Hormone Measurements Research Articles

Near-Infrared Autofluorescence or Intraoperative Parathyroid Hormone Determination as a Surgical Support Tool in Primary Hyperparathyroidism: Too Close to Call?

Background: This study investigates the use of near-infrared fluorescence imaging (NIFI) as an alternative to intraoperative parathyroid hormone (ioPTH) measurement in patients with primary hyperparathyroidism (PHP) due to parathyroid adenoma (PA) with two preoperative imaging examinations in agreement on the position of the altered parathyroid gland. Methods: Fifty patients who underwent minimally invasive parathyroidectomy (MIP) from March 2021 to April 2024 were enrolled. MIPs utilised both NIFI and ioPTH, comparing the time to adenoma excision with NIFI against the total surgical time, including ioPTH measurement wait time. Results: Results showed successful MIPs in all patients, with a mean excision time of 44.7 min (SD ± 25.2) and a total procedure time of 85.5 min (SD ± 37.1). The median of the duration of the surgical procedure until adenoma excision was 35.0 min (IQR 38.8), while the median duration of the entire procedure was 74.5 min (IQR 40.5). This difference in medians is statistically significant using the Wilcoxon non-parametric test for paired samples (p < 0.001). The average wait for ioPTH results post-excision was 37 min (SD ± 12.2). Three fluorescence patterns were identified: “cap” (46%), heterogeneous (30%), and homogeneous (24%). Conclusions: If preoperative imaging confirms PA location, NIFI could replace ioPTH reducing surgical time without compromising success rates.

ULTRASOUND APPEARANCE OF THE ENDOMETRIUM AND CLINICAL CORRELATION IN A CASE OF AMENORRHEA

The subject of this paper is the presentation of a clinical case of amenorrhea in a young woman with regular menstrual cycles and a positive pregnancy test. The ultrasound examination of the uterus at 5 weeks and 1 day after the last menstruation showed the absence of a gestational sac and a thin and hypoechoic endometrium. Since the pregnancy test was positive, the ultrasound examination was repeated after 7 days. During this interval, the patient presented a hemorrhage. The ultrasound performed 4 days after showed a thin endometrium with medium echogenicity. Repeating at 48 hours the measurement of the human chorionic gonadotrophin hormone showed low values that decreased, which correlated with the clinical and ultrasound aspects confirmed the diagnosis of early pregnancy failure.

The Effects of Single Testosterone Administration and Stress Induction on Steroid Hormone Levels in Hair

Hair steroid analysis is increasingly recognized for its ability to capture cumulative hormone secretion, thought to reflect an individual’s response to long-term environmental conditions. Despite its growing use, the influence of single, isolated events on hair steroid concentrations remains underexplored. Our study therefore examined the effects of a single-dose transdermal testosterone administration (150mg) and acute laboratory stress induction on hair testosterone and cortisol levels in a randomized, between-subject, placebo-controlled, and double-blind design. Liquid chromatography-tandem mass spectrometry analysis revealed a significant increase in hair testosterone levels seven weeks post-testosterone administration, underscoring the efficacy of hair analysis in detecting single-use hormone intake. Moreover, we observed a significant elevation in hair cortisol levels seven weeks post-exposure to the laboratory somatic stressor (Cold pressor test), highlighting the efficacy of hair analysis in capturing experimentally induced hormonal responses. The exploration of contextual factors revealed that individuals in committed relationships exhibited lower levels of both hair cortisol and testosterone compared to those who were single or in uncommitted relationships.Our study provides new insights into the sensitivity of hair analysis for detecting hormonal changes following single-dose hormone administration and experimentally induced short-term stress events. The exploratory findings emphasize the importance of individual contextual factors in influencing hair hormone concentrations and lay the groundwork for further investigation into the dynamics of cumulative hair hormone measurements.

The predictive role of intraoperative parathyroid hormone measurement on postoperative parathyroid function in patients undergoing total thyroidectomy

Hypocalcemia is a common complication of thyroidectomy, particularly total thyroidectomy. The higher incidence of hypocalcemia following total thyroidectomy is primarily due to inadvertent damage to the parathyroid glands during surgery. This study aims to investigate the predictive value of intraoperative parathyroid hormone (IOPTH) in determining hypocalcemia during hospitalization and the recovery of parathyroid function after total thyroidectomy, as well as the factors that may influence IOPTH. In this retrospective study, we analyzed a cohort of 164 patients who underwent total thyroidectomy at our institution between 2018 and 2019. IOPTH was measured either 15 min after bilateral thyroidectomy combined with area VI lymph node dissection, or 15 min after bilateral thyroidectomy alone. We plotted ROC curves for IOPTH and ΔPTH% in relation to hypocalcemia during hospitalization and recovery of parathyroid function. Additionally, we explored risk factors for the development of hypocalcemia during hospitalization and factors that may influence IOPTH. IOPTH and ΔPTH% demonstrated good specificity and sensitivity for predicting hypocalcemia during hospitalization and for assessing recovery of parathyroid function. Lower preoperative PTH levels, chronic lymphocytic thyroiditis, and intraoperative parathyroid auto-transplantation were identified as risk factors for IOPTH < 15 pg/mL. IOPTH and ΔPTH% are predictive of hypocalcemia during the postoperative hospital stay and recovery of parathyroid function. Patients with chronic lymphocytic thyroiditis, intraoperative parathyroid auto-transplantation, and low preoperative PTH levels should be closely monitored for the recovery of parathyroid function after surgery.

