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C in Maternal Serum during Human Pregnancy
Department of Obstetrics and Gynecology, Monash University (P.T., E.W.), Clayton, 3168 Australia; Oxford Brookes University (N.G.), Oxford OX3 OBP, United Kingdom; and Prince Henry’s Institute of Medical Research (T.S., J.F., P.G.S., D.M.R.), Clayton, Victoria 3168, Australia
Address all correspondence and requests for reprints to: David Robertson, Ph.D., Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: david.robertson{at}med monash.edu.au.
Abstract
Maternal serum pools obtained from healthy women throughout normal
pregnancy were fractionated by a combined immunoaffinity
chromatography, preparative PAGE, and electroelution procedure. Inhibin
A and the pro-
C region of the inhibin -subunit were determined in
the eluted fractions by specific ELISAs, and the profiles of
immunoactivity characterized in terms of molecular weight and percent
recovery. The molecular weight patterns of inhibin A and pro-C in
serum during early pregnancy (<19 wk gestation) showed peaks between
25–40K and approximately 60K, consistent with the presence of known
mature and larger precursor inhibin forms. However, during late
pregnancy (>19 wk gestation), an increase in the proportion of smaller
molecular weight forms (from 2% to 
25%) of inhibin A and pro-C
of unknown structure were observed in the less than 30K and less than
25K regions, respectively. To assess whether this change in molecular
weight distribution in late pregnancy was related to the method of
serum collection, serum and plasma from women during early and late
pregnancy were collected and snap-frozen. Three pools [one from early
pregnancy (12–15 wk), two from late pregnancy (28–39 wk)] of serum
and plasma were then fractionated as described above. No differences in
molecular weight patterns of inhibin A and pro-C were observed
between serum and plasma pools obtained in early pregnancy. However, in
late pregnancy there was a reduction in the proportion of low molecular
weight forms between serum (25% inhibin A, 35% pro-C) and plasma
(12% and 17%, respectively), but not to the low levels seen in early
pregnancy. Incubation of iodinated 30K human inhibin A with serum
or plasma obtained from early or late pregnancy showed no evidence of
cleavage, suggesting that 30K inhibin A is not the cleavage
precursor. It is speculated that the formation of small molecular
weight forms of both inhibin A and pro-C is attributed to
proteolytic changes, in part induced in the circulation during late
gestation and in part by the placenta before secretion. It is concluded
that inhibin A and pro-C are processed in late pregnancy by more
than one mechanism to form low molecular weight circulating forms of
unknown structure.
INHIBIN, A MEMBER of the TGFß superfamily
is composed of two subunits, and ßA or
ßB (forming inhibin A or B). Inhibin plays an
established role in the suppression of FSH and secretion
(1, 2, 3). Inhibin is synthesized as two precursor chains
that are linked by disulfide bonds before subsequent processing through
several recognized steps to form the mature 30K form. Studies to date
have highlighted two main proteolytic cleavage sites in the -subunit
dividing the -subunit into three regions, pro [amino acids (aa)
19–61], N (aa 62–232), and C (aa 233–366) regions
(4). One cleavage site has been identified within the
ßA-subunit giving two regions, pro-ß (aa
21–310) and ßA (aa 311–426)
(4).
Studies exploring the forms of inhibin in biological fluids have
identified inhibins ranging in size between the precursor and mature
30K (C/ß) forms, largely consistent with combinations of the above
- and ß-subunit subregions. In addition, a processed form of the
free -subunit, pro-C, has been identified in high concentrations
in biological fluids (5). The precursor forms of inhibin
are intrinsically less bioactive compared with 30K inhibin or inactive,
requiring cleavage of the precursor sequences for bioactivity to be
evident (6). Studies have shown the presence of these
various forms of inhibin in human serum and plasma and human follicular
fluid (hFF) (7, 8). Little, however, is known about the
inhibin form(s) present in human maternal serum. Inhibin A in maternal
serum increases markedly during late pregnancy in parallel with serum
E2 and progesterone (9). Inhibin B levels are low
throughout gestation (10, 11), whereas -subunit
(pro-C) levels are biphasic, with an initial peak early in pregnancy
followed by a rise in late pregnancy similar to that seen with inhibin
A (11). It is believed that inhibin A throughout early and
late pregnancy is primarily of fetal placental origin (10, 12), whereas pro-C is a product of the corpus luteum during
early pregnancy and the fetal placental unit during late pregnancy
(12, 13, 14).
