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3. Steroid biomarkers for identifying non-classic adrenal hyperplasia due to 21-hydroxylase deficiency in a population of PCOS with suspicious levels of 17OH-progesterone

Author

Oriolo, C., Fanelli, F., Castelli, S., Mezzullo, M., Altieri, P., Corzani, F., Pelusi, C., Repaci, A., Di Dalmazi, G., Vicennati, V., Baldazzi, L., Menabò, S., Dormi, A., Nardi, E., Brillanti, G., Pasquali, R., Pagotto, U., and Gambineri, A.

Abstract

Objective: We aimed at defining the most effective routine immunoassay- or liquid chromatography-tandem mass spectrometry (LC–MS/MS)-determined steroid biomarkers for identifying non-classic adrenal hyperplasia due to 21-hydroxylase deficiency (21-NCAH) in a PCOS-like population before genotyping. Methods: Seventy PCOS-like patients in reproductive age with immunoassay-determined follicular 17OH-progesterone (17OHP) ≥ 2.00 ng/mL underwent CYP21A2gene analysis and 1–24ACTH test. Serum steroids were measured by immunoassays at baseline and 60 min after ACTH stimulation; basal steroid profile was measured by LC–MS/MS. Results: Genotyping revealed 23 21-NCAH, 15 single allele heterozygous CYP21A2mutations (21-HTZ) and 32 PCOS patients displaying similar clinical and metabolic features. Immunoassays revealed higher baseline 17OHP and testosterone, and after ACTH stimulation, higher 17OHP (17OHP60) and lower cortisol, whereas LC–MS/MS revealed higher 17OHP (17OHPLC-MS/MS), progesterone and 21-deoxycortisol and lower corticosterone in 21-NCAH compared with both 21-HTZ and PCOS patients. Steroid thresholds best discriminating 21-NCAH from 21-HTZ and PCOS were estimated, and their diagnostic accuracy in identifying 21-NCAH from PCOS was established by ROC analysis. The highest accuracy was observed for 21-deoxycortisol ≥ 0.087 ng/mL, showing 100% sensitivity, while the combination of 17OHPLC-MS/MS≥ 1.79 ng/mL and corticosterone ≤ 8.76 ng/mL, as well as the combination of ACTH-stimulated 17OHP ≥ 6.77 ng/mL and cortisol ≤ 240 ng/mL by immunoassay, showed 100% specificity. Conclusions: LC–MS/MS measurement of basal follicular 21-deoxycortisol, 17OHP and corticosterone seems the most convenient method for diagnosing 21-NCAH in a population of PCOS with a positive first level screening, providing high accuracy and reducing the need for ACTH stimulation test.

Published
2024
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5. Shorter androgen receptor polyQ alleles protect against life-threatening COVID-19 disease in European males

Author

Baldassarri, M, Picchiotti, N, Fava, F, Fallerini, C, Benetti, E, Daga, S, Valentino, F, Doddato, G, Furini, S, Giliberti, A, Tita, R, Amitrano, S, Bruttini, M, Croci, S, Meloni, I, Pinto, A, Iuso, N, Gabbi, C, Sciarra, F, Venneri, M, Gori, M, Sanarico, M, Crawley, F, Pagotto, U, Fanelli, F, Mezzullo, M, Dominguez-Garrido, E, Planas-Serra, L, Schluter, A, Colobran, R, Soler-Palacin, P, Lapunzina, P, Tenorio, J, Pujol, A, Castagna, M, Marcelli, M, Isidori, A, Renieri, A, Frullanti, E, Mari, F, Montagnani, F, Di Sarno, L, Tommasi, A, Palmieri, M, Fabbiani, M, Rossetti, B, Zanelli, G, Sestini, F, Bergantini, L, D'Alessandro, M, Cameli, P, Bennett, D, Anedda, F, Marcantonio, S, Scolletta, S, Franchi, F, Mazzei, M, Guerrini, S, Conticini, E, Cantarini, L, Frediani, B, Tacconi, D, Spertilli, C, Feri, M, Donati, A, Scala, R, Guidelli, L, Spargi, G, Corridi, M, Nencioni, C, Croci, L, Caldarelli, G, Spagnesi, M, Piacentini, P, Bandini, M, Desanctis, E, Cappelli, S, Canaccini, A, Verzuri, A, Anemoli, V, Ognibene, A, Vaghi, M, Monforte, A, Merlini, E, Miraglia, F, Mondelli, M, Mantovani, S, Ludovisi, S, Girardis, M, Venturelli, S, Sita, M, Cossarizza, A, Antinori, A, Vergori, A, Emiliozzi, A, Rusconi, S, Siano, M, Gabrieli, A, Riva, A, Francisci, D, Schiaroli, E, Paciosi, F, Scotton, P, Andretta, F, Panese, S, Baratti, S, Scaggiante, R, Gatti, F, Parisi, S, Castelli, F, Quiros-Roldan, E, Antoni, M, Zanella, I, Monica, M, Piscopo, C, Capasso, M, Russo, R, Andolfo, I, Iolascon, A, Fiorentino, G, Carella, M, Castori, M, Merla, G, Aucella, F, Raggi, P, Marciano, C, Perna, R, Bassetti, M, Di Biagio, A, Sanguinetti, M, Masucci, L, Valente, S, Mencarelli, M, Lo Rizzo, C, Bargagli, E, Mandala, M, Giorli, A, Salerni, L, Zucchi, P, Parravicini, P, Menatti, E, Trotta, T, Giannattasio, F, Coiro, G, Lena, F, Coviello, D, Mussini, C, Bosio, G, Martinelli, E, Mancarella, S, Tavecchia, L, Crotti, L, Parati, G, Aguilera-Albesa, S, Albu, S, Casasnovas, C, Velez-Santamaria, V, Horcajada, J, Villar, J, Rodriguez-Palmero, A, Ruiz, M, Seijo, L, Troya, J, Valencia-Ramos, J, Gut, M, Baldassarri M., Picchiotti N., Fava F., Fallerini C., Benetti E., Daga S., Valentino F., Doddato G., Furini S., Giliberti A., Tita R., Amitrano S., Bruttini M., Croci S., Meloni I., Pinto A. M., Iuso N., Gabbi C., Sciarra F., Venneri M. A., Gori M., Sanarico M., Crawley F. P., Pagotto U., Fanelli F., Mezzullo M., Dominguez-Garrido E., Planas-Serra L., Schluter A., Colobran R., Soler-Palacin P., Lapunzina P., Tenorio J., Pujol A., Castagna M. G., Marcelli M., Isidori A. M., Renieri A., Frullanti E., Mari F., Montagnani F., Di Sarno L., Tommasi A., Palmieri M., Fabbiani M., Rossetti B., Zanelli G., Sestini F., Bergantini L., D'Alessandro M., Cameli P., Bennett D., Anedda F., Marcantonio S., Scolletta S., Franchi F., Mazzei M. A., Guerrini S., Conticini E., Cantarini L., Frediani B., Tacconi D., Spertilli C., Feri M., Donati A., Scala R., Guidelli L., Spargi G., Corridi M., Nencioni C., Croci L., Caldarelli G. P., Spagnesi M., Piacentini P., Bandini M., Desanctis E., Cappelli S., Canaccini A., Verzuri A., Anemoli V., Ognibene A., Vaghi M., Monforte A. D., Merlini E., Miraglia F. G., Mondelli M. U., Mantovani S., Ludovisi S., Girardis M., Venturelli S., Sita M., Cossarizza A., Antinori A., Vergori A., Emiliozzi A., Rusconi S., Siano M., Gabrieli A., Riva A., Francisci D., Schiaroli E., Paciosi F., Scotton P. G., Andretta F., Panese S., Baratti S., Scaggiante R., Gatti F., Parisi S. G., Castelli F., Quiros-Roldan E., Antoni M. D., Zanella I., Monica M. D., Piscopo C., Capasso M., Russo R., Andolfo I., Iolascon A., Fiorentino G., Carella M., Castori M., Merla G., Aucella F., Raggi P., Marciano C., Perna R., Bassetti M., Di Biagio A., Sanguinetti M., Masucci L., Valente S., Mencarelli M. A., Lo Rizzo C., Bargagli E., Mandala M., Giorli A., Salerni L., Zucchi P., Parravicini P., Menatti E., Trotta T., Giannattasio F., Coiro G., Lena F., Coviello D. A., Mussini C., Bosio G., Martinelli E., Mancarella S., Tavecchia L., Crotti L., Parati G., Aguilera-Albesa S., Albu S., Casasnovas C., Velez-Santamaria V., Horcajada J. P., Villar J., Rodriguez-Palmero A., Ruiz M., Seijo L. M., Troya J., Valencia-Ramos J., Gut M., Baldassarri, M, Picchiotti, N, Fava, F, Fallerini, C, Benetti, E, Daga, S, Valentino, F, Doddato, G, Furini, S, Giliberti, A, Tita, R, Amitrano, S, Bruttini, M, Croci, S, Meloni, I, Pinto, A, Iuso, N, Gabbi, C, Sciarra, F, Venneri, M, Gori, M, Sanarico, M, Crawley, F, Pagotto, U, Fanelli, F, Mezzullo, M, Dominguez-Garrido, E, Planas-Serra, L, Schluter, A, Colobran, R, Soler-Palacin, P, Lapunzina, P, Tenorio, J, Pujol, A, Castagna, M, Marcelli, M, Isidori, A, Renieri, A, Frullanti, E, Mari, F, Montagnani, F, Di Sarno, L, Tommasi, A, Palmieri, M, Fabbiani, M, Rossetti, B, Zanelli, G, Sestini, F, Bergantini, L, D'Alessandro, M, Cameli, P, Bennett, D, Anedda, F, Marcantonio, S, Scolletta, S, Franchi, F, Mazzei, M, Guerrini, S, Conticini, E, Cantarini, L, Frediani, B, Tacconi, D, Spertilli, C, Feri, M, Donati, A, Scala, R, Guidelli, L, Spargi, G, Corridi, M, Nencioni, C, Croci, L, Caldarelli, G, Spagnesi, M, Piacentini, P, Bandini, M, Desanctis, E, Cappelli, S, Canaccini, A, Verzuri, A, Anemoli, V, Ognibene, A, Vaghi, M, Monforte, A, Merlini, E, Miraglia, F, Mondelli, M, Mantovani, S, Ludovisi, S, Girardis, M, Venturelli, S, Sita, M, Cossarizza, A, Antinori, A, Vergori, A, Emiliozzi, A, Rusconi, S, Siano, M, Gabrieli, A, Riva, A, Francisci, D, Schiaroli, E, Paciosi, F, Scotton, P, Andretta, F, Panese, S, Baratti, S, Scaggiante, R, Gatti, F, Parisi, S, Castelli, F, Quiros-Roldan, E, Antoni, M, Zanella, I, Monica, M, Piscopo, C, Capasso, M, Russo, R, Andolfo, I, Iolascon, A, Fiorentino, G, Carella, M, Castori, M, Merla, G, Aucella, F, Raggi, P, Marciano, C, Perna, R, Bassetti, M, Di Biagio, A, Sanguinetti, M, Masucci, L, Valente, S, Mencarelli, M, Lo Rizzo, C, Bargagli, E, Mandala, M, Giorli, A, Salerni, L, Zucchi, P, Parravicini, P, Menatti, E, Trotta, T, Giannattasio, F, Coiro, G, Lena, F, Coviello, D, Mussini, C, Bosio, G, Martinelli, E, Mancarella, S, Tavecchia, L, Crotti, L, Parati, G, Aguilera-Albesa, S, Albu, S, Casasnovas, C, Velez-Santamaria, V, Horcajada, J, Villar, J, Rodriguez-Palmero, A, Ruiz, M, Seijo, L, Troya, J, Valencia-Ramos, J, Gut, M, Baldassarri M., Picchiotti N., Fava F., Fallerini C., Benetti E., Daga S., Valentino F., Doddato G., Furini S., Giliberti A., Tita R., Amitrano S., Bruttini M., Croci S., Meloni I., Pinto A. M., Iuso N., Gabbi C., Sciarra F., Venneri M. A., Gori M., Sanarico M., Crawley F. P., Pagotto U., Fanelli F., Mezzullo M., Dominguez-Garrido E., Planas-Serra L., Schluter A., Colobran R., Soler-Palacin P., Lapunzina P., Tenorio J., Pujol A., Castagna M. G., Marcelli M., Isidori A. M., Renieri A., Frullanti E., Mari F., Montagnani F., Di Sarno L., Tommasi A., Palmieri M., Fabbiani M., Rossetti B., Zanelli G., Sestini F., Bergantini L., D'Alessandro M., Cameli P., Bennett D., Anedda F., Marcantonio S., Scolletta S., Franchi F., Mazzei M. A., Guerrini S., Conticini E., Cantarini L., Frediani B., Tacconi D., Spertilli C., Feri M., Donati A., Scala R., Guidelli L., Spargi G., Corridi M., Nencioni C., Croci L., Caldarelli G. P., Spagnesi M., Piacentini P., Bandini M., Desanctis E., Cappelli S., Canaccini A., Verzuri A., Anemoli V., Ognibene A., Vaghi M., Monforte A. D., Merlini E., Miraglia F. G., Mondelli M. U., Mantovani S., Ludovisi S., Girardis M., Venturelli S., Sita M., Cossarizza A., Antinori A., Vergori A., Emiliozzi A., Rusconi S., Siano M., Gabrieli A., Riva A., Francisci D., Schiaroli E., Paciosi F., Scotton P. G., Andretta F., Panese S., Baratti S., Scaggiante R., Gatti F., Parisi S. G., Castelli F., Quiros-Roldan E., Antoni M. D., Zanella I., Monica M. D., Piscopo C., Capasso M., Russo R., Andolfo I., Iolascon A., Fiorentino G., Carella M., Castori M., Merla G., Aucella F., Raggi P., Marciano C., Perna R., Bassetti M., Di Biagio A., Sanguinetti M., Masucci L., Valente S., Mencarelli M. A., Lo Rizzo C., Bargagli E., Mandala M., Giorli A., Salerni L., Zucchi P., Parravicini P., Menatti E., Trotta T., Giannattasio F., Coiro G., Lena F., Coviello D. A., Mussini C., Bosio G., Martinelli E., Mancarella S., Tavecchia L., Crotti L., Parati G., Aguilera-Albesa S., Albu S., Casasnovas C., Velez-Santamaria V., Horcajada J. P., Villar J., Rodriguez-Palmero A., Ruiz M., Seijo L. M., Troya J., Valencia-Ramos J., and Gut M.