Phenotypic impact of CFTR mutations on male reproductive tract agenesis in a Chinese cohort with congenital absence of the vas deferens.

To investigate the genotype-phenotype correlations of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and their impact on male reproductive tract development in a cohort of Chinese patients with congenital absence of the vas deferens (CAVD). A total of 121 Chinese CAVD patients underwent genetic testing for CFTR and ADGRG2 mutations, semen analysis, scrotal and transrectal ultrasound examinations, and reproductive hormone measurements. The genotype-phenotype correlations were analyzed, focusing on the impact of CFTR variants on the presence or absence of the epididymis, vas deferens, seminal vesicles, and other related structures. CFTR mutations were identified in 72.7% (88/121) of CAVD patients, with the IVS9-5T variant being the most prevalent (54.5%, 66/121). Six novel CFTR variants (CFTR: L218Ffs*15, V1007Ffs*40, V938M, A566V, S605P, H949P) were identified in Chinese men. Patients with CFTR homozygous IVS9-5T variants had a significantly lower rate of epididymal absence compared to those with one 5T and one non-5T variant or two non-5T variants (p = 0.016). Notably, patients carrying at least one non-5T variant were associated with an 8.17-fold increased risk of epididymal partial absence compared to those having the homozygous 5T mutation (95% confidence interval 1.52-59.58, p = 0.009). This study provides novel insights into the genotype-phenotype correlations of CFTR variants in Chinese CAVD patients, highlighting the differential impact of 5T and non-5T variants on male reproductive tract development. These findings provide additional information that may be helpful for genetic counseling, clinical management, and the development of personalized diagnostic and therapeutic strategies for CAVD patients.

Germ Cell Dysfunction is Universal in Male Patients with β-Thalassemia Following Hematopoietic Stem Cell Transplantation During Childhood and Adolescence.

To assess gonadal function in adolescent male patients with β-thalassemia who underwent successful hematopoietic stem cell transplantation (HSCT) during childhood or adolescence. Fifty-two male patients with β-thalassemia, aged ≥10 years, who had undergone HSCT ≥2 years were included. Clinical data, such as age, genital Tanner (GT) stage at HSCT and enrollment, serum ferritin levels, and cumulative doses of alkylating agents, were collected. Gonadal function was evaluated through measurements of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, inhibin B levels, and semen analysis. Age at enrollment and HSCT were 17 (10-31) and 9 (1-19) years, respectively. The duration from HSCT to enrollment was 7.5 (2-20) years. Of 52 patients, 46 (88%) exhibited Sertoli cell dysfunction. Thirty-one patients had relatively small testes for their GT stage, 34 of 44 with GT V had elevated FSH of ≥5 IU/L, and 20 of 49 with GT stages II-V had low serum inhibin B levels. None of the patients with GT stage V showed Leydig cell dysfunction or gonadotropin deficiency. Serum FSH ≥8 IU/L showed the best diagnostic accuracy for detecting oligo- and azoospermia. All 39 patients who underwent semen analysis had >1 abnormal parameters. Having relatively small testes for GT stage and serum FSH ≥8 IU/L were associated with oligo- and azoospermia (p <0.01). Male patients with β-thalassemia after HSCT experienced universal spermatogenesis impairment and frequent Sertoli cell dysfunction but their Leydig cell function appears to be preserved. The high likelihood of future subfertility should be informed before HSCT.

Longitudinal assessment of thyroid function in dogs with hypoadrenocorticism: Clinical outcomes and prevalence of autoantibodies.

