Diagnostic utility of a low-dose gonadotropin-releasing hormone test in the context of puberty disorders

OBJECTIVES: The 10-microg gonadotropin-releasing hormone (GnRH) test assesses pituitary gonadotroph responsiveness, whereas the 100-microg dose assesses maximal secretory capacity. Our aims were to establish normative data for the low-dose test in children and to evaluate the test in diagnosing common pubertal disorders. METHODS: We retrospectively classified 107 children who underwent 10-microg GnRH tests into normal prepubertal (20 boys, 10 girls), normal early pubertal (10 boys, 16 girls), constitutional delay of puberty (CDP, 13 prepubertal boys >12 years), hypogonadotropic hypogonadism (HH, 5 prepubertal boys >12 years), central precocious puberty (CPP, 19 girls) or premature thelarche/variant (13 girls). RESULTS: Peak LH response was higher in prepubertal boys >12 years compared with younger boys (p < 0.01) but showed no further change in early puberty. CDP boys had LH responses similar to prepubertal boys >12 years. HH boys showed an absent LH response which diagnosed HH with 100% sensitivity and 96% specificity. Thelarche girls had LH:FSH peak ratios lower than normal prepubertal (p = 0.001), pubertal (p < 0.05) or CPP (p = 0.001) girls. CONCLUSIONS: We have established normative values for the low-dose GnRH test in children. The test successfully differentiated HH from CDP in boys, and contributed to the differential diagnosis of CPP and premature thelarche in girls.     

Thyroxine-binding globulin, triiodothyronine, thyroxine and thyrotropin in newborn infants and children.

Thyroxine-binding globulin (TBG), triiodothyronine (T3), thyroxine (T4) and thyrotropin (TSH) have been determined by radioimmunoassay in plasma of newborn infants and throughout childhood until puberty. Mean maternal TBG concentration was 1.65 +/- 0.09 mg/100 ml (SEM) and significantly higher (p less than 0.01) than cord blood levels of TBG (1.16 +/- 0.08 mg/100 ml (SEM). Throughout infancy and childhood TBG remained significantly elevated (p less than 0.01) compared to a middle age control group of healthy blood donors. T3, T4 and TSH concentrations behaved postnatally as known from previous studies. The T3 and T4 increase observed immediately after birth was not a secondary phenomenon due to changes in TBG concentration since this globulin did not change significantly during this period.     

Steroidal and non-steroidal factors in plasma sex hormone binding globulin regulation.

Sex hormone binding globulin (SHBG) is a specific steroid-binding plasma glycoprotein regulated by several factors. Sex steroids are currently considered to be the main physiological regulators of this protein. SHBG levels, in fact, increase during estrogen treatment and decrease after androgen administration. It is well known, however, that in many physiological and pathological conditions SHBG concentrations cannot be explained only on the basis of steroidal control mechanisms. The regulation of SHBG levels is in fact more complex and other factors can also affect its plasma values. Between the steroidal factors our attention was focused on the role of androgens, of glandular and peripheral origin, in their capacity to lower SHBG plasma levels. We studied hyperandrogenic conditions in prepubertal (65 subjects with precocious adrenarche and 16 girls with prepubertal hypertrichosis, aged between 4 and 8 years) and in adult age (51 hirsute patients aged between 14-35 years and 51 acneic patients aged between 15-40 years). The effects of dexamethasone and ACTH administration on SHBG plasma levels were also evaluated. The results obtained showed that in adult hyperandrogenic patients SHBG levels, significantly lower than in controls, were not always inversely correlated with androgen levels, which, on the contrary, were higher than in controls. In patients with precocious adrenarche we found an inverse correlation only between SHBG, which was significantly lower than normal, and body mass index or bone age but not with androgens, suggesting that in this condition other factors may be more relevant than steroids in SHBG regulation. Between the non-steroidal factors our attention focused on insulin. We studied 40 non-obese hyperandrogenic patients with or without ultrasonographic evidence of polycystic ovaries, aged 18-39 years, and 35 obese patients, aged 19-37 years, with or without hyperandrogenism or evidence of PCO. Low levels of SHBG were found not only in hyperandrogenic obese patients but also in obese patients with normal androgens. It is possible to conclude that (1) several factors (calorie intake, energy balance and growth factors), other than steroids, may be involved in the regulation of SHBG levels in plasma; and (2) each regulating factor may act to a different extent depending on the various periods of the life cycle.     

