1 Department of Obstetrics and Gynaecology, Royal Free-UCL Medical School, 8696 Chenies Mews, London WC1E 6HX, 2 Academic Department of Obstetrics and Gynaecology, St. Marys Hospital, Imperial College Medical School, London and 3 School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane, Headington, Oxford, UK
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Abstract |
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Key words: activin A/early pregnancy/follistatin/inhibin A/miscarriage
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Introduction |
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Miscarriage is the most common complication of pregnancy. Early pregnancy loss occurs in 1020% of clinically recognized gestations (Alberman, 1992). Of these, a proportion will present to medical practitioners as missed miscarriages.
As distinct to recurrent miscarriage, cases of sporadic/missed miscarriage have no defined past history of miscarriage. In missed miscarriage, also referred to as missed abortion in the literature, failure of pregnancy is identified before expulsion of the fetal and placental tissue. The diagnosis is made on ultrasound by either failure to identify fetal heart action or by the presence of an empty gestational sac where early embryonic death has been followed by resorption of the fetal tissue, thus giving rise to the more recent description of missed miscarriage as early pregnancy failure (EPF). These empty gestation sacs were in the past described as anembryonic pregnancies, this being rather misleading in its inference that there was total non-development of the fetus. Isolated cases of EPF presenting at random to the early pregnancy unit with a history of bleeding per vaginam or with a complaint of diminishing pregnancy symptoms will be referred to in this study as sporadic miscarriage.
Recurrent miscarriage is defined as three or more consecutive pregnancy losses and affects 1% of couples trying to conceive (Regan, 1997). Sporadic miscarriages are essentially linked with chromosomal abnormalities of the conceptus (Hassold, 1986
) whereas recurrent miscarriages have been associated mainly with karyotypic abnormalities in either partner, or uterine anomalies such as fibroids and congenital malformation of the mesonephric systems. More recently primary antiphospholipid syndrome, thrombophilic states including activated protein C resistance and Factor V Leiden have been found in association with recurrent miscarriage (Regan, 1997
). The predictive value of human chorionic gonadotrophin (hCG), estradiol and progesterone has been investigated extensively in predicting sporadic miscarriage (Nygren et al., 1973
; Confino et al., 1986
; Whittaker et al., 1989
; Johnson et al., 1993
). Inhibin A has been shown to be low in non-viable IVF (Lockwood et al., 1997
) and spontaneous pregnancies with symptoms of miscarriage (Phipps et al., 2000
) compared with ongoing pregnancies. However, there is only one study of the circulatory levels of immuno reactive (ir) inhibin [dimeric inhibins (A and B) + monomeric
subunit] in recurrent miscarriage (France et al., 1996
). These authors found that ir-inhibin concentrations are not significantly altered in women before miscarriage but the assay used in their study was not specific for biologically active dimeric inhibin A or inhibin B, because monomeric
subunit is produced by several tissues including the ovaries. The aim of the present study was to further investigate circulating levels of inhibin A, inhibin B, activin A and follistatin in women with sporadic or recurrent miscarriages. We have evaluated whether circulating levels of inhibin A, activin A and follistatin are endocrine markers of early pregnancy loss in patients with a history of recurrent miscarriage and are linked with other serum markers such as estradiol, progesterone and hCG.
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Patients and methods |
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Study 2
Longitudinal samples were collected weekly or fortnightly between 6 and 12 weeks gestation in 12 women referred to the recurrent miscarriage clinic at St Marys Hospital. In addition, at 67 weeks gestation, samples were collected from 18 women attending the clinic. None of these women had any infection. All women were investigated according to the clinic protocol. The mean maternal age of the group with live births (33.8 ± 6.5 years; mean ± SD; range 2341) and recurrent miscarriages (36.8 ± 5.1 years; mean ± SD; range 2944) were not significantly different. Each group (live/recurrent) included five nulliparous and four parous (one/two) women. Patients on any medication other than folic acid were excluded. Women and their partners were tested for peripheral blood karyotype abnormalities. Women were also tested for antiphospholipid antibodies (IgG and IgM anticardiolipin antibodies and lupus anticoagulant), early follicular gonadotrophin and testosterone levels, and had pelvic ultrasound scans to determine ovarian and uterine morphology. In addition, blood was taken for activated protein C resistance ratio and restriction fragment length polymorphism analysis for factor V Leiden. Unexplained recurrent miscarriage was diagnosed when no cause for recurrent miscarriage was found. Women were subsequently followed-up in the early pregnancy clinic for weekly or fortnightly ultrasound scans and consultations in their next pregnancy. For patients with unexplained recurrent miscarriage, apart from folic acid (Department of Health guidelines) no other medication was administered. Pregnancy outcome, including a subsequent miscarriage or a live birth, was determined prospectively in each patient by way of a return questionnaire.
In all groups, gestational age was determined from the first day of the last menstrual period and confirmed by ultrasound measurement of the fetal crownrump length in ongoing pregnancies. Cytogenetic analysis was obtained from placental tissue in all cases of miscarriages and in controls.
