Difference in prevalence of congenital cryptorchidism in infants between two Nordic countries.

BACKGROUND: Several investigators have shown striking differences in semen quality and testicular cancer rate between Denmark and Finland. Since maldescent of the testis is a shared risk factor for these conditions we undertook a joint prospective study for the prevalence of congenital cryptorchidism. METHODS: 1068 Danish (1997-2001) and 1494 Finnish boys (1997-99) were consecutively recruited prenatally. We also established prevalence data for all newborns at Turku University Central Hospital, Finland (1997-99, n=5798). Testicular position was assessed by a standardised technique. All subtypes of congenital cryptorchidism were included, but retractile testes were considered normal. FINDINGS: Prevalence of cryptorchidism at birth was 9.0% (95% CI 7.3-10.8) in Denmark and 2.4% (1.7-3.3) in Finland. At 3 months of age, prevalence rates were 1.9% (1.2-3.0) and 1.0% (0.5-1.7), respectively. Significant geographic differences were still present after adjustment for confounding factors (birthweight, gestational age, being small for gestational age, maternal age, parity, mode of delivery); odds ratio (Denmark vs Finland) was 4.4 (2.9-6.7, p<0.0001) at birth and 2.2 (1.0-4.5, p=0.039) at three months. The rate in Denmark was significantly higher than that reported 40 years ago. INTERPRETATION: Our findings of increasing and much higher prevalence of congenital cryptorchidism in Denmark than in Finland contribute evidence to the pattern of high frequency of reproductive problems such as testicular cancer and impaired semen quality in Danish men. Although genetic factors could account for the geographic difference, the increase in reproductive health problems in Denmark is more likely explained by environmental factors, including endocrine disrupters and lifestyle.

Inhibin A, inhibin B, follicle-stimulating hormone, luteinizing hormone, estradiol, and sex hormone-binding globulin levels in 473 healthy infant girls.

The early postnatal regulation of reproductive hormones seems to be more complex in girls than in boys. The aim of this study was to describe inhibins A and B, FSH, LH, estradiol, and SHBG in a large prospective cohort of 473 unselected, healthy, 3-month-old girls. In full term, appropriate-for- gestational-age girls (n = 355) hormones showed a marked interindividual variation, with concentrations up to pubertal values [medians (95% confidence intervals): inhibin B, 82 pg/ml (<20-175); FSH, 3.8 IU/liter (1.2-18.8); LH, 0.07 IU/liter (<0.05-1.07); estradiol, 31 pM (<18-83); SHBG, 137 nM (72-260)]. In 38%, FSH levels exceeded 4.5 IU/liter. Weight at 3 months had significant inverse relationships with estradiol and SHBG (P = 0.048 and P = 0.001, respectively). Gestational age was negatively correlated to estradiol (P = 0.001), with a similar trend for LH, FSH, and inhibin B. Inhibin B was higher in premature girls [126 pg/ml (<20-265)] than in term [80 pg/ml (<20-181), P = 0.002] and postmature girls [59 pg/ml (<20-152), P = 0.012]. Likewise, estradiol levels in prematures were higher than in mature girls [51 pM (<18-128) vs. 31 pM (<18-85), P = 0.009]. Estradiol was also higher in small-for-gestational-age than in appropriate-for-gestational-age girls (P = 0.046), with inhibin B and LH, but not FSH, showing a similar trend. In conclusion, reproductive hormones showed a large variation, and concentrations corresponded to those observed in puberty. Our findings support the concept of a minipuberty in infant girls similar to that in boys.

The birth rate of hypospadias in the Turku area in Finland.

