Influence of ethnicity on biochemical markers of health and disease in the CALIPER cohort of healthy children and adolescents.

Background Accurate pediatric reference intervals (RIs) for laboratory tests determined in a healthy pediatric population are essential for correct laboratory test interpretation and clinical decision-making. In pediatrics, RIs require partitioning by age and/or sex; however, the need for partitioning based on ethnicity is unclear. Here, we assessed the influence of ethnicity on biomarker concentrations in the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) cohort of healthy children and adolescents and compared the results with the National Health and Nutrition Examination Survey (NHANES). Methods A total of 52 biomarkers were measured in a multiethnic population of 846-1179 healthy children (aged 5 to <19 years) upon informed consent. Biomarker concentrations were retrospectively compared between four major ethnic groups (i.e. Black, Caucasian, East Asian, and South Asian, determined by parental ethnicity). Retrospective results were verified prospectively using an additional 500 healthy pediatric samples with equal sample size across ethnicities. Ethnic-specific differences were assessed based on statistical significance and biological and analytical variations. Appropriate age-, sex-, and ethnic-specific RIs were calculated. Results Ethnic-specific differences were not observed for 34 biomarkers examined in the retrospective analysis, while 18 demonstrated statistically significant ethnic differences. Among these, seven analytes demonstrated ethnic-specific differences in the prospective analysis: vitamin D, amylase, ferritin, follicle-stimulating hormone (FSH), immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM). Analysis of select NHANES data confirmed CALIPER findings. Conclusions This is the first comprehensive Canadian pediatric study examining ethnic-specific differences in common biomarkers. While the majority of biomarkers did not require ethnic partitioning, ethnic-specific RIs were established for seven biomarkers showing marked differences. Further studies in other populations are needed to confirm our findings.

Establishing reference intervals for sex hormones and SHBG in apparently healthy Chinese adult men based on a multicenter study.

BACKGROUND: Measuring sex hormones is essential in diagnosing health issues such as testicular dysfunction, male infertility and feminization syndrome. However, there are no reports on reference intervals (RIs) in Chinese men. We conducted a nationwide multicenter study to establish RIs for seven sex hormones (luteinizing hormone [LH], follicle-stimulating hormone [FSH], prolactin [PRL], total testosterone [TT], free testosterone [FT], bioavailable testosterone [BAT] and estrogen [E2]), as well as sex hormone-binding globulin (SHBG). METHODS: In 2013, 1043 apparently healthy adult men from five representative cities in China (Beijing, Hangzhou, Guangzhou, Dalian and Urumqi) were recruited; hormones were measured using an automated immunoassay analyzer. Multiple regression analysis (MRA) was performed to identify sources of variation (SVs) that might influence the hormone serum levels. RIs were computed using the parametric method. RESULTS: Dalian and Hangzhou had significantly higher E2 values than other cities; age was a major source of variation for FSH, LH, PRL, SHBG, FT and BAT. FSH, LH and SHBG increased significantly with age, while PRL, FT and BAT decreased with age. TT showed no significant age-related changes. Median (RIs) derived without partition by age were as follows: FSH, 5.6 (1.9-16.3) IU/L; LH, 4.2 (1.6-10.0) IU/L; PRL, 189 (88-450) mIU/L; E2, 85 (4.7-195) pmol/L; SHBG, 29.4 (11.5-66.3) nmol/L; TT, 15.6 (7.4-24.5) nmol/L; FT, 0.31 (0.16-0.52) nmol/L; and BAT, 8.0 (3.7-13.2) nmol/L. RIs were also derived in accordance with between-city and between-age differences. CONCLUSIONS: RIs were established for sex hormones and SHBG in apparently healthy Chinese men in consideration of age.

Lowered reference limits for hCG improve follow-up of patients with hCG-producing tumors.

