Circulating cell free DNA testing: are some test failures informative?

OBJECTIVE: The proportion of circulating cell free DNA derived from the feto-placental unit (fetal fraction or FF) correlates with test success and interpretation reliability. Some fetal disorders are associated with systematically lower FF, sometimes resulting in noninformative results. METHODS: We analyzed results from pregnancies tested in a nested case/control study derived from a cohort of 4664 high-risk pregnancies. Low FF was defined before and after adjusting for maternal weight and gestational age. RESULTS: Compared with euploid pregnancies, the median FF was significantly higher in Down syndrome pregnancies (ratio 1.17) and significantly lower in trisomy 18 and triploid pregnancies (ratios 0.71 and 0.19, respectively). Among 2157 pregnancies tested, 13 (0.6%) had FF <3.0% (all noninformative), including three trisomy 18 and three triploidy fetuses. After adjustment, 16 pregnancies (0.7%) had FF <0.3 multiples of the median (six informative), including one trisomy 18 and three triploidy fetuses. Modeled positive predictive values for low and high-risk populations were 7% and 30%, respectively. CONCLUSION: Among women with noninformative results attributable to low FF, trisomy 18 and/or triploidy risk are sufficiently high to warrant offering additional assessments (e.g. ultrasound). If the testing indication is ultrasound abnormality, amniocentesis and karyotype/microarray should be considered. © 2014 John Wiley & Sons, Ltd.

The influence of depression, body mass index, and smoking on serum inhibin B levels in late reproductive-aged women.

CONTEXT: Women experiencing depression have difficult psychosocial functioning, and recent data suggest an earlier onset of menopause. Understanding the biological mechanism for the impairment of reproductive function associated with depression is important. OBJECTIVE: The objective of the study was to determine whether a lifetime history of depression is associated with reduced ovarian reserve as reflected in serum levels of the granulosa cell product, inhibin B. DESIGN: Residual serum samples from a subset of patients in the Harvard Study of Cycles and Moods were collected. SETTING: Patients were recruited from seven Boston-area communities. PATIENTS: Women with or without a history of major depression, based on structured clinical interviews for Diagnostic and Statistical Manual of Mental Disorders, fourth edition, were enrolled. A subset of patients who had provided an early follicular phase blood specimen at study enrollment and two or more other samples over the first 18-month period of follow-up were included. INTERVENTION: There were no interventions. MAIN OUTCOME MEASURE: Serum inhibin B levels were measured. RESULTS: Serum FSH levels were higher in women with a history of depression, whereas inhibin B levels did not differ between groups. Body mass index and age were significantly and inversely related to serum inhibin B levels. Smoking history was noted, for the first time, to have a significant negative association with inhibin B levels. CONCLUSIONS: Smoking has a direct negative effect on ovarian reserve, as suggested by decreased serum inhibin B levels. In contrast, effects of depression on the reproductive axis may occur at the level of the pituitary and/or hypothalamus rather than at the gonadal level, as suggested by increased serum FSH levels.

Extragonadal alpha-inhibin precursor proteins circulate in human male serum.