Reports based on national registers of congenital malformations have suggested that the birth rate of hypospadias has increased during the last few decades. Register-based information may, however, have pitfalls because of changes in diagnostics, reporting accuracy and registration system. The aim of this study was to determine the current birth rate of hypospadias in Turku University Central Hospital (TUCH) in Finland. This was a prospective study on live-born boys born in TUCH from 1997 to 1999. In the total birth cohort (n=5,798) as well as in a special subcohort group (n=1,505) 0.3% of boys had hypospadias. Only one scrotal hypospadias was found in a boy who had a chromosomal anomaly. Other hypospadias were glandular or coronal. No increase was found in the birth rate of hypospadias when comparing our result with register-based data of boys born in Finland during the years 1970 to 1986 and surgically treated for hypospadias by the age of 8 years. No difference was found either from malformation register-based data concerning the nationwide birth rate of hypospadias during the years 1993 to 1998. Due to differences in national registration systems between countries, prospective studies with equal assessment criteria are needed in order to make reliable international comparisons.

Disparate response of wild-type and variant forms of LH to GnRH stimulation in individuals heterozygous for the LHbeta variant allele.

There is a common genetic variant of LH due to two amino acid changes in the LHbeta subunit, Trp(8)Arg and Ile(15)Thr. In order to compare the relative activities of wild type (wt-LH) and variant LHbeta (v-LHbeta) genes in LH production and secretion, we performed gonadotrophin-releasing hormone (GnRH) stimulation tests for healthy females (n = 7) and males (n = 10) heterozygous for the v-LHbeta allele. Blood samples were drawn up to 180 min after injection of GnRH. The serum samples were subjected to two immunofluorometric assays, one detecting wt hormone, the other detecting equally both LH types. The wt/total ratio increased significantly (P < or = 0.016) after GnRH injection in males. This indicates that the proportion of wt-LH increases in the circulation in men but not in women, and that women consequently secrete relatively more v-LH. An in-vitro bioassay was performed on 0 and 60 min samples, and the bio/immunoreactivity (B/I) ratio decreased in both sexes (P = 0.010-0.012). This supports the previously reported lower B/I ratio of wt. than v-LH, since wt-LH is expected to accumulate in circulation because of its longer half-life. In conclusion, these findings demonstrate that wt.- and v-LH respond differently to GnRH stimulation in men and women heterozygous for v-LHbeta. These results are in agreement with previously documented differences of the two forms in circulation, as well as with different promoter activities of the two LHbeta alleles.

Recombinant forms of rat and human luteinizing hormone and follicle-stimulating hormone; comparison of functions in vitro and in vivo.

We have previously described the preparation, purification and partial characterization of recombinant (rec) forms of rat luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In the present study, the special functional features of these hormones were studied further, in vitro and in vivo, and compared with human recLH and recFSH, as well as with human urinary choriongonadotropin (hCG) and rat pituitary LH (NIDDK-RP3). In radioreceptor assay, the affinity of hCG binding to rat testis membranes was 5-fold higher than that of human recLH and 100-fold higher than that of rat recLH. In in vitro bioassay, using dispersed adult mouse interstitial cells or a mouse Leydig tumor cell line (BLT-1), hCG and human recLH were 10- to 20-fold more potent than rat recLH. Correspondingly, rat pituitary LH was about 10-fold less potent than rat recLH, and evoked a maximum testosterone response that was about half of that elicited by the other LH/CG preparations. Rat recFSH was about 10-fold less potent than human recFSH in stimulating cAMP production of a mouse Sertoli cell line (MSC-1) expressing the recombinant rat FSH receptor. The circulating half-times (T1/2) of rat and human rec hormones were assessed after i.v. injections into adult male rats rendered gonadotropin-deficient by treatment with a gonadotropin-releasing hormone antagonist. A novel immunometric assay was used for the rat FSH measurements. In the one-component model the T1/2 values of rat and human recLH were 18.2 +/- 1.9 min (n = 7) and 44.6 +/- 3.1 min (n = 7) respectively and those of rat and human recFSH were 88.4 +/- 10.7 min (n = 6) and 55.0 +/- 4.2 min (n = 6) respectively; the two-component models revealed similar differences between the rec hormone preparations. Collectively, rat recLH was eliminated significantly faster from the circulation than human recLH (P < 0.0001). In contrast, the elimination of rat recFSH was significantly slower than that of human recFSH (P = 0.02). In conclusion, rat recFSH and rat recLH display lower biopotencies per unit mass than the respective human hormones in vitro, and also in vivo for LH. This is paralleled by shorter T1/2 of rat recLH than the respective human hormone in the circulation, whereas human recFSH has a shorter T1/2 than human FSH. The special functional features of the rat rec gonadotropins emphasize the use of these preparations on studies of gonadotropin function in the rat, an important animal model for reproductive physiology.

