Bioactive FSH Levels Research Articles

In vitro biological-to-immunological ratio of serum gonadotropins throughout male puberty in children with insulin-dependent diabetes mellitus

Information on the impact of prolonged deficient glycemic control in the quality of the gonadotropin signal delivered by the pituitary gland during puberty in children with insulin-dependent diabetes mellitus (IDDM) is scarce. In the present study, we examined the impact of deficient glycemic control on bioactive LH and FSH concentrations and their corresponding biological-to-immunological (B:I) ratio in boys with poorly controlled, but systemically uncomplicated IDDM. Dual control groups comprising patients with well-controlled IDDM and healthy boys of comparable age and body mass index were included for appropriate comparisons within and between each pubertal stage. Patients with poorly controlled and well-controlled IDDM exhibited serum bioactive FSH levels and B:I FSH ratio similar to those showed by the healthy control group. In contrast, in early and mid-pubertal boys with poorly controlled IDDM bioactive LH levels were normal, but its B:I LH relationship was significantly (P < 0.05) decreased. This attenuation in the quality of the LH signal did not affect total serum T concentrations, and apparently, progression of puberty. Long-standing uncontrolled diabetes and the consequent metabolic disturbances and/or complications may aggravate the reproductive axis dysfunction and eventually provoke pubertal arrest.

Bioactive FSH May Have Value as a Marker for Ovarian Reserve in Patients With Elevated Serum FSH and Normal Basal Antral Follicle Counts

ObjectiveTo evaluate the bioactivity of FSH in women with paradoxically high serum immunoactive basal FSH levels and otherwise “normal” ovarian reserve as determined by ultrasound screening and response to gonadotropin stimulation.DesignCase Series at a large academic reproductive center.Materials and methodsWe compared immunoactive basal FSH levels (immulite assay (DPC)) and bioactive FSH levels in CHO cells transfected with the FSH receptor in two groups of women. Group 1 consisted of two patients with an elevated basal serum immunoactive FSH (> 13 mIU/mL), a normal basal antral follicle count (bAFC >8), and a robust response to gonadotropin stimulation (> 10 follicles) during IVF-ET therapy. Group 2 consisted of five patients with elevated basal serum FSH levels and either abnormal bAFC (< 6 total in both ovaries) or poor response to gonadotropin stimulation (< 5 mature follicles).ResultsTabled 1Tabled 1ConclusionsFSH with disproportionately lower bioactivity compared with immunoactivity on assay testing may account for some paradoxically abnormal basal FSH assessments in women with otherwise normal ovarian reserve and response to fertility therapy. Patients with normal antral follicle counts and elevated serum FSH by immunoassays can be candidates for a trial of IVF-ET, and based on our small series, can anticipate a reasonable reproductive outcome. Larger trials are required to assess if a basal bioactive FSH assessment can prospectively predict which of these patients are good IVF-ET candidates, and what cutoff (if any) of FSH bioactivity is most appropriate to use clinically. Presented with an abnormal FSH, but a normal bAFC, bioactivity testing may reassure that ovarian reserve is intact. ObjectiveTo evaluate the bioactivity of FSH in women with paradoxically high serum immunoactive basal FSH levels and otherwise “normal” ovarian reserve as determined by ultrasound screening and response to gonadotropin stimulation. To evaluate the bioactivity of FSH in women with paradoxically high serum immunoactive basal FSH levels and otherwise “normal” ovarian reserve as determined by ultrasound screening and response to gonadotropin stimulation. DesignCase Series at a large academic reproductive center. Case Series at a large academic reproductive center. Materials and methodsWe compared immunoactive basal FSH levels (immulite assay (DPC)) and bioactive FSH levels in CHO cells transfected with the FSH receptor in two groups of women. Group 1 consisted of two patients with an elevated basal serum immunoactive FSH (> 13 mIU/mL), a normal basal antral follicle count (bAFC >8), and a robust response to gonadotropin stimulation (> 10 follicles) during IVF-ET therapy. Group 2 consisted of five patients with elevated basal serum FSH levels and either abnormal bAFC (< 6 total in both ovaries) or poor response to gonadotropin stimulation (< 5 mature follicles). We compared immunoactive basal FSH levels (immulite assay (DPC)) and bioactive FSH levels in CHO cells transfected with the FSH receptor in two groups of women. Group 1 consisted of two patients with an elevated basal serum immunoactive FSH (> 13 mIU/mL), a normal basal antral follicle count (bAFC >8), and a robust response to gonadotropin stimulation (> 10 follicles) during IVF-ET therapy. Group 2 consisted of five patients with elevated basal serum FSH levels and either abnormal bAFC (< 6 total in both ovaries) or poor response to gonadotropin stimulation (< 5 mature follicles). ResultsTabled 1Tabled 1 ConclusionsFSH with disproportionately lower bioactivity compared with immunoactivity on assay testing may account for some paradoxically abnormal basal FSH assessments in women with otherwise normal ovarian reserve and response to fertility therapy. Patients with normal antral follicle counts and elevated serum FSH by immunoassays can be candidates for a trial of IVF-ET, and based on our small series, can anticipate a reasonable reproductive outcome. Larger trials are required to assess if a basal bioactive FSH assessment can prospectively predict which of these patients are good IVF-ET candidates, and what cutoff (if any) of FSH bioactivity is most appropriate to use clinically. Presented with an abnormal FSH, but a normal bAFC, bioactivity testing may reassure that ovarian reserve is intact. FSH with disproportionately lower bioactivity compared with immunoactivity on assay testing may account for some paradoxically abnormal basal FSH assessments in women with otherwise normal ovarian reserve and response to fertility therapy. Patients with normal antral follicle counts and elevated serum FSH by immunoassays can be candidates for a trial of IVF-ET, and based on our small series, can anticipate a reasonable reproductive outcome. Larger trials are required to assess if a basal bioactive FSH assessment can prospectively predict which of these patients are good IVF-ET candidates, and what cutoff (if any) of FSH bioactivity is most appropriate to use clinically. Presented with an abnormal FSH, but a normal bAFC, bioactivity testing may reassure that ovarian reserve is intact.

