Maternal serum concentrations of inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC-related inhibin forms, total follistatin, steroids and gonadotrophins were measured longitudinally in six normal singleton pregnancies. Maternal venous blood was collected randomly during a spontaneous follicular phase prior to donor insemination, at 5, 7, 9, 11, 16, 20, 24, 28, 32 and 36 weeks after the first missed menses and in the early puerperium. Steroid and gonadotrophin profiles conformed to previous reports. While at week 5 of gestation inhibin-A, activin-A and follistatin concentrations were similar to those at the follicular phase, all three increased progressively (P < 0.001) to maximal concentrations in week 36: approximately 48-fold (3740 +/- 1349 ng inhibin-A/ml), approximately 22-fold (6109 +/- 1443 ng activin-A/ml) and approximately 10-fold (3563 +/- 418 ng follistatin/ml) higher. Pro-alphaC concentrations reached a maximum in weeks 5 (approximately 5-fold, P < 0.001) and 36 (1027 +/- 174 pg/ml, P < 0.01). Inhibin-B (71 +/- 23 pg/ml prior to pregnancy) was undetectable (<12 pg/ml) between week 5-16 of gestation but increased slightly in the third trimester (26 +/- 7 pg/ml in week 36). Activin-AB was undetectable throughout pregnancy. Post-partum concentrations of inhibin-A (41 +/- 12 ng/ml), inhibin-B (<12 pg/ml), activin-A (950 +/- 149 pg/ml), pro-alphaC (128 +/- 22 pg/ml) and follistatin (990 +/- 79 ng/ml) were substantially lower than at week 36 of gestation. The activin-A:follistatin ratio increased from 0.5 in week 5 to 1.8 in week 36, suggesting that more free activin-A is available in the maternal circulation during late pregnancy.

Recent studies have found follistatin to be an important regulator of activin bioactivity. Whilst a number of assay formats have been described, all are of limited sensitivity and require the use of isotopes. Many use polyclonal antibodies. Furthermore, a wide range of follistatin preparations have been used as standards, complicating inter-laboratory comparison.We now describe an ultra-sensitive two-site enzyme immunoassay using a pair of mouse monoclonal antibodies raised against follistatin 288. The presence of sodium deoxycholate and Tween 20 in the diluent gave results for total (free and activin-dissociated) follistatin. The assay had a detection limit of <19 pg/ml and recovery of spiked follistatin 288 from amniotic fluid, serum, seminal plasma, human follicular fluid and granulosa cell conditioned medium averaged 100·7 7·5%, 89·1 5·5%, 98 4·9%, 96 7·2% and 123·9 11% respectively. The intra-and interplate coefficients of variation were <5%. An excess of activin-A (50 ng/ml) prior to assay did not affect follistatin recovery. Inhibin-A, inhibin-B, activin-A, activin-B and activin-AB had minimal cross-reactivity (<0·3%). However, follistatin 315 had a significant cross-reaction (9·9%).Serially diluted human samples gave dose-response curves parallel to the standard. Pooled human follicular fluid contained high concentrations of follistatin (242 ng/ml). Follistatin was also found in maternal serum during pregnancy (first trimester 0·8 ng/ml, third trimester 2·8 ng/ml), normal male serum (0·45 ng/ml), amniotic fluid (sixteen week 3·63 ng/ml, term 0·89 ng/ml), seminal plasma (2·4-30 ng/ml) and human granulosa cell conditioned media (0·44 ng/ml). Serial serum samples taken throughout the menstrual cycle of ten women showed fluctuating follistatin concentrations (0·62 ng/ml) with no apparent relationship to the stage of the cycle. Interestingly, pooled serum from postmenopausal women appeared to have higher follistatin levels than any of the normal women (1·4 ng/ml).The possible presence in certain samples of mixtures of follistatin isoforms with different immunoreactivities poses major problems of interpretation in this and all other current follistatin immunoassays. Further work is needed to identify the major immunoreactive forms in different tissues and fluids. Nevertheless, the new assay has a number of advantages over previous assays and should prove a useful tool for various clinical and physiological studies.

