Volume 33, Issue 11

Short stature, ovarian dysgenesis, infertility, and cardiovascular malformations are classic features in Turner syndrome (TS), but the phenotypical spectrum is wide. Through early diagnosis and appropriate treatment, TS patients have a chance to achieve satisfactory adult height and sexual development. The doses of recombinant growth hormone (rGH) used are usually higher than the substitution dose. The safety aspects of this therapy are very important, especially in terms of the cardiovascular system. The presented study aimed to analyze how the rGH therapy may influence the cardiovascular system in TS based on current literature data. We conducted a systematic search for studies related to TS, cardiovascular system, and rGH therapy. The results show that rGH seems to have a positive effect on lipid parameters, reducing the risk of ischemic disease. It is additionally optimized by estradiol therapy. Although rGH may increase insulin resistance, the metabolic derangement is rare, probably due to lower fat content and an increase in lean body mass. Several studies showed that rGH treatment could cause aorta widening or increase the aorta growth rate. IGF-1 can be independently associated with increased aortic diameters. The studies analyzing the impact of GH on blood pressure show conflicting data. The proper cardiovascular imaging before and during rGH treatment and detecting the known risk factors for aorta dissection in every individual is very important. The long-term effects of growth hormone treatment on the heart and arteries are still not available and clearly estimated and have to be monitored in the future.

Background Girls with Turner syndrome (TS) are at an increased risk of primary ovarian insufficiency (POI). Good correlation between serum and urinary gonadotrophins exists in children assessed for disorders of puberty, but there is little evidence of their reliability in hypergonadotropic states. Objectives To determine whether there was a correlation between serum and urinary Luteinising Hormone (uLH) and Follicle-Stimulating Hormone (uFSH) in hypergonadotrophic states, and whether uFSH could suggest an ovarian failure in TS as Anti-Mullerian Hormone (AMH). Patients and Methods Retrospective cohort study of 37 TS girls attending the paediatric TS clinic in Glasgow between February 2015 and January 2019, in whom 96 non-timed spot urine samples were available with a median age at time of sample of 12.89 years (3.07–20.2 years). uLH and uFSH were measured by chemiluminescent microparticle immunoassay. Simultaneous serum gonadotrophins and AMH were available in 30 and 26 girls, respectively. AMH <4 pmol/L was considered indicative of ovarian failure. Results A strong correlation was found between serum LH and uLH (r 0.860, P<0.001) and serum FSH and uFSH (r 0.905, p<0.001). Among patients≥10 years not on oestrogen replacement, ROC curve identified uFSH as a reasonable marker for AMH<4 pmol/L uFSH of >10.85 U/L indicates an AMH <4 pmol/L with 75% sensitivity and 100 % specificity (AUC 0.875)with similar ability as serum FSH (AUC 0.906). Conclusion uLH and uFSH are non-invasive, useful and reliable markers of ovarian activity in hypergonadotropic states as TS. uFSH could provide an alternative to AMH (in centres which are limited by availability or cost) in revealing ovarian failure and requirement for oestrogen replacement in pubertal induction.

Background Sclerostin and osteoprotegerin (OPG) are new markers of chronic kidney disease (CKD) mediated mineral bone disease (CKD-MBD) which were extensively evaluated in adult population. We aimed to evaluate the associations between serum levels of sclerostin/OPG and parameters of bone turnover and compare the serum levels of sclerostin/OPG in different stages of CKD in children. Methods 70 children with CKD stage 1-5, aged 2-21 years were examined. Serum levels of alkaline phosphatase (ALP), creatinine, total calcium, phosphorus , intact parathyroid hormone (iPTH) and vitamin D were measured. Serum sclerostin and OPG levels were measured in children with different levels of CKD stage and their association with bone turnover parameters were noted. Results We did not observe any significant correlation between serum levels of sclerostin and OPG and stages of CKD. A negative relationship was present between serum sclerostin and 25-OH vitamin D levels. Osteoprotegerin was positively and significantly correlated with ALP but serum sclerostin was negatively correlated with ALP. Conclusion Our study, which includes only children and adolescents with a growing skeleton under uremic conditions and excluding diabetes and atherosclerosis interference, is very valuable. We couldn't find any significant relationship between either sclerostin or OPG levels among different stages of CKD. Also our study demonstared a strong negative relationship between ALP and sclerostin levels and a strong positive relationship between ALP and OPG levels, reminding the importance of ALP levels to predict the bone-mineral status of the children with CKD.

