Estudios del laboratorio de diversos parámetros orientados a tratar de dilucidar la fisiopatología del síndrome de ovarios poliquísticos (SOP). Parte 2 / Laboratory studies of several parameters aimed to elucidate the pathophysiology of polycystic ovary syndrome. Second part

RESUMEN Diversos estudios bioquímicos adicionales a la evaluación de Testosterona total (TT), biodisponible (Tbio) y libre (TL) han sido realizados a los efectos que pudieran resultar de mayor utilidad para el diagnóstico de patologías concomitantes en el SOP, entre otros. En la hormona anti Mülleriana, cuando la concentración supera a los 3,0 ng/ml existen evidencias de que el 79% de las mismas pueden ser identificadas correctamente como SOP. El Antígeno Prostático Específico (PSA), marcador de singular importancia en pacientes con cáncer de Próstata, con técnicas ultrasensibles ha podido ser detectado en más del 50% en mujeres. En un grupo de pacientes con SOP, los niveles circulantes de PSA fueron significativamente mayores que en las mujeres sin SOP. El Kiss-1 aislado de la placenta y demostrado en otros tejidos, presenta niveles aumentados que correlacionan con la LH, TT, TL y resistencia a la insulina (RI) en adolescentes con SOP versus adolescentes sin SOP, sugiriendo que el Kiss-1 podría estar involucrado en el desarrollo del SOP en estas pacientes. Algunas pacientes con SOP están asociadas a patologías relevantes, de las cuales han sido comunicadas el aumento del BMI, mayor grado de dislipemia, adiposidad central, RI y Síndrome Metabólico (SMe). En las pacientes con un fenotipo clásico (hiperandrogenismo, alteración del ciclo menstrual y ovarios poliquísticos), estas patologías son de mayor frecuencia y severidad que en los otros fenotipos, particularmente aquellos sin hiperandrogenismo. Otras determinaciones como TNFα, interleuquinas, test de tolerancia a la glucosa, ApoB, partículas pequeñas de LDL e Inhibidor del Activador del Plasminógeno-1 han sido comunicados que podrían ser de utilidad para tener mayor sensibilidad en la definición de patología concomitantes en el SOP. Actualmente se ha comenzado a evaluar otros marcadores como el Fetuin-A; Quemerina, Nesfatina-1, Neopterina y Endocannabinoides, cuyos resultados preliminares parecerían ser un aporte importante para evaluar SMe y RI en paciente con SOP y tratar de definir su prevalencia en los distintos fenotipos de esta patología.

ABSTRACT Several biochemical studies in addition to the evaluation of total Testosterone (TT), bioavailable (bioT) and free (FT) have been performed to the effects that could be of greater use for the diagnosis of concomitant pathologies in the PCOS, among others. The anti-Müllerian hormone whose concentration when exceeds 3.0 ng/ml, there is evidence that 79% of these patients can be correctly identified as PCOS. The Prostate-Specific Antigen (PSA), a marker of singular importance in patients with prostate cancer, with ultra-sensitive techniques, has been detected in more than 50% of women. In a group of patients with PCOS, circulating levels of PSA are significantly higher than in women without PCOS. The Kiss-1 isolated from the placenta and demonstrated in other tissues, has increased levels that correlate with LH, TT, TL and insulin resistance (IR) in adolescents with PCOS respect to adolescents without PCOS, suggesting that Kiss-1 could be involved in the development of the PCOS in these patients. In some patients with PCOS, they are associated with relevant pathologies, of which the increase in BMI, higher degree of dyslipidemia, central adiposity, IR and Metabolic Syndrome (MeS) have been reported. Those that show a classic phenotype (hyperandrogenism, alteration of the menstrual cycle and polycystic ovaries) these characteristics are of greater frequency and severity than in the other phenotypes, particularly those without hyperandrogenism. Other determinations such as TNFα, interleukins, glucose tolerance test, ApoB, small particles of LDL and Plasminogen Activator Inhibitor-1 have been reported that could be useful to have greater sensitivity in the definition of concomitant pathology in the PCOS. Currently, other markers such as Fetuin-A, Chemerin, Nesfatin-1 Neopterin and Endocannabinoids have been evaluated. The preliminary results suggest to be an important contribution to define MeS and IR in patient with PCOS and to try to determine its prevalence in the different phenotypes of this pathology.

Estudios de laboratorio de diversos parámetros orientados a tratar de dilucidar la fisiopatología del síndrome de ovarios poliquísticos. Parte 1 / Laboratory studies of various parameters aimed at trying to elucidate the pathophysiology of polycystic ovary syndrome. Part 1

