Predictive value of hyperglycosylated human chorionic gonadotropin for pregnancy outcomes in threatened abortion in first-trimester viable pregnancies / Valor predictivo de la hCG hiperglosilada para predecir resultado de la gestación en amenaza de aborto de primer trimestre en gestación viable

BACKGROUND: To investigate the value of a single determination of hyperglycosylated hCG (hCG-H) for predicting the clinical outcome of patients with threatened abortion in the first trimester of pregnancy. METHODS: Prospective study performed on 86 consecutively selected women with a diagnosis of threatened abortion and viable intrauterine pregnancy in the first trimester of pregnancy, conducted in two tertiary care hospitals. All patients underwent a single blood sample to determine hCG-H and total hCG serum levels and a transvaginal ultra-sound 12-24 hours after diagnosis. Patients were monitored to determine whether the outcome was a miscarriage before the 20th week of pregnancy. RESULTS: Forty-three women (50%) had a miscarriage during the follow-up. We observed a very high correlation between hCG-H and total hCG (r = 0.91, p < 0.001). Median hCG-H and total hCG from pregnancies with normal outcome was significantly higher than those ending in abortion. hCG-H and total hCG were very similar predictors of pregnancy outcomes (AUC: 0.90 and 0.89, respectively). The ratio hCG-H / total hCG was a poor predictor (AUC: 0.64). CONCLUSION: A single hCG-H assay is helpful for predicting pregnancy outcomes in women with first trimester threatened abortion and viable or potentially viable pregnancy at the time of presentation. However, hCG-H is not a better predictor than total hCG

FUNDAMENTO: Investigar el valor de una única determinación de hCG hiperglicosilada (hCG-H) para predecir el resultado clínico de pacientes con amenaza de aborto en el primer trimestre del embarazo. MÉTODOS: Estudio prospectivo realizado en 86 mujeres, seleccionadas consecutivamente, con diagnóstico de amenaza de aborto y embarazo intrauterino viable en el primer trimestre de embarazo, realizado en dos hospitales de tercer nivel. A todas las pacientes se les realizó una única extracción sanguínea para determinar los niveles séricos de hCG-H y hCG total, y una ecografía transvaginal 12-24 horas después del episodio de sangrado. Se realizó seguimiento de las pacientes para determinar si el resultado fue un aborto espontáneo antes de la semana 20 de embarazo. RESULTADOS: Cuarenta y tres mujeres (50%) sufrieron un aborto espontáneo durante el seguimiento. Se observó una correlación muy alta entre hCG-H y hCG total (r = 0,91, p < 0,001). La mediana de hCG-H y hCG total de los embarazos con resultado normal fue significativamente mayor que la de aquellos que terminaron en aborto. La hCG-H y la hCG total fueron predictores muy similares del resultado del embarazo (AUC: 0,90 y 0,89, respectivamente). La relación hCG-H / hCG total fue un mal predictor (AUC: 0,64). CONCLUSIÓN: La determinación única de hCG-H es útil para predecir el resultado del embarazo en mujeres con amenaza de aborto en el primer trimestre y embarazo viable en el momento de la presentación clínica. Sin embargo, la hCG-H no es mejor predictor que la hCG total

Soluble LH-HCG receptor and oestradiol as predictors of pregnancy and live birth in IVF.