Objectives Osteogenesis imperfecta (OI) is a skeletal dysplasia characterized by recurrent fractures due to congenital bone fragility. The only bisphosphonate approved for OI in Japan is pamidronate (PAM). To investigate whether monthly intravenous alendronate (ALN) infusions can maintain bone strength in OI children following cyclical PAM treatment. Methods A prospective and non-inferiority study was conducted. Eight school-age OI patients aged 8.5±2.0 years who were treated with cyclical PAM for 6.0±2.3 years were enrolled and switched to monthly intravenous ALN (0.030 mg/kg/month). Changes in L1-4 bone mineral density (BMD) Z-scores, fracture rates, and bone turnover markers for 12 months were analyzed. Results Average BMD Z-scores were −3.0±1.9, −2.9±2.0, and −2.2±2.0 in 12 months before enrollment, at enrollment, and after 12 months of ALN treatment, respectively. BMD Z-scores increased significantly during treatment with both PAM and ALN (p=0.012), and the effect of ALN was not inferior to that of PAM (p=0.67). There was no change in fracture rates (p=0.86) and bone turnover markers during the 12 months before and after enrollment. Additionally, ALN showed no remarkable side effects. Conclusions Our results suggest that monthly intravenous ALN can maintain bone strength after primary usage of cyclical PAM. We concluded that monthly intravenous ALN as a maintenance treatment following cyclical PAM administration can be an option for OI children.

Objectives The is no consensus on the early patterns of lipid-based cardiovascular disease (CVD) risk in youth with either type 1 diabetes (T1D) or type 2 diabetes (T2D). The aim was todetermine the differences in CVD risk, using lipid profiles, in children and adolescents with either T1D or T2D at the time of their first lipid assessment, after stratifying the T1D cohort into remitters and non-remitters based on their honeymoon history. Methods A cross-sectional study of 249 subjects consisting of 73 controls, 53 T2D subjects, and 123 T1D subjects stratified into remitters (n=44), and non-remitters (n=79). Partial clinical remission (PCR) was defined as insulin-dose adjusted HbA1c of ≤9. Pubertal status was determined by Tanner staging. Results After adjusting for age, sex, BMI, race, and pubertal status, T2D patients had significantly higher LDL-C compared to the controls (p=0.022), the remitters (p=0.029), but not the non-remitters (103.1 ± 5.9 mg/dL vs. 91.4 ± 4.2 mg/dL, p=0.49). Similarly, T2D patients had significantly higher non-HDL-C compared to the controls (p=0.006), the remitters (p=0.0002), but not the non-remitters (137.6 ± 7.1 mg/dL vs. 111.71 ± 5.0 mg/dL, p=0.053). Total cholesterol was also significantly higher in T2D patients compared to the controls (p=0.0005), the remitters (p=0.006) but not the non-remitters (183.5 ± 6.6 mg/dL vs. 166.2 ± 4.8 mg/dL, p=0.27). Conclusions Lack of the honeymoon phase in children and adolescents with T1D confers early and significantly increased lipid-based cardiovascular risk to these patients that is similar to the elevated cardiovascular risk seen in T2D.

Background The thyroid gland of patients with congenital heart disease may be exposed to large doses of iodine from various sources. We assessed the thyroid response after iodine exposure during conventional angiography in cardiac catheterization and angiographic computer tomography in childhood. Methods Retrospective mid- to long-term follow-up of 104 individuals (24% neonates, 51% infants, 25% children) with a median age and body weight of 104 days [0–8 years] and 5.3 kg [1.6–20]. Serum levels of thyroid-stimulating hormone, free triiodthyronine and free thyroxine were evaluated at baseline and after excess iodine. We also assessed risk factors that may affect thyroid dysfunction. Results Baseline thyroidal levels were within normal range in all patients. The mean cumulative iodinate contrast load was 6.6 ± 1.6 mL/kg. In fact, 75% had experienced more than one event involving iodine exposure, whose median frequency was three times per patient [1–12]. During the median three years follow-up period [0.5–10], the incidence of thyroid dysfunction was 15.4% (n=16). Those patients developed acquired hypothyroidism (transient n=14, long-lasting n=2 [both died]) with 10 of them requiring temporary replacement therapy for transient thyroid dysfunction, while four patients recovered spontaneously. 88 individuals (84.6%) remained euthyroid. Repeated cardiac interventions, use of drugs that interfere with the thyroid and treatment in the intensive care unit at the index date were strong predictors for acquired thyroid dysfunction. Conclusions The incidence of acquired hypothyroidism after iodine excess was 15.4%. However, most patients developed only transient hypothyroidism. Systemic iodine exposure seems to be clinically and metabolically well tolerated during long-term follow-up.

