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Measurement of inhibin A and inhibin pro-αC in early human pregnancy and their role in the prediction of pregnancy outcome in patients with recurrent pregnancy loss
FERTILITY AND STERILITY威 VOL. 80, NO. 6, DECEMBER 2003 Copyright ©2003 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.
Measurement of inhibin A and inhibin pro-␣c in early human pregnancy and their role in the prediction of pregnancy outcome in patients with recurrent pregnancy loss Majedah Al-Azemi, M.D.,a William L. Ledger, M.D.,b,d Michael Diejomaoh, M.D.,a Mohamed Mousa, Ph.D.,c Ma’asoumah Makhseed, M.D.,a and Alexander Omu, M.D.a Faculty of Medicine, Kuwait University, Safat, Kuwait, and University of Sheffield, Sheffield, United Kingdom
Received August 22, 2002; revised and accepted April 25, 2003. This study was supported by facilities provided by Project Grant MO 02/00 of the Kuwait University Research Administration, Kuwait. Reprint requests: Majedah Al-Azemi, M.D., Department of Obstetrics and Gynecology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait (FAX: 965-5338906; E-mail: [email protected]. edu.kw). a Department of Obstetrics and Gynecology, Faculty of Medicine, Kuwait University. b Academic Department of Obstetrics and Gynecology, University of Sheffield. c Community Medicine Department, Faculty of Medicine, Kuwait University. d Head of Section of Reproductive and Developmental Medicine, Obstetrics and Gynecology, University of Sheffield. 0015-0282/03/$30.00 doi:10.1016/S0015-0282(03) 02215-5
Objective: To examine the temporal relationship among inhibin A, -hCG, and pro-␣C in early pregnancy and to determine whether the measurement of these hormones has any role in prediction of pregnancy outcome in patients with recurrent spontaneous miscarriage. Design: Prospective descriptive study. Setting: A tertiary referral center for recurrent miscarriage. Patient(s): Thirty-six pregnant women with previous history of recurrent spontaneous pregnancy loss. Intervention(s): Serial blood samples were collected prospectively at 6, 8, 10, and 12 weeks of gestation and were analyzed for inhibin A and inhibin pro-␣c using a two-site enzyme-linked immunosorbent assay as well as for -hCG using the microparticle enzyme immunoassay. Main Outcome Measure(s): Serum levels of inhibin A, inhibin pro-␣c, and -hCG. Result(s): The patients were allocated to two groups according to the pregnancy outcome: group 1 consisted of patients whose pregnancy continued beyond 20 weeks (control group); and group 2 consisted of patients who spontaneously aborted (aborted group). There was a significant difference in inhibin A concentrations between the control and aborted groups at 8, 10, and 12 weeks’ gestation. Significant differences in -hCG concentrations between the two groups is evident only at 10 weeks’ gestation. There were no significant differences in inhibin pro-␣c concentrations between the two groups at any gestational age. Assessment of the trend in the control group over the study period showed a significant increase in inhibin A and -hCG but not inhibin pro-␣c levels. Conclusion(s): Low serum levels of inhibin A at early gestational age in pregnancies destined to miscarry suggest a role for this glycoprotein as a marker for early pregnancy viability. Its measurement at the time of the first pregnancy test might be able to predict pregnancy outcome. (Fertil Steril威 2003;80:1473–9. ©2003 by American Society for Reproductive Medicine.) Key Words: Inhibin A, inhibin pro-␣c, pregnancy outcome, recurrent miscarriage
Recurrent spontaneous miscarriage, usually defined as a loss of three or more consecutive pregnancies, affects 1% of pregnant women (1). It is a worldwide obstetric problem, which has a variety of etiological factors. The etiology of recurrent spontaneous miscarriage requires comprehensive evaluation and investigations and poses a major challenge to the treating obstetrician.
factor in 16%, and infectious factor in 0.5%. Forty percent were classified as unexplained. Counseling is an important part in the management and should include an explanation of the possible underlying causes of the condition. More important is the prognosis of the current pregnancy. Research into the methods for prediction and early detection of pregnancy failure has generated great interest.
In a large prospective cohort study by Stephenson et al. (2), genetic factor was identified in 3.5%, endocrine factor in 20%, anatomical
Current available methods for detection of pregnancy failure include ultrasound scan and serum -hCG estimation. Transvaginal ultra1473
sound has become an important tool for the examination of early pregnancy viability. Indeed, the use of high-frequency transducers has improved the image quality and facilitated the measurement of various parameters of the embryo structures. However, it is possible to diagnose potentially abnormal growth only when a significant departure from normal measurements is found, which usually occurs over weeks.
abnormalities in early pregnancy concentrations of this glycoprotein might allow recognition of a group of patients with luteal phase defect as a cause of their recurrent spontaneous miscarriages and who therefore might benefit from luteal phase support. These factors have not been investigated previously in this high-risk group of patients.
Another method for detection of early pregnancy failure is monitoring serum -hCG. This hormone reflects trophoblastic activity rather than fetal demise, and although it does fall with failing pregnancy, it is not completely reliable. Evaluation of CA-125 level as well has been reported to improve its predictive value (3). In addition, -hCG has a slow total elimination rate and it is detectable in the serum 21 days postpartum (4). An ideal alternative would be a substance secreted mainly by the viable fetoplacental unit, probably mainly fetal, with a short half-life. Could inhibin be this alternative?
MATERIALS AND METHODS
Recently, significant research has focused on inhibins and their roles in female reproduction, including pregnancy failure. Inhibins are glycoproteins that belong to the transforming growth factor- (TGF-) superfamily (5). Inhibins are heterodimers consisting of disulphide-linked ␣ (mol.wt 18 K Da) and  (mol.wt 14 K Da) subunits: inhibin A (␣-A) and inhibin B (␣-B). Only the dimeric forms of inhibin are bioactive, although the ␣-subunits circulate in vast excess as biologically inert monomers. Serum concentration of immunoreactive (ir) inhibin has been reported to be higher during various stages of pregnancy compared with nonpregnant subjects (6 –10). In those studies, ir-inhibin was measured using radioimmunoassay, which cross-reacted extensively with different inhibin ␣-subunit forms. Consequently, the contribution of the biologically active dimeric inhibin forms was unknown. Development of specific and sensitive assays for dimeric inhibins has facilitated the measurement of these proteins throughout human pregnancy (11). Inhibin A is the major circulating form of inhibin in human pregnancy. Many efforts were made to identify the source of different forms of inhibins in early pregnancy. It was found that the fetoplacental unit is the major source of inhibin A (12, 13). Inhibin pro-␣c, on the other hand, circulates in great excess as a functionally inactive monomer with its major source being corpus luteum (14). In this study, our aim was to investigate a possible role for inhibins as markers of early pregnancy viability in patients with recurrent spontaneous miscarriage. Early diagnosis of a complicated or poor pregnancy outcome could aid in counseling and management of this group of patients. In addition, this study compared concentrations of inhibin A with -hCG in early pregnancy since -hCG is another pregnancy hormone that correlates with pregnancy viability. Finally, as inhibin pro-␣c is mainly of corpus luteum origin, identifying 1474 Al-Azemi et al.