Esta revisión fue realizada con el fin de evaluar nuestros resultados de laboratorio así como aquellos de la literatura que constituyen, a nuestro entender, aportes significativos en el síndrome de ovarios poliquísticos (SOP). Nuestro especial énfasis será presentar las limitaciones de las metodologías empleadas por nuestro grupo, comparativamente a las reportadas por otros investigadores. La determinación de andrógenos, en particular de Testosterona (TT), es quizá la de mayor complejidad dado que los resultados con los diferentes inmunoensayos empleados en nuestro medio producen resultados muy variables por los diferentes métodos y aún entre laboratorios que usan la misma metodología. La técnica de referencia es la cromatografía líquida en tándem con espectrometría de masa (LC-MSMS), de difícil aplicación en laboratorios de análisis clínicos debido a su alto costo y la imposibilidad de resolver numerosas muestras. En estudios previos demostramos que de los métodos habitualmente usados para evaluar la TT circulante, solo en 2 inmunoensayos los resultados obtenidos fueron satisfactoriamente validados indirectamente según el criterio del Consenso de los Centros para el Control y Prevención de Enfermedades (CDC, USA) contra LC-MSMS, los cuales fueron comparables a dicha metodología con niveles superiores a 0,5 ng/ml. El SOP puede presentar factores de riesgo aumentados para la enfermedad cardiovascular y la diabetes II. Estos factores no están debidamente categorizados en función de los distintos fenotipos del SOP. Se evaluarán los principales analitos empleados con este objetivo y los nuevos que aporten elementos de mayor especificidad en este sentido

This review was performed in order to evaluate our laboratory results as well as those of the literature that constitute, in our opinion, significant contributions in these pathophysiologies. Our special emphasis will be on presenting the limitations of the methodologies used by our group, compared to those reported by other researchers. The determination of androgens, in particular Testosterone (TT), is perhaps the most complex since the results with the different immunoassays used in our environment produce very variable results by the different methods and even between laboratories that use the same methodology. The reference technique is LC-MSMS, difficult to apply in clinical analysis laboratories because of its high cost and the inability to solve numerous samples. In previous studies, we demonstrated that, in comparison to LC-MSMS with the usual methods for evaluating circulating TT, the results obtained in only 2 immunoassays were satisfactorily validated indirectly according to the criteria of CDC against LC-MSMS, which were comparable to that methodology with levels higher than 0.5 ng/ml. PCOS may have increased risk factors for cardiovascular disease and diabetes II. These factors are not properly categorized according to the different phenotypes of PCOS. The main analytes used for this purpose will be evaluated and new ones that contribute elements of greater specificity in this sense

Evaluation of the hypothalamic-pituitary-gonadal axis in eugonadal men with type 2 diabetes mellitus.

Men with type 2 diabetes mellitus (DM2) have lower testosterone levels and a higher prevalence of hypogonadism. It still remains unclear the mechanism by which there is a relationship between hypogonadism and DM2. The objective was to evaluate the hypothalamic-pituitary-gonadal axis at different levels in eugonadal patients with DM2. Fourteen patients with DM2 (DM2 group) and 15 subjects without DM2 (normal glucose tolerance test) as control group (CG) were included. We assessed: (i) fasting glucose, insulin, Homeostasis Model Assessment (HOMA); (ii) luteinizing hormone (LH) pulsatility through blood collections every 10 min for 4 h; (iii) gonadotropin-releasing hormone (GnRH) test: basal LH and 30, 60 and 90 min after 100 µg of i.v. GnRH; (iv) human chorionic gonadotropin (hCG) test: basal total testosterone (TT), bioavailable testosterone (BT), free testosterone (FT), estradiol (E2), bioavailable E2 (BE2) and sex hormone-binding globulin (SHBG) and 72 h post 5000 IU of i.m. hCG. There were no differences in age, body mass index and waist circumference between groups. Glucose was higher in the DM2 group vs. CG: 131.1 ± 25.5 vs. 99.1 ± 13.6 mg/dL, p = 0.0005. There were no difference in basal insulin, HOMA, TT, BT, FT, E2, BE2, SHBG and LH levels between groups. The DM2 group had lower LH pulse frequency vs. CG: 0.8 ± 0.8 vs. 1.5 ± 0.5 pulses, p = 0.009. Differences in LH pulse amplitude were not found. A negative correlation was found between the number of LH pulses and glucose, r: -0.39, p = 0.03. There were no differences in the response of LH to GnRH between groups nor in the response of sexual steroids and SHBG to hCG. Patients with DM2 showed lower hypothalamic pulse frequency without changes in the pituitary response to GnRH nor testicular response to hCG. Glucose levels negatively correlated with the number of LH pulses which suggests a negative effect of hyperglycaemia in the hypothalamic secretion of GnRH.

Quantification of Testosterone (T) by 8 Immunoassays and by Liquid Chromatography -Tandem Mass Spectrometry (LC-MSMS) in Normal and Hirsute Women: A Multicenter Study / Cuantificación de testosterona (T) por 8 inmunoensayos y por cromatografía líquida en tandem con espectrometría de masa (LC MS/MS): En mujeres normales e hirsutas. Estudio multicéntrico