The role of inhibin as a negative feedback regulator of pituitary FSH secretion in men remains controversial inasmuch as serum inhibin and FSH levels are often not correlated, in part because of the known alpha-inhibin subunit cross-reactivity of the most widely used inhibin antiserum (Monash #1989). The objective of this study was to identify the nature of these alpha-inhibin proteins in male serum using antisera specific for the alpha-inhibin precursor. Three polyclonal antisera were raised against synthetic peptide fragments from the proregion (amino acids 21-35 = precursor alpha inhibin (PIN) 1 and 42-56 = PIN 2) and alpha-N segment (113-127 = PIN 3) of the human alpha-inhibin precursor protein. These antisera were then used in individual RIAs with the homologous peptide as both standard and radioligand. Because pure human alpha-inhibin subunit proteins are not available, recombinant alpha-inhibin medium and porcine follicular fluid were used as reference preparations in the PIN assays. All assays were specific for alpha-inhibin proteins, i.e. 1) they showed no significant cross-reactivity with other alpha-inhibin peptide fragments, dimeric 32-kDa inhibin, recombinant activin, FSH, human albumin, or the inhibin binding proteins alpha-2 macroglobulin and follistatin, and 2) they recognized native protein in alpha-inhibin precursor-containing porcine follicular fluid and in medium from a recombinant alpha-inhibin-only secreting cell line. Furthermore, immunoreactivity of all serum proteins seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with the PIN antisera was abolished or significantly reduced (40-100%) by preincubation of each PIN antiserum with the homologous peptide. Serial dilutions of serum from normal, GnRH-deficient, and castrate males exhibited equivalent displacement curves in each precursor (PIN) assay, and there were no significant group differences in PIN 2 immunoreactivity between normal (n = 14), GnRH-deficient (n = 8), and castrate men (n = 3). Western blotting of serum samples from a normal and a GnRH-deficient male revealed immunoreactive proteins of approximately 57 and 29 kDa under reducing conditions with all three PIN antisera. An additional 40-kDa protein was observed with the pro-alpha-inhibin antisera, PIN 1 and 2, and a protein of more than 97 kDa was seen with the PIN 2 antiserum.(ABSTRACT TRUNCATED AT 400 WORDS)

Human follicular fluid contains pro- and C-terminal immunoreactive alpha-inhibin precursor proteins.

The majority of immunoactive inhibin in human follicular fluid (hFF) is devoid of pituitary cell bioactivity and is hypothesized to contain alpha-inhibin monomeric proteins known to cross-react with an inhibin antiserum (Monash 1989). The aim of this study was to define more precisely the nature of these inhibin-immunoreactive proteins using alpha-inhibin sequence-specific antisera. First, a polyclonal antiserum was raised to precursor amino acids 21-35 (PIN-1) and was used in a RIA to measure pro-alpha-inhibin-immunoreactive proteins. Western blotting was used to confirm these findings. Secondly, the binding epitope of the Monash antiserum was defined by peptide analysis to be located within the C-terminus (precursor amino acids 326-341) of alpha-inhibin, and this assay was then used to monitor the presence of C-terminal sequences. Similar levels of pro-alpha-inhibin immunoreactivity (PIN-1 RIA; N-terminus alpha-inhibin precursor) were detected in hFF collected from women with normal menstrual cycles during the follicular phase and from multiple follicles from a woman undergoing ovarian hyperstimulation during an in vitro fertilization (IVF) protocol. Western blotting with the PIN-1 antibody confirmed the presence of immunoreactive proteins of 57,000 and 29,000 mol wt in the follicular fluids of both normal cycle and IVF follicles. However, ovarian hyperstimulation elevated intrafollicular C-terminal immunoreactivity (Monash RIA) compared to that in normal cycle hFF. Furthermore, intrafollicular estradiol and progesterone were significantly correlated to C-terminal activity in follicles from IVF, but not in normal cycles. These data show that 1) both pro- and C-terminal alpha-inhibin proteins are secreted into follicular fluids from normal and IVF cycles, suggesting that alpha-inhibin precursor proteins may be physiologically relevant in the process of folliculogenesis; and 2) IVF and normal cycle follicular fluids differ in their production and processing of inhibin.

Frequency modulation of follicle-stimulating hormone (FSH) during the luteal-follicular transition: evidence for FSH control of inhibin B in normal women.