BACKGROUND: Human Chorionic Gonadotropin (hCG) is produced by germ cell tumors, but can also be elevated in benign conditions such as primary hypogonadism, where hCG is produced by the pituitary gland. In our experience, the reference limits for hCG (Elecsys hCG+ß-assay, Roche Diagnostics), were unnecessarily high and did not reflect levels encountered in clinical practice. We wanted to establish new reference limits to increase the clinical utility of the hCG-assay. METHODS: We analysed hCG in serum samples from a healthy adult population and in a cohort of testicular cancer survivors. The gonadotropins LH and FSH were measured in the cohort and in a selection of the reference population to assess gonadal function. RESULTS: We found low hCG levels for all men and women <45years (97.5 percentiles 0.1 and 0.2IU/L, respectively) from the healthy population (n=795) having normal FSH and LH. Due to assay limitations, we suggest a common reference limit of <0.3IU/L. For the age group ≥45, the 97.5 percentiles in the healthy population were 0.5IU/L for men and 6.0IU/L for women. In all subjects from both the reference population and the cohort (n=732), hCG levels exceeding the reference limit could be fully explained by reduced gonadal function indicated by elevated LH and FSH levels. CONCLUSION: The Elecsys hCG+ß-assay should have lower reference limits than recommended by the manufacturer, with important implications for tumor follow-up. Elevated hCG is rare with intact gonadal function, both in a normal population and among survivors of testicular cancer, and should lead to further investigations when encountered in clinical practice.

Operating characteristics of follicle-stimulating hormone in azoospermic men.

OBJECTIVE: To validate factors predictive of nonobstructive azoospermia (NOA) and to determine the operating characteristics of FSH for predicting NOA. DESIGN: Retrospective cohort study. SETTING: Tertiary care military treatment facility. PATIENT(S): One hundred forty azoospermic males undergoing infertility evaluation. INTERVENTION(S): Standard evaluation included history and physical, hormonal workup, and genetic evaluation. Diagnostic testicular biopsy was offered to characterize patients as obstructive azoospermia (OA) or NOA. MAIN OUTCOME MEASURE(S): Semen volume, semen fructose, FSH, T, E2, PRL, testicular atrophy. RESULT(S): Seventy-eight of 140 azoospermic patients underwent a biopsy. The ability to predict NOA based on logistic regression was statistically significant for FSH and testicular atrophy. On multivariate analysis, only FSH remained predictive of NOA. The area under the FSH receiver operating characteristic curve was 0.847, which is significant. The cut point of FSH with the highest likelihood ratio of predicting NOA on biopsy was ≥12.3 mIU/mL. CONCLUSION(S): FSH remains the best predictor of NOA. With full knowledge of the operating characteristics of FSH in this population, a patient can be properly educated and treatment can be individualized, based on the specific risk associated with that subject's measured FSH.

Standardization of therapeutic, urinary gonadotrophins: an update on the use and availability of International Standards for Menotrophin.

The potencies of therapeutic preparations of gonadotrophins of human, urinary origin, which comprise a heterogenous mix of isoforms with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) bioactivities, are standardized by WHO International Standards (IS). We report here, the evaluation, through an international collaborative study, of a candidate preparation, coded 10/286, to replace the 4th IS, 98/704, for human, urinary FSH and LH (Menotrophin) which has been used for many years for the potency assignment of therapeutic preparations using bioassays. The mean FSH and LH bioactivities of 10/286, determined by in vivo bioassays in terms of 98/704, were 183 IU per ampoule (95% confidence limits 165-202) and 177 IU per ampoule (95% confidence limits 159-197), respectively.

Appropriate cut-off value for follicle-stimulating hormone in azoospermia to predict spermatogenesis.

