Testosterone, free testosterone, and free androgen index in women: Reference intervals, biological variation, and diagnostic value in polycystic ovary syndrome.

OBJECTIVE: The objective of our study was to determine reference intervals and biologic variation for testosterone (T), free testosterone (fT), and free androgen index (FAI) in women with accurate methods and to test the discriminative value of these parameters in a polycystic ovary syndrome (PCOS)-population. METHODS: Serum was obtained daily during a normal menstrual cycle from 25 healthy women (677 data-points). A single serum sample was obtained from 44 PCOS-patients. T was measured by LC­MS/MS and by Architect® 2nd generation T Immunoassay. Sex hormone-binding globulin was measured to calculate fT and FAI. Results: Reference intervals which were established in healthy women with an ovulatory menstrual cycle were T = 0.3-1.6 nmol/L and 0.5-2.0 nmol/L, fT = 5.2-26 pmol/L and 7.2-33 pmol/L, and FAI = 0.4-2.9 and 0.6-4.4, by LC-MS/MS and immunoassay, respectively. T, fT and FAI were higher in PCOS patients than in controls (p b 0.0001). The areas under the curve of receiver operator characteristic (ROC) plots were not different for T, fT, or FAI when T was measured by LC­MS/MS versus immunoassay based on prediction of PCOS. FAI and fT were the strongest predictors of PCOS. CONCLUSIONS: When based upon the appropriate reference intervals and ROC analysis, LC-MS/MS and second generation immunoassay have equivalent clinical utility for the diagnosis of PCOS.

Compensatory Increase in Ovarian Aromatase in Older Regularly Cycling Women.

CONTEXT: Serum estradiol (E2) levels are preserved in older reproductive-aged women with regular menstrual cycles despite declining ovarian function. OBJECTIVE: The objective of the study was to determine whether increased granulosa cell aromatase expression and activity account for preservation of E2 levels in older, regularly cycling women. DESIGN: The protocol included daily blood sampling and dominant follicle aspirations at an academic medical center during a natural menstrual cycle. SUBJECTS: Healthy, regularly cycling older (36-45 y; n = 13) and younger (22-34 y; n = 14) women participated in the study. MAIN OUTCOME MEASURES: Hormone levels were measured in peripheral blood and follicular fluid aspirates and granulosa cell CYP19A1 (aromatase) and FSH-R mRNA expression were determined. RESULTS: Older women had higher FSH levels than younger women during the early follicular phase with similar E2 but lower inhibin B and antimullerian hormone levels. Late follicular phase serum E2 did not differ between the two groups. Follicular fluid E2 [older (O) = 960.0 [interquartile range (IQR) 765.0-1419.0]; younger (Y) = 994.5 [647.3-1426.5] ng/mL, P = 1.0], estrone (O = 39.6 [29.5-54.1]; Y = 28.8 [22.5-42.1] ng/mL, P = 0.3), and the E2 to testosterone (T) ratio (O = 109.0 ± 41.9; Y = 83.0 ± 18.6, P = .50) were preserved in older women. Granulosa cell CYP19A1 expression was increased 3-fold in older compared with younger women (P < .001), with no difference in FSH-R expression. CONCLUSIONS: Ovarian aromatase expression increases with age in regularly cycling women. Thus, up-regulation of aromatase activity appears to compensate for the known age-related decrease in granulosa cell number in the dominant follicle to maintain ovarian estrogen production in older premenopausal women.

Evidence that increased ovarian aromatase activity and expression account for higher estradiol levels in African American compared with Caucasian women.