To isolate the impact of GnRH pulse frequency on FSH secretion and to examine the effect of differing levels of FSH on inhibin B secretion during the luteal-follicular transition, exogenous GnRH was administered to GnRH-deficient women using one of two regimens, and the results were compared to those in normal women. In the GnRH-deficient women, the GnRH pulse frequency was increased from every 4 h in the late luteal phase to every 90 min on the day of menses to mimic normal cycling women (physiological frequency transition; n = 8 studies) or the GnRH pulse frequency was kept constant at a late luteal phase frequency of every 4 h through the first 6 days of the subsequent early follicular phase of cycle 2 (slow frequency transition; n = 6 studies). The differential rise in FSH secretion induced in these studies allowed us to examine the subsequent contribution of varying levels of FSH to inhibin B secretion. A physiological regimen of GnRH during the luteal-follicular transition resulted in a rise in FSH and inhibin B levels that did not differ from that in normal cycling women and a normal follicular phase length. On the other hand, maintaining a luteal frequency of GnRH for 6 days into the subsequent early follicular phase produced FSH levels significantly lower than those in the physiological transition (P < 0.05), with the greatest difference seen on the day after menses (9.1 +/- 1.0 vs. 16.4 +/- 1.4 IU/L for the slow and physiological transition groups, respectively; P < 0.005), but no difference in LH. This slower rise of FSH secretion in the slow frequency group was associated with significantly lower inhibin B levels (43.3 +/- 21.5 vs. 140.0 +/- 24.4 pg/mL, mean days 1, 3, and 5; P < 0.02), a later doubling of estradiol from baseline (day 9.6 +/- 0.9 vs. day 5.6 +/- 0.1; P < 0.02), and a longer follicular phase length (16.0 +/- 1.4 vs. 11.6 +/- 0.9 days; P < 0.05) compared with those in the physiological transition group. In conclusion, during the luteal-follicular transition, the GnRH pulse frequency contributes to but is not solely responsible for the FSH rise that initiates folliculogenesis. Alteration of FSH dynamics induced by changes in GnRH pulse frequency in GnRH-deficient women provides evidence that FSH stimulates inhibin B production in the human. Timely follicular development indicated by both estradiol and inhibin B secretion appears to be dependent on the pattern of increase in FSH during the luteal-follicular transition.

Relatively low levels of dimeric inhibin circulate in men and women with polycystic ovarian syndrome using a specific two-site enzyme-linked immunosorbent assay.

The endocrine feedback role of dimeric inhibin on FSH secretion from the pituitary has been well established in many species; however, evidence that inhibin is an important endocrine regulator of FSH in the human is more tenuous. One potential explanation for the equivocal data may be that the inhibin immunoassay used most widely in the human is a heterologous assay with an antiserum that exclusively recognizes the inhibin alpha-subunit in both its monomeric form and in inhibin dimers. The aim of the present study was to quantify serum inhibin levels in a variety of fertile and infertile men and women using a new ultrasensitive enzyme-linked immunosorbent assay that is specific for the dimeric form (alpha/beta) only. The specificity of the present assay was demonstrated by the absence of significant cross-reactivity with Mullerian inhibiting substance, transforming growth factor-beta, activin, FSH, LH, hCG, TSH, and hCG alpha and with the alpha-subunit of inhibin. The assay was sensitive to 1 pg/mL, and serial dilutions of human male and female serum samples paralleled the recombinant 32-kilodalton (kDa) dimeric inhibin standard curve. Complete recovery of exogenous recombinant 32-kDa inhibin added to serum was obtained. Mean serum inhibin levels ranged from a low of 5.7 +/- 0.6 pg/mL in the early follicular phase to 49.0 +/- 11.2 pg/mL in the midluteal phase of the normal menstral cycle and were elevated during ovulation induction (1250 pg/mL) and pregnancy (500 pg/mL). Interestingly, mean levels of dimeric inhibin in women with polycystic ovarian syndrome (PCOS) were indistinguishable from normal follicular phase. The most striking observation was the extremely low mean inhibin levels (< 2 pg/mL) found in normal men, GnRH-deficient men before any pulsatile GnRH treatment, and men with Klinefelter's syndrome, all of which were indistinguishable from levels observed in postmenopausal women. These observations in the male raise the possibly that 1) some forms of circulating and bioactive inhibin in the human are not detected by this assay due to either their conformation or the presence of a unique binding protein for endogenous inhibin that does not bind to recombinant 32-kDa inhibin; or 2) dimeric inhibin is not a major endocrine regulator of FSH in the human male. In addition, the relatively high dimeric inhibin levels at midcycle and in the luteal phase of the normal menstrual cycle, after gonadotropin stimulation, and during pregnancy suggest that dimeric inhibin is predominantly produced by dominant follicles, corpora lutea, and placental tissues.

