The steroid response to human chorionic gonadotropin (hCG) stimulation in men with Klinefelter syndrome does not change using immunoassay or mass spectrometry.
OBJECTIVE
Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) was developed in parallel to Immunoassays (IAs) and today is proposed as the "gold standard" for steroid assays. Leydig cells of men with Klinefelter syndrome (KS) are able to respond to human chorionic gonadotropin (hCG) stimulation, even if testosterone (T) production was impaired. The aim was to evaluate how results obtained by IAs and LC-MS/MS can differently impact on the outcome of a clinical research on gonadal steroidogenesis after hCG stimulation.


METHODS
A longitudinal, prospective, case-control clinical trial. (clinicaltrial.gov NCT02788136) was carried out, enrolling KS men and healthy age-matched controls, stimulated by hCG administration. Serum steroids were evaluated at baseline and for 5 days after intramuscular injection of 5000 IU hCG using both IAs and LC-MS/MS.


RESULTS
13 KS patients (36 ± 9 years) not receiving T replacement therapy and 14 controls (32 ± 8 years) were enrolled. T, progesterone, cortisol, 17-hydroxy-progesterone (17OHP) and androstenedione, were significantly higher using IAs than LC-MS/MS. IAs and LC-MS/MS showed direct correlation for all five steroids, although the constant overestimation detected by IAs. Either methodology found the same 17OHP and T increasing profile after hCG stimulation, with equal areas under the curves (AUCs).


CONCLUSIONS
Although a linearity between IA and LC-MS/MS is demonstrated, LC-MS/MS is more sensitive and accurate, whereas IA shows a constant overestimation of sex steroid levels. This result suggests the need of reference intervals built on the specific assay. This fundamental difference between these two methodologies opens a deep reconsideration of what is needed to improve the accuracy of steroid hormone assays.
Department of Laboratory Medicine and Pathology Anatomy, Azienda USL of Modena, Modena, Italy.
L
L
Roli
Unit of Endocrinology, NOCSAE, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Via Giardini 1355, 41126, Modena, Italy. santi.daniele@gmail.com.|Department of Medicine, Endocrinology, Metabolism and Geriatrics, Azienda Ospedaliero-Universitaria of Modena, Modena, Italy. santi.daniele@gmail.com.
D
http://orcid.org/0000-0001-6607-7105
D
Santi
Unit of Endocrinology, NOCSAE, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Via Giardini 1355, 41126, Modena, Italy.
S
S
Belli
Department of Laboratory Medicine and Pathology Anatomy, Azienda USL of Modena, Modena, Italy.
S
S
Tagliavini
Laboratory of Clinical and Endocrinological Analysis, Arcispedale S. Maria Nuova, Reggio Emilia, Italy.
S
S
Cavalieri
Department of Laboratory Medicine and Pathology Anatomy, Azienda USL of Modena, Modena, Italy.
M C
MC
De Santis
Department of Laboratory Medicine and Pathology Anatomy, Azienda USL of Modena, Modena, Italy.
E
E
Baraldi
Endocrinology Unit and Centre for Applied Biomedical Research, Department of Medical and Surgical Sciences, Alma Mater University of Bologna, S. Orsola-Malpighi Hospital, 40138, Bologna, Italy.
F
F
Fanelli
Endocrinology Unit and Centre for Applied Biomedical Research, Department of Medical and Surgical Sciences, Alma Mater University of Bologna, S. Orsola-Malpighi Hospital, 40138, Bologna, Italy.
M
M
Mezzullo
Department of Medicine, Endocrinology, Metabolism and Geriatrics, Azienda Ospedaliero-Universitaria of Modena, Modena, Italy.
A R
AR
Granata
Endocrinology Unit and Centre for Applied Biomedical Research, Department of Medical and Surgical Sciences, Alma Mater University of Bologna, S. Orsola-Malpighi Hospital, 40138, Bologna, Italy.
U
U
Pagotto
Endocrinology Unit and Centre for Applied Biomedical Research, Department of Medical and Surgical Sciences, Alma Mater University of Bologna, S. Orsola-Malpighi Hospital, 40138, Bologna, Italy.
R
R
Pasquali
Unit of Endocrinology, NOCSAE, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Via Giardini 1355, 41126, Modena, Italy.|Department of Medicine, Endocrinology, Metabolism and Geriatrics, Azienda Ospedaliero-Universitaria of Modena, Modena, Italy.
V
V
Rochira
Unit of Endocrinology, NOCSAE, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Via Giardini 1355, 41126, Modena, Italy.
C
C
Carani
Unit of Endocrinology, NOCSAE, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Via Giardini 1355, 41126, Modena, Italy.|Department of Medicine, Endocrinology, Metabolism and Geriatrics, Azienda Ospedaliero-Universitaria of Modena, Modena, Italy.|Center for Genomic Research, University of Modena and Reggio Emilia, Modena, Italy.
M
M
Simoni
Department of Laboratory Medicine and Pathology Anatomy, Azienda USL of Modena, Modena, Italy.
T
T
Trenti
D019326:17-alpha-Hydroxyprogesterone / Q000097:blood; D000293:Adolescent; D000328:Adult; D016022:Case-Control Studies; D006063:Chorionic Gonadotropin / Q000627:therapeutic use*; D012739:Gonadal Steroid Hormones / Q000097:blood*; D006801:Humans; D006854:Hydrocortisone / Q000097:blood; D007118:Immunoassay / Q000379:methods*; D007713:Klinefelter Syndrome / Q000097:blood* / Q000188:drug therapy; D008137:Longitudinal Studies; D008297:Male; D008875:Middle Aged; D011374:Progesterone / Q000097:blood; D011446:Prospective Studies; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013739:Testosterone / Q000097:blood; D055815:Young Adult
D006063:Chorionic Gonadotropin; D012739:Gonadal Steroid Hormones; D013739:Testosterone; D011374:Progesterone; D019326:17-alpha-Hydroxyprogesterone; D006854:Hydrocortisone
Immunoassay; Klinefelter syndrome; Mass spectrometry; hCG test