Inhibins and recurrent pregnancy loss
Subject Selection and Serum Samples Blood samples were collected from 36 pregnant women with a previous history of three or more consecutive first trimester spontaneous miscarriages. Serial blood samples were collected prospectively at 6, 8, 10, and 12 weeks of gestation. The blood was allowed to clot. The sera were separated (centrifugation for 10 minutes at 3,000 rpm) and stored at ⫺80°C before analysis. Ultrasound scan was performed at each time of blood collection for viability and then at 20 weeks’ gestation for anomaly scan. All patients received luteal phase support in the form of P pessaries, 400 mg twice a day per vaginum (Cyclogest, Shire, Andover, UK) in addition to low-dose aspirin (80 mg daily) with or without heparin. The study was approved by the ethics committees of the Faculty of Medicine and the Maternity Hospital. Informed consent was obtained from the subjects according to the guidelines of the Hospital Ethics Committee. Stored samples were later assayed for inhibin A, inhibin pro-␣c, and -hCG estimations.
Hormone Assays Inhibin A Serum inhibin A was measured using a two-site enzymelinked immunosorbent assay (ELISA) (14). Before the assay, patient samples and standards were pretreated with sodium dodecyl sulphate, heated to 100°C and exposed to hydrogen peroxide to enhance the specificity and sensitivity. Wells of a microtitre plate were dry coated with a monoclonal antibody specific for the A subunit of inhibin. Samples (including standards, appropriate controls, etc.) were incubated overnight at room temperature for the antigenantibody reaction. The wells were washed repeatedly to remove any excess unbound antigen. This was followed by the addition of a monoclonal, second antibody specific for the ␣ subunit of inhibin coupled with alkaline phosphatase and incubated at room temperature for 1 more hour. Any unreacted material was then removed by washing before the detection of alkaline phophatase using a sensitive amplified substrate reaction included in the kit. The resulting red color was measured in a spectrophotometer at 490 nm, the intensity of which was directly proportional to the concentration of inhibin A present in the original sample. Vol. 80, No. 6, December 2003
The intra- and interassay coefficients of variation were 4.2% and 4.9%, respectively. The assay was highly specific for the dimeric molecules with minimal cross-reactivity to the C subunit, inhibin B, or activins. The minimum detection limit of this assay was 3.9 pg/mL (inhibin A: dimer assay [ultrasensitive]; Oxford Bio-Innovation, Oxfordshire, UK). Inhibin Pro-␣c
TABLE 1 Patient characteristics. Features
Minimum Maximum
Mean ⫾ SD or median
This was measured using a Microparticle Enzyme Immunoassay (AXSYM system) (Reagent Pack: 7A59-22; Abbott Laboratories, IL). The minimum detection limit of this assay was 2 mIU/mL. The intra- and interassay coefficients of variation were 3.6% and 3%, respectively.
Age (yrs) Group 1 Group 2 Gestational age (wks) Group 1 Group 2 No. of previous deliveries Group 1 Group 2 No. of previous abortions Group 1 Group 2 Birth weight (grams) Group 1
Statistical Analysis
Note: Group 1 ⫽ control group; group 2 ⫽ aborted group.
The patients were allocated to two groups according to the outcome of their pregnancy: group 1 (control group ⫽ 24) consisted of patients whose pregnancy continued beyond 20 weeks; group 2 (aborted group ⫽ 12) consisted of patients who aborted before 20 weeks
Al-Azemi. Inhibins and recurrent pregnancy loss. Fertil Steril 2003.
Serum inhibin pro-␣c was also measured using a two-site enzyme-linked immunosorbent assay as above (15). The minimum detection limit of this assay was 2 pg/mL. The intra- and interassay coefficients of variation were 5.5% and 8.9%, respectively (inhibin pro-␣c, Oxford Bio-Innovation).
-hCG
Data were processed using the SPSS software package, version 11. The cutoff level for statistical significance was Pⱕ.05. Since the frequency distributions of the three measured glycoproteins were not normally distributed, we described our data as median and range (minimum, maximum) rather than mean and SD. Significance between the levels of these glycoproteins in aborters and controls at different gestational week points (6, 8, 10, and 12) was assessed using the nonparametric Mann-Whitney U test. The trend for change of their levels throughout the study period (6 –12 weeks’ gestation) was measured by the Friedman test for dependent repeated measures.
P
25 23
40 40
31.83 ⫾ 4.72 31.25 ⫾ 5.67
NS
29 9
41 19
38 12
⬍.0001
0 0
5 4
2 1
NS
3 3
10 18
5 5
NS
1,800
4,150
2,945.83 ⫾ 508.72
15 patients delivered vaginally, while nine delivered by cesarean section. There was one case of congenital abnormality (polycystic kidney). All pregnancies (24 women) were represented at all time points in the control group, while the aborted group was represented till the time of miscarriage (that is, 12, 12, 7, and 4 women were represented at 6, 8, 10, and 12 weeks’
FIGURE 1 Number of women in group 1 (solid bar) and group 2 (open bar) at different time points during the study period.
To determine sensitivity and specificity of inhibin A and -hCG levels, a cutoff level of a multiple of 0.5 of the median of the control levels was used. If the level at a particular gestational week was below this cutoff level, there is a risk for abortion (sensitivity), while if the level exceeded the cutoff level, there is no risk for abortion (specificity).
RESULTS There was no significant difference between the two groups in age (31.83 ⫾ 4.64 vs. 31.25 ⫾ 5.48 years), number of previous deliveries or number of previous abortions, and onset of conception (all were spontaneous conceptions). The median gestational age at which the control group delivered was 38 weeks (range: 29 – 41 weeks), and the median gestational age at which the aborted group miscarried was 12 weeks (range: 9 –19 weeks) (Table 1). In the control group, FERTILITY & STERILITY威
Al-Azemi. Inhibins and recurrent pregnancy loss. Fertil Steril 2003.