Inhibins and activins in human fetal abnormalities.

To date, the only routine clinical application of inhibin or activin measurement in testing for fetal abnormalities has been the use of inhibin A in prenatal screening for trisomy 21 (Down syndrome). Second trimester maternal serum levels of inhibin A are, on average, two-fold higher in Down syndrome than in unaffected pregnancies. Although the biology of altered second trimester maternal serum analyte levels in Down syndrome pregnancy cannot yet be explained, it seems that fetal products tend to be decreased, while placental products tend to be increased. This pattern holds true for inhibin A because maternal serum levels appear to be derived from placental rather than fetal sources. Therefore, the measurement of inhibins and activins in maternal fluids, although clinically useful and relatively easy to obtain, may not be helpful in studying their role in human fetal development. Studies in transgenic mice indicate a role for activin, follistatin, and activin receptor type IIA in development of the palate and craniofacial region. Cleft palate is a common birth defect and is associated with serious feeding and respiratory complications in newborns. We have begun to investigate the potential role of activin in human craniofacial development by examining the spatial and temporal expression of inhibin/activin subunits, follistatin and the activin receptors in the fetal palate. Palate tissues were collected at autopsy from fetuses ranging in gestational age from 9 to 42 weeks, and 8 week embryonic tissues were also examined. Tissues were either stored in paraffin for immunocytochemistry or were frozen for RT-PCR examination of the expression of inhibin/activin proteins or mRNAs, respectively. To date, betaA subunit, follistatin, and activin receptor, but not alpha and betaB subunit, mRNAs are present in palate tissues and inhibin/activin betaA immunoreactivity has been consistently observed in developing bone. Expression of the activin A subunit and its receptors in the human fetal palate are consistent with a developmental role. Studies are ongoing to determine whether altered activin biosynthesis is associated with cleft palate. Future studies of fetal tissues may help to elucidate other roles for the TGF-beta family in human development.

Second trimester levels of maternal serum total activin A and placental inhibin/activin alpha and betaA subunit messenger ribonucleic acids in Down syndrome pregnancy.

OBJECTIVES: Previous data have shown that inhibin A (alpha/betaA) is increased about twofold in maternal serum samples from Down syndrome pregnancy. Our objectives were to determine whether activin A (betaA/betaA) was similarly increased in maternal serum from pregnancies affected with fetal Down syndrome, and to investigate whether increased expression of each inhibin/activin subunit occurred in placental tissue from cases of fetal Down syndrome. DESIGN AND METHODS: Maternal serum total activin A levels were measured in 20 cases of fetal Down syndrome and 100 unaffected pregnancy samples. In addition, analysis of inhibin/activin alpha and betaA subunit mRNA levels was performed in placental tissue extracts from six cases of fetal Down syndrome and six tissues with a normal karyotype. RESULTS: The median total activin A level in the Down syndrome cases was 0.82 MoM (multiples of the median); values did not differ significantly (P = 0.36, Mann-Whitney U analysis) from those in unaffected pregnancies. The inhibin alpha subunit/GAPDH mRNA ratio, but not that of betaA subunit/GAPDH mRNA, was significantly greater (P < 0.01, ANOVA) in placental tissue from Down syndrome than in control placental tissue. CONCLUSIONS: Unlike inhibin A, activin A is not significantly increased in Down syndrome relative to unaffected pregnancy. Furthermore, increased amounts of maternal serum inhibin A in Down syndrome pregnancy probably result from increased placental expression of inhibin alpha, but not betaA, subunit.

Progesterone, inhibin, and hCG multiple marker strategy to differentiate viable from nonviable pregnancies.

