Quantification of urinary gonadotropins by specific assays may improve the evaluation of sex-specific hormonal changes in early infancy.

OBJECTIVE: The aim of this study was to investigate the feasibility of different gonadotropin assays for determining total and intact luteinizing hormone (LH), and follicle-stimulating hormone (FSH) immunoreactivity in urine (U-LH-ir and U-FSH-ir, respectively) during early infancy. DESIGN, PATIENTS AND MEASUREMENTS: Morning urine samples were obtained from 31 infants, aged between 0 and 6 months, to study the age-related course of urinary gonadotropins. Additionally, we investigated bi-hourly urine samples of a 5-day-old male neonate for 24 h to observe the course of urinary gonadotropins during a daily cycle. We employed different immunofluorometric assays for measuring total and intact U-LH-ir, and U-FSH-ir. RESULTS: In neonates up to 21 days of age, the U-LH-ir levels measured by the regular LH assay (also detecting hCG) were significantly higher than those determined by the total (specific) LH assays (p = .004). U-FSH-ir was higher in girls than boys during both the first and the next 5 months (p = .02 and p < .001, respectively), whereas total U-LH-ir was higher in boys until 6 months of age (p < .001). Total U-LH-ir/U-FSH-ir ratio was significantly higher in boys than girls across the first half-year (p < .001). CONCLUSIONS: The assessment of total U-LH-ir and U-FSH-ir, and their respective ratio constitutes a noninvasive, practical and scalable tool to investigate sex-specific changes during early infancy, with the ratio being significantly higher in boys than girls. Only highly specific LH assays detecting beta-subunit and its core fragment in addition to intact LH should be used for determining U-LH-ir in the neonatal period to avoid potential cross-reactivity with hCG of placental origin.

Quantification of overnight urinary gonadotropin excretion predicts imminent puberty in girls: a semi-longitudinal study.

PURPOSE: We explored the alternative of using overnight fold change in gonadotropin levels by comparing the last-night-voided (LNV) and first-morning-voided (FMV) urine concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) as a conceptual analogy to the invasive gonadotropin-releasing hormone (GnRH) stimulation test setting. METHODS: We investigated the nocturnal changes in the immunoreactivity levels of urinary gonadotropins between early and late prepubertal stages as well as between early and late pubertal stages in FMV and LNV urine samples from 30 girls, of whom those who were prepubertal were further investigated through follow-up visits within the 1-year period from the start of the study. RESULTS: ROC analysis revealed that the FMV total U-LH and FMV U-FSH concentrations at or above 0.3 IU/L and 2.5 IU/L, respectively, were excellent predictors of forthcoming onset of puberty within 1 year (100% sensitivity, 100% specificity, AUC: 1.00, and n = 10, for both). FMV total U-LH concentration at or above 0.8 IU/L represented the cut-off for clinical signs of puberty. FMV/LNV total U-LH and FMV/LNV U-FSH ratios at or below 4.11 and 1.38, respectively, were also good predictors of the onset of clinical puberty within 1 year. An overnight increase (FMV/LNV ratio) in total U-LH concentrations and in the U-LH/U-FSH ratio at or below 1.2-fold in pubertal girls was associated with the postmenarcheal pubertal stage. CONCLUSION: FMV total U-LH and U-FSH above 0.3 IU/L and 2.5 IU/L, respectively, can be used as cut-off values to predict the manifestation of the clinical signs of puberty within 1 year. FMV total U-LH concentrations 0.3-0.8 IU/L and 0.6 IU/L may represent the range and the threshold, respectively, that reflect the loosening of the central brake on the GnRH pulse generator. An overnight increase of 20% or less in total U-LH concentrations and in the U-LH/U-FSH ratio in an early pubertal girl may serve as an indicator of imminent menarche, a presumed timing of which can be unraveled by future longitudinal studies.

Urine Sampling Protocol Recommendations for Reliable Determination of Total Urinary Luteinizing Hormone Immunoreactivity in the Pediatric Population.

This study investigates day-to-day variations in urinary luteinizing hormone (U-LH) concentrations in children, focusing on potential minimization or correction methods. 95 children and adolescents (51 boys, 44 girls, ages 5-17) provided daytime and evening urine samples for U-LH determinations over three consecutive days. No consistent day-to-day differences in U-LH levels were observed, although random variations, particularly in adolescents aged 13 or older, were noted. The net inter-assay CV% for U-LH changes over three days showed high variability, averaging 24.6% to 28.0% for boys and 21.6% to 27.3% for girls, independent of sex, collection time, or U-LH level. To reliably determine total urinary luteinizing hormone immunoreactivity in the pediatric population, it is advisable to collect multiple first-morning voided samples for at least three consecutive days as an interim solution, pending the development of a standardized protocol or correction method for varying urine composition. Strict adherence, especially for adolescents aged 13 or older, is vital.