Circulating Bioactive and Immunoreactive Recombinant Human Follicle-Stimulating Hormone (Org 32489) After Administration to Gonadotropin-Deficient Subjects

Objective: To study the bioactivity of recombinant and urinary human FSH after single IM injection into gonadotropin-deficient subjects. Design: Serum FSH levels were measured by immature rat granulosa cell bioassay and immunofluorometric assay. The isohormone distributions of injected FSH materials were analyzed by chromatofocusing. Serum samples were collected before, and 6, 24, and 72 hours after 300 IU of recombinant or urinary FSH. Volunteers: Fifteen gonadotropin-deficient subjects (8 women and 7 men) received recombinant FSH and 8 of them (4 women and 4 men) received an equal dose of urinary FSH. Result(s): No significant differences were apparent between the bioactive FSH levels after recombinant and urinary FSH treatments (n = 8). The immunoreactive FSH levels at 72 hours after urinary FSH were significantly higher than after recombinant FSH injection with values (median and range) of 3.80 (2.76 to 5.75) IU/L (IRP 78/549) and 3.10 (1.78 to 4.95) IU/L, respectively. There were no significant changes in the bioactive to immunoreactive ratios of FSH within time and between sexes after either recombinant FSH (n = 15) or urinary FSH (n = 8). However, the bioactive to immunoreactive ratio of the FSH material injected and of the post-treatment serum samples were both higher after recombinant FSH than after urinary FSH injection. Chromatofocusing revealed that injected recombinant FSH contained more activity in the basic fractions than urinary FSH. Conclusion(s): Recombinant human FSH maintains its biological activity when injected into gonadotropin-deficient subjects. The bioactive to immunoreactive ratio of recombinant FSH was higher than that of urinary FSH indicating that recombinant FSH contains relatively more basic isohormones, and this finding was strengthened by chromatofocusing.

Gonadotropin requirements of the developing follicle

To evaluate follicular FSH and LH requirements during suppression of endogenous gonadotropins with the GnRH antagonist Nal-Glu and whether LH-like activity could be supplied by administering subcutaneous hCG. Randomized clinical trial. Thirty-two normally cycling females in the late follicular phase (dominant follicle mean diameter > or = 14 mm). Twelve normal women were randomized to receive 150 IU IM FSH with or without 75 IU SC hCG; 11 subjects were randomized to receive 225 IU FSH with or without 50 IU SC hCG; 9 women received 150 or 225 IU IM hMG. Subjects returned the next day for repeat blood sample and ultrasound. Continued follicular maturation, as evidenced by rising E2 levels, correlated with serum immunoactive and bioactive FSH levels and was unrelated to bioactive LH-hCG. Two hundred twenty-five international units of exogenous FSH consistently supported follicular maturation. There was a similar increase in mean follicular diameter in women with an E2 rise versus those with a plateau or fall. In subjects receiving SC mini-dose hCG, serum bioactive LH-hCG levels were increased significantly and were similar to levels before Nal-Glu. During administration of a GnRH-a, the maturing follicle appears to require only FSH support. In markedly hypogonadotropic women, mini-dose hCG may be a more practical alternative to recombinant LH to promote normal follicle maturation.