Abstract

Background: While SARS-CoV-2 similarly infects men and women, COVID-19 outcome is less favorable in men. Variability in COVID-19 severity may be explained by differences in the host genome. Methods: We compared poly-amino acids variability from WES data in severely affected COVID-19 patients versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects. Findings: Shorter polyQ alleles (≤22) in the androgen receptor (AR) conferred protection against severe outcome in COVID-19 in the first tested cohort (both males and females) of 638 Italian subjects. The association between long polyQ alleles (≥23) and severe clinical outcome (p = 0.024) was also validated in an independent cohort of Spanish men <60 years of age (p = 0.014). Testosterone was higher in subjects with AR long-polyQ, possibly indicating receptor resistance (p = 0.042 Mann-Whitney U test). Inappropriately low serum testosterone level among carriers of the long-polyQ alleles (p = 0.0004 Mann-Whitney U test) predicted the need for intensive care in COVID-19 infected men. In agreement with the known anti-inflammatory action of testosterone, patients with long-polyQ and age ≥60 years had increased levels of CRP (p = 0.018, not accounting for multiple testing). Interpretation: We identify the first genetic polymorphism that appears to predispose some men to develop more severe disease. Failure of the endocrine feedback to overcome AR signaling defects by increasing testosterone levels during the infection leads to the polyQ tract becoming dominant to serum testosterone levels for the clinical outcome. These results may contribute to designing reliable clinical and public health measures and provide a rationale to test testosterone as adjuvant therapy in men with COVID-19 expressing long AR polyQ repeats. Funding: MIUR project “Dipartimenti di Eccellenza 2018-2020” to Department of Medical Biotechnologies University of Siena, Italy (Italian D.L. n.18 March 17, 2020) and “Bando Ricerca COVID-19 Toscana” project

Published
2021

6. Shorter androgen receptor polyQ alleles protect against life-threatening COVID-19 disease in European males

Author

Baldassarri M., Picchiotti N., Fava F., Fallerini C., Benetti E., Daga S., Valentino F., Doddato G., Furini S., Giliberti A., Tita R., Amitrano S., Bruttini M., Croci S., Meloni I., Pinto A. M., Iuso N., Gabbi C., Sciarra F., Venneri M. A., Gori M., Sanarico M., Crawley F. P., Pagotto U., Fanelli F., Mezzullo M., Dominguez-Garrido E., Planas-Serra L., Schluter A., Colobran R., Soler-Palacin P., Lapunzina P., Tenorio J., Pujol A., Castagna M. G., Marcelli M., Isidori A. M., Renieri A., Frullanti E., Mari F., Montagnani F., Di Sarno L., Tommasi A., Palmieri M., Fabbiani M., Rossetti B., Zanelli G., Sestini F., Bergantini L., D'Alessandro M., Cameli P., Bennett D., Anedda F., Marcantonio S., Scolletta S., Franchi F., Mazzei M. A., Guerrini S., Conticini E., Cantarini L., Frediani B., Tacconi D., Spertilli C., Feri M., Donati A., Scala R., Guidelli L., Spargi G., Corridi M., Nencioni C., Croci L., Caldarelli G. P., Spagnesi M., Piacentini P., Bandini M., Desanctis E., Cappelli S., Canaccini A., Verzuri A., Anemoli V., Ognibene A., Vaghi M., Monforte A. D., Merlini E., Miraglia F. G., Mondelli M. U., Mantovani S., Ludovisi S., Girardis M., Venturelli S., Sita M., Cossarizza A., Antinori A., Vergori A., Emiliozzi A., Rusconi S., Siano M., Gabrieli A., Riva A., Francisci D., Schiaroli E., Paciosi F., Scotton P. G., Andretta F., Panese S., Baratti S., Scaggiante R., Gatti F., Parisi S. G., Castelli F., Quiros-Roldan E., Antoni M. D., Zanella I., Monica M. D., Piscopo C., Capasso M., Russo R., Andolfo I., Iolascon A., Fiorentino G., Carella M., Castori M., Merla G., Aucella F., Raggi P., Marciano C., Perna R., Bassetti M., Di Biagio A., Sanguinetti M., Masucci L., Valente S., Mencarelli M. A., Lo Rizzo C., Bargagli E., Mandala M., Giorli A., Salerni L., Zucchi P., Parravicini P., Menatti E., Trotta T., Giannattasio F., Coiro G., Lena F., Coviello D. A., Mussini C., Bosio G., Martinelli E., Mancarella S., Tavecchia L., Crotti L., Parati G., Aguilera-Albesa S., Albu S., Casasnovas C., Velez-Santamaria V., Horcajada J. P., Villar J., Rodriguez-Palmero A., Ruiz M., Seijo L. M., Troya J., Valencia-Ramos J., Gut M., Institut Català de la Salut, [Baldassarri M, Fallerini C] Medical Genetics, University of Siena, Italy. Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Italy. [Picchiotti N] University of Siena, DIISM-SAILAB, Siena, Italy. Department of Mathematics, University of Pavia, Pavia, Italy. [Fava F] Medical Genetics, University of Siena, Italy. Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Italy. Genetica Medica, Azienda Ospedaliero-Universitaria Senese, Italy. [Benetti E] Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Italy. [Colobran R] Servei d’Immunologia, Àrea de Genètica, Vall d'Hebron Hospital Universitari, Barcelona, Spain. Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain. [Soler-Palacin P] Unitat de Patologia Infecciosa i Immunodeficiències de Pediatria, Vall d'Hebron Hospital Universitari, Barcelona, Spain. Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain, Vall d'Hebron Barcelona Hospital Campus, Baldassarri M., Picchiotti N., Fava F., Fallerini C., Benetti E., Daga S., Valentino F., Doddato G., Furini S., Giliberti A., Tita R., Amitrano S., Bruttini M., Croci S., Meloni I., Pinto A.M., Iuso N., Gabbi C., Sciarra F., Venneri M.A., Gori M., Sanarico M., Crawley F.P., Pagotto U., Fanelli F., Mezzullo M., Dominguez-Garrido E., Planas-Serra L., Schluter A., Colobran R., Soler-Palacin P., Lapunzina P., Tenorio J., Pujol A., Castagna M.G., Marcelli M., Isidori A.M., Renieri A., Frullanti E., Mari F., Montagnani F., Di Sarno L., Tommasi A., Palmieri M., Fabbiani M., Rossetti B., Zanelli G., Sestini F., Bergantini L., D'Alessandro M., Cameli P., Bennett D., Anedda F., Marcantonio S., Scolletta S., Franchi F., Mazzei M.A., Guerrini S., Conticini E., Cantarini L., Frediani B., Tacconi D., Spertilli C., Feri M., Donati A., Scala R., Guidelli L., Spargi G., Corridi M., Nencioni C., Croci L., Caldarelli G.P., Spagnesi M., Piacentini P., Bandini M., Desanctis E., Cappelli S., Canaccini A., Verzuri A., Anemoli V., Ognibene A., Vaghi M., Monforte A.D., Merlini E., Miraglia F.G., Mondelli M.U., Mantovani S., Ludovisi S., Girardis M., Venturelli S., Sita M., Cossarizza A., Antinori A., Vergori A., Emiliozzi A., Rusconi S., Siano M., Gabrieli A., Riva A., Francisci D., Schiaroli E., Paciosi F., Scotton P.G., Andretta F., Panese S., Baratti S., Scaggiante R., Gatti F., Parisi S.G., Castelli F., Quiros-Roldan E., Antoni M.D., Zanella I., Monica M.D., Piscopo C., Capasso M., Russo R., Andolfo I., Iolascon A., Fiorentino G., Carella M., Castori M., Merla G., Aucella F., Raggi P., Marciano C., Perna R., Bassetti M., Di Biagio A., Sanguinetti M., Masucci L., Valente S., Mencarelli M.A., Lo Rizzo C., Bargagli E., Mandala M., Giorli A., Salerni L., Zucchi P., Parravicini P., Menatti E., Trotta T., Giannattasio F., Coiro G., Lena F., Coviello D.A., Mussini C., Bosio G., Martinelli E., Mancarella S., Tavecchia L., Crotti L., Parati G., Aguilera-Albesa S., Albu S., Casasnovas C., Velez-Santamaria V., Horcajada J.P., Villar J., Rodriguez-Palmero A., Ruiz M., Seijo L.M., Troya J., Valencia-Ramos J., Gut M., Baldassarri, M., Picchiotti, N., Fava, F., Fallerini, C., Benetti, E., Daga, S., Valentino, F., Doddato, G., Furini, S., Giliberti, A., Tita, R., Amitrano, S., Bruttini, M., Croci, S., Meloni, I., Pinto, A. M., Iuso, N., Gabbi, C., Sciarra, F., Venneri, M. A., Gori, M., Sanarico, M., Crawley, F. P., Pagotto, U., Fanelli, F., Mezzullo, M., Dominguez-Garrido, E., Planas-Serra, L., Schluter, A., Colobran, R., Soler-Palacin, P., Lapunzina, P., Tenorio, J., Pujol, A., Castagna, M. G., Marcelli, M., Isidori, A. M., Renieri, A., Frullanti, E., Mari, F., Montagnani, F., Di Sarno, L., Tommasi, A., Palmieri, M., Fabbiani, M., Rossetti, B., Zanelli, G., Sestini, F., Bergantini, L., D'Alessandro, M., Cameli, P., Bennett, D., Anedda, F., Marcantonio, S., Scolletta, S., Franchi, F., Mazzei, M. A., Guerrini, S., Conticini, E., Cantarini, L., Frediani, B., Tacconi, D., Spertilli, C., Feri, M., Donati, A., Scala, R., Guidelli, L., Spargi, G., Corridi, M., Nencioni, C., Croci, L., Caldarelli, G. P., Spagnesi, M., Piacentini, P., Bandini, M., Desanctis, E., Cappelli, S., Canaccini, A., Verzuri, A., Anemoli, V., Ognibene, A., Vaghi, M., Monforte, A. D., Merlini, E., Miraglia, F. G., Mondelli, M. U., Mantovani, S., Ludovisi, S., Girardis, M., Venturelli, S., Sita, M., Cossarizza, A., Antinori, A., Vergori, A., Emiliozzi, A., Rusconi, S., Siano, M., Gabrieli, A., Riva, A., Francisci, D., Schiaroli, E., Paciosi, F., Scotton, P. G., Andretta, F., Panese, S., Baratti, S., Scaggiante, R., Gatti, F., Parisi, S. G., Castelli, F., Quiros-Roldan, E., Antoni, M. D., Zanella, I., Monica, M. D., Piscopo, C., Capasso, M., Russo, R., Andolfo, I., Iolascon, A., Fiorentino, G., Carella, M., Castori, M., Merla, G., Aucella, F., Raggi, P., Marciano, C., Perna, R., Bassetti, M., Di Biagio, A., Sanguinetti, M., Masucci, L., Valente, S., Mencarelli, M. A., Lo Rizzo, C., Bargagli, E., Mandala, M., Giorli, A., Salerni, L., Zucchi, P., Parravicini, P., Menatti, E., Trotta, T., Giannattasio, F., Coiro, G., Lena, F., Coviello, D. A., Mussini, C., Bosio, G., Martinelli, E., Mancarella, S., Tavecchia, L., Crotti, L., Parati, G., Aguilera-Albesa, S., Albu, S., Casasnovas, C., Velez-Santamaria, V., Horcajada, J. P., Villar, J., Rodriguez-Palmero, A., Ruiz, M., Seijo, L. M., Troya, J., Valencia-Ramos, J., Gut, M., Baldassarri, M, Picchiotti, N, Fava, F, Fallerini, C, Benetti, E, Daga, S, Valentino, F, Doddato, G, Furini, S, Giliberti, A, Tita, R, Amitrano, S, Bruttini, M, Croci, S, Meloni, I, Pinto, A, Iuso, N, Gabbi, C, Sciarra, F, Venneri, M, Gori, M, Sanarico, M, Crawley, F, Pagotto, U, Fanelli, F, Mezzullo, M, Dominguez-Garrido, E, Planas-Serra, L, Schluter, A, Colobran, R, Soler-Palacin, P, Lapunzina, P, Tenorio, J, Pujol, A, Castagna, M, Marcelli, M, Isidori, A, Renieri, A, Frullanti, E, Mari, F, Montagnani, F, Di Sarno, L, Tommasi, A, Palmieri, M, Fabbiani, M, Rossetti, B, Zanelli, G, Sestini, F, Bergantini, L, D'Alessandro, M, Cameli, P, Bennett, D, Anedda, F, Marcantonio, S, Scolletta, S, Franchi, F, Mazzei, M, Guerrini, S, Conticini, E, Cantarini, L, Frediani, B, Tacconi, D, Spertilli, C, Feri, M, Donati, A, Scala, R, Guidelli, L, Spargi, G, Corridi, M, Nencioni, C, Croci, L, Caldarelli, G, Spagnesi, M, Piacentini, P, Bandini, M, Desanctis, E, Cappelli, S, Canaccini, A, Verzuri, A, Anemoli, V, Ognibene, A, Vaghi, M, Monforte, A, Merlini, E, Miraglia, F, Mondelli, M, Mantovani, S, Ludovisi, S, Girardis, M, Venturelli, S, Sita, M, Cossarizza, A, Antinori, A, Vergori, A, Emiliozzi, A, Rusconi, S, Siano, M, Gabrieli, A, Riva, A, Francisci, D, Schiaroli, E, Paciosi, F, Scotton, P, Andretta, F, Panese, S, Baratti, S, Scaggiante, R, Gatti, F, Parisi, S, Castelli, F, Quiros-Roldan, E, Antoni, M, Zanella, I, Monica, M, Piscopo, C, Capasso, M, Russo, R, Andolfo, I, Iolascon, A, Fiorentino, G, Carella, M, Castori, M, Merla, G, Aucella, F, Raggi, P, Marciano, C, Perna, R, Bassetti, M, Di Biagio, A, Sanguinetti, M, Masucci, L, Valente, S, Mencarelli, M, Lo Rizzo, C, Bargagli, E, Mandala, M, Giorli, A, Salerni, L, Zucchi, P, Parravicini, P, Menatti, E, Trotta, T, Giannattasio, F, Coiro, G, Lena, F, Coviello, D, Mussini, C, Bosio, G, Martinelli, E, Mancarella, S, Tavecchia, L, Crotti, L, Parati, G, Aguilera-Albesa, S, Albu, S, Casasnovas, C, Velez-Santamaria, V, Horcajada, J, Villar, J, Rodriguez-Palmero, A, Ruiz, M, Seijo, L, Troya, J, Valencia-Ramos, J, and Gut, M