Knowledge about primary hypoadrenocorticism coexisting with immune-mediated thyroiditis (Schmidt's syndrome) in dogs is limited. To evaluate thyroid function in dogs with naturally occurring hypoadrenocorticism before and during treatment. Sixty-six client-owned dogs. Measurement of canine thyroid stimulating hormone (cTSH), total thyroxine (T4), free thyroxine, and autoantibodies against thyroglobulin, T4, and total triiodothyronine. Thirty-eight dogs were assessed before and 28 during treatment. Follow-up data were available for 24/38 and 17/28 dogs, with median follow-up duration of 3.8 years (range, <1.0-8.8 years) and 4 years (range, 1.1 weeks to 10.5 years), respectively. Canine thyroid stimulating hormone was above the reference range at the time of diagnosis of hypoadrenocorticism in 10 of 38 dogs but decreased into the reference range in 7 for which follow-up data was available. Hypothyroidism was confirmed in 5 dogs at a median age of 11 years (range, 7-15 years). In 4 dogs, the condition was diagnosed after a median treatment duration of 5.75 years (range, 2.6-10 years), while in 1 dog, the diagnosis was made concurrently. One dog had detectable thyroid autoantibodies. Hypothyroidism occurs as a rare concurrent condition in dogs with hypoadrenocorticism, potentially at any phase of treatment. Close monitoring of cTSH levels in these dogs could be beneficial, as early changes might indicate the onset of hypothyroidism. The low prevalence of detectable thyroid autoantibodies suggests that nonimmune mechanisms might contribute to thyroid dysfunction.

Trends in the use of thyroid diagnostics and treatments between 2008 and 2019 in Germany

Following implementation of a national prevention program against iodine deficiency-related disorders in 1993 in Germany, the prevalence of thyroid disorders declined, but aggregated drug dispensation data suggested a recent increase in thyroid hormone use. We aimed to systematically investigate trends in the use of thyroid diagnostic and treatment measures between 2008 and 2019 in Germany. Using the GePaRD database (claims data from ~ 20% of the German population), we determined the annual age-specific and age-standardized prevalence of the use of thyroid diagnostics (thyroid-stimulating hormone (TSH) measurement, thyroid ultrasound, scintiscan, biopsy) and treatment (medication, surgery, radioiodine therapy) from 2008 to 2019 stratified by sex. Between 2008 and 2019, the age-standardized prevalence of TSH measurement increased by 44% in males (from 165 to 238 per 1,000 persons) and by 31% in females (from 134 to 176 per 1,000). The prevalence of thyroid ultrasound use showed no clear time trend, while there was a decrease in the prevalence of scintiscan use (males/females: decrease by 42/43%) and biopsy (males/females: decrease by 42/34%). The prevalence of levothyroxine use increased by ~ 31% both in males (from 36 to 47 per 1,000) and females (from 134 to 175 per 1,000), while the prevalence of thionamide use, iodine use, thyroidectomy, and radioiodine therapy decreased. Our study suggests a continued decline in the prevalence of clinically significant thyroid disorders between 2008 and 2019. The substantial increase in the prevalence of TSH measurement and levothyroxine use during this time period might indicate overuse of diagnostics and overtreatment, warranting further investigation.

Identification of novel inhibitors of plant GH3 IAA-amido synthetases through molecular docking studies.

Auxins play a critical role in several plant developmental processes and their endogenous levels are regulated at multiple levels. The enzymes of the GRETCHEN HAGEN 3 (GH3) protein family catalyze the conjugation of amino acids to indoleacetic acid (IAA), the major endogenous auxin. The GH3 proteins are encoded by multiple redundant genes in plant genomes, making it difficult to perform functional genetic studies to understand their role in auxin homeostasis. To address these challenges, we used a chemical approach that exploits the reaction mechanism of GH3 proteins to identify small molecule inhibitors of their activity from a defined chemical library. The study evaluated receptor-ligand complexes based on their binding energy and classified them accordingly. Docking algorithms were used to correct any deviations, resulting in a list of the most important inhibitory compounds for selected GH3 enzymes based on a normalized sum of energy. The study presents atomic details of protein-ligand interactions and quantifies the effect of several of the identified small molecule inhibitors on auxin-mediated root growth processes in Arabidopsis thaliana. The direct effect of these compounds on endogenous auxin levels was measured using appropriate auxin sensors and endogenous hormone measurements. Our study has identified novel compounds of the flavonoid biosynthetic pathway that are effective inhibitors of GH3 enzyme-mediated IAA conjugation. These compounds play a versatile role in hormone-regulated plant development and have potential applications in both basic research and agriculture.

Recurrence patterns in differentiated thyroid cancer (DTC) following adjuvant radioiodine therapy.

