Aberrations of early trophoblast differentiation predispose to pregnancy failure: lessons from the anti-phospholipid syndrome.

OBJECTIVES: The epithelium of the human placenta comprises an inner cytotrophoblast (CT) which proliferates and fuses with the outer differentiated syncytiotrophoblast (ST). Turnover has been studied focussing on second and third trimester placentas but with a paucity of data describing the normal first trimester trophoblast. The aim of this study was to compare the nuclear CT:ST ratio in normal and pathological pregnancy and thus establish the relationship between cytotrophoblast and syncytiotrophoblast nuclear number during early gestation. METHODS: Archival first trimester material from placentas from healthy pregnancy and recurrent miscarriage (anti-phospholipid syndrome) was stained with H&E, cytokeratin-7 and Mib-1. The area of trophoblast as a fraction of total villous area was calculated and the number of sectioned cytotrophoblast and syncytiotrophoblast nuclei as well as the number of proliferating cytotrophoblast was evaluated. RESULTS: Normal features of trophoblast development during the first trimester (rise in trophoblast area, increase in number of syncytiotrophoblast nuclei, increase in number of proliferating cytotrophoblast, decrease in the nuclear CT:ST ratio) are absent/reversed in tissues from recurrent miscarriage (decreasing trophoblast area, constant number of syncytiotrophoblast nuclei, decreasing number of proliferating trophoblast, constant nuclear CT:ST ratio). CONCLUSIONS: Proliferation of cytotrophoblast in early gestation provides a pool of trophoblast stem cells critical for ongoing placental development. Premature cytotrophoblast differentiation in favour of syncytial fusion results in deficiencies of cytotrophoblast and rarification of villous trophoblast. Abnormal trophoblast differentiation in early gestation may be due to a premature onset of maternal perfusion of the placenta and may be a likely antecedent for conditions associated with failure of placentation such as recurrent miscarriage.

Thromboelastography, whole-blood haemostasis and recurrent miscarriage.

BACKGROUND: Some cases of recurrent miscarriage have a thrombotic basis. Thromboelastography is a rapid, reproducible test of whole-blood haemostasis. METHODS: Thromboelastography was performed in 494 consecutive, non-pregnant women (median age 35 years; range 21-48) with a history of miscarriages at <12 weeks gestation (median 4; range 3-12) and 55 parous women (median age 33 years; range 20-41) with no history of pregnancy loss. The prospective outcome of untreated pregnancies amongst 108 women with recurrent miscarriage was studied. RESULTS: The maximum clot amplitude (MA) (median 66.0 mm; range 48.0-76.0) was significantly higher and the rate of clot lysis (LY30) (median 2.5%; range 0.5-7.8) significantly lower amongst women with recurrent miscarriage compared with controls (MA 61.5 mm; range 50.0-67.0; P = 0.01; LY30 4.9%; range 2.9-9.7; P = 0.01). The pre-pregnancy MA was significantly higher amongst women who subsequently miscarried (median 66.0 mm; range 54.0-73.0) compared with those whose had a live birth (median 61.7 mm; 48.0-71.5; P < 0.01). A pre-pregnancy MA >or=64 mm has a sensitivity of 68% and specificity of 82% to predict miscarriage. CONCLUSIONS: Thromboelastography identifies a subgroup of women with recurrent miscarriage to be in a prothrombotic state outside of pregnancy. Women in such a state are at increased risk of miscarriage in future untreated pregnancies.

Reproducibility of three-dimensional ultrasound diagnosis of congenital uterine anomalies.

OBJECTIVE: To examine the reproducibility of the diagnosis of congenital uterine anomalies and the repeatability of measurements of uterine cavity dimensions using three-dimensional (3D) ultrasound. METHODS: The reproducibility of diagnosis of congenital uterine anomalies was examined by re-slicing stored 3D ultrasound volumes. Each data set was evaluated by two independent observers. Assessment of uterine morphology was performed in a standardized plane, with the interstitial portions of the Fallopian tubes used as reference points. Additionally, in 35 cases of congenital uterine anomalies the width of the uterine cavity (W), fundal distortion (F) and the length of unaffected uterine cavity (C) were measured. Intraobserver and interobserver variabilities were evaluated by each observer performing all three measurements twice. RESULTS: Eighty-three 3D ultrasound volumes were examined. Both operators classified 27 uteri as normal, 33 as arcuate, 19 as subseptate and three as unicornuate. A single case of uterine anomaly was described as arcuate uterus by one operator and subseptate by another (kappa 0.97). The intraobserver variability for each of the three measurements (W, F and C) was satisfactory with limits of agreement ranging from +/-1.43 to +/-2.51 mm. The examination of the interobserver variability showed no significant differences between the two observers (F = 0.484, P > 0.05). CONCLUSION: 3D ultrasound is a reproducible method for the diagnosis of congenital uterine anomalies and for the measurement of uterine cavity dimensions.

Placental pathology, antiphospholipid antibodies, and pregnancy outcome in recurrent miscarriage patients.