Background The relationship between growth hormone (GH)-replacement therapy and the thyroid axis in GH-deficient (GHD) children remains controversial. Furthermore, there have been few reports regarding non-GHD children. We aimed to determine the effect of GH therapy on thyroid function in GHD and non-GHD children and to assess whether thyrotropin-releasing hormone (TRH) stimulation test is helpful for the identification of central hypothyroidism before GH therapy. Methods We retrospectively analyzed data from patients that started GH therapy between 2005 and 2015. The free thyroxine (FT4) and thyroid-stimulating hormone (TSH) concentrations were measured before and during 24 months of GH therapy. The participants were 149 children appropriate for gestational age with GHD (IGHD: isolated GHD) (group 1), 29 small for gestational age (SGA) children with GHD (group 2), and 25 short SGA children (group 3). Results In groups 1 and 2, but not in group 3, serum FT4 concentration transiently decreased. Two IGHD participants exhibited central hypothyroidism during GH therapy, and required levothyroxine (LT4) replacement. They showed either delayed and/or prolonged responses to TRH stimulation tests before start of GH therapy. Conclusions GH therapy had little pharmacological effect on thyroid function, similar changes in serum FT4 concentrations were not observed in participants with SGA but not GHD cases who were administered GH at a pharmacological dose. However, two IGHD participants showed central hypothyroidism and needed LT4 replacement therapy during GH therapy. TRH stimulation test before GH therapy could identify such patients and provoke careful follow-up evaluation of serum FT4 and TSH concentrations.

Objectives Graves’ disease (GD) is an autoimmune disease involving intimate response of both T cells and B cells. Immunophenotyping of peripheral blood lymphocyte subsets in GD children with different clinical characteristics can provide further information of the pathogenesis of GD. Methods We studied the lymphocyte subsets in peripheral blood of 141 children with GD. We repeatedly divided the patients into two groups in accordance with different clinical characteristics (abnormal or normal alanine aminotransferase (ALT) levels, the presence or absence of Graves’ orbitopathy (GO), and the presence or absence of hematuria. Then we compared the lymphocyte subsets measurements between two paired groups. Results We found that serum ALT levels correlated positively with CD3+CD8+ T cell percentages in children with GD. Moreover, we detected higher percentages of CD3−CD19+ cells and higher ratio of CD4/CD8 in patients with GO. However, no correlation was found between GO status and lymphocyte subsets after excluding confounding effect of TRAb. No difference of lymphocyte subset percentages was found between groups with or without hematuria. Conclusions Serum ALT levels correlate positively with cytotoxic T cell percentages in the peripheral blood of children with GD. The cytotoxic T cell may play a role in the pathogenesis of hepatic dysfunction in children with GD.

Objectives The aims were to analyze the clinical features, response to treatment, prognostic factors and long-term follow-up of children and adolescents with differentiated thyroid carcinoma (DTC). Methods Eighty patients with DTC were studied retrospectively. All underwent total or near-total thyroidectomy, and in 75 cases, ablative iodine therapy was recommended. Patients were assessed periodically by tests for serum thyroglobulin levels and whole-body iodine scans. Age, gender, initial clinical presentation, histology, tumor stage, postoperative complications, radioiodine treatment protocol, treatment response, thyroglobulin (Tg), recurrence and long-term disease progression were evaluated. Results Seventy patients completed >2 years of follow-up (23 males, 47 females; median age: 14 years; range: 3–18 years). Sixty-two patients showed papillary DTC and eight, follicular DTC. Sixty-five percent presented nodal metastasis and 16%, pulmonary metastasis at diagnosis. Six months after first radioiodine treatment, 36.2% of patients were free of disease. Seven recurrences were documented. At the end of follow-up, overall survival was 100%, and 87.2% of patients were in complete remission. Nine patients had persistent disease. We found a significant association between stage 4 and persistent disease. Hundred percent of patients with negative Tg values at 6 months posttreatment were documented free of disease at the end of the follow-up. The analysis of disease-free survival based on radioiodine treatment protocols used showed no statistically significant differences. Conclusions DTC in children and adolescents is frequently associated with presence of advanced disease at diagnosis. Despite this, complete remission was documented after treatment in most cases, with a good prognosis in the long-term follow-up. Negative posttreatment thyroglobulin and stage 4 at diagnosis were significant prognostic variables.