With an increasing incidence of differentiated thyroid cancer (DTC) diagnosis, questions emerge about the optimal duration of follow-up for detecting recurrent disease and its outcomes. The objective of this retrospective research was to assess the clinical course of differentiated thyroid cancer after radioiodine adjuvant treatment in patients monitored over an extended period. Special attention was paid to the analysis of the time from treatment to recurrence. We also assessed patient outcomes after recurrence. A total of 650 patients with DTC after total/near-total thyroidectomy and adjuvant radioiodine post-recombinant human thyrotropin (post-rhTSH) stimulation were evaluated. All patients were followed up with neck ultrasound, serum thyroid-stimulating hormone (TSH), thyroglobulin (Tg), and antithyroglobulin antibody (anti-Tg) measurements at intervals of 6 to 18 months. Only structural recurrences were considered. They were defined as locoregional recurrence confirmed by biopsy or distant metastases [confirmed by computed tomography (CT) or magnetic resonance imaging (MRI), or abnormal foci on radioiodine scintigraphy or 18F-gluorodeoxyglucose positron emission tomography [18 F] FDG-PET scan], regardless of thyroglobulin (Tg) or anti-Tg levels. The median follow-up was 12 years (5-15.5). Structural recurrence was observed in 47 out of 650 patients (7%). All but 3 locoregional recurrences were suitable for surgery. The median time to structural recurrence was 16 months, with only 9 (1.4%) patients presenting with recurrence after more than 60 months. At the time of the database closure, 601 patients (92%) had an excellent response, including 20 out of 47 (42%) patients with structural recurrence. Eighty-one out of 650 patients had died (12.5%) before the database closure. The median age at the last follow-up of the patients who died was 72 years (range 20-88). A second recurrence was diagnosed in 10 out of 650 patients (1.5%), corresponding to 21% (10 out of 47) of patients who had already experienced a recurrence. The median time from radioiodine (RAI) therapy to the second structural recurrence was 108 months. Structural recurrences in DTC are uncommon, with most patients showing a favourable response to treatment. Improved understanding of recurrence timing may define the duration of patient surveillance at reference centres that can be safely discontinued after 5 years in low- and intermediate-risk groups, as indicated in our study.

The combined analysis of transcriptome and phytohormone provides new insights into signaling mechanism for lateral root formation of tea plant (Camellia sinensis)

The growth and development of lateral roots (LRs) determine the nutrient absorption and environmental adaptability of tea plant, playing a crucial role in the economic efficiency of tea plantations. However, the molecular mechanisms underlying the formation of LRs in tea plant remain unreported. In this study, we established a LRs induction system in tea plants by exogenously adding N-1-naphthylphthalamic acid (NPA) and naphthaleneacetic acid (NAA). The molecular mechanisms of LRs formation were analyzed through transcriptomics and endogenous hormone measurements. Results showed that NAA treatment could counteract the inhibitory effect of NPA on LRs formation and promote the generation of LRs in tea plant. The addition of exogenous NAA reduced the levels of auxin and zeatin in the root tips while increasing the content of abscisic acid (ABA). Transcriptomic analysis at four time points (2, 6, 12, and 24 h) after NAA vs. MOCK treatment revealed 78 different expression genes (DEGs) associated with plant hormone signaling pathways. Among these, the auxin signaling pathway was the most significant in responding to the LRs formation in tea plant. Weighted Gene Co-Expression Network Analysis (WGCNA) identified the Mitogen-Activated Protein Kinases (MAPK) signaling pathway as responsive to NAA treatment, participating in the formation of LRs in tea plant. This study provides potential signaling pathways for the investigation of LRs formation in tea plant and identifies candidate genes for further research into tea plant root system architecture (RSA).

No effect of infant nursery rearing on laboratory rhesus monkey dams’ social behavior or long-term cortisol profiles

While the behavioral and physiological impacts of nursery rearing in laboratory-housed infant monkeys have been well characterized, to date no studies have examined the impact on nonhuman primate dams of their infants being removed for rearing in the nursery. Despite the lack of evidence on the topic, anti-animal research groups often cite mother-infant separation and infant nursery rearing (NR) as a welfare concern for laboratory monkey mothers. As such, important policy decisions regarding research activities may result without adequate evidence. Therefore, we designed this study to examine behavioral and hormonal responses in laboratory monkey dams, who were part of independent long-term longitudinal studies, in response to their infants being NR or mother-peer-reared (MPR). We assessed social, self-care, environmental exploration, and abnormal behaviors for 30 days before and 30 days after parturition in rhesus monkey dams (Macaca mulatta, N=49). Infants were randomly assigned to be either NR (N = 27) or MPR (N = 22). We also analyzed hair samples for cortisol concentrations three times across the study period (Pregnancy, Neonatal Period, Peak Lactation). Dams of NR infants showed no gross behavioral differences relative to dams of MPR infants (all p’s>0.05). Dams of MPR infants showed expected increases in social grooming and social contact, and concomitant decreases in foraging and locomotion, in the 30 days post-partum compared to the 30 days pre-partum (p<0.01). Dams whose infants were NR or MPR showed no differences in hair cortisol concentrations across the study period (p>0.05). We conclude that, with respect to the behavioral and endocrine measures we assessed, nursery rearing of their infants is not detrimental to the welfare of laboratory-housed macaque dams.

Neonatal reference intervals for serum steroid hormone concentrations measured by LC-MS/MS.