Sex-independent timing of the onset of central puberty revealed by nocturnal luteinizing hormone concentrations.

OBJECTIVE: We designed a longitudinal study to investigate the association between the ages of central pubertal activation and the appearance of clinical signs of puberty and determined total luteinizing hormone (LH) immunoreactivity in daytime- and nocturnal sleeptime-excreted urine samples. PATIENTS AND MEASUREMENTS: Thirty healthy volunteers (17 boys and 13 girls, aged 3.4-15.2 years and 4.3-14.3 years, respectively, at the beginning of the study) were included. Male and female subjects were followed for an average of 15 visits during 5.5 and 5.8 years on average, respectively. At each visit, subjects provided 24-h urine samples divided into nocturnal sleeptime and waketime portions according to the participant's sleep-and-wake rhythm. Total urinary LH (U-LH) concentrations were measured in duplicate by Delfia® IFMA (Wallac), which has been designed specifically to detect intact LH as well as the beta subunit and its core fragment, but not the human chorionic gonadotropin. RESULTS: The initial increases in nocturnal sleeptime total U-LH concentrations over the cutoff value of 0.7 IU/L occurred at around the same time (around 9-10 years of age) in both sexes, which could not be detected in waketime urine samples. The mean first age for the nocturnal sleeptime total U-LH concentrations to reach or surpass the cutoff was 10.7 years (range: 10.2-11.6 years) in boys and 11.8 years (range: 10.7-13.4 years) in girls, showing no statistically significant difference between the sexes (p = .15). The mean time span from the age at which sleeptime total U-LH concentration first exceeded the 0.7 IU/L level to observing pubertal stage 2 was 1.5 years in boys and 0.1 years in girls. CONCLUSIONS: Findings in our population with a limited sample size suggest that the timing of central pubertal activation is a sex-independent phenomenon, which can be observed by monitoring the nocturnal sleeptime total LH concentrations in urine. The lag time from central pubertal activation of gonadotropin secretion to the clinical onset of puberty is significantly longer in boys.

The negative impact of levothyroxine treatment on urinary luteinizing hormone measurements in pediatric patients with thyroid disease.

Objectives: Previous studies suggest urinary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) measurements by immunofluorometric assays (IFMA) as noninvasive alternatives to serum assays for puberty assessment. However, these studies excluded patients with other endocrine disorders and those taking medications. Besides, the recent discontinuation of IFMA manufacturing is a concern. We explored the utility of luminometric assays (LIA) for urinary gonadotropins and thyroid-stimulating hormone (TSH) determinations in euthyroid patients with thyroid pathologies. Methods: We used LIA and IFMA assays to measure serum and first-morning-voided (FMV) urine LH, FSH, and TSH concentrations in euthyroid patients with various thyroid disorders. Of the 47 euthyroid patients with normal serum TSH (S-TSH) levels, 14 were receiving levothyroxine therapy. Results: FMV total urinary LH (U-LH) concentrations correlated significantly with those measured in serum using either LIA (r=0.67, P<.001) or IFMA (r=0.83, P=.003) in patients not receiving levothyroxine treatment; however, no significant correlation could be detected in patients receiving levothyroxine regardless of the assay method (for LIA: r=0.50, P=.08 and IFMA r=0.44, P=.15). Urinary TSH (U-TSH) concentrations correlated poorly with those in serum in both the untreated and the treated groups (r=-0.13, P=.49, and r=-0.45, P=.11, respectively). Conclusion: FMV total U-LH determinations by LIA can be used to assess pubertal development in patients with thyroid pathology, provided the euthyroid patient is not on levothyroxine treatment. U-TSH measurements by LIA cannot replace invasive S-TSH measurements at least in patients with normal S-TSH levels. Further research may reveal the utility of U-TSH determinations in patients with elevated S-TSH levels.

Quantification of urinary total luteinizing hormone immunoreactivity may improve the prediction of ovulation time.