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TABLE 2 Inhibin A (pg/mL), -hCG (mIU/mL), and inhibin pro-␣c (pg/mL) concentrations throughout the study period in the control and aborted groups presented as median (minimum, maximum). Variable Inhibin A: Group 1 Group 2 P -hCG: Group 1 Group 2 P Pro-␣c: Group 1 Group 2 P
6 Weeks
429.4 (26.9, 837.5) 234.5 (3.0, 474.0) .067 16,752.9 (372.1, 130,350.9) 6,318.7 (3,216.0, 27,883.5) NS 401.9 (2.0, 1,809.7) 694.8 (2, 867.9) NS
8 Weeks
10 Weeks
725.0 (3.0, 1,612.5) 162.5 (32.2, 387.5) ⬍.001
875.0 (120, 2,150.0) 121.3 (66.3, 387.5) .002
727.5 (117.5, 1,687.5) 135.0 (100.0, 262.5) .006
121,445.5 (45,286.5, 200,000) 65,112.8 (62.6, 120,721.4) .011
82,889.4 (33,996.1, 200,000) 83,435.7 (147.2, 200,000) .972
106,208.3 (2852.6, 199,661.8) 49,071.4 (47.7, 2,000,000) NS 509.7 (2, 2,013.6) 245.1 (2, 622.9) NS
12 Weeks
403.7 (2, 2,354.2) 295 (2, 633.7) NS
462.1 (2, 2,698) 354.3 (2, 540.3) NS
Note: Group 1 ⫽ control group; group 2 ⫽ aborted group. Al-Azemi. Inhibins and recurrent pregnancy loss. Fertil Steril 2003.
gestation, respectively). Figure 1 shows the total number of women who were enrolled and the dropout due to miscarriage at different time points. There was a significant difference in inhibin A concentrations between control and aborted groups at 8 (P⬍.001), 10 (P ⫽ .002), and 12 weeks’ gestation (P ⫽ .006). At 6 weeks’ gestation, there was a difference; however, this did not reach statistical significance (P ⫽ .067). Assessment of the difference in -hCG concentrations between control and aborted groups revealed a statistically significant difference only at 10 weeks’ gestation (P ⫽ .011). There was no significant difference in inhibin pro-␣c concentrations between control and aborted groups at each gestational age (Table 2). In the control group, there was a significant increase in inhibin A (P ⫽ .005), and -hCG (P⬍.001) over the study period (Friedman test). However, there was no statistically significant increase in inhibin pro-␣c concentrations over the study period. As we were losing cases in the aborted group, testing for a trend was not feasible because of the small number of cases (four cases at 12 weeks) (Figs. 2 and 3). In an attempt to determine a cutoff (threshold) level for inhibin A and -hCG to be used as a marker for predicting pregnancy outcome, we used a multiple of 0.5 of the median of the control level at each gestational age and we checked the sensitivity and specificity (Table 3). The inhibin A threshold gave better sensitivity and the same specificity at 8 weeks’ gestation (75% and 81%, respectively) than the -hCG threshold (50% and 81%, respectively). We also attempted to identify the point in our assay where we could identify abnormal pregnancies before an ultrasound does. Hence, when we further analyzed our data in the aborted group (group 2) at 10 weeks, we found that 5 of the 1476 Al-Azemi et al.
Inhibins and recurrent pregnancy loss
12 pregnancies had absent fetal heart sound on ultrasound. When we compared the inhibin A values of these 5 women at 6 and 8 weeks’ gestation to our cutoff level (0.5 multiple of the median (MOM) of controls), we found that 40% of these women had inhibin A levels ⬍0.5 MOM of controls at 6 weeks’ gestation (4 weeks earlier than ultrasound) and 80% of these women had inhibin A levels ⬍0.5 MOM of controls at 8 weeks’ gestation (2 weeks earlier than ultrasound).
DISCUSSION This study demonstrates that women with a history of recurrent spontaneous miscarriage (at least three previous consecutive first trimester pregnancy losses) have consistently lower concentrations of inhibin A in serum as early as 6 weeks’ gestation if the current pregnancy is destined to miscarry. Measurement of inhibin A, but not inhibin pro-␣c, in early pregnancy serum therefore offers the possibility of
TABLE 3 Sensitivity and specificity for cutoff level (0.5 ⫻ median) for inhibin A and -hCG at different gestational week points.
Inhibin A Sensitivity Specificity -hCG Sensitivity Specificity
Week 6
Week 8
Week 10
Week 12
50 73.3
75 81
85.7 82.6
100 77.3
66.7 66.7
50 81
42.9 87
25 95.5
Al-Azemi. Inhibins and recurrent pregnancy loss. Fertil Steril 2003.
Vol. 80, No. 6, December 2003
FIGURE 2 Distribution of inhibin A levels between group 1 (control group) (crosses and dashed line) and group 2 (aborted group) (bullets and dot-dashed line).
Al-Azemi. Inhibins and recurrent pregnancy loss. Fertil Steril 2003.
identifying viable and nonviable pregnancies earlier than transvaginal ultrasound. Although serum concentrations of -hCG prove to be lower in the pregnancy loss group, this difference was less striking than that observed for inhibin A and only reached statistical significance at 10 weeks’ gestation. We failed to observe any significant differences in concentrations of inhibin pro-␣c between the two groups. The findings of this study are consistent with those from previous studies. Inhibin concentrations in early singleton, multiple, and failing pregnancies were examined by Norman et al. (16). His study showed that in singleton pregnancies there was a significant increase in ir-inhibin concentrations in early pregnancy. Significantly higher concentrations were found in multiple pregnancies. Patients destined to abort showed no increase in ir-inhibin early in pregnancy. In that study, ir-inhibin was measured using radioimmunoassay, which cross-reacted extensively with different inhibin ␣-subunit forms. The use of the more specific and sensitive assay for inhibin A in our study facilitates the measurement of the biologically active form of this glycoprotein. To identify the source of inhibin A in early pregnancy, Birdsall et al. used the donor-egg model in conjunction with specific ELISA for inhibin A (13). They concluded that the FERTILITY & STERILITY威
fetoplacental unit was the major source of these polypeptides in early pregnancy. Lockwood et al. (12) investigated the source of inhibins in early pregnancies conceived in vitro with and without corpus luteum function. That study confirmed that the main source of dimeric inhibin A in early pregnancy is the fetoplacental unit, since comparable concentrations of inhibin A were found in singleton pregnancies arising from spontaneous conception and after IVF treatment with both fresh and frozen ETs. In the first case, a single corpus luteum would be expected; in the second case, multiple corpora lutea are routinely observed and in downregulated frozen embryo replacement, no cycle’s corpus luteum is present. The significantly higher concentration of inhibin A found in multiple pregnancies is further evidence for the production by the fetoplacental unit. The significant differences between concentrations of inhibin A in viable, nonviable, and multiple IVF pregnancies detectable as early as 13 days after ET in that study are further evidence for a fetoplacental source of inhibin A in early pregnancy and suggest that the estimations of inhibin A may be helpful in the management of early pregnancy (12). In a recent study, maternal serum inhibin A and -hCG concentrations in very early IVF pregnancy (16 days after 1477
FIGURE 3 Distribution of -hCG levels between group 1 (control group) (crosses and dashed line) and group 2 (aborted group) (bullets and dot-dashed line).