Subjects
0301 basic medicine, Oncology, Male, lcsh:Medicine, Disease, COVID-19 (Malaltia), Severity of Illness Index, Androgen, Hormones, Hormone Substitutes, and Hormone Antagonists::Hormones::Gonadal Hormones::Gonadal Steroid Hormones::Testosterone Congeners::Testosterone [CHEMICALS AND DRUGS], 0302 clinical medicine, Risk Factors, Receptors, virosis::infecciones por virus ARN::infecciones por Nidovirales::infecciones por Coronaviridae::infecciones por Coronavirus [ENFERMEDADES], Testosterone, Other subheadings::/therapeutic use [Other subheadings], Amino Acids, Peptides, and Proteins::Proteins::Receptors, Cytoplasmic and Nuclear::Receptors, Steroid::Receptors, Androgen [CHEMICALS AND DRUGS], Testosterona, Androgen receptor gene, COVID-19, LASSO logistic regression, Viral infection and host genome, WES, Aged, Case-Control Studies, Critical Care, Female, Genome, Human, Humans, Middle Aged, Peptides, Polymorphism, Single Nucleotide, Receptors, Androgen, SARS-CoV-2, Spain, lcsh:R5-920, Genome, Virus Diseases::RNA Virus Infections::Nidovirales Infections::Coronaviridae Infections::Coronavirus Infections [DISEASES], General Medicine, Single Nucleotide, Testosterona - Ús terapèutic, Homes -- Malalties, androgen receptor gene, testosterone, viral infection and host genome, 030220 oncology & carcinogenesis, Cohort, Peptide, aminoácidos, péptidos y proteínas::proteínas::receptores citoplásmicos y nucleares::receptores de esteroides::receptores de andrógenos [COMPUESTOS QUÍMICOS Y DROGAS], Androgens, lcsh:Medicine (General), Case-Control Studie, Research Paper, hormonas, sustitutos de hormonas y antagonistas de hormonas::hormonas::hormonas gonadales::hormonas esteroides gonadales::congéneres de la testosterona::testosterona [COMPUESTOS QUÍMICOS Y DROGAS], Human, medicine.medical_specialty, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Andrògens - Receptors, Internal medicine, Severity of illness, medicine, Adjuvant therapy, Allele, Polymorphism, business.industry, Otros calificadores::/uso terapéutico [Otros calificadores], Public health, Risk Factor, lcsh:R, Case-control study, Androgen receptor, 030104 developmental biology, business, Andrògens
Abstract

Coronavirus SARS-CoV-2; COVID-19; 2019-nCoV; Gen receptor d'andrògens; Infecció vírica i genoma de l’hoste Coronavirus SARS-CoV-2; COVID-19; 2019-nCoV; Gen receptor de andrógenos; Infección viral y genoma del huésped Coronavirus SARS-CoV-2; COVID-19; 2019-nCoV; Androgen receptor gene; Viral infection and host genome Background While SARS-CoV-2 similarly infects men and women, COVID-19 outcome is less favorable in men. Variability in COVID-19 severity may be explained by differences in the host genome. Methods We compared poly-amino acids variability from WES data in severely affected COVID-19 patients versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects. Findings Shorter polyQ alleles (≤22) in the androgen receptor (AR) conferred protection against severe outcome in COVID-19 in the first tested cohort (both males and females) of 638 Italian subjects. The association between long polyQ alleles (≥23) and severe clinical outcome (p = 0.024) was also validated in an independent cohort of Spanish men

Published
2021

7. Health status is related to testosterone, estrone and body fat: Moving to functional hypogonadism in adult men with HIV

Author

De Vincentis, S., Decaroli, M. C., Fanelli, F., Diazzi, C., Mezzullo, M., Morini, F., Bertani, D., Milic, J., Carli, F., Cuomo, G., Santi, D., Tartaro, G., Tagliavini, S., De Santis, M. C., Roli, L., Trenti, T., Pagotto, U., Guaraldi, G., Rochira, V., and De Vincentis S, Decaroli MC, Fanelli F, Diazzi C, Mezzullo M, Morini F, Bertani D, Milic J, Carli F, Cuomo G, Santi D, Tartaro G, Tagliavini S, De Santis MC, Roli L, Trenti T, Pagotto U, Guaraldi G, Rochira V

Subjects
Adult, Male, Estrone, Health Status, health status, HIV Infections, frailty, comorbidities, Health Statu, Absorptiometry, Photon, HIV, male hypogonadism, testosterone, estradiol, frailty, comorbidities, health status, functional hypogonadism, body fat, estrone, estradiol, Antiretroviral Therapy, Highly Active, Health Status Indicators, Humans, HIV Infection, Testosterone, Health Status Indicator, Prospective Studies, functional hypogonadism, Cross-Sectional Studie, Frailty, Hypogonadism, male hypogonadism, HIV, Multimorbidity, Middle Aged, estrone, body fat, Prospective Studie, Cross-Sectional Studies, Adipose Tissue, testosterone, Body Composition, Human
Abstract

Objective: Hypogonadism is common in HIV-infected men. The relationship between health status, sex steroids and body composition is poorly known in HIV. The aim was to investigate the association between health status (comorbidities/frailty), body composition, and gonadal function in young-to-middle-aged HIV-infected men. Design: Prospective, cross-sectional, observational study. Methods: HIV-infected men aged

Published
2021

8. Testosterone (T) is poorly related to sexual desire and Erectile Dysfunction (ED) in Young/Middle Aged Human immunodeficiency virus (HIV)-Infected Men

Author

De Vincentis, S., Decaroli, M. C., Diazzi, C, Morini, F., Bertani, D., Fanelli, F., Mezzullo, M., Santi, D., Tartaro, G., Baraldi, E., Tagliavini, S., Pagotto, U., Guaraldi, G., and Rochira, V.

Subjects
IIEF-15, erectile dysfunction, sexual dysfunction, testosterone, HIV, hypogonadism, HIV, testosterone, erectile dysfunction, sexual dysfunction, IIEF-15, hypogonadism
Published
2020

9. Low Serum Testosterone (T) Is Associated with Poor Health Status in Young to Middle-Aged Human Immunodeficiency Virus (HIV)-Infected Men

Author

De Vincentis, S., Decaroli, M. C., Diazzi, C., Morini, F., Bertani, D., Fanelli, F., Mezzullo, M., Santi, D., Tartaro, G., Baraldi, E., Tagliavini, S., Pagotto, U., Guaraldi, G., and Rochira, V.

Subjects
AIDS, sex steroids, mass spectrometry, AIDS, HIV, hypogonadism, testosterone, health status, comorbidities, testosterone, HIV, hypogonadism, sex steroids, health status, comorbidities, mass spectrometry
Published
2020

10. Gonadal Function in Human Immunodeficiency Virus (HIV)-Infected Men Assessed by Isotopic Dilution-Liquid Chromatography-Tandem Mass Spectrometry (ID-LC-MS/MS) and Chemiluminescent Immunoassay

Author

De Vincentis, S., Decaroli, M. C., Diazzi, C., Morini, F., Bertani, D., Fanelli, F., Mezzullo, M., Santi, D., Baraldi, E., Tagliavini, S., Pagotto, U., Guaraldi, G., and V. Rochira.

Subjects
Chemiluminescent Immunoassay, testosterone, HIV, sex steroids, mass spectrometry, sex steroids, testosterone, HIV, ID-LC-MS/MS, Chemiluminescent Immunoassay, ID-LC-MS/MS, mass spectrometry
Published
2019

11. Gonadal function in human immunodeficiency virus (HIV)-infected men assessed by isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) and chemiluminescent assay

Author

De Vincentis, S., Decaroli, M. C., Diazzi, C., Morini, F., Bertani, D., Fanelli, F., Mezzullo, M., Santi, D., Baraldi, E., Tagliavini, S., Pagotto, U., Guaraldi, G., and Rochira, Vincenzo

Subjects
MALE HYPOGONADISM, MASS SPECTROMETRY, ANDROGENS, ESTROGENS, HIV, SEX STEROIDS, ESTROGENS, ANDROGENS, GONADOTROPINS, HIV, MALE HYPOGONADISM, ID-LC-MS/MS, IMMUNOMETRY, MASS SPECTROMETRY, SEX STEROIDS, GONADOTROPINS, ID-LC-MS/MS, IMMUNOMETRY
Published
2018

13. La risposta degli steroidi allo stimolo con gonadotropina corionica (hCG) nei soggetti con sindrome di Klinefelter non cambia utilizzando gli immunodosaggi o la spettrometria di massa

Author

Roli, L., Santi, D., Tagliavini, S., Cavalieri, S., De Santis, M. C., Baraldi, E., Fanelli, F., Mezzullo, M., Granata, A. R., Pagotto, U., Pasquali, R., Rochira, V., Carani, C., Simoni, M., and Trenti, T.

Subjects
Immunoassay, Klinefelter Syndrome, Sex steroids, hCG, Androgens, Testosterone, Klinefelter Syndrome, Immunoassay, Mass Spectrometry, Sex steroids, Testosterone, Androgens, hCG, Mass Spectrometry
Published
2018

17. Will steroid measurements affect the outcomes of clinical trials? Comparison between immunoassayand mass spectrometry in men with Kinefelter Syndrome undergoing human corionic gonadotropin stimulation test

Author

Santi, Daniele, Roli, L., Belli, Serena, Tagliavini, Silvia, Cavalieri, Silvia, De Santis, M. C., Baraldi, Enrica, Fanelli, F., Mezzullo, M., Granata, A. R., Pagotto, U., Pasquali, R., Rochira, Vincenzo, Carani, Cesare, Trenti, T., and Simoni, Manuela

Subjects
Klinefelter Syndrome, testosterone, methods of steroid assays, sex steroids, Klinefelter Syndrome, sex steroids, testosterone, methods of steroid assays
Published
2016

18. P3012 Deconstructing the pig genome-metabolome functional interactions

Author

Fontanesi, L., primary, Bovo, S., additional, Schiavo, G., additional, Mazzoni, G., additional, Ribani, A., additional, Utzeri, V. J., additional, Dall’Olio, S., additional, Bertolini, F., additional, Fanelli, F., additional, Mezzullo, M., additional, Galimberti, G., additional, Calò, D. G., additional, Trevisi, P., additional, Martelli, P. L., additional, Casadio, R., additional, Pagotto, U., additional, and Bosi, P., additional

Published
2016
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19. Human chorionic gonadotropin stimulation gives evidence of differences in testicular steroidogenesis in Klinefelter syndrome, as assessed by liquid chromatography–tandem mass spectrometry

Author

Belli, S, primary, Santi, D, additional, Leoni, E, additional, Dall’Olio, E, additional, Fanelli, F, additional, Mezzullo, M, additional, Pelusi, C, additional, Roli, L, additional, Tagliavini, S, additional, Trenti, T, additional, Granata, A R, additional, Pagotto, U, additional, Pasquali, R, additional, Rochira, V, additional, Carani, C, additional, and Simoni, M, additional

Published
2016
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21. Deconstructing the pig sex metabolome: Targeted metabolomics in heavy pigs revealed sexual dimorphisms in plasma biomarkers and metabolic pathways1

Author

Bovo, S., primary, Mazzoni, G., additional, Calò, D. G., additional, Galimberti, G., additional, Fanelli, F., additional, Mezzullo, M., additional, Schiavo, G., additional, Scotti, E., additional, Manisi, A., additional, Samoré, A. B., additional, Bertolini, F., additional, Trevisi, P., additional, Bosi, P., additional, Dall'Olio, S., additional, Pagotto, U., additional, and Fontanesi, L., additional

Published
2015
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22. Comparison of three patterns of feed supplementation with live Saccharomyces cerevisiae yeast on postweaning diarrhea, health status, and blood metabolic profile of susceptible weaning pigs orally challenged with Escherichia coli F4ac1

Author

Trevisi, P., primary, Colombo, M., additional, Priori, D., additional, Fontanesi, L., additional, Galimberti, G., additional, Calò, G., additional, Motta, V., additional, Latorre, R., additional, Fanelli, F., additional, Mezzullo, M., additional, Pagotto, U., additional, Gherpelli, Y., additional, D'Inca, R., additional, and Bosi, P., additional

Published
2015
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25. Comparison of three patterns of feed supplementation with live Saccharomyces cerevisiaeyeast on postweaning diarrhea, health status, and blood metabolic profile of susceptible weaning pigs orally challenged with Escherichia coliF4ac1

Author

Trevisi, P., Colombo, M., Priori, D., Fontanesi, L., Galimberti, G., Calò, G., Motta, V., Latorre, R., Fanelli, F., Mezzullo, M., Pagotto, U., Gherpelli, Y., D'Inca, R., and Bosi, P.

Abstract

The development of effective feeding strategies to reduce the detrimental effect of enterotoxigenic Escherichia coliF4ac (ETEC) plays a crucial role in reducing the occurrence of therapeutic intervention with antibiotics in livestock. The ability of Saccharomyces cerevisiaeCNCM I-4407 (SCC), supplied in different patterns to counteract ETEC infection in weaned pigs, was evaluated. Fifty pigs weaned at 24 d were then divided into 5 groups: control (CO), CO + colistin (AB), CO + 5 × 1010cfu of SCC/ kg feed, from d 0 to 21 (PR), CO + 5 × 1010cfu of SCC/ kg feed from d 7 to 11 (CM), and CO + 1 shot of 2 × 1011cfu of SCC when the first diarrhea appeared (CU). On d 7 postweaning, all the pigs were orally challenged with 108cfu of ETEC. Blood samples were taken from the pigs (d 7, 8, 12, and 21) while the fecal excretion of ETEC was assessed on d 7 and 10. Fecal consistency was scored from 12 h before infection to 144 h postinfection (p.i.). On d 21, the pigs were sacrificed. The in vitro adhesion test on the intestinal villi confirmed individual susceptibility to ETEC, excluding the presence of resistant pigs. Growth performance did not differ between the treatments. Mortality was reduced in the AB group (P< 0.01) and, marginally, in the PR group (P= 0.089) when compared to the CO group. The CO group had a higher fecal score than AB in the period of observation (from P= 0.01 to P< 0.001). Yeast administration reduced the fecal score when compared to the CO group 12 and 48 h p.i. (P= 0.04). Total IgA never differed among the treatments, but the ETEC-specific IgA concentration was lower in the AB group than in CO (P= 0.04) at d 12. Four days p.i., the pigs fed live yeast had reduced ETEC excretion compared with the CO pigs (P= 0.05). Blood concentrations of dodecenoyl-L-carnitine (P< 0.01), glutaryl-L-carnitine/hydroxyhex¬anoyl-L-carnitine, phosphatidylcholine diacyl and phosphatidylcholine diacyl (P= 0.01 and P< 0.01, respectively), and α-amino adipic acid (P< 0.01) were reduced in the AB group compared to the CO group; PR + CM reduced the concentration of sphingomyelin-ceramide (P= 0.02) and increased the concentration of decadienyl-L-carnitine (C10:2; P= 0.02) vs. CO. The CM group had an increased concentration of C10:2 (P< 0.01) compared to the PR group. In conclusion, the administration of live yeast, even in concomitance with ETEC infections, reduces pig illness and mortality. The strain of SCC tested did not show a therapeutic effect.