Blood samples were collected in tubes containing EDTA from women with a history of unexplained recurrent miscarriages and centrifuged to separate blood cells and platelets from plasma. Serum and plasma was then stored at 80°C and analysed later for inhibin A, inhibin B, activin A, follistatin, progesterone, estradiol and hCG. Written consent was obtained from each woman after receiving complete information about the procedure. Approval for this study was obtained from the local research ethics committees.
Hormone assays
Inhibin A was measured using a two-site ELISA (enzyme linked immunosorbent assay) that has been previously validated for human serum (Muttukrishna et al., 1994). The minimum detection limit of this assay for human recombinant inhibin A (National Institute for Biological Standards, Potters Bar, Herts, UK) was 2 pg/ml. Intra- and interassay variations were 4.5 and 5.1% respectively.
Inhibin B was measured using an enzyme immunoassay as described in detail elsewhere (Groome et al., 1996). An in-house standard preparation (partially purified human follicular fluid) was standardized against human recombinant inhibin B (Genentech, San Francisco, CA, USA) and was used as the assay standard. The minimum detection limit of the assay for human recombinant inhibin B was 15 pg/ml. The mean intra and interassay coefficients of variation (CV) were 6.2 and 7.2% respectively.
Activin A was measured using a two-site ELISA specific for total activin A (follistatin bound + unbound activin A) as previously described (Muttukrishna et al., 1996). The detection limit of this assay for human recombinant activin A (Genentech) was 50 pg/ml. Intra- and interassay variations were 8.5 and 9.8% respectively.
Follistatin was measured using a two-site ELISA as previously described (Evans et al., 1998). The sensitivity of this assay was 20 pg/ml. Intra- and interassay variations were 6.8 and 9.15% respectively.
hCG was measured by an immuno-radiometric assay (IRMA) using a commercial kit purchased from EURO DPC (Llanberis, UK). Intra- and interassay CV were <10%. The minimum detection limit of the assay was 0.03 mIU/ml.
Estradiol and progesterone were measured by radioimmunoassay (following ether extraction and using tritium labelled tracers) as previously described (Darne et al., 1989). Intra- and interassay CV for both assays were <10%. The minimum detection limit of the estradiol RIA was <80 pmol/l and progesterone was 1 nmol/l.
Statistical analysis
Data were log transformed to obtain a normal distribution. In the missed miscarriage study, an unpaired Students t-test was used to analyse differences between the groups. In the recurrent miscarriage group, analysis of variance was carried out to study the time course changes in the longitudinal study and paired sample t-tests were used to study the difference between the women with subsequent miscarriages and women with a normal outcome. Pearsons correlation analysis was carried out to study the relationship between the different hormones measured. The SPSS statistical package was used for the analysis (SPSS Inc., Chicago, Illinois, USA). P was considered significant at <0.05.
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Results |
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In the recurrent miscarriage group, the data regarding women who had another miscarriage were analysed separately from those with a live birth. In the women with a live birth, plasma inhibin A showed a positive correlation with activin A (r = 0.563, P < 0.01), estradiol (r = 0.61, P < 0.01) and hCG (r = 0.66, P = 0.001). Plasma activin A had a negative correlation with follistatin (r = 0.41, P = 0.05) and a positive correlation with hCG (r = 0.557, P < 0.01). Plasma estradiol had a positive correlation with hCG (r = 0.473, P < 0.05).
In women with a subsequent miscarriage, plasma inhibin A was positively correlated with activin A (r = 0.511, P < 0.05), progesterone (r = 0.55, P < 0.05), estradiol (r = 0.45, P < 0.05) and hCG (r = 0.989, P < 0.001). Plasma activin A was also positively correlated with progesterone (r = 0.55, P < 0.05), estradiol (r = 0.58, P < 0.01) and hCG (r = 0.511, P < 0.05). Plasma progesterone levels were positively correlated with estradiol (r = 0.714, P < 0.001) and hCG (r = 0.49, P < 0.05).