Effects of corticosterone and testosterone on pituitary gonadotropin content, secretion, bioactivity and messenger RNA levels in the presence or absence of GnRH in male rats

The effects of corticosterone (B) and testosterone (T) on pituitary and serum bioactive and immunoreactive gonadotropins and on gonadotropin hormone subunit messenger RNA levels were compared in the absence of GnRH. Male rats were implanted with pellets of either cholesterol, B or T. At implantation, 2 and 4 days later half of each group received GnRH antagonist and animals were killed 5 days after implantation. As expected, GnRH antagonist lowered bioactive and immunoreactive serum FSH and LH, pituitary FSH, LHβ and FSHβ mRNA. B treatment alone lowered bioactive and immunoreactive serum FSH and immunoreactive serum LH. B reversed the antagonist effect on bioactive and immunoreactive pituitary FSH and FSHβ mRNA. T alone lowered bioactive and immunoreactive serum FSH and LH levels. T reversed the antagonist effect on bioactive and immunoreactive pituitary FSH. T lowered bioactive and immunoreactive pituitary LH and LHβ mRNA and partially reversed the antagonist effect on FSHβ mRNA. The data suggest that either B or T enhance FSH synthesis by acting directly at the gonadotrope, but that B does not affect LH variables to the same extent as T. The results suggest that in stressed animals, when T levels are reduced, B can substitute for T in sustaining FSH synthesis.

Circulating bioactive and immunoreactive recombinant human follicle stimulating hormone (Org 32489) after administration to gonadotropin-deficient subjects

To study the bioactivity of recombinant and urinary human FSH after single IM injection into gonadotropin-deficient subjects. Serum FSH levels were measured by immature rat granulosa cell bioassay and immuno-fluorometric assay. The isohormone distributions of injected FSH materials were analyzed by chromatofocusing. Serum samples were collected before, and 6, 24, and 72 hours after 300 IU of recombinant or urinary FSH. Fifteen gonadotropin-deficient subjects (8 women and 7 men) received recombinant FSH and 8 of them (4 women and 4 men) received an equal dose of urinary FSH. No significant differences were apparent between the bioactive FSH levels after recombinant and urinary FSH treatments (n = 8). The immunoreactive FSH levels at 72 hours after urinary FSH were significantly higher than after recombinant FSH injection with values (median and range) of 3.80 (2.76 to 5.75) IU/L (IRP 78/549) and 3.10 (1.78 to 4.95) IU/L, respectively. There were no significant changes in the bioactive to immunoreactive ratios of FSH within time and between sexes after either recombinant FSH (n = 15) or urinary FSH (n = 8). However, the bioactive to immunoreactive ratio of the FSH material injected and of the post-treatment serum samples were both higher after recombinant FSH than after urinary FSH injection. Chromatofocusing revealed that injected recombinant FSH contained more activity in the basic fractions than urinary FSH. Recombinant human FSH maintains its biological activity when injected into gonadotropin-deficient subjects. The bioactive to immunoreactive ratio of recombinant FSH was higher than that of urinary FSH indicating that recombinant FSH contains relatively more basic isohormones, and this finding was strengthened by chromatofocusing.

Serum Bioactive Luteinizing and Follicle-Stimulating Hormone Concentrations in Girls Increase during Puberty

FSH plays an essential role in folliculogenesis and ovarian growth. However, cross-sectional studies have not shown an increase in bioactive FSH (B-FSH) during puberty. To eliminate intersubject variability, we used a longitudinal design and tested the hypothesis that B-FSH increases during puberty. Thirty normal, healthy girls were enrolled in a longitudinal study from pubertal stages I to IV. The subjects were evaluated at 6-mo intervals; each visit consisted of pubertal staging, bone age determination by x-ray, measurements of serum immunoreactive FSH (I-FSH) and B-FSH (n = 14) or immunoreactive LH (I-LH) and bioactive LH (B-LH) (n = 18), and adrenal and ovarian steroids. All girls had clinical and hormonal characteristics of puberty. Both I-FSH and B-FSH levels were relatively elevated before puberty, whereas serum I-LH and B-LH were low. From pubertal stages I to III, there was a modest yet significant rise in serum I-FSH (p < 0.001) and serum B-FSH (p < 0.01). Serum I-LH and B-LH concentrations showed the expected increases with puberty (p < 0.001), with serum B-LH concentrations exhibiting a greater rise than I-LH (p < 0.001). Our results demonstrate that serum B-FSH and I-FSH increase during puberty. Relatively elevated B-FSH concentrations from early to midpuberty may be an important factor for ovarian growth while circulating LH and estrogen are still low. As puberty progresses, the continued and selective increase in LH induces a rise in estradiol and ultimately leads to ovulation.