Objective: To compare T results in normal and hirsute women, obtained by different laboratories employing the same or different methods, including an in-home RIA, and the gold standard method LC-MS/MS. In addition, T results were referred to a curve obtained by 6 different pools that had been prepared on the basis of LC-MS/MS results. Design: Prospective study Setting: Hormone Determination Laboratory, Hospital Italiano, La Plata, and private practice of each participant laboratory. Patient(s): Blood samples were obtained from 78 individuals sorted into 3 groups, namely, normal men (n:39), normal women (n:24) and hirsute women (n:15) Interventions(s): None Main Outcome Measure(s): To evaluate if the results obtained in each lab for each serum sample by the methods currently employed in our country are significantly different from those obtained by LC-MS/MS (Gold standard) Result(s) One out of the 24 NW showed high T values by LC - MS/MS. In each lab, except in 1 (Architect) T results of this serum sample were normal. Two out of the 15 hirsute patients showed normal T values (LC - MS/MS). Method and number of labs -shown between brackets- and percentages of normal T results (false negatives) are described for each method as follows: Chemiluminescence: Axsym - Abbott (Axn) - (3) 85, Architect - Abbott - (Arch); (2) 70; Immulite - Siemmens - (IMM); (2) 42; Electrochemiluminescence - Elecsys - Roche- ((EQL); (4) 52; Fluorescent enzymatic - Vidas - Bio-Merieux - (Vidas) (1) 69; Manual coated tube radioimmunoassay (RIA): RIA - Siemmens Coat-a-Count (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; and in-Home RIA (in-H) (1) 12. Statistically significant differences were obtained between different methods and against LC MS/MS. In-H method is the one that comes closest to 1 on the Weighted Deming regression and closest to zero on the SD intercept, (standard deviation of the constant in the straight line equation) indicating that the values match those obtained by LC - MS/MS. The values recorded by the various methods employed showed no significant modifications when plotted against a secondary standard curve. Conclusion(s) This indicates that the techniques in current use in our area underestimate hyperandrogenemia in these patients. Discrepancies are not due to the various calibration curves proposed in the corresponding commercial kits. The fact that the In-H technique affords finer results while employing a larger serum volume suggests that the disparities among the various commercial methods result from their limited sensitivity to the sample volumes they process.

El diagnóstico de hiperandrogenemia requiere la demostración de niveles aumentados de Testosterona Total (TT) en suero. Los inmuno ensayos comerciales dan resultados divergentes a niveles bajos de TT como los obtenidos en mujeres. Valoramos los niveles de TT en 24 mujeres normales (MN) y 15 hirsutas (MH) en 18 laboratorios por métodos comúnmente empleados en nuestro medio. Los métodos y número de laboratorio que emplearon cada método se muestra entre paréntesis así como los porcentajes de T normal (falsos negativos) obtenidos en cada método fueron: Quimioluminiscencia: Axsym - Abbott (Axn)- (3) (85), Architect - Abbott - (Arch); (2) 70; Immulite - Siemmens - (IMM); (2) 42; Electroquimioluminiscencia - Elecsys - Roche- ((EQL); (4) 52; Enzimático acoplado a fluorescencia Vidas - Bio-Merieux - (Vidas) (1) 69, Radioinmunoiensayo en tubo recubierto (RIA): RIA - Siemmens (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; y un método desarrollado en uno de los laboratorios (in-H) (1) 12. Comparativamente a LC MS/MS los niveles fueron en todas las muestras significativamente más bajos por Axn y en 18 de las 24 MN por DiaS. En 7 casos; 3 por RIA S, 2 por IMM y 1 por EQL y Arch los valores de TT fueron superiores al límite superior de sus respectivos métodos. En todos los casos se obtuvo una gran variación entre los mismos y con diferentes métodos. Trece de las 15 MH tuvieron niveles altos de TT por LC MS/MS. De las MH con TT aumentada de acuerdo a la determinación por LC MS/MS entre el 12 y el 85 % de las mismas por los distintos métodos fueron normales, indicando que en la mayoría de los métodos habitualmente utilizados en nuestro medio subvaloran la hipernadrogenemia en estas pacientes. Estas diferencias se hacen más notorias a niveles más bajos de TT (Se obtuvieron valores normales en el 71 % de los casos con valores de TT entre 0,47 y 0,74 ng/ml y en el 38 % de los casos, con niveles de TT mayor a 0,98 ng/ml). ...

Quantification of Testosterone (t) by 8 Immunoassays and by Liquid Chromatography -Tandem mass Spectrometry (lc -msms) in Normal and Hirsute Women: A Multicenter Study / Cuantificación de testosterona (t) por 8 inmunoensayos y por cromatografía líquida en tandem con espectrometría de masa (Lc ms/ms): En mujeres normales e hirsutas. Estudio multicéntrico