Al-Azemi. Inhibins and recurrent pregnancy loss. Fertil Steril 2003.
oocyte retrieval) were compared with pregnancy outcomes and treatment protocols in 237 women undergoing IVF and ET (17). Consistent with other studies, concentrations of inhibin A were found to be significantly higher in women with ongoing pregnancies than in nonpregnant women. However, contrary to others, the study demonstrated significantly lower concentrations of inhibin A in the absence of functioning ovaries, suggesting that the corpus luteum is a major source of circulating inhibin A in very early pregnancy. This could possibly be explained by the relatively small size of trophoblast mass at such an early gestation with a small contribution to maternal circulating inhibin A. Additionally, the study also showed, consistent with our findings, that there was no significant difference in -hCG concentrations between those with ongoing pregnancies and those later diagnosed as biochemical pregnancies, which suggests that low concentrations of serum inhibin A may be useful in identifying -hCG–positive preclinical “biochemical” pregnancies. Illingworth et al. (18) in 1996 showed that inhibin A levels rose steadily during the conception luteal phase to an initial peak 12 days after ovulation (4 weeks), then rose rapidly to a further peak 43 days after ovulation (8 weeks). 1478 Al-Azemi et al.
Inhibins and recurrent pregnancy loss
Similar observations were made in our study in the control group with the peak in our study at 10 weeks gestation. Illingworth et al. (18) found that exogenous hCG resulted in a significant rise in the concentrations of inhibin A and inhibin pro-␣c. None of our patients received hCG for luteal phase support. Inhibin pro-␣c circulates in great excess as a functionally inactive monomer. The corpus luteum is the major source of this form during pregnancy (12). The presence of comparable levels of inhibin pro-␣c in both control and aborted groups in our study makes it unlikely that the fetoplacental unit is a major contributor to circulating levels of this glycoprotein in pregnancy and supports the hypothesis that this form is mainly produced by the corpus luteum. Interestingly, in a recent study it was found that inhibin pro-␣c is secreted from cultured explants from chorion leave and placenta (19). However, how much these sources contribute to the maternal serum concentration of this glycoprotein in early pregnancy is still to be determined. Our study showed no significant differences in the level of -hCG at 6 and 8 weeks between the aborted and control groups. The difference was evident only at 10 weeks’ gesVol. 80, No. 6, December 2003
tation. In comparison, inhibin A levels are significantly lower between the aborted and control groups as early as 6 weeks and onward. This can be explained by the longer half-life of -hCG compared with inhibin A. Lower concentrations of inhibin A as early as 6 weeks’ gestation implies that serial blood testing may not be cost-effective, and single measurement at 6 weeks’ gestation may be equally effective in predicting pregnancy outcome. These findings were also found by Norman et al. (16), who examined the endocrine changes in very early pregnancy. There were no statistical differences in -hCG levels early in pregnancy between viable singleton pregnancies and those pregnancies destined to miscarry. The biological roles of inhibin A and inhibin pro-␣c in early pregnancy are unknown. Inhibin A has been shown to regulate GnRH, hCG, and P secretion from human placental cells in vitro (20). The function is probably related to cellular differentiation and embryogenesis. Activins were found to induce mesoderm in embryonic explants (21). Inhibins probably have some role in directing cell lineage. The continued secretion of inhibin A from the fetoplacental unit throughout pregnancy (22) suggests the possibility of inhibin A having different functions throughout pregnancy. In conclusion, our study demonstrates a possible role of inhibin A as a marker for predicting pregnancy outcome in high-risk pregnancy. It fails to demonstrate any role for inhibin pro-␣c as a prognostic tool. The expanding knowledge of the role of inhibins since the development of specific and sensitive assays has opened the field for their potential uses in clinical practice. Further research on these glycoproteins in diagnosis, prediction, and monitoring of pathological human pregnancies is required. Improvement in pregnancy outcome in patients with recurrent miscarriage has been shown to follow early ultrasound demonstration of fetal heart activity (23). Measurement of serum inhibin A at the time of the first positive pregnancy test might be able to predict pregnancy outcome (12). Very early identification of a potentially viable pregnancy might further enhance the benefits of such knowledge to pregnancy outcome. A randomized trial of this intervention is planned.
Acknowledgments: The authors thank Tunde Fatinikum, Rawia Abdulmonem, and Susan George for collecting and processing blood samples. The assistance of Mrs. Asiya Tasnim in preparing the manuscript is gratefully acknowledged.
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References 1. Clifford K, Rai R, Watson H, Regan L. An informative protocol for the investigation of recurrent miscarriage: preliminary experience of 500 consecutive cases. Hum Reprod 1994;9:1328 –32. 2. Stephenson MD. Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril 1996;66:24 –9. 3. Predanic M. Differentiating tubal abortion from viable ectopic pregnancy with serum CA-125 and -human chorionic gonadotropin determinations. Fertil Steril 2000;73:522–5. 4. Korhonen J, Alfthan H, Ylostalo P, Veldhuis J, Stenman UH. Disappearance of human chorionic gonadotropin and its alpha- and betasubunits after term pregnancy. Clin Chem 1997;43:2155–63. 5. Kingsley DM. The TGF- superfamily: new members, new receptors and new genetic tests of function in different organisms. Genes Dev 1994;8:133–46. 6. Mclachlan RI, Healy DI, Robertson DM, Burger HG, de Kretser DM. Circulating immunoreactive inhibin in the luteal phase and early gestation in women undergoing ovulation induction. Fertil Steril 1987;48: 1011–5. 7. Abe Y, Hasegawa Y, Miyamoto K, Yamaguchi M, Andoh A, Ibuki Y, et al. High concentrations of plasma immunoreactive inhibin during normal pregnancy in women. J Clin Metab 1990;71:133–7. 8. Tabie T, Ochiai K, Terashima Y, Takanashi N. Serum levels of inhibin in maternal and umbilical blood during pregnancy. Am J Obstet Gynaecol 1991;164:896 –900. 9. Baird DT, Smith KB. Inhibin and related peptides in the regulation of reproduction. Oxf Rev Reprod Biol 1993;15:191–232. 