BACKGROUND: This study was undertaken to determine the optimal cut-off value for FSH to predict the presence of spermatogenesis in patients with non-obstructive azoospermia. METHODS: A total of 206 non-obstructive azoospermic men were enrolled in this prospective study. By using receiver operating characteristic (ROC) curves, we determined the optimal cut-off value for FSH and evaluated whether the test could adequately predict successful sperm retrieval. RESULTS: There were 108 non-obstructive azoospermic patients who had evidence of spermatogenesis (group A) and achieved success in sperm retrieval. Another 98 non-obstructive azoospermic patients (group B) failed in sperm retrieval. The mean value of serum FSH in group B was significantly higher than in group A (28.03 +/- 14.56 mIU/mL vs 7.94 +/- 4.95 mIU/mL, p < 0.01; respectively). The area under the receiver operating characteristic curves were 0.939 +/- 0.02 and a cut-off value of 19.4 mIU/mL discriminated between group A and B with a sensitivity of 70%. The positive predictive value for failed sperm retrieval (group B) can reach 100%. CONCLUSIONS: Elevated plasma levels of FSH of more than 19.4 mIU/mL could be used as a reliable criterion for a trial of sperm retrieval from testes in artificial reproductive techniques.

Analytical identification of additional impurities in urinary-derived gonadotrophins.

Advances in proteomic technology have enabled contaminant proteins to be identified from complex protein mixtures. The purity of two purified urinary gonadotrophin products, human menopausal gonadotrophin (u-HMG) and human FSH (u-hFSH), was compared with a preparation of recombinant human FSH (r-hFSH). After separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis showed that the recombinant preparation contained only FSH, whereas the urine-derived preparations exhibited several non-FSH or LH-related bands. These urinary components were further investigated by a proteomic approach using two-dimensional SDS-PAGE followed by mass spectrometric identification. The proteomic approach detected a total of 23 non-gonadotrophin-related proteins, at variable levels in different batches of the urine-derived preparations. Of these, 16 co-purified proteins have not been previously reported to be present in urine-derived gonadotrophins. These results indicate that the process used to purify urinary gonadotrophins may not remove all non-gonadotrophin proteins. By using a comprehensive proteomic approach, it has been shown that the recombinant FSH preparation has greater purity than either of the urine-derived preparations.

Reference intervals for follicle-stimulating hormone, luteinizing hormone and prolactin in children and young adults on the bioMérieux Mini-Vidas system.

We measured serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin concentrations on a bioMérieux Mini Vidas system in a pediatric population ranging in age from 1 to 19 years. Reference intervals were established separately for females and males, with stratification by age group and by Tanner's pubertal stage. FSH values were higher in females than in males, and were lowest in both sexes of age class 2 (4-8 years), increasing thereafter to the upper limit for stage PIV (females) and stage PV (males). LH values showed a similar pattern of change: concentrations were lowest for class 1 (1-3 years) and class 2 (4-8 years), and highest for stage PII (females) and stage PV (males). No significant difference was observed according to gender. Prolactin values did not differ markedly according to gender or pubertal status.

Biochemical reference intervals for sex hormones with a new AutoDelfia method in aged men.

BACKGROUND: Our aim was to establish sex hormone reference intervals measured with a new AutoDelfia immunoassay method for aged men free of medication and/or conditions known to influence sex hormone levels. METHODS: The reference population consisted of 466 individuals between 64 and 97 years (mean 72 years) and a mean body mass index (BMI) of 26.9 kg/m(2). RESULTS AND CONCLUSIONS: Because age correlated significantly with most sex hormones studied, we calculated reference intervals for three age groups (64-69, 70-74 and > or =75 years). In clinical practice, single ranges can be used for men aged 64 years or over for testosterone, estradiol and follicle-stimulating hormone (FSH) with the AutoDelfia method. For free testosterone and luteinizing hormone (LH), separate reference intervals should be used for men aged 64-74 years and those aged 75 years or over. For sex hormone-binding globulin, two separate reference intervals by age (64-69 and > or =70 years) are also needed for aged men. LH and FSH reference ranges should be judged with caution, because they may be too high due to cases of subclinical hypogonadism included in the reference population.

A comparison between follitropin alpha filled by mass and follitropin alpha filled by bioassay in the same egg donors.