Objective: To compare T results in normal and hirsute women, obtained by different laboratories employing the same or different methods, including an in-home RIA, and the gold standard method LC-MS/MS. In addition, T results were referred to a curve obtained by 6 different pools that had been prepared on the basis of LC-MS/MS results. Design: Prospective study Setting: Hormone Determination Laboratory, Hospital Italiano, La Plata, and private practice of each participant laboratory. Patient(s): Blood samples were obtained from 78 individuals sorted into 3 groups, namely, normal men (n:39), normal women (n:24) and hirsute women (n:15) Interventions(s): None Main Outcome Measure(s): To evaluate if the results obtained in each lab for each serum sample by the methods currently employed in our country are significantly different from those obtained by LC-MS/MS (Gold standard) Result(s) One out of the 24 NW showed high T values by LC - MS/MS. In each lab, except in 1 (Architect) T results of this serum sample were normal. Two out of the 15 hirsute patients showed normal T values (LC - MS/MS). Method and number of labs -shown between brackets- and percentages of normal T results (false negatives) are described for each method as follows: Chemiluminescence: Axsym - Abbott (Axn) - (3) 85, Architect - Abbott - (Arch); (2) 70; Immulite - Siemmens - (IMM); (2) 42; Electrochemiluminescence - Elecsys - Roche- ((EQL); (4) 52; Fluorescent enzymatic - Vidas - Bio-Merieux - (Vidas) (1) 69; Manual coated tube radioimmunoassay (RIA): RIA - Siemmens Coat-a-Count (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; and in-Home RIA (in-H) (1) 12. Statistically significant differences were obtained between different methods and against LC MS/MS. In-H method is the one that comes closest to 1 on the Weighted Deming regression and closest to zero on the SD intercept, (standard deviation of the constant in the straight line equation) indicating that the values match those obtained by LC - MS/MS. The values recorded by the various methods employed showed no significant modifications when plotted against a secondary standard curve. Conclusion(s) This indicates that the techniques in current use in our area underestimate hyperandrogenemia in these patients. Discrepancies are not due to the various calibration curves proposed in the corresponding commercial kits. The fact that the In-H technique affords finer results while employing a larger serum volume suggests that the disparities among the various commercial methods result from their limited sensitivity to the sample volumes they process. No financial conflicts of interest exist.

El diagnóstico de hiperandrogenemia requiere la demostración de niveles aumentados de Testosterona Total (TT) en suero. Los inmuno ensayos comerciales dan resultados divergentes a niveles bajos de TT como los obtenidos en mujeres. Valoramos los niveles de TT en 24 mujeres normales (MN) y 15 hirsutas (MH) en 18 laboratorios por métodos comúnmente empleados en nuestro medio, Quimioluminiscencia: Axsym - Abbott (Axn)- (3 ), Architect - Abbott - (Arch); (2); Immulite - Siemmens - (IMM); (2); Electroquimioluminiscencia - Elecsys - Roche- ((EQL); (4); Enzimático acoplado a fluorescencia Vidas - Bio-Merieux - (Vidas) (1), Radioinmunoiensayo en tubo recubierto (RIA): RIA - Siemmens (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; y un metodo desarrollado en uno de los laboratorios (in-H) (1).El número entreparéntesis indica elnúmero de laboratorios que emplearon la misma técnica,y comparamos los resultados por LC MS/MS. Comparativamente a LC MS/MS los niveles fueron en todas las muestras significativamente más bajos por AXS y en 18 de las 24 MN por DiaS. En 7 casos; 3 por RIA S, 2 por IMM y 1 por EQL y Arch los valores de TT fueron superiores al límite superior de sus respectivos métodos. En todos los casos se obtuvo una gran variación entre los mismos y con diferentes métodos. Trece de las 15 MH tuvieron niveles altos de TT por LC MS/MS. De las MH con TT aumentada de acuerdo a la determinación por LC MS/MS entre el 12 y el 85 % de las mismas por los distintos métodos fueron normales, indicando que en la mayoría de los métodos habitualmente utilizados en nuestro medio subvaloran la hipernadrogenemia en estas pacientes. Estas diferencias se hacen más notorias a niveles más bajos de TT (Se obtuvieron valores normales en el 71 % de los casos con valores de TT entre 0.47 y 0.74 ng/ml y en el 38 % de los casos, con niveles de TT mayor a 0.98 ng/ml). En 9 muestras se determinó la TT empleando una curva en el rango de 0.21 a 6.44 ng/ml preparada con de una mezcla de 78 sueros cuyos valores fueron obtenidos por LC MS/MS. No se obtuvo una modificación significativa de los valores indicando que la diferencia entre los distintos métodos no es debida a las diferentes curvas de calibración de los kit comerciales. En conclusión ninguno de los métodos mayormente empleados en nuestro medio son aceptables para la evaluación de niveles menores a 1.5 ng/ml.

Analysis of molecular heterogeneity of prolactin in human systemic lupus erythematosus.

Hyperprolactinemia without clinical manifestations has been reported in some patients with systemic lupus erythematosus (SLE) because an increase of prolactin (PRL) is produced due to the BIG/BIG molecular variant (molecular variant < 150 kD). This research project aimed to determine levels of PRL: its bioactive form, the little nonglycosylated form (NGPRL) and variants with decreased bioactivity such as the BIG/BIG and the little glycosylated (GPRL), in 29 women and five men with SLE. PRL was assayed by IRMA with a kit from Immunotech Laboratory, the BIG/BIG form by precipitation with polyethyleneglycol 6000, and the NGPRL and GPRL by chromatography on Concanavalin-A- Sepharose. Increased PRL was detected in seven patients (20.6%) of whom three had increased BIG/BIG, six had increased GPRL and only four had increased NGPRL. The three cases with increased BIG/BIG were contrasted by chromatography on Sephadex G-100. No increased PRL or any of the other variants assayed were found in men. Results were similar when PRL was evaluated in the same blood samples by a different IRMA (DPC Laboratory). The etiology of the hyperprolactinemia in some of these patients is unknown, but their lack of symptoms (galactorrhea or amenorrhea) could be due to the BIG/BIG forms and basically to the glycosylation of the hormone. As for the relation between PRL and SLE activity, we found that hyperprolactinemic patients were younger, had a shorter history of illness, although it was not statistically significant, and a higher SLEDAI score. This would indicate a relation between hyperprolactinemia and lupus activity. The patients with increased BIG/BIG form also had a very active illness at the time of the study.