10. Tovanabutra S, Illingworth PJ, Ledger WL, Glasier AF, Baird DT. The relationship between peripheral immunoreactive inhibin, human chorionic gonadotrophin, oestradiol and progesterone during human pregnancy. Clin Endocrinol 1993;38:101–7. 11. Muttukrishna S, George L, Fowler PA, Groome NP, Knight PG. Measurement of serum concentrations of inhibin A during human pregnancy. Clin Endocrinol (Oxf) 1995;42:391–7. 12. Lockwood GM, Ledger WL, Barlow DH, Groome NP, Muttukrishna S. Measurement of inhibin and activin in early pregnancy: demonstration of fetoplacental origin and role in prediction of early pregnancy outcome. Biol Reprod 1997;57:1490 –4. 13. Birdsall M, Ledger WL, Groome NP, Abdulla H, Muttukrishna S. Inhibin A and activin A in the first trimester of human pregnancy. J Clin Endocrinol Metab 1997;82:1557–60. 14. Muttukrishna S, Fowler PA, Groome NP, Michell GG, Robertson WR, Knight PG. Serum concentration of dimeric inhibin during the spontaneous human menstrual cycle and after treatment with exogenous gonadotrophin. Hum Reprod 1994;9:1634 –42. 15. Groome NP, Illingworth PJ, O⬘Brien M, Priddle J, Weaver K. Quantification of inhibin pro-␣ C containing forms in human serum by a new ultrasensitive enzyme linked immunosorbent assay. J Clin Endocrinol Metab 1995;80:2926 –32. 16. Norman RJ, Mcloughlin JW, Borthwick GM, Yohkaichiya T, Matthews CD, MacLennan AH, et al. Inhibin and relaxin concentrations in early singleton, multiple, and failing pregnancy: relationship to gonadotropin and steroid profiles. Fertil Steril 1993;59:130 –7. 17. Treetampinich C, O⬘Conner AE, MacLachen V, Groome NP, de Kretser DM. Maternal serum inhibin A concentrations in early pregnancy after IVF and embryo transfer reflect the corpus luteum contribution and pregnancy outcome. Hum Reprod 2000;15:2028 –32. 18. Illingworth PJ, Groome NP, Duncan WC, Grant V, Tovanbutra S, Baird DT, et al. Measurement of circulating inhibin forms during the establishment of pregnancy. J Clin Endocrinol Metab 1996;81:1471–5. 19. Riley SC, Leask R, Balfour C, Brenand JE, Groome NP. Production of inhibin forms by the fetal membranes, deciduas, placenta and fetus at parturition. Hum Reprod 2000;15:578 –83. 20. Petraglia F, Sawchenko P, Lim AT, Rivier J, Vale W. Localisation, secretion and action of inhibin in the human placenta. Science 1987; 237:187–9. 21. Hemmati-Brivanlou A, Melton DA. A truncated activin receptor inhibits mesoderm induction and formation of axial structures in Xenopus embryos. Nature 1992;359:586 –7. 22. Muttukrishna S, Child TJ, Groome NP, Ledger WL. Feto-placental unit is the major source of inhibin A and activin A in early pregnancy. Hum Reprod 1996;11:155 (abstract). 23. Clifford K, Rai R, Regan L. Future pregnancy outcome in unexplained recurrent first trimester miscarriage. Hum Reprod 1997;12:387–9.
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Measurement of inhibin A and inhibin pro-␣c in early human pregnancy and their role in the prediction of pregnancy outcome in patients with recurrent pregnancy loss Majedah Al-Azemi, M.D.,a William L. Ledger, M.D.,b,d Michael Diejomaoh, M.D.,a Mohamed Mousa, Ph.D.,c Ma’asoumah Makhseed, M.D.,a and Alexander Omu, M.D.a Faculty of Medicine, Kuwait University, Safat, Kuwait, and University of Sheffield, Sheffield, United Kingdom
Received August 22, 2002; revised and accepted April 25, 2003. This study was supported by facilities provided by Project Grant MO 02/00 of the Kuwait University Research Administration, Kuwait. Reprint requests: Majedah Al-Azemi, M.D., Department of Obstetrics and Gynecology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait (FAX: 965-5338906; E-mail: [email protected]. edu.kw). a Department of Obstetrics and Gynecology, Faculty of Medicine, Kuwait University. b Academic Department of Obstetrics and Gynecology, University of Sheffield. c Community Medicine Department, Faculty of Medicine, Kuwait University. d Head of Section of Reproductive and Developmental Medicine, Obstetrics and Gynecology, University of Sheffield. 0015-0282/03/$30.00 doi:10.1016/S0015-0282(03) 02215-5
Objective: To examine the temporal relationship among inhibin A, -hCG, and pro-␣C in early pregnancy and to determine whether the measurement of these hormones has any role in prediction of pregnancy outcome in patients with recurrent spontaneous miscarriage. Design: Prospective descriptive study. Setting: A tertiary referral center for recurrent miscarriage. Patient(s): Thirty-six pregnant women with previous history of recurrent spontaneous pregnancy loss. Intervention(s): Serial blood samples were collected prospectively at 6, 8, 10, and 12 weeks of gestation and were analyzed for inhibin A and inhibin pro-␣c using a two-site enzyme-linked immunosorbent assay as well as for -hCG using the microparticle enzyme immunoassay. Main Outcome Measure(s): Serum levels of inhibin A, inhibin pro-␣c, and -hCG. Result(s): The patients were allocated to two groups according to the pregnancy outcome: group 1 consisted of patients whose pregnancy continued beyond 20 weeks (control group); and group 2 consisted of patients who spontaneously aborted (aborted group). There was a significant difference in inhibin A concentrations between the control and aborted groups at 8, 10, and 12 weeks’ gestation. Significant differences in -hCG concentrations between the two groups is evident only at 10 weeks’ gestation. There were no significant differences in inhibin pro-␣c concentrations between the two groups at any gestational age. Assessment of the trend in the control group over the study period showed a significant increase in inhibin A and -hCG but not inhibin pro-␣c levels. Conclusion(s): Low serum levels of inhibin A at early gestational age in pregnancies destined to miscarry suggest a role for this glycoprotein as a marker for early pregnancy viability. Its measurement at the time of the first pregnancy test might be able to predict pregnancy outcome. (Fertil Steril威 2003;80:1473–9. ©2003 by American Society for Reproductive Medicine.) Key Words: Inhibin A, inhibin pro-␣c, pregnancy outcome, recurrent miscarriage
Recurrent spontaneous miscarriage, usually defined as a loss of three or more consecutive pregnancies, affects 1% of pregnant women (1). It is a worldwide obstetric problem, which has a variety of etiological factors. The etiology of recurrent spontaneous miscarriage requires comprehensive evaluation and investigations and poses a major challenge to the treating obstetrician.
factor in 16%, and infectious factor in 0.5%. Forty percent were classified as unexplained. Counseling is an important part in the management and should include an explanation of the possible underlying causes of the condition. More important is the prognosis of the current pregnancy. Research into the methods for prediction and early detection of pregnancy failure has generated great interest.