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Discussion |
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Several studies have shown that estradiol, progesterone and hCG can predict pregnancy loss (Check et al., 1992; Ogasawara et al., 1997
; Ong et al., 2000
; Phipps et al., 2000
; Zayed et al., 2001
). Phipps et al.(2000) have shown that inhibin A levels are low in those patients with clinical symptoms of spontaneous miscarriage. After 67 weeks gestation the trophoblast is considered to be the principal source of estrogens and progesterone (Csapo and Pulkkinen, 1978
). Thus, a pregnancy failure after this time can mainly be linked to placental dysfunction either secondary to a fetal abnormality or as the result of primary trophoblast dysfunction. In the present study, women with miscarriages all presented after 8 weeks gestation and four out of 10 of those presenting with a sporadic miscarriage had a fetal chromosomal abnormality, suggesting placental dysfunction as the main cause of miscarriage. Anatomical studies of the placental bed in women with pre-eclampsia and miscarriage have shown a similar defect in the trophoblastic infiltration of the placentadecidual interface (Hustin et al., 1990
). In both types of pregnancy complications, this leads to an incomplete transformation of the utero-placental arteries and abnormal villous development. In miscarriage, this defect of placentation is often very severe and associated with the premature entry of a large amount of maternal blood inside the placenta, which progressively dislocates the entire gestational sac (Jauniaux et al., 1994
). Serum inhibin A was
3 fold less in women presenting with a sporadic miscarriage compared with controls, however, activin A and follistatin levels were not significantly altered at 812 weeks gestation in cases of miscarriage. In early pregnancy, inhibin A levels gradually rise up to 12 weeks and then decline to a nadir and remain low in the second trimester (Muttukrishna et al., 1995
). Activin A levels are similar in the first and second trimester (Muttukrishna et al., 1996
) whereas follistatin levels progressively rise throughout pregnancy (Fowler et al., 1998
). Although inhibin A and activin A are in circulation in pregnancy, a precise biological role for these proteins in pregnancy is, as yet, unclear. Studies in other species suggest that activin A is important for early embryonic development (Asashima et al., 1990
; Green and Smith, 1990
; Ariizumi et al., 1991
; Jenkin et al., 1995
).
Serum progesterone (2 fold), estradiol (
3.5 fold) and hCG (
4 fold) levels were also significantly lower in miscarriage patients compared with controls. Serum progesterone and hCG were significantly correlated with levels of inhibin A suggesting similar source(s) and mechanisms regulating these hormones. Activin A and follistatin have several sources such as the placenta, peripheral mononuclear cells (Muttukrishna et al., 2001
), vascular endothelial cells (Phillips et al., 2001
) and bone marrow (Mather et al., 1997
). The lack of difference in the levels of activin A and follistatin in miscarriage patients could be due to other sources for these hormones, possibly masking the reflection of any changes in production by the fetoplacental unit.
In women with recurrent miscarriages all samples were collected before the onset of the clinical symptoms. In this group, time dependent changes in inhibin A and hCG levels were significantly lower in women who had another miscarriage compared with those who had live births. At 67 weeks, women who had a subsequent miscarriage had significantly lower levels of inhibin A (4 fold), estradiol (
2 fold) and hCG (
4 fold) compared with those whose pregnancy ended with a live birth. These data indicate that inhibin A and hCG levels are consistently lower in all cases of miscarriage and are positively and significantly correlated with each other. Serum hCG levels rise until 89 weeks in normal early pregnancy and then decrease and remain low during the second and third trimesters. Whilst inhibin A levels rise in early pregnancy and decrease in the second trimester, inhibin A increases again in the third trimester of pregnancy.
It has been previously reported that lower levels of inhibin A are found in the maternal circulation in IVF missed abortions (Lockwood et al., 1997) and spontaneous non-viable pregnancies with clinical symptoms of miscarriage (Phipps et al., 2000
). Our missed miscarriage data confirm these previous studies with lower maternal circulatory levels of inhibin A in miscarriage patients compared with the controls. Consistent with our current observation in sporadic missed miscarriages, activin A levels were not different in IVF missed abortions (Lockwood et al., 1997
). These data suggest that inhibin A is a more specific marker of placental function than activin A or follistatin, as these are also produced by several other tissues. Our previous study investigating the clearance of inhibin A, activin A and hCG after pregnancy termination suggested that inhibin A concentrations clear from circulation within hours (Muttukrishna et al., 1997
), unlike hCG which has a longer half life. This indicates that any variation in inhibin A production will quickly be reflected in the circulation, rendering it a sensitive marker of early placental dysfunction.
A previous study has reported ir-inhibin in recurrent miscarriage patients and found there was no correlation between hCG and ir-inhibin in these patients (France et al., 1996). In our study, we report a strong positive correlation between inhibin A and hCG (r = 0.989, P < 0.001) in the recurrent miscarriage group. The discrepancy between the two studies could be due to the specificity of the inhibin assay. We have specifically measured dimeric inhibin A, which is the bioactive dimeric inhibin form in the circulation of pregnant women, whilst in another study (France et al., 1996
) a radioimmunoassay was used for inhibin which crossreacted with dimeric inhibins (A and B) and monomeric
subunit. It is clear from previous studies that inhibin A is the predominant molecular form of inhibin in the circulation during pregnancy (Muttukrishna, 2001
).
In conclusion, this pilot study shows that inhibin A in combination with hCG could be useful in monitoring unexplained recurrent miscarriage patients to predict a subsequent miscarriage as early as 6 weeks gestation. However, larger studies with different groups of recurrent miscarriage patients are needed to confirm this observation and the potential clinical application. Future studies should focus on the role of inhibins in early pregnancy in order to inform therapeutic interventions.
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Acknowledgements |
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Notes |
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References |
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Submitted on June 27, 2002; accepted on September 17, 2002.