Objectives Hypocalcemia following total thyroidectomy (TT) is relatively common. It may result in significant morbidity, prolonged hospital stay, and increased costs. Treatment with intravenous (IV) calcium gluconate may also carry significant risks. In pediatrics, management consensus guidelines are lacking. Methods At Phoenix Children’s Hospital, a team of pediatric endocrinologists, surgeons and otolaryngologists developed a clinical pathway for patients undergoing TT. It was a Quality Improvement (QI) project with the primary aim of decreasing IV calcium gluconate use from a baseline of 68% to less than 40% over 15 months. Secondary aims included reducing hypocalcemia and length of hospitalization. Interventions included sending weekly pathway reminder emails, starting pre-operative calcium, and pathway implementation into the electronic health record. Results Twenty-seven patients underwent TT over 15 months. IV calcium gluconate use dropped to 48%. Hypocalcemia and length of hospitalization were 96% and 52.7 h (range 21.1–115.7) respectively. Pathway adherence improved after targeted interventions. Eleven (73%) of the 15 patients whose post-operative parathyroid hormone (PTH) nadir was below 15 pg/mL required IV calcium gluconate vs. two (17%) out of 12 with levels above this threshold. Conclusions Standardizing care allowed for objective outcome analysis. We learned that post-operative serum PTH level was the main risk factor for requiring IV calcium gluconate. Implementing the pathway as a QI project allows for revisions based on outcomes, ultimately resulting in a pathway that best utilizes our infrastructure to optimize care. Other pediatric institutions may face similar challenges and can potentially learn from our experience.

Objectives Adequate treatment of congenital hypothyroidism (CH) is required for normal growth and sexual development. To evaluate pubertal development in patients with permanent CH detected by a statewide Neonatal Screening Program of Paraná and, secondly, to evaluate adult height (AH) in a subgroup of patients. Methods Clinical, laboratory, and auxological data obtained from medical records of 174 patients (123 girls). Results Median chronological age (CA) at treatment initiation was 24 days, and mean initial levothyroxine dose was 11.7 ± 1.9 μg/kg/day; mean CA at puberty onset was 11.5 ± 1.3 years (boys) and 9.7 ± 1.2 years (girls); mean CA in girls who underwent menarche (n=81) was 12.1 ± 1.1 years. Thyroid-stimulating hormone (TSH) values above the normal range were observed in 36.4% of the boys and 32.7% of the girls on puberty onset, and in 44.6% around menarche. Among 15 boys and 66 girls who had reached the AH, the median height z-score value was significantly greater than the target height (TH) z-score value in boys (p=0.01) and in girls (p<0.001). Boys with normal TSH values at puberty onset had greater mean AH z-score compared with boys with TSH values above the normal range (p=0.04). Conclusions In this group, pubertal development in girls with CH was not different from that reported in healthy girls in the general Brazilian population. Boys with higher TSH at puberty onset may have an increased risk of not reaching their potential height compared with those with normal TSH during this period. In a subgroup who attained AH, the median AH z-score was greater than the median TH z-score.