RESEARCH QUESTION: Circulating soluble LH-HCG receptor (sLHCGR) is a first-trimester marker for screening pregnancy pathologies and predicts premature or multiple births before fertility treatment. Oestradiol per oocyte at ovulation induction predicts IVF treatment outcomes. We asked whether sLHCGR levels are stable during fertility treatment and whether, alone or with oestradiol, they could improve prediction of fertility treatment outcomes. DESIGN: Serum sLHCGR, anti-Müllerian hormone [AMH] and oestradiol were measured in patients undergoing IVF. Antral follicle count before ovarian stimulation and oocyte yield were used to establish sLHCGR- oocyte ratio (SOR), sLHCGR- antral follicle ratio (SAR), oestradiol at trigger per oocyte (oestradiol-oocyte ratio [EOR]) and oestradiol at trigger per antral follicle (oestradiol-antral follicle ratio [EAR]). RESULTS: The relatively stable sLHCGR was negatively related to AMH when oocyte yield was high. The sLHCGR levels were proportional (r = 0.49) to oestradiol at early cycle (day-3). Pregnancy and live birth were highest at low sLHCGR (≤1.0 pmol/ml) and SOR (≤ 0.1 pmol/ml/oocyte). A total of 86-89% of live births in IVF treatment were within the cut-off parameters of SAR and SOR (0.5 pmol/ml) and EAR and EOR (380 pg/ml). For failed pregnancy, age, SOR and EOR together had positive and negative predictive values of 0.841 and 0.703, respectively. CONCLUSIONS: sLHCGR levels are negatively related to AMH when oocyte yield is high. High early cycle sLHCGR is associated with elevated day-3 oestradiol. Low sLHCGR and SOR are indicators of increased clinical pregnancy and live birth rates. Patient age and SOR, combined with EOR, might improve prediction of IVF treatment outcomes.

Seasonal expression of luteinizing hormone receptor and follicle stimulating hormone receptor in testes of the wild ground squirrels (Citellus dauricus Brandt).

The objective of this study was to evaluate whether luteinizing hormone (LH), follicle stimulating hormone (FSH) and their receptors luteinizing hormone receptor (LHR) and follicle stimulating hormone receptor (FSHR) play roles in the seasonal spermatogenesis of the wild ground squirrels. To that end, we characterized the testicular immunolocalization of LHR and FSHR, their expression on both mRNA and protein levels, as well as serum concentrations of LH and FSH in male wild ground squirrels throughout the annual reproductive cycle. Histologically, all types of spermatogenic cells including mature spermatozoa were identified in the breeding season (April), while spermatogonia and primary spermatocytes were observed in the non-breeding season (June), and spermatogonia, primary spermatocytes and secondary spermatocytes were found in pre-hibernation (September). LHR was present in Leydig cells during the whole periods with more intense staining in the breeding season; Stronger immunostaining of FSHR was observed in Sertoli cells during the breeding season compared to the non-breeding season and pre-hibernation. Consistently, the mRNA and protein levels of LHR and FSHR were higher in testes of the breeding season, and then decreased to a relatively lower level in the non-breeding season and pre-hibernation. Meanwhile, serum LH and FSH concentrations were significantly higher in the breeding season than those in the non-breeding season and pre-hibernation. These results suggested that gonadotropins and its receptors, LHR and FSHR may be involved in the regulation of seasonal changes in testicular functions of the wild ground squirrels.

Anti-Muellerian hormone, inhibin A, gonadotropins, and gonadotropin receptors in bull calves after partial scrotal resection, orchidectomy, and Burdizzo castration.

Eight-week-old calves were either castrated by partial scrotal resection (SR) without removing the testes (n = 10), Burdizzo (BZ) clamp (n = 10), orchidectomy (OR; n = 10), or were left gonad intact as controls (CO; n = 10). Concentrations of anti-Muellerian hormone (AMH), inhibin A, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in plasma were determined from 16 to 48 weeks of age. At 18 months, testes of SR, BZ, and CO bulls were obtained and the immunolocalization of LH and FSH receptors and AMH analyzed. Concentration of AMH in plasma of CO and SR bulls decreased with increasing age (P < 0.001). A similar AMH profile in CO and SR indicates that SR did not induce a true cryptorchid state. In groups OR and BZ, AMH was undetectable. Plasma inhibin concentration was higher in groups CO and SR than BZ and OR (P < 0.001). Plasma LH and FSH concentrations decreased over time (P < 0.001) and were higher in groups BZ and OR than SR and CO (P < 0.001). In the testes, immunolabeling for AMH existed in Sertoli cells of CO and SR but not BZ bulls. FSH receptors were localized in Sertoli cells, Leydig cells, spermatocytes, and the epididymis of CO and SR animals, whereas LH receptors were restricted to Leydig cells. In BZ animals, FSH and LH receptors and AMH were absent, indicating complete testicular degeneration. In conclusion, AMH is a more reliable marker for the presence of testicular tissue in bulls than inhibin. Scrotal resection did not induce a true inguinal cryptorchid state but affected testicular responsiveness to gonadotropic stimulation.