Published
2015
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27. Exploring the human chorionic gonadotropin induced steroid secretion profile of mouse Leydig tumor cell line 1 by a 20 steroid LC-MS/MS panel

Author

Flaminia Fanelli, Matteo Magagnoli, Marco Mezzullo, Monica Lispi, Silvia Limoncella, Alessia Tommasini, Carla Pelusi, Daniele Santi, Manuela Simoni, Uberto Pagotto, Livio Casarini, Fanelli F., Magagnoli M., Mezzullo M., Lispi M., Limoncella S., Tommasini A., Pelusi C., Santi D., Simoni M., Pagotto U., and Casarini L.

Subjects
History, Androgen, Backdoor pathway, Leydig cells, Liquid chromatography – tandem mass spectrometry, Progesterone, Polymers and Plastics, Endocrinology, Diabetes and Metabolism, Clinical Biochemistry, Cell Biology, Biochemistry, Industrial and Manufacturing Engineering, Leydig cell, Endocrinology, Molecular Medicine, Business and International Management, Molecular Biology
Abstract

The canonical androgen synthesis in Leydig cells involves Delta 5 and Delta 4 steroids. Besides, the backdoor pathway, eompassing 5 alpha and 5 alpha,3 alpha steroids, is gaining interest in fetal and adult pathophysiology. Moreover, the role of androgen epimers and progesterone metabolites is still unknown. We developed a liquid chromatographytandem mass spectrometry (LC-MS/MS) method for measuring 20 steroids and used it to investigate the steroid secretion induced by human chorionic gonadotropin (hCG) in the mouse Leydig tumor cell line 1 (mLTC1). Steroids were extracted from 500 mu L supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 x 3 mm, 3 mu m column, with 100 mu M NH4F and methanol as mobile phases, and analyzed by positive electrospray ionization and multiple reaction monitoring. Sensitivity ranged within 0.012-38.0 nmol/L. Intra-assay and inter-assay imprecision were < 9.1% and 10.0%, respectively. Trueness, recovery and matrix factor were within 93.4-122.0, 55.6-104.1 and 76.4-106.3%, respectively. Levels of 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, 17OH-progesterone, androstenedione, epitestosterone, dihydrotestosterone, progesterone, androsterone and 17OH-allopregnanolone were effectively measured. Traces of 17OH-dihydroprogesterone, androstanediol and dihydroprogesterone were found, whereas androstenediol, 17OH-pregnenolone, dehydroepiandrosterone, pregnenolone and allopregnanolone showed no peak. hCG induced an increase of 80.2-102.5 folds in 16OH-progesterone, androstenedione and testosterone, 16.6 in dihydrotestosterone, 12.2-27.5 in epitestosterone, progesterone and metabolites, 8.1 in 17OH-allopregnanolone and

Published
2023

28. Report from the HarmoSter study:Impact of calibration on comparability of LC-MS/MS measurement of circulating cortisol, 17OH-progesterone and aldosterone

Author

Flaminia Fanelli, Marco Cantù, Anastasia Temchenko, Marco Mezzullo, Johanna M. Lindner, Mirko Peitzsch, James M. Hawley, Stephen Bruce, Pierre-Alain Binz, Mariette T. Ackermans, Annemieke C. Heijboer, Jody Van den Ouweland, Daniel Koeppl, Elena Nardi, Finlay MacKenzie, Manfred Rauh, Graeme Eisenhofer, Brian G. Keevil, Michael Vogeser, Uberto Pagotto, Endocrinology Laboratory, AGEM - Amsterdam Gastroenterology Endocrinology Metabolism, Laboratory Medicine, Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam Reproduction & Development (AR&D), Fanelli F., Cantu M., Temchenko A., Mezzullo M., Lindner J.M., Peitzsch M., Hawley J.M., Bruce S., Binz P.-A., Ackermans M.T., Heijboer A.C., Van Den Ouweland J., Koeppl D., Nardi E., Mackenzie F., Rauh M., Eisenhofer G., Keevil B.G., Vogeser M., Pagotto U., and Analytical Chemistry and Forensic Science (HIMS, FNWI)

Subjects
17OH-progesterone, aldosterone, Hydrocortisone, inter-laboratory performance, steroid hormones, Biochemistry (medical), Clinical Biochemistry, Reproducibility of Results, General Medicine, cortisol, calibration, external quality control, Tandem Mass Spectrometry, method comparison, harmonization, Humans, liquid chromatography-tandem mass spectrometry, Progesterone, Chromatography, Liquid
Abstract

Objectives Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration. Methods Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials. Results Using CAL1, intra-laboratory CVs ranged between 2.8–7.4%, 4.4–18.0% and 5.2–22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p Conclusions Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.

Published
2022

29. Impact of age, body weight and metabolic risk factors on steroid reference intervals in men

Author

Uberto Pagotto, Margherita Baccini, Alessia Fazzini, Flaminia Fanelli, Marco Mezzullo, Alessandra Gambineri, Carla Pelusi, Valentina Vicennati, Guido Di Dalmazi, Andrea Repaci, Mezzullo M., Di Dalmazi G., Fazzini A., Baccini M., Repaci A., Gambineri A., Vicennati V., Pelusi C., Pagotto U., and Fanelli F.

Subjects
Male, Hydrocortisone, Endocrinology, Diabetes and Metabolism, medicine.medical_treatment, Body Mass Index, chemistry.chemical_compound, 0302 clinical medicine, Endocrinology, Risk Factors, Tandem Mass Spectrometry, Corticosterone, Age Factor, Multivariate Analysi, Testosterone, 2. Zero hunger, 17-alpha-Hydroxyprogesterone, Age Factors, Dihydrotestosterone, General Medicine, 030220 oncology & carcinogenesis, Human, medicine.drug, medicine.medical_specialty, Cortodoxone, Dehydroepiandrosterone, 030209 endocrinology & metabolism, Estrone, 03 medical and health sciences, Insulin resistance, Internal medicine, medicine, Humans, Obesity, Cross-Sectional Studie, business.industry, Risk Factor, Insulin, Body Weight, Androstenedione, Overweight, medicine.disease, Cross-Sectional Studies, chemistry, Multivariate Analysis, business, Chromatography, Liquid
Abstract

Objective To evaluate the independent impact of age, obesity and metabolic risk factors on 13 circulating steroid levels; to generate reference intervals for adult men. Design Cross-sectional study. Methods Three hundred and fifteen adults, drug-free and apparently healthy men underwent clinical and biochemical evaluation. Thirteen steroids were measured by LC-MS/MS and compared among men with increasing BMI. Moreover, the independent impact of age, BMI and metabolic parameters on steroid levels was estimated. Upper and lower reference limits were generated in steroid-specific reference sub-cohorts and compared with dysmetabolic sub-cohorts. Results We observed lower steroid precursors and testosterone and increase in estrone levels in men with higher BMI ranges. By multivariate analysis, 17-hydroxyprogesterone and dihydrotestosterone decreased with BMI, while cortisol decreased with waist circumference. Estrone increased with BMI and systolic blood pressure. Testosterone decreased with worsening insulin resistance. 17-hydroxypregnenolone and corticosterone decreased with increasing total/HDL-cholesterol ratio. Age-related reference intervals were estimated for 17-hydroxypregnenolone, DHEA, 17-hydroxyprogesterone, corticosterone, 11-deoxycortisol, cortisol and androstenedione, while age-independent reference intervals were estimated for progesterone, 11-deoxycorticosterone, testosterone, dihydrotestosterone, estrone and estradiol. Testosterone lower limit was 2.29 nmol/L lower (P = 0.007) in insulin resistant vs insulin sensitive men. Furthermore, the upper limits for dihydrotestosterone (−0.34 nmol/L, P = 0.045), cortisol (−87 nmol/L, P = 0.045–0.002) and corticosterone (−10.1 nmol/L, P = 0.048–0.016) were lower in overweight/obese, in abdominal obese and in dyslipidaemic subjects compared to reference sub-cohorts, respectively. Conclusions Obesity and mild unmedicated metabolic risk factors alter the circulating steroid profile and bias the estimation of reference limits for testosterone, dihydrotestosterone, cortisol and corticosterone. Applying age-dependent reference intervals is mandatory for steroid precursors and corticosteroids.

Published
2020

30. Age-related changes in human Leydig cell status

Author

Valentina Mularoni, Uberto Pagotto, Carla Pelusi, Flaminia Fanelli, Elena Vicini, Niels Jørgensen, G. Spadetta, Anne Jørgensen, Marco Mezzullo, Carla Boitani, Valentina Esposito, Pasquale Berloco, John E Nielsen, Sara Di Persio, Ewa Rajpert-De Meyts, Mularoni V., Esposito V., Di Persio S., Vicini E., Spadetta G., Berloco P., Fanelli F., Mezzullo M., Pagotto U., Pelusi C., Nielsen J.E., Rajpert-De Meyts E., Jorgensen N., Jorgensen A., and Boitani C.

Subjects
Adult, Male, steroidogenesis, Cell type, endocrine system, medicine.drug_class, Somatic cell, Biology, Andrology, 03 medical and health sciences, Young Adult, 0302 clinical medicine, Leydig cell, Tandem Mass Spectrometry, Testis, medicine, ageing, human, leydig cells, testis, Humans, Insulin, Spermatogenesis, Testosterone, steroidogenesi, 030304 developmental biology, Aged, Aged, 80 and over, 0303 health sciences, 030219 obstetrics & reproductive medicine, Sertoli Cells, Rehabilitation, Obstetrics and Gynecology, Leydig Cells, Proteins, Middle Aged, Androgen, Sertoli cell, Androgen secretion, medicine.anatomical_structure, Reproductive Medicine, Chromatography, Liquid
Abstract

STUDY QUESTION What are the consequences of ageing on human Leydig cell number and hormonal function? SUMMARY ANSWER Leydig cell number significantly decreases in parallel with INSL3 expression and Sertoli cell number in aged men, yet the in vitro Leydig cell androgenic potential does not appear to be compromised by advancing age. WHAT IS KNOWN ALREADY There is extensive evidence that ageing is accompanied by decline in serum testosterone levels, a general involution of testis morphology and reduced spermatogenic function. A few studies have previously addressed single features of the human aged testis phenotype one at a time, but mostly in tissue from patients with prostate cancer. STUDY DESIGN, SIZE, DURATION This comprehensive study examined testis morphology, Leydig cell and Sertoli cell number, steroidogenic enzyme expression, INSL3 expression and androgen secretion by testicular fragments in vitro. The majority of these endpoints were concomitantly evaluated in the same individuals that all displayed complete spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS Testis biopsies were obtained from 15 heart beating organ donors (age range: 19–85 years) and 24 patients (age range: 19–45 years) with complete spermatogenesis. Leydig cells and Sertoli cells were counted following identification by immunohistochemical staining of specific cell markers. Gene expression analysis of INSL3 and steroidogenic enzymes was carried out by qRT-PCR. Secretion of 17-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone by in vitro cultured testis fragments was measured by LC-MS/MS. All endpoints were analysed in relation to age. MAIN RESULTS AND THE ROLE OF CHANCE Increasing age was negatively associated with Leydig cell number (R = −0.49; P LIMITATIONS, REASONS FOR CAUTION In vitro androgen production analysis could not be correlated with in vivo hormone values of the organ donors. In addition, the number of samples was relatively small and there was scarce information about the concomitant presence of potential confounding variables. WIDER IMPLICATIONS OF THE FINDINGS This study provides a novel insight into the effects of ageing on human Leydig cell status. The correlation between Leydig cell number and Sertoli cell number at any age implies a connection between these two cell types, which may be of particular relevance in understanding male reproductive disorders in the elderly. However aged Leydig cells do not lose their in vitro ability to produce androgens. Our data have implications in the understanding of the physiological role and regulation of intratesticular sex steroid levels during the complex process of ageing in humans. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from Prin 2010 and 2017. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

Published
2020

31. Steroid biomarkers for identifying non-classic adrenal hyperplasia due to 21-hydroxylase deficiency in a population of PCOS with suspicious levels of 17OH-progesterone

Author

S Castelli, Flaminia Fanelli, G. Di Dalmazi, Alessandra Gambineri, Valentina Vicennati, Lilia Baldazzi, G Brillanti, Uberto Pagotto, Andrea Repaci, Paola Altieri, Soara Menabo, C. Pelusi, Renato Pasquali, Marco Mezzullo, Elena Nardi, A Dormi, Francesca Corzani, Claudia Oriolo, Oriolo C., Fanelli F., Castelli S., Mezzullo M., Altieri P., Corzani F., Pelusi C., Repaci A., Di Dalmazi G., Vicennati V., Baldazzi L., Menabo S., Dormi A., Nardi E., Brillanti G., Pasquali R., Pagotto U., and Gambineri A.