Sex hormone-binding globulin (SHBG) and estradiol cross-talk in breast cancer cells.

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that regulates the action of steroid hormones at several levels. SHBG regulates the availability of free androgens and estradiol to hormone-responsive tissues. Moreover, SHBG is also part of a novel steroid signaling system. We report here on the mechanism of action and the biological effects of SHBG in breast cancer cells, especially distinguishing cross-talk between membrane-initiated SHBG and estradiol pathways. After interacting with a specific binding site on breast cancer cell membranes, SHBG activates a specific pathway, and by cAMP induction, inhibits estradiol-mediated activation of ERK. Both estradiol and SHBG membrane-initiated pathways involve cross-talk at MAP kinase level with the ultimate result of inhibiting estradiol-mediated cell growth and antiapoptosis. On the basis of reported evidence, we suggest that SHBG is one of the regulators of growth and apoptosis of estrogen-dependent breast cancer cells.     

Sex hormone-binding globulin, its membrane receptor, and breast cancer: a new approach to the modulation of estradiol action in neoplastic cells

The role of human Sex Hormone-Binding Globulin (SHBG), the plasma carrier of sex steroids, and its membrane receptor, SHBG-R, in estrogen-dependent breast cancer has been investigated in our laboratory in the past few years. SHBG-R is expressed in MCF-10 A cells (not neoplastic mammary cells), MCF-7 cells (breast cancer, ER positive) and in tissue samples from patients affected with ER positive breast cancer, but not in estrogen-insensitive MDA-MB 231 cells. The SHBG/SHBG-R interaction, followed by the binding of estradiol to the complex protein/receptor, causes a significant increase of the intracellular levels of cAMP, but does not modify the amount of estradiol entering MCF-7 cells. The estradiol-induced proliferation of MCF-7 cells is inhibited by SHBG, through SHBG-R, cAMP and PKA. Similarly, the proliferation rate of tissue samples positive for SHBG-R was significantly lower than the proliferation rate of negative samples. SHBG and SHBG-R could thus trigger a ‘biologic’ anti-estrogenic pathway. In order to get a more detailed knowledge of this system, we first examined the frequence of the reported mutated form of SHBG in 255 breast cancer patients. The mutated SHBG is characterized by a point mutation (Asp 327→Asn) causing an additional N-glycosylation site, which does not affect the binding of steroids to SHBG. The frequence of the mutation was significantly higher (24.5%) in estrogen-dependent breast cancers than in healthy control subjects (11.6%). This observation confirms the close relationship between SHBG and estrogen-dependent breast cancer and suggests that the mutation could modify SHBG activity at cell site. Lastly, the possibility of using SHBG to modulate the estradiol action in breast cancer was further studied by transfecting MCF-7 cells with an expression vector carrying the SHBG cDNA (study in collaboration with G.L. Hammond). Transfected cells are able to produce significant amount of SHBG in their medium, but their SHBG-R is reduced to undetectable levels. The SHBG produced by transfected MCF-7 cells is, however, able to inhibit estradiol-induced proliferation of MCF-7 cells expressing a functional receptor. Thus, the local production of SHBG obtained with transfection could be a useful tool to control cell growth in estrogen-dependent breast cancer.     