CONTEXT: Serum estradiol levels are significantly higher across the menstrual cycle in African American (AAW) compared with Caucasian women (CW) in the presence of similar FSH levels, yet the mechanism underlying this disparity is unknown. OBJECTIVE: The objective of the study was to determine whether higher estradiol levels in AAW are due to increased granulosa cell aromatase mRNA expression and activity. DESIGN: The design of the study included daily blood sampling and dominant follicle aspirations at an academic medical center during a natural menstrual cycle. SUBJECTS: Healthy, normal cycling AAW (n = 15) and CW (n = 14) aged 19-34 years participated in the study. MAIN OUTCOME MEASURES: Hormone levels in peripheral blood and follicular fluid (FF) aspirates and aromatase and FSH receptor mRNA expression in granulosa cells were measured. RESULTS: AAW had higher FF estradiol [1713.0 (1144.5-2032.5) vs 994.5 (647.3-1426.5) ng/mL; median (interquartile range); P < .001] and estrone [76.9 (36.6-173.4) vs 28.8 (22.5-42.1) ng/mL; P < .001] levels than CW, independent of follicle size. AAW also had lower FF androstenedione to estrone (7 ± 1.8 vs 15.8 ± 4.1; mean ± SE; P = .04) and T to estradiol (0.01 ± 0.002 vs 0.02 ± 0.005; P = .03) ratios, indicating enhanced ovarian aromatase activity. There was a 5-fold increase in granulosa cell aromatase mRNA expression in AAW compared with CW (P < .001) with no difference in expression of FSH receptor. FSH, inhibin A, inhibin B, and AMH levels were not different in AAW and CW. CONCLUSIONS: Increased ovarian aromatase mRNA expression, higher FF estradiol levels, and decreased FF androgen to estrogen ratios in AAW compared with CW provide compelling evidence that racial differences in ovarian aromatase activity contribute to higher levels of estradiol in AAW across the menstrual cycle. The absence of differences in FSH, FSH receptor expression, and AMH suggest that population-specific genetic variation in CYP19, the gene encoding aromatase, or in factors affecting its expression should be sought.

Estrogen levels are higher across the menstrual cycle in African-American women compared with Caucasian women.

CONTEXT: Previous studies have suggested that estrogen levels may be higher in African-American women (AAW) compared with Caucasian women (CW), but none have systematically examined estrogen secretion across the menstrual cycle or in relation to other reproductive hormones. OBJECTIVE: The objective of the study was to compare estradiol (E2), progesterone (P), gonadotropins, androstenedione (a'dione), inhibins, and SHBG levels between AAW and CW across the menstrual cycle. DESIGN, SETTING, AND SUBJECTS: Daily blood samples were collected from regularly cycling AAW (n = 27) and CW (n = 27) for a full menstrual cycle, and serial ultrasounds were performed. MAIN OUTCOME MEASURES: Comparison of E2, P, LH, FSH, SHBG, inhibin A, inhibin B, and a'dione levels. RESULTS: AAW and CW were of similar age (27.2 ± 0.6 yr, mean ± sem) and body mass index (22.7 ± 0.4 kg/m(2)). All subjects grew a single dominant follicle and had comparable cycle (25-35 d) and follicular phase (11-24 d) lengths. E2 levels were significantly higher in AAW compared with CW (P = 0.02) with the most pronounced differences in the late follicular phase (225.2 ± 14.4 vs. 191.5 ± 10.2 pg/ml; P = 0.02), midluteal phase (211.9 ± 22.2 vs.150.8 ± 9.9, P < 0.001), and late luteal phase (144.4 ± 13.2 vs. 103.5 ± 8.5, P = 0.01). Although LH, FSH, inhibins A and B, P, a'dione, and SHBG were not different between the two groups, the a'dione to E2 ratio was lower in AAW (P < 0.001). CONCLUSIONS: Estradiol is higher in AAW compared with CW across the menstrual cycle. Higher estradiol in the face of similar androstenedione and FSH levels suggests enhanced aromatase activity in AAW. Such differences may contribute to racial disparities in bone mineral density, breast cancer, and uterine leiomyomas.

Maternal, prenatal and perinatal characteristics and first trimester maternal serum hormone concentrations.

In uncomplicated pregnancies, first trimester androgen, oestrogen and prolactin concentrations were higher in nulliparous (n=160) than parous (n=260) mothers. Androgens and estrogens were higher in younger than older mothers. These data are consistent with elevated hormone concentrations mediating the breast cancer protection from a first pregnancy and pregnancies occurring at younger ages.

Androgens may mediate a relative preservation of IGF-I levels in overweight and obese women despite reduced growth hormone secretion.