Congenital adrenal hyperplasia (CAH) is a rare, inherited disorder of adrenal steroid synthesis. In many countries it is part of the neonatal screening program enabling early diagnosis and treatment. In case of an abnormal neonatal screening result or when other differences of sexual development (DSD) are suspected, measurement of serum steroid hormones using liquid chromatography coupled to mass spectrometry (LC-MS/MS) is needed for further diagnosis. However, reliable age- and sex-specific reference intervals (RIs) for serum steroid hormones during the neonatal period are missing. We therefore aimed to establish LC-MS/MS based RIs for serum steroid hormones in neonates. Serum was obtained from healthy term neonates at two time points: 130 samples at day 3-8 (T1, time of the neonatal screening) and 126 samples at day 13-15 (T2,two weeks old). Concentrations of cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, testosterone, androstenedione, and 17-hydroxyprogesterone (17-OHP) were measured using LC-MS/MS. RIs (in nmol/L) were established for T1 and T2: cortisone (19.3-215;18.0-212), cortisol (10.0-407;8.4-446), corticosterone (<31;<50), 11-deoxycortisol (0.73-4.6;0.70-3.6), 17-OHP (<4.9;<5.1), androstenedione (0.3-1.8;0.3-2.7), 11-deoxycorticosterone (<0.2;<0.2), and 21-deoxycortisol (<1;<1), respectively. Testosterone differed between boys and girls: RIs at T1 and T2 for boys were 0.27-4.3 and 0.63-13.9, and for girls<0.30 and <0.47, respectively. We established LC-MS/MS based RIs for cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, testosterone, androstenedione, and 17-OHP in neonates in the first and second week of life.

Associations of body composition measures with circulating insulin-like growth factor-I, testosterone, and sex hormone-binding globulin concentrations in 16,000 men

BackgroundAdiposity is positively associated with risk of some cancer sites and other health conditions in men; however, it is unclear if endogenous hormones play a role in these associations. We examined how body composition, measured from magnetic resonance imaging (MRI) and common measures of adiposity (e.g., body mass index (BMI)), are related to hormone concentrations in men from the UK Biobank study.MethodsUp to 16,237 men with available body composition data (including visceral, subcutaneous, and liver fat, muscle fat infiltration (MFI), lean tissue, and common adiposity measures) and serum hormone measurements (insulin-like growth factor-I (IGF-I), total testosterone, sex hormone-binding globulin (SHBG), and calculated free testosterone) were included. Multivariable-adjusted linear regression models were used to determine the geometric mean hormone and SHBG concentrations across categories of each exposure.ResultsCommon measurements of adiposity were highly correlated with MRI measures of central and total adiposity (r = 0.76–0.91), although correlations with ectopic fat (liver fat and MFI) were lower (r = 0.43–0.54). Most adiposity measurements showed an inverse U- or J-shaped association with circulating IGF-I and free testosterone; however, MFI was linearly inversely associated, and lean tissue volume was positively associated with both IGF-I and free testosterone concentrations. All body composition measures were inversely associated with total testosterone and SHBG concentrations (relative geometric mean difference between Q5 vs. Q1: 20–30%).ConclusionOur results show that common adiposity and most MRI measures of adiposity relate similarly to serum hormone concentrations; however, associations with ectopic fat (particularly MFI) and lean tissue were different.

7864 Accurate Machine Learning-Based Diagnosis of At-Risk MASH and MASLD Subtypes Using Categorical Gradient Boosting and Select Clinical, Biochemical, and Metabolomic Measurements: Building Highly Robust Models With Few Variables Through a Multinational, Multi-Center, Biopsy-Proven Cohort

Abstract Disclosure: K. Stefanakis: None. E. Axarloglou: None. C.S. Mantzoros: None. Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by intrahepatic lipid accumulation, may precipitate steatohepatitis (MASH) and liver fibrosis, and is a significant cardiometabolic risk factor. Liver biopsy, though invasive and costly, is the diagnostic gold standard, thus creating an unmet clinical need for accurate and reliable non-invasive diagnostic methods to diagnose MASLD, particularly the combination of MASH and significant fibrosis, classified as “at-risk MASH” and known to markedly increase morbidity and mortality. We aimed to leverage novel machine-learning (ML) algorithms to create lightweight but highly precise models that may accurately diagnose at-risk MASH and related histological outcomes through a top-down approach. This is a study of 443 participants from two Gastroenterology-Hepatology Clinics (Greece, Australia) and one Metabolic Surgery Clinic (Italy), including healthy controls and MASLD patients across the entire spectrum of the disease. All participants underwent liver biopsies, anthropometric, standard biochemical and additional hormonal measurements, and global serum metabolomics using ultra-high-performance liquid chromatography-tandem mass spectrometry, followed by robust batch correction and quality control, eventually identifying 687 known and 152 unknown metabolites. The dataset was randomly divided into a discovery (n=353) and a validation cohort (n=90) ensuring a balanced distribution of at-risk MASH and clinic. Our main ML pipeline implemented Categorical Gradient Boosting Machines using the CatBoost package for R. We pre-specified domain knowledge variables and implemented recursive feature elimination, among additional techniques, to build models through an iterative, algorithm-based process. The models were tested in the validation and entire cohort using k-fold and leave-one-out cross-validation with random resampling, and further assessed on relevant subgroups (MASLD status, obesity, diabetes, clinic, country). The main ML model consists of three routine clinical and biochemical variables, supplemented by two aminoacid metabolites (full model to be shown), and can detect the presence of at-risk MASH with a mean AUROC of 93% and mean accuracy of 92% in the validation cohort, and AUCS&amp;gt;93% with very high sensitivities and specificities per J-statistic in all subgroups. We likewise present ML models for the detection of MASLD, MASH, and fibrosis grades, with AUCs of over 90% in the validation cohort and all subgroups. Our models significantly outperform all applicable biomarker-based indices per DeLong’s tests. Further enhancement with lipidomic and proteomic variables will fully delineate the circulating biochemical snapshot of MASLD and likely lead to even more accurate, similarly structured models. Presentation: 6/1/2024