This study assessed the ovarian stimulation characteristics of recombinant follitropin alpha filled by mass (rFSH-fbm) versus recombinant follitropin alpha filled by conventional bioassay (rFSH-bio) in the same egg donor patients. Eleven egg donors, who had two ovarian stimulation cycles for oocyte retrieval (total of 23 cycles), one with rFSH-bio (Gonal-f Multidose) and the second one with rFSH-fbm (Gonal-f RFF), were evaluated. The protocol of ovarian stimulation was exactly the same in both cycles, consisting of GnRH suppression (luteal phase) followed by exclusive stimulation with rFSH. Despite no differences in the number of days of rFSH treatment and in the total dosage of rFSH administered, the number of follicles >14 mm and the number of oocytes retrieved were significantly higher in the rFSH-fbm group (P = 0.01 and 0.04 respectively). The mean oestradiol peak values showed a trend in favour of rFSH-fbm (3123 versus 2405 pg/ml respectively). These results suggest that the consistency in dosing provided by follitropin alpha filled by mass as opposed to follitropin alpha filled by bioassay offers added value for the ovarian stimulation of oocyte donor patients.

Comparative assessment of the consistency and quality of a highly purified FSH extracted from human urine (urofollitropin) and a recombinant human FSH (follitropin alpha).

The revolutionary development of biotechnology-derived therapeutic proteins has provided the expected improvements in quality, purity and consistency, as demonstrated in recombinant human FSH (rhFSH). However, the development of urine-derived gonadotrophins has not always shown comparable improvements. More recently, highly purified urine-derived FSH (uFSH-HP) products have become widely available. The relative purity, level of urine-derived contaminants, and consistency of one such highly purified human uFSH (uhFSH) (urofollitropin) has been assessed and directly compared with rhFSH (follitropin alpha). It has been demonstrated that the highly purified urofollitropin contains variable levels of urine-derived contaminant proteins and demonstrates a variable level of FSH purity, FSH isoforms, and delivered dose. These variable factors may contribute to the control of ovarian stimulation. The relative purity, variable consistency and the presence of contaminants indicates that the urofollitropin is, at best, a partially purified uFSH that is not able to meet the quality attributes of follitropin alpha (rhFSH).

High-resolution reference ranges for estradiol, luteinizing hormone, and follicle-stimulating hormone in men and women using the AxSYM assay system.

OBJECTIVES: High-resolution reference ranges are essential for reproductive hormones due to the significant day-to-day variation seen with these analytes. DESIGN AND METHODS: The performance of AxSYM assays for estradiol, luteinizing hormone (LH), and follicle-stimulating hormone was evaluated. RESULTS: These studies validate the performance of each assay, permitting high-resolution reference ranges to be established. CONCLUSIONS: The high-resolution reference range data provided herein for both normally cycling females and males should be applicable to a wide variety of clinical situations.

Factors influencing response to ovarian stimulation.

Ovarian stimulation is an integral part of assisted reproduction treatments. Ovarian response to gonadotrophin treatment, besides other factors, determines the outcome of treatment, as the number and quality of oocytes retrieved are related to the chance of achieving a pregnancy. A number of factors have been identified that might predict ovarian response, such as age of the patient and antral follicle count. In addition, it has been shown that genetic factors such as the patient's FSH-receptor genotype also determine individual response to FSH treatment. Besides patient-related factors, the choice of drugs for ovarian stimulation plays a significant role. Until recently, biopotency of gonadotrophin preparations was tested by an in-vivo bioassay with an intrinsic variability up to 20%. Due to a superior manufacturing technique, follitropin alpha can now be filled by mass. This allows assessment of FSH with a precise SE-HPLC assay and variability of the FSH content between production lots has now been estimated at 1.6%. Results of recent studies indicate that treatment with follitropin alpha filled by mass results in consistent ovarian response, fewer treatment days and fewer cancelled cycles. This is an important step towards further minimizing drug-related variability of ovarian response to FSH treatment.

Reactivity of different LH and FSH standards and preparations in the world health organization matched reagents for enzyme-linked immunoassays of gonadotrophins.