Persistence of biological activity of biotinylated human chorionic gonadotropin and its use for visualization of rat luteinizing hormone receptors in tissue sections.

Biotinylation of antibodies is an established method for producing systems for detection of antigens. We currently aim to develop liposomal targeting vectors for gene transfer into transgenic gonadal tumor cells expressing the luteinizing hormone (LH) receptor (R). We have biotinylated (B) human chorionic gonadotrophin (hCG) to obtain a selective targeting molecule to be attached to biotinylated liposomes via an avidin-streptavidin bridge. The biotinylation was performed by combining biotin isothiocyanate (BITC) and hCG in alkaline reaction buffer in a 100:1 (BITC:hCG) molar ratio. B-hCG maintained its ability to bind specifically to rat testicular membranes and was also bound to streptavidin-coated polypropylene wells. cAMP production was induced in BLT-1 Leydig tumor cells in vitro after stimulation with B-hCG, as a sign of persistent bioactivity. Frozen sections of rat testicular and ovarian tissues and skeletal muscle were labeled by incubating for 2 hr at 37 degrees C with 10 ng/microliter B-hCG. The binding was subsequently visualized by the avidin-biotin-peroxidase system, followed by silver enhancement of Ni-DAB staining. In rat testicular and ovarian sections, labeling was observed in structures known to strongly express the LH-R, i.e., Leydig cells, corpora lutea, and blood vessels. The labeling was blocked by preincubation with a 100-fold excess of the native hormone, and by injecting the rats sc with a high dose of hCG (1000 IU/kg) 48 hr before sacrifice. Skeletal muscle, used as negative control, was not labeled. These data demonstrate that the bioactivity of hCG is relatively well preserved after biotinylation. The biotinylated gonadotropin offers a new nonradioactive alternative for visualization of bioactive LH receptors in tissue sections.

Fetal development of Leydig cell activity in the mouse is independent of pituitary gonadotroph function.

During fetal development the testes secrete anti-Mullerian hormone and testosterone to induce formation of the male phenotype. Adult Leydig cells secrete testosterone under the control of LH, but the role of the fetal pituitary in regulating fetal Leydig cell function is unclear. To study the early relationship between pituitary and Leydig cell function, we have examined the development of fetal pituitary LH levels and Leydig cell function in normal mice and in hypogonadal (hpg) mice that lack GnRH and, thus, circulating gonadotropins. In normal and hpg mice, pituitary LH content was barely detectable until embryonic day 17 (E17), when levels began to increase significantly in both groups. Pituitary levels of LH in hpg mice were, however, only about 10% of normal at all ages. Full-length LH receptor transcripts were first detectable in fetal testes on E16 in both normal and hpg mice. In normal mice, levels of testicular messenger RNA (mRNA) encoding cytochrome P450 side-chain cleavage and 17alpha-hydroxylase increased from E13 to reach a peak around birth. In hpg mice, levels of mRNA encoding these enzymes were normal until around birth, at which time there was a significant decline. Levels of testicular mRNA encoding 3beta-hydroxysteroid dehydrogenase type I were similar in normal and hpg mice and showed little change during development. Intratesticular testosterone reached a peak on E18 in normal animals before declining again after birth. In hpg mice, intratesticular testosterone levels were normal throughout fetal development and on the day of birth, but were barely detectable by postnatal day 5. Results show 1) that fetal Leydig cell function in the mouse is normal in the absence of endogenous circulating gonadotropins; 2) that Leydig cells become dependent on gonadotropins shortly after birth; and 3) that pituitary LH synthesis can start in the absence of GnRH but is dependent on LH for a normal level of synthesis and secretion.

