Natalie D. Shaw

Obese Children Who Are Referred to the Pediatric Endocrinologist: Characteristics and Outcome

Objective. The objective of this study was to characterize a population of obese children who were referred to a pediatric endocrinology clinic and to analyze the outcome of the referral. Methods. Children who were referred for obesity and seen at least twice between January 1984 and July 2003 were included in this retrospective study. Demographic, clinical, and, when available, laboratory (fasting glucose and insulin, random cholesterol, and liver function tests) and bone age data were abstracted from the medical record at the first and last endocrine evaluations. Self-reported parental height, weight, and family history of diabetes were also entered in the database. Records from the pediatrician were used to identify the age of onset of obesity. Results. The database included 587 children (mean age at referral: 9.5 years) with a preponderance of non-Hispanic white children (75.6%) and female gender (57.6%). Growth records in 251 children indicated that 80.6% of the children had become obese before the age of 6 years and were referred 4.3 ± 2.9 years after having become obese. At follow-up, 38% of the children exhibited a decreased percentage of overweight compared with the baseline visit, but only 5 children had an age- and gender-specific BMI below the 95th percentile. Cholesterol and fasting insulin were elevated in 174 of 311 and 72 of 94 children, respectively. Bone age radiographic records for 157 patients showed significant advancement in 46%. Half of the mothers and 57.7% of fathers were obese, and type 2 diabetes was self-reported in 11.4% parents. Conclusions. In the majority of this predominantly non-Hispanic white referred sample of obese children, overweight started in the preschool years. The referral to the endocrinologist, occurring after a prolonged interval from the obesity onset, was ineffective in treating obesity. Hyperinsulinemia and hypercholesterolemia are often present also at a young age. These obesity comorbidities in association with high prevalence of parental obesity and type 2 diabetes expose these youths to high risk for developing type 2 diabetes and coronary heart disease. Our data underscore the need for early family-based behavioral-lifestyle intervention programs to be made available to pediatricians, enabling them to target the “at risk for overweight” preschool children and their likely overweight parents.

Prioritizing genetic testing in patients with Kallmann syndrome using clinical phenotypes.

CONTEXT The complexity of genetic testing in Kallmann syndrome (KS) is growing and costly. Thus, it is important to leverage the clinical evaluations of KS patients to prioritize genetic screening. OBJECTIVE The objective of the study was to determine which reproductive and nonreproductive phenotypes of KS subjects have implications for specific gene mutations. SUBJECTS Two hundred nineteen KS patients were studied: 151 with identified rare sequence variants (RSVs) in 8 genes known to cause KS (KAL1, NELF, CHD7, HS6ST1, FGF8/FGFR1, or PROK2/PROKR2) and 68 KS subjects who remain RSV negative for all 8 genes. MAIN OUTCOME MEASURES Reproductive and nonreproductive phenotypes within each genetic group were measured. RESULTS Male KS subjects with KAL1 RSVs displayed the most severe reproductive phenotype with testicular volumes (TVs) at presentation of 1.5 ± 0.1 mL vs 3.7 ± 0.3 mL, P < .05 vs all non-KAL1 probands. In both sexes, synkinesia was enriched but not unique to patients with KAL1 RSVs compared with KAL1-negative probands (43% vs 12%; P < .05). Similarly, dental agenesis and digital bone abnormalities were enriched in patients with RSVs in the FGF8/FGFR1 signaling pathway compared with all other gene groups combined (39% vs 4% and 23% vs 0%; P < .05, respectively). Hearing loss marked the probands with CHD7 RSVs (40% vs 13% in non-CHD7 probands; P < .05). Renal agenesis and cleft lip/palate did not emerge as statistically significant phenotypic predictors. CONCLUSIONS Certain clinical features in men and women are highly associated with genetic causes of KS. Synkinesia (KAL1), dental agenesis (FGF8/FGFR1), digital bony abnormalities (FGF8/FGFR1), and hearing loss (CHD7) can be useful for prioritizing genetic screening.