CIRCULATING BIOACTIVE FOLLICLE STIMULATING HORMONE AND IMMUNOREACTIVE INHIBIN LEVELS DURING THE NORMAL HUMAN MENSTRUAL CYCLE

To examine the relationship between circulating levels of bioactive FSH (B-FSH) and immunoactive inhibin and oestradiol we studied five women during ovulatory cycles. Daily blood samples were collected from each subject during one menstrual cycle. B-FSH was measured using a modified, highly sensitive in-vitro rat granulosa cell bioassay. The inclusion of IGF-1 (10 micrograms/l) and transferrin (50 mg/l) in the assay system enhanced granulosa cell responsiveness to FSH and resulted in increased assay sensitivity. Inhibin was measured by a heterologous radioimmunoassay (RIA) using an antibody raised against 31 kDa bovine inhibin. Bioactive FSH (B-FSH) levels were closely correlated to those of immunoactive FSH (I-FSH, r = 0.79, P less than 0.001) throughout the cycle. Peak levels of B-FSH were observed during the early follicular phase (day -13, 44.7 +/- 9.6 IU/l, mean +/- SEM) and during the midcycle surge (35.2 +/- 6.2 IU/l); lowest levels occurring during the luteal phase (nadir 3.9 +/- 0.27 IU/l). Plasma oestradiol levels increased significantly during the follicular phase (P less than 0.001) to a peak on day -1 and were negatively correlated with B-FSH during the late follicular phase (day -8 to -1; r = -0.45, P less than 0.02). There was no change in the concentration of inhibin (range 55.3-72.3 U/l) during the follicular phase until day -2 after which an increase to a midcycle peak of 139 +/- 10.6 U/l was observed. No correlation was observed between inhibin and B-FSH during the follicular phase. A second increase in the concentration of inhibin was seen during the luteal phase; peak levels occurred by day 6 (311 +/- 25.8 U/l), remained elevated until day 12, and were negatively correlated with B-FSH (r = -0.53, P less than 0.001). No correlation was observed between oestradiol and inhibin or B-FSH during the luteal phase. We conclude that (1) oestradiol secretion from the growing follicle is primarily responsible for the negative feedback regulation of B-FSH resulting in a change from peak levels in the early follicular phase to basal levels in the late follicular phase; (2) significant and sustained increase in peripheral inhibin concentrations occur mostly during the luteal phase; and (3) regulation of FSH secretion by inhibin occurs primarily in the luteal phase. These results suggest a temporal relationship between oestradiol and inhibin in the negative feedback regulation of FSH in vivo.

BIOACTIVE AND IMMUNOREACTIVE FSH IN SERUM OF NORMAL AND OLIGOSPERMIC MEN

In men suffering from idiopathic oligospermia and azoospermia the hypothesis was tested that decreased spermatozoa production could be due in part to inadequate hormonal stimulation of spermatogenesis. In 53 healthy male subjects with normal spermatozoa production (n = 10), varying degrees of oligospermia (n = 40), and azoospermia (n = 3), serum immunoreactive LH, testosterone (T), and oestradiol (E2), and immunoreactive and bioactive FSH concentrations were determined. FSH bioactivity was estimated using the rat granulosa cell aromatase bioassay. Mean LH, T, and E2 levels were similar in the control group (spermatozoa concentration greater than 40 x 10(6)/ml) compared with men exhibiting mild (10-20 x 10(6)/ml), moderate (5-10 x 10(6)/ml), or severe oligospermia (1-5 x 10(6)/ml), and in the azoospermia group. An inverse correlation was found between immunoreactive FSH levels and spermatozoa concentration (P less than 0.05), with elevated levels (twofold increase) of FSH in the combined severe oligospermia and azoospermia (P less than 0.01) groups. Moreover, augmented (P less than 0.01) bioactive FSH levels were also observed in this group of patients. The mean bioactive to immunoreactive FSH ratio was also negatively correlated with sperm counts (P less than 0.005). In addition, T/LH ratios were inversely correlated with immunoreactive (P less than 0.05) and bioactive (P less than 0.05) FSH, which may indicate that altered Leydig cell function is involved in the augmented secretion of FSH. The data presented in this study indicate that immunoreactive FSH, as measured in oligospermia and azoospermia, does not exhibit decreased bioactivity.

Diagnostic value of bioactive FSH in male infertility

Bioactive FSH and immunoreactive FSH were determined in 193 infertile men and in 23 men with proven fertility using the Sertoli cell aromatase bioassay for bioactive FSH measurement and a two-site fluoroimmunoassay for immunoreactive FSH measurement. Overall bioactive and immunoreactive FSH levels correlated well (r = 0.74, p less than 0.001) but were significantly different from fertile men (bioactive FSH: 6.2 +/- 0.3 U/l; immunoreactive FSH: 4.1 +/- 0.4 U/l) in patients with Klinefelter's syndrome (24.1 +/- 6.1; 26.9 +/- 3.0), non-obstructive azoospermia (25.1 +/- 4.3; 22.2 +/- 4.0), maldescended testes (12.5 +/- 4.6; 14.6 +/- 1.6), and patients with severe oligozoospermia (11.9 +/- 1.2; 11.2 +/- 1.0). Infertile men with moderate oligozoospermia (8.9 +/- 1.5; 8.0 +/- 1.1) and normal sperm counts (9.6 +/- 1.1; 7.6 +/- 1.0) had insignificantly elevated bioactive FSH and immunoreactive FSH levels. Bioactive to immunoreactive FSH ratios were significantly reduced in all patient groups except for patients with normal sperm counts when compared with fertile men. A considerable number of patients exhibited elevated immunoreactive FSH concomitant with normal bioactive FSH levels. We conclude that 1. determination of immunoreactive FSH suffices for classification of patients; 2. bioactive to immunoreactive FSH ratios are reduced in infertile men; 3. some men might secrete immunoreactive FSH with reduced bioactivity.

