Next-generation sequencing refines the genetic architecture of Greek GnRH-deficient patients.

Isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD) is a rare disease with a wide spectrum of reproductive and non-reproductive clinical characteristics. Apart from the phenotypic heterogeneity, IGD is also highly genetically heterogeneous with >35 genes implicated in the disease. Despite this genetic heterogeneity, genetic enrichment in specific subpopulations has been described. We have previously described low prevalence of genetic variation in the Greek IGD cohort discovered with utilization of Sanger sequencing in 14 known IGD genes. Here, we describe the expansion of genetic screening in the largest IGD Greek cohort that has ever been studied with the usage of whole-exome sequencing, searching for rare sequencing variants (RSVs) in 37 known IGD genes. Even though Sanger sequencing detected genetic variation in 21/81 IGD patients in 7/14 IGD genes without any evidence of oligogenicity, whole exome sequencing (WES) revealed that 27/87 IGD patients carried a rare genetic change in a total of 15 genes with 4 IGD cases being oligogenic. Our findings suggest that next-generation sequencing (NGS) techniques can discover previously undetected variation, making them the standardized method for screening patients with rare and/or more common disorders.

Gene variants associated with age at menopause are also associated with polycystic ovary syndrome, gonadotrophins and ovarian volume.

STUDY QUESTION: Is there a relationship between the genetic risk for polycystic ovary syndrome (PCOS) and genetic variants that influence timing of menopause? SUMMARY ANSWER: The genetic risk score, which sums the contribution of variants at all menopause loci, was associated with PCOS. WHAT IS ALREADY KNOWN: Ovarian parameters and anti-Mullerian hormone levels suggest that women with PCOS should have a later age at menopause. STUDY DESIGN, SIZE, DURATION: The study was a case-control examination of genetic variants associated with age at menopause in a discovery cohort of women with PCOS (n = 485) and controls (n = 407) from Boston recruited from 2003 to 2012. Replication was performed in women from Greece (cases, n = 884 and controls, n = 311). PARTICIPANTS/MATERIALS, SETTINGS, METHODS: PCOS was defined by the National Institutes of Health criteria in Boston and Greece (n = 783), with additional subjects fulfilling the Rotterdam criteria (hyperandrogenism, polycystic ovary morphology and regular menses) in Greece (n = 101). Controls in Boston and Greece had regular menstrual cycles and no hyperandrogenism. Allele frequencies for variants previously associated with age at menopause were examined in PCOS cases and controls, along with the relationship to quantitative traits. MAIN RESULTS AND ROLE OF CHANCE: The variant rs11668344-G was associated with decreased risk of PCOS (odds ratio: 0.77 [0.59-0.93]; P = 0.004). There was a strong relationship between the late menopause allele rs12294104-T and increased LH levels (ß ± SE; 0.26 ± 0.06; P = 5.2 × 10(-5)) and the LH:FSH ratio (0.28 ± 0.06; P = 2.7 × 10(-6)). The minor allele at rs10852344-T was associated with smaller ovarian volume (-0.16 ± 0.05; P = 0.0012). A genetic risk score calculated from 16 independent variants associated with age at menopause was also associated with PCOS (P < 0.02), LH and the LH:FSH ratio (both P < 0.05). LIMITATIONS, REASONS FOR CAUTION: The variant rs11668344 was not associated with PCOS in the Greek cohort, but results exhibited the same direction of effect as the Boston cohort. However, it is possible that the individual association was a false positive in the Boston cohort. WIDER IMPLICATIONS OF THE FINDINGS: The study demonstrates that gene variants known to influence age at menopause are also associated with risk for PCOS. Further, our data suggest that the relationship between age at menopause and PCOS may be explained, at least in part, by effects on LH levels and follicle number. The data point to opposing influences of the genetic variants on both menopausal age and PCOS. STUDY FUNDING/COMPETING INTERESTS: The project was supported by award number R01HD065029 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development, award number 1 UL1 RR025758, Harvard Clinical and Translational Science Center, from the National Center for Research Resources and award 1-10-CT-57 from the American Diabetes Association. C.K.W. is a consultant for Takeda Pharmaceuticals. TRIAL REGISTRATION NUMBER: NCT00166569.

Han Chinese polycystic ovary syndrome risk variants in women of European ancestry: relationship to FSH levels and glucose tolerance.