CONTEXT: Obesity is characterized by reduced GH secretion, but data regarding IGF-I levels and their determinants are conflicting. OBJECTIVES: The objectives were to determine whether IGF-I levels are reduced and to investigate determinants of GH and IGF-I in healthy overweight and obese women. DESIGN: A cross-sectional study was performed. SETTING: The study was conducted at a General Clinical Research Center. STUDY PARTICIPANTS: Thirty-four healthy women without pituitary/hypothalamic disease participated, including 11 lean [body mass index (BMI) <25 kg/m(2)], 12 overweight (BMI > or =25 kg/m(2) and <30 kg/m(2)), and 11 obese (BMI > or =30 kg/m(2)) women of comparable age (overall mean age, 30.7 +/- 7.8 yr). INTERVENTION: There was no intervention. MAIN OUTCOME MEASURES: The main outcome measures were frequent sampling (every 10 min for 24 h) for GH, peak GH after GHRH-arginine stimulation, IGF-I, IGF binding protein-3, estrone, estradiol, testosterone, free testosterone, SHBG, homeostasis model assessment of insulin resistance, and abdominal fat. RESULTS: Mean 24-h GH and peak stimulated GH were lower in overweight than lean women and lowest in obese women. Mean IGF-I levels trended lower in obese, but not overweight, compared with lean women. Free testosterone was positively associated with IGF-I (R = 0.36, P = 0.04) but not with GH measures. Visceral fat was the only determinant of mean 24-h GH (R(2) = 0.66, P < 0.0001) and of peak stimulated GH (R(2) = 0.63, P < 0.0001), and mean 24-h GH accounted for 39% of the variability of IGF-I (P = 0.0002), with an additional 28% (P < 0.0001) attributable to free testosterone levels. CONCLUSIONS: Despite a linear decrease in GH secretion and peak stimulated GH levels with increasing BMI in healthy overweight and obese women, IGF-I levels were not commensurately reduced. Androgens may contribute to this relative preservation of IGF-I secretion in overweight and obese women despite reduced GH secretion.

Breed differences in clearance of porcine FSH in hypophysectomized rats.

Extracts of anterior pituitary (AP) glands were infused i.v. into hypophysectomized male rats followed by sequential sampling of blood for 120 min. Determination of follicle-stimulating hormone (FSH) concentrations established that FSH from Chinese Meishan males decreased in the circulation of rats more slowly than FSH in extracts of AP from crossbred occidental pigs (P<0.003). Additionally, FSH from AP extracts of castrated males disappeared somewhat more slowly (P<0.06) than FSH from extracts of boars. Evaluation of FSH by bioassay and radioimmunoassay yielded similar concentrations in AP from Meishan and crossbred boars. Serum testosterone concentrations increased with time through 90 min after infusion of AP, but the rate of increase of testosterone was not related to amount of luteinizing hormone (LH) that was administered indicating LH receptor saturation. Unexpectedly, the rate of increase in testosterone was more rapid with AP extracts from boars than with extracts from castrated males. Observations from the current study imply structural alterations of FSH in the AP of Meishan males relative to crossbred males allowing sustained concentrations in the circulation, and this FSH possesses similar activation of the FSH receptor. The amount of LH in the AP extracts saturated the LH receptors of the hypophysectomized male rats, but some factor in extracts of boars differed from those of castrated males.

Control of estradiol secretion in reproductive ageing.

BACKGROUND: Estradiol (E(2)) concentration is preserved in older reproductive-aged women despite a decrease in follicle number and androstenedione (AD) levels. We hypothesized that increased aromatase activity accounts for E(2) preservation in older women. METHODS: Older (36-46 years; n = 11) and younger (21-35 years; n = 10) women with 25- to 35-day menstrual cycles participated in a parallel design study. Daily blood samples were drawn starting at menses, and recombinant human FSH (rhFSH), 150 IU, was administered when the dominant follicle's diameter was > or =16 mm. FSH, LH, E(2), estrone (E(1)), AD and the AD/E(1) ratio were compared. RESULTS: E(2) and E(1) concentrations and the E(1)/E(2) ratio were similar across the follicular phase in older compared with younger women, whereas AD and the AD/E(1) ratio were lower. Older women had higher FSH concentrations in the early follicular phase and fewer small follicles. RhFSH-stimulated changes in E(1) were similar between older and younger subjects despite the smaller number of follicles. CONCLUSIONS: These findings suggest that E(2) secretion is maintained by increased aromatase function in older compared with younger reproductive-aged women, whereas there is no apparent difference in 17beta-hydroxysteroid dehydrogenase activity. The increased aromatase is probably driven by increased FSH in the early follicular phase and compensates for the decreased follicle number in older reproductive-aged women.