In a large prospective cohort study by Stephenson et al. (2), genetic factor was identified in 3.5%, endocrine factor in 20%, anatomical
Current available methods for detection of pregnancy failure include ultrasound scan and serum -hCG estimation. Transvaginal ultra1473
sound has become an important tool for the examination of early pregnancy viability. Indeed, the use of high-frequency transducers has improved the image quality and facilitated the measurement of various parameters of the embryo structures. However, it is possible to diagnose potentially abnormal growth only when a significant departure from normal measurements is found, which usually occurs over weeks.
abnormalities in early pregnancy concentrations of this glycoprotein might allow recognition of a group of patients with luteal phase defect as a cause of their recurrent spontaneous miscarriages and who therefore might benefit from luteal phase support. These factors have not been investigated previously in this high-risk group of patients.
Another method for detection of early pregnancy failure is monitoring serum -hCG. This hormone reflects trophoblastic activity rather than fetal demise, and although it does fall with failing pregnancy, it is not completely reliable. Evaluation of CA-125 level as well has been reported to improve its predictive value (3). In addition, -hCG has a slow total elimination rate and it is detectable in the serum 21 days postpartum (4). An ideal alternative would be a substance secreted mainly by the viable fetoplacental unit, probably mainly fetal, with a short half-life. Could inhibin be this alternative?
MATERIALS AND METHODS
Recently, significant research has focused on inhibins and their roles in female reproduction, including pregnancy failure. Inhibins are glycoproteins that belong to the transforming growth factor- (TGF-) superfamily (5). Inhibins are heterodimers consisting of disulphide-linked ␣ (mol.wt 18 K Da) and  (mol.wt 14 K Da) subunits: inhibin A (␣-A) and inhibin B (␣-B). Only the dimeric forms of inhibin are bioactive, although the ␣-subunits circulate in vast excess as biologically inert monomers. Serum concentration of immunoreactive (ir) inhibin has been reported to be higher during various stages of pregnancy compared with nonpregnant subjects (6 –10). In those studies, ir-inhibin was measured using radioimmunoassay, which cross-reacted extensively with different inhibin ␣-subunit forms. Consequently, the contribution of the biologically active dimeric inhibin forms was unknown. Development of specific and sensitive assays for dimeric inhibins has facilitated the measurement of these proteins throughout human pregnancy (11). Inhibin A is the major circulating form of inhibin in human pregnancy. Many efforts were made to identify the source of different forms of inhibins in early pregnancy. It was found that the fetoplacental unit is the major source of inhibin A (12, 13). Inhibin pro-␣c, on the other hand, circulates in great excess as a functionally inactive monomer with its major source being corpus luteum (14). In this study, our aim was to investigate a possible role for inhibins as markers of early pregnancy viability in patients with recurrent spontaneous miscarriage. Early diagnosis of a complicated or poor pregnancy outcome could aid in counseling and management of this group of patients. In addition, this study compared concentrations of inhibin A with -hCG in early pregnancy since -hCG is another pregnancy hormone that correlates with pregnancy viability. Finally, as inhibin pro-␣c is mainly of corpus luteum origin, identifying 1474 Al-Azemi et al.
Inhibins and recurrent pregnancy loss
Subject Selection and Serum Samples Blood samples were collected from 36 pregnant women with a previous history of three or more consecutive first trimester spontaneous miscarriages. Serial blood samples were collected prospectively at 6, 8, 10, and 12 weeks of gestation. The blood was allowed to clot. The sera were separated (centrifugation for 10 minutes at 3,000 rpm) and stored at ⫺80°C before analysis. Ultrasound scan was performed at each time of blood collection for viability and then at 20 weeks’ gestation for anomaly scan. All patients received luteal phase support in the form of P pessaries, 400 mg twice a day per vaginum (Cyclogest, Shire, Andover, UK) in addition to low-dose aspirin (80 mg daily) with or without heparin. The study was approved by the ethics committees of the Faculty of Medicine and the Maternity Hospital. Informed consent was obtained from the subjects according to the guidelines of the Hospital Ethics Committee. Stored samples were later assayed for inhibin A, inhibin pro-␣c, and -hCG estimations.
Hormone Assays Inhibin A Serum inhibin A was measured using a two-site enzymelinked immunosorbent assay (ELISA) (14). Before the assay, patient samples and standards were pretreated with sodium dodecyl sulphate, heated to 100°C and exposed to hydrogen peroxide to enhance the specificity and sensitivity. Wells of a microtitre plate were dry coated with a monoclonal antibody specific for the A subunit of inhibin. Samples (including standards, appropriate controls, etc.) were incubated overnight at room temperature for the antigenantibody reaction. The wells were washed repeatedly to remove any excess unbound antigen. This was followed by the addition of a monoclonal, second antibody specific for the ␣ subunit of inhibin coupled with alkaline phosphatase and incubated at room temperature for 1 more hour. Any unreacted material was then removed by washing before the detection of alkaline phophatase using a sensitive amplified substrate reaction included in the kit. The resulting red color was measured in a spectrophotometer at 490 nm, the intensity of which was directly proportional to the concentration of inhibin A present in the original sample. Vol. 80, No. 6, December 2003
The intra- and interassay coefficients of variation were 4.2% and 4.9%, respectively. The assay was highly specific for the dimeric molecules with minimal cross-reactivity to the C subunit, inhibin B, or activins. The minimum detection limit of this assay was 3.9 pg/mL (inhibin A: dimer assay [ultrasensitive]; Oxford Bio-Innovation, Oxfordshire, UK). Inhibin Pro-␣c
TABLE 1 Patient characteristics. Features
Minimum Maximum
Mean ⫾ SD or median
This was measured using a Microparticle Enzyme Immunoassay (AXSYM system) (Reagent Pack: 7A59-22; Abbott Laboratories, IL). The minimum detection limit of this assay was 2 mIU/mL. The intra- and interassay coefficients of variation were 3.6% and 3%, respectively.
Age (yrs) Group 1 Group 2 Gestational age (wks) Group 1 Group 2 No. of previous deliveries Group 1 Group 2 No. of previous abortions Group 1 Group 2 Birth weight (grams) Group 1
Statistical Analysis
Note: Group 1 ⫽ control group; group 2 ⫽ aborted group.
The patients were allocated to two groups according to the outcome of their pregnancy: group 1 (control group ⫽ 24) consisted of patients whose pregnancy continued beyond 20 weeks; group 2 (aborted group ⫽ 12) consisted of patients who aborted before 20 weeks
Al-Azemi. Inhibins and recurrent pregnancy loss. Fertil Steril 2003.
Serum inhibin pro-␣c was also measured using a two-site enzyme-linked immunosorbent assay as above (15). The minimum detection limit of this assay was 2 pg/mL. The intra- and interassay coefficients of variation were 5.5% and 8.9%, respectively (inhibin pro-␣c, Oxford Bio-Innovation).