The aim of this study was to examine the forms of inhibin A and
pro-C in maternal serum and plasma of women with normal pregnancies
using an improved fractionation procedure in combination with specific
ELISAs to provide baseline inhibin profiles for subsequent comparisons
with the inhibin profiles of abnormal pregnancies.
Materials and Methods
Preparations
Recombinant human 30K inhibin A was a gift from Biotech Australia P/L (Roseville, Australia). hFF and in vitro fertilization (IVF) serum (obtained from women undergoing gonadotropin stimulation as part of an IVF program) were obtained as described previously (7).
Maternal serum samples were collected at various stages of pregnancy at the Antenatal Clinic, Monash Medical Centre (1998–1999). The women were all healthy with singleton pregnancies. Blood samples were collected in plain serum-gel tubes (Greiner Labortechnik, Kremsmunster, Austria), stored at 4 C, and centrifuged within 48 h of collection. The serum was then stored frozen at -20 C. From this batch, 7 serum pools, covering various gestational stages (<9, 10–12, 13–18, 19–24, 25–30, 31–36, and 37–40 wk) were prepared. Each pool consisted of equal aliquots from more than 20 women. A pregnancy serum pool, covering samples from all gestational periods, was also prepared, which was used for establishing the reproducibility of the fractionation procedure.
Serum and plasma were collected under more defined conditions from early pregnancy (eight women, 12–15 wk), and late pregnancy [two separate collections, 28–39 wk (n = 15) and 31–36 wk (n = 20)]. Blood was collected in serum-gel tubes (Greiner) on ice, allowed to clot at 4 C, and centrifuged within 2 h. Serum was stored frozen at -80 C. Plasma was collected on ice in EDTA-coated tubes (Greiner) and centrifuged within 2 h at 4 C, and the plasma was stored at -80 C.
The study was approved by the research and ethics committee of Monash Medical Centre. All women gave informed consent.
Fractionation procedure
Serum was fractionated by previously described methods (7, 8) with modifications. The procedure consists of an initial
immunoaffinity fractionation using an immobilized antiserum raised to
the -subunit of inhibin, followed by a preparative PAGE (Prep-PAGE)
procedure (largely as previously described), followed by a modified
electroelution procedure. This latter step was introduced to increase
the practicability of the procedure. In a typical fractionation run,
serum or plasma (30 ml) was rapidly thawed in the presence of 20 ml 200
mmol/liter phosphate buffer, pH 7.4, containing 20 mmol/liter EDTA, 2
mmol/liter phenylmethylsulfonylfluoride, and 1.67% Triton X-100
(Merck, Kilsyth, Australia). The mixture (50 ml) was added to 60 ml
gel-immobilized IgG fraction from antiserum raised in sheep
against a human inhibin C-subunit fusion protein
(7) and incubated at 4 C overnight on a wheel. The gel
suspension was distributed into 10-ml columns, washed with 100
mmol/liter phosphate buffer (pH 7.4) and 10 mmol/liter EDTA, and
brought to room temperature before elution with 6 mol/liter guanidine
hydrochloride (GnHCl). The GnHCl fraction (30 ml) was diluted with 30
ml water, divided into 10-ml lots, and fractionated on six disposable
C18 reverse phase cartridges (Vydac Bio-Select extraction columns,
Separations Group, Hesperia, CA) that had been prewashed with 100%
acetonitrile (Acn; 3 ml) followed by 5% AcN/0.1% trifluoroacetic acid
(TFA; 3 ml). The columns were eluted with 60% AcN/0.1% TFA (3 ml),
the six eluates were pooled (18 ml), SDS (final concentration, 0.5%)
was added, and the columns were lyophilized for 5 d to ensure
removal of all TFA. Aliquots for assessment of recoveries were
lyophilized separately in BSA.