Serum prolactin and TSH in an in vitro fertilization population: is there a link between fertilization and thyroid function?

PURPOSE: Measurements of TSH and prolactin are generally included in the evaluation of female infertility, but their value in women coming to in vitro fertilization (IVF) has been questioned. METHODS: In this study, we sought to investigate whether prolactin or TSH, measured in 509 specimens collected prior to therapy, predicted outcome in a prospective study of couples undergoing IVF between 1994 and 2001. RESULTS: TSH was higher in women whose fertility problem was attributed to a male factor, and prolactin was lower if the measurement was taken during menses. TSH and prolactin were positively correlated (p < 0.0001). Neither TSH nor prolactin levels correlated with overall IVF outcome; however, TSH levels were significantly higher among women who produced oocytes that failed to be fertilized and this finding persisted after adjustment for several covariates, including sperm motility. Among women who had a least one oocyte inseminated, the likelihood that they would have fewer than 50% of their eggs fertilized was significantly related to higher TSH levels in a multivariate model. CONCLUSION: We conclude that TSH may predict poor fertilization in IVF and reflect the importance of thyroid hormones in oocyte physiology.

Early follicular phase hormone levels in relation to patterns of alcohol, tobacco, and coffee use.

OBJECTIVE: To examine the effects of alcohol, caffeine, and tobacco use on early follicular phase FSH, LH, E2, and sex hormone-binding globulin (SHBG). DESIGN: Cross-sectional study. SETTING: Academic medical center. PATIENT(S): Four hundred ninety-eight women selected from the general population, ages 36-45, who were not currently pregnant, breast feeding, or using exogenous hormones. INTERVENTION(S): A general questionnaire assessing demography, anthropometry, and smoking habits and a standardized dietary questionnaire assessing food and beverage frequencies, including sources of alcohol and caffeine. MAIN OUTCOME MEASURE(S): FSH, LH, E2, and SHBG levels measured during the early follicular phase of the menstrual cycle. RESULT(S): Significant associations observed in a univariate analysis included age > or =40 and current smoking associated with higher FSH; higher body mass index (BMI) associated with lower SHBG levels; and daily alcohol use, cholesterol consumption greater than the median, and coffee use >1 cup/d associated with higher E2 levels. In a multivariate model, total caffeine use was significantly associated with E2 levels after adjustment for age, BMI, total calories, current smoking, alcohol, cholesterol consumption, and day of sampling. Early follicular phase E2 increased from 28.2 pg/mL for women consuming < or =100 mg of caffeine to 45.2 pg/mL for women consuming > or =500 mg of caffeine per day, about a 70% increase. CONCLUSION(S): Coffee consumption and total caffeine use may increase early follicular phase E2 levels independent of related habits of alcohol or tobacco use.

Müllerian Inhibiting Substance lowers testosterone in luteinizing hormone-stimulated rodents.

Müllerian Inhibiting Substance (MIS) expression is inversely proportional to the serum concentration of testosterone in males after birth and in vitro studies have shown that MIS can lower testosterone production by Leydig cells. Also, mice overexpressing MIS exhibited Leydig cell hypoplasia and lower levels of serum testosterone, but it is not clear whether this is a result of MIS affecting the development of Leydig cells or their capacity to produce testosterone. To examine the hypothesis that MIS treatment will result in decreased testosterone production by mature Leydig cells in vivo, we treated luteinizing hormone (LH)-stimulated adult male rats and mice with MIS and demonstrated that it can lead to a several-fold reduction in testosterone in serum and in testicular extracts. There was also a slight decrease in 17-OH-progesterone compared to the more significant decrease in testosterone, suggesting that MIS might be regulating the lyase activity of cytochrome P450c17 hydroxylase/lyase (Cyp17), but not its hydroxylase activity. Northern analysis showed that, in both MIS-treated rats and mice, the mRNA for Cyp17, which catalyzes the committed step in androgen synthesis, was down-regulated. In rats, the mRNA for cytochrome P450 side-chain cleavage (P450scc) was also down-regulated by MIS. This was not observed in mice, indicating that there might be species-specific regulation by MIS of the enzymes involved in the testosterone biosynthetic pathway. Our results show that MIS can be used in vivo to lower testosterone production by mature rodent Leydig cells and suggest that MIS-mediated down-regulation of the expression of Cyp17, and perhaps P450scc, contributes to that effect.