The immunoreactivity of various LH and FSH calibration standards and recombinant preparations in the enzyme-linked immunoassay (EIA) systems for gonadotrophins developed for the Special Programme of Research in Human Reproduction of the World Health Organization (WHO) were compared. The preparations tested included three LH and two FSH pituitary standards (calibrated against LH 80/552 and 68/40 and FSH 78/549 respectively) provided with the EIA or radioimmunoassay WHO matched reagent kits, the pituitary preparation LER-907, and recombinant human LH (rhLH) and FSH (rhFSH). Simultaneous curve fitting of the EIA dose-response curves revealed no significant differences among the slopes generated by the WHO LH standards and LER-907; in contrast, no parallelism was found between the curves of rhLH and the pituitary-derived LH standards. No significant differences were found among the slopes of the curves elicited by the pituitary and recombinant FSH preparations. Each LH preparation exhibited a high degree of charge heterogeneity. Considerable variations in charge isoform distribution among the WHO LH standards, rhLH and LER-907 were also evident. In contrast, the FSH preparations were less heterogeneous and exhibited minor differences in charge distribution. Despite the existing differences in charge isoform distribution, all the pituitary-derived preparations as well as rhFSH seem appropriate for using as calibration standards in this particular EIA system.

Superovulation induction in Japanese Black cattle by a single intramuscular injection of hMG or FSH dissolved in polyvinylpyrrolidone.

The efficacy of a single intramuscular dose of 450 or 600 international units (IU) of human menopausal gonadotropin (hMG) or 30 mg of follicle stimulating hormone (FSH), each dissolved in 30% polyvinylpyrrolidone K-30 (PVP), for superovulation treatment was compared to that of superovulation induction by administration of a total dose of 600 IU hMG given in declining doses twice daily over a 3-day period. A total of 48 Japanese Black cows were used for the investigation. Oestrus was observed within 60 h after PGF2alpha administration in all cows in the hMG groups. In the hMG group that received a single dose of 600 IU hMG (n = 12), oestrus was observed less than 36 h after treatment in one cow. In contrast, oestrus was not observed in 3 of the 12 cows (25%) in the FSH group. Neither the average number of recovered ova/embryos nor the number of transferable embryos per collection differed significantly among the hMG groups. However, the average number of transferable embryos was not significantly higher in cows treated with a single dose of 600 IU of hMG than in cows treated with a single 30 mg dose of FSH (7.5+/-4.5 vs. 2.1+/-2.8). The number of cows from which more than three excellent grade embryos were collected was highest in the group that received a single dose of 600 IU hMG (9/12, 75%) and lowest in the group that received a single 30 mg dose of FSH (2/9, 22%). The differences between groups in the percentages of cows with three or more excellent embryos between treatments were not statistically significant. The proportion of recovered ova/embryos classified as excellent was highest in the group that received 600 IU hMG in declining doses and lowest in the group that received a single 30 mg dose of FSH (55.2% vs. 30.2%; P < 0.05). The recovery rate of unfertilized ova was lowest in the group that received a single dose of 600 IU hMG and highest in the group received a single 30 mg dose of FSH (18.3% vs. 48.8%; P < 0.05). Although the differences in recovery results between the groups were not statistically significant, the recovery rates in hMG groups were higher than that the FSH group. These findings suggest that superovulation can be induced adequately in Japanese Black cows using one injection of 450 to 600 IU hMG dissolved in PVP.

Characterisation, calibration and comparison by international collaborative study of international standards for the calibration of therapeutic preparations of FSH.