OBJECTIVE: To determine whether a combination of serum and urine biomarkers drawn from symptomatic pregnant women will help early differentiation of viable from nonviable pregnancies. METHODS: We conducted a prospective cohort study of 220 women who presented in the first trimester of pregnancy with complaints of pain, cramping, bleeding, or spotting. Serum samples for progesterone, inhibin A, and hCG, and urine beta-core hCG, were collected at presentation. To evaluate whether those biomarkers could predict viable and nonviable outcomes in pregnancy, we used likelihood ratios to compare operating characteristics of single and multiple biomarker strategies. RESULTS: Of 220 pregnancies studied, 98 were viable and 122 nonviable. Among single biomarkers, progesterone alone appears to have the greatest utility (area under the receiver operator characteristic curve = 0.923). Among dual-biomarker strategies, progesterone plus hCG and progesterone plus inhibin A improved specificity but not sensitivity. At 95% sensitivity, the combination of progesterone and hCG improved specificity from 0.29 to 0.66 (improvement = 0.37 [95% confidence interval 0.23, 0.52]). A triple-biomarker combination did not show substantial improvement over the dual-biomarker strategy. Also, combinations that used urine beta-core hCG did not improve diagnostic accuracy. CONCLUSION: Serum progesterone appeared to be the single most specific biomarker for distinguishing viable from nonviable pregnancies. When a dual-biomarker strategy was applied, combining serum progesterone with hCG, specificity improved significantly, which suggests that a multiple biomarker strategy might help distinguish viable from nonviable pregnancies in early gestation.

Serum inhibin levels are lower in ectopic than intrauterine spontaneously conceived pregnancies.

OBJECTIVE: To determine if serum inhibin concentrations are lower in ectopic (EP) versus intrauterine pregnancies (IUPs) that are conceived spontaneously. DESIGN: Case-control study. SETTING: Academic clinical practice. PATIENTS: Serum samples were obtained from 19 women who had EP confirmed at surgery and by pathology. For comparison, serum samples were collected from 24 women of similar chronological and gestational age with sonographic evidence of an IUP. MAIN OUTCOME MEASURE: Serum dimeric inhibin-A, total inhibin, P, and hCG. RESULTS: Serum total and dimeric inhibin concentrations in women with EP were < 60% of the concentrations for women with single IUPs. Total inhibin, but not dimeric inhibin-A, was elevated in maternal serum before week 8 of gestation relative to normal menstrual cycle levels. CONCLUSIONS: Serum inhibin concentrations are lower in EP as compared with IUPs that are spontaneously conceived and the relative amounts of dimeric inhibin-A, B, and alpha inhibin subunit in maternal serum may change throughout gestation.

Second-trimester levels of maternal serum human chorionic gonadotropin and inhibin a as predictors of preeclampsia in the third trimester of pregnancy.

OBJECTIVE: To determine whether second-trimester maternal serum levels of inhibin A, human chorionic gonadotropin (hCG), unconjugated estriol (uE3), and alpha-fetoprotein (AFP) are predictive of the later onset of preeclampsia in pregnancy. METHODS: Retrospective evaluation of serum analyte levels in 60 women with preeclampsia compared with 300 controls. Levels of each analyte were compared in women with preeclampsia and controls using matched rank analysis. Analytes that were significantly different between groups were examined with univariate and bivariate Gaussian distribution analysis. RESULTS: Second-trimester inhibin A (1.36 multiples of the median [MoM]) and hCG (1.40 MoM) levels were significantly but modestly elevated in women who later developed preeclampsia. A combination test of maternal age plus inhibin A and hCG predicted 23% of cases of preeclampsia with 95% specificity. There was a statistically significant trend for inhibin A, but not hCG, levels to be higher when the onset of preeclampsia occurred within a shorter (<17 weeks) interval after collection of the second-trimester screening sample. CONCLUSIONS: Second-trimester serum levels of inhibin A and hCG are modest predictors of the later onset of preeclampsia. Inhibin A may be a better predictor of early-onset preeclampsia, which is associated with a higher maternal and perinatal morbidity and mortality, than preeclampsia at or near term.

Participation in maternal serum screening following screen positive results in a previous pregnancy.