Follistatin: essential role for the N-terminal domain in activin binding and neutralization.

Follistatin is recognized to be an important regulator of cellular differentiation and secretion through its potent ability to bind and bioneutralize activin with which it is colocalized in many tissue systems. The 288-residue follistatin molecule is comprised of a 63-residue N-terminal segment followed by three repeating 10-cysteine "follistatin domains" also represented in several extracellular matrix proteins. We have used chemical modifications and mutational analyses to define structural requirements for follistatin bioactivity that previously have not been investigated systematically. Mutant follistatins were stably expressed from Chinese hamster ovary cell cultures and assayed for activin binding in a solid-phase competition assay. Biological activities were determined by inhibition of activin-mediated transcriptional activity and by suppression of follicle-stimulating hormone secretion by cultured anterior pituitary cells. Deletion of the entire N-terminal domain, disruption of N-terminal disulfides, and deletion of the first two residues each reduced activin binding to <5 % of expressed wild-type follistatin and abolished the ability of the respective mutants to suppress activin-mediated responses in both bioassay systems. Hence, the three follistatin domains inherently lack the ability to bind or neutralize activin. Activin binding was impaired after oxidation of at least one tryptophan, at position 4, in FS-288. Mutation of Trp to Ala or Asp at either positions 4 or 36 eliminated activin binding and bioactivity. Mutation of a third hydrophobic residue, Phe-52, reduced binding to 20%, whereas substitutions for the individual Lys and Arg residues in the N-terminal region were tolerated. These results establish that hydrophobic residues within the N-terminal domain constitute essential activin-binding determinants in the follistatin molecule. The correlation among the effects of mutation on activin binding, activin transcriptional responses, and follicle-stimulating hormone secretion substantiates the concept that, at least in the pituitary, the biological activity of follistatin is attributable to its ability to bind and bioneutralize activin.

Analysis of human follistatin structure: identification of two discontinuous N-terminal sequences coding for activin A binding and structural consequences of activin binding to native proteins.

A primary physiological function of follistatin is the binding and neutralization of activin, a transforming growth factor-beta family growth factor, and loss of function mutations are lethal. Despite the critical biological importance of follistatin's neutralization of activin, the structural basis of activin's binding to follistatin is poorly understood. The purposes of these studies were 1) to identify the primary sequence(s) within the N-terminal domain of the follistatin coding for activin binding, and 2) to determine whether activin binding to the native protein causes changes in other structural domains of follistatin. Synthetic peptide mimotopes identified within a 63-residue N-terminal domain two discontinuous sequences capable of binding labeled activin A. The first is located in a region (amino acids 3-26) of follistatin, a site previously identified by directed mutagenesis as important for activin binding. The second epitope, predicted to be located between amino acids 46 and 59, is newly identified. Although the sequences 3-26 and 46-59 code for activin binding, native follistatin only binds activin if disulfide bonding is intact. Furthermore, pyridylethylation of Cys residues followed by N-terminal sequencing and amino acid analysis revealed that all of the Cys residues in follistatin are involved in disulfide bonds and lack reactive free sulfhydryl groups. Specific ligands were used to probe the structural effects of activin binding on the other domains of the full-length molecule, comprised largely of the three 10-Cys follistatin module domains. No effects on ligand binding to follistatin-like module I or II were observed after the binding of activin A to native protein. In contrast, activin binding diminished recognition of domain III and enhanced that of the C domain by their respective monoclonal antibody probes, indicating an alteration of the antigenic structures of these regions. Thus, subsequent to activin binding, interactions are likely to occur between regions of follistatin located in different domains and separated by considerable lengths of linear sequence. Such interactions could have important functional significance with respect to the structural heterogeneity of naturally occurring follistatins.