STUDY QUESTION: Are PCOS risk variants identified in women of Han Chinese ethnicity also associated with risk of PCOS or the phenotypic features of PCOS in European women? SUMMARY ANSWER: One variant, rs2268361-T, in the intron of FSHR was associated with PCOS and lower FSH levels, while another variant rs705702-G near the RAB5B and SUOX genes was associated with insulin and glucose levels after oral glucose testing in women with PCOS of European ethnicity. WHAT IS KNOWN ALREADY: Three of the eleven variants associated with PCOS in the Han Chinese genome-wide association studies were also associated with PCOS in at least one European population when corrected for multiple testing (DENND1A, THADA and YAP1). However, additional replication is needed to establish the importance of these variants in European women and to determine the relationship to PCOS phenotypic traits. STUDY DESIGN, SIZE, DURATION: The study was a case-control examination in a discovery cohort of women with PCOS (n = 485) and controls (n = 407) from Boston (Boston 1). Replication was performed in women from Greece (cases n = 884 and controls n = 311) and an additional cohort from Boston (Boston electronic medical record (EMR); n = 350 cases and n = 1258 controls). PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Women had PCOS defined by the National Institutes of Health criteria in Boston 1 and Greece (n = 783), with additional subjects fulfilling the Rotterdam criteria (hyperandrogenism, polycystic ovary morphology and regular menses) in Greece (n = 101). Controls in Boston and Greece had regular menstrual cycles and no hyperandrogenism. The second cohort from Boston was defined using the EMR and natural language processing. Allele frequencies for variants associated with PCOS in Han Chinese women were examined in PCOS cases and controls, along with the relationship to quantitative traits. MAIN RESULTS AND THE ROLE OF CHANCE: A variant rs2268361-T in an intron of FSHR was associated with PCOS (0.84 [0.76-0.93], OR [95% CI]; P = 0.002). The rs2268361-T was associated with lower FSH levels (-0.15 ± 0.05; P = 0.0029). A variant rs705702-G near RAB5B and SUOX was associated with insulin (-0.16 ± 0.05, P = 0.0029) and glucose levels (-0.20 ± 0.05, P = 0.0002) 120 min after an oral glucose test. LIMITATIONS, REASONS FOR CAUTION: The study was large and contained replication cohorts, but was limited by a small number of controls in the Greek cohort and a small number of cases in the second Boston cohort. The second Boston group was identified using electronic medical record review, but was validated for the cardinal features of PCOS. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrates a cross-ethnic PCOS risk locus in FSHR in women of European ancestry with PCOS. The variant may influence FSH receptor responsiveness as suggested by the associated change in FSH levels. The relationship between a variant near RAB5B and SUOX and glucose stimulated insulin and glucose levels suggests an influence of one of these genes on glucose tolerance, but the absence of a relationship with PCOS points to potential differences in the international PCOS patient populations. STUDY FUNDING/COMPETING INTERESTS: The project was supported by Award Number R01HD065029 from the Eunice Kennedy Shriver National Institute Of Child Health & Human Development, Award Number 1 UL1 RR025758, Harvard Clinical and Translational Science Center, from the National Center for Research Resources, award 1-10-CT-57 from the American Diabetes Association and the Partners Healthcare Center for Personalized Genetics Project Grant. C.K.W. is a consultant for Takeda Pharmaceuticals. TRIAL REGISTRATION NUMBER: NCT00166569.

The influence of intensive physical training on salivary adipokine levels in Elite Rhythmic Gymnasts.

Exercise challenges homeostasis and establishes a new dynamic equilibrium. Elite Rhythmic Gymnasts (RG's) begin exercise at an early age, undergo physical and psychological stress, and adopt negative energy balance to retain a lean physique. The aim of the present study was to evaluate the effect of negative energy balance, acute and chronic exercise on salivary adiponectin, resistin and visfatin levels and their interaction with salivary cortisol, and insulin levels in elite RG's. This study is unique in character, as all variables were assessed on the field of competition. The study included 51 elite RG's participating in "Kalamata 2010 World Cup" in Kalamata, Greece on April 2010. Twenty-seven healthy age-matched girls were used as controls. Anthropometric values were assessed; baseline and post exercise salivary cortisol, insulin, adiponectin, resistin, and visfatin levels were measured. Comparisons regarding hormonal features between RG's and controls were adjusted for BMI and body fat percentage. Salivary adiponectin levels were higher (p<0.05) and visfatin lower (p=0.094) in RG's compared with controls, while no significant changes were observed regarding salivary cortisol, insulin, and resistin levels. In elite RG's acute intensive anaerobic exercise led to increased salivary insulin levels (p<0.001), reduced salivary adiponectin (p<0.001) and visfatin levels (p<0.05), and no changes in salivary resistin levels. Moreover, diurnal variation of salivary cortisol was lost. In elite RG's salivary adiponectin is upregulated and salivary visfatin is downregulated after chronic intensive exercise and negative energy balance, while both salivary adiponectin and visfatin levels are suppressed after short term intensive anaerobic exercise.