OBJECTIVE: To determine whether women who have had a positive serum screening result in one pregnancy have a lower rate of participation in screening in their next pregnancy. SETTING: The Women and Infants Hospital triple marker screening programme. METHODS: Pregnancy and screening information was collected from laboratory and hospital databases to compare subsequent screening participation in women who were screen negative and screen positive for risk of Down's syndrome (DS) or neural tube defect (NTD) pregnancy. RESULTS: In an age matched comparison, 108 women who had a previous screen positive result were significantly less likely than 108 women who were screen negative to participate in maternal serum screening in their next pregnancy. When examined according to type of screen positive result, the effect was significant for both those who were screen positive for DS and those who were screen positive for NTD. The degree of risk in screen positive women did not significantly affect their uptake of screening in the next pregnancy. CONCLUSIONS: Anxiety related to a screen positive result probably causes decreased participation in maternal serum screening in the next pregnancy. Reducing the screen positive rate in prenatal serum screening would alleviate maternal anxiety and would probably lead to more stable participation.

Second trimester levels of maternal serum inhibin A, total inhibin, alpha inhibin precursor, and activin in Down's syndrome pregnancy.

OBJECTIVE: To determine the levels of various biochemical forms of the placental protein, inhibin (total inhibin, inhibin A, and alpha inhibin precursor) and activin in maternal serum samples from fetal Down's syndrome, and to determine which of these analytes most effectively identifies samples from affected pregnancies. METHODS: Maternal serum samples were collected from 100 unaffected pregnancies and 20 cases of fetal Down's syndrome during gestational weeks 15-20 for routine triple marker screening, and were stored frozen after clinical assay. Levels of inhibin A, total inhibin, alpha inhibin precursor (pro-alphaC), and activin were compared retrospectively in the Down's syndrome cases and control samples. RESULTS: There was no association of the inhibin or activin levels with gestational age or length of freezer storage, and therefore single median values were determined for the unaffected pregnancies for each analyte. Multiples of the unaffected median (MoM) values were calculated for all cases, showing that inhibin A (1.95 MoM) provided the best discrimination between cases and controls, followed by total inhibin (1.37 MoM). Mann-Whitney U analysis showed significant group differences in inhibin A (P = 0.0001) and total inhibin (P = 0.0005). In contrast, alpha inhibin precursor (0.81 MoM) and activin (1.16 MoM) levels in Down's syndrome cases were not significantly different from those in unaffected patients. CONCLUSIONS: Levels of inhibin A and total inhibin, but not alpha inhibin precursor or activin, are significantly raised in maternal serum from cases of fetal Down's syndrome. These data, taken together, indicate that inhibin A levels are specifically raised in Down's syndrome pregnancy. 45% of the inhibin A levels in the Down's syndrome samples were above the 90th centile of unaffected levels, indicating that inhibin A may be as good a marker as human chorionic gonadotrophin, the most informative serum marker currently in use.

Clinical validation of a new dimeric inhibin-A assay suitable for second trimester Down's syndrome screening.

OBJECTIVE: To compare the Down's syndrome screening performance of a simplified dimeric inhibin-A assay (Diagnostic Systems Laboratories (DSL)) with an assay whose clinical utility has been established (Serotec). SETTING: A case control set consisting of 51 Down's syndrome and 245 matched unaffected pregnancies collected as part of an earlier multicentre cohort study. METHODS: Sera were assayed for dimeric inhibin-A using the DSL assay and Serotec reference assay. Data analysis included a method comparison of mass values, fit of data to a logarithmic Gausian distribution, and determination of detection and false positive rates. In addition, 234 fresh sera were assayed using the simplified method. RESULTS: The two assays showed a high correlation (r = 0.93) but average concentrations of the DSL assay were 48% higher. However, the differences were basically proportional over the range of values important for screening. The detection rate was essentially equivalent for the DSL assay whether analysed univariately or in combination with other markers (for example, 79% v 75% at a 5% false positive rate for the DSL and Serotec assays for the combination of alpha fetoprotein, unconjugated oestriol, human chorionic gonadotrophin, and dimeric inhibin-A, respectively). The 234 dimeric inhibin-A values measured on fresh sera fitted a logarithm Gaussian distribution for the DSL assay, as indicated by the fit to a probability plot. CONCLUSIONS: The Down's syndrome screening performance of a simplified dimeric inhibin-A immunoassay was equivalent to a more labour intensive established dimeric inhibin-A assay.