Inhibin A and inhibin B responses to gonadotropin withdrawal depends on stage of follicle development.

Previous studies demonstrate that inhibin A and B are differentially secreted across the menstrual cycle. To test the hypothesis that the responses of inhibin A and inhibin B to partial gonadotropin withdrawal are influenced by the stage of follicular development, a maximally suppressive dose of the Nal-Glu GnRH antagonist (150 microg/kg) was administered to normal cycling women during the midfollicular (MFP; n = 8), late follicular (LFP; n = 6), and midluteal phase (MLP; n = 5) to assess ovarian responsiveness over a broad range of developmental stages. Administration of the GnRH antagonist resulted in a significant decrease in LH (75 +/- 5%, 63 +/- 3%, and 84 +/- 7%; P < 0.05) and FSH (23 +/- 9%, 32 +/- 5%, and 39 +/- 6%; P < 0.04) during the MFP, LFP, and MLP, respectively. During the MFP, partial withdrawal of gonadotropins resulted in disappearance of the dominant follicle on ultrasound accompanied by a decrease in estradiol (E2; 64.9 +/- 11.4 to 23.9 +/- 3.3 pg/mL; P < 0.02) and inhibin B levels (121.6 +/- 14.8 to 28.4 +/- 4.8 pg/mL; P < 0.002) from baseline to near the limit of detection. Inhibin A was near the limit of detection in this cycle stage (0.8 +/- 0.1 IU/mL). When gonadotropins were withdrawn during the LFP, the size of the dominant follicle remained stationary in four of five subjects, and inhibin B (84.1 +/- 14.1 to 22.2 +/- 3.4 pg/mL; 71 +/- 5%; P < 0.02), inhibin A (4.4 +/- 1.1 to 1.3 +/- 0.5 IU/mL; 71 +/- 7%; P < 0.02), and E2 (236.8 +/- 48.2 to 95.6 +/- 39.0 pg/mL; 61 +/- 12%; P < 0.02) decreased similarly. Time to ovulation was shorter in association with higher inhibin A (r = -0.67; P < 0.02) and E2 (r = -0.79; P < 0.003), but there was no relation to inhibin B. During the MLP, decreased gonadotropin levels resulted in the disappearance of corpus luteum function with a significant decrease in inhibin A (9.0 +/- 0.4 to 0.7 +/- 0.1 IU/mL; 92 +/- 1%; P < 0.0001) in combination with decreased E2 (150.4 +/- 22.9 to 23.8 +/- 4.2 pg/mL; 83 +/- 3%; P < 0.005) and progesterone (12.6 +/- 2.6 to 0.9 +/- 0.2 ng/mL; 92 +/- 2%; P < 0.01). Administration of a GnRH antagonist at precise stages of the menstrual cycle provides further evidence that differential regulation of inhibin A and inhibin B is critically dependent on the stage of follicular development. Inhibin B secretion is exquisitely sensitive to gonadotropin withdrawal during the MFP when inhibin A has not yet risen. Inhibin A and inhibin B decrease equally after GnRH antagonist administration during the LFP. However, before antagonist administration, the positive correlation between estradiol and inhibin A and time to ovulation and the lack of such a correlation with inhibin B suggest that the source of inhibin B secretion is different from that of inhibin A and E2. The decrease in inhibin A secretion after antagonist administration during the luteal phase confirms gonadotropin-dependent secretion of dimeric inhibin A by the corpus luteum.

Identification of an activin-follistatin growth modulatory system in the human prostate: secretion and biological activity in primary cultures of prostatic epithelial cells.