Longitudinal reproductive hormone profiles in infants: peak of inhibin B levels in infant boys exceeds levels in adult men.

The gonads are usually considered quiescent organs in infancy and childhood. However, during the first few postnatal months of life, levels of gonadotropins and sex hormones are elevated in humans. Recent epidemiological evidence suggests that environmental factors operating perinatally may influence male reproductive health in adulthood. The early postnatal activity of the Sertoli cell, a testicular cell type that is supposed to play a major role in sperm production in adulthood is largely unknown. Recently, the peptide hormone inhibin B was shown to be a marker of Sertoli cell function in the adult male. In the adult woman, inhibin B is secreted by the granulosa cells. Longitudinal serum levels of inhibin B were measured in healthy boys (n = 15) and girls (n = 15), in cord blood, and every third month during the first 2 yr of life. In addition, serum levels of FSH, LH, and testosterone (boys) were measured in the same group of children. In boys, inhibin B, FSH, LH, and testosterone levels were all elevated at 3 months of age. However, the peak of inhibin B was unexpectedly high, into the supraadult range (mean +/- SE, 378 +/- 23 pg/mL) and persisted much longer than the elevation of FSH, LH, and testosterone. Thus, although levels of FSH, LH, and testosterone decreased into the range observed later in childhood by the age of 6-9 months, serum inhibin B levels remained elevated up to at least the age of 15 months. In girls, the hormonal pattern was generally more complex, with a high interindividual variation in levels of inhibin B, FSH, and LH within each age. In conclusion, the sustained elevation of inhibin B to supraadult levels in infant boys indicates that the neonatal period may be a developmental window important for Sertoli cell proliferation and maturation. Thus, the gonads may be potentially vulnerable to exogenous endocrine interference, e.g. from environmental factors during this period of life. Measurement of serum levels of inhibin B in infants may give clinical clues about developmental deficiencies in the gonads that otherwise only become apparent around puberty or later in life.

Sensitive immunoassay and in vitro bioassay demonstrate constant bioactive/immunoreactive ratio of luteinizing hormone in healthy boys during the pubertal maturation.

The quality of serum LH was assessed during pubertal maturation in boys by measuring immunoreactive (I) LH by a time-resolved immunofluorometric assay (IFMA, Delfia), and bioactive (B) LH by a sensitized in vitro bioassay. Seven samples were collected at 3-mo intervals from 14 healthy boys (median starting age 11.8 y) during pubertal maturation from Tanner stage I-III or II-IV (n = 7 for each). The mouse Leydig cell in vitro bioassay was sensitized 10-fold, to 0.05-0.1 IU/L, by including 1.5 mumol/L of forskolin in the incubation medium. The I- and B-LH levels showed good linear correlation throughout the concentration range analyzed. Mean I-LH increased between the pubertal stages I-IV from 0.42 to 2.24 IU/L and that of B-LH from 1.35 to 5.04 IU/L. No concomitant change occurred in the B-LH/I-LH (B/I) ratio, which was 2.84 +/- 0.54 in stage I and 2.58 +/- 0.48 in stage IV (mean +/- SEM, n = 7). Although the B/I ratios of LH varied from 0.59 to 5.85 in the samples analyzed, the intraindividual variation was small (mean coefficient of variance, 22%). In conclusion, IFMA and sensitized in vitro bioassay showed in healthy boys a similar 4-5-fold increase in the mean LH concentration during pubertal maturation, with no concomitant change in the B/I ratio. The sensitized in vitro bioassay of LH is useful for analysis of the low peripubertal LH levels. The good correlation between the I-LH and B-LH levels, and the lack of change in LH B/I ratio, indicate that IFMA correctly estimates the LH levels upon evaluation of pubertal maturation.