Activin A levels are elevated in maternal serum of pregnant women with hypertensive disturbances. Because follistatin is a circulating binding protein for activin A, the present study was designed to evaluate whether serum follistatin and activin A levels also change in patients with hypertensive disorders in the last gestational trimester. The study design was a controlled survey performed in the setting of an academic prenatal care unit. Healthy pregnant women (controls, n=38) were compared with patients suffering from pregnancy-induced hypertension (PIH, n=18) or pre-eclampsia (n=16). In addition, the study included a subset of patients with pre-eclampsia associated with intrauterine growth restriction (IUGR, n=5). Maternal blood samples were withdrawn at the time of diagnosis (patients) or in a random prenatal visit (controls), and serum was assayed for follistatin and activin A levels using specific enzyme immunoassays. Hormone concentrations were corrected for gestational age by conversion to multiples of median (MoM) of the healthy controls of the same gestational age. Follistatin levels were not different between controls and patients, while activin A levels were significantly increased in patients with PIH (1·8 MoM), pre-eclampsia (4·6 MoM), and pre-eclampsia+IUGR (3·2 MoM, P<0·01, ANOVA). The ratio between activin A and follistatin was significantly increased in patients with PIH (1·5 MoM) and was further increased in patients with pre-eclampsia (4·5 MoM) and in the group with preeclampsia+IUGR (2·6 MoM). Follistatin levels were positively correlated with gestational age in control subjects (r=0·36, P<0·05) and in patients with PIH (r=0·46, P<0·05) or pre-eclampsia (r=0·61, P<0·01), while activin A correlated with gestational age only in the healthy control group (r=0·69, P<0·0001). The finding of apparently normal follistatin and high activin A levels in patients with PIH and pre-eclampsia suggests that unbound, biologically active, activin A is increased in women with these gestational diseases.

Objective To examine changes in maternal serum levels of activin A and follistatin during pregnancy and labour Design In three cross sectional and three longitudinal studies venous blood was collected from women during pregnancy, spontaneous labour, labour induction and prior to elective caesarean section for the measurement of activin A and follistatin.Setting Monash Medical Centre, Clayton, Victoria, Australia.Population One hundred and twenty-three women participated in a cross sectional study in pregnancy, 18 women in two longitudinal pregnancy studies, 36 women in a cross sectional labour study, nine women in a longitudinal study of labour induction. Ten women undergoing elective caesarean section were also studied.Methods Activin A and follistatin were measured using two sensitive and specific enzyme-linked immunosorbent assays. ResultsIn the cross sectional study of pregnancy, mean (SEM) maternal serum activin A and follistatin levels increased towards term (2.4 ng/mL (0.3) and 1.8 ng/mL (0.3) in first trimester to 18.9 ng/mL (3.8) and 5.3 ng/mL (0.9) at term, respectively), but the longitudinal study revealed that levels plateau in the last three weeks of pregnancy (16.0 ng/mL (2-6) and 6-2 ng/mL (1.4) at 37 weeks and 16-6 ng/mL (3.5) and 6.2 n g / d (0.5) before labour for activin A and follistatin, respectively). There was no difference in levels of activin A and follistatin between women delivered by caesarean section and labouring women at term (14.9 ng/mL (2.8) vs 11.0 ng/mL (0.93) and 5.95 ng/mL (0-67) vs 5.71 ng/mL (0.63), respectively) and levels of both proteins did not alter throughout spontaneous or induced labour.Conclusions We believe that these data argue against activin A playing an acute role in the initiation or regldation of human parturition.

Active immunization of ewes against inhibin (IMM) consistently increases ovulation rate but this response is not always accompanied by the expected rise in plasma FSH. Inhibin-related molecules also have local auto/paracrine effects within the ovary and the ovulatory response to IMM could be due to neutralization of one of these effects, independent of changing FSH levels. To investigate this, ovaries were collected from long-term IMM (n=6) and control (CON; n=8) ewes killed 48 h after progestagen withdrawal (late follicular phase) and all follicles _3 mm were recovered to determine intrafollicular levels of inhibin A, activin A and follistatin by specific two-site immunoassay and oestradiol and testosterone by radioimmunoassay. Blood samples were collected to assess plasma FSH, oestradiol and inhibin antibody titres. Although plasma FSH levels were similar in IMM and CON ewes, IMM ewes had 3-fold more follicles _3 mm (P<0·0001) and 3-fold more oestrogenic follicles (P<0·001) than CON ewes. Compared with CON ewes, follicles from IMM ewes had much higher concentrations of activin A (6-fold; P<0·001) and inhibin A (3-fold; P<0·001) but only slightly more follistatin (1·4-fold; not significant). The activin A:follistatin ratio in follicles from IMM ewes (1:1) was significantly higher (P<0·001) than in follicles from CON ewes (0·3:1). Levels of inhibin antibody measured in follicular fluid (FF) from IMM ewes were similar to plasma levels. Given that activin A has been shown previously to up-regulate FSH receptors and aromatase activity in rat granulosa cells, the increase in intrafollicular activin A, unaccompanied by a rise in the concentration of its binding protein (follistatin), could explain how long-term IMM enhances follicle development and ovulation rate without necessarily promoting a sustained increase in FSH secretion.