Subjects
Adult, medicine.medical_specialty, Adolescent, Genotyping Techniques, Endocrinology, Diabetes and Metabolism, Population, DNA Mutational Analysis, Steroid profiling, CYP21A2 genotyping, ACTH test, Cohort Studies, Diagnostic Techniques, Endocrine, Basal (phylogenetics), chemistry.chemical_compound, Young Adult, Endocrinology, Corticosterone, Tandem Mass Spectrometry, Internal medicine, Follicular phase, Medicine, Humans, Testosterone, education, education.field_of_study, medicine.diagnostic_test, biology, Adrenal Hyperplasia, Congenital, business.industry, 17-alpha-Hydroxyprogesterone, ACTH stimulation test, 21-Hydroxylase, Reproducibility of Results, Hyperplasia, Non-classic adrenal hyperplasia due to 21-hydroxylase deficiency, medicine.disease, 1–24, chemistry, biology.protein, Female, Steroids, Steroid 21-Hydroxylase, business, Biomarkers, Blood Chemical Analysis, Chromatography, Liquid, Polycystic Ovary Syndrome
Abstract

Objective: We aimed at defining the most effective routine immunoassay- or liquid chromatography-tandem mass spectrometry (LC–MS/MS)-determined steroid biomarkers for identifying non-classic adrenal hyperplasia due to 21-hydroxylase deficiency (21-NCAH) in a PCOS-like population before genotyping. Methods: Seventy PCOS-like patients in reproductive age with immunoassay-determined follicular 17OH-progesterone (17OHP) ≥ 2.00 ng/mL underwent CYP21A2 gene analysis and 1–24ACTH test. Serum steroids were measured by immunoassays at baseline and 60 min after ACTH stimulation; basal steroid profile was measured by LC–MS/MS. Results: Genotyping revealed 23 21-NCAH, 15 single allele heterozygous CYP21A2 mutations (21-HTZ) and 32 PCOS patients displaying similar clinical and metabolic features. Immunoassays revealed higher baseline 17OHP and testosterone, and after ACTH stimulation, higher 17OHP (17OHP60) and lower cortisol, whereas LC–MS/MS revealed higher 17OHP (17OHPLC-MS/MS), progesterone and 21-deoxycortisol and lower corticosterone in 21-NCAH compared with both 21-HTZ and PCOS patients. Steroid thresholds best discriminating 21-NCAH from 21-HTZ and PCOS were estimated, and their diagnostic accuracy in identifying 21-NCAH from PCOS was established by ROC analysis. The highest accuracy was observed for 21-deoxycortisol ≥ 0.087 ng/mL, showing 100% sensitivity, while the combination of 17OHPLC-MS/MS ≥ 1.79 ng/mL and corticosterone ≤ 8.76 ng/mL, as well as the combination of ACTH-stimulated 17OHP ≥ 6.77 ng/mL and cortisol ≤ 240 ng/mL by immunoassay, showed 100% specificity. Conclusions: LC–MS/MS measurement of basal follicular 21-deoxycortisol, 17OHP and corticosterone seems the most convenient method for diagnosing 21-NCAH in a population of PCOS with a positive first level screening, providing high accuracy and reducing the need for ACTH stimulation test.

Published
2019

32. Female and male serum reference intervals for challenging sex and precursor steroids by liquid chromatography - tandem mass spectrometry

Author

Alessandra Gambineri, Uberto Pagotto, Flaminia Fanelli, Marco Mezzullo, Carla Pelusi, Andrea Repaci, Guido Di Dalmazi, Alessia Fazzini, Mezzullo M., Pelusi C., Fazzini A., Repaci A., Di Dalmazi G., Gambineri A., Pagotto U., and Fanelli F.

Subjects
0301 basic medicine, Male, 17-hydroxypregnenolone, Endocrinology, Diabetes and Metabolism, Clinical Biochemistry, Tandem mass spectrometry, Biochemistry, chemistry.chemical_compound, 0302 clinical medicine, Endocrinology, Liquid chromatography–mass spectrometry, Reference Values, Tandem Mass Spectrometry, estrogen, Aged, 80 and over, Estradiol, Dihydrotestosterone, Middle Aged, 3. Good health, Menopause, 030220 oncology & carcinogenesis, Molecular Medicine, Female, medicine.drug, Adult, medicine.medical_specialty, Adolescent, medicine.drug_class, Estrone, 17-alpha-Hydroxypregnenolone, 03 medical and health sciences, Young Adult, Internal medicine, medicine, Endocrine system, Humans, immunoassay, LC-MS/MS, Molecular Biology, Aged, business.industry, Selected reaction monitoring, Cell Biology, medicine.disease, 030104 developmental biology, chemistry, reference intervals, Estrogen, business, Chromatography, Liquid
Abstract

Measuring some sex and precursor steroids is still challenging even by liquid chromatography – tandem mass spectrometry (LC-MS/MS), and few normal values are available. We developed a LC-MS/MS method for estradiol, estrone, dihydrotestosterone and 17-hydroxypregnenolone measurement, compared it with direct immunoassays, and generated sex, age, menopausal and menstrual status specific reference intervals. Liquid-liquid extraction was optimized on 300 μL serum spiked with isotopic internal standards. A 2D-LC system allowed on-line purification and separation in 11 min run. Electrospray ionization was enhanced by ammonium fluoride. MS-detection was obtained by multiple reaction monitoring. Direct ECLIA for estradiol (n = 80) and RIA for estrone (n = 41) were compared with LC-MS/MS. Reference values were estimated in healthy, lean women in reproductive age (n = 118), menopausal women (n = 33) and men (n = 159). The assay showed satisfying imprecision, trueness, recovery and selectivity. Adequate functional sensitivity was achieved for measuring estrone (18.1 pmol/L) and 17-hydroxypregnenolone (117 pmol/L) in all subjects, and estradiol (35.9 pmol/L) and dihydrotestosterone (134 pmol/L) in women in reproductive age and men, but not in menopausal women. Compared with LC-MS/MS, immunoassays showed good agreement for estradiol but severe disagreement for estrone. Estrogens exhibited sex, menopausal and menstrual variations. Dihydrotestosterone and 17-hydroxypregnenolone depended on sex and menopause, the latter also declining with age in men. Strictly defined reference intervals in the adult female and male population were generated for challenging steroids such as estrogens, dihydrotestosterone and 17-hydroxypregnenolone by a novel LC-MS/MS method. Our achievement can be used to deepen the comprehension of several endocrine diseases.

Published
2019

33. The Steroid Profile of Adrenal Incidentalomas: Subtyping Subjects With High Cardiovascular Risk

Author

Carla Pelusi, Flaminia Fanelli, Rita Golfieri, Claudio Borghi, Paola Altieri, Uberto Pagotto, Alessandra Gambineri, Guido Di Dalmazi, Guido Zavatta, Eugenio Roberto Cosentino, Renato Pasquali, Andrea Repaci, Caterina Balacchi, Marco Mezzullo, Cristina Mosconi, Silvia Ricci Bitti, Valentina Vicennati, Di Dalmazi G., Fanelli F., Zavatta G., Ricci Bitti S., Mezzullo M., Repaci A., Pelusi C., Gambineri A., Altieri P., Mosconi C., Balacchi C., Golfieri R., Cosentino E.R., Borghi C., Vicennati V., Pasquali R., and Pagotto U.

Subjects
Cortisol secretion, Male, medicine.medical_specialty, Adenoma, Hydrocortisone, Endocrinology, Diabetes and Metabolism, medicine.medical_treatment, Clinical Biochemistry, Cortodoxone, Adrenal Gland Neoplasms, Dehydroepiandrosterone, Biochemistry, Gastroenterology, Steroid, chemistry.chemical_compound, Endocrinology, Corticosterone, Risk Factors, Tandem Mass Spectrometry, Internal medicine, medicine, Humans, Aged, business.industry, Biochemistry (medical), Hazard ratio, Hyperplasia, Middle Aged, Adrenal Incidentalomas: Cardiovascular Risk, medicine.disease, chemistry, Cardiovascular Diseases, Female, business, Cohort study, Chromatography, Liquid
Abstract

Context Steroid profiling by mass spectrometry has shown implications for diagnosis and subtyping of adrenal tumors. Objectives To investigate steroid profiles and their cardiovascular correlates in a large cohort of patients with nonsecreting (NS) adrenal incidentalomas and autonomous cortisol secretion (ACS). Design Cohort study. Setting University hospital. Patients Patients (n = 302) with incidentally discovered adrenal masses, divided into unilateral adenoma and hyperplasia with ACS (n = 46 and n = 52, respectively) and NS (n = 120 and n = 84, respectively). Post–dexamethasone suppression test (DST) cortisol 50 nmol/L defined NS and ACS, respectively. Intervention Analysis of 10-steroid panel by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and clinical data (mean follow-up 39 months). Main Outcome Measures Difference in baseline and post-DST steroid profiles between groups. Correlation with cardiovascular profile. Results Patients with unilateral adenomas and ACS showed higher cortisol, 11-deoxycortisol, and corticosterone and lower dehydroepiandrosterone than those with NS adenomas. Patients with ACS hyperplasia showed higher cortisol and lower androgens in women than those with NS. Patients with ACS had reduced suppression of post-DST cortisol, 11-deoxycortisol, and corticosterone, irrespective of adrenal morphology. Post-DST cortisol and corticosterone were associated with higher prevalence of severe/resistant hypertension. Patients with ACS unilateral adenomas showed higher incidence of worsening of hypertensive disease and novel cardiovascular events than those with NS, with post-DST cortisol [hazard ratio (HR) 1.02; 95% CI, 1.01 to 1.03; P < 0.001] and baseline corticosterone (HR 1.06; 95% CI, 1.01 to 1.12; P = 0.031) among the main predictors. Conclusions Patients with adrenal incidentalomas showed different steroid profiles, depending on functional status and adrenal morphology, with implications for their cardiovascular status.

Published
2019

34. Disease-Specific Derangement of Circulating Endocannabinoids and N-Acylethanolamines in Myeloproliferative Neoplasms

Author

Flaminia Fanelli, Uberto Pagotto, Marina Martello, Michele Cavo, Giuseppe Auteri, Francesca Palandri, Daniela Bartoletti, Emanuela Ottaviani, Dorian Forte, Marco Mezzullo, Antonio Curti, Carolina Terragna, Martina Barone, Giulia Corradi, Lucia Catani, Forte D., Fanelli F., Mezzullo M., Barone M., Corradi G., Auteri G., Bartoletti D., Martello M., Ottaviani E., Terragna C., Curti A., Pagotto U., Palandri F., Cavo M., and Catani L.

Subjects
Male, 0301 basic medicine, Metabolite, Myeloproliferative neoplasm, Oleic Acids, lcsh:Chemistry, chemistry.chemical_compound, 0302 clinical medicine, Polycythemia vera, Tandem Mass Spectrometry, Platelet, lcsh:QH301-705.5, Spectroscopy, Aged, 80 and over, essential thrombocythemia, Myelofibrosi, food and beverages, General Medicine, Middle Aged, Endocannabinoid system, Computer Science Applications, Haematopoiesis, Ethanolamines, 030220 oncology & carcinogenesis, Female, lipids (amino acids, peptides, and proteins), N-acylethanolamines, Thrombocythemia, Essential, Adult, medicine.medical_specialty, Polyunsaturated Alkamides, Mutation, Missense, myelofibrosis, Arachidonic Acids, Palmitic Acids, Article, myeloproliferative neoplasms, Catalysis, Glycerides, Inorganic Chemistry, 03 medical and health sciences, polycythemia vera, Internal medicine, medicine, Humans, endocannabinoids, Physical and Theoretical Chemistry, Myelofibrosis, Molecular Biology, Endocannabinoid, Aged, Myeloproliferative Disorders, Essential thrombocythemia, business.industry, Organic Chemistry, N-acylethanolamine, Cancer, Janus Kinase 2, medicine.disease, Amides, 030104 developmental biology, Endocrinology, lcsh:Biology (General), lcsh:QD1-999, chemistry, Primary Myelofibrosis, business, Chromatography, Liquid
Abstract

Growing evidence highlights the endocannabinoid (EC) system involvement in cancer progression. Lipid mediators of this system are secreted by hematopoietic cells, including the ECs 2-arachidonoyl-glycerol (2AG) and arachidonoyl-ethanolamide (AEA), the 2AG metabolite 1AG, and members of N-acylethanolamine (NAE) family&mdash, palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). However, the relevance of the EC system in myeloproliferative neoplasms (MPN) was never investigated. We explored the EC plasma profile in 55 MPN patients, including myelofibrosis (MF, n = 41), polycythemia vera (PV, n = 9), and essential thrombocythemia (ET, n = 5) subclasses and in 10 healthy controls (HC). AEA, PEA, OEA, 2AG, and 1AG plasma levels were measured by LC&ndash, MS/MS. Overall considered, MPN patients displayed similar EC and NAE levels compared to HC. Nonetheless, AEA levels in MPN were directly associated with the platelet count. MF patients showed higher levels of the sum of 2AG and 1AG compared to ET and PV patients, higher OEA/AEA ratios compared to HC and ET patients, and higher OEA/PEA ratios compared to HC. Furthermore, the sum of 2AG and 1AG positively correlated with JAK2V617F variant allele frequency and splenomegaly in MF and was elevated in high-risk PV patients compared to in low-risk PV patients. In conclusion, our work revealed specific alterations of ECs and NAE plasma profile in MPN subclasses and potentially relevant associations with disease severity.

Published
2020

35. Normative Basal Values of Hormones and Proteins of Gonadal and Adrenal Functions from Birth to Adulthood

Author

Uberto Pagotto, Federico Baronio, Rita Ortolano, Antonio Balsamo, Flaminia Fanelli, Alessandra Cassio, Marco Mezzullo, and Fanelli F, Baronio F, Ortolano R, Mezzullo M, Cassio A, Pagotto U, Balsamo A.

Subjects
Adult, Male, Embryology, Adolescent, Endocrinology, Diabetes and Metabolism, Physiology, 030209 endocrinology & metabolism, 030204 cardiovascular system & hematology, Peptide hormone, Biology, 03 medical and health sciences, Basal (phylogenetics), Young Adult, 0302 clinical medicine, Reference Values, Adrenal Glands, medicine, Humans, Reference population, Disorders of sex development, Child, Gonads, Testosterone, Infant, Newborn, Infant, Proteins, Middle Aged, medicine.disease, Hormones, Reference intervals, normative values, hormones, gonadal function, adrenal function, Child, Preschool, Normative, Female, Developmental Biology, Hormone
Abstract

In clinical practice, it is fundamental to compare the results of hormonal examinations obtained in the laboratory with reliable reference values. This is particularly difficult when faced with rare conditions, such as disorders of sex development, where not routinely assayed peptide hormones as well as intermediate steroid metabolites are often needed and local reliable reference values are not available. There are considerable differences among techniques and assays used in clinical and research laboratories. In fact, laboratory hormonology is undergoing a critical transition between techniques for quantitative determination: established immunoassays and mass spectrometry. Harmonizing results from different laboratories is a major challenge along the path leading to the establishment of consensus reference intervals for steroid hormones. Most of the efforts are being concentrated on testosterone, with very encouraging results being provided by the harmonization of liquid chromatography-tandem mass spectrometry results. However, this goal is still far from being achieved for the other steroid and small-molecule hormones, and a much more challenging perspective is foreseeable for protein hormones. In addition to technical issues, the importance of the definition and of the characterization of the reference population as well as sampling and processing methodology should not be underestimated, as these aspects may impact on hormonal axis and compound stability. The aim of the present review is to provide a comprehensive overview of the circulating reference values in basal condition of the hormones and proteins involved in sex development reported to date in the peer-reviewed literature. We present a series of tables where we have collected the reference intervals for each specific hormone and protein.