The lyophilized sample was dissolved in 1200 µl 1 mol/liter Tris (pH 8.0), 2 mmol/liter phenylmethylsulfonylfluoride, 0.1% Tween 40, and bromophenol blue (assisted by sonication), and applied to a 10% polyacrylamide gel (20 x 20 cm) in Tris/Tricine/SDS buffers (15). Colored molecular weight standards (6.5–205K, lot 59H9306, Sigma-Aldrich Corp., St Louis, MO) were applied either with the sample or in an outside lane. The gel was run overnight at 35mA until the 13.5K molecular weight standard reached the bottom of the gel. At completion of the run, the polarity was reversed for 1 min.
The samples were electroeluted from the gel using an elution device
(Whole Gel Eluter, Bio-Rad Laboratories, Inc., Hercules,
CA). Preliminary experiments were undertaken to optimize this
procedure. To maximize recoveries, the elution process was continued
for longer (150 min) than recommended, requiring an increase in buffer
strength [270 mmol/liter Tris, 300 mmol/liter HEPES (pH 8.0), and
0.05% SDS] and the inclusion of 0.1% BSA in the collection vessel to
prevent sticking of the eluted protein to the eluter surfaces. The
electroelution process was undertaken at 250 mA and continued until the
voltage increased to 35 V. At higher voltages the pH declined rapidly
as the buffering capacity of the buffer was exhausted, heat was
generated, and inhibin recoveries decreased. Thirty fractions (3–4 ml)
were collected into weighed tubes containing 500 µl 0.75%
NaN3, 800 mmol/liter NaCl, and 5% BSA. The
recovered fractions were assayed for inhibin A and pro-C by
ELISA.
Validation of the fractionation method
The following studies were undertaken to establish whether
modifications to the molecular weight profiles of inhibin occurred by
either proteolysis or differential losses through the immunoaffinity
step or electroelution step. To establish whether inhibin was processed
during the immunoaffinity procedure, hFF was fractionated either
directly by Prep-PAGE or by mixing with inhibin-free serum, obtained by
immunoabsorption of normal nonpregnant female serum, and fractionated
through the immunoaffinity/Prep-PAGE procedure. Highly comparable
patterns of inhibin immunoactivity were obtained (data not shown) by
the two methods, indicating that processing was not evident during this
step. Recoveries of inhibin A and pro-C immunoactivities for the
series of sera and plasma are presented in Table 1
. These recoveries are comparable with
those obtained with the earlier fractionation method (7, 8).
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Inhibin A (ßA) ELISA. The method of Groome and O’Brien
(16) was employed with the human recombinant inhibin A
reference preparation (WHO 91/624) as standard, with modifications
(17). The ßA-subunit-directed
monoclonal antibody (Mab E4, aa 82–114 of the mature
ßA-subunit) was used as coating antibody,
whereas the Fab-alkaline phosphatase conjugate of monoclonal antibody
(R1) to the -subunit peptide (aa 1–32) was used as tracer. The
alkaline phosphatase activity was amplified using the AMPAK kit
(DAKO Corp., Carpinteria, CA). The assay procedure was
modified to measure inhibin A in electroelution buffer. Sample (100
µl) in electroelution buffer, buffer (100 µl), Groome assay buffer
(17), or standard (100 µl in buffer), followed by
hydrogen peroxide (10 µl, 30%), was incubated overnight at room
temperature. The steps following in the assay protocol remained
unchanged.