Clinical study on increased serum thyroxine-binding globulin in cancerous state

Serum thyroxine-binding globulin (TBG) in 169 patients with various cancers was determined by radioimmunoassay (RIA). Eleven patients showed a high serum TBG level (greater than 35 micrograms/ml). Two of them had been treated with estrogen for prostate cancer. One patient had high serum TBG with serum hepatitis. Another 8 cases had normal liver function and also normal levels serum estrogen. Thus, about 4.7% (8/169) of the cancer patients had high serum TBG and mild hyperthyroxinemia caused by unknown mechanisms. The high TBG level in these patients continued until just before death, or in some cases decreased to normal after removal of cancer tumors by operation. Cancer is occasionally associated with an increase in serum TBG. Although the mechanism is not clear, the increased TBG in the cancerous state in interesting and has significance as a tumor marker.     

Gonadotropin pulsatile secretion in girls with premature menarche

Five prepubertal girls (2.3-8.1 years old) were studied for isolated or recurrent vaginal bleeding in the absence of other signs of precocious puberty (premature menarche). Four of these girls with recurrent vaginal bleeding were studied for pulsatile gonadotropin secretory patterns. During sleep 3 girls showed luteinizing hormone (LH) pulses with low amplitude and a pubertal pattern of frequency whereas follicle-stimulating hormone (FSH) increased without demonstrable episodic secretion. Luteinizing hormone-releasing hormone (LHRH) tests demonstrated that FSH responses are greater than the LH responses, as in prepuberty. In 3 cases estradiol levels had augmented above normal prepubertal range. The menses spontaneously stopped during the follow-up. A reevaluation of the gonadotropin pattern, having the menses stopped for 6 months, in one of the girls with pulsatile LH secretion showed an apulsatile prepubertal LH pattern. Also estradiol levels returned to prepubertal range. A follow-up of 10-66 months of these patients did not show any growth and bone acceleration or signs of precocious puberty. Our data suggest that in premature menarche a partial and transient activation of hypothalamo-pituitary axis could be present. Premature menarche seems to be a benign and self-limiting condition and one of the girls had a normal onset of puberty during follow-up.     

Serum 5-androstene-3 beta,17 beta-diol sulphate, sex hormone binding globulin and free androgen index in girls with premature adrenarche

Serum sex hormone binding globulin (SHBG), testosterone (T), DHEA sulphate (DHEA-S), androstenedione (AD) and delta 5-androstene-3 beta,17 beta-diol sulphate (5-ADIOL-S) levels were measured by specific radioimmunoassay in 16 girls presenting with premature adrenarche (PA) and in 14 normal girls. Mean levels of steroids measured were elevated, and SHBG significantly depressed, in the girls with PA, with values (mean +/- SE) for DHEA-S (1.73 +/- 0.17 vs 0.25 +/- 0.06 mumol/l), 5-ADIOL-S (104 +/- 8 vs 31 +/- 4 nmol/l), AD (0.89 +/- 0.06 vs 0.62 +/- 0.04 nmol/l), and T (0.49 +/- 0.03 vs 0.23 +/- 0.06 nmol/l). SHBG levels were 68 +/- 6 vs 108 +/- 5 nmol/l, and the free androgen index [100 x T (nmol/l) divided by SHBG (nmol/l)] was 0.89 +/- 0.17 vs 0.22 +/- 0.01. These studies show that SHBG is depressed in girls with premature adrenarche; with the increased testosterone levels, this results in a markedly elevated free androgen index, a measure of testosterone which is bioavailable to target tissue. This may be compounded by the elevated levels of 5-ADIOL-S in girls with PA since its role may be as a prohormone for more potent androgens (testosterone, 5 alpha-dihydrotestosterone) in target tissues such as pubic skin.     

The role of estrogen in bone growth and maturation during childhood and adolescence