Objectives: Most of the currently available ovulation prediction kits provide a relatively rough estimation of ovulation time with a short fertility window. This is due to their focus on the maximum probability of conception occurring one day before ovulation, with no follow-up after LH surge until ovulation nor during the subsequent days thereafter. Earlier studies have shown that urine of reproductive age women contains at least 3 different molecular forms of luteinizing hormone (LH); 1) intact LH, 2) LH beta-subunit (LHß) and a 3) small molecular weight fragment of LHß, LHß core fragment (LHßcf). The proportion of these LH forms in urine varies remarkably during the menstrual cycle, particularly in relation to the mid-cycle LH surge. In this exploratory study, we studied the potential implications of determining the periovulatory course of total LH immunoreactivity in urine (U-LH-ir) and intact LH immunoreactivity in serum (S-LH-ir) in the evaluation of the fertility window from a broader aspect with emphasis on the post-surge segment. Methods: We determined total U-LH-ir in addition to intact S-LH-ir, follicle-stimulating hormone (FSH), progesterone, and estradiol in 32 consecutive samples collected daily from 10 women at reproductive age. Inference to the non-intact U-LH-ir levels was made by calculating the proportion of total U-LH-ir to intact S-LH-ir. Results: Total U-LH-ir increased along with LH surge and remained at statistically significantly higher levels than those in serum for 5 consecutive days after the surge in S-LH-ir. S-LH-ir returned to follicular phase levels immediately on the following day after the LH surge, whereas the same took 7 days for total U-LH-ir. Conclusions: The current exploratory study provides preliminary evidence of the fact that U-LH-ir derived from degradation products of LH remains detectable at peak levels from the LH surge until ovulation and further during the early postovulatory period of fecundability. Thus, non-intact (or total) U-LH-ir appears to be a promising marker in the evaluation of the post-surge segment of the fertility window. Future studies are needed to unravel if this method can improve the prediction of ovulation time and higher rates of fecundability in both natural and assisted conception.

Clinical Utility of Respiratory Scores at Admission for Estimating the Definitive Microbiological Diagnosis in Lower Respiratory Tract Infections in Infants.

We aimed at determining the clinical utility of respiratory scores and the durations of wheezing or respiratory distress during hospitalization in infants with lower respiratory tract infections (LRTI) at admission for estimating the definitive microbiological diagnosis. We obtained data from a study population of 201 patients, 79 girls and 122 boys. There was a significant divide in the causative agents of LRTI among patients younger and older than 6 months of age (P = .002), and significantly different respiratory score findings were determined in infants with viral LRTI: a low respiratory score in a younger-than-6 month infant suggests Adenovirus as the causative agent and a high respiratory score suggests Parainfluenza 1 or 2; as for infants of 6 months of age or older, a low respiratory score indicates Influenza A or B or a mixed infection, whereas a high respiratory score is likely an indication of Parainfluenza 3 or RSV.

Effect of Breastfeeding and Preterm Births on the Severity of Lower Respiratory Tract Infections and Associated Risk of Hospitalization in Infants and Toddlers.

We studied the effect of duration of breastfeeding and history of prematurity on the duration of hospitalization in infants with lower respiratory tract infections (LRTI) because these may reflect the severity of illness as well as sizable direct and indirect healthcare costs. One hundred twenty-five patients (49 girls, 76 boys; aged 1-24 months) were hospitalized for LRTI during a period of 102 days and studied prospectively. We found a significant difference (P = .045) between the durations of hospitalization of the 92 patients breastfed for at least six months, compared to the other group of 33 patients who were breastfed for less than six months. The durations of hospitalization among the groups with and without a history of prematurity were not statistically different (P = .78). A history of breastfeeding for more than six months had significant effect on the duration of hospitalization, but this was not true for children with a history of preterm birth.

Identification of the LH surge by measuring intact and total immunoreactivity in urine for prediction of ovulation time.

OBJECTIVES: In our earlier study, we separated three different molecular forms of urinary LH-ir (U-LH-ir) by gel filtration and identified them by immunoassay in urine from regularly menstruating women on periovulatory days. U-LH-ir is composed of intact luteinizing hormone (LH), its free beta-subunit (LHß), and the core fragment of LHß (LHßcf), the latter two establishing the non-intact portion of LH-ir. The aim was to determine whether timing of ovulation can be improved by detecting different molecular forms of U-LH-ir in women of reproductive age. METHODS: We determined intact and total U-LH-ir in 14 regularly menstruating women on consecutive periovulatory days during the menstrual cycle. Non-intact LH-ir was calculated as the arithmetic difference between total and intact LH-ir. In addition, LH-ir was determined in both serum and urine from four of the women throughout the menstrual cycle. RESULTS: During the LH surge, U-LH-ir consisted mainly of intact LH and presented with an abrupt increase. Intact U-LH-ir dropped rapidly within 1 day after the surge, reaching baseline levels at the end of the luteal phase. In contrast, LHßcf in urine increased further 1 day after the surge. After this, most of the U-LH-ir consisted of LHßcf and it remained strongly elevated (over fivefold compared to intact LH) for the first 3 days after the LH surge, moderately elevated (over threefold) thereafter until day + 5, and mildly elevated until day + 7. CONCLUSIONS: Total and non-intact LH-ir are potential add-on characteristics which can be utilized in ovulation predictor kits to measure LH-ir in urine beyond the LH surge during a broader time frame, thereby paving the way for more precise prediction of the timing of ovulation than that obtained with currently available products.