Pro-C-subunit ELISA
The method of Groome et al. (18) was
used. The monoclonal antibody INPRO directed against the entire pro
region was used as capture antibody, and Fab-complexed monoclonal
antibody (R1) to the -subunit was used as label. The assay procedure
used was similar to that presented for the inhibin A ELISA (above)
without the addition of hydrogen peroxide.
C immunofluorometric assay (IFMA)
The C IFMA, which detects all -subunit-containing forms,
has been used previously in serum inhibin fractionation studies
(8). In the present study it was noted that the
electroelution buffer interfered in the C IFMA with a marked loss of
assay sensitivity. Each fraction was dialyzed against water (3 d at 4
C), followed by a reverse phase HPLC step to remove the SDS. The sample
was then lyophilized, reconstituted in assay buffer, and assayed using
30K inhibin A (WHO 91/624) as standard.
Serum/plasma incubation with iodinated 30K inhibin A
Serum (4 µl) or plasma (4 µl) was incubated with 100 mmol/liter phosphate buffer, 0.1% BSA, and 0.1% NaN3, pH 7.2 (36 µl), containing 30,000 cpm iodinated 30K inhibin A for 24, 48, or 72 h at room temperature. These serum/plasma samples had been collected and stored under the same conditions described above. An aliquot was then fractionated on SDS-PAGE in Tris/Tricine buffers using a Mini Protean Apparatus (Bio-Rad Laboratories, Inc.). The gel was dried, and the molecular weight pattern of radioactivity was visualized using a phosphoautoradiograph and phosphorimaging system (Molecular Dynamics, Inc., Sunnyvale, CA).
Data analysis
The molecular weights of the eluted Prep-PAGE fractions were calculated using protein standard markers (soybean trypsin inhibitor, 23.5K; bovine carbonic anhydrase, 29.5K; BSA, 82K; Sigma), which bracketed the 20–90K molecular weight range of interest. Regression analysis (fraction number vs. log molecular weight) of these three markers gave correlation coefficients greater than 0.995. The resulting line of best fit was used to determine the molecular weight of the eluted fractions. As a measure of precision, the molecular weight determinations of the above protein standard markers when measured against the line of best fit were within 5% of their stated value.
The profiles of immunoactivity were assessed for clear evidence of
peaks. Shoulders were not considered, and only major peaks (>10%
of recovered activity) were considered. The molecular weight
values of peak tubes of immunoactivity from multiple runs were combined
(mean ± SD), and from their distribution activity
regions were defined (Table 2). The
immunoactivity levels/fraction are also presented as a percentage of
recovered activity (Table 2 and
Figs. 2–7). Changes in the proportions
of inhibin A and pro-C in the less than 30K and more than 40K, and
the less than 25K and more than 40K molecular weight regions,
respectively, were analyzed using Spearman’s rank correlation
(StatView 4.1, Abacus Concepts, Inc., Berkeley, CA).
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Validation of the fractionation procedure
The molecular weight profiles of inhibin A and pro-C
immunoactivity in recombinant human 30K inhibin A, IVF serum, and the
pregnancy serum pool after fractionation by the new procedure are
presented in Fig. 1. The patterns of
immunoactivity seen with IVF serum were similar to those published
previously (7, 8), showing various molecular weight forms
consistent with partially processed and processed forms of inhibin A
and inhibin -subunit containing the pro-C fragments. Some
differences were observed between studies, particularly in the high
molecular region; however, these differences are thought to be
encompassed within the normal distribution of inhibin molecular wt
forms for different IVF serum samples.
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C in the pregnancy
serum pool showed primarily the mature 30–36K inhibin A and 25–36K
pro-C forms, with differences in molecular weight within inhibin
forms attributable to glycosylation differences in the -subunit.
