Underwent Stimulation Testing Research Articles

OR18-1 The Impact of Makorin Ring Finger Protein 3 (MKRN3) Overexpression in the Arcuate Nucleus of Postpubertal Female Mice on the HPG Axis

Abstract Background and Objective Makorin Ring Finger Protein 3 (MKRN3), an inhibitor of the KISS1 and TAC3 promoters in the hypothalamic arcuate nucleus (ARC), is an important player in age of menarche and etiology of central precocious puberty. However, its potential role in delayed puberty and/or hypogonadism is not defined. Mkrn3 expression in the ARC declines prior to the onset of puberty and remains low in adulthood, supporting its role as an inhibitor of pubertal onset. Neonatal hypothalamic overexpression of Mkrn3 leads to delayed puberty in female mice; this study aimed to explore the effects of MKRN3 overexpression in the postpubertal mouse. We hypothesized that MKRN3 overexpression in the ARC of postpubertal female mice would result in suppression of the HPG axis, manifested as hypogonadotropic hypogonadism. Methods and Results Adult female C57BL/6 mice were stereotaxically injected into the ARC with a recombinant adeno-associated virus (rAAV-Mkrn3-IRES-EGFP;n=12) to overexpress Mkrn3, or with a control virus (rAAV-EGFP;n=9). Daily vaginal smears collected six weeks post-injection showed that rAAV-Mkrn3 injected mice spent significantly more time (62.7%) in diestrus compared to controls (41.3%;P<0.01). Mean body weight did not statistically differ between groups. Eight weeks after viral injection, gonad-intact females underwent stimulation tests while in diestrus. Tail vein blood samples were collected at baseline and 20, 40 and 60 minutes after stimulation. In response to the NK3R agonist, senktide, evaluated at two doses (5 nmol and 10 nmol ip, respectively), Mkrn3-overexpressing mice had a reduced peak (i.e., T20) LH response compared to controls (P<0.001 for both dosing paradigms). In contrast, in response to kisspeptin-10 (1 nmol ip), mice responded with similar peak serum LH levels. Subsequently, tests were repeated seven days after ovariectomy and estradiol capsule replacement (OVX+E2). Peak LH levels (T20) were again significantly lower in response to senktide (5 nmol ip) in the rAAV-Mkrn3 injected females (P<0.001). Peak serum LH levels (T20) were not different in response to kisspeptin-10 (1 nmol) or GnRH (1.25 ng/g body weight). Additionally, there were no significant differences in serum LH levels between groups 7 days after OVX or OVX + E2. Conclusions In this novel mouse model, developed to investigate the mechanism of action of MKRN3, an important neuroendocrine controller of puberty initiation, these findings suggest that MKRN3 can exert inhibitory effects on the reproductive axis in female mice beyond the juvenile period. In these mice, response to kisspeptin and GnRH was preserved but the response to senktide was blunted. These findings suggest that Mkrn3 may act in ARC kisspeptin neurons to inhibit kisspeptin synthesis or secretion and/or potentially the action of neurokinin B via its receptor. Modulation of MKRN3 expression may be a future therapeutic target for reproductive disorders. Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.

Precocious puberty in Korean girls with and without exposure to endocrine-disrupting chemicals in toy slime: a comparative analysis

BackgroundToy slime is popular in Korea, and in parallel, pre-pubertal girls visit hospitals for early pubertal signs. Thus far, numerous studies have investigated the association of endocrine-disrupting chemicals (EDCs) with precocious puberty (PP). However, there is a lack of studies on the clinical manifestations and sex hormones. We aimed to investigate early pubertal development in Korean girls with or without a history of toy slime exposure and determine changes in bone age, Tanner stage, and sex hormones.MethodsIn this study, 140 girls underwent stimulation tests at Kyungpook National University Children’s Hospital Endocrinology Department, during January 2018 and December 2020. Patients were divided into two groups for gonadotropin-releasing hormone (GnRH) stimulation test and frequency of exposure to toy slime (EDCs). GnRH stimulation test was conducted after an intravenous injection of 100 µg of luteinizing hormone-releasing hormone. Slime exposure was defined as Slime ≥ 3 times/week for ≥ 3 months.ResultsHistory of slime exposure was found in 14 of 58 and 65 of 82 patients in the central PP (CPP) and non-CPP groups, respectively. Slime-exposed patients had advanced bone age, although their Tanner stage was low. Patients with a history of toy slime exposure were 5.5 times more likely to be diagnosed with non-CPP than patients without slime exposure (p < 0.05).ConclusionsExposure to toy slime in prepubertal girls may be associated with rapid clinical advancement of pubertal development and bone age, and the patients appear more likely to be diagnosed with non-CPP.

