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.

Cosecretion of glycosylated prolactin and non-glycosylated prolactin from childhood to the end of puberty.

BACKGROUND: Glycosylated prolactin (G-PRL) is considered as the major post-translational modification of prolactin (PRL) showing reduced lactotropic and mitogenic activities compared to non-glycosylated prolactin (NG-PRL). AIM: To evaluate the evolution of G-PRL in normoprolactinemic children and adolescents and to analyze possible variations in glycosylated/total prolactin (T-PRL) ratios. METHODS: T-PRL, G-PRL and NG-PRL were evaluated in 111 healthy female and male children and adolescents (4.1-18 years), classified as group 1 (Tanner I), group 2 (Tanner II-III) and group 3 (Tanner IV-V). G-PRL and NG-PRL were identified by chromatography on concanavalin-A-Sepharose. RESULTS: G-PRL/T-PRL (median-range): females, group 1: 0.59 (0.17-0.77), group 2: 0.56 (0.31-0.78), group 3: 0.60 (0.38-0.79); males, group 1: 0.64 (0.39-0.80), group 2: 0.61 (0.24-0.79), group 3: 0.62 (0.35-0.90); the p value is not significant among the different groups in both genders. G-PRL/T-PRL ratios do not change when comparing low (first quartile) versus high (third quartile) T-PRL levels in the different groups. CONCLUSION: Our study would appear to support cosecretion of G-PRL and NG-PRL from childhood to the end of puberty. Such cosecretion would not be dependent on sex steroid levels. It is important to point out that puberty does not change the proportions of G-PRL and NG-PRL.

Perfil androgénico en mujeres hirsutas: Modificaciones de sus niveles en la fase lútea / Androgenic profile in hirsute women: Modifications of the levels in lutheal phase

Introducción: El objetivo de este estudio fue evaluar Los niveles de Testosterona (T), T libre TL, DHEAs y Androstanodiol glucuronidato (A2G) mujeres hirsutas con ciclos menstruales (CM) regulares en la fase folicular (FF) y en una muestra tomada entre -5 a -10 días premenstrual (FL) a los efectos de 1) poder definir bioquímicamente el tipo de hirsutismo y 2) determinar si el aumento de Progesterona modifica los niveles de los andrógenos. Materiales y Métodos: En 65 mujeres hirsutas con CM regulares se determinó en FF los niveles de T, A2G, y DHEAs por RIE y TL calculada por la ecuación de la ley de acción de masas, y en la FL los niveles de P4. En 28 de las 65 pacientes, en la FL se repitió el perfil androgénico Resultados: Los niveles de T correlacionaron, en todos los casos, con los de TL. En 51 de las pacientes los niveles de P4 fueron ovulatorios, 25 de las cuales tuvieron normales los andrógenos evaluados (Hirsutismo Idiopático) De las 26 pacientes restantes, en 2 tenían T aumentada, en 4 la DHEAS. Se obtuvieron 2 parámetros aumentados en los siguientes casos; en 2 la DHEAs y el A2G, en 1 la T y la DHEAs y en1 la T y el A2G. En 4 pacientes se obtuvieron incremento de los 3 parámetros. Estas pacientes corresponden a Hiperandrogénicas ovulatorias. Las 12 restantes de estas 26 hirsutas tenían solamente el A2G aumentados. Dado que éste constituye la expresión periférica de la 5alfa reductasa, las mismas podrían incluirse en el grupo de hiperandrogénicas ovulatorias por aumento local de DHT. En 14 de las 65 pacientes los niveles de P4 fueron compatibles con ciclos anovulatorios correspondiendo a pacientes con Síndrome de Ovario Poliquístico (SOP). En 6 de ellas se constató aumento de 1, 2 o los 3 parámetros evaluados (SOP hiperandrogénicos), en las restantes 6 pacientes los niveles androgénicos fueron normales (SOP con hirsutismo clínico). El A2G aumentó significativamente en FL en las mujeres con ciclos ovulatorios (4.89±2.19 vs 3.36±2.38 ng/ml en FL y FF, respectivamente). En las anovulatorias las diferencias no fueron significativas (4.32±3.16 vs 4.69±4.54 ng/ml en FL y FF, respectivamente. Estos resultados indican que la P4 podría inducir un incremento del A2G. Dado que la T no se modificó en la FL respecto a FF (0.28±0.22 vs 0.30±0.25ng/ml en hirsutas ovulatorias y 0.47±0.32 vs 0.42±0.23 en hirsutas anovulatorias) es posible que la P4 aumente el A2G por un camino distinto a la de la T y DHT Conclusiones: En base a estos resultados podemos concluir que la determinación de A2G podría ser empleada como parámetro complementario en el estudio del hiperandrogenismo debiendo realizarse en FF dado que en FL podría ser el resultado del metabolismo de hormonas no androgénicas.