Hyperprolactinemia in asymptomatic patients is related to high molecular weight posttranslational variants or glycosylated forms.

Circulating human Prolactin (PRL) exists in different variants related to posttranslational modifications, dimerization or association with other serum proteins. Compared to monomeric prolactin these variants usually have little or no biologic activity and include BigBig (BB PRL), Big (B PRL), and Glycosylated forms (G PRL). The aim of the present study was to assess levels of BB PRL, B PRL, little PRL (L PRL) and G PRL in hyperprolactinemic patients with no menstrual alterations or galactorrhea. L PRL, B PRL, and BB PRL were identified by gel filtration chromatography on Sephadex G-100; G PRL and NG PRL were identified by chromatography on Concanavalin A Sepharose. PRL was measured by IRMA DPC. Eleven women, aged 22-50 yrs, were studied for: breast dysplasia (1), controlled hypothyroidism (3), dysmenorrhea (3), microadenoma follow-up (2), and gynecological control (2). Pituitary MRI was normal in all but one patient, who had a microadenoma discovered by Magnetic Resonance Imaging. Six patients had normal L PRL levels, and their hyper PRL was due to excess BPRL or BB PRL. Five patients had increased L PRL levels, but excess G PRL. Patients harboring molecular PRL variants do not present the symptoms typical of the hyperprolactinemic syndrome. Furthermore in patients with clinically controlled prolactinomas the presence of PRL variants should be ruled out to avoid an unnecessary increase of dopamine agonist dosage.

Hypothalamic-pituitary-testicular axis and seminal parameters in hyperthyroid males.

Information on the effect of abnormal thyroid function on male reproduction is less available than that for the female. To assess the effects of hyperthyroidism on hypothalamic-pituitary-testicular axis and on spermogram parameters, 25 male patients (19-47 years old) suffering from active Graves' disease were studied. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin (PRL) were measured before and after administration of 100 microg GnRH plus 200 microg thyrotropin-releasing hormone (TRH). Testosterone (T), estradiol (E2), and 17-hydroxyprogesterone (17-OHP) were determined before and after 5000 IU human chorionic gonadotropin (HCG) administration. Serum sex hormone-binding globulin (SHBG), cortisol-binding globulin (CBG), androstenedione and bioavailable testosterone (bioT), and bioavailable estradiol (bioE2) were also measured. Spermograms according to World Health Organization (WHO) criteria were determined in 21 patients. Hormonal and seminal studies were repeated in six patients after 7 to 19 months of euthyroidism achieved after treatment for hyperthyroidism. As a control group, 10 normal men were evaluated. Impaired sexual function, gynecomastia, and low testicular volume were found in 12, 6, and 3 hyperthyroid patients. Mean basal LH was significantly higher than the control group (7.8 +/- 4.7 vs. 5.0 +/- 1.9 mIU/mL, respectively, p < 0.02), with hyperresponse to GnRH. The response of PRL to TRH was lower in patients versus control group (30 minutes: 3.9 +/- 3.4 and 12.0 +/- 2.8 ng/mL, p < 0.01). Basal levels of steroids and SHBG were significantly higher in patients than in normal men (T: 9.3 +/- 3.3 vs. 5.4 +/- 1.6 ng/mL, p < 0.005; E2: 62.2 +/- 25.2 vs. 32.1 +/- 11.0 pg/mL, p < 0.005; 17-OHP: 2.4 +/- 0.9 vs. 1.1 +/- 0.5 ng/mL, p < 0.001; SHBG: 102.3 +/- 37.3 vs. 19.0 +/- 5.0 nmol/L, p < 0.01). The maximal increment of T and 17-OHP after HCG was lower in hyperthyroid patients than in normal men (p < 0.019 and p < 0.001, respectively). Basal bioT was lower in patients than controls (1.7 +/- 0.8 and 3.1 +/- 1.9 ng/mL, p < 0.02). The following incidence of abnormal semen parameters was found: asthenospermia 85.7%, hypospermia 61.9%, oligospermia 42.9%, necrospermia 42.9% and teratospermia 19.0%. In euthyroidism, a normalization of 85% of seminal alterations was observed in the limited number of patients evaluated. Our results confirm that hyperthyroidism causes marked alterations of the gonadotropic and PRL axis and dramatically affects spermatic function. BioT measurement was useful to identify hypoandrogenism in these patients in spite of the high concentration of total testosterone. The restoration of most semen parameters when euthyroidism was achieved suggests that the alterations were induced by the Graves' disease.

Altered luteinizing hormone pulsatility in infertile patients with idiopathic oligoasthenozoospermia.