SAT-197 Unilateral Adrenalectomy Can Induce Control in ARMC5 Mutations Patients

Primary macronodular hyperplasia (PMAH) is a rare cause of endogenous Cushing’s syndrome characterized by functioning adrenal macronodules and variable cortisol secretion. ARMC5 is the most frequent gene responsible for PMAH. Genetic mutations including inactivating germline mutations in armadillo repeat-containing 5 (ARMC5) gene have been identified. The occurrence of several other non adrenal tumors (meningioma, breast, colon, thyroid and parathyroid) has also been associated with PBMAH, suggesting a possible role of ARMC5 for the development of other neoplasias. The best treatment to this condition is not established. Clinical case: 64-year-old man, referred due to bilateral adrenal incidentalomas on a CT scan with characteristics of adenoma/hyperplasia. He had type-2 diabetes, hypertension and dyslipidemia for about 4 years. He was being treated with 4 different anti-hypertensive medications, gliclazide and simvastatin. Physical examination revealed thin and dry skin, obesity with centripetal fat distribution, multiple ecchymosis and facial erythrosis. He had a son and a daughter. No known familial relevant diseases. His laboratory workup revealed ACTH-independent hypercortisolism: failure to suppress on the overnight dexametasone suppression test - cortisol 27.6ug/dL- and on the low-dose dexamethasone test- cortisol 24ug/dL- associated to morning ACTH <1ng/L. He underwent stimulation tests with tetracosactide, LHRH, TRH, vasopressin, metoclopramide, glucagon anddeambulation test, which were overall negative. It was decided to proceed to bilateral adrenalectomy. Due to surgical complications, only right adenalectomy was performed. Histology revealed adrenal nodular hyperplasia. Molecular study in DNA extracted from peripheral blood leucocyte and in the adrenal gland revealed the presence in heterozygosity of the pathogenic mutation c.1379T>C in the ARMC5 gene. The patient underwent a cerebral CT scan which showed a meningioma in the posterior left temporal convexity. Genetic testing was also performed on the daughter and son of the patient, which revealed the same mutation. They were also tested for hypercortisolism and underwent adrenal and cerebral CT scan, which showed no abnormality. The adrenal CT scan of the daughter showed enlargement of the left adrenal gland, with aspects suggestive of nodular hyperplasia. Her blood tests revealed no sign of hypercortisolism - overnight dexametasone test- cortisol 1.0 ug/dL, morning ACTH 19.1 ng/L. The patient is currently with cortisol hypersecretion controlled. Conclusion: All patients with PMAH should be tested for ARMC5 mutations and if they are found, family screening is mandatory in this autosomal dominant disorder. Unilateral adrenalectomy could control the cortisol hypersecretion, however some of these patients can have subclinical Cushing Syndrome.

SAT0159 Inhibition of Adrenal Axis in Elderly Patients under Treatment with Low Dosis of Corticosteroids: Should We Supplement in Stressful Situations?

BackgroundIn some rheumatic diseases low dosis of glucocorticoids (2-5 mg) are used for long periods of time. It is not clear if there is an inhibition of the adrenal axis...

Pelvic Ultrasonography in the Evaluation of Central Precocious Puberty: Comparison with Leuprolide Stimulation Test

To compare the ovarian and uterine structure demonstrated sonographically with baseline and leuprolide-stimulated luteinizing hormone (LH) and estradiol values in females with suspected precocious puberty. Retrospective chart review. Fifty females (age 3.1 to 9.5 years) underwent stimulation testing with leuprolide (20 μg/kg) and pelvic ultrasonography. Subjects were grouped as (1) prepubertal (baseline and stimulated LH and estradiol in prepubertal range); (2) early pubertal (baseline LH and estradiol in prepubertal range but stimulated LH or estradiol in pubertal range); and (3) pubertal (baseline and stimulated LH or estradiol in pubertal range). Sonographic data were compared with baseline and leuprolide-stimulated LH and estradiol. Baseline and stimulated LH and stimulated estradiol significantly correlated with ovarian and uterine volumes. Ovarian and uterine volumes were significantly higher in females in the pubertal group than in females in the prepubertal group. No significant differences were noted in the ovarian or uterine dimensions between the prepubertal and early pubertal groups. There was significant overlap in ovarian and uterine volumes among females in all three groups. Contrary to leuprolide stimulation, pelvic ultrasonography alone cannot distinguish between prepubertal females and those in the early stages of puberty.