Premature and multiple births in IVF are associated with pretreatment circulating LH/hCG receptor concentration.

The luteinizing hormone (LH) and pregnancy hormone, human chorionic gonadotrophin (hCG), share a common receptor: LH/hCG-R or LHCGR. In this prospective study involving 290 patients undergoing in vitro fertilization (IVF) and embryo transfer, we have examined whether pretreatment circulating LHCGR (sLHCGR) influences the course of pregnancy and perinatal outcome after embryo transfer. The blood samples were collected before the fertility treatment began and sLHCGR concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) test. We demonstrate that extreme pretreatment sLHCGR concentrations (low & high) were linked to abnormal birth weights for singleton births, while very low concentrations of sLHCGR were associated with premature delivery (≤34 weeks) of singletons and multiple births following transfer of ≥2 embryos.

Downregulation of adiponectin system in granulosa cells and low levels of HMW adiponectin in PCOS.

PURPOSE: The purpose of the study was to investigate changes in adiponectin system expression in granulosa cells (GCs) and high molecular weight adiponectin levels in serum and follicular fluid (FF) of 40 women with polycystic ovary syndrome (PCOS) compared to those in 40 women with normal ovary function. METHODS: Adiponectin (Adipo), adiponectin receptor 1 (AdipoR1), and adiponectin receptor 2 (AdipoR2) messenger RNA (mRNA) expression levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR). High molecular weight (HMW) adiponectin protein concentration was evaluated by ELISA method. Data were analyzed using Student's t test and one-way ANOVA in SPSS 21 software. At oocyte retrieval, FF was aspirated and GCs were obtained from a pooled collection of FF per each patient. RESULTS: PCR results showed expression of adiponectin, AdipoR1, AdipoR2, follicle-stimulating hormone receptor (FSHR), and luteinizing hormone receptor (LHR) in GCs. After controlling body mass index (BMI) values, qRT-PCR demonstrated a decreased expression of adiponectin system in GCs of PCOS patients compared to those in controls (p = 0.001). There was a strong positive correlation among AdipoR1 and AdipoR2 expression and also among FSH and LH receptor expression. (Both r = 0.8, p = 0.001). There were low levels of high molecular weight adiponectin in the serum of PCOS patients with controlled ovarian hyperstimulation (30.19 ± 4.3 ng/ml) compared to the controls (48.47 ± 5.9 ng/ml) and in the FF of PCOS patients with controlled ovarian hyperstimulation (7.86 ± 1.44 ng/ml) compared to the controls (14.22 ± 2.01 ng/ml; p = 0.02). CONCLUSIONS: Lower expression of adiponectin and its receptors in GCs might be an important manifestation in gonadotropin-stimulated PCOS patients which could influence the physiologic adiponectin roles such as interaction with insulin and LH in induction of GC gene expression.

[Morinda Officinalis How improves cellphone radiation-induced abnormality of LH and LHR in male rats].

OBJECTIVE: To investigate the effects of Morina Officinalis How (MOH) on the abnormal levels of serum luteotrophic hormone (LH) and LH receptor (LHR) in the testis tissue induced by cellphone radiation (CPR) in rats. METHODS: Fifty adult male SD rats were randomly divided into five groups of equal number: sham CPR, untreated CPR, negative double distilled water (DDW) control, aqueous MOH extract, and alcohol MOH extract. All the animals were exposed to mobile phone radiation except those of the sham CPR group. Then, the rats of the latter two groups were treated intragastrically with MOH at 20 g per kg of the body weight per day in water and alcohol, respectively. After 2. weeks of treatment, all the rats were sacrificed for measurement of the levels of serum LH and LHR in the testis tissue. RESULTS: The levels of serum LH and LHR were 30.15 ± 8.71 and 33.28 ± 6.61 in the aqueous MOH group and 0.96 ± 0.06 and 0.94 ± 0.08 in the alcohol MOH group, both significantly decreased as compared with the negative DDW controls (P < 0.05), but with no remarkable difference between the two MOH groups (P > 0.05). CONCLUSION: MOH can improve CPR-induced abnormality of LH and LHR in adult male rats.