In a previous study, we demonstrated that oligoasthenozoospermic (OAZ) patients had two types of testosterone response to human chorionic gonadotrophin (HCG) administration: group 1 (OAZ-1) had an altered, monophasic (no first peak) response, and group 2 (OAZ-2) had a normal biphasic response. The objective of the present work was to study the luteinizing hormone (LH) pulsatility in OAZ-1 compared with both OAZ-2 and men of proven fertility (PF), in order partly to determine the possible aetiology of the blunted acute testosterone response to HCG in these patients. LH pulsatility was measured in 10 PF, 10 OAZ-1 and 10 OAZ-2 patients, in blood samples taken every 5 min for 6 h in PF, and for 4 h in OAZ patients. LH values were determined by a time-resolved immunofluorometric assay. Frequency and amplitude of the LH pulses were determined by a computer program. LH pulse frequency, expressed as pulses/4 h, was significantly lower in OAZ-1 (1.5+/-0.97) than in PF (2.4+/-0.63) and OAZ-2 (2.4+/-0.84) patients. In six OAZ-1 and two OAZ-2 patients, LH pulsatility was diminished, as they showed less than two pulses/4 h. No statistically significant differences in LH pulse amplitude were found. These results, together with a higher number of OAZ-1 cases found with decreased LH pulsatility, suggest that, at least in a subset of these men, quantitative and/or qualitative alterations of LH secretion might have occurred.

Recombinant human follicle-stimulating hormone administration increases testosterone production in men, possibly by a Sertoli cell-secreted nonsteroid factor.

We previously showed that recombinant human FSH (R-FSH) in males increased the testosterone (T) concentration in spermatic venous blood (SB). To investigate the effect of R-FSH on spermatic steroid levels and the action of steroid- and LH-free SB on isolated Leydig cells, nine normospermic males were studied during spermatic cord surgery. Peripheral blood and SB samples were collected before and 30 min after iv administration of 150 U R-FSH to measure LH, FSH, T, estradiol, 17alpha-hydroxyprogesterone, and sex hormone-binding globulin, and in SB, androstenedione (delta4) and dehydroepiandrosterone (DHEA) were also measured. LH bioactivity was assessed by in vitro production of T in isolated Leydig cells. The actions of R-FSH and SB (steroid and LH free) were analyzed in the bioassay. Data are expressed as the mean +/- SE. FSH in peripheral blood and SB increased by 411% and 477% after R-FSH administration. R-FSH induced a significant increase in spermatic T (basal vs. 30 min, 326.4 +/- 98.5 vs. 732.4 +/- 152.8 ng/mL; P < 0.047) and in spermatic estradiol (289.5 +/- 66.9 vs. 535.6 +/- 83.4 pg/mL; P < 0.036). The T/delta4 ratio (36.9 +/- 9.2 vs. 74.5 +/- 13.3; P < 0.019) and the T/DHEA ratio (10.8 +/- 1.1 vs. 22.4 +/- 4.9; P < 0.024) increased significantly. In isolated Leydig cells, R-FSH did not change T production, but the SB (steroid and LH free) after R-FSH administration induced an increase in T production (3.3 +/- 0.6 vs. 4.9 +/- 0.6 ng/tube; P < 0.04). LH-like activity was found in a more than 50,000-Da fraction after centrifugation in Amicon filters, even in the presence of anti-LH. These results suggest that R-FSH increases the production of T by Leydig cells through a Sertoli cell-released nonsteroid factor with a molecular mass greater than 50 kDa. The increase in the T/delta4 and T/DHEA ratios indicates that this factor would act by amplifying the LH response through the delta5 pathway and the 17beta-hydroxysteroid dehydrogenase enzyme.

Serum luteinizing hormone pulsatility and intratesticular testosterone and oestradiol concentrations in idiopathic infertile men with high and normal follicle stimulating hormone serum concentrations.

The purpose of the study was to evaluate pulsatile luteinizing hormone (LH) release and intratesticular concentrations of testosterone and oestradiol in infertile men, to determine if alterations in gonadotrophin secretion are associated with changes in the testicular concentrations of steroids. Patients with idiopathic oligo/azoospermia were divided into a high follicle stimulating hormone (FSH) group (n = 5) and a normal FSH group (n = 6). Blood samples were taken every 15 min for 6 h to determine LH, FSH, testosterone, oestradiol, sex hormone binding globulin, bioactive LH and bioavailable testosterone. The patients underwent a bilateral testicular biopsy for histological assessment and to determine testosterone and oestradiol concentrations. Serum measurements were compared with those of seven fertile men. The high FSH group had a higher concentration of serum LH and oestradiol than normal men (P < 0.01) and showed a lower frequency of LH pulses than the normal FSH group and control men (P < 0.01). Intratesticular oestradiol was higher in the high FSH group (P < 0.001), with a lower testosterone/oestradiol ratio (P < 0.01). Patients showed a negative correlation between the serum testosterone/LH ratio and FSH (r = -0.75; P < 0.01) and a positive correlation between the testicular oestradiol concentration and serum FSH (r = 0.86; P < 0.01). The histopathological examination only showed a smaller tube diameter in the high FSH group (P < 0.05). These data seem to indicate that a higher intratesticular concentration of oestradiol with a lower testosterone/oestradiol ratio in the high FSH group could have a deleterious effect on spermatogenesis.