Expanding the Phenotype and Genotype of Female GnRH Deficiency

CONTEXT GnRH deficiency is a rare genetic disorder of absent or partial pubertal development. The clinical and genetic characteristics of GnRH-deficient women have not been well-described. OBJECTIVE To determine the phenotypic and genotypic spectrum of a large series of GnRH-deficient women. DESIGN, SETTING, AND SUBJECTS Retrospective study of 248 females with GnRH deficiency evaluated at an academic medical center between 1980 and 2010. MAIN OUTCOME MEASURES Clinical presentation, baseline endogenous GnRH secretory activity, and DNA sequence variants in 11 genes associated with GnRH deficiency. RESULTS Eighty-eight percent had undergone pubarche, 51% had spontaneous thelarche, and 10% had 1-2 menses. Women with spontaneous thelarche were more likely to demonstrate normal pubarche (P = 0.04). In 27% of women, neuroendocrine studies demonstrated evidence of some endogenous GnRH secretory activity. Thirty-six percent (a large excess relative to controls) harbored a rare sequence variant in a gene associated with GnRH deficiency (87% heterozygous and 13% biallelic), with variants in FGFR1 (15%), GNRHR (6.6%), and PROKR2 (6.6%) being most prevalent. One woman had a biallelic variant in the X-linked gene, KAL1, and nine women had heterozygous variants. CONCLUSIONS The clinical presentation of female GnRH deficiency varies from primary amenorrhea and absence of any secondary sexual characteristics to spontaneous breast development and occasional menses. In this cohort, rare sequence variants were present in all of the known genes associated with GnRH deficiency, including the novel identification of GnRH-deficient women with KAL1 variants. The pathogenic mechanism through which KAL1 variants disrupt female reproductive development requires further investigation.

Estrogen Negative Feedback on Gonadotropin Secretion: Evidence for a Direct Pituitary Effect in Women

CONTEXT Studies in humans and animals indicate that estrogen negative feedback occurs at the level of the hypothalamus, but it is unclear whether estrogen also exerts an inhibitory effect directly at the pituitary. OBJECTIVES The aim of the study was to determine whether estrogen has a direct negative feedback effect at the pituitary and whether this varies with aging. DESIGN AND SETTING A GnRH antagonist and graded doses of GnRH were used to isolate pituitary responsiveness before and after estrogen administration in Clinical Research Center studies at an academic medical center. SUBJECTS Subjects were healthy postmenopausal women aged 48-56 yr (n = 8) or 70-75 yr (n= 8). INTERVENTIONS A suppressive dose of the NAL-GLU GnRH antagonist was administered, followed by graded doses of GnRH before and after 1 month of estrogen administration. RESULTS LH and FSH responses to GnRH decreased after estrogen administration (P = 0.01 and P = 0.0001, respectively). The ratio of FSH to LH amplitudes decreased in response to estrogen (P = 0.04) indicating a greater sensitivity of FSH than LH to inhibition by estrogen. The inhibitory effect of estrogen on FSH was attenuated with aging (P = 0.02), but was maintained for LH (P = 0.4). CONCLUSIONS Studies that control for endogenous GnRH and estradiol demonstrate a direct pituitary site of estrogen negative feedback on LH and FSH responsiveness to GnRH in women. The effect of estrogen on FSH responsiveness is greater than on LH and is attenuated with aging. These studies indicate that estrogen negative feedback occurs directly at the pituitary and contributes to the differential regulation of FSH and LH secretion.

Aging Attenuates the Pituitary Response to Gonadotropin-Releasing Hormone

CONTEXT Complex changes in GnRH secretion occur with aging in women, but little is known about the effect of aging on the pituitary per se. OBJECTIVE The aim of the study was to determine whether pituitary responsiveness to GnRH is attenuated with aging. DESIGN AND SETTING A GnRH antagonist and graded doses of GnRH were used to isolate pituitary responsiveness in Clinical Research Center studies at an academic medical center. SUBJECTS Subjects were healthy postmenopausal women (PMW) aged 48-57 yr (n = 10) or 70-77 yr (n= 9). INTERVENTIONS A suppressive dose of the NAL-GLU GnRH antagonist (150 microg/kg sc) was administered and was followed by GnRH doses of 25, 75, 250, or 750 ng/kg iv every 4 h. RESULTS The LH response to GnRH was attenuated with aging (P = 0.05) with an interaction between age and dose (P = 0.01) such that the LH amplitude was less in older PMW at the higher doses (250 ng/kg, 50 +/- 9 vs. 29 +/- 4.9 IU/liter, for young and old PMW, respectively, P = 0.02; and 750 ng/kg, 97.7 +/- 11 vs. 70.2 +/- 9.3 IU/liter, P = 0.002), but not the lower doses of GnRH. The FSH response to GnRH was also attenuated with aging in PMW (P = 0.005). CONCLUSIONS In studies that isolated the pituitary from endogenous GnRH stimulation, aging attenuated the LH and FSH responses to exogenous GnRH in PMW. These studies indicate that the pituitary plays a role in the decline in gonadotropin levels with aging, further supporting the potential contribution of age-associated changes in both hypothalamic and pituitary function to reproductive senescence.