Therapeutic preparations of FSH, used primarily for treatment of infertility, are calibrated by in vivo bioassay against international standards (IS) derived from different sources deemed appropriate to their use according to pharmacopoeial monographs. Menotrophins, which have been used for several decades to treat infertility, have been calibrated against the IS for urinary FSH and LH (ISU) but are now being replaced by highly purified urinary FSH or rDNA-derived FSH (rFSH). The aim of this study was to evaluate two preparations of human rFSH and one preparation of highly purified urinary FSH as candidate WHO IS for bioassay in an international collaborative study by 27 laboratories in 12 countries, and to characterise them in a range of in vitro bioassays and immunoassays. The biological activity of the three candidate standards was confirmed by all laboratories using all assays contributed to the study. Dose-response relationships by in vivo bioassay for any of the candidate standards did not differ significantly from that for the ISU. Dose-response relationships obtained in in vitro bioassays and immunoassays were also broadly similar among these preparations although dose-response lines for some preparations appeared to be non-parallel in some immunoassays. For each of the three candidate IS, estimates of the relative potency in terms of ISU by in vivo bioassay did not differ significantly between laboratories. In contrast estimates by immunoassays and in vitro bioassays showed significant differences between laboratories. Estimates of relative potency of the highly purified candidate IS materials in terms of one another exhibited less inter-laboratory variability than estimates in term of ISU. Each of the candidate standards showed adequate stability to serve as an IS. On the basis of the results of this study rFSH (code 92/642) was established as the first IS for FSH, human, recombinant for bioassay with an assigned unitage of 138 IU per ampoule and urinary FSH (code 92/512) was established as the first IS for FSH, human, urinary (urofollitropin) for bioassay with an assigned unitage of 121 IU per ampoule, based on their respective calibration by in vivo bioassay in terms of ISU. These assignments of unitage maintain continuity of unitage for preparations in therapeutic use and also appear to be consistent with one another.

FSH bioactivity in commercial preparations of gonadotropins.

During the past 2 decades, commercial preparations of FSH have been extensively used to superovulate cattle. The problems that have been encountered in superovulation of cattle include high variability in the ovulation rate and subsequent yield of viable embryos. The lack of predictability in superovulatory trials has been attributed to difficulties in standardizing the potency of commercial FSH preparations. Traditionally, FSH potency has been tested in bioassays that utilize specific responses in whole animals or primary cell cultures. Whole animal bioassays lack sensitivity, while primary cell culture bioassays, which use fresh cells, have inherent variability within each preparation. An FSH bioassay that employed a stable chimeric cell line expressing the human FSH-R was used to provide an accurate measurement of FSH bioactivity. The hormonal potency of 2 commercial preparations of FSH used to superovulate cattle was determined using FSH immuno- and bioassays. Commercial FSH preparations differed in potency. One commercial product, prepared in 4 different years, showed no difference in the immunoactive levels of FSH. In the same product stored under identical conditions, FSH bioactivity varied from year to year. There was variability in FSH bioactivity both between and within commercial products. The lack of correlation between bioactivity and immunoactivity of commercial FSH preparations may explain, in part, the variability observed in superovulation of cattle.

Recombinant follicle-stimulating hormone (follitropin beta, Puregon) yields higher pregnancy rates in in vitro fertilization than urinary gonadotropins.

OBJECTIVE: To assess ongoing pregnancy rates (PRs) in IVF after treatment with recombinant FSH (follitropin beta, Puregon; NV Organon, Oss, The Netherlands) as compared with urinary gonadotropins. DESIGN: A combined analysis of three prospective, multicenter, randomized, comparative trials. SETTING: Twenty-five IVF centers in 13 countries. PATIENT(S): Six hundred ninety-seven infertile women receiving recombinant FSH and 463 women receiving hMG or urinary FSH and undergoing one cycle of controlled ovarian hyperstimulation and IVF-ET. INTERVENTION(S): A center-based and study-based analysis weighing the treatment differences in individual centers and studies, respectively. MAIN OUTCOME MEASURES(S): Pregnancy rate at least 12 weeks after ET per started cycle. RESULTS(S): In the center-based analysis, the ongoing PR was 22.9% for recombinant FSH and 17.9% for urinary gonadotropins. The 5.0% treatment difference (95% confidence interval [CI], 0.2% to 9.7%) was significant. When the results of the cryoprogram were included, the treatment difference increased to 6.4% (95% CI, 1.4% to 11.3%). Also in the study-based analysis, significantly higher PRs were seen after follitropin beta treatment. CONCLUSION(S): Follitropin beta (Puregon) used for controlled ovarian hyperstimulation in IVF yields significantly higher PRs compared with urinary gonadotropins.