Twenty years ago it was believed that pubertal growth was stimulated by testicular androgen in boys and by adrenal androgen in girls. Estrogen, which was used to inhibit growth in excessively tall girls, was not thought to have growth-promoting effects. We hypothesized that estrogen has a biphasic effect on epiphyseal growth, with maximal stimulation at low levels. We showed that the administration of low doses of estrogen, corresponding to a serum estradiol level of about 4 pg/ml (15 pmol/l) caused more than a 60% increase over the prepubertal growth rate in both boys and girls. To test the hypothesis that estrogen is the principal mediator of the pubertal growth spurt in boys, we administered the aromatase inhibitor, testolactone, to boys with familial male-limited precocious puberty. Testolactone produced near normalization of both growth velocity and bone maturation, despite levels of serum testosterone that remained within the adult male range. The observation that low levels of estrogen stimulate growth and bone maturation suggested that estrogen might explain the more rapid epiphyseal maturation of prepubertal girls compared to boys. To determine whether prepubertal girls have higher estrogen levels than prepubertal boys, we developed an ultrasensitive recombinant cell bioassay for estrogen with a sensitivity of 0.02 pg/ml (0.07 pmol/l) estradiol equivalents. Prepubertal girls had approximately eight-fold higher levels of serum estradiol than did prepubertal boys (0.6 ± 0.6 pg/ml (SD) (2.2 ± 2.2 pmol/l) vs 0.08 ± 0.2 pg/ml (0.29 ± 0.73 pmol/l), P < 0.05). We concluded that the pubertal growth spurt of both sexes is driven primarily by estrogen, and that the more rapid epiphyseal maturation of prepubertal girls (vs boys) may be explained by their higher estradiol levels.     

Differences in infantile growth patterns in Turner syndrome girls with and without spontaneous puberty.

OBJECTIVE: The role of prepubertal estrogen in child growth was modeled using Turner's syndrome, comparing growth patterns of girls who later did or did not enter puberty spontaneously. The hypothesis was that TS patients with normal prepubertal estrogen levels would have a different growth pattern from those with subnormal estrogen levels. STUDY DESIGN: Growth data from 78 full-term patients with Turner's syndrome were collected retrospectively. 24/78 later developed spontaneous puberty, (+Pub), and their growth data were compared to TS patients without spontaneous puberty (-Pub). A nonlinear mixed model was fitted using the bi-exponential model. RESULTS: The growth velocity difference between the -Pub and +Pub groups suggests an early infantile growth advantage in the -Pub group, which disappears before the end of the first year of life; growth velocity remains similar (+/- 1 cm/y) for the next 6 years and declines at age 7-8 years in the +Pub group faster than it does in the -Pub group. Bi-exponential analysis showed that both the 1st (restrictive) and 2nd exponent (forward) were different (p = 0.0003). CONCLUSIONS: Comparison of girls with or without spontaneous puberty suggests a role for estrogen in child growth. Estrogens restrict infantile growth, as well as growth during the mid-childhood spurt.     

Changes in serum sex hormone-binding globulin and in serum non-sex hormone-binding globulin-bound testosterone during prepuberty in boys

Much evidence suggests that sex hormone-binding globulin (SHBG) influences the delivery of sex steroids to cells, probably by playing an important role in the distribution of serum sex hormones between SHBG-bound, albumin (HSA)-bound and free fractions. Recent evidence also suggests that HSA-bound testosterone (T), the major constituent of non-sex hormone-binding globulin-bound T, is biologically important. To examine the potential exposure of peripheral tissues to T during prepubertal years, the serum concentration of SHBG as well as the distribution of serum T in SHBG-bound, HSA-bound, free and non-SHBG-bound fractions was studied in 80 normal boys aged 0.5-14 yr, all at Tanner's stage G1 of sexual development. A gradual decrease in serum SHBG as a function of age was found without significant changes in the Ka of SHBG-dihydrotestosterone association. While regression analysis of serum total T vs age showed a 2.6-fold increase from 0.5 to 14 yr of age, those of non-SHBG-found, HSA-bound and free T vs age showed 8- to 9-fold increases during the same period. On the other hand, SHBG-bound T had only a 1.9-fold increase. Expressed as a function of serum total T, non-SHBG-bound T increased from 6.6 to 30.4%, the relative increment being greater for HSA-bound T than for free T. It is concluded that, with advancing age, there is a progressive increase in the T exposure of all tissues in normal prepubertal boys. It is speculated that, at the level of the central nervous system, this increase in serum bioavailable T could induce maturative changes in brain cells that result in the onset of puberty in normal boys.     