Altered testicular hormone production in infertile patients with idiopathic oligoasthenospermia.

We studied the kinetics of testicular response to human chorionic gonadotropin (hCG) in oligoasthenospermic and asthenospermic patients (OAZ-AZ). The responses of testosterone (T), androstenedione (A), 17 OH-progesterone (17OHP), and estradiol (E2) were evaluated in 60 OAZ-AZ patients and compared to those of 10 normal men. The responses of T, A, and 17OHP to hCG in the control group displayed a biphasic pattern with an initial peak at 4 hours and a second peak after 24 hours. The E2 response showed a single peak between 24 and 48 hours after hCG administration. OAZ-AZ patients had two types of T responses: group 1 (n = 40) had no first peak and group 2 (n = 20) had a normal response pattern. The response of A was similar to that of T, and the E2 response was normal in both groups. There were three types of 17OHP responses in group 1 (low, high, or normal); however, the 17OHP response was normal in group 2. Treatment of group 1 with aromatase inhibitors (aminoglutethimide or testolactone) induced an improvement of the acute T response only in patients with high or normal 17OHP response to hCG, whereas no effects were observed in patients with low 17OHP response. In group 2, the aromatase inhibitors induced no changes in the T response. These results demonstrate that in some OAZ-AZ patients (group 1, blunted T response) testicular hormone production is altered. They also suggest the presence of two enzyme blocks: one at the 17,20 desmolase level, mediated by E2, and another at early biosynthetic steps, not mediated by E2.

Ontogenic screening of aldosterone in the South American toad Bufo arenarum (Hensel).

The content of aldosterone in the South American toad Bufo arenarum embryos developed in Holtfreter's 10% solutions and in distilled water was measured. The screening was carried out on embryos from the following periods of their development: 15 min after fertilization; gray crescent-32 cells; mid cleavage-late gastrula; neural plate-neural tube; tail bud-heart beat; gill circulation-tail fin circulation and operculum fold-complete operculum. The aldosterone content of embryos incubated both in Holtfreter and in distilled water decreased gradually up to gill circulation-tail fin circulation stages. The steroid content of both groups of embryos rose sharply at the end of their development (operculum fold-complete operculum); the observed increase was significantly higher in distilled water animals.

Preliminary study of hormone determinations during aminoglutethimide therapy for advanced breast cancer.

The aim of this study is to report on Aminoglutethimide-induced hormonal modifications in advanced breast cancer. Estradiol (E2), Testosterone (T), Dehydroepiandrosterone sulphate (DHEA(s] and Aldosterone (A) were determined before, and once every two weeks during treatment with Aminoglutethimide plus Hydrocortisone in 13 menopausal women with advanced breast cancer. The patients were selected either for their E2 and P4-receptor-positive in the original tumor or in metastases or by presenting objective clinical improvement to prior endocrine treatment. On the basis of the response to treatment the patients may be classified in two groups: 1) responders (n = 7) and 2) non-responders. No significant modifications of T concentrations were obtained in group 1 until after the first 8 months of treatment. One spontaneous menopausal patient with a T basal value of 0.80 ng/ml was evaluated during 12 months of treatment. From month 8, T diminished to values below 0.30 ng/ml, indicating a direct action of Aminoglutethimide, hydrocortisone or both drugs on ovarian steroidogenesis. The results obtained from the remaining hormonal parameters, evaluated in all the cases beginning from the second week of treatment, remained unchanged throughout the entire period of study. They were as follows: 1) E2 diminished with respect to basal values between 36 and 60%, thus confirming Aminoglutethimide inhibitory effect upon peripheral aromatization; 2) DHEA(s) diminished between 80 and 90%, indicating an adrenal inhibition due to the combined effect of both drugs, and 3) Aldosterone diminished to values between 80 and 110 pg/ml, these values being within the normal lower range.(ABSTRACT TRUNCATED AT 250 WORDS)

LH receptor in testosterone-producing ovarian tumor. In vivo and in vitro HCG-stimulated testosterone production.