Published
2018

36. Deconstructing the pig sex metabolome: Targeted metabolomics in heavy pigs revealed sexual dimorphisms in plasma biomarkers and metabolic pathways

Author

S, Bovo, G, Mazzoni, D G, Calò, G, Galimberti, F, Fanelli, M, Mezzullo, G, Schiavo, E, Scotti, A, Manisi, A B, Samoré, F, Bertolini, P, Trevisi, P, Bosi, S, Dall'Olio, U, Pagotto, L, Fontanesi, Bovo, S, Mazzoni, G, Calò, D G, Galimberti, G, Fanelli, F, Mezzullo, M, Schiavo, G, Scotti, E, Manisi, A, Samoré, A B, Bertolini, F, Trevisi, P, Bosi, P, Dall'Olio, S, Pagotto, U, and Fontanesi, L

Subjects
Male, Sex Characteristics, Swine, Discriminant Analysis, Lipids, Metabolomics, pig sex metabolome, Gene Expression Regulation, Metabolome, Animals, Metabolomics, Female, Least-Squares Analysis, Biomarkers, Metabolic Networks and Pathways
Abstract

Metabolomics has opened new possibilities to investigate metabolic differences among animals. In this study, we applied a targeted metabolomic approach to deconstruct the pig sex metabolome as defined by castrated males and entire gilts. Plasma from 545 performance-tested Italian Large White pigs (172 castrated males and 373 females) sampled at about 160 kg live weight were analyzed for 186 metabolites using the Biocrates AbsoluteIDQ p180 Kit. After filtering, 132 metabolites (20 AA, 11 biogenic amines, 1 hexose, 13 acylcarnitines, 11 sphingomyelins, 67 phosphatidylcholines, and 9 lysophosphatidylcholines) were retained for further analyses. The multivariate approach of the sparse partial least squares discriminant analysis was applied, together with a specifically designed statistical pipeline, that included a permutation test and a 10 cross-fold validation procedure that produced stability and effect size statistics for each metabolite. Using this approach, we identified 85 biomarkers (with metabolites from all analyzed chemical families) that contributed to the differences between the 2 groups of pigs ( < 0.05 at the stability statistic test). All acylcarnitines and almost all biogenic amines were higher in castrated males than in gilts. Metabolites involved in tryptophan catabolism had the largest differences (i.e., delta = 20% for serotonin) between castrated males (higher) and gilts (lower). The level of several AA (Ala, Arg, Gly, His, Lys, Ser, Thr, and Trp) was higher in gilts (delta was from approximately 1.0 to approximately 4.8%) whereas products of AA catabolism (taurine, 2-aminoadipic acid, and methionine sulfoxide) were higher in castrated males (delta was approximately 5.0-6.0%), suggesting a metabolic shift in castrated males toward energy storage and lipid production. Similar general patterns were observed for most sphingomyelins, phosphatidylcholines, and lysophosphatidylcholines. Metabolomic pathway analysis and pathway enrichment identified several differences between the 2 sexes. This metabolomic overview opened new clues on the biochemical mechanisms underlying sexual dimorphism that, on one hand, might explain differences in terms of economic traits between castrated male pigs and entire gilts and, on the other hand, could strengthen the pig as a model to define metabolic mechanisms related to fat deposition.

Published
2015

37. Comparison of three patterns of feed supplementation with live Saccharomyces cerevisiae yeast on postweaning diarrhea, health status, and blood metabolic profile of susceptible weaning pigs orally challenged with Escherichia coli F4ac

Author

P, Trevisi, M, Colombo, D, Priori, L, Fontanesi, G, Galimberti, G, Calò, V, Motta, R, Latorre, F, Fanelli, M, Mezzullo, U, Pagotto, Y, Gherpelli, R, D'Inca, P, Bosi, Trevisi, P, Colombo, M, Priori, D, Fontanesi, L, Galimberti, G, Calò, G, Motta, V, Latorre, R, Fanelli, F, Mezzullo, M, Pagotto, U, Gherpelli, Y, D'Inca, R, and Bosi, P

Subjects
Diarrhea, Swine Diseases, Swine, Health Status, Saccharomyces cerevisiae, Weaning, Animal Feed, Anti-Bacterial Agents, Diet, Feces, Yeast, Dried, Dietary Supplements, Escherichia coli, Metabolome, blood metabolic profile, Escherichia coli F4ac, health status, pig, Saccharomyces cerevisiae CNCM I-4407, Animals, Escherichia coli Infections
Abstract

The development of effective feeding strategies to reduce the detrimental effect of enterotoxigenic F4ac (ETEC) plays a crucial role in reducing the occurrence of therapeutic intervention with antibiotics in livestock. The ability of CNCM I-4407 (SCC), supplied in different patterns to counteract ETEC infection in weaned pigs, was evaluated. Fifty pigs weaned at 24 d were then divided into 5 groups: control (CO), CO + colistin (AB), CO + 5 × 10(10) cfu of SCC/ kg feed, from d 0 to 21 (PR), CO + 5 × 10(10) cfu of SCC/ kg feed from d 7 to 11 (CM), and CO + 1 shot of 2 × 10(11) cfu of SCC when the first diarrhea appeared (CU). On d 7 postweaning, all the pigs were orally challenged with 10(8) cfu of ETEC. Blood samples were taken from the pigs (d 7, 8, 12, and 21) while the fecal excretion of ETEC was assessed on d 7 and 10. Fecal consistency was scored from 12 h before infection to 144 h postinfection (p.i.). On d 21, the pigs were sacrificed. The in vitro adhesion test on the intestinal villi confirmed individual susceptibility to ETEC, excluding the presence of resistant pigs. Growth performance did not differ between the treatments. Mortality was reduced in the AB group (P< 0.01) and, marginally, in the PR group (P = 0.089) when compared to the CO group. The CO group had a higher fecal score than AB in the period of observation (from P = 0.01 to P< 0.001). Yeast administration reduced the fecal score when compared to the CO group 12 and 48 h p.i. (P = 0.04). Total IgA never differed among the treatments, but the ETEC-specific IgA concentration was lower in the AB group than in CO (P = 0.04) at d 12. Four days p.i., the pigs fed live yeast had reduced ETEC excretion compared with the CO pigs (P = 0.05). Blood concentrations of dodecenoyl-L-carnitine (P < 0.01), glutaryl-L-carnitine/hydroxyhex¬anoyl-L-carnitine, phosphatidylcholine diacyl and phosphatidylcholine diacyl (P = 0.01 and P< 0.01, respectively), and α-amino adipic acid (P < 0.01) were reduced in the AB group compared to the CO group; PR + CM reduced the concentration of sphingomyelin-ceramide (P = 0.02) and increased the concentration of decadienyl-L-carnitine (C10:2; P= 0.02) vs. CO. The CM group had an increased concentration of C10:2 (P < 0.01) compared to the PR group. In conclusion, the administration of live yeast, even in concomitance with ETEC infections, reduces pig illness and mortality. The strain of SCC tested did not show a therapeutic effect.

Published
2015

38. Revisiting hyper- and hypo-androgenism by tandem mass spectrometry

Author

Carla Pelusi, Flaminia Fanelli, Alessandra Gambineri, Uberto Pagotto, Valentina Vicennati, Renato Pasquali, Marco Mezzullo, Fanelli F, Gambineri A, Mezzullo M, Vicennati V, Pelusi C, Pasquali R, and Pagotto U

Subjects
medicine.medical_specialty, medicine.drug_class, Endocrinology, Diabetes and Metabolism, Biochemical diagnosis, Bioinformatics, Tandem mass spectrometry, LC-MS/MS, Testosterone, Androgens, Hyperandrogenism, Hypogonadism, Endocrinology, Tandem Mass Spectrometry, Internal medicine, medicine, Humans, Steroid Measurement, Biochemical testing, Testosterone, business.industry, Hypogonadism, Hyperandrogenism, Androgen, medicine.disease, Reference intervals, Androgens, business, Chromatography, Liquid
Abstract

Modern endocrinology is living a critical age of transition as far as laboratory testing and biochemical diagnosis are concerned. Novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for steroid measurement in biological fluids have abundantly demonstrated their analytical superiority over immunometric platforms that until now have dominated the world of steroid hormones determination in clinical laboratories. One of the most useful applications of LC-MS/MS is in the hypogonadism and hyperandrogenism field: LC-MS/MS has proved particularly suitable for the detection of low levels of testosterone typical of women and children, and in general more reliable in accurately determining hypogonadal male levels. This technique also offers increased informative power by allowing multi-analytical profiles that give a more comprehensive picture of the overall hormonal asset. Several LC-MS/MS methods for testosterone have been published in the last decade, some of them included other androgen or more comprehensive steroid profiles. LC-MS/MS offers the concrete possibility of achieving a definitive standardization of testosterone measurements and the generation of widely accepted reference intervals, that will set the basis for a consensus on the diagnostic value of biochemical testing. The present review is aimed at summarizing technological advancements in androgen measurements in serum and saliva. We also provide a picture of the state of advancement of standardization of testosterone assays, of the redefinition of androgen reference intervals by novel assays and of studies using LC-MS/MS for the characterization and diagnosis of female hyperandrogenism and male hypogonadism.

Published
2013

39. Leucine supplementation protects from insulin resistance by regulating adiposity levels

Author

Caroline André, E. Binder, Melissa Elie, llaria Belluomo, Sophie Layé, Samantha Clark, Uberto Pagotto, Gilles Mithieux, Flaminia Fanelli, Agnès Aubert, Marco Mezzullo, Thierry Leste-Lasserre, Francisco Javier Bermúdez-Silva, Daniela Cota, A. Duchampt, Silvana Y. Romero-Zerbo, Neurocentre Magendie, Physiopathologie de la plasticité neuronale, U862, Institut National de la Santé et de la Recherche Médicale (INSERM), Hospital Carlos Haya, Nutrition et Neurobiologie intégrée (NutriNeuro), Institut National de la Recherche Agronomique (INRA)-Université Sciences et Technologies - Bordeaux 1-Centre National de la Recherche Scientifique (CNRS), Alma Mater Studiorum Università di Bologna [Bologna] (UNIBO), Marcia B. Aguila, Elke Binder, Francisco J. Bermúdez-Silva, Caroline André, Melissa Elie, Silvana Y. Romero-Zerbo, Thierry Leste-Lasserre, llaria Belluomo, Adeline Duchampt, Samantha Clark, Agnes Aubert, Marco Mezzullo, Flaminia Fanelli, Uberto Pagotto, Sophie Layé, Gilles Mithieux, Daniela Cota, ProdInra, Migration, Institut National de la Recherche Agronomique (INRA)-Université Sciences et Technologies - Bordeaux 1 (UB)-Centre National de la Recherche Scientifique (CNRS), [Binder,E, Bermúdez-Silva,FJ, André,C, Elie,M, Romero-Zerbo,SY, Lester-Lasserre,T, Belluomo,I, Clark,S, Cota,D] INSERM, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, Bordeaux, France. [Binder,E, Belluomo,I, Clark,S] Université de Bordeaux, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, Bordeaux, France. [Bermúdez-Silva,FJ, Romero-Zerbo,SY] IBIMA-Hospital Carlos Haya, Laboratorio de Investigación, Malaga, Spain. [Duchampt,A, Mithieux,G] INSERM, Lyon, France. [Duchampt,A, Mithieux,G, Cota,D] Université de Lyon, Lyon, France. [Duchampt,A, Mithieux,G] Université Lyon 1, Villeurbanne, France. [Aubert,A, Layé,S] Nutrition et Neurobiologie Intégrée, Université de Bordeaux, Bordeaux, France. INRA, Nutrition et Neurobiologie Intégrée, Bordeaux, France. [Mezzullo,M, Fanelli,F] Endocrinology Unit and Centro di Ricerca Biomedica Applicata, Department of Clinical Medicine, S.Orsola-Malpighi Hospital, Alma Mater University of Bologna, Bologna, Italy., This work was supported by INSERM, Aquitaine Region, Ajinomoto 3ARP research program, ANR-2010-1414-01 and EquipEx OptoPath ANR-10-EQPX-08 (to DC), European Community’s Seventh Framework Programme FP7-People2009-IEF-251494 (DC and EB) and Fondation Recherche Médicale. FJBS is recipient of a research contract from the National System of Health (Instituto de Salud Carlos III, and CP07/00283) and of a BAE from Instituto de Salud Carlos III (BA09/90066).