Bioactive follicle-stimulating hormone responses to intravenous gonadotropin-releasing hormone in boys with idiopathic hypogonadotropic hypogonadism.

To test the hypothesis that exogenous pulsatile administration of GnRH will increase serum bioactive FSH (bFSH) levels, we studied four boys with suspected idiopathic hypogonadotropic hypogonadism (IHH). These boys presumably secreted relatively little GnRH. By virtue of their low baseline serum gonadotropin levels yet responsive pituitary gonadotrophs, these boys with IHH proved to be an excellent clinical model to test this hypothesis. Administration of GnRH (0.025 microgram/kg.dose) iv at 1- or 2-h intervals for 3-5 days resulted in an increase in serum bFSH after 91% of the GnRH doses. Serum immunoreactive FSH (iFSH) and LH (iLH) levels increased after 42% and 64% of the GnRH doses, respectively. Ninety percent of the iLH responses were concordant with bFSH responses, but only 33% of the iLH responses were concordant with iFSH responses. The serum bFSH responses occurred consistently within 20 min after GnRH administration and resulted in an increased serum bioactive to immunoreactive FSH ratio. By 60 min, serum bFSH levels had returned to preinjection levels. Serum testosterone and estradiol levels did not change during the period of GnRH administration in three of the four boys. We conclude that pulsatile, low dose iv GnRH administration in boys with IHH elicits significant serum bFSH increases by 20 min; the newly secreted FSH is preferentially enriched with increased in vitro FSH bioactivity, and it is rapidly cleared from serum (60 min). Therefore, serum bFSH measurements may provide a sensitive index of GnRH effects on the gonadotrophs.

Decreased serum inhibin levels in normal elderly men: evidence for a decline in Sertoli cell function with aging.

Compared to young men, normal elderly men have decreased sperm production despite elevated serum gonadotropin levels. To determine whether the seminiferous tubule defect in elderly men includes decreased Sertoli cell function, we measured serum immunoreactive inhibin concentrations in young and elderly men before and after clomiphene citrate (CC) administration. Thirty-eight healthy men, 19 young (aged 22-35 yr) and 19 elderly (aged 65-85 yr), were studied before CC administration. The mean baseline serum inhibin level was significantly lower (P less than 0.001) in the elderly men than in the young men [416 +/- 22 (+/- SE) vs. 588 +/- 30 U/L], while serum immunoreactive FSH and LH levels were higher in the older men, and bioactive FSH levels were similar in the two age groups. Eleven young men and 13 elderly men were studied after 1 week of CC administration. The mean serum inhibin level increased by 71%, from 566 +/- 36 to 970 +/- 82 U/L, in the young men, but it increased by only 24%, from 421 +/- 26 to 520 +/- 38 U/L, in the elderly men. Serum immunoreactive LH and bioactive and immunoreactive FSH concentrations increased to similar levels in both groups after CC administration. We conclude that the seminiferous tubule defect of elderly men includes decreased Sertoli cell function.

Maintenance of the ratio of bioactive to immunoreactive follicle-stimulating hormone in normal men during chronic luteinizing hormone-releasing hormone agonist administration.

Chronic administration of LHRH agonist analogs to humans reduces gonadal function through pituitary desensitization. Serum immunoreactive gonadotropin levels are modestly reduced, whereas serum bioactive LH levels are drastically suppressed. The effects on bioactive FSH levels, however, are not known. In this study, serum bioactive FSH was measured using an in vitro granulosa cell aromatase bioassay in four normal men given a LHRH agonist, [D-Trp6,Pro9-NEt]LHRH (LHRHA; 500 micrograms/day for 16 weeks), by sc infusion and testosterone enanthate (TE; 100 mg, im every 2 weeks) and in five men given 500 micrograms/day LHRHA by daily sc injection for 20 weeks and TE (100 mg every 2 weeks) from weeks 10 through 20. During the first study, serum immunoreactive FSH levels (IR-FSH) decreased by 56.5 +/- 4.8% (+/- SEM), and serum bioactive FSH (Bio-FSH) level decreased by 57.6 +/- 6.4%. The ratio of Bio-FSH to IR-FSH did not change. During the second study, both serum IR-FSH and Bio-FSH levels followed a triphasic pattern, decreasing slightly but not significantly immediately after initiation of LHRHA administration, progressively increasing to a peak (P less than 0.5 vs, baseline) at week 10, and then, after addition of TE to this regimen, decreasing slightly again. The Bio-FSH to IR-FSH ratio, as in the first study, did not change. When serum obtained at week 10 during the second study, just before initiation of TE, was chromatographed on a Sephadex G-100 column, IR-LH eluted in two distinct peaks, while IR-FSH eluted as a single peak. These results demonstrate that in normal men chronic LHRHA administration alone for up to 10 weeks or LHRHA plus TE for up to 16 weeks does not alter the qualitative characteristics of secreted FSH, since there was no dissociation between serum IR- and Bio-FSH levels.

