecsys (Roche). Passing Bablok regression was used to compare assay methods. Spearman's correlation was used to correlate AMH levels and TFC. RESULTS: Strong interassay correlations were present over the total range of AMH levels (0.81-0.94). Stratification in subgroups, revealed an AMH level-dependent interassay correlation with strong interassay correlations in the low (<2.80 ng/mL) and high (>7.04 ng/mL) subgroups (0.62-0.86). However, the correlation in the mid-AMH subgroup (2.80-7.04 ng/mL) was only moderate (0.28-0.56). A strong correlation was present between the total range of AMH levels and TFC (0.57-0.62). However, in all 3 AMH subgroups the correlation became moderate at best, independently of assay method (0.11-0.45). CONCLUSION: In conclusion, both the interassay correlation and the correlation between AMH level and follicle count depend on the range of serum AMH levels. This once more emphasizes the need of a standardization of AMH measurement for an accurate interpretation of AMH in clinical practice.

Ghrelin Levels in Children With Intestinal Failure Receiving Long-Term Parenteral Nutrition.

Background: Children with intestinal failure (IF) require parenteral nutrition (PN). Transition to oral and enteral nutrition (EN) can be difficult also due to abnormal gastrointestinal motility. The gut hormone ghrelin is increased in states of negative energy balance, functioning to preserve euglycemia, and also has appetite stimulating and prokinetic properties. We aimed to evaluate and compare ghrelin levels in children with IF, and to assess the relationship with PN-dependency. Methods: In this exploratory prospective multicenter study, plasma acylated (AG) and unacylated (UAG) ghrelin levels were measured in children with short bowel syndrome (SBS) and with functional IF (pseudo-obstruction or any enteropathy) and compared with healthy control subjects. Spearman's rho (rs) was used to assess correlations of AG and UAG with PN-dependency (%PN) and parenteral glucose intake. Results: Sixty-four samples from 36 IF-patients were analyzed. Median baseline AG and UAG levels were respectively 279.2 and 101.0 pg/mL in children with SBS (n = 16), 126.4 and 84.5 pg/mL in children with functional IF (n = 20) and 82.4 and 157.3 pg/mL in healthy children (n = 39). AG levels were higher in children with SBS and functional IF than in healthy children (p = 0.002 and p = 0.023, respectively). In SBS, AG positively correlated with %PN (rs = 0.5, p = 0.005) and parenteral glucose intake (rs = 0.6, p = 0.003). These correlations were not observed in functional IF. Conclusion: Children with IF had raised AG levels which could be related to starvation of the gut. The positive correlation between AG and glucose infusion rate in SBS suggests an altered glucoregulatory function.

Association between an AMH promoter polymorphism and serum AMH levels in PCOS patients.

STUDY QUESTION: Do polymorphisms in the anti-Müllerian hormone (AMH) promoter have an effect on AMH levels in patients with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: We have identified a novel AMH promoter polymorphism rs10406324 that is associated with lower serum AMH levels and is suggested to play a role in the mechanism of regulation of AMH gene expression in women. WHAT IS KNOWN ALREADY: Follicle number is positively correlated with serum AMH levels, reflected by elevated AMH levels in women with PCOS. In addition, it is suggested that AMH production per follicle is higher in women with PCOS than in normo-ovulatory women, implying an altered regulation of AMH in PCOS. STUDY DESIGN, SIZE, DURATION: A discovery cohort of 655 PCOS women of Northern European ancestry and both an internal and external validation PCOS cohort (n = 458 and n = 321, respectively) were included in this study. Summary-level data of an AMH genome-wide association study meta-analysis including 7049 normo-ovulatory women was included as a control cohort. A genetic approach was taken through association analysis and in silico analysis of the associated variants in the AMH promoter. In vitro analysis was performed to investigate the functional mechanisms. PARTICIPANTS/MATERIALS, SETTING, METHODS: All common two-allelic single-nucleotide polymorphisms (SNPs) in the region Chr19:2 245 353-2 250 827 bp (Build 37) were selected for the analysis. Linear regression analyses were performed to determine the association between SNPs in the AMH promoter region and serum AMH levels. For the in silico analysis, the webtools 'HaploReg' v4.1 for ENCODE prediction weight matrices and 'atSNP' were used. In vitro analysis was performed using KK1 cells, a mouse granulosa cell line and COV434 cells, a human granulosa tumor cell line. Cells were transfected with the reference or the variant human AMH promoter reporter construct together with several transcription factors (TFs). Dual-Glo® Luciferase Assay was performed to measure the luciferase activity. MAIN RESULTS AND THE ROLE OF CHANCE: Polymorphism rs10406324 was significantly associated with serum AMH levels in all three PCOS cohorts. Carriers of the minor allele G had significantly lower log-transformed serum AMH levels compared to non-carriers (P = 8.58 × 10-8, P = 1.35 × 10-3 and P = 1.24 × 10-3, respectively). This result was validated in a subsequent meta-analysis (P = 3.24 × 10-12). Interestingly, rs10406324 was not associated with follicle count, nor with other clinical traits. Also, in normo-ovulatory women, the minor allele of this variant was associated with lower serum AMH levels (P = 1.04 × 10-5). These findings suggest that polymorphism rs10406324 plays a role in the regulation of AMH expression, irrespective of clinical background. In silico analysis suggested a decreased binding affinity of the TFs steroidogenenic factor 1, estrogen-related receptor alpha and glucocorticoid receptor to the minor allele G variant, however in vitro analysis did not show a difference in promoter activity between the A and G allele. LIMITATIONS, REASONS FOR CAUTION: Functional analyses were performed in a mouse and a human granulosa cell line using an AMH promoter reporter construct. This may have limited assessment of the impact of the polymorphism on higher order chromatin structures. Human granulosa cells generated from induced pluripotent stem cells, combined with gene editing, may provide a method to elucidate the exact mechanism behind the decrease in serum AMH levels in carriers of the -210 G allele. We acknowledge that the lack of follicle number in the external validation and the control cohort is a limitation of the paper. Although we observed that the association between rs10406324 and AMH levels was independent of follicle number in our discovery and internal validation PCOS cohorts, we cannot fully rule out that the observed effects on serum AMH levels are, in part, caused by differences in follicle number. WIDER IMPLICATIONS OF THE FINDINGS: These results suggest that variations in serum AMH levels are not only caused by differences in follicle number but also by genetic factors. Therefore, the genetic context should be taken into consideration when assessing serum AMH levels in women. This may have clinical consequences when serum AMH levels are used as a marker for the polycystic ovarian morphology phenotype. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used. J.S.E.L. has received consultancy fees from the following companies: Ferring, Roche Diagnostics and Ansh Labs and has received travel reimbursement from Ferring. J.A.V. has received royalties from AMH assays, paid to the institute/lab with no personal financial gain. The other authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.