Prevalence of an immunological LH beta-subunit variant in a UK population of healthy women and women with polycystic ovary syndrome.

OBJECTIVE: An immunological LH beta-subunit variant has been described, which is undetectable using monoclonal antibodies directed to the intact LH molecule alone. Subjects have been found homozygous or heterozygous for nucleotide mutations within codons 8 and 15 in the LH beta-subunit gene. The prevalence of the variant LH beta-subunit has been estimated in a healthy UK population of women of reproductive age and in women with polycystic ovary syndrome (PCOS). The relationship of the variant molecule to the clinical and hormonal parameters of the subjects has been evaluated. DESIGN: The control and PCOS subjects were screened for the presence of the mutation by using a ratio of two immunofluorometric assays using monoclonal antibodies (Mab). One assay, not detecting the LH variant, uses a Mab directed to the intact LH molecule and a beta-specific Mab. The other assay, detecting both the variant and wild-type LH, uses two beta-subunit specific Mabs. The mutations in the LH beta-subunit gene were confirmed by restriction fragment length polymorphism. The relationship of the presence of the variant to the clinical and hormonal parameters was assessed by ANOVA. PATIENTS: Two hundred and twelve normal ovulatory women, of whom 66 (31%) were obese (body mass index > 25) and 146 (69%) non-obese, and 153 women with PCOS, 115 (75%) obese and 38 (25%) non-obese participated in the study. RESULTS: The variant LH was detected in 31 (15%) controls and 32 (21%) PCOS subjects (P = 0.124) using specific Mab. Obese PCOS had a higher incidence of the heterozygous LH variant compared to obese controls (odds ratio 2.5, P = 0.03), and compared to non-obese PCOS (odds ratio 6.3, P = 0.01). The previously described two mutations in codon 8 and codon 15 were present in all subjects detected to be mutant hetero of homo-zygous by RFLP. There was no relationship between the presence of the variant LH and the clinical and hormonal parameter in the PCOS subjects; however, in the controls the presence of the variant LH was associated with a higher serum total testosterone (P = 0.046), oestradiol (P = 0.03) and SHBG (P = 0.002). CONCLUSIONS: The results of this study show that the variant LH beta-subunit is a common polymorphism occurring in 15% of a healthy UK population of women. The prevalence was not higher in women with PCOS, though it was over represented in obese women with PCOS. The presence of the variant did not alter the clinical or hormonal expression of the disorder in women with PCOS. Its presence in the controls was however associated with higher serum oestradiol and probably secondary elevation of SHBG and testosterone, suggesting that the variant form of LH may be associated with subtle changes in the function of the hypothalamic-pituitary-gonadal axis.

Occurrence and biological properties of a common genetic variant of luteinizing hormone.