Forms of Circulating Luteinizing Hormone Human Chorionic Gonadotropin Receptor for the Prediction of Early and Late Preeclampsia in the First Trimester of Pregnancy.

OBJECTIVE: To explore the value of circulating luteinizing human chorionic gonadotropin receptor (LHCGR) forms for the prediction of preeclampsia (PE) in the first trimester of pregnancy. METHODS: Case-control study, based on a cohort of 5,759 pregnancies, including 20 early PE, 20 late PE, and 300 controls. We recorded/measured maternal characteristics, mean arterial pressure (MAP), uterine artery (UtA) Doppler, placental growth factor (PlGF), soluble Fms-like tyrosine kinase-1 (sFtl-1), and LHCGR forms (hCG-LHCGR and soluble LHCGR), and their independent predictive values were analyzed by logistic regression. RESULTS: For early PE, the model included black ethnicity, chronic hypertension, previous PE, MAP, UtA Doppler, PlGF, sFlt-1, and LHCGR forms, achieving detection rates (DR) of 83% at 10% of false-positive rates (FPR) [AUC: 0.961 (95% CI: 0.921-1)]. For late PE, the model included body mass index, previous PE, UtA Doppler, PlGF, sFlt-1, and LHCGR forms, with DR of 75% at 10% of FPR [AUC: 0.923 (95% CI: 0.871-0.976)]. In both early and late PE, LHCGR forms improved DR by 6-15%. CONCLUSIONS: LHCGR forms improved the prediction for early and late PE. These results should be confirmed in larger prospective studies.

The utility of circulating LHCGR as a predictor of Down's syndrome in early pregnancy.

BACKGROUND: Previous studies showed that soluble LHCGR/hCG-sLHCGR concentrations in serum or plasma combined with PAPP-A and free ßhCG significantly increased the sensitivity of Down's syndrome screen at early pregnancy without altering the false positive rate. The goal of the present study was to further examine the role of sLHCGR forms as combinatorial markers and to investigate whether sLHCGR could serve as an independent biomarker for Down's syndrome in first trimester pregnancy screens. METHODS: The PAPP-A, free ßhCG, and hCG-sLHCGR concentrations together with nuchal translucency (NT) were measured in 40 Down's and 300 control pregnancies. The sLHCGR concentration was analysed in 40 Down's and 206 control pregnancies. RESULTS: The hCG-LHCGR in combination with PAPP-A and free ßhCG increased the detection rate (DR) by 35% without altering the false positive rate (FPR). The sLHCGR: hCG-sLHCGR ratio alone detected 80% of Down's pregnancies in first trimester screening, with a false positive rate of 0.5%. CONCLUSIONS: While measurement of sLHCGR forms in combination with PAPP-A and free ßhCG significantly increases the detection rate of Down's syndrome at first trimester, the ratio of sLHCGR: hCG-sLHCGR acts as an independent marker with a detection rate that is significantly higher than the existing biochemical markers individually for prenatal first trimester screening of Down's syndrome.

Quantitative ELISAs for serum soluble LHCGR and hCG-LHCGR complex: potential diagnostics in first trimester pregnancy screening for stillbirth, Down's syndrome, preterm delivery and preeclampsia.