-hCG
Data were processed using the SPSS software package, version 11. The cutoff level for statistical significance was Pⱕ.05. Since the frequency distributions of the three measured glycoproteins were not normally distributed, we described our data as median and range (minimum, maximum) rather than mean and SD. Significance between the levels of these glycoproteins in aborters and controls at different gestational week points (6, 8, 10, and 12) was assessed using the nonparametric Mann-Whitney U test. The trend for change of their levels throughout the study period (6 –12 weeks’ gestation) was measured by the Friedman test for dependent repeated measures.
P
25 23
40 40
31.83 ⫾ 4.72 31.25 ⫾ 5.67
NS
29 9
41 19
38 12
⬍.0001
0 0
5 4
2 1
NS
3 3
10 18
5 5
NS
1,800
4,150
2,945.83 ⫾ 508.72
15 patients delivered vaginally, while nine delivered by cesarean section. There was one case of congenital abnormality (polycystic kidney). All pregnancies (24 women) were represented at all time points in the control group, while the aborted group was represented till the time of miscarriage (that is, 12, 12, 7, and 4 women were represented at 6, 8, 10, and 12 weeks’
FIGURE 1 Number of women in group 1 (solid bar) and group 2 (open bar) at different time points during the study period.
To determine sensitivity and specificity of inhibin A and -hCG levels, a cutoff level of a multiple of 0.5 of the median of the control levels was used. If the level at a particular gestational week was below this cutoff level, there is a risk for abortion (sensitivity), while if the level exceeded the cutoff level, there is no risk for abortion (specificity).
RESULTS There was no significant difference between the two groups in age (31.83 ⫾ 4.64 vs. 31.25 ⫾ 5.48 years), number of previous deliveries or number of previous abortions, and onset of conception (all were spontaneous conceptions). The median gestational age at which the control group delivered was 38 weeks (range: 29 – 41 weeks), and the median gestational age at which the aborted group miscarried was 12 weeks (range: 9 –19 weeks) (Table 1). In the control group, FERTILITY & STERILITY威
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TABLE 2 Inhibin A (pg/mL), -hCG (mIU/mL), and inhibin pro-␣c (pg/mL) concentrations throughout the study period in the control and aborted groups presented as median (minimum, maximum). Variable Inhibin A: Group 1 Group 2 P -hCG: Group 1 Group 2 P Pro-␣c: Group 1 Group 2 P
6 Weeks
429.4 (26.9, 837.5) 234.5 (3.0, 474.0) .067 16,752.9 (372.1, 130,350.9) 6,318.7 (3,216.0, 27,883.5) NS 401.9 (2.0, 1,809.7) 694.8 (2, 867.9) NS
8 Weeks
10 Weeks
725.0 (3.0, 1,612.5) 162.5 (32.2, 387.5) ⬍.001
875.0 (120, 2,150.0) 121.3 (66.3, 387.5) .002
727.5 (117.5, 1,687.5) 135.0 (100.0, 262.5) .006
121,445.5 (45,286.5, 200,000) 65,112.8 (62.6, 120,721.4) .011
82,889.4 (33,996.1, 200,000) 83,435.7 (147.2, 200,000) .972
106,208.3 (2852.6, 199,661.8) 49,071.4 (47.7, 2,000,000) NS 509.7 (2, 2,013.6) 245.1 (2, 622.9) NS
12 Weeks
403.7 (2, 2,354.2) 295 (2, 633.7) NS
462.1 (2, 2,698) 354.3 (2, 540.3) NS
Note: Group 1 ⫽ control group; group 2 ⫽ aborted group. Al-Azemi. Inhibins and recurrent pregnancy loss. Fertil Steril 2003.
gestation, respectively). Figure 1 shows the total number of women who were enrolled and the dropout due to miscarriage at different time points. There was a significant difference in inhibin A concentrations between control and aborted groups at 8 (P⬍.001), 10 (P ⫽ .002), and 12 weeks’ gestation (P ⫽ .006). At 6 weeks’ gestation, there was a difference; however, this did not reach statistical significance (P ⫽ .067). Assessment of the difference in -hCG concentrations between control and aborted groups revealed a statistically significant difference only at 10 weeks’ gestation (P ⫽ .011). There was no significant difference in inhibin pro-␣c concentrations between control and aborted groups at each gestational age (Table 2). In the control group, there was a significant increase in inhibin A (P ⫽ .005), and -hCG (P⬍.001) over the study period (Friedman test). However, there was no statistically significant increase in inhibin pro-␣c concentrations over the study period. As we were losing cases in the aborted group, testing for a trend was not feasible because of the small number of cases (four cases at 12 weeks) (Figs. 2 and 3). In an attempt to determine a cutoff (threshold) level for inhibin A and -hCG to be used as a marker for predicting pregnancy outcome, we used a multiple of 0.5 of the median of the control level at each gestational age and we checked the sensitivity and specificity (Table 3). The inhibin A threshold gave better sensitivity and the same specificity at 8 weeks’ gestation (75% and 81%, respectively) than the -hCG threshold (50% and 81%, respectively). We also attempted to identify the point in our assay where we could identify abnormal pregnancies before an ultrasound does. Hence, when we further analyzed our data in the aborted group (group 2) at 10 weeks, we found that 5 of the 1476 Al-Azemi et al.
Inhibins and recurrent pregnancy loss
12 pregnancies had absent fetal heart sound on ultrasound. When we compared the inhibin A values of these 5 women at 6 and 8 weeks’ gestation to our cutoff level (0.5 multiple of the median (MOM) of controls), we found that 40% of these women had inhibin A levels ⬍0.5 MOM of controls at 6 weeks’ gestation (4 weeks earlier than ultrasound) and 80% of these women had inhibin A levels ⬍0.5 MOM of controls at 8 weeks’ gestation (2 weeks earlier than ultrasound).
DISCUSSION This study demonstrates that women with a history of recurrent spontaneous miscarriage (at least three previous consecutive first trimester pregnancy losses) have consistently lower concentrations of inhibin A in serum as early as 6 weeks’ gestation if the current pregnancy is destined to miscarry. Measurement of inhibin A, but not inhibin pro-␣c, in early pregnancy serum therefore offers the possibility of
TABLE 3 Sensitivity and specificity for cutoff level (0.5 ⫻ median) for inhibin A and -hCG at different gestational week points.
Inhibin A Sensitivity Specificity -hCG Sensitivity Specificity
Week 6
Week 8
Week 10
Week 12
50 73.3
75 81
85.7 82.6
100 77.3
66.7 66.7
50 81
42.9 87
25 95.5
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Vol. 80, No. 6, December 2003
FIGURE 2 Distribution of inhibin A levels between group 1 (control group) (crosses and dashed line) and group 2 (aborted group) (bullets and dot-dashed line).