Serum bioactive and immunoreactive follicle-stimulating hormone in prostatic cancer patients during gonadotropin-releasing hormone agonist treatment and after orchidectomy.

Serum bioactive and immunoreactive FSH levels were measured in five prostatic cancer patients during treatment for 6 months with the GnRH agonist analog buserelin (Hoechst; 600 micrograms, intranasally, 3 times per day) and for up to 12 weeks after subsequent orchidectomy. FSH bioactivity was measured using a sensitive specific in vitro granulosa cells aromatase bioassay. Before buserelin treatment, mean serum FSH bioactivity and immunoreactivity were 19.7 +/- 4.1 (+/- SE) IU/L (n = 5) and 13.7 +/- 3.8 IU/L, respectively, with a bioactivity to immunoactivity (B/I) ratio of 1.7 +/- 0.2. After the initiation of treatment with the GnRH agonist, FSH bio- and immunoactivities both transiently increased for 1-3 days. The increase in bioactivity was greater and prolonged, and the B/I ratio increased nearly 7-fold in 2 weeks. Serum FSH immunoreactivity declined to below the pretreatment level in 5 days and remained low for the rest of the treatment period. In contrast, serum FSH bioactivity did not decrease significantly below the pretreatment level during the 6-month treatment period, although the B/I ratio returned slowly toward the pretreatment value. After orchidectomy, both FSH activities increased dramatically, and the B/I ratio rose transiently from 1.5 to 7 in 2 weeks. Interestingly, serum FSH bioactivity and immunoreactivity decreased significantly (P less than 0.05) 1 day after orchidectomy in the buserelin-treated patients. In contrast, serum FSH immunoreactivity increased during the same period (P less than 0.05) in patients treated only by orchidectomy (FSH bioactivity was not measured). In conclusion, serum FSH bioactivity increases acutely more than FSH immunoreactivity after initiation of GnRH agonist treatment or orchidectomy. In the former case, serum FSH bioactivity subsequently returned to the pretreatment range. A clear decline during long term agonist treatment occurred only in serum FSH immunoreactivity, in contrast to the concomitant decline in serum LH bio- and immunoreactivities reported previously. The persistence of bioactive FSH may explain the inconsistent effects of GnRH agonist treatment on the suppression of spermatogenesis. The acute decrease in serum FSH after orchidectomy in the buserelin-treated men suggests that the testes may produce a factor that stimulates pituitary FSH secretion.

The dependency of bioactive follicle-stimulating hormone secretion on gonadotropin-releasing hormone in hypogonadal and cycling women.

An antagonist analog of GnRH, (Ac-delta 3-Pro1,p-F-D-Phe2,D-Trp3,6)GnRH (4F-antagonist), was administered to normal women and women with hypergonadotropic hypogonadism. Serum FSH levels were determined by both the granulosa cell aromatase bioassay and RIA. The constant infusion of 4F-antagonist (30 micrograms/kg.h) to the four hypogonadal women resulted in a more pronounced decline in bioactive FSH (62%) than in immunoreactive FSH levels (30%), and the FSH bioactive to immunoreactive ratio decreased significantly (P less than 0.05). Infusion of 4F-antagonist in normal women in the midfollicular phase revealed a similar pattern of suppression of bioactive (64%) and immunoreactive FSH (29%). When 4F-antagonist was administered sc at a dose of 80 micrograms/kg twice daily for 3 days to normal women in the midfollicular phase of their cycles, the bioactive FSH response was biphasic, with the maximal decrease on the second day, followed by return to basal levels on the third day. Correspondingly, there was a precipitous decline in serum estradiol (apparent demise of the dominant follicle), followed by a progressive rise in estradiol levels. Thus, in contrast to immunoreactive FSH levels, bioactive FSH more clearly reflects the biological action of FSH on the follicle in response to GnRH antagonist administration in women. The disparity in the quantitative decline between serum bioactive and immunoreactive FSH levels after presumed blockade of the GnRH receptor may reflect the microheterogeneity of the FSH molecule and suggests that alterations in the biological activity of secreted FSH may be GnRH dependent.