BACKGROUND: Soluble LH/hCG receptor (sLHCGR) released from placental explants and transfected cells can be detected in sera from pregnant women. To determine whether sLHCGR has diagnostic potential, quantitative ELISAs were developed and tested to examine the correlation between pregnancy outcome and levels of serum sLHCGR and hCG-sLHCGR complex. METHODS: Anti-LHCGR poly- and monoclonal antibodies recognizing defined LHCGR epitopes, commerical anti-hCGbeta antibody, together with recombinant LHCGR and yoked hCGbeta-LHCGR standard calibrators were used to develop two ELISAs. These assays were employed to quantify serum sLHCGR and hCG-sLHCGR at first trimester human pregnancy. RESULTS: Two ELISAs were developed and validated. Unlike any known biomarker, sLHCGR and hCG-sLHCGR are unique because Down's syndrome (DS), preeclampsia and preterm delivery are linked to both low (less than or equal to 5 pmol/mL), and high (equal to or greater than 170 pmol/mL) concentrations. At these cut-off values, serum hCG-sLHCGR together with PAPP-A detected additional DS pregnancies (21%) which were negative by free hCGbeta plus PAPP-A screening procedure. Therefore, sLHCGR/hCG-sLHCGR has an additive effect on the current primary biochemical screening of aneuploid pregnancies. More than 88% of pregnancies destined to end in fetal demise (stillbirth) exhibited very low serum hCG-sLHCGR(less than or equal to 5 pmol/mL) compared to controls (median 16.15 pmol/mL, n = 390). The frequency of high hCG-sLHCGR concentrations (equal to or greater than 170 pmol/mL) in pathological pregnancies was at least 3-6-fold higher than that of the control, suggesting possible modulation of the thyrotropic effect of hCG by sLHCGR. CONCLUSIONS: Serum sLHCGR/hCG-sLHCGR together with PAPP-A, have significant potential as first trimester screening markers for predicting pathological outcomes in pregnancy.

Influence of standing estrus before an injection of GnRH during a beef cattle fixed-time AI protocol on LH release, subsequent concentrations of progesterone, and steriodogenic enzyme expression.

Beef cows that exhibit estrus before fixed-time AI have been reported to have increased pregnancy success and increased concentrations of progesterone during the subsequent estrous cycle. Therefore, these experiments were conducted to evaluate if initiation of standing estrus before an injection of GnRH during a fixed-time AI protocol affected LH pulses, subsequent concentrations of progesterone, and luteal steroidogenic enzyme expression. In Experiments 1 and 2, cows were treated with the CO-Synch protocol (100 µg GnRH day -9, 25 mg PGF(2α) day -2, and 100 µg GnRH day 0) and allotted to one of two treatments: 1) cows that initiated estrus before GnRH on day 0 (estrus; n = 5) or 2) cows that did not initiate estrus and were induced to ovulate by the GnRH on day 0 (no estrus; n = 5). In Experiment 1, blood samples were collected at 15-min intervals from 0 to 6 (bleed 1), 12 to 20 (bleed 2), 26 to 34 (bleed 3), and 40 to 48 (bleed 4) h after GnRH. Daily blood samples were collected for 17 d. Initiation of estrus before the GnRH injection had no effect on LH release or the pattern of progesterone increase; however, cows detected in estrus had overall increased (P = 0.002) concentrations of progesterone compared with cows not in estrus. In Experiment 2, estrus was detected with the HeatWatch system. Location and size of the ovulatory follicle was determined on day 0 by transrectal ultrasonography at time of injection with GnRH. Blood samples were collected on days 3, 4, 5, 7, and 9; luteal tissue was collected on day 10 (n = 4 estrus and n = 9 no estrus) from corpus luteum (CL) originating from similar-sized follicles (13.0 to 16.0 mm). Total cellular RNA was extracted, and relative mRNA levels were determined by real-time reverse transcription PCR and corrected for GAPDH. There was no effect of estrus on CL weight or concentrations of progesterone. In addition, there was no effect of estrus, follicle size, or CL weight on luteal expression of LH receptor, StAR, CYP11A1, or 3ßHSD. However, there was a correlation between follicle size and CL weight (P = 0.01; R(2) = 0.43); for every increase of 1 mm in follicle size, CL weight increased by 1.5 g. In summary, estrus did not influence release of LH, CL weight, progesterone concentrations, or expression of steriodogenic enzymes. However, as follicle size increased, CL weight increased; therefore, both follicle size and CL weight were associated with progesterone concentrations.

Circulating LH/hCG receptor (LHCGR) may identify pre-treatment IVF patients at risk of OHSS and poor implantation.