Interrelations between sex hormone-binding globulin (SHBG), plasma lipoproteins and cardiovascular risk

The incidence of coronary artery disease is significantly higher in men than in women, at least until menopause. This gender difference could be explained by the action of sex steroids on the lipoprotein profile. In prepubertal children, high-density lipoprotein (HDL) cholesterol and triglyceride levels are similar between sexes, while adult men have generally lower HDL cholesterol and higher triglyceride levels than premenopausal adult women. Most cross-sectional studies have reported that sex hormone binding globulin (SHBG) and testosterone levels correlate positively with HDL cholesterol levels between sexes. Thus SHBG by modulating the balance in the biodisposal of testosterone and estradiol, might have a profound effect on the risk of cardiovascular disease. However, adjustment for body weight and body fat distribution weakens the association between SHBG, testosterone and HDL cholesterol. The negative correlation of fasting insulin with SHBG and HDL cholesterol levels in both sexes, and some evidence that insulin is an inhibitor of SHBG production in vitro, has suggested that hyperinsulinism might negatively regulate SHBG and HDL levels. It remains to be determined whether the inverse relationship between SHBG and insulin levels is coincidental or has a causal effect on the increase of atherosclerosis. Decreased SHBG has been shown to be predictive of the incidence of non-insulin-dependent diabetes mellitus in women but not in men, and of subsequent development of cardiovascular disease and overall mortality in postmenopausal women. SHBG is an index of androgenism in women and of insulin-resistance in both sexes, and might be useful in epidemiological studies of cardiovascular risk. However, in men, SHBG is not predictive of the occurrence of cardiovascular disease. Whether SHBG might have an intrinsic protective effect on the arterial wall through SHBG-receptors is still highly speculative.     

A thyroxine-binding globulin of major biological significance in the serum of mice: demonstration and ontogenesis

We demonstrate in the mouse serum a hitherto unrecognized major thyroxine binding globulin (TBG), analogous to human TBG or to the recently discovered rat TBG. Our demonstration is based on equilibrium dialysis, electrophoresis, immunoelectrodiffusion and autoradiography techniques. Mouse TBG displays a remarkable ontogenic pattern, with 2-3 times higher activity in foetal than in maternal serum, and a further dramatic increase after birth. Between 1 and 5 days, the T4 binding to serum reaches peak levels 7-10 times more elevated than those measured in normal or pregnant adults. We also present for the first time the ontogenesis of the thyroxine binding prealbumin (TBPA), considered until now as the only specific T4 carrier of the murine species. We show that throughout development it is the TBG, not the TBPA, which crucially governs the level of the T4-serum interactions.     

Effect of estrogen on the synthesis and secretion of thyroxine-binding globulin by a human hepatoma cell line, Hep G2

Hyperestrogenemia in humans increases both the concentration of serum T4-binding globulin (TBG) by 2- to 3-fold and the proportion having anodal mobility on isoelectric focusing (IEF). As TBG is synthesized in the liver, we studied the effect of estrogen on TBG synthesis, secretion, and degradation by cultured human hepatocarcinoma cells (Hep G2). beta-Estradiol in concentrations in the range found in pregnancy (10(-7) M) had no effect on the accumulation of immunoreactive TBG in medium over 4 days. The absence of fetal calf serum or phenol red did not alter these findings. The amount of [35S]TBG accumulated 6 h after addition of [35S]methionine was not influenced by exposure to estrogen or to serum obtained from pregnant women. However, 10(-5) M beta-estradiol suppressed TBG more severely than albumin synthesis (34% vs. 9%). The lack of an estrogen effect on TBG synthesis and secretion was supported by experiments showing no effect of estrogen on the disappearance of TBG added to the medium or the accumulation of cytoplasmic TBG mRNA. The same cultures responded to estrogen by a 10-fold increase in nuclear estrogen receptor binding sites and a 2-fold increase in apolipoprotein CII. As TBG in serum, the rate of heat denaturation was not altered in TBG synthesized by Hep G2 cells in the presence of estrogen. In contrast to the effect on TBG in serum, in Hep G2 cells estrogen did not produce an anodal shift on IEF, or increased its proportion not bound to Concanavalin A, nor reduced its clearance rate when injected into rats. However, even untreated Hep G2 cells synthesized TBG with a larger number of anodal IEF bands and proportion of Concanavalin A excluded material than TBG in pregnancy serum. Results support our hypothesis, based on analysis of TBG in pregnancy, that estrogen-induced serum TBG elevation may not be mediated through an increase in synthesis. The failure to observe estrogen induced changes in oligosaccharide structure does not exclude estrogen responsivity of Hep G2 cells. Such effect could be masked by the marked constitutive increase in number of oligosaccharide chain antennae typical in this and other neoplastic tissues.     