Case report of an anaphylactoid reaction to arginine

Arginine is an agent commonly used to evaluate adequacy of growth hormone (GH) secretion. Because arginine is a simple amino acid, it is considered safe and rarely causes adverse reactions. To report the second anaphylactoid reaction to arginine in a child undergoing stimulation testing with arginine for assessing GH secretion. Allergy skin testing to arginine was performed with a protocol similar to penicillin testing 4 weeks after the anaphylactoid reaction. Testing revealed a positive response to the arginine. The use of intravenous arginine as a test of GH reserve remains safe and effective, but it is prudent to have the equipment and medication available to treat an allergic reaction.

Bladder Inhibition by Penile Nerve Stimulation in Spinal Cord Injury Patients

Detrusor hyperreflexia causing voiding dysfunction in spinal cord injury patients is a difficult problem and is not always treated effectively by anticholinergic agents. We have been investigating electrical stimulation methods to inhibit hyperreflexia and dorsal penile nerve stimulation is the most promising. Six chronic suprasacral spinal cord injury men (average age 36 years) underwent stimulation testing with water cystometry before, during and after stimulation. Dorsal penile stimulation was done with carbon rubber butterfly electrodes (Medtronic) with parameters of 5 pulses per second, 0.35 msec. pulse duration, and current at a level above the threshold for pelvic twitching activity and adjusted for optimal bladder effect (range 25 to 70 mamp.). In all 6 patients the cystometrogram during stimulation showed an increase in bladder volume over the prestimulation cystometrogram (range 27 to 150%). In 2 patients there was no detrusor activity after filling to 500 cc. Stimulation was then stopped and a spontaneous contraction occurred. The cystometrogram conducted after the stimulus also had less volume than that performed during stimulation but it was larger than the prestimulation volume. Penile nerve stimulation was painless with no side effects. Penile nerve electrical stimulation is effective for inhibiting bladder hyperreflexia and should be easily adaptable for chronic home use as an alternative to current therapy.

Effects of dexamethasone on growth hormone (GH)-releasing hormone, arginine- and dopaminergic stimulated GH secretion, and total plasma insulin-like growth factor-I concentrations in normal male volunteers.

In man, glucocorticoid treatment and endogenous corticosteroid excess generally suppress stimulated GH release. However, such effects are not entirely consistent and depend on both the duration of pituitary exposure to steroids and the secretagogue employed. To further evaluate the effects of glucocorticoids in man, we have studied the response to four different GH stimulation tests before and after treatment with dexamethasone (DEX; 2 mg twice daily during 84 h). Twelve healthy male volunteers were divided into two groups of six subjects (groups A and B). Group A underwent stimulation tests with arginine (500 mg/kg, i.v.) and GH-releasing hormone (GHRH, 100 micrograms, i.v.) before and after DEX treatment. Group B were subjected to stimulation tests with two dopaminergic agents, a novel nonergot D2-dopamine agonist CV205-502 (CV; 10 micrograms, i.v.) and dopamine (4 micrograms/kg.min, i.v.), before and after DEX. Within each group, the effect of DEX on the different secretagogues was studied 4 weeks apart. GHRH-stimulated GH release was significantly blunted by DEX treatment [median peak GH value, 34.2 micrograms/L; 25-75th percentiles, 22.1-56.2), control, vs. 19.8 (9.7-34.5), DEX; P less than 0.05; integrated GH secretion expressed as the area under the curve (AUC) was 48% lower after DEX; P less than 0.01]. In the same group, DEX treatment significantly enhanced the response to arginine [10.6 (8.0-22.8), control, vs 26.1 (15.1-38.6), DEX; P less than 0.01; with an increase in AUC of 72%; P less than 0.01]. In group B, under control conditions before glucocorticoid administration, the GH response to CV was significantly greater than that to dopamine in terms of both peak response [25.1 (8.6-30.9), CV, vs. 11.8 (5.5-16.4), dopamine; P less than 0.05] and AUC [2406 +/- 654 (CV) vs. 658 +/- 125 (dopamine); P less than 0.01], suggesting that CV may be a useful adjunct in the diagnosis of GH deficiency. After DEX administration, responses to both dopaminergic agents were suppressed [CV, 6.7 (4.0-21.2); P less than 0.01 vs. control response; and dopamine, 5.3 (4.8-7.9); P less than 0.05 vs. control response]. When compared with the effects of dexamethasone on the GH response to arginine, the results with dopaminergic agents highlight important differences in the mechanisms of action of these indirectly acting GH secretagogues. Moreover, this may be of physiological importance, because in contrast to the inhibitory effect of glucocorticoid on GHRH-stimulated GH release, DEX treatment significantly increased basal plasma GH levels [1.4 (0.5-5.1) vs. control 0.3 (0.1-0.6) microgram/L; P less than 0.001].(ABSTRACT TRUNCATED AT 400 WORDS)