The influence of obesity on androstenedione to testosterone ratio in women with polycystic ovary syndrome (PCOS) and hyperandrogenemia.

The aim of the present study was to evaluate the impact of obesity and insulin resistance on testosterone formation from androstenedione and its contribution to biochemical hyperandrogenemia in all different phenotypic subgroups of PCOS patients. The case-control study included 1087 PCOS women and 206 regularly menstruating, ovulatory controls. The main clinical measurements included anthropometric and basal hormonal characteristics and evaluation of hyperandrogenic and insulin resistance-related features. The results were the following: In PCOS women with biochemical hyperandrogenemia, obesity significantly lowers serum A levels and increases T to A ratio. These findings were not present in PCOS women with clinical hypeandrogenemia and in normal ovulatory controls.

Diabetes insipidus and two consecutive pregnancies: a case report and review of the literature.

We report a case of a woman with a preexisting diabetes insipidus (DI), who had two consecutive uncomplicated pregnancies. Both pregnancies resulted after spontaneous conception and had a similar uneventful course. At the time of conception the patient was receiving 1-desamino-8D-arginine-vasopressin (DDAVP) 30 microg/d which maintained a urinary volume of 2-3 l/day. Pre-existing DI can be handled carefully and result in an uncomplicated pregnancy. In such cases careful monitoring of the patient's fluid balance and liver enzymes, as well as monitoring for pre-eclampsia and oligohydramnios during pregnancy are essential.

Adiponectin levels in women with polycystic ovary syndrome: a systematic review and a meta-analysis.

BACKGROUND: Conflicting results regarding adiponectin levels in women with polycystic ovary syndrome (PCOS) have been reported. To evaluate adiponectin levels in PCOS, a systematic review of all studies comparing adiponectin levels in women with PCOS with healthy controls and a meta-analysis of those involving women with similar body mass index (BMI) were performed. The influence of possible effect modifiers, such as insulin resistance (IR) and testosterone, was investigated. The influence of obesity was investigated through a 'nested' meta-analysis after within-study BMI stratification and appropriate pooling. METHODS: Literature search was conducted through MEDLINE, EMBASE, Cochrane CENTRAL (through June 2008), references from relevant studies and personal contact with the authors. Thirty-one studies, reporting data on 3469 subjects, were reviewed and 16 included in the main meta-analysis. RESULTS: Women with PCOS demonstrated significantly lower adiponectin values [weighted mean difference (95% confidence interval) -1.71 (-2.82 to -0.6), P < 10(-4)], yet with significant between-study heterogeneity. Lower adiponectin levels are associated with the IR observed in women with PCOS, compared with controls. IR, but not total testosterone, was found significant among biological parameters explored in the meta-regression model. Hypoadiponectinaemia was present in both lean and obese women with PCOS when compared with non-PCOS counterparts. Data on high molecular weight (HMW) adiponectin are limited (three studies). CONCLUSIONS: After controlling for BMI-related effects, adiponectin levels seem to be lower in women with PCOS compared with non-PCOS controls. Low levels of adiponectin in PCOS are probably related to IR but not to testosterone. Total adiponectin should not be used as a biomarker of PCOS severity. Further investigation is needed for HMW adiponectin levels in PCOS.

Polycystic ovarian syndrome, insulin resistance and thickness of the endometrium.

INTRODUCTION: Disorders possibly associated with insulin resistance (IR) are hyperandrogenemia, hirsutism, irregular menstrual cycles, central obesity and polycystic ovarian syndrome (PCOS). It is known that PCOS is related to a high risk of endometrial hyperplasia after many years of estrogen stimulation from anovulation. OBJECTIVES: The purpose of the study was to estimate the thickness of the endometrium in women with IR without a diagnosis of PCOS and in women with PCOS without IR. STUDY DESIGN: Three groups of women included in the study: 15 women diagnosed as IR without PCOS, 16 women diagnosed as PCOS without IR and 20 women used as controls matched for age and body mass index (BMI) with the previous patients. Thickness of the endometrium was estimated in all cases with a transvaginal ultrasound in three consecutive measures during a cycle. RESULTS: The mean thickness of the endometrium was statistically higher in the PCOS group (11.1mm), and in the IR group (9.6mm), compared with the control group (6.2mm) (F=13.1, p<0.001). CONCLUSIONS: It is concluded that both in women diagnosed as having insulin resistance without PCOS, and in women with PCOS without insulin resistance, the ultrasonographically estimated thickness of the endometrium is relatively high and a closer follow-up of these women is required in order to detect those in risk to develop hyperplasia and/or atypia.