Subjects
Male, food intake, Ratones, [SDV]Life Sciences [q-bio], medicine.medical_treatment, Obesidad, Mice, Obese, tissu adipeux, Type 2 diabetes, test de tolérance, souris, Weight Gain, Mice, 0302 clinical medicine, Brown adipose tissue, Homeostasis, Insulin, Uncoupling protein, Aminoácidos, Adiposity, 2. Zero hunger, 0303 health sciences, Multidisciplinary, Diseases::Pathological Conditions, Signs and Symptoms::Signs and Symptoms::Body Weight::Overweight::Obesity [Medical Subject Headings], Fatty Acids, digestive, oral, and skin physiology, santé humaine, Lipids, Pérdida de peso, 3. Good health, [SDV] Life Sciences [q-bio], adiposité, obésité, Phenotype, medicine.anatomical_structure, Medicine, Leucine, leucine, Colesterol, Oxidation-Reduction, Research Article, diabète, medicine.medical_specialty, Science, 030209 endocrinology & metabolism, Carbohydrate metabolism, Biology, masse graisseuse, Diet, High-Fat, Alimentación rica en grasa, 03 medical and health sciences, Phenomena and Processes::Physiological Phenomena::Nutritional Physiological Phenomena::Diet::Diet, High-Fat [Medical Subject Headings], Insulin resistance, Leptina, pcr, Internal medicine, medicine, Animals, Diseases::Pathological Conditions, Signs and Symptoms::Signs and Symptoms::Body Weight::Body Weight Changes::Weight Loss [Medical Subject Headings], Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Amino Acids [Medical Subject Headings], baisse de poids, Chemicals and Drugs::Hormones, Hormone Substitutes, and Hormone Antagonists::Hormones::Peptide Hormones::Adipokines::Leptin [Medical Subject Headings], insuline, 030304 developmental biology, Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Rodentia::Muridae::Murinae::Mice [Medical Subject Headings], medicine.disease, Chemicals and Drugs::Lipids::Sterols::Cholesterol [Medical Subject Headings], Mice, Inbred C57BL, Glucose, Endocrinology, Dietary Supplements, Insulin Resistance, Energy Metabolism, Phenomena and Processes::Physiological Phenomena::Physiological Processes::Homeostasis [Medical Subject Headings]
Abstract

International audience; Background Leucine supplementation might have therapeutic potential in preventing diet-induced obesity and improving insulin sensitivity. However, the underlying mechanisms are at present unclear. Additionally, it is unclear whether leucine supplementation might be equally efficacious once obesity has developed. Methodology/Principal Findings Male C57BL/6J mice were fed chow or a high-fat diet (HFD), supplemented or not with leucine for 17 weeks. Another group of HFD-fed mice (HFD-pairfat group) was food restricted in order to reach an adiposity level comparable to that of HFD-Leu mice. Finally, a third group of mice was exposed to HFD for 12 weeks before being chronically supplemented with leucine. Leucine supplementation in HFD-fed mice decreased body weight and fat mass by increasing energy expenditure, fatty acid oxidation and locomotor activity in vivo. The decreased adiposity in HFD-Leu mice was associated with increased expression of uncoupling protein 3 (UCP-3) in the brown adipose tissue, better insulin sensitivity, increased intestinal gluconeogenesis and preservation of islets of Langerhans histomorphology and function. HFD-pairfat mice had a comparable improvement in insulin sensitivity, without changes in islets physiology or intestinal gluconeogenesis. Remarkably, both HFD-Leu and HFD-pairfat mice had decreased hepatic lipid content, which likely helped improve insulin sensitivity. In contrast, when leucine was supplemented to already obese animals, no changes in body weight, body composition or glucose metabolism were observed. Conclusions/Significance These findings suggest that leucine improves insulin sensitivity in HFD-fed mice by primarily decreasing adiposity, rather than directly acting on peripheral target organs. However, beneficial effects of leucine on intestinal gluconeogenesis and islets of Langerhans's physiology might help prevent type 2 diabetes development. Differently, metabolic benefit of leucine supplementation is lacking in already obese animals, a phenomenon possibly related to the extent of the obesity before starting the supplementation.

Published
2013

40. Metabolomics evidences plasma and serum biomarkers differentiating two heavy pig breeds

Author

Paolo Trevisi, Giuliano Galimberti, Samuele Bovo, Uberto Pagotto, Giuseppina Schiavo, Marco Mezzullo, Paolo Bosi, Stefania Dall'Olio, Gianluca Mazzoni, Daniela Giovanna Calo, Flaminia Fanelli, Luca Fontanesi, Annamaria Manisi, Bovo, S., Mazzoni, G., Galimberti, G., Calò, D.G., Fanelli, F., Mezzullo, M., Schiavo, G., Manisi, A., Trevisi, P., Bosi, P., Dall'Olio, S., Pagotto, U., and Fontanesi, L

Subjects
Male, 0301 basic medicine, molecular phenotypes, Stability test, Swine, metabolite, pig breeding, Breeding, Biology, SF1-1100, Mass Spectrometry, 03 medical and health sciences, Metabolomics, Serum biomarkers, Biogenic amine, Animals, Amino Acids, metabolites, chemistry.chemical_classification, Discriminant Analysis, production traits, Large white, metabolic pathway, Mass spectrometric, Breed, Animal culture, Pig breeding, molecular phenotype, Phenotype, 030104 developmental biology, Italy, chemistry, Biochemistry, Female, Animal Science and Zoology, Biomarkers
Abstract

In pigs, many production traits are known to vary among breeds or lines. These traits can be considered end phenotypes or external traits as they are the final results of complex biological interactions and processes whose fine biological mechanisms are still largely unknown. This study was designed to compare plasma and serum metabolomic profiles between animals of two heavy pig breeds (12 Italian Large White and 12 Italian Duroc), testing indirectly the hypothesis that different genetic backgrounds might be the determining factors of differences observed on the level of metabolites in the analyzed biofluids between breeds. We used a targeted metabolomic approach based on mass spectrometric detection of about 180 metabolites and applied a statistical validation pipeline to identify differences in the metabolomic profiles of the two heavy pig breeds. Blood samples were collected after jugulation at the slaughterhouse and prepared for metabolomics analysis that was carried out using the Biocrates AbsoluteIDQ p180 Kit, covering five different biochemical classes: glycerophospholipids, amino acids, biogenic amines, hexoses and acylcarnitines. A statistical pipeline that included the selection of the most relevant metabolites differentiating the two breeds by sparse Partial Least Squares Discriminant Analysis (sPLS-DA) was coupled with a stability test and significance test determined with leave one out and permutation procedures. sPLS-DA plots clearly separated the pigs of the two investigated breeds. A few metabolites (a total of five metabolites considering the two biofluids) involved in key metabolic pathways largely contributed to these differences between breeds. In particular, a higher level of the sphingomyelins SM (OH) C14:1 (both in plasma and serum), SM (OH) C16:1 (in serum) and SM C16:0 (in serum) were observed in Italian Duroc than in Italian Large White pigs and the inverse was for the biogenic amine kynurenine (in plasma). The level of another biogenic amine (acetylornithine) was higher in Italian Large White than in Italian Duroc pigs in both analysed biofluids. These results provided biomarkers that could be important to understand the biological differences between these two heavy pig breeds. In particular, according to the functional role played by sphingomyelins in obesity-induced inflammatory responses, it could be possible to speculate that a higher level of sphingomyelins in Italian Duroc might be related to the higher interrmuscular fat deposition of this breed compared with the Italian Large White. Additional studies will be needed to evaluate the relevance of these biomarkers for practical applications in pig breeding and nutrition.

41. Monitoring adrenal insufficiency through salivary steroids: a pilot study.

Author

Tucci L, Fanelli F, Improta I, Bissi V, Lena C, Galante G, Mezzullo M, Magagnoli M, Lalumera AB, Colombin G, Coscia K, Rotolo L, Vicennati V, Pagotto U, and Di Dalmazi G

Subjects
Humans, Glucocorticoids adverse effects, Hydrocortisone analysis, Pilot Projects, Saliva chemistry, Adrenal Insufficiency chemically induced, Adrenal Insufficiency diagnosis, Adrenal Insufficiency drug therapy, Cortisone therapeutic use, Cortisone analysis
Abstract

Background: Various glucocorticoid replacement therapies (GRTs) are available for adrenal insufficiency (AI). However, their effectiveness in restoring glucocorticoid rhythm and exposure lacks adequate biochemical markers. We described the diurnal salivary cortisol (SalF) and cortisone (SalE) rhythm among different GRTs and analysed the associations between saliva-derived parameters and life quality questionnaires., Methods: Control subjects (CSs, n = 28) and AI patients receiving hydrocortisone (HC, n = 9), cortisone acetate (CA, n = 23), and dual-release hydrocortisone once (DRHC-od, n = 10) and twice a day (DRHC-td, n = 6) collected 9 saliva samples from 07:00 to 23:00. Patients compiled Pittsburgh Sleep Quality Index, Hospital Anxiety and Depression Scale, and Addison disease-specific quality-of-life questionnaires. SalE and SalF were measured by liquid chromatography-mass spectrometry. Exposure was monitored using SalE for HC and DRHC and SalF for CA. Area under the curve (AUC) was computed. Different GRTs were compared by Z-scores calculated from saliva-derived parameters. Questionnaire results predictors were evaluated with multiple regression analysis., Results: Compared with controls, all GRTs resulted in glucocorticoid overexposure in the morning. Hydrocortisone, CA, and DRHC-td caused overexposure also in afternoon and evening. Compared with other treatments, CA determined increased Z-score-07:00 (P < .001), DRHC-td determined increased Z-score-AUC07:00→14:00 (P = .007), and DRHC-od induced lower Z-score-AUC14:00→23:00 (P = .015). Z-scores-AUC14:00→16:00 ≥ .619 best predicted questionnaire scores., Conclusions: None of the GRTs mimics normal glucocorticoid rhythmicity and exposure. SalE, SalF, and Z-score may be useful markers for monitoring and comparing different GRTs. Excess glucocorticoid in early afternoon best associated with depressive symptoms and worse life and sleep quality., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Endocrinology.)

Published
2024
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42. Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword.

Author

Fanelli F, Peitzsch M, Bruce S, Cantù M, Temchenko A, Mezzullo M, Lindner JM, Hawley JM, Ackermans MT, Van den Ouweland J, Koeppl D, Nardi E, MacKenzie F, Binz PA, Rauh M, Keevil BG, Vogeser M, Eisenhofer G, Heijboer AC, and Pagotto U

Subjects
Female, Humans, Male, Middle Aged, Calibration, Liquid Chromatography-Mass Spectrometry methods, Liquid Chromatography-Mass Spectrometry standards, Tandem Mass Spectrometry standards, Tandem Mass Spectrometry methods, Androstenedione blood, Androstenedione analysis, Dehydroepiandrosterone Sulfate blood, Dehydroepiandrosterone Sulfate analysis, Dehydroepiandrosterone Sulfate standards, Testosterone blood, Testosterone analysis, Testosterone standards
Abstract

Objectives: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification., Methods: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued., Results: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively., Conclusions: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Published
2024
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43. Determinants of sexual function in men living with HIV younger than 50 years old: Focus on organic, relational, and psychological issues.

Author

De Vincentis S, Decaroli MC, Milic J, Fanelli F, Tartaro G, Diazzi C, Mezzullo M, De Santis MC, Roli L, Trenti T, Santi D, Pagotto U, Guaraldi G, and Rochira V

Subjects
Humans, Male, Female, Middle Aged, Dihydrotestosterone, Cross-Sectional Studies, Testosterone therapeutic use, Estradiol, Erectile Dysfunction etiology, Sexual Dysfunction, Physiological epidemiology, Sexual Dysfunction, Physiological drug therapy, HIV Infections complications, HIV Infections epidemiology
Abstract

Background: Sexual dysfunctions, particularly erectile dysfunction, are common in men living with HIV, whose organic and psychological components remain to be clarified. The aim of the study is to investigate the impact of risk factors of sexual dysfunctions, including organic, relational, and psychological determinants of erectile function, in men living with HIV younger than 50 years old., Methods: A cross-sectional, observational study was conducted in men living with HIV < 50 years. The questionnaire International Index of Erectile Function-15 was used to assess the prevalence and degree of erectile dysfunction. The structured interview of erectile dysfunction was used to explore the organic (Scale 1), relational (Scale 2), and psychological (Scale 3) components of erectile dysfunction. Total testosterone, estradiol, and dihydrotestosterone were measured by liquid chromatography-tandem-mass spectrometry; free testosterone was calculated by the Vermeulen equation., Results: A total of 313 consecutive men living with HIV were prospectively enrolled (median age 47.0 years; median HIV-infection duration 16.2 years). 187 patients (59.7%) had erectile dysfunction, with a higher prevalence of non-heterosexual (138 out of 187, 73.8%) than heterosexual patients (p = 0.003). Patients with erectile dysfunction showed a worse score of structured interview of erectile dysfunction scale 3 compared to patients without erectile dysfunction (p = 0.025); the International Index of Erectile Function-15 was inversely related to structured interview of erectile dysfunction scale 3 (p = 0.042). No difference was found for sex steroids (total testosterone, estradiol, free testosterone, and dihydrotestosterone) between men living with HIV with and without erectile dysfunction. In the multivariate analysis sexual orientation, and lack of stable relationships were major determinants for erectile dysfunction. Only 35 of 187 patients with erectile dysfunction (18.7%) reported the use of erectile dysfunction medications., Conclusions: Within the multidimensional network of erectile dysfunction in men living with HIV, the psychological component is predominant, highlighting the contribution of peculiar factors related to HIV distress (e.g., fear of virus transmission, stigma) rather than gonadal status and other classical risk factors. In contrast to the high prevalence, only a few patients reported the use of erectile dysfunction medications suggesting a general under-management of such issues., (© 2022 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Published
2023
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44. Exploring the human chorionic gonadotropin induced steroid secretion profile of mouse Leydig tumor cell line 1 by a 20 steroid LC-MS/MS panel.

Author

Fanelli F, Magagnoli M, Mezzullo M, Lispi M, Limoncella S, Tommasini A, Pelusi C, Santi D, Simoni M, Pagotto U, and Casarini L

Subjects
Humans, Animals, Mice, Cell Line, Tumor, Male, Chromatography, Liquid methods, Tandem Mass Spectrometry methods, Chorionic Gonadotropin pharmacology, Leydig Cells drug effects, Leydig Cells metabolism, Gonadal Steroid Hormones analysis, Gonadal Steroid Hormones metabolism
Abstract

The canonical androgen synthesis in Leydig cells involves Δ5 and Δ4 steroids. Besides, the backdoor pathway, eompassing 5α and 5α,3α steroids, is gaining interest in fetal and adult pathophysiology. Moreover, the role of androgen epimers and progesterone metabolites is still unknown. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 20 steroids and used it to investigate the steroid secretion induced by human chorionic gonadotropin (hCG) in the mouse Leydig tumor cell line 1 (mLTC1). Steroids were extracted from 500 µL supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 × 3 mm, 3 µm column, with 100 µM NH4F and methanol as mobile phases, and analyzed by positive electrospray ionization and multiple reaction monitoring. Sensitivity ranged within 0.012-38.0 nmol/L. Intra-assay and inter-assay imprecision were < 9.1% and 10.0%, respectively. Trueness, recovery and matrix factor were within 93.4-122.0, 55.6-104.1 and 76.4-106.3%, respectively. Levels of 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, 17OH-progesterone, androstenedione, epitestosterone, dihydrotestosterone, progesterone, androsterone and 17OH-allopregnanolone were effectively measured. Traces of 17OH-dihydroprogesterone, androstanediol and dihydroprogesterone were found, whereas androstenediol, 17OH-pregnenolone, dehydroepiandrosterone, pregnenolone and allopregnanolone showed no peak. hCG induced an increase of 80.2-102.5 folds in 16OH-progesterone, androstenedione and testosterone, 16.6 in dihydrotestosterone, 12.2-27.5 in epitestosterone, progesterone and metabolites, 8.1 in 17OH-allopregnanolone and ≤ 3.3 in 5α and 5α,3α steroids. In conclusion, our LC-MS/MS method allows exploring the Leydig steroidogenesis flow according to multiple pathways. Beside the expected stimulation of the canonical pathway, hCG increased progesterone metabolism and, to a low extent, the backdoor route., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Published
2023
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45. Primary, secondary and compensated male biochemical hypogonadism in people living with HIV (PLWH): relevance of sex hormone-binding globulin (SHBG) measurement and comparison between liquid chromatography-tandem mass spectrometry (LC-MS/MS) and chemiluminescent immunoassay for sex steroids assay.