Are estrogen receptor content in breast cancer and effects of tamoxifen on sex hormone-binding globulin markers for individual estrogen sensitivity?

Individual women differ with respect to their sensitivity to estrogen and serum levels of sex hormone-binding globulin (SHBG) may reflect the individual response. We found a significant correlation between estrogen receptor (ER) concentrations in breast cancer tissue and SHBG levels during tamoxifen treatment. Estrogen sensitivity may be a general characteristic common to various organs and different between individual women.     

Estrogen and androgen regulation of sex hormone binding globulin secretion by a human liver cell line

Both estrogens and androgens have been shown to stimulate sex hormone binding globulin (SHBG) secretion in vitro in the hepatocellular carcinoma cell line, Hep G2, in contrast to the expected inhibition by androgens from in vivo studies. However, such in vitro stimulation was only demonstrated at high steroid doses, generally in serum-containing medium, with added Phenol Red. In the present study, Hep G2 cells were grown in serum-free medium, without Phenol Red, under the influence of testosterone (T) (0, 0.5-500 nM) and ethinyl estradiol (EE2) (0, 50 pM-500 nM). Levels of secreted SHBG and albumin were correlated with androgen receptors in cytosolic (ARc) and nuclear (ARn) fractions and with DNA levels. In the presence of increasing T levels, SHBG levels fell to 39% of control values at 5 nM T (P = 0.047), rising to 97% of control at 500 nM. Conversely, incubation with EE2 produced a rise in SHBG secretion of more than 100% at 0.5 nM (P less than 0.02) which was sustained to 50 nM (P less than 0.005). DNA levels did not change with the addition of testosterone or EE2, with the exception of a 15% reduction at 5 nM EE2 (P less than 0.05). Albumin levels in the medium were not significantly altered by either steroid. However, in response to T, androgen receptor (AR) levels were reduced in cytosolic (42% of control) and nuclear (22%) fractions at 5 nM, and these changes in ARc and ARn correlated with SHBG levels over the range of T concentrations (P = 0.04 and P = 0.017, respectively). Nuclear estrogen receptor (ER) increased over 10-fold at 5 and 50 pM EE2 (P less than 0.001) and maintained 50 nM (P less than 0.001). Cytosolic ER was reduced at 0.5 and 5 nM but recovered at 50 nM, correlating with SHBG levels (P less than 0.001). These findings are consistent with the hypothesis that estrogens and androgens regulate SHBG synthesis in man by direct, specific, probably receptor-mediated effects on hepatocytes. Hep G2 cells grown in serum-free medium are a suitable experimental system for further study of this phenomenon.     

Characterization of obese women with reduced sex hormone-binding globulin concentrations