The aim of the present work is to report in vivo and in vitro hormonal studies performed on a 71 year old virilized woman due to a Leydig cell tumor of the ovary. Testosterone (T), Cortisol, DHEA(s) and ACTH concentrations were determined in blood samples taken every 4 h throughout 24 h previous to surgery. T average concentration from the 6 samples was 3.3 ng/ml (range: 2.5-4.2 ng/ml). DHEA(s) was normal; Cortisol and ACTH levels were normal and their circadian rhythms were present. T value obtained during Dexamethasone administration (2 mg daily/3 days) was 2.4 ng/ml. This value was significantly higher than those obtained from postcorticoid normal women (0.3 +/- 0.1 ng/ml), suggesting an extraadrenal source. T concentration was 5.4, 6.0 and 6.6 ng/ml at 24, 48 and 72 h after hCG injection (5000 IU). After tumor removal, T values decreased progressively up to 6.0 ng/ml values, 5 days later, and remained steady on the following days. The studies performed in vitro were: determination of T in the tumor cytosol, specific binding of LH to ovarian tumor cell membrane fraction and T production in tissue culture in both with and without added hCG conditions. Normal ovarian tissue from the same patient under similar experimental conditions was used as control. The T concentration expressed as ng/mg of protein in the tumor and normal ovarian cytosol was 9.1 and 1.1, respectively. Scatchard analysis of specific 125I-hCG binding to tumor and normal ovarian cells indicated 53 pg and 28 pg of labeled hCG bound/mg of membrane protein, respectively, suggesting that this Leydig cell tumor of the ovary contained LH (hCG) receptors. The amounts of T, expressed as ng/mg of tissue/6 days, generated by tumoral and normal tissue ere 4.1 and 0.3, respectively. The addition of hCG elicited a response of 6.3 and 0.6 ng/mg protein/6 days in both preparation, respectively. These results demonstrate in vivo and in vitro hCG-stimulated T production in this particularly masculinizing ovarian tumor and suggest tumoral LH dependence.

Binding of prolactin by fetal human lung cell membrane fractions.

PRL binding capacity to cell membrane fraction was studied in lung tissues obtained from 4 human fetuses or newborn infants. One of the fetuses was a stillborn delivered at 33 weeks of gestation. The newborn infants died for unknown causes within 24 h after birth. The gestational age was 20, 39 and 41 weeks. The cell membrane fraction was prepared by ultracentrifugation. binding capacity and affinity constants were calculated according to athe Scatchard method. No significant specific binding of PRL to lung tissue from the stillborn fetus was observed, while for the other 3 newborn infants the binding capacity was 5.7, 7.4 and 9.6 fmol PRL bound/mg of membrane protein, respectively. The affinity constants were in the order of 10(10) M-1. these preliminary results show that human neonatal lung has receptors for PRL and suggest that PRL itself may be involved in the lung maturation.

Heterogeneous results of serum testosterone obtained in multiple blood samples from women with idiopathic hirsutism in both basal and post-dexamethasone conditions.

Serum radioimmunoassayable testosterone (T), cortisol and luteinizing hormone (LH) were determined before and after dexamethasone (DXM) administration in 35 patients with idiopathic hirsutism (IH). Blood samples were taken at 15-min intervals during 1 hour in both basal and post-DXM conditions. Testosterone values obtained in 5 normal women in the same conditions during the early follicular phase were (mean +/- SD): baseline, 2.26 +/- 0.49 nmol/l; post-DXM, 0.80 +/- 0.35 nmol/l. Serum T levels in the whole group of patients with IH were significantly higher than those in the control group (mean +/- SD): baseline, 3.30 +/- 1.80nmol/l; post-DXM, 1.67 +/- 1.49nmol/l. Patients with IH were divided into 4 groups according to T results in the DXM test (mean +/- SD in both basal and post-DXM conditions, respectively): group 1 (n = 13) 1.67 +/- 0.66 and 0.62 +/- 0.35nmol/l; group 2 (n = 11) 3.89 +/- 1.63 and 3.09 +/- 1.49nmol/l; group 3 (n = 6) 3.96 +/- 1.46 and 0.87 +/- 0.73nmol/l; and group 4 (n = 5) 5.45 +/- 1.25 and 2.05 +/- 0.38nmol/l. In all cases, maximal adrenal inhibition, as judged by serum cortisol, was obtained. No LH modifications after DXM were obtained in any of the cases. Our results demonstrate that there is no common androgenic abnormality in IH. It is possible to obtain normal or high circulating T levels. The findings of this study also suggest that the adrenals, to ovary or both may be the sources of high T levels.

Prolactin, estriol and progesterone levels in frequent blood samples throughout normal pregnancy (lack of prolactin pulsatile secretion).

The aim of this paper was to study the episodic fluctuations of circulating prolactin (PRL), estriol (E3) and progesterone (P4) concentrations throughout pregnancy. We examined 24 pregnant women; 21 were between the 28th and 40th week of gestation, and the other 3 in the 12th, 16th and 20th week of gestation. Blood samples were drawn every 5 min for half an hour, and every 15 min for one and a half hour. Blood samples were taken in two and three different weeks of gestation in 11 and 2 of the cases, respectively. Two normal non-pregnant women were also studied and used as controls. PRL, E3 and P4 were determined by radioimmunoassay in all the samples. The coefficients of variation of PRL values were 40 and 22.6%, respectively, in the two control women, 8, 12 and 9.8% in the pregnant women studied at the 12th, 16th and 20th week of gestation, respectively, while in the 21 cases studied during the third trimester the coefficient of variation was 8 +/- 3% (mean +/- SD). The coefficients of variation of the values obtained for E3 and P4 in women studied in he third trimester were 26 +/- 15 and 16 +/- 6% (Mean +/- SD), respectively. There was an increase in the average concentration of the three hormones in all the cases at two or three different weeks. We can conclude that E3 and P4 have a pulsatile secretion pattern throughout pregnancy, and that PRL looses its pulsatile secretion as from an early gestational age. Our results suggested that central mechanisms regulating PRL episodic fluctuations were altered during pregnancy.