Spinocerebellar ataxia and hypergonadotropic hypogonadism associated with familial sensorineural hearing loss.

Cerebellar ataxia has been described as being associated with hypogonadism for almost 100 years. In the majority of cases, hypogonadism is hypogonadotropic. The association of cerebellar ataxia with hypergonadotropic hypogonadism is a rare genetic disorder with a recessive mode of inheritance. Cerebellar ataxia and hypogonadism can also occur associated with a large spectrum of additional clinical manfestations, including mental retardation, sensorineural deafness, choroidal dystrophy, ectodermal dysplasia and short stature, and polyneuropathy. We report the case of a woman with early-onset spinocerebellar ataxia, primary amenorrhea due to hypergonadotropic hypogonadism, and late-onset sensorineural hearing loss. Additional family members from the father's side are affected with late-onset hearing loss, suggesting a dominant mode of inheritance.

Improvement of iodine deficiency after iodine supplementation in schoolchildren of Azerbaijan was accompanied by hypo and hyperthyrotropinemia and increased title of thyroid autoantibodies.

OBJECTIVE: In the mountainous areas of Azerbaijan the schoolchildren suffer from severe Iodine Deficiency (ID) with median Urinary iodine excretion (UIE) 36 mcg/l and prevalence of goiter 99% (estimated by US). In a population of 293,000 schoolchildren aged 8-14 y.o. we administered capsules containing 190 mg of iodized oil (Lipiodol-Guerbet, Cedex, France) twice yearly in 6 months apart (total 380 mg). The aim of the present study was to evaluate the efficacy, the benefits, as well as the possible side-effects in a follow-up period of 6 and 12 months after the initial administration of iodized oil. METHODS: Six and 12 months after the initial administration of iodide, two representative samples of 391 and 326 children respectively were examined. The evaluation included: estimation of goiter by US, determination of UIE and serum measurements of T3, T4, TSH, Tg, autoantibodies against thyroid peroxidase (anti-TPO) and thyroglobulin (anti-Tg). RESULTS: There was an improvement in median UIE which increased from 36 mcg/l to 68 and 81 mcg/l after 6 and 12 months of treatment respectively. The prevalence of goiter decreased from 99% to 54% and 26% respectively. Tg was decreased at 6 and 12 months from the first administration, whereas TSH remained unchanged at 6 months and decreased at 12 months when compared to the latter value. Hypothyroidism was detected in 7% of children after iodide administration both at 6 and 12 months, but overt hypothyroidism was observed only in 0.5% at 12 months. Subclinical hyperthyroidism was detected in 2% and 6% after iodide administration both at 6 and 12 months. There was a significant increase in the title of thyroid auto antibodies in 6 months which was retained and increased in 12 months. There was no relation between the appearance of thyroid dysfunction and the positive thyroid auto antibodies. CONCLUSION: The dose of 190 mg iodide administered twice yearly, improved iodine deficiency and endemic goiter in schoolchildren. The increase of UIE resulted from iodide administration, was accompanied by an increased title of thyroid auto-antibodies and an increased prevalence of hyper- and hypothyrotropinemia apparently of no autoimmune etiology.

Height velocity and skeletal maturation in elite female rhythmic gymnasts.

Rhythmic gymnasts performing under conditions of high intensity are exposed to particularly high levels of psychological stress and intense physical training, factors that can contribute to the observed delay in skeletal maturation and pubertal development, and alter optimal growth. The study was conducted in the field, during the International, European, and World Rhythmic Sports Gymnastics Championships of the years 1997-2000, and included 104 elite female rhythmic gymnasts, aged 12-23 yr. The study included height and weight measurements, estimation of body fat and skeletal maturation, and registration of parental height. Height, weight, target height, and predicted adult height were expressed as the SD score of the mean height and weight for age, according to Tanner's standards. Gymnasts were taller and thinner than average for age, with height velocity SD score for each age group above the 50th percentile for all age groups (n = 140, mean = 1.9 +/- 2.5). Interestingly, although height velocity in normal girls comes to an end by the age of 15, in our examined rhythmic gymnasts it continues up to the age of 18. There was a delay of skeletal maturation of 1.8 yr (n = 72, r = 0.730, P < 0.001), compensated by an acceleration of height velocity toward the end of puberty. The final adult height was identical to the estimated predicted height at first evaluation, and significantly higher than the genetically determined target height (n = 35, r = 0.58, P < 0.001), denoting that genetic predisposition to final height is not only achieved, but even exceeded. Using multiple regression analysis, target height was the only independent parameter that has been proven to influence positively the height velocity SD score (b = 0.233, t = 2.215, P = 0.029), denoting that genetic predisposition remains the main driving force for the observed efficient catch up growth. In conclusion, the elite rhythmic gymnasts compensate for their loss of pubertal growth spurt by a late acceleration of linear growth. Despite the delay in skeletal maturation, genetic predisposition of growth is not only preserved, but even exceeded.