Al-Azemi. Inhibins and recurrent pregnancy loss. Fertil Steril 2003.
identifying viable and nonviable pregnancies earlier than transvaginal ultrasound. Although serum concentrations of -hCG prove to be lower in the pregnancy loss group, this difference was less striking than that observed for inhibin A and only reached statistical significance at 10 weeks’ gestation. We failed to observe any significant differences in concentrations of inhibin pro-␣c between the two groups. The findings of this study are consistent with those from previous studies. Inhibin concentrations in early singleton, multiple, and failing pregnancies were examined by Norman et al. (16). His study showed that in singleton pregnancies there was a significant increase in ir-inhibin concentrations in early pregnancy. Significantly higher concentrations were found in multiple pregnancies. Patients destined to abort showed no increase in ir-inhibin early in pregnancy. In that study, ir-inhibin was measured using radioimmunoassay, which cross-reacted extensively with different inhibin ␣-subunit forms. The use of the more specific and sensitive assay for inhibin A in our study facilitates the measurement of the biologically active form of this glycoprotein. To identify the source of inhibin A in early pregnancy, Birdsall et al. used the donor-egg model in conjunction with specific ELISA for inhibin A (13). They concluded that the FERTILITY & STERILITY威
fetoplacental unit was the major source of these polypeptides in early pregnancy. Lockwood et al. (12) investigated the source of inhibins in early pregnancies conceived in vitro with and without corpus luteum function. That study confirmed that the main source of dimeric inhibin A in early pregnancy is the fetoplacental unit, since comparable concentrations of inhibin A were found in singleton pregnancies arising from spontaneous conception and after IVF treatment with both fresh and frozen ETs. In the first case, a single corpus luteum would be expected; in the second case, multiple corpora lutea are routinely observed and in downregulated frozen embryo replacement, no cycle’s corpus luteum is present. The significantly higher concentration of inhibin A found in multiple pregnancies is further evidence for the production by the fetoplacental unit. The significant differences between concentrations of inhibin A in viable, nonviable, and multiple IVF pregnancies detectable as early as 13 days after ET in that study are further evidence for a fetoplacental source of inhibin A in early pregnancy and suggest that the estimations of inhibin A may be helpful in the management of early pregnancy (12). In a recent study, maternal serum inhibin A and -hCG concentrations in very early IVF pregnancy (16 days after 1477
FIGURE 3 Distribution of -hCG levels between group 1 (control group) (crosses and dashed line) and group 2 (aborted group) (bullets and dot-dashed line).
Al-Azemi. Inhibins and recurrent pregnancy loss. Fertil Steril 2003.
oocyte retrieval) were compared with pregnancy outcomes and treatment protocols in 237 women undergoing IVF and ET (17). Consistent with other studies, concentrations of inhibin A were found to be significantly higher in women with ongoing pregnancies than in nonpregnant women. However, contrary to others, the study demonstrated significantly lower concentrations of inhibin A in the absence of functioning ovaries, suggesting that the corpus luteum is a major source of circulating inhibin A in very early pregnancy. This could possibly be explained by the relatively small size of trophoblast mass at such an early gestation with a small contribution to maternal circulating inhibin A. Additionally, the study also showed, consistent with our findings, that there was no significant difference in -hCG concentrations between those with ongoing pregnancies and those later diagnosed as biochemical pregnancies, which suggests that low concentrations of serum inhibin A may be useful in identifying -hCG–positive preclinical “biochemical” pregnancies. Illingworth et al. (18) in 1996 showed that inhibin A levels rose steadily during the conception luteal phase to an initial peak 12 days after ovulation (4 weeks), then rose rapidly to a further peak 43 days after ovulation (8 weeks). 1478 Al-Azemi et al.
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Similar observations were made in our study in the control group with the peak in our study at 10 weeks gestation. Illingworth et al. (18) found that exogenous hCG resulted in a significant rise in the concentrations of inhibin A and inhibin pro-␣c. None of our patients received hCG for luteal phase support. Inhibin pro-␣c circulates in great excess as a functionally inactive monomer. The corpus luteum is the major source of this form during pregnancy (12). The presence of comparable levels of inhibin pro-␣c in both control and aborted groups in our study makes it unlikely that the fetoplacental unit is a major contributor to circulating levels of this glycoprotein in pregnancy and supports the hypothesis that this form is mainly produced by the corpus luteum. Interestingly, in a recent study it was found that inhibin pro-␣c is secreted from cultured explants from chorion leave and placenta (19). However, how much these sources contribute to the maternal serum concentration of this glycoprotein in early pregnancy is still to be determined. Our study showed no significant differences in the level of -hCG at 6 and 8 weeks between the aborted and control groups. The difference was evident only at 10 weeks’ gesVol. 80, No. 6, December 2003
tation. In comparison, inhibin A levels are significantly lower between the aborted and control groups as early as 6 weeks and onward. This can be explained by the longer half-life of -hCG compared with inhibin A. Lower concentrations of inhibin A as early as 6 weeks’ gestation implies that serial blood testing may not be cost-effective, and single measurement at 6 weeks’ gestation may be equally effective in predicting pregnancy outcome. These findings were also found by Norman et al. (16), who examined the endocrine changes in very early pregnancy. There were no statistical differences in -hCG levels early in pregnancy between viable singleton pregnancies and those pregnancies destined to miscarry. The biological roles of inhibin A and inhibin pro-␣c in early pregnancy are unknown. Inhibin A has been shown to regulate GnRH, hCG, and P secretion from human placental cells in vitro (20). The function is probably related to cellular differentiation and embryogenesis. Activins were found to induce mesoderm in embryonic explants (21). Inhibins probably have some role in directing cell lineage. The continued secretion of inhibin A from the fetoplacental unit throughout pregnancy (22) suggests the possibility of inhibin A having different functions throughout pregnancy. In conclusion, our study demonstrates a possible role of inhibin A as a marker for predicting pregnancy outcome in high-risk pregnancy. It fails to demonstrate any role for inhibin pro-␣c as a prognostic tool. The expanding knowledge of the role of inhibins since the development of specific and sensitive assays has opened the field for their potential uses in clinical practice. Further research on these glycoproteins in diagnosis, prediction, and monitoring of pathological human pregnancies is required. Improvement in pregnancy outcome in patients with recurrent miscarriage has been shown to follow early ultrasound demonstration of fetal heart activity (23). Measurement of serum inhibin A at the time of the first positive pregnancy test might be able to predict pregnancy outcome (12). Very early identification of a potentially viable pregnancy might further enhance the benefits of such knowledge to pregnancy outcome. A randomized trial of this intervention is planned.
Acknowledgments: The authors thank Tunde Fatinikum, Rawia Abdulmonem, and Susan George for collecting and processing blood samples. The assistance of Mrs. Asiya Tasnim in preparing the manuscript is gratefully acknowledged.
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