Introduction: The aim of the present study was to evaluate the circulating levels of Testosterone (T), free T (TL), DHEAs and Androstanediol glucuronide (A2G) in hirsute women with regular menstrual cycles (CM) in follicular phase (FF), and in a samples obtained 5 to 10 days before the next menstrual bleeding (FL), in order to 1) biochemically define type of hirsutism and 2) determine whether the increase in progesterone (P4) induces changes in androgen levels. Materials and Methods: Sixty five hirsute women with regular CM were studied. FF levels of T, A2G and DHEAs were determined by RIA, and TL by mass law calculation. FL levels of P4 were measured by RIA. In 28 of the 65 patients the androgen profile was also evaluated in FL. Results: The levels of T correlated in every case with those of TL. In 51 patients P4 levels were ovulatory. Twenty five of them showed normal androgen levels (Idiopathic hirsutism). From the remaining 26 patients, 2 had increased T, and 4 had increased DHEAs. Two parameters were found increased in the following cases: DHEAs and A2G in 2, T and DHEAs in 1, and T and A2G in 1. All the 3 parameters were found increased in 4 cases. These patients were ovulatory hiperandrogenic women. The remaining 12 of these 26 hirsute women had only A2G increased. Since this steroid is the peripheral expression of the 5alpha reductase activity, these women could be included in the ovulatory hiperandrogenic group because of a local increase in DHT. In 14 of the 65 patients the levels of P4 correlated with anovulatory cycles corresponding to Polycystic Ovarian Syndrome (SOP). In 6 of them an increase of 1, 2 or the 3 parameters were observed (Hiperandrogenic SOP); in the remaining 6 patients androgen levels were normal (SOP with clinical hirsutism). FL A2G significantly increased in women with ovulatory cycles (4.89±2.19 vs 3.36±2.38 ng/ml in FL and FF, respectively. Differences were no significant in the anovulatory patients (4.32±3.16 vs 4.69±4.54 ng/ml in FL and FF, respectively. These results indicate that P4 could induce an increase in A2G. Since T did not change in FL respect to FF (0.28±0.22 vs 0.30±0.25ng/ml in ovulatory hirsute and 0.47±0.32 vs 0.42±0.23 in anovulatory hirsute) it is possible that P4 increases A2G through a pathway different than that of T and DHT. Conclusions: Based on these results we conclude that A2G could be used as a complementary parameter in the study of hiperandrogenism, only in FF since in FL, it could be the result of the metabolism of non-androgenic hormones.

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.

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.

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.

Failure of the müllerian regression factor in two patients with complete androgen insensitivity syndrome.

The present paper describes the histological and endocrinologic features of 2 subjects with 46,XY karyotype affected by complete androgen insensitivity syndrome (AIS) with müllerian structures. Both patients had fallopian tubes, but only one had also uterus and presented a seminoma. Serum levels of luteinizing hormone, testosterone and estradiol were high or in the upper part of normal limits, whereas levels of follicle-stimulating hormone were normal. The association between AIS and the presence of müllerian structures observed in these 2 patients might be explained by an impaired synthesis of müllerian regression factor or by a failure in its mechanism of action.

Radioimmunoassays of human luteinizing and follicle-stimulating hormones using the same radioactive tracer.