PURPOSE: To determine if the activin/follistatin system is present in human prostate tissue and primary cultures of prostatic epithelium and if these growth factors play a role in the control of epithelial cell growth. MATERIALS AND METHODS: Cells derived directly from human prostates were studied to determine: a) if they secrete activin and follistatin, and b) if they are growth inhibited by activin. Primary cell cultures were established from tissues removed from 13 unselected prostate carcinoma patients in order to examine the secretion of activin and follistatin and test the effects of these proteins on cell proliferation. RESULTS: Both primary explant cells and epithelial cells isolated and sub-cultured from explant cultures secreted activin A and follistatin. Treatment of cultured cells with recombinant human activin A resulted in a dose-dependent inhibition of thymidine incorporation, with an IC50 of 0.22 nM. Recombinant follistatin neutralized the inhibitory effects of activin A on cell proliferation whilst adding follistatin alone enhanced thymidine incorporation, suggesting a similar neutralizing effect on the endogenous activin produced by these cells. CONCLUSION: These results demonstrate that cells derived from human prostate tissue secrete activin and follistatin and, as observed in human prostate cancer cell lines, activin inhibited the growth of prostatic epithelial cells. Also consistent with our earlier studies of prostate cancer cell lines, the biological activity of activin was neutralized by follistatin. These observations support the hypothesis that the activin/follistatin system plays an important role in the local regulation of human prostate cell growth.

Identification of naturally occurring follistatin complexes in human biological fluids.

Follistatin (FS) binds activin and inhibin proteins. Many organs are sensitive to activin and inhibin; thus the formation of FS-activin/inhibin complexes is important to our understanding of ligand activity. Other investigators studying FS have detected large molecular weight immunoreactive FS bands (greater than the expected molecular weight of FS alone) that have not been well characterized. The goal of this study was to identify naturally occurring FS monomers and FS-activin/inhibin complexes in several organ systems. The pituitary, ovary, kidney, and urine were chosen for this investigation. Molecular masses were assigned to in vitro assemblies of complexes containing recombinant inhibin or activin with FS for comparison with naturally occurring FS forms. The recombinant complex of FS-activin was primarily 97-kDa size, while FS-inhibin complexes were detected in a range of molecular sizes from 66 kDa to 97 kDa, 133 kDa, and > 220 kDa. FS-containing complexes of 66-kDa, 97-kDa, and 133-kDa were identified in the tissues examined and in pregnant urine. Our study points to the assembly of a series of FS-activin/inhibin complexes in a variety of organ systems that may impact upon the available amount of free versus bound (or "complexed") ligand, which must be considered when investigating the biology of activin- or inhibin-responsive cells. In addition, urine may be an important biological fluid that can be used to measure significant changes in circulating FS complexes.

A two-site chemiluminescent assay for activin-free follistatin reveals that most follistatin circulating in men and normal cycling women is in an activin-bound state.

Follistatin (FS) is a monomeric protein that binds and regulates the bioavailability of activin. Previously, we found circulating levels of total FS to be similar in men and cycling women. Because relative amounts of activin-bound and free FS are important considerations in determining activin bioavailability, we asked here whether the relative proportions of these two changed during different physiologic states. For this, we developed a two-site, solid-phase, immunochemiluminescent assay for free FS. The assay recognizes the 288 or 315 amino acid variants of human FS and has a detectable limit of 1 ng/mL. Inhibin, transforming growth factor-beta, or alpha-2-macroglobulin do not cross-react or interfere in this assay. Preincubation of FS with activin results in dose-dependent loss of immunoreactivity, confirming specificity of the assay for free FS. Human follicular fluid, pituitary extract, and serum with added FS dilute parallel with the recombinant human FS-288 standard. Recovery of recombinant human FS-288 from serum is quantitative. Using this assay, we found circulating concentrations of free FS to be at or below the detection limit of the assay throughout the menstrual cycle. Comparison of circulating total and free FS levels in postmenopausal or cycling women and normal men suggested that at least 90% is activin-bound. In contrast, measurable quantities of free FS were found in follicular fluid and pituitary extracts. The results of this study, showing that most circulating FS is normally activin-bound, argue against an endocrine role for FS and suggest that a major role of circulating FS is to bind and neutralize the bioactivity of circulating activin. The roles of FS as a local autocrine or paracrine regulator of activin in target tissues, where FS exists in free form, or as an endocrine regulator in human pathophysiology, warrants further investigation.

Pituitary follistatin regulates activin-mediated production of follicle-stimulating hormone during the rat estrous cycle.