Objectives Thyroid cancer is the most common endocrine neoplasm in childhood. There are few studies characterizing pediatric population in Colombia. We intend to detail the clinical, histological characteristics, BRAFV600E mutational status and NIS (sodium-iodine symporter) expression of children with papillary thyroid carcinoma (PTC) managed at Hospital de La Misericordia. Methods Medical records of the Department of Pediatric Surgery and Pathology from 2009 to 2018 were scrutinized in search of cases of differentiated thyroid carcinoma. A descriptive analysis was made. Paraffin embedded tumoral tissue was recovered to assess BRAF V600E mutational status by PCR and NIS expression by immunohistochemistry. Results Sixteen patients were selected, 81.2% were girls. Average age of presentation was 11.8 years. Only one patient had previous radiation exposure. Most frequent symptom was cervical adenopathy with a mean time of 29.2 weeks before diagnosis. 93.7% underwent total thyroidectomy and lymphadenectomy. 62.5% were PTC combining both classic and follicular pattern. 6.25% cases had BRAFV600E mutation and 25% showed NIS focal reactivity. Conclusions We found greater female predominance, lower percentage of risk factors described and a high percentage of patients requiring aggressive surgical treatment. We consider important to contemplate thyroid cancer as a differential diagnosis of cervical lymph node enlargement in children. Diagnosis can be challenging in benign and indeterminate categories of the FNA cytology and biomolecular profiles such as BRAF and NIS could be determinant in guiding treatment. More studies with larger sample size, complete genetic analysis, evaluation to iodine response and long term follow up are required.

Objectives The primary objective of this study was to determine the prevalence of BRAF V600E and TERT promoter mutations in paediatric and young adult patients with papillary thyroid carcinoma (PTC) and the secondary objective, to assess their association with clinicopathological features. Methods Patients ≤20 years who underwent surgery for differentiated thyroid cancer (DTC) from 2005 to 2018 were consecutively enrolled for BRAF V600E and TERT promoter mutations analysis and records analysed for the association of aggressive features. Univariate analysis and multivariate logistic regression were used to identify the independent predictors of BRAF V600E mutations. Results Among 100 patients with DTC, 68 patients were ≤18 years and the remaining 30 patients were >18 years of age with a median age of 17 years (IQR 14–19 years) 98 patients had PTC and 2 had FTC. BRAF V600E mutation was present in 14/98 (14.3%) PTC and TERT promoter mutation noted in none. Multivariate analysis identified RAI refractoriness (OR:10.57, 95% CI: 2.6 to 41.6, P-0.0008) as an independent factor associated with BRAF V600E mutation. 17 patients with distant metastases were negative for both BRAF V600E or TERT promoter mutation. No significant association was observed between age, gender, PTC variants, extra-thyroidal extension, lymphovascular invasion, multifocality, RAI administration and event rate with BRAF V600E mutation. Irrespective of BRAF V600E mutation, radioiodine refractory status (p-0.0001) had a reduced EFS probability. Conclusion In paediatric & young adult PTC, TERT promoter mutation is absent and BRAF V600E mutation is not associated with distant metastasis. The prevalence rate of the BRAF V600E mutation is much lower compared to adult PTC patients.

Objectives The objective of this paper is to report a peculiar case of a patient with pseudohypoparathyroidism type 1b (PHP1B). Pseudohypoparathyroidism (PHP) refers to a group of disorders characterized by hypocalcemia, hyperphosphatemia, and elevated parathyroid hormone (PTH) concentrations as the result of end-organ unresponsiveness to PTH. Case presentation We present a 14-year-old boy, who was admitted with severe symptomatic hypocalcaemia, absence of dysmorphic features and Albright's hereditary osteodystrophy features. Laboratory investigations revealed markedly low serum calcium, high phosphate, markedly elevated PTH levels and vitamin D insufficiency, while magnesium, albumin, ALP and TSH were normal. The clinical and laboratory findings were consistent with PHP1B. Molecular analysis revealed loss of methylation at the AB DMR of the GNAS locus, confirming the diagnosis. Yet no STX16 deletion was detected. Conclusions It is possible that delSTX16- patients carry a defect in an element that controls the methylation both at the GNAS-A/B DMR and at the GNAS-AS2. This rare case emphasizes the need of individualized molecular analysis in PHP1B patients in order to elucidate the possible molecular defect.