The National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) provides the hFSH-I-3 preparation, to be employed as a tracer in the radioimmunoassay (RIA) of human follicle-stimulating hormone (hFSH). The contaminating LH contained in that preparation led us to study whether the iodination of such a material could render it a suitable tracer for RIA of both LH and FSH. hFSH-I-3 was labelled with 125I by the chloramine-T method and was further purified on Sephadex G-75 column. The LH-RIA was performed using this preparation and anti-LH at a final dilution of 1:37,500, with a sensitivity of 3 mIU LH/ml (2nd international reference pattern). The method was validated by comparing the LH values obtained in different serum samples with those obtained using the standard RIA (125I-LH/anti-LH); the correlation coefficient (r) was equal to 0.9988. No LH overestimation due to the putative cross-reaction with FSH was found. This was demonstrated by testing serum samples containing high (greater than 100 mIU/ml) and low (less than 10 mIU/ml) concentrations of FSH before and after the treatment with anti-LH. Under these conditions, serum samples from postmenopausal women, pregnant women, normal men and women in basal conditions and after the LH-RH administration, and from a patient with Klinefelter's syndrome, were evaluated. In conclusion, NIDDK 125I-hFSH-I-3 can be used as a tracer for the radioimmunological quantitation of both hLH and hFSH, which results not only inexpensive, but also allows to reduce the amount of the stored radioactive materials.

Sleep-dependent hyperprolactinemia and Corpus luteum pathogenesis.

The results obtained in wake and sleep conditions by PRL determinations performed in 6 normoprolactinemic infertile women with luteal phase deficiency (LPD) are reported. Infertility was apparently due to LPD. PRL levels were determined by RIA in blood samples collected at 20-min intervals from 18(00) to 08(00). LPD has been previously demonstrated by endometrial biopsy, basal temperature and circulating progesterone determinations. PRL levels were also determined in 5 normal women used as control subjects under the same experimental conditions. The results obtained, expressed as means +/- SD of LPD vs. control group, were 15.9 +/- 4.6 vs. 11.6 +/- 3.3 ng/ml (p greater than 0.1) in wake conditions and 31.9 +/- 5.9 vs. 21.4 +/- 5.7 ng/ml (p less than 0.01) in sleep conditions. PRL values during the highest pulse (HP) in sleep and wake conditions were 20.9 +/- 5.2 vs. 17.0 +/- 3.3 ng/ml (p less than 0.1) and 51.1 +/- 17.1 vs. 34.3 +/- 2.4 ng/ml (p less than 0.01), respectively. In 2 out of the 6 patients mean PRL values were 22.0 and 26.5 ng/ml during sleep, and 26.0 and 33.0 ng/ml during HP. These values were not statistically significant when compared with those obtained in the control group. The results obtained show that 4 out of the 6 patients with LPD and normal PRL levels in wake conditions had sleep-dependent hyperprolactinemia due to the pulses with a more significant amplitude. These findings suggest that in some cases sleep-induced hyperprolactinemia might be involved in LPD pathogenesis.

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.

Inherited male pseudohermaphroditism due to gonadotrophin unresponsiveness.

An inherited form of incomplete male pseudohermaphroditism was studied in two post-pubertal and one pre-pubertal sibling. All patients presented a 46XY karyotype infantile female external genitalia, lack of breast development and sexual hair. Persistently elevated serum levels of gonadotrophins with normal pituitary responsiveness to LRH were found. Serum 17-OH progesterone, androstenedione, and testosterone levels were extremely low before and after gonadal stimulation with hCG. Laparotomy revealed absence of Wolffian and Mullerian derivatives. Testes were small and cryptorchidic. Microscopic and ultrastructural examination revealed seminiferous tubules with absence of spermatogenesis and normal Sertoli cells. The interstitial spaces were mainly occupied by poorly differentiated cells although in the post-pubertal patients there were small and randomly distributed nodules of Leydig cells without crystaloids. Incubation of testicular tissue from one post-pubertal patient with [14C]acetate showed lack of 14C-incorporation into appropriate steroid carriers. These data were interpreted as demonstrating that gonadotrophin resistance was the underlying abnormality of this syndrome, representing the human counter part of the "vet" pseudohermaphroditic rat.

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.