Follistatin, an activin-binding protein, plays a key role in the modulation of activin-dependent functions. In the anterior pituitary, activin stimulates the synthesis and secretion of FSH. In the current study, we assessed the roles of locally produced activin and follistatin in the control of FSH gene expression and secretion. The anterior pituitary gland follistatin content was measured at frequent intervals during the rat estrous cycle. Follistatin protein levels were high before the primary gonadotropin surges, decreased by 50% on proestrous evening, and rebounded to a peak at midnight on proestrus before returning to presurge levels on estrus morning. Changes in pituitary follistatin protein content were preceded by parallel changes in pituitary follistatin messenger RNA (mRNA). The trough in follistatin protein content on proestrus coincided with a peak in circulating levels of free activin A (not bound to follistatin) and a sharp rise in FSHbeta mRNA levels, suggesting that decreased pituitary follistatin leads to increased available activin. To quantitate the contribution of pituitary free activin to pituitary expression of FSHbeta mRNA and the primary and secondary serum FSH surges, rats were infused through carotid catheters with saline or recombinant human follistatin (288-amino acid isoform; rhFS-288) at different times during the estrous cycle. Infusion of rhFS-288 on diestrus did not affect FSH production. In contrast, infusion of rhFS-288 during the secondary FSH surge decreased the peaks in FSHbeta mRNA and serum FSH by 63% and 47%, respectively, relative to those in saline-infused control animals. Infusion of rhFS-288 during the primary FSH surge decreased serum FSH to a lesser degree (24%). These data indicate a physiological role for pituitary follistatin in the control of activin-mediated FSH synthesis and secretion during the rat estrous cycle.

Activin A and follistatin are dynamically regulated during human pregnancy.

Activin A (beta A-beta A) and activin B (beta B-beta B) are related dimeric proteins that regulate numerous cellular activities. Activin activity is bioneutralized by follistatin, a specific and high-affinity binding protein. Recently, our group developed specific and sensitive enzyme-linked immunosorbent activin assays that do not detect either activin isoform when bound to follistatin, therefore, the assays are specific for biologically relevant ligands. Activin A is measurable in the serum of pregnant women (cross-sectional sample collection), while activin B is not detected in maternal serum. However, activin B is measurable in amniotic fluid and cord blood sera. The purpose of this study was to measure serum activin A, activin B, and follistatin prospectively in longitudinally collected samples during pregnancy. This study design offered observations of relative changes in serum hormone concentration with each person serving as an internal reference. Serum samples were collected bimonthly from seven pregnant women beginning within the second month of gestation, and up to, but not including, the onset of labor. Six of the seven women had normal labor and delivery. One patient required pitocin (an oxytocin agonist) for induction of labor which led to delivery. Activin A, activin B, total follistatin, free follistatin, human chorionic gonadotropin, estradiol, progesterone, FSH, and LH were measured in maternal serum samples using specific assays. Serum activin A levels increased in the final month of pregnancy in the six patients who delivered following normal labor (< 0.78 ng/ml (first trimester) to 1-6 ng/ml (term)). Activin B was not detected in any serum sample (< 0.78 pg/ml). Total serum follistatin (free follistatin, follistatin-activin, and follistatin-inhibin) increased 10- to 45-fold in the final month of pregnancy in four of the women undergoing normal labor (10 ng/ml (first trimester) to 100-450 ng/ml (final month)). Total follistatin was high and variable in two women throughout pregnancy. Total follistatin returned to basal serum concentration in three of the patients during the last 2 weeks of pregnancy. Free follistatin was detected throughout pregnancy (range < 2-35 ng/ml). Free follistatin represented a small percentage of the total follistatin throughout the time of pregnancy and did not rise coincident with the rise in total follistatin. Serum activin A and activin B were not detected during the entire course of pregnancy in the one patient who did not have normal labor and total follistatin did not rise in the last trimester of pregnancy. Gonadotropin and steroid hormones were measured in all patients and were within normative ranges for human pregnancy (inclusive of the non-laboring patient). The results suggest that immunodetectable activin A is present in the third trimester of pregnant women who have normal onset labor. The total follistatin assay results suggest that follistatin-activin (or -inhibin) complexes are upregulated during the third trimester of pregnancy. Importantly, activin A production exceeds the binding capacity of circulating follistatin. Because binding protein free activin A is biologically active we conclude that the activin A detected in late pregnancy is biologically relevant. The findings are consistent with our hypothesis that activin A is an endocrine factor during the last trimester of human pregnancy and may be involved in normal labor.