Ovulation induction with pulsatile gonadotropin-releasing hormone (GnRH) or gonadotropins in a case of hypothalamic amenorrhea and diabetes insipidus.

Hypothalamic amenorrhea is a treatable cause of infertility. Our patient was presented with secondary amenorrhea and diabetes insipidus. Cortisol and prolactin responded normally to a combined insulin tolerance test (ITT) and thyrotropin-releasing hormone (TRH) challenge, while thyroid-stimulating hormone (TSH) response to TRH was diminished, and no response of growth hormone to ITT was detected. Both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels increased following gonadotropin-releasing hormone (GnRH) challenge. No response of LH to clomiphene citrate challenge was detected. Magnetic resonance imaging findings demonstrated a midline mass occupying the inferior hypothalamus, with posterior lobe not visible and thickened pituitary stalk. Ovulation induction was carried out first with combined human menopausal gonadotropins (hMG/LH/FSH) (150 IU/day) and afterwards with pulsatile GnRH (150 ng/kg/pulse). Ovulation was achieved with both pulsatile GnRH and combine gonadotropin therapy. Slightly better results were achieved with the pulsatile GnRH treatment.

Iodine deficiency in Azerbaijan after the discontinuation of an iodine prophylaxis program: reassessment of iodine intake and goiter prevalence in schoolchildren.

The goal of this study was to assess the prevalence of iodine deficiency (ID) in Azerbaijan after the discontinuation of an iodine prophylaxis program by assessing the prevalence of goiter, iodine intake, and thyroid function. The study included 942 schoolchildren (475 boys and 467 girls) ages 8-14 years, from 13 distinct regions. The survey included the following: (1) clinical evaluation; (2) assessment of thyroid volume both by ultrasound and by palpation; (3) determination of iodide in a morning urine specimen using the classic Sandel-Kolthoff reaction in 347 schoolchildren; (4) determinations of thyrotropin (TSH), triiodothyronine (T3), thyroxine (T4), thyroglobulin (Tg), and anti-thyroid peroxidase (TPO) in serum (n = 165) and TSH in whole blood spotted on filter paper (n = 942). The prevalence of goiter for the whole country was determined by ultrasound (US) to be 86% and by palpation 66%, reaching 100% in the mountainous regions of Caucasus. The median urinary iodine excretion (UIE) was 54 microg/L, reaching level of 26 and 39 microg/L in the Caucasus region. In conclusion, according to the World Health Organization (WHO) classification, Azerbaijan now has mild to moderate ID (median UIE, 54 microg/L) and in the mountainous regions with severe ID. The high prevalence of goiter and the low UIE emphasizes the need for urgent medical reintervention. An iodination program is now implemented by our team in the mountainous regions under the auspice of the government of Azerbaijan.

Genetic heterogeneity evidenced by low incidence of KAL-1 gene mutations in sporadic cases of gonadotropin-releasing hormone deficiency.

Isolated GnRH deficiency is a heritable condition characterized by a functional deficit in GnRH secretion. Familial cases with different modes of inheritance have been described, and the gene responsible for the X-linked form (KAL-1) has been identified. However, sporadic cases with no documented family history of GnRH deficiency account for the majority of the affected patients. For this reason, we sought to determine the frequency with which KAL-1 gene mutations occur in patients with sporadic GnRH deficiency. Only 1 of 21 patients with sporadic GnRH deficiency was found to bear a defect in the KAL-1 gene (a deletion of 14 bases starting at codon 464). Three types of polymorphic single base substitutions with no apparent correlation with GnRH deficiency were also detected in several patients. In each of 3 different patients with an X-linked mode of inheritance, 3 genetic defects, 2 point mutations and a small intragenic deletion, were detected. These defects consist of a single base mutation introducing a stop codon at position 328, a single base mutation resulting in a phenylalanine to leucine substitution at position 517, and a 9-base deletion at the 3'-exon-intron splice site of exon 8, respectively. All identified genetic defects occur within the fibronectin type III repeats of the predicted protein encoded by the KAL-1 gene. In conclusion, our study indicates that the incidence of genetic defects within the coding region of the KAL-1 gene in patients with sporadic GnRH deficiency is low (5-8%), thus supporting the idea that the X-linked form of inheritance represents the least common form of the disease.