We have characterized the frequency and selected biological properties of a variant form of LH caused by two point mutations in the gene of the LH beta-subunit. Detection of the LH variant (or polymorphism) is based on aberrant immunoreactivity; it is not detected by a monoclonal antibody (Mab) recognizing a specific epitope in the LH alpha/beta-dimer (assay 1), but an assay using two LH beta-specific Mab recognizes this LH form normally (assay 2). Hence, the ratio of LH measured by assays 1 and 2 is 1.18-2.10 (range of mean +/- 2 SD) in wild-type subjects, 0.54-0.98 in heterozygotes, and below 0.15 in homozygotes with regard to the mutant LH beta allele. Analysis of sera from 249 healthy male and female subjects of Finnish origin revealed a frequency of 24.1% heterozygotes and 3.6% homozygotes for the mutation, with similar proportions in each sex. The ratio of in vitro bioactivity to immunoreactivity (assay 2) of the variant LH was significantly (P < 0.01) increased (2.9 +/- 0.1; n = 11) compared to that of wild-type LH (2.2 +/- 0.1; n = 13). No difference was observed in LH pulsatility, measured from blood samples collected at 5-min intervals for 5 h, between three male and three female subjects homozygous for the LH variant and three matched male and three female controls with wild-type LH. Likewise, the responses of LH immunoreactivity (assay 2) to GnRH stimulation were similar with both types of LH. The half-time of the variant LH in rat circulation from both sexes was significantly shorter than that of LH from control subjects (males, 25.5 +/- 3.8 vs. 48.3 +/- 2.7 min, respectively; P < 0.01; n = 3). Upon isoelectric focusing of peripheral serum samples, the isoform distribution of the variant LH was similar to that of wild-type LH. In conclusion, the LH variant discovered by us appears to occur with high frequency in the Finnish population (28% homo- or heterozygotes). It has increased in vitro bioactivity and a decreased half-time in vivo. These differences are compatible with a putative extra carbohydrate chain in the LH beta-chain, as one of the two mutations introduces an extra glycosylation signal. The subjects homozygous for the LH polymorphism are apparently healthy. However, the altered bioactivity and in vivo kinetics of the LH variant may induce subtle changes in LH action, either predisposing the affected individuals to or protecting them from disease conditions related to LH action.

Effects of oestrogen treatment on serum gonadotrophin bioactivity, immunoreactivity and isohormone distribution, and on immunoreactive inhibin levels, in prostatic cancer patients.

OBJECTIVE AND DESIGN: No data are available on effects of long-term exposure to oestrogen on bioactivity of gonadotrophins in men. We studied the effects of a 6-month oestrogen therapy on serum FSH and LH bioactivity (B), immunoreactivity (I) and isohormone distribution, and on serum I-inhibin levels, in patients with prostatic carcinoma. PATIENTS: Eleven men with advanced prostatic cancer were studied, each receiving 160 mg of polyoestradiol phosphate (Estradurin) once a month intramuscularly for 6 months. MEASUREMENTS: Serum samples were collected before, and after 2 and 6 months of oestrogen treatment. Serum B- and I-FSH levels were measured by immature rat granulosa cell bioassay and immunofluorometric (IFMA, Delfia) assay, respectively, and those of B- and I-LH by mouse interstitial cell bioassay and IFMA, respectively. Serum oestradiol (E2) concentrations were measured by IFMA assay, and serum testosterone (T) and inhibin levels by radioimmunoassay. Isoelectric focusing was used for fractionation of the FSH and LH isoforms. RESULTS: The pretreatment levels of B-FSH and I-FSH were 84.7 +/- 21.6 and 11.4 +/- 3.2 IU/l (mean +/- SEM), respectively, and the B/I ratio of FSH was 8.3 +/- 1.0. The pretreatment levels of B-LH and I-LH were 23.5 +/- 3.2 and 10.1 +/- 2.3 IU/l, respectively, and the B/I ratio was 3.0 +/- 0.4. After 6 months of oestrogen therapy, B-FSH and I-FSH decreased to 37.5 +/- 8.1 (P < 0.05) and 1.3 +/- 0.3 IU/l (P < 0.01), respectively, but the B/I ratio of FSH increased to 28.5 +/- 4.2 (P < 0.05). B- and I-LH levels decreased in 6 months to 7.4 +/- 0.9 and 2.3 +/- 0.5 IU/l (P < 0.01), respectively, but no change was found in the B/I ratio of LH. Serum T levels decreased from 19.0 +/- 2.6 to 2.7 +/- 0.9 nmol/l (P < 0.01) during the 6-month treatment, and the respective E2 levels increased from 0.2 +/- 0.01 to 4.4 +/- 0.5 nmol/l (P < 0.01). Serum I-inhibin levels were analysed from eight patients. The levels at 0, 2 and 6 months were 0.81 +/- 0.09, 0.50 +/- 0.03 and 0.54 +/- 0.01 microgram/l, respectively. Gonadotrophins in the pretreatment and 6-month samples of four patients were analysed by isoelectric focusing. In FSH of all subjects, and in LH of three subjects, a shift from acidic to more basic isoforms occurred after oestrogen therapy. This is in keeping with the increase of the B/I ratio of FSH. With LH, the isoform shift occurred between fractions with similar B/I ratios, and hence there was no shift in the overall B/I ratio. CONCLUSIONS: Oestrogen therapy of men suppressed bioactive and immunoreactive levels of gonadotrophins. The B/I ratio of FSH increased, and this increase was associated with a shift in the isohormone profile to more basic forms. In contrast, no change occurred in the B/I ratio of LH, even though changes in the isohormone profile were observed. Hence, not all changes in the isohormone distribution of gonadotrophins result in changes of the intrinsic in-vitro bioactivity.