7701 Comparative Analysis of Dried Blood Spot and Serum ELISAs for AMH, FSH, and LH in Patients Seeking IVF Treatment

Abstract Disclosure: T. Kumar: None. R. Chauhan: None. B. Kalra: None. A.S. Patel: None. S. Chauhan: None. A. Kumar: None. Relevance: Dried Blood Spot (DBS) technology has not been widely implemented in the diagnostic industry due to its limitations in obtaining reliable, quantitative data from small blood samples. However, recent advancements in methods, i.e., sensitive antibodies, homogeneous and reproducible paper quality, and painless needles have renewed the interest in the DBS collection technique. Objective: The aim of the study is to validate the hormone measurements using DBS technology and compare the correlation matrix between AMH, LH, FSH, inhibin B, Antral Follicle Counts (AFC), age, and BMI in patients seeking infertility workup. Materials and Methods: Matched serum and DBS samples from thirty-nine patients between the ages of 18 and 45 years were collected on day 3 of the cycle. The subjects were visiting the clinic for initial fertility workup and subjects on medications for ovarian suppression were excluded. The samples were tested on Ansh Labs DBS AMH (AL-129), FSH (AL-187), and LH (AL-190), PCOCheck AMH (N-N terminus, AL-196), picoAMH (C-N terminus, AL-124-r), US Inhibin B (AL-195) ELISAs. Baseline AFC obtained via transvaginal ultrasound, BMI, and age were collected. Spearman’s correlations, and regression analysis using Passing and Bablok were calculated to examine the relationship between AFC, AMH, inhibin B, FSH LH, age, and BMI. Results: DBS samples were highly correlated to their matched serum specimens. The slope and Spearman’s rank correlation (rs) between matched serum and DBS specimens for AMH, FSH, and LH were 1.17x, 1.07x, 1.07x, and 0.98, 0.93, and 0.97, respectively. DBS AMH strongly correlated with AFC. The Spearman’s rank correlation (rs) between AFC and DBS AMH, DBS LH, and DBS FSH were 0.80, -0.11, and -0.31, respectively. A strong correlation was observed between age and AMH, LH and FSH. A weak correlation was observed between BMI and AMH, LH and FSH. The Spearman’s rank correlation (rs) between age and DBS AMH, LH, and FSH were -0.34, 0.28, and 0.25, respectively. DBS AMH, LH, and FSH correlation with BMI were -0.06, 0.05, and 0.09, respectively. Inhibin B had a strong correlation to age (rs -0.33), BMI (rs -0.67), AFC (rs 0.44), DBS AMH (rs 0.40), DBS FSH (rs -0.19), DBS LH (rs-0.11), respectively. AMH method comparison between N-N and N-C terminal ELISA using 39 serum samples yielded slopes of 0.85x and Spearman’s rank correlation of 0.97. Conclusions: DBS and serum measurements for AMH, LH, and FSH are very accurate and precise. They can be used interchangeably. The strong relationship between BMI and Inhibin B will require further investigation and may open new avenues in obesity and reproductive endocrinology. Presentation: 6/1/2024