BACKGROUND: Successful pregnancy via in vitro fertilization (IVF) depends on the recovery of an adequate number of healthy oocytes and on blastocyst implantation following uterine transfer. Two hormones, LH and hCG, utilize a common LH/hCG receptor (LHCGR), variations in which have profound implications in human reproduction. Soluble LHCGR (sLHCGR) is released from experimental cell lines and placental explants and it can be detected in the follicular fluid and serum. METHODS: To evaluate the impact of circulating soluble LHCGR (sLHCGR) in fertility treatment, we measured sLHCGR and LH-sLHCGR complex in serum from women seeking IVF using specifically developed quantitative enzyme-linked immunosorbent assays (ELISA). Following an IVF cycle of treatment, patients were grouped according to oocyte yield into low (lower than or equal to 7 oocytes), intermediate (8-14 oocytes) and high (greater than or equal to 15 oocytes) responders and pregnancy outcome noted. RESULTS: Pre-treatment sLHCGR identified many women at risk of ovarian hyperstimulation. Low levels of sLHCGR were associated with pregnancy in both high and low responders but sLHCGR did not significantly affect the treatment outcome of intermediate responders. Low responders who failed to become pregnant had high levels of circulating sLHCGR bound to LH (LH-sLHCGR). CONCLUSIONS: Pre-treatment measurement of sLHCGR could be used to tailor individual fertility treatment programs and improve outcomes by avoiding ovarian hyperstimulation and poor embryo implantation.

Early hCG addition to rFSH for ovarian stimulation in IVF provides better results and the cDNA copies of the hCG receptor may be an indicator of successful stimulation.

A simple, safe and cost-effective treatment protocol in ovarian stimulation is of great importance in IVF practice, especially in the case of previous unsuccessful attempts. hCG has been used as a substitute of LH because of the degree of homology between the two hormones. The main aim of this prospective randomized study was to determine, for the first time, whether low dose hCG added to rFSH for ovarian stimulation could produce better results compared to the addition of rLH in women entering IVF-ET, especially in those women that had previous IVF failures. An additional aim was to find an indicator that would allow us to follow-up ovarian stimulation and, possibly, modify it in order to achieve a better IVF outcome; and that indicator may be the cDNA copies of the LH/hCG receptor. Group A patients (n = 58) were administered hCG and Group B rLH (n = 56) in addition to rFSH in the first days of ovarian stimulation. The number of follicles and oocytes and, most importantly, implantation and pregnancy rates were shown to be statistically significantly higher in the hCG group. This study has also determined, for the first time to our best knowledge, m-RNA for LH/hCG receptors in the lymphocytes of peripheral blood 40 h before ovum pick-up. cDNA levels of the hCG receptor after ovarian stimulation were significantly higher among women receiving hCG compared to those receiving LH. In addition, higher levels were encountered among women with pregnancy compared to those without, although this was not statistically significant due to the small number of pregnancies. It seems that hCG permits a highly effective and more stable occupancy of rLH/hCG receptors and gives more follicles and more oocytes. The determination of cDNA copies could be, in the future, a marker during ovulation induction protocols and of course a predictor for the outcome of ART in the special subgroup of patients with previous failures.

The human chorionic gonadotropin molecule from patients with trophoblastic diseases has a high thyrotropic activity but is less active in the ovary.

To examine the pathogenesis of hyperthyroidism in women with trophoblastic diseases, the biological activity of human chorionic gonadotropin (hCG) molecules in women with normal pregnancy (n = 85) and in women with trophoblastic diseases (vesicular mole, n = 30; and choriocarcinoma, n = 12) was compared. Hyperthyroidism (thyroid stimulating hormone (TSH) < 0.3 mIU/l) was observed more frequently in women with trophoblastic diseases. All the sera were then subjected to Chinese hamster ovary cells transfected with the human TSH receptor (CHO-hTSHr cells) and cAMP production was compared. Sera from the women with choriocarcinoma showed the highest cAMP production. Interestingly, significant correlation between serum hCG level and cAMP production in CHO-hTSHr cells was observed only in women with trophoblastic disease. All the sera were then applied to CHO cells transfected with hCG/luteinizing hormone (LH) receptor (CHO-hCG/LHr cells). In contrast to the findings with the TSH receptor, sera from the women with normal pregnancy showed the highest cAMP production in these cells. Correlation between serum hCG level and cAMP production in CHO-hCG/LHr cells was significant only in normal pregnancy. These results indicate that the hCG molecule from women with trophoblastic diseases displays enhanced thyrotropic activity.