Serum bioactive follicle-stimulating hormone in men with idiopathic azoospermia and oligospermia.

Using an in vitro granulosa cell aromatase bioassay (GAB), serum bioactive FSH (bio-FSH) levels were measured in 20 fertile men and 74 men with idiopathic azoospermia or oligospermia. The serum bio-FSH levels measured by the GAB assay and the immunoreactive FSH (immuno-FSH) levels measured by RIA were positively correlated (r = 0.93). Compared to normal men, serum bio-FSH and immuno-FSH levels were elevated in patients with idiopathic azoospermia associated with severe germinal epithelium damage; the bioactive to immunoreactive ratio (B:I ratio) of FSH in these men [mean, 1.5 +/- 0.5 (+/- SD)] was significantly lower than that in fertile men (2.7 +/- 0.8). Similarly, in men with moderate and severe oligospermia, the B:I ratios of FSH were decreased (1.4 +/- 0.4 and 1.7 +/- 0.3, respectively). Although serum immuno-FSH levels correlated weakly with mean sperm concentrations in the normal and oligospermic men (r = -0.35), no relationship was found between serum bio-FSH and sperm concentrations. The B:I ratio of FSH correlated weakly with sperm concentration (r = 0.46). These findings suggest that the B:I ratio of FSH measured by the GAB assay decreases in patients with low sperm concentrations and germinal cell failure.

Serum bioactive and immunoreactive follicle-stimulating hormone levels and the response to clomiphene in healthy young and elderly men.

Testicular function declines with normal aging, while serum immunoreactive LH and FSH levels increase. Since there are reports of an age-related decrease in the ratio of bioactivity to immunoreactivity (B/I ratio) for LH, we used a newly available bioassay for FSH to assess age-associated changes in the bioactivity and B/I ratio of FSH in man. Thirty-nine healthy men (23 young and 16 elderly) had single blood samples drawn. In addition, a subset of these men (12 young and 13 elderly) underwent frequent blood sampling for 24 h, both before and after 7 days of clomiphene citrate (CC) administration. Hourly blood samples from the 24-h sampling were pooled, and these, along with the single samples, were assayed for FSH by an in vitro bioassay system, using estrogen production by immature rat granulosa cells as the end point, and by RIA. Baseline single sample mean FSH, as measured by bioassay, was similar in young and elderly men [386 +/- 98 (+/- SEM) and 342 +/- 77 ng/mL, respectively]. Baseline mean FSH, measured by RIA, was significantly higher (P less than 0.001) in elderly men (234 +/- 31 ng/mL) than in young men (122 +/- 12 ng/mL). The baseline FSH B/I ratio based on single sampling was significantly lower (P less than 0.01) in elderly men (1.4 +/- 0.2) than in young men (2.7 +/- 0.3). In the men given CC and sampled for 24 h, mean bioactive FSH levels increased significantly in both the young (1180 +/- 282 ng/mL) and the elderly (992 +/- 227 ng/mL; P less than 0.01 for both values compared to baseline). Mean FSH by RIA also increased to similar levels in these young (217 +/- 34 ng/mL) and elderly (258 +/- 45 ng/mL) men. The FSH B/I ratio was 4.8 +/- 0.8 in young and 4.7 +/- 1.1 in elderly men after CC administration. We conclude that serum bioactive FSH levels are similar in elderly and young men, suggesting that the age-related decline in testicular function in man cannot be explained by a chronic deficiency in FSH stimulation; elderly men have a lower serum FSH B/I ratio than young men, which may reflect changes in the circulating form of FSH with aging; and administration of CC to young and elderly men increases both bioactive and immunoreactive serum FSH, implying preserved hypothalamic-pituitary responsiveness in the elderly.

Estrogen-producing ovarian granulosa cells: use of the granulosa cell aromatase bioassay (GAB) to monitor FSH levels in body fluids.

FSH is a key inducer of ovarian follicular and testicular tubule developments. During reproductive life, FSH is released in an orchestrated fashion subject to endocrine control. However, the microheterogeneity of the FSH molecule suggests that studies on circulating FSH bioactivity are essential to understanding gonadal development and function. The study of potential paracrine modulators (estrogens, androgens and growth factors) that could enhance FSH action in granulosa cells and therefore have potential physiologic roles in the development of the dominant follicle led to the development of an in vitro bioassay for FSH. The combined action of enhancing hormones and factors resulted in a granulosa cell assay highly sensitive to the aromatase-inducing action of FSH. This assay is hormone-specific and sensitive, and has been applied to the measurement of FSH bioactivity in serum samples through the use of pre-treatment of serum with polyethylene glycol to remove substances inhibitory in the granulosa cell culture. Recent applications of this assay to human and animal serum and urine samples reveal that physiologic levels of FSH can be measured by this assay. Although bioactive FSH levels in serum and urine samples throughout the normal menstrual cycle generally reflect changes in immunoreactive FSH in serum, substantial decreases in B/I ratio of serum FSH were detected in aging men and normal males treated with a GnRH antagonist. Further applications of the Granulosa Cell Aromatase Bioassay (GAB) in different animals species as well as to other physiologic and pathophysiologic states in humans should be rewarding.