A-070 Evaluation of Plain and Serum-Separator Blood Collection Tubes for the Measurement of Steroid Hormones by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Abstract Background Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is the preferred method for quantifying steroids. Commonly measured steroids 11-deoxycortisol, androstenedione, testosterone, 17-hydroxyprogesterone and dehydroepiandrosterone (DHEA). Although serum separator blood collection tubes may not be recommended but their frequent use is attributed to the ease of serum separation. Moreover, the serum separator tube may be the only available option for analysis, without the need for additional sample collection. Under such conditions, from certain serum separator tubes, we observed occasional interference in LC-MS/MS steroid method. We pursued to evaluate the performance of blood collection tubes without and those with serum separators, for the quantification of specified steroids by LC-MS/MS. Methods Blood samples were collected from 10 volunteers using both plain and serum separator blood collection tubes procured from two major suppliers (Greiner and BD). The specified steroids were measured by LC-MS/MS method. The procedure involved addition of labeled internal standards to the samples and extraction of steroids in methyl tert-butyl ether. Subsequently, the extract dried under a stream of nitrogen, and the residue was reconstituted in the mobile phase. LC-MS/MS analysis involved multiple reaction monitoring (MRM) for the specified steroids and their internal standards. Results The figure shows a sample chromatogram from a subject with blood samples collected in a plain (A) and serum separator tubes from Greiner (B) and BD (C1 and C2, a zoomed of C1). In this sample, from the plain tube, the concentrations of 11-deoxycortisol, androstenedione, testosterone, 17-hydroxyprogesterone and DHEA were 27, 55, 690, 99 and 283 ng/mL respectively. No significant interference was observed in Greiner serum separator tube. Whereas BD serum separator tube exhibited several erroneous peaks making the quantitation of several steroids challenging or inaccurate. Conclusions In the described LC-MS/MS method, BD serum-separator tubes exhibited erroneous peaks. These peaks were not seen in Greiner serum-separator tubes.

A-061 Analytical Performance and R55G Mutant Detection Ability of an Improved Thyroid-Stimulating Hormone Assay, TSH3-Ultra II, on the ADVIA Centaur Immunoassay System and the Atellica IM Analyzer

Abstract Background Measurement of thyroid stimulating hormone (TSH) is a critical tool for the investigation of thyroid disorders. Previous studies have demonstrated that some immunoassays are unable to detect TSH from patients with the R55G mutation in the TSHβ subunit. This study evaluated the analytical performance of the ADVIA Centaur® and Atellica® IM TSH3-Ultra II (TSH3ULII) assay and its ability to detect the R55G TSH mutant. Methods Limit of quantitation (LoQ) and linearity were determined as described in CLSI EP17-A2 and EP06, respectively. Precision and method comparison (MC) studies were performed with three reagent lots according to CLSI EP05-A3 and CLSI EP09c using native and contrived human serum and plasma samples. For precision, one aliquot of each sample was tested in duplicate in two runs per day &amp;gt;2 hours apart on each analyzer for ≥20 days. MC studies were performed by testing native human serum samples using ADVIA Centaur TSH3-UL assay (current TSH assay) and the newer ADVIA Centaur TSH3ULII assay. An additional MC was performed by testing native human serum samples with the TSH3ULII assay on both the ADVIA Centaur XP and the Atellica IM Analyzer. Samples from 61 patients with TSH results below the LoQ when using TSH3-UL were sequenced and confirmed to have the R55G mutant. Each of these samples was tested in duplicate using the ADVIA Centaur TSH3ULII assay. Results LoQ on both the ADVIA Centaur XP and the Atellica IM Analyzer was estimated to be 0.010 μIU/mL (mIU/L). Linearity was verified from 0.010 to 150.000 μIU/mL (mIU/L). ADVIA Centaur TSH3ULII repeatability %CVs ranged from 1.4 to 3.6% at 0.099 and 91.993 μIU/mL (mIU/L), and within-lab %CVs ranged from 2.3 to 6.2% at 4.908 and 91.993 μIU/mL, respectively. Atellica IM TSH3ULII repeatability %CVs ranged from 1.1 to 3.4% at 4.800 and 93.097 μIU/mL, and within-lab %CVs ranged from 2.2 to 4.6% at 0.103 and 93.097 μIU/mL, respectively. For the MC studies, slopes determined by the Passing-Bablock regression model were approximately 1 (0.96-0.97 range) across the sample interval tested. All TSH3ULII results from the 61 mutant patient samples were greater than LoQ, ranging from 0.864 to 23.443 μIU/mL (mIU/L). Conclusions Evaluation of the TSH3ULII assay using the ADVIA Centaur system demonstrated performance comparable to the commercially available TSH3-UL assay for LoQ, linearity, and precision, when using normal patient samples. TSH3ULII results on the Atellica IM Analyzer were comparable to the results obtained on the ADVIA Centaur XP. All 61 TSH mutant samples used in this study that had TSH values below the LoQ with the TSH3-UL assay, were detectable with the TSH3ULII assay. The data presented here demonstrate the ability of the TSH3ULII assay to detect the TSH in samples from patients with the R55G mutation.