A supersensitive immunofluorometric assay for rat luteinizing hormone.

We have developed an immunofluorometric assay (IFMA) for rat (r) LH, which is based on two monoclonal antibodies, one to bovine and the other to human LH. Signal detection occurs by time-resolved fluorescence evoked by a europium label (Delfia, Wallac). The method is fast in comparison to the standard RIA with the NIDDK reagents (4 h vs. 3 days). The sensitivity of the IFMA assay (0.75 pg/tube; NIDDK rLH RP-2) is over 30-fold higher than that of the NIDDK RIA (usual detection limit, 20-30 pg/tube). Using 25-microliters serum samples, the sensitivity of IFMA is 0.03 micrograms/liter; with 100-microliters samples, it is 0.0075 micrograms/liter. The cross-reactivity of the IFMA assay is 0.3% with rFSH, 3% with rTSH, and less than 0.05% with rGH, rPRL, and the rat alpha-subunit. A linear correlation between IFMA and RIA values is seen at serum levels above 0.4 micrograms/liter. Below this level, only IFMA is able to detect concentration differences between samples. In practice, this means that only IFMA is able to provide meaningful measurements of suppressed levels of serum LH. The correlation coefficient between IFMA and the mouse interstitial cell in vitro bioassay for LH in randomly selected rat pituitary homogenates was 0.93 (n = 47). The serum concentration of LH determined by IFMA is 0.57 +/- 0.10 micrograms/liter in intact adult male rats (mean +/- SEM; n = 12) and 0.41 +/- 0.10 micrograms/liter (n = 10) in randomly cycling females. The level in hypophysectomized rat serum is 0.035 +/- 0.0033 micrograms/liter (n = 8), if the limit of sensitivity (0.03 microgram/liter) is assigned to unmeasurable levels. One-week treatment of male rats with 2-cm Silastic implants containing testosterone suppressed serum LH, measured by IFMA, from 0.56 +/- 0.057 to 0.086 +/- 0.057 micrograms/liter (P < 0.01). The suppression of LH measured in the same samples by RIA was lower, from 0.73 +/- 0.057 to 0.44 +/- 0.048 micrograms/liter (P < 0.01). A 5-day starvation of intact male rats suppressed serum LH from 0.57 +/- 0.10 to 0.30 +/- 0.05 microgram/liter by IFMA (P < 0.01), whereas the decrease determined by RIA was not significant (0.80 +/- 0.07 vs. 0.66 +/- 0.13 micrograms/liter).(ABSTRACT TRUNCATED AT 400 WORDS)

The bio/immuno ratio of serum luteinizing hormone increases after orchiectomy in prostatic cancer patients and is associated with decreased molecular weight and appearance of isohormones with alkaline pI values.