SMCHD1 mutations associated with a rare muscular dystrophy can also cause isolated arhinia and Bosma arhinia microphthalmia syndrome

Arhinia, or absence of the nose, is a rare malformation of unknown etiology that is often accompanied by ocular and reproductive defects. Sequencing of 40 people with arhinia revealed that 84% of probands harbor a missense mutation localized to a constrained region of SMCHD1 encompassing the ATPase domain. SMCHD1 mutations cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) via a trans-acting loss-of-function epigenetic mechanism. We discovered shared mutations and comparable DNA hypomethylation patterning between these distinct disorders. CRISPR/Cas9-mediated alteration of smchd1 in zebrafish yielded arhinia-relevant phenotypes. Transcriptome and protein analyses in arhinia probands and controls showed no differences in SMCHD1 mRNA or protein abundance but revealed regulatory changes in genes and pathways associated with craniofacial patterning. Mutations in SMCHD1 thus contribute to distinct phenotypic spectra, from craniofacial malformation and reproductive disorders to muscular dystrophy, which we speculate to be consistent with oligogenic mechanisms resulting in pleiotropic outcomes.

Insights into Puberty: The Relationship between Sleep Stages and Pulsatile LH Secretion

CONTEXT During the pubertal transition, LH secretion initially increases only during sleep; however, its relationship to sleep stage is unknown. OBJECTIVES Our objective was to determine whether the initiation of LH pulses is related to a specific sleep stage in pubertal children. DESIGN AND SETTING Frequent blood sampling and polysomnographic studies were performed in a Clinical Research Center. SUBJECTS Fourteen studies were performed in nine healthy pubertal children, ages 9.9-15.6 yr. INTERVENTIONS Subjects underwent one to two overnight studies with polysomnography and blood sampling for LH at 10-min intervals. RESULTS Alignment of polysomnographic records and LH pulses demonstrated that LH pulses (n = 58) occurred most frequently during slow-wave sleep (SWS) (1.1 pulse/h, n = 30) compared with all other sleep stages or periods of wake after sleep onset (P < 0.001). There was also a significant increase in the amount of SWS in the 15 min preceding and the 5 min following each pulse compared with the amount of SWS seen across the study night (P < 0.01). CONCLUSIONS During puberty, the majority of LH pulses that occur after sleep onset are preceded by SWS, suggesting that SWS is intimately involved in the complex control of pubertal onset. These studies raise concerns about the potential hormonal repercussions of the increasing prevalence of sleep disturbances in adolescents.

Responsiveness to a Physiological Regimen of GnRH Therapy and Relation to Genotype in Women With Isolated Hypogonadotropic Hypogonadism

CONTEXT Isolated hypogonadotropic hypogonadism (IHH) is caused by defective GnRH secretion or action resulting in absent or incomplete pubertal development and infertility. Most women with IHH ovulate with physiological GnRH replacement, implicating GnRH deficiency as the etiology. However, a subset does not respond normally, suggesting the presence of defects at the pituitary or ovary. OBJECTIVES The objective of the study was to unmask pituitary or ovarian defects in IHH women using a physiological regimen of GnRH replacement, relating these responses to genes known to cause IHH. DESIGN, SETTING, AND SUBJECTS This study is a retrospective analysis of 37 IHH women treated with iv pulsatile GnRH (75 ng/kg per bolus). MAIN OUTCOME MEASURES Serum gonadotropin and sex steroid levels were measured, and 14 genes implicated in IHH were sequenced. RESULTS During their first cycle of GnRH replacement, normal cycles were recreated in 60% (22 of 37) of IHH women. Thirty percent of women (12 of 37) demonstrated an attenuated gonadotropin response, indicating pituitary resistance, and 10% (3 of 37) exhibited an exaggerated FSH response, consistent with ovarian resistance. Mutations in CHD7, FGFR1, KAL1, TAC3, and TACR3 were documented in IHH women with normal cycles, whereas mutations were identified in GNRHR, PROKR2, and FGFR1 in those with pituitary resistance. Women with ovarian resistance were mutation negative. CONCLUSIONS Although physiological replacement with GnRH recreates normal menstrual cycle dynamics in most IHH women, hypogonadotropic responses in the first week of treatment identify a subset of women with pituitary dysfunction, only some of whom have mutations in GNRHR. IHH women with hypergonadotropic responses to GnRH replacement, consistent with an additional ovarian defect, did not have mutations in genes known to cause IHH, similar to our findings in a subset of IHH men with evidence of an additional testicular defect.