Author

De Vincentis S, Decaroli MC, Fanelli F, Diazzi C, Mezzullo M, Tartaro G, Tagliavini S, De Santis MC, Roli L, Milic J, Trenti T, Pagotto U, Guaraldi G, and Rochira V

Subjects
Chromatography, Liquid, Cross-Sectional Studies, Humans, Immunoassay, Male, Middle Aged, Prospective Studies, Tandem Mass Spectrometry, Testosterone, HIV Infections complications, Hypogonadism complications, Sex Hormone-Binding Globulin analysis
Abstract

Background: Data about classification of hypogonadism and estrogen deficiency in male people living with HIV (PLWH) are scanty., Aim: To investigate the prevalence and characterization of biochemical hypogonadism and relative estrogen deficiency in male PLWH aged < 50 comparing liquid chromatography-tandem mass spectrometry (LC-MS/MS) with chemiluminescent immunoassay (CI), and combining gonadotropin, sex hormone-binding globulin (SHBG) and serum estradiol (E2) measurements., Methods: Prospective, cross-sectional, observational study. Serum total testosterone (TT), E2, gonadotropins, SHBG were measured by CI. TT and E2 were also assessed by LC-MS/MS. Free testosterone (cFT) was calculated by Vermeulen equation., Results: A total of 316 PLWH (45.3 ± 5.3 years) were enrolled. TT and cFT by LC-MS/MS were lower compared to CI ( p  < 0.0001). The prevalence of biochemical hypogonadism was higher with LC-MS/MS than CI, both for TT (5.1% vs 3.2%, p  < 0.0001) or cFT (9.5% vs 7%, p  < 0.0001). The prevalence of hypogonadism (overt + compensated) was 17.1% for cFT using LC-MS/MS. Secondary form of hypogonadism was more prevalent than primary. The prevalence of relative estrogen deficiency was of 30.0% among hypogonadal patients and 15.5% among eugonadal., Conclusions: The prevalence of male hypogonadism results underestimated by CI compared to LC-MS/MS in PLWH, both for TT and cFT. SHBG and gonadotropins are essential for detecting T deficiency.

Published
2022
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46. Report from the HarmoSter study: inter-laboratory comparison of LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone.

Author

Fanelli F, Bruce S, Cantù M, Temchenko A, Mezzullo M, Lindner JM, Peitzsch M, Binz PA, Ackermans MT, Heijboer AC, Van den Ouweland J, Koeppl D, Nardi E, Rauh M, Vogeser M, Eisenhofer G, and Pagotto U

Subjects
Humans, Chromatography, Liquid methods, Cortodoxone, Corticosterone, Tandem Mass Spectrometry methods, Reproducibility of Results, Cortisone
Abstract

Objectives: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels that include glucocorticoid-related steroids are increasingly used to characterize and diagnose adrenal cortical diseases. Limited information is currently available about reproducibility of these measurements among laboratories. The aim of the study was to compare LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone at eight European centers and assess the performance after unification of calibration., Methods: Seventy-eight patient samples and commercial calibrators were measured twice by laboratory-specific procedures. Results were obtained according to in-house and external calibration. We evaluated intra-laboratory and inter-laboratory imprecision, regression and agreement against performance specifications derived from 11-deoxycortisol biological variation., Results: Intra-laboratory CVs ranged between 3.3 and 7.7%, 3.3 and 11.8% and 2.7 and 12.8% for corticosterone, 11-deoxycortisol and cortisone, with 1, 4 and 3 laboratories often exceeding the maximum allowable imprecision (MAI), respectively. Median inter-laboratory CVs were 10.0, 10.7 and 6.2%, with 38.5, 50.7 and 2.6% cases exceeding the MAI for corticosterone, 11-deoxycortisol and cortisone, respectively. Median laboratory bias vs. all laboratory-medians ranged from -5.6 to 12.3% for corticosterone, -14.6 to 12.4% for 11-deoxycortisol and -4.0 to 6.5% for cortisone, with few cases exceeding the total allowable error. Modest deviations were found in regression equations among most laboratories. External calibration did not improve 11-deoxycortisol and worsened corticosterone and cortisone inter-laboratory comparability., Conclusions: Method imprecision was variable. Inter-laboratory performance was reasonably good. However, cases with imprecision and total error above the acceptable limits were apparent for corticosterone and 11-deoxycortisol. Variability did not depend on calibration but apparently on imprecision, accuracy and specificity of individual methods. Tools for improving selectivity and accuracy are required to improve harmonization., (© 2022 the author(s), published by De Gruyter, Berlin/Boston.)

Published
2022
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47. Report from the HarmoSter study: impact of calibration on comparability of LC-MS/MS measurement of circulating cortisol, 17OH-progesterone and aldosterone.

Author

Fanelli F, Cantù M, Temchenko A, Mezzullo M, Lindner JM, Peitzsch M, Hawley JM, Bruce S, Binz PA, Ackermans MT, Heijboer AC, Van den Ouweland J, Koeppl D, Nardi E, MacKenzie F, Rauh M, Eisenhofer G, Keevil BG, Vogeser M, and Pagotto U

Subjects
Aldosterone, Calibration, Chromatography, Liquid methods, Humans, Reproducibility of Results, Tandem Mass Spectrometry methods, Hydrocortisone, Progesterone
Abstract

Objectives: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration., Methods: Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials., Results: Using CAL1, intra-laboratory CVs ranged between 2.8-7.4%, 4.4-18.0% and 5.2-22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from -6.6 to 6.9%, -17.2 to 7.8% and -12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation., Conclusions: Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements., (© 2022 Flaminia Fanelli et al., published by De Gruyter, Berlin/Boston.)

Published
2022
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48. Impact of Clomiphene Citrate on the Steroid Profile in Dysmetabolic Men with Low Testosterone Levels.

Author

Pelusi C, Fanelli F, Baccini M, De Pergola G, Triggiani V, Mezzullo M, Fazzini A, Di Dalmazi G, Petrovic B, Paterini P, Labate AMM, Pagotto U, and Giagulli VA

Subjects
Adult, Chromatography, Liquid, Cross-Over Studies, Double-Blind Method, Humans, Male, Middle Aged, Obesity metabolism, Steroids metabolism, Tandem Mass Spectrometry, Transcortin analysis, Clomiphene pharmacology, Steroids blood, Testosterone blood
Abstract

Clomiphene citrate (CC) in male hypogonadism increases testosterone (T) and estrogen levels by stimulating pituitary gonadotropin release. Our group confirmed these hormonal changes in a randomized, cross-over, double-blind trial of CC versus placebo in addition to metformin, conducted in 21 obese dysmetabolic men with low T levels. However, we hypothesize that based on its mechanism of action, CC may directly or indirectly affect adrenal steroidogenesis. The aim of this sub-study was to better understand the changes in steroid levels and metabolism induced by CC treatment. We assessed 17α-hydroxypregnelone (17αOH-P5), dehydroepiandrosterone (DHEA), progesterone (P4), 17α-hydroxyprogesterone (17αOH-P4), androstenedione (A), T, dihydrotestosterone (DHT), estrone (E1), 17β-estradiol (E2), 11-deoxycortisol (11 S), cortisol (F), and cortisone (E) by LC-MS/MS, and corticosteroid binding globulin (CBG) by ELISA, before and after each treatment. In addition, free-F and steroid product/precursor ratios were calculated. We observed a significant change in serum levels induced by CC compared with placebo for 17αOH-P4, DHT, T, E2, E1, F, E, and CBG, but not free-F. In addition, compared to placebo, CC induced higher 17αOH-P4/P4, E2/E1, 17αOH-P4/17αOH-P5, A/17αOH-P4, T/A, E1/A, F/11 S, and F/E ratios. Therefore, besides the CC stimulating effect on testis steroidogenesis, our study showed increased F, E, but not free-F, levels, indicating changes in steroid metabolism rather than adrenal secretion stimulation. The steroid profiling also revealed the CC stimulation of the Δ5 rather than the Δ4 pathway, thus indicating considerable testicular involvement in the increased androgen secretion., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Published
2021
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49. Shorter androgen receptor polyQ alleles protect against life-threatening COVID-19 disease in European males.

Author

Baldassarri M, Picchiotti N, Fava F, Fallerini C, Benetti E, Daga S, Valentino F, Doddato G, Furini S, Giliberti A, Tita R, Amitrano S, Bruttini M, Croci S, Meloni I, Pinto AM, Iuso N, Gabbi C, Sciarra F, Venneri MA, Gori M, Sanarico M, Crawley FP, Pagotto U, Fanelli F, Mezzullo M, Dominguez-Garrido E, Planas-Serra L, Schlüter A, Colobran R, Soler-Palacin P, Lapunzina P, Tenorio J, Pujol A, Castagna MG, Marcelli M, Isidori AM, Renieri A, Frullanti E, and Mari F

Subjects
Aged, Case-Control Studies, Critical Care statistics & numerical data, Female, Genome, Human genetics, Humans, Male, Middle Aged, Polymorphism, Single Nucleotide genetics, Risk Factors, SARS-CoV-2, Severity of Illness Index, Spain, Testosterone blood, COVID-19 pathology, Peptides genetics, Receptors, Androgen genetics
Abstract

Background: While SARS-CoV-2 similarly infects men and women, COVID-19 outcome is less favorable in men. Variability in COVID-19 severity may be explained by differences in the host genome., Methods: We compared poly-amino acids variability from WES data in severely affected COVID-19 patients versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects., Findings: Shorter polyQ alleles (≤22) in the androgen receptor (AR) conferred protection against severe outcome in COVID-19 in the first tested cohort (both males and females) of 638 Italian subjects. The association between long polyQ alleles (≥23) and severe clinical outcome (p = 0.024) was also validated in an independent cohort of Spanish men <60 years of age (p = 0.014). Testosterone was higher in subjects with AR long-polyQ, possibly indicating receptor resistance (p = 0.042 Mann-Whitney U test). Inappropriately low serum testosterone level among carriers of the long-polyQ alleles (p = 0.0004 Mann-Whitney U test) predicted the need for intensive care in COVID-19 infected men. In agreement with the known anti-inflammatory action of testosterone, patients with long-polyQ and age ≥60 years had increased levels of CRP (p = 0.018, not accounting for multiple testing)., Interpretation: We identify the first genetic polymorphism that appears to predispose some men to develop more severe disease. Failure of the endocrine feedback to overcome AR signaling defects by increasing testosterone levels during the infection leads to the polyQ tract becoming dominant to serum testosterone levels for the clinical outcome. These results may contribute to designing reliable clinical and public health measures and provide a rationale to test testosterone as adjuvant therapy in men with COVID-19 expressing long AR polyQ repeats., Funding: MIUR project "Dipartimenti di Eccellenza 2018-2020" to Department of Medical Biotechnologies University of Siena, Italy (Italian D.L. n.18 March 17, 2020) and "Bando Ricerca COVID-19 Toscana" project to Azienda Ospedaliero-Universitaria Senese. Private donors for COVID-19 research and charity funds from Intesa San Paolo., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Published
2021
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50. Steroid reference intervals in women: influence of menopause, age and metabolism.

Author

Mezzullo M, Gambineri A, Di Dalmazi G, Fazzini A, Magagnoli M, Baccini M, Vicennati V, Pelusi C, Pagotto U, and Fanelli F

Subjects
Adolescent, Adult, Age Factors, Aged, Aged, 80 and over, Aging metabolism, Blood Chemical Analysis methods, Chromatography, Liquid standards, Cross-Sectional Studies, Diagnostic Techniques, Endocrine standards, Female, Gonadal Steroid Hormones analysis, Gonadal Steroid Hormones standards, Humans, Menopause metabolism, Middle Aged, Reference Values, Sex Factors, Tandem Mass Spectrometry standards, Young Adult, Aging blood, Blood Chemical Analysis standards, Energy Metabolism physiology, Gonadal Steroid Hormones blood, Menopause blood
Abstract

Objective: To investigate the impact of age, obesity and metabolic parameters on 13 circulating steroids in reproductive and menopausal age. To define reference intervals (RIs)., Design: Cross-sectional., Methods: Three hundred and twenty five drug-free, healthy and eumenorrheic women were selected from the general population. Independent relationships of LC-MS/MS-determined steroid levels with age, BMI and metabolic parameters were estimated. Reference sub-cohorts were defined for calculating upper and lower limits in reproductive age, menstrual phases and menopause, and these were compared with limits in dysmetabolic sub-cohorts., Results: Lower androgens, pro-androgens and estrogens, but higher cortisol and metabolites were found in menopausal compared to reproductive age women. Androgens and precursors decreased during reproductive age (P < 0.001-P = 0.002) but not after menopause. 17OH-progesterone decreased with BMI (P = 0.006) and glucocorticoids with waist circumference (P < 0.001P = 0.002) in reproductive age, but increased with triglycerides (P=0.011P=0.038) after menopause. Inverse associations of dihydrotestosterone with BMI (P=0.004) and HDL-cholesterol (P=0.010), estrone with total cholesterol (P=0.033) and estradiol with triglycerides (P=0.011) were found in reproductive age. After menopause, estrone increased with waist circumference (P<0.001) and decreased with insulin resistance (P=0.012). Ovarian steroid RIs were estimated in menstrual phases and menopause. Age- and reproductive status-specific RIs were generated for androgens, precursors and corticosteroids. Lower limits for reproductive age cortisol (P=0.020) and menopausal 11-deoxycortisol (P=0.003) in dysmetabolic sub-cohorts were reduced and increased, respectively, compared to reference limits., Conclusions: Obesity and dysmetabolism differently influence circulating steroids in reproductive and menopausal status. Age, menstrual and menopausal status-specific RIs were provided by LC-MS/MS for a broad steroid panel.

Published
2021
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