Sertoli-Leydig cell tumor of the ovary, a rare cause of precocious puberty in a 12-month-old infant.

We report a 12-month-old infant who presented with a 4-month history of isosexual precocious puberty secondary to an estrogenizing Sertoli-Leydig cell tumor of the ovary. Total serum immunoreactive inhibin and subunits A and B were markedly elevated before surgical resection and subsequently decreased 7 wk later into the normal prepubertal range. Twenty weeks following surgical removal, the patient presented again with central precocious puberty; inhibin B levels were raised on this occasion, a luteinizing releasing hormone stimulation test confirmed central precocious puberty. This is the youngest reported occurrence of this rare sex cord stromal neoplasm. The prognosis of this extremely rare tumor presenting at this early juvenile stage is uncertain. This report illustrates the usefulness of serum inhibin as a tumor marker during therapeutic suppression with leuprorelin acetate for central precocious puberty. Analysis of genomic and tumor DNA revealed a normal nucleotide sequence for the LH receptor and the Galpha(s) gene. To understand the molecular pathogenesis of this tumor we analyzed mRNA levels for the inhibin A and B subunits, FSH receptor, LH receptor aromatase, steroidogenic factor-1 and the ER beta genes. Molecular characterization reveals the presence of genes specific for granulosa and Leydig cells; the relative expression of these genes, in addition to its histologic characteristics, suggests that this tumor may result from a dysdifferentiation of a primordial follicle.

The inhibitory effect of hCG on counter current transfer of GnRH and the presence of LH/hCG receptors in the perihypophyseal cavernous sinus--carotid rete vascular complex of ewes.

The existence of the hormone passage from venous blood into arterial blood in the area of the perihypophyseal vascular complex has been demonstrated in some mammals, but its mechanism has not been defined. To study the regulatory mechanism we infused hCG into perihypophyseal cavernous sinus of ovariectomized, conscious ewes to test if the hCG would affect putative LH/hCG receptors and inhibit counter-current transfer of GnRH from the venous cavernous sinus to the arterial carotid rete. The latter study was done on an isolated head model. Ewes were ovariectomized in mid-anestrus and, after 4 to 5 wk were used in the experiments. On the day of experiment ewes were treated intramuscularly with estradiol benzoate or oil vehicle, and 18 to 20 h later were infused either with a multielectrolyte solution or hCG for 2 h via the venae angularis oculi. Immediately thereafter the ewes were anesthetized and exanguinated, and subsequently decapitated. The isolated head was perfused with Dextran in multielectrolyte. The 125I-GnRH was infused into the cavernous sinus via the venae angularis oculi for 5 min; contemporaneous samples were taken from the carotid rete and both jugular veins at 1-min intervals. Transfer of 125I-GnRH from the cavernous sinus to the carotid rete was inhibited by hCG in ewes pretreated with estradiol benzoate but not with oil (P<0.005). We collected tissue samples from the vascular complex of the cavernous sinus and carotid rete of cyclic ewes to determine the presence of LH/hCG receptors. In situ hybridization showed the presence of LH/hCG receptor mRNA transcripts in the walls of both arterial and venous compartments of the cavernous sinus-carotid rete complex, and immunohistochemistry revealed the presence of receptor proteins. These novel findings confirm previously obtained data suggesting that LH is a modulatory factor for the counter-current transfer of neuropeptides from the venous blood of the cavernous sinus to the arterial blood supplying the brain and hypophysis. The LH could modulate 125I-GnRH transfer acting directly on the vascular smooth muscle.

Development and validation of a radioligand receptor assay for measurement of luteinizing hormone in human serum.