Evidence That Increased Ovarian Aromatase Activity and Expression Account for Higher Estradiol Levels in African American Compared With Caucasian Women

CONTEXT Serum estradiol levels are significantly higher across the menstrual cycle in African American (AAW) compared with Caucasian women (CW) in the presence of similar FSH levels, yet the mechanism underlying this disparity is unknown. OBJECTIVE The objective of the study was to determine whether higher estradiol levels in AAW are due to increased granulosa cell aromatase mRNA expression and activity. DESIGN The design of the study included daily blood sampling and dominant follicle aspirations at an academic medical center during a natural menstrual cycle. SUBJECTS Healthy, normal cycling AAW (n = 15) and CW (n = 14) aged 19-34 years participated in the study. MAIN OUTCOME MEASURES Hormone levels in peripheral blood and follicular fluid (FF) aspirates and aromatase and FSH receptor mRNA expression in granulosa cells were measured. RESULTS AAW had higher FF estradiol [1713.0 (1144.5-2032.5) vs 994.5 (647.3-1426.5) ng/mL; median (interquartile range); P < .001] and estrone [76.9 (36.6-173.4) vs 28.8 (22.5-42.1) ng/mL; P < .001] levels than CW, independent of follicle size. AAW also had lower FF androstenedione to estrone (7 ± 1.8 vs 15.8 ± 4.1; mean ± SE; P = .04) and T to estradiol (0.01 ± 0.002 vs 0.02 ± 0.005; P = .03) ratios, indicating enhanced ovarian aromatase activity. There was a 5-fold increase in granulosa cell aromatase mRNA expression in AAW compared with CW (P < .001) with no difference in expression of FSH receptor. FSH, inhibin A, inhibin B, and AMH levels were not different in AAW and CW. CONCLUSIONS Increased ovarian aromatase mRNA expression, higher FF estradiol levels, and decreased FF androgen to estrogen ratios in AAW compared with CW provide compelling evidence that racial differences in ovarian aromatase activity contribute to higher levels of estradiol in AAW across the menstrual cycle. The absence of differences in FSH, FSH receptor expression, and AMH suggest that population-specific genetic variation in CYP19, the gene encoding aromatase, or in factors affecting its expression should be sought.

Compensatory Increase in Ovarian Aromatase in Older Regularly Cycling Women

CONTEXT Serum estradiol (E2) levels are preserved in older reproductive-aged women with regular menstrual cycles despite declining ovarian function. OBJECTIVE The objective of the study was to determine whether increased granulosa cell aromatase expression and activity account for preservation of E2 levels in older, regularly cycling women. DESIGN The protocol included daily blood sampling and dominant follicle aspirations at an academic medical center during a natural menstrual cycle. SUBJECTS Healthy, regularly cycling older (36-45 y; n = 13) and younger (22-34 y; n = 14) women participated in the study. MAIN OUTCOME MEASURES Hormone levels were measured in peripheral blood and follicular fluid aspirates and granulosa cell CYP19A1 (aromatase) and FSH-R mRNA expression were determined. RESULTS Older women had higher FSH levels than younger women during the early follicular phase with similar E2 but lower inhibin B and antimullerian hormone levels. Late follicular phase serum E2 did not differ between the two groups. Follicular fluid E2 [older (O) = 960.0 [interquartile range (IQR) 765.0-1419.0]; younger (Y) = 994.5 [647.3-1426.5] ng/mL, P = 1.0], estrone (O = 39.6 [29.5-54.1]; Y = 28.8 [22.5-42.1] ng/mL, P = 0.3), and the E2 to testosterone (T) ratio (O = 109.0 ± 41.9; Y = 83.0 ± 18.6, P = .50) were preserved in older women. Granulosa cell CYP19A1 expression was increased 3-fold in older compared with younger women (P < .001), with no difference in FSH-R expression. CONCLUSIONS Ovarian aromatase expression increases with age in regularly cycling women. Thus, up-regulation of aromatase activity appears to compensate for the known age-related decrease in granulosa cell number in the dominant follicle to maintain ovarian estrogen production in older premenopausal women.