Granulosa Cell Aromatase Bioassay for Follicle-Stimulating Hormone: Validation and Application of the Method*

Stimulation of rat granulosa cell aromatase activity by FSH has recently been used as a sensitive biological end point to develop an in vitro FSH bioassay. The present report provides a detailed validation and application of this assay. In the presence of androstenedione and diethylstilbestrol, FSH stimulated estrogen production in a dose-dependent manner. Although addition of high doses of a phosphodiesterase inhibitor [1-methyl-3-isobutyl xanthine (MIX)] decreased maximal estrogen production, treatment with 0.125 mM MIX increased the sensitivity of granulosa cells to FSH, presumably by minimizing endogenous cAMP breakdown. Addition of insulin and human CG (hCG) further synergistically enhanced granulosa cell sensitivity to FSH. Although inclusion of gonadotropin-free serum obtained from hypophysectomized male rats decreased the assay sensitivity, pretreatment of serum with polyethylene glycol [(PEG) 10-14%] resulted in a dose-dependent decrease in the serum-interfering effect. Studies using exogenous [125I]iodo-rat FSH or RIA measurement indicated recovery of 94-98% FSH after pretreatment of serum with 12% PEG. In the presence of the PEG-pretreated gonadotropin-free serum (4%), ovine, rat, and human FSH preparations induced parallel dose-response curves for estrogen production with minimal detectable doses of 0.12 ng, 0.12 ng, and 0.12 mIU/culture, respectively. In contrast, treatment with GH, PRL, TSH, and ACTH did not affect estrogen production. The apparent stimulatory effect of high doses (greater than 60 ng/culture) of LH and hCG could be attributed to FSH contamination or intrinsic FSH activity in these preparations. Changes in serum bioactive FSH levels were studied in adult male rats after GnRH administration. GnRH (5 micrograms/rat) treatment significantly elevated FSH levels within 30 min after injection. Maximal increases (approximately 2.8-fold) in serum bioactive FSH were observed between 60-120 min. At 8 h after treatment, FSH levels decreased to control levels. Comparison between granulosa cell aromatase bioassay and RIA results indicated no apparent changes in the bio- to immuno- ratio of FSH after GnRH treatment. In conclusion, extreme sensitivity of the bioassay allows the measurement of circulating levels of bioactive FSH. Since rat granulosa cells respond to FSH preparations from different species, the in vitro assay should also provide valuable information on FSH levels in many animal species including those lacking a specific RIA. Measurement of serum levels of bioactive FSH should provide insight regarding the role of FSH in various physiological and pathological conditions.

Serum bioactive follicle-stimulating hormone during the human menstrual cycle and in hyper- and hypogonadotropic states: application of a sensitive granulosa cell aromatase bioassay.

A sensitive in vitro assay based on the stimulation of estrogen production by cultured rat granulosa cells was recently developed for the measurement of biologically active FSH. This bioassay system is specific for FSH, highly sensitive, and capable of measuring basal FSH levels in rat serum. The granulosa cell aromatase bioassay was improved by the use of additives known to enhance FSH activity and by pretreatment of serum with 12% polyethylene glycol to remove inhibitory substances. We applied this method to the measurement of bioactive FSH levels in serum samples from human subjects. As determined in daily blood samples during ovulatory menstrual cycles in seven women, bioactive FSH levels exhibited a pattern closely resembling that of immunoreactive FSH. The mean bioactive serum FSH levels were 29.9, 20.5, 39.2, and 14.8 mIU/ml for the early follicular phase, late follicular phase, preovulatory surge, and luteal phase, respectively. The bio- to immunoratio (B:I) throughout the menstrual cycle ranged from 1.4-3.4, with a mean of 2.5. The ratios for early follicular phase, late follicular phase, preovulatory surge, and luteal phase were 2.7, 2.3, 1.4, and 2.6, respectively. The correlation coefficient (r) of the serum FSH values obtained by bioassay and RIA was 0.91. FSH bioactivity was also measured in patients in each of the following categories with the following mean values: oral contraceptive pill users (undetectable), hypothalamic amenorrhea (18.7 mIU/ml; B:I, 2.6), premature ovarian failure (163 mIU/ml; B:I, 1.7), and postmenopausal women (191 mIU/ml; B:I, 1.6). These findings suggest that measurement of immunoreactive FSH levels correctly reflects the biological activity of FSH in serum of cycling women and patients in certain hyper- and hypogonadotropic states. The granulosa cell aromatase bioassay represents a new tool for future assessments of biologically active FSH in physiological and pathophysiological conditions.