Serum concentrations of bioactive (B) and immunoreactive (I) luteinizing hormone (LH) were determined in six patients with prostatic cancer before castration and at frequent intervals after the operation up to 6 mo. B-LH increased in 6 mo from 11 +/- 1 to 90 +/- 9 (mean +/- SEM, n = 6) IU/liter (p less than 0.01), and I-LH from 9 +/- 1 to 37 +/- 5 IU/liter (p less than 0.01). Accordingly, a significant increase in the B/I ratio of LH occurred at the same time, from 1.3 +/- 0.1 to 2.4 +/- 0.2 (p less than 0.01). To elucidate the molecular basis of the B/I ratio change, serum samples obtained before and 2-6 mo after orchiectomy were fractionated by gel filtration and chromatofocusing, and the eluted fractions were analyzed for B-LH and I-LH. In gel filtration, the fractions with the highest B-LH and I-LH contents were eluted later in the post-castration samples than in the pretreatment samples (mean Ve/Vo 1.31-1.32 vs. 1.26-1.28; p less than 0.02-0.01), indicating a small reduction in the average Mr of the circulating LH after castration. In chromatofocusing, a single major peak of immunoreactivity with a pI value of 7.4 was identified before castration, but in post-castration samples, a significantly large proportion of the immunoreactivity was eluted in the alkaline pI range 7.4-9 (22.2 +/- 2.4% before, 56.5 +/- 5.2% after castration, p less than 0.05). These findings indicate that after castration, the increased B/I ratio of serum LH is explained by a preferential increase in isohormones with slightly reduced molecular weights and alkaline pI values.

LH measurements by in vitro bioassay and a highly sensitive immunofluorometric assay improve the distinction between boys with constitutional delay of puberty and hypogonadotropic hypogonadism.

The basal and gonadotropin-releasing hormone (GnRH) stimulated levels of LH were measured in 21 boys with delayed puberty using conventional RIA, mouse interstitial cell in vitro bioassay (B-LH), and a highly sensitive immunofluorometric method (F-LH). On the basis of subsequent clinical follow-up, the subjects were diagnosed as idiopathic constitutional delay of puberty (CD, n = 13) or hypogonadotropic hypogonadism (HH, n = 8). The basal RIA LH levels were similar in the two diagnostic groups (HH, 2.92 +/- 0.76, CD 3.53 +/- 1.37 IU/L). In contrast, the mean basal B-LH was significantly lower in boys with HH than with CD (1.10 +/- 0.45 versus 2.91 +/- 1.23 IU/L; p less than 0.01). A similar finding was made by F-LH measurements which were clearly lower in the HH than the CD group (0.073 +/- 0.04 versus 1.71 +/- 0.97 IU/L, p less than 0.01). Also upon GnRH stimulation (3.5 micrograms/kg i.v.), the distinction between the CD and HH groups was better with the B-LH and F-LH measurements. The basal B/I ratio of the CD group (0.90 +/- 0.43) was more than that of the HH group (0.42 +/- 0.25, p less than 0.01) and this ratio increased significantly (more than 2-fold, p less than 0.01) during GnRH stimulation in the CD group, but not in the HH patients. Such differences were not found between the B/F ratios of the CD and HH groups. Measurements of basal and GnRH stimulated B- and F-LH levels clearly improved the distinction between CD and HH in comparison to the conventional RIA method, due to the low sensitivity and likely cross-reactions with some non-LH constituents of serum in the latter assay. This problem is, to a great extent, eliminated by better sensitivity and specificity of B-LH and F-LH measurements. For the same reasons, the difference in B/I ratios between the CD and HH samples, and the increased B/I ratio after GnRH stimulation in the CD group, were not observed in B/F ratios. In conclusion, the measurements of basal and GnRH-stimulated concentrations of serum B-LH and F-LH clearly improve the differential diagnostics between CD and HH. The discrepancies measured between the B/I and B/F ratios in these samples call for reevaluation of the bio/immuno ratios of circulating LH.