Factors influencing sex-hormone binding globulin (SHBG) concentrations in obesity are poorly understood. Preliminary observations suggest that dietary lipids may be involved and there are data confirming a direct inhibiting effect of insulin. Since only some obese subjects show lowered SHBG levels, we performed this study with the aim of defining obese women with low SHBG (LSO) (2 SD above normal values) in comparison with those presenting normal globulin concentrations (NSO). These groups were selected from a larger group of obese women with a history of normal menses and aged less than 40 years. An age-matched group of normal weight healthy women served as controls. Both LSO and NSO had similar body mass index and percentage body fat, but the waist to hip girth ratio (WHR), an index of body fat distribution, was significantly higher in LSO (0.88 +/- 0.04) than in NSO (0.81 +/- 0.09; P less than 0.05). Gonadotropin and androgen concentrations were similar in both groups, whereas estrone (E1) levels were higher in LSO (32.8 +/- 15.8 pg/ml) than in NSO (19.4 +/- 6.2 pg/ml; P less than 0.05; controls: 23.5 +/- 7.8 pg/ml; P less than 0.05). Moreover, compared to NSO, LSO women had significantly higher glucose-stimulated insulin and C-peptide levels. Partial regression analysis revealed significant correlation coefficients between SHBG, stimulated insulin values (r = -0.38; P less than 0.05) and WHR (r = 0.40; P less than 0.005). Therefore, compared to NSO, LSO women have distinctive clinical and endocrine characteristics, namely more pronounced hyperinsulinemia, higher E1 concentrations and a central type body fat distribution.     

Low polarity ligands of sex hormone-binding globulin in pregnancy. Part I--Characterization

Certain previously unidentified substances of low polarity binding to the sex hormone binding globulin (SHBG) in pregnancy were investigated. This material consisted of four major peaks (designated 1a, 1b, 2 and 3) as defined in maternal serum on Sephadex LH-20 chromatography. They were characterized with respect to changes in their concentration at various gestational ages and at premature and term labour in maternal serum, cord serum, placenta and maternal urine. Levels were much higher in placenta than in serum, suggesting that all four peaks were of placental origin. In the serum of mothers not in labour, while there was a significant increase in the mean serum concentration of peak (1a + 1b) and peak 3 for 12-24 weeks to 30-38 weeks gestation, there was no significant difference in the mean level of peak 2. However, there was a significant decrease in the serum levels of peak 2, but not peaks (1a + 1b) and 3, from 30-38 weeks gestation to 39-41 weeks gestation (P less than or equal to 0.05). In the serum of mothers in premature labour (30-38 weeks gestation), the levels of peak 2 but not those of peak (1a + 1b) and 3, were decreased (P less than or equal to 0.01) compared to those not in labour. These findings are consistent with the identification of peak 1a as 5 alpha-dihydroprogesterone, peak 1b as progesterone and peak 3 as 2-methoxyestrone as described in part 2. The decrease in the levels of peak 2 (for which the identification remains unconfirmed) in association with premature and impending term spontaneous labour suggest that it may be involved in the maintenance of pregnancy.     

Low serum allopregnanolone levels in girls with precocious pubarche

Allopregnanolone, a neuroactive steroid, increases during pubertal development and high concentrations are present in subjects with precocious puberty. The aim of the present study was to evaluate serum allopregnanolone levels in girls with precocious pubarche (PP). Basal gonadotropins and steroid hormones were assessed in 17 girls with PP, 22 girls with central precocious puberty (CPP), 25 girls with normal puberty at the same pubertal stage of CPP ones, and 17 prepubertal girls. Adrenocorticotropin hormone (ACTH) and gonadotropin-releasing hormone (GnRH) stimulation tests were performed in all subjects with PP, and in 12 out of 22 with CPP. All girls with normal puberty underwent to GnRH test, while ACTH test was performed in 17 out of 25. Basal dehydroepiandrosterone sulfate (DHEAS) concentrations resulted significantly higher in PP and normal pubertal girls than in prepubertal ones. Allopregnanolone, gonadotropins and estradiol levels were significantly lower in PP group with respect to CPP (P<0.05), while they were comparable among PP, normal pubertal and prepubertal groups. After ACTH administration, allopregnanolone concentrations significantly increased in all groups (P<0.05). After GnRH stimulation, its levels significantly increased in CPP and normal pubertal controls (P<0.05), while no incremental rise was found in PP girls. In conclusion, our study shows that in girls with PP basal and GnRH-stimulated levels of allopregnanolone are significantly lower than in CPP girls. These data suggest that this neurosteroid may be considered a new marker of pubertal development.