The Partial Adrenocortical Hydroxylase Deficiency Syndrome in Infertile Women

Eighteen infertile women desiring conception were selected for additional diagnostic studies on the basis of clinical evidence of hyperandrogenism characterized by acne and/or facial hirsutism. These 18 patients underwent stimulation tests with adrenocorticotropic hormone which demonstrated the presence of partial adrenocortical hydroxylase deficiences. All patients were then placed on glucocorticoid suppression therapy. Ten patients conceived and, of these, six required no therapy other than prednisone. Four conceived using a combination of prednisone and clomiphene therapy. Of othe eight patients who did not conceive, seven had other significant infertility factors. Therefore, of the patients without other significant fertility factors, 91% conceived. This study demonstrates that partial adrenocortical enzyme defects play a significant role in some women with clinical evidence of hyperandrogenism, and, in such women, either glococorticoid therapy alone or in combination with clomiphene is indicated.

FSH: III. Evidence for a possible prepubertal regulation of its secretion by the seminiferous tubules in cryptorchid boys.

Two groups of prepubertal unilateral cryptorchid boys, 42▵12 to 12 years of age, underwent stimulation tests with 1 μg (n = 10) or 25 μg (n = = 8) of LHRH. Two groups of prepubertal bilateral cryptorchid boys, 52▵12 to H9▵12 years of age, were similarly studied (1 μg, n = 6; 25 μg, n = 8). For comparison, control groups of both prepubertal boys, 66▵12 to 119▵12 Via years of age (1 μg, n = 9; 25 μg, n = 11), and pubertal boys, 12 5/12 to 14 years of age (1 μg, n = 11), were also tested for response to LHRH. All patients had surgical descents of the cryptorchid testes with bilateral biopsy of the testes at that time. The number of spermatogonia per 50 transverse tubular sections was determined. Unilateral cryptorchid groups exhibited FSH responses to 1 and 25 μg of LHRH similar to those of the prepubertal control groups. The numbers of spermatogonia were low (means ± SE of 12 ± 6 to 25 ± 12) on the cryptorchid side, and in the normal range (greater than 50 per 50 tubular transverse sections) on the normal s...

FSH: Evidence for a possible prepubertal regulation of its secretion by the germinal epithelium in cryptorchid boys

Two groups of prepubertal unilateral cryptorchid boys (UCT) underwent stimulation tests with 1 μg (n=10) or 25 μg (n=8) of LHRH. Two groups of prepubertal bilateral cryptorchid boys (BCT) were similary studied (1 μg, n=6; 25 μg, n=8). The number of spermatogonia was determined in both sides in each group after testicular biopsy. UCT groups exhibited FSH and LH responses to LHRH similar to those of prepubertal control groups. Mean numbers of spermatogonia were low on the cryptorchid side (12 ± 6 to 25 ± 12) and normal on the normal side (>50). Both groups of BCT had very low counts of spermatogonia on both sides (means of 7 ± 4 to 14 ± 8). Their FSH responses to LHRH at both dosages were significantly higher than those of the normal prepubertal controls. LH responses were identical. These data suggest that the negative feedback mechanism between FSH secretion and the spermatogonia observed in the adult male is also operative in the prepubertal boy.