As a measure of the reproducibility and precision of the fractionation
procedure, five replicate immunoaffinity/Prep-PAGE/electroelution runs
of the same pregnancy pool were undertaken as quality control samples
at various stages throughout the study. To compare chromatograms, the
inhibin A and pro-C patterns were divided into regions based on the
molecular weight of the peak regions, and the immunoactivity values
were calculated as a percentage of the recovered activity (Fig. 2
). The combined analysis of data from
the five runs showed a high reproducibility, with the coefficient of
variation of recovered activity for the various molecular weight
regions ranging from 10–20% (Fig. 2). The coefficient of variation of
molecular weight calculated for the peak tubes of immunoactivity for
the 5 runs was less than 10% (Table 2).
Pregnancy serum
The patterns of inhibin A and pro-C immunoactivity were
determined in seven individual serum pools collected at specific stages
throughout normal pregnancy (Fig. 3).
Similar molecular weight forms of inhibin A and pro-C were
identified throughout pregnancy (as presented in
Table 2), although inhibin A forms with apparent molecular weight less
than 30K and pro-C forms less than 25K (i.e. molecular
weight less than the recognized molecular weight for 30K recombinant
human inhibin and pro-C) were identified in late pregnancy (>19 wk;
Fig. 3 and Table 2). The profile of -subunit-containing forms in one
pool of late pregnancy serum, as determined by the C IFMA, also
showed elevated levels of immunoactivity in the low molecular weight
regions (Fig. 4).
To investigate the change in molecular weight distribution across
pregnancy, the molecular weight profile for inhibin A and pro-C was
divided into three molecular weight regions [inhibin A (<30K,
30–40K, >40K) and pro-C (<25K, 25–40K, >40K); Figs. 5 and 6]. A
marked change in the proportion of forms
less than 30K inhibin A and less than 25K pro-C in midpregnancy was
observed, with the proportion of immunoactivity recovered increasing
from less than 5% before 19 wk to approximately 25% after 19 wk
(Figs. 5 and 6). The data suggest that the small molecular weight
inhibin A and pro-C forms originate from the more than 40K region in
both cases. A Spearman’s rank correlation was undertaken comparing the
less than 30K and more than 40K inhibin A, and the less than 25K and
more than 40K pro-C forms, respectively. A significant correlation
(r = -0.89; P = 0.012) was observed for inhibin
A, but not pro-C (r = 0.04; P = 0.93). Based on
this apparent shift in molecular weight at 19 wk, the molecular weight
patterns for serum pools obtained before and after 19 wk of gestation
were separately combined, and the pooled data are presented in Fig. 4.
Pregnancy serum vs. plasma
One explanation for the apparent reduction in molecular weight of
inhibin A and pro-C during late pregnancy is the effect of
proteolysis induced during serum collection and storage. The pregnancy
serum pools studied were collected as part of a clinical service and
hence stored at 4 C for up to 48 h before storage of serum at -20
C. Under these storage conditions, cleavage may occur. To explore this
possibility further, fresh serum and plasma obtained under rapid and
chilled collection conditions were obtained from women during early
(13–18 wk) and late (two separate pools, 28–39 and 31–36 wk)
pregnancy and fractionated as described above. As seen in
Figs. 5–7, inhibin A and pro-C profiles in
early pregnancy serum and plasma showed little change in molecular
weight pattern. However, in late pregnancy, plasma showed lower levels
of less than 30K inhibin A and pro-C compared with the original
serum profiles, but not as low as that seen during early pregnancy
(
Figs. 5–7). The proportion of more than 40K forms was less affected
between serum and plasma.
In an attempt to confirm that serum contains proteases that cleave inhibin, iodinated 30K inhibin was incubated with serum or plasma obtained from early and late pregnancy under various conditions. No changes in molecular weight of iodinated inhibin were seen under any of the incubation conditions used (data not shown).
Discussion
The objective of this study was to establish whether the molecular
weight profiles of inhibin A and pro-C in serum were modified during
human pregnancy. The results show that there is a major increase in the
proportion of small molecular weight forms of inhibin A and pro-C in
maternal serum during late pregnancy compared with early pregnancy. The
observation that there is less of the small molecular weight forms in
plasma collected under more stringent conditions suggests that a late
pregnancy blood protease is responsible in part.