Endocrine analytes in multiple-marker screening.

Prenatal serum screening is based on the observation that secretory products of the placenta and fetus are altered in maternal serum of pregnancies affected with certain birth defects. Most of these products are hormones that have been previously characterized, although the pathophysiologic basis of the altered maternal serum levels of these products that occurs in association with fetal chromosome for the most part is unknown. Prenatal serum screening is in a period of transition, with the relatively recent advance of second trimester triple-marker screening (AFP, uE3, hCG) and now the improved performance of the four-marker test that adds inhibin. A. Proposed first trimester screening and other new second trimester serum markers may dramatically change prenatal screening practices as we enter the next century.

Expression of inhibin/activin proteins and receptors in the human hypothalamus and basal forebrain.

The inhibin/activin family of proteins is known to have a broad distribution of synthesis and expression in many species, as well as a variety of functions in reproductive and other physiological systems. Yet, our knowledge regarding the production and function of inhibin and activin in the central nervous system is relatively limited, especially in humans. The present study aimed to explore the distribution of inhibin/activin protein subunits and receptors in the adult human brain. The human hypothalamus and surrounding basal forebrain was examined using post-mortem tissues from 29 adults. Immunocytochemical studies were conducted with antibodies directed against the inhibin/activin α, ßA, and ßB subunits, betaglycan and the activin type IIA and IIB receptors. An immunoassay was also utilised to measure dimeric inhibin A and B levels in tissue homogenates of the infundibulum of the hypothalamus. Robust ßA subunit immunoreactivity was present in the paraventricular, supraoptic, lateral hypothalamic, infundibular, dorsomedial and suprachiasmatic nuclei of the hypothalamus, in the basal ganglia, and in the nucleus basalis of Meynert. A similar staining distribution was noted for the ßB subunit, betaglycan and the type II receptor antibodies, whereas α subunit staining was not detected in any of the major anatomical regions of the human brain. Inhibin B immunoreactivity was present in all tissues, whereas inhibin A levels were below detectable limits. These studies show for the first time that the inhibin/activin protein subunits and receptors can be co-localised in the human brain, implicating potential, diverse neural functions.

Inhibin A measurement using an automated assay platform.

BACKGROUND: Second-trimester measurement of maternal serum inhibin A is widely used for Down syndrome screening. To date, only a manual enzyme-linked immunosorbent assay (ELISA) produced by Diagnostic Systems Laboratories, Inc (DSL) has been available. The objective of this study was to compare the DSL assay with a new automated assay produced by Beckman Coulter, Inc (Access). METHODS: Residual serum samples from 570 women, who were receiving routine screening for Down syndrome, were retrieved from storage. The Access assay sensitivity, linearity and reproducibility were determined and a method comparison was performed. Inhibin A levels were measured using both assays. Twenty samples from women with confirmed Down syndrome pregnancy were also tested. RESULTS: The Access assay had coefficients of variation of less than 10% across the range of values tested, and a sensitivity below 1 pg/mL. The DSL and Access inhibin A assay values were highly correlated (r = 0.961, r(2) = 0.923), with no apparent outliers. Inhibin A values from the Access assay were a constant 23% lower (95% CI 1-41%) than corresponding values from the DSL assay. Median values from 15 to 20 completed weeks' gestation were computed and found to be consistent with expectations. The weight-adjusted multiples of the median (MoM) levels in the unaffected pregnancies fit a log Gaussian distribution well between at least the 5th and 95th percentiles with corresponding log standard deviations of 0.1960 and 0.1919 for DSL and Access, respectively. CONCLUSIONS: With median inhibin A levels appropriately calculated for the Access assay, Down syndrome screening performance is expected to be comparable to that obtained with the manual DSL assay.