A-046 Quantification of Corticosteroids and Androgens in Serum, utilizing Waters MassTrak™ Steroid Serum Sets 2 &amp; Sets 3 for Clinical Research

Abstract Background Steroid hormones encompass a large class of small molecules that play a central role in metabolic processes, such as the regulation of sexual characteristics, blood pressure, and inflammation. Enzymes that form part of the steroid biosynthetic pathway are pivotal in these metabolic processes, and their dysfunction can be examined through the correct measurement of steroid hormones in a clinical research setting. The availability of lyophilized calibrators and QCs reduces sample preparation time, aids in method harmonization, and assists with metrological traceability in accordance with ISO15189:2022, when used alongside an analytically selective chromatographic method. Methods A quantitative clinical research method utilizing Waters MassTrak Steroid Serum Sets 2 and 3, Cals &amp; QCs and Waters Oasis™ PRiME HLB µElution plate technology for the extraction of testosterone, androstenedione, 17-hydroxyprogesterone (17- OHP), dehydroepiandrosterone sulfate (DHEAS), Cortisol, 11-deoxycortisol and 21-deoxycortisol from human serum samples. Chromatographic separation was performed on an Waters ACQUITY™ UPLC™ I Class Plus (FL-I) System, using an ACQUITY UPLC HSS T3 Column, accompanied by a Xevo™ TQ-S micro Mass Spectrometer. Results Accuracy (±6%) and precision (±10%) have been confirmed through comparison to External Quality Assurance (EQA) LC-MS/MS schemes, panels and QC material for all seven steroid hormones. All analytes were assessed at the low, medium and high concentrations for each MassTrak Steroid Serum Set, which yielded excellent results across the range (±10%). Deming and Linear regression analysis were performed. No statistically significant bias was observed for each compound, with a mean method bias of ±1.3% for Set 2 and ±1.0% for Set 3. The clinical research method was shown to be linear for all analytes over the calibration ranges specified, furthermore, calibration lines created using Set 2 and Set 3 were analysed over a five-day period and were linear with a co-efficient of determination of (r2) &amp;gt; 0.999 for all analytes. Analytical sensitivity using Signal:Noise (S/N) of the low calibrator (Calibrator 1) of each set, was &amp;gt;10:1 for each analyte across several analytical runs. Conclusions This evaluation has demonstrated that the MassTrak Steroid Serum Calibrator and Quality Control Sets 2 and 3 can provide precise and accurate quantification of steroid hormones in serum. An analytically sensitive and selective clinical research method has been developed for the analysis of testosterone, androstenedione, 17- OHP, DHEAS, cortisol, 11-deoxycortisol and 21-deoxycortisol in serum using Waters I-Class Xevo TQ-S micro system. For Research Use Only. Not for Use in Diagnostic Procedures.

A-048 Reference Interval and Clinical Performance Studies for Vitros® Immunodiagnostic Products Intact PTH II Assay to Aid in Diagnosis of Calcium and Parathyroid Disorders and Intraoperative Testing

Abstract Background Intact parathyroid hormone (iPTH) measurements aid in the differential diagnosis of hypercalcemia, hypocalcemia, and parathyroid disorders. The National Academy of Clinical Biochemistry has established guidelines for intraoperative PTH testing to monitor surgical success during parathyroidectomies. Studies were conducted to define the reference interval and clinical performance of the Vitros Immunodiagnostic Products Intact PTH II (Vitros iPTH II) assay, using samples from healthy individuals and patients with conditions commonly requiring iPTH testing, including intraoperative cases. Methods The reference interval study was conducted in accordance with Clinical and Laboratory Standards Institute (CLSI) guideline EP28-A3c in serum samples from 134 healthy individuals. For the clinical performance study, serum samples from individuals with hypoparathyroidism, primary hyperparathyroidism, chronic renal failure (secondary or tertiary hyperparathyroidism), hypercalcemia, hypercalcemia of malignancies, and hypocalcemia were tested. Intraoperative samples used were collected from parathyroidectomy patients in series at each surgical timepoint along with the standard plasma samples used to determine surgical success. Results The reference samples were composed of 134 serum donors that met all inclusion/exclusion criteria for healthy donors and had a normal test result on calcium, TSH, alkaline phosphatase, creatinine, and phosphate assays. The reference interval for iPTH was determined to be 14.2-75.2 pg/mL. The results in various disease states demonstrated the clinical performance of the Vitros iPTH II assay. Of 32 intraoperative sample series included in the study, three samples demonstrated no intraoperative decrease in iPTH due to surgical failures. Consistent with the goal of the parathyroidectomy procedure, at least a 50% decrease in iPTH was observed between the presurgical and post-excision samples in all other sample series. The clinical concordance with the Roche PTH assay used intraoperatively in these procedures was 100%. Conclusions Defining the reference interval for a normal healthy population and results from the intended use population demonstrated that the Vitros iPTH II assay can be used as an aid in the diagnosis of calcium and parathyroid disorders and intraoperative testing.