Objectives Holocarboxylase synthetase deficiency (HCSD) (OMIM #253270) is a rare inborn error of metabolism with an estimated annual incidence of 1 in 200,000 people. Typical manifestations of HCSD include eczema, alopecia, lactic acidosis and hyperammonemia. Diagnosis is made through genetic analysis. Case presentation Patient 1 was a 7-year-old girl with normal growth and development, presenting with severe hypoglycemia and metabolic acidosis. Her family reported that she was diagnosed as having ketotic hypoglycemia; she had five episodes of hypoglycemia and metabolic acidosis in past 4 years when her oral intake decreased during acute illness. Patient 2 was a 6-month-old female infant with normal growth and development, presenting with progressive generalized eczema and metabolic acidosis for the first time. We found that they both had hyperammonemia, hyperlactatemia, hyperketonemia, organic acids detected in urine and elevated C5OH acylcarnitine level by tandem mass spectrometry. HLCS gene analysis showed a homozygous pathogenic variant p.V363D in patient 1 and a pathogenic variant p.R508W compound with a novel splice site pathogenic variant c.2010-1G>A in patient 2. They have been on biotin treatment (10 mg/day for both of them) for more than 2 years and no more symptoms have occurred. Conclusions HCSD is a rare disease, and it can be fatal if severe metabolic acidosis occurs without timely management. Once the diagnosis is made, most of the patients with HCSD have good prognosis and normal life expectancy with biotin treatment.

Objectives Transcobalamin II (TC) is an essential plasma protein for the absorption, transportation, and cellular uptake of cobalamin. TC deficiency presents in the first year of life with failure to thrive, hypotonia, lethargy, diarrhea, pallor, mucosal ulceration, anemia, pancytopenia, and agammaglobulinemia. Herein, we present TC deficiency diagnosed in two cases (twin siblings) with a novel variant in the TCN2 gene. Case presentation 4-month-old twins were admitted with fever, respiratory distress, vomiting, diarrhea, and failure to thrive. Physical examination findings revealed developmental delay and hypotonia with no head control, and laboratory findings were severe anemia, neutropenia, and hypogammaglobulinemia. Despite normal vitamin B12 and folate levels, homocysteine and urine methylmalonic acid levels were elevated in both patients. Bone marrow examinations revealed hypocellular bone marrow in both cases. The patients had novel pathogenic homozygous c.241C>T (p.Gln81Ter) variant in the TCN2 gene. In both cases, with intramuscular hydroxycobalamin therapy, laboratory parameters improved, and a successful clinical response was achieved. Conclusions In infants with pancytopenia, growth retardation, gastrointestinal manifestations, and immunodeficiency, the inborn error of cobalamin metabolism should be kept in mind. Early diagnosis and treatment are crucial for better clinical outcomes. What is new? In literature, to date, less than 50 cases with TC deficiency were identified. In this report, we presented twins with TCN2 gene mutation. Both patients emphasized that early and aggressive treatment is crucial for achieving optimal outcomes. In this report, we identified a novel variation in TCN2 gene.

Background Aldosterone deficiency (hypoaldosteronism) or aldosterone resistance (pseudohypoaldosteronism) both result in defective aldosterone activity. Case presentation A 42-day-old man presented with failure to thrive, hyponatremia, high urine sodium output, severe hyperkalemia and high plasma renin activity and aldosterone levels. NR3C2 , SCNN1A , B and G sequencing showed no variants. Exclusive sodium supplementation resulted in clinical stabilization and growth normalization. His younger sibling had similar clinical and laboratory features, except for low-normal aldosterone. Both patients showed compound heterozygous mutations in CYP11B2 (c.C554T/2802pbE1-E2del). The younger patient needed transient fludrocortisone treatment and higher sodium supplementation, recuperating his weight and a normal growth velocity, although below his brother’s and target height (c.10th vs. c.50th). Conclusions On a suggestive clinical picture, high aldosterone plasma levels in early infancy do not rule out aldosterone insufficiency and might mislead differential diagnosis with pseudohypoaldosteronism. Therapeutic requests and growth impairment in hypoaldosteronism vary even with a common genetic background.

Introduction Primary adrenal insufficiency is a potentially life-threatening condition that can have many underlying causes. Mutations in the steroidogenic acute regulatory protein (StAR) gene produce lipoid congenital adrenal hyperplasia (LCAH) which usually presents in the infantile period with severe symptoms of adrenal insufficiency. Less commonly, a non-classical form is identified which may present at a later age in affected individuals. Till date, around 30 individuals with the non-classical form have been described. Case presentation We describe a 4-year-old 46, XX Indian girl who presented with hypoglycemic seizures and was subsequently diagnosed as non-classical LCAH on genetic analysis, with homozygous R188C mutation in the StAR gene. Conclusions StAR mutations may have a variety of clinical presentations and are likely under-diagnosed. Genetic diagnosis is important for treatment as well as monitoring of reproductive function.