Intact luteinizing hormone (LH), LHß, and LHß core fragment in urine of menstruating women.

OBJECTIVES: We examined different molecular forms of luteinizing hormone (LH) in urine samples taken during periovulatory days with the aim of revealing different forms of LH immunoreactivity (LH-ir) in normally menstruating women. METHODS: Serum and first-morning-voided urine serum samples were obtained from six healthy, 22 to 38 years old, regularly menstruating women during their periovulatory days based on their previous menstrual cycles. The day of the LH surge was determined on the basis of serum LH concentrations and confirmed by an at least two-fold increase in urinary concentrations of intact LH on consecutive days. Different molecular forms of LH-ir were identified by gel filtration of first-morning-voided urine samples obtained from regularly menstruating women on periovulatory days. RESULTS: Different forms of LH immunoreactivity (LH-ir) were distinguished as intact LH, its free beta-subunit (LHß), and the core fragment of LHß (LHßcf) according to their molecular sizes. The latter two are also called non-intact LH. Intact LH was the dominating form on the day before and on the day of LH surge while LHßcf was the major form of LH immunoreactivity after the LH surge for the following 5-7 days. LHß was detected on the day of the LH surge as well as on the following day. CONCLUSIONS: These results indicate that LH is degraded in the kidneys and excreted as LHß, and mainly as LHßcf for 7 days following the LH peak.

Urinary gonadotropin measurements by enhanced luminometric assays (LIA) for the evaluation of pubertal development.

OBJECTIVES: Determination of LH in urine has proved to be a reliable method for evaluation of pubertal development. The human LH assay based on time-resolved immunofluorometric (IFMA) technology (AutoDELFIA, PerkinElmer, Wallac) has been found to be suitable for this purpose thanks to its high sensitivity but other assays have not been evaluated. We have analyzed our data obtained by another potentially sensitive detection technique, enhanced luminometric assay (LIA) with the objective of finding a viable alternative to IFMA since these may not be available in the future. METHODS: LIA was used to measure LH and FSH in serum and urine samples from 100 healthy subjects of each Tanner stage and both genders, whose pubertal development has been determined. RESULTS: Urinary gonodotropin concentrations measured by LIA correlated well with Tanner stage [(r=0.93 for girls, r=0.81 for boys; p<0.01 for LH) and (r=0.81 for girls, r=0.73 for boys; p<0.01 for FSH)]. LIA determinations revealed the increase in U-LH concentrations during the transition from Tanner stage 1-2 in both girls and boys (p<0.001), whereas U-FSH and S-LH were able to detect the increase from Tanner stage 1-2 only in boys or girls, respectively (both p<0.001). CONCLUSIONS: Measurement of urinary gonadotropin concentrations by LIA may be useful for the evaluation of overall pubertal development and also in the detection of transition from prepuberty to puberty.

First Morning Voided Urinary Gonadotropin Measurements as an Alternative to the GnRH Test.

AIMS: We studied whether first morning voided (FMV) urinary gonadotropin measurements could be used as a noninvasive alternative to the GnRH test in the assessment of the hypothalamic-pituitary-gonadal function in children. METHODS: In a single-center study, we compared FMV urinary gonadotropin concentrations with basal and GnRH-stimulated serum gonadotropin levels in 274 children and adolescents (78 girls, 196 boys) aged 5-17 years referred for growth and pubertal disorders. The concordance between FMV urinary gonadotropin concentrations and GnRH test results was assessed. RESULTS: FMV urinary LH (U-LH), urinary FSH (U-FSH) and their ratios correlated well with the corresponding basal and GnRH-stimulated serum parameters (r ≥ 0.66, p < 0.001). Receiver operating characteristic curve analyses using urinary and serum LH and FSH concentrations showed that FMV U-LH and U-LH/U-FSH performed equally well as the GnRH test in the differentiation of early puberty (Tanner stage 2) from prepuberty (Tanner stage 1) (area under the curve 0.768-0.890 vs. 0.712-0.858). FMV U-LH and U-LH/U-FSH performed equally well as basal serum LH in predicting a pubertal GnRH test result (area under the curve 0.90-0.93). CONCLUSION: FMV U-LH determination can be used for the evaluation of pubertal development and its disorders, reducing the need for invasive GnRH stimulation tests.