Studies of circulating LH physiology and pathophysiology are dependent upon measurements of immuno- and bioactivity, both of which have methodologic limitations. We have developed and validated a RRA which allows direct measurement of receptor-bindable LH in human serum. Using a cultured Leydig tumor cell line (MA-10) known to express the CG/LH receptor as the receptor source and polyacrylamide-gel electrophoresis purified hCG as the radioligand, we have established an assay system with the requisite sensitivity (0.04 ng/tube) to measure circulating LH, without significant alteration in total specific binding upon addition of up to 150 microL gonadotropin-free serum when compared to no serum. Standard curves of hLH diluted in gonadotropin-free serum were not statistically different in slope or ED50 from buffer curves. Dilutions of human serum from postmenopausal women and men with Klinefelter's syndrome containing LH measured in the assay were parallel to the standard curve. Further validation of the RRA included measurement of LH by RRA and RIA in daily serum samples from normal women across the menstrual cycle (n = 6) where there was excellent correlation (P less than 0.001) between RRA and RIA measurements with the exception of the mid-cycle surge where the RRA/RIA ratio fell to 0.5. This LH RRA will be useful in further studies of the physiology and biochemistry of LH in human serum.

Enhanced testosterone secretion in adult rams after establishment of a high-frequency, low-amplitude pattern of LH pulses in the nonbreeding season occurs without changes in the number or binding affinity of testicular LH receptors.

When the LH signal in the ram is changed from one of large and infrequent pulses to one of small and frequent pulses, the testes quickly become more responsive to LH and testosterone secretion is elevated, perhaps because the number and (or) binding affinity of testicular LH receptors have increased. An experiment was undertaken in the nonbreeding season (July) with 10 adult Dorset x Leicester x Suffolk rams that were about 3.5 years of age and 69 +/- 2 kg in body weight. Rams were given injections into the jugular vein of either 5 micrograms NIH-LH-S24 (in 1 ml saline) or vehicle every 80 min for 6 days. LH treatment produced a series of LH pulses that occurred three times more frequently and were 70% less in amplitude than pulses in the control rams, without causing mean LH concentration to increase. Endogenously produced LH pulses were not evident in the treated rams after LH injection began. The modified LH-pulse pattern elevated mean testosterone concentration by 150% (assessed on days 2 and 5), and caused the cumulative testosterone response to LH pulses, estimated by multiplying testosterone-pulse amplitude by frequency per 6 h, to increase progressively by 180% (days -2 through 5). Enhanced testicular steroidogenic activity, presumably due to greater enzymatic activity and cholesterol availability within Leydig cells, was not associated with increases in either the concentration or affinity of LH-binding sites in the testis (assessed on days 3 and 6).

Inability to demonstrate an ultrashort loop feedback mechanism for luteinizing hormone in humans.

hCG has biological properties similar to those of LH, but can be measured separately from LH by current radioimmunometric assays. To investigate the possible existence of an autoregulatory mechanism for LH in humans, we compared the basal LH concentrations and the LH response to a GnRH stimulus with and without prior administration of hCG. On two separate occasions, at least 1 week apart, six normal (eugonadal) males and six normal postmenopausal females were given, in random order, either 10,000 IU hCG or saline followed by iv injection of a 200-micrograms bolus of GnRH. Blood samples were then taken 30, 60, 90, 120, 180, 240, and 300 min after GnRH. Serum concentrations of LH and hCG were measured at each time by two monoclonal antibody sandwich assays developed in our laboratory. After exogenous hCG, serum hCG concentrations rose rapidly to 200-500 IU/L (15,000-35,000 pg/mL) in both the men and women, remaining at this high level throughout the study. In the men, sex steroid concentrations did not change in response to the hCG during the 9 study hours. Compared to saline-treated controls, hCG had no significant effect in either men or postmenopausal women on the basal LH concentration or the response to a GnRH bolus, as determined by peak response and area under the LH/time curve between 0-300 min after GnRH. We conclude that an ultrashort loop feedback mechanism for LH on its own secretion does not exist in humans, as assessed by the present protocol.