The structures of the small molecular weight forms of inhibin A and
pro-C present in late pregnancy are unclear. The similar changes in
molecular weight pattern during pregnancy for inhibin A and pro-C
suggest that there is a common mechanism probably related to cleavage
of the -subunit. Mature 30K inhibin consists of two known forms (32K
and 34K), the differences attributed to mono- and diglycosylated forms
of the - subunit (**ßA and
*ßA) (4, 19). In the present
study these forms, with apparent molecular weight of 31–32K and 37K,
were present throughout pregnancy. Similarly, pro-C, with apparent
molecular weight of 32K and 27K, most likely refer to mono- and
diglycosylated pro-C forms. What are the likely structures of the
smaller forms? One possibility is that the inhibin A form with apparent
molecular weight of 27.2K found in late pregnancy serum is
deglycosylated inhibin A. However, there is no ready explanation for
the much smaller inhibin A form with apparent molecular weight of
21.7K. It should be noted that these forms were determined by an
inhibin A ELISA using as one of the two antibodies, an
-subunit-directed monoclonal antibody (R1), where the epitope is
located at the N-terminus of the C region (17), and the
ßA-subunit- directed antibody. Although
<30K inhibin forms have been isolated from human and testicular
sources in which the terminal 17 aa of the N-terminus of the C
region has been deleted (20, 21), the reduction in length
is insufficient to account for the reduced size. Furthermore, if a
larger portion of the C region was excised (i.e. terminal
32 aa), the epitope region for the - subunit monoclonal antibody
also would be excised. A similar situation applies with pro-C where
apparent molecular weight forms of 22.3K and 20.1K were identified. One
would need to postulate that to account for the marked reduction in
size of both inhibin A and pro-C, either an internal sequence of the
C region has been deleted or a large portion of the N-terminal
region of the -subunit has been cleaved, and the -subunit epitope
is still present at least in part. Cleavage of the pro sequence, the
carboxyl-terminus (although disulfide linked), and
ßA-subunit cannot be excluded. Reduced
glycosylation by the placenta leading to deglycosylated, and therefore
reduced, sizes of inhibin -subunit is also a possibility, but is
unproven.
It is of interest to note that the low molecular weight -subunit
forms were readily detected by the C IFMA. This assay employed
polyclonal antiserum directed to multiple epitopes on the -subunit
(22) and may detect forms not seen with the ELISAs.
Evidence of small molecular weight forms of inhibin have been detected
previously in fractionated postmenopausal plasma (8).
These forms were attributed in part to the presence of the free
-subunit, although it may also represent small molecular weight
forms as found in pregnancy plasma.
Studies assessing the forms of inhibin in placental extracts after extensive purification (23) identified 32K and 33K dimeric inhibin as the major forms with no evidence of smaller molecular weight forms. These data suggest that the smaller inhibins observed in the present study may be produced in the circulation. Fractionation of pregnancy serum by gel filtration (9) showed that the majority of inhibin A immunoactivity was present in the 30K range, although this fractionation method has limited resolution and may not readily detect relatively low levels of smaller molecular weight forms.
A number of studies have shown the presence of increased proteolytic activity in pregnancy serum. A serum protease that cleaved IGF-binding proteins 4 and 5 was stimulated by pregnancy with highest levels in late gestation (24, 25). Similarly, an hCG ß-subunit nicking enzyme (26) responsible for the formation of degraded hCG has been specifically detected in pregnancy serum. Although microbial cleavage in stored samples (27) is possible, why this should change through gestation is not clear.
The normal cleavage of inhibin at dibasic peptide sites (pro-N,
N-C, proß-ß regions) is believed to be caused by
intracellular furin-like serine peptidases. Thus, the cleavage of
precursor forms (>40K) observed in late pregnancy may be attributed to
the effects of placental furins. Previous studies by this group
(28) showed that 58K bovine inhibin A (N-C/ßA) was
cleaved in serum to form 30K inhibin by serine peptidase-like activity
with pH and inhibitor specificity similar to those of furin
(29), but that 30K inhibin itself was not further
metabolized by serum. The observation that 30K inhibin was not cleaved
in that study is similar to that of the present study. It is also
interesting to note that a significant correlation was observed between
the less than 30K and more than 40K regions for inhibin A, but not
pro-C, suggesting that the larger molecular weight forms of inhibin,
rather than the 30K inhibin forms, are the precursors to the less than
25K inhibin forms. Thus, the decrease in high molecular weight forms of
inhibin observed in late pregnancy may occur in either placenta or
circulation.
The possibility that artifacts in the fractionation procedure may be
responsible for formation of the small molecular weight forms across
pregnancy was countered by the addition of serine protease inhibitors
to the initial serum/plasma samples and at various stages throughout
the fractionation procedure. To test whether cleavage of
inhibin/pro-C may occur during the initial immunoaffinity step, a
study was undertaken in which hFF was fractionated either directly
through Prep-PAGE or added to inhibin-depleted serum and processed
through the immunoaffinity column followed by Prep-PAGE. A similar
molecular weight pattern was observed in both cases, indicating that
this possibility was unlikely.
The overall recovery of inhibin activity throughout the various purifications steps ranged from 25–32% with recoveries of 50–60% in the immunoaffinity step and 45–58% in Prep-PAGE/electroelution step. These reduced recoveries are not attributed to differential losses of particular inhibin forms at particular steps. For example, undetectable inhibin immunoactivity was observed in the immunodepleted serum, indicating that more than 95% of the inhibin was bound to the immunosupport. The losses at the Prep-PAGE step have been previously shown (30) to be similar for both high and low molecular weight forms. The observed losses are thus attributed to nonspecific/irreversible binding to the various supports and the difficulty in obtaining quantitative recoveries in the electroelution step due to limitations in the methodology.
Future studies will need to address the biological significance of
these small molecular weight forms during pregnancy. At this stage, it
is unclear whether the increase in low molecular weight forms may be
attributed to the normal progression of pregnancy or some other
undefined clinical difference in the patient groups that is associated
with altered inhibin processing. It is also unclear whether the small
molecular weight forms of inhibin A are bioactive, nor is it clear
whether these forms of both inhibin A and pro-C are adequately
detected by current immunoassay methods.
Acknowledgments
We thank Biotech Australia for providing the recombinant human inhibin A.
Footnotes
This work was supported by a program grant (98/3212) from the National Health and Medical Research Council of Australia and the Sylvia and Charles Viertel Charitable Foundation (E.W.).
Abbreviations: aa, Amino acids; AcN, acetonitrile; hFF, human follicular fluid; IFMA, immunofluorometric assay; IVF, in vitro fertilization; Prep-PAGE, preparative PAGE; TFA, trifluoroacetic acid.
Received October 18, 2000.
Accepted August 23, 2001.
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implications for radioimmunoassay. J Clin Endocrinol Metab 70:1208–1212[Abstract]
-ßA dimer) during human pregnancy. Clin
Endocrinol (Oxf) 42:391–397[Medline]
C and follistatin during
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and activin A in early pregnancy. Hum Reprod 12:1089–1093
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forms in human serum by a new ultrasensitive two-site enzyme-linked
immunosorbent assay. J Clin Endocrinol Metab 80:2926–2932 subunit ELISA with broad specificity. Mol Cell Endocrinol 180:79–86[CrossRef][Medline]
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) collected routinely in
an antenatal clinic and three pools of matched serum (
) and plasma
(
) collected under defined conditions. The pools were fractionated
through the combined immunoaffinity/Prep-PAGE procedure.
) samples, collected
under defined conditions (see Materials and Methods).
The pools were fractionated through the combined
immunoaffinity/Prep-PAGE procedure. The vertical dashed
line refers to the molecular weight of 31K recombinant inhibin
A, which was used as reference.