OBJECTIVE: To examine whether there are characteristic histological features in placentas from ongoing pregnancies of patients with a history of recurrent miscarriage, with and without primary antiphospholipid antibody syndrome, in relation to clinical pregnancy outcome. METHODS: Patients attending a recurrent miscarriage clinic were investigated and treated according to an established protocol. One hundred twenty-one consecutive patients achieving a potentially viable pregnancy (at least 24 completed weeks' gestation), including 60 primary antiphospholipid antibody syndrome-positive cases and 61 primary antiphospholipid antibody syndrome-negative cases were included. After delivery, placental pathologic examination was carried out by a pathologist unaware of the clinical details. Histological sections were examined by two pathologists independently. Pregnancy outcome and placental findings were reviewed in relation to the maternal antiphospholipid antibody status. RESULTS: Pregnancy outcome was similar in primary antiphospholipid antibody syndrome-positive and primary antiphospholipid antibody syndrome-negative groups regarding gestation at delivery and antepartum obstetric complications. Several histological placental abnormalities were identified in both groups, but most pregnancies were clinically uncomplicated, with no significant placental abnormalities. In cases with pregnancy complications, the placental pathology was primarily that of uteroplacental vasculopathy, such as placental infarction and preeclampsia, but there were no specific placental lesions or patterns of abnormalities characteristic of primary antiphospholipid antibody syndrome-positive patients. A small subgroup of primary antiphospholipid antibody syndrome-positive patients may be at increased risk of development of maternal floor infarction or massive perivillus fibrin deposition. CONCLUSION: There are no specific histopathologic placental abnormalities characteristic of treated patients with antiphospholipid antibody syndrome and poor reproductive history, but complications of uteroplacental disease are more common.

A comparative study of the morphology of congenital uterine anomalies in women with and without a history of recurrent first trimester miscarriage.

BACKGROUND: The true impact of congenital uterine anomalies on reproductive outcomes is unknown. The aim of this study was to examine differences in the morphology of uterine anomalies found in women with and without a history of recurrent miscarriage. METHODS: A total of 509 women with a history of unexplained recurrent miscarriage and 1976 low risk women were examined for the presence of congenital uterine anomalies by three-dimensional ultrasound. The anomalies were classified according to the American Fertility Society classification. In addition, the size of fundal distortion (F) and the length of the remaining uterine cavity (C) were measured to calculate a distortion ratio (F/F+C). The findings were compared with the measurements obtained in low risk women with an incidental finding of uterine anomaly. RESULTS: In all, 121 anomalies were detected in the recurrent miscarriage group and 105 in low risk women. There was no significant difference in relative frequency of various anomalies or depth of fundal distortion between the two groups. However, with both arcuate and subseptate uteri, the length of remaining uterine cavity was significantly shorter (P < 0.01) and the distortion ratio was significantly higher (P < 0.01) in the recurrent miscarriage group. CONCLUSION: The distortion of uterine anatomy is more severe in congenital anomalies, which are found in women with a history of recurrent first trimester miscarriage.

Placental massive perivillous fibrin deposition associated with antiphospholipid antibody syndrome.

We present three pregnancies in which massive perivillous fibrous deposition (MPVFD) and maternal floor infarction (MFI) occurred in patients with primary antiphospholipid antibody syndrome (PAPS) attending a recurrent miscarriage clinic, and who were treated with low dose aspirin and heparin. We hypothesise that PAPS may be a predisposing factor to the development of this condition. The increased prevalence of late pregnancy complications in PAPS patients with a history of early miscarriage suggests that aspirin and heparin therapy does not eradicate the underlying pathological process but merely reduces the severity. Therefore, untreated early pregnancy losses may be converted into treated pregnancies with late antenatal complications. Some patients with PAPS may therefore be prone to suffer either the previously reported complications of the uteroplacental vasculature, such as pre-eclampsia, and/or specific complications related to the environment of the intervillus space, such as MPVFD/MFI.

Defective endovascular trophoblast invasion in primary antiphospholipid antibody syndrome-associated early pregnancy failure.

BACKGROUND: Primary antiphospholipid antibody syndrome (PAPS) is an established cause of recurrent pregnancy loss, traditionally presumed to be due to 'intraplacental thromboses'. This study examines products of conception (POC) from early pregnancy failures to investigate the mechanism of pregnancy loss. METHODS: POC from patients attending a recurrent miscarriage clinic and from terminations of pregnancy for non-medical reasons were examined histologically with particular regard to the presence or absence of vascular or intervillous thromboses and decidual endovascular trophoblast invasion. RESULTS: There were 31 PAPS-positive, 50 PAPS-negative, 34 aneuploid and 20 control cases at 6-14 weeks gestation. Villous morphology and frequency of intervillous thrombosis were not different among groups. Normal intradecidual endovascular trophoblast invasion was identified significantly less frequently in PAPS+ cases (24%), compared with controls (75%), aneuploid (53%), or PAPS- cases (61%; Z = -3.0, P < 0.01). In all cases there was apparently normal interstitial extravillous trophoblast invasion. CONCLUSIONS: Defective decidual endovascular trophoblast invasion, rather than excessive intervillous thrombosis, is the most frequent histological abnormality in PAPS+ associated early pregnancy loss. Furthermore, endovascular trophoblast invasion is not significantly reduced in the majority of fetal aneuploidy-associated pregnancy failures.

Factor V Leiden and recurrent miscarriage-prospective outcome of untreated pregnancies.

BACKGROUND: Some cases of recurrent miscarriage and later pregnancy complications have a thrombotic basis. Factor V Leiden is a common thrombophilic mutation. METHODS: The prospective outcome of untreated pregnancies amongst 25 women heterozygous for the Factor V Leiden allele who had a history of either recurrent early miscarriages only (three or more miscarriages at <12 weeks gestation; n = 19) or of late miscarriage (>12 weeks gestation; n = 9) was studied. Control groups of women with a similar pregnancy history but who had a normal Factor V genotype were also studied. RESULTS: The live birth rate was significantly lower amongst women with a history of recurrent early miscarriage who carried the Factor V Leiden allele (6/16; 37.5%) compared with that amongst those with a normal Factor V genotype (106/153; 69.3%; odds ratio 3.75, 95% confidence intervals 1.3-10.9). The live birth rate was 11.1% (1/9) amongst those with a history of late miscarriage carrying the Factor V Leiden allele and 48.9% (22/45) amongst those with a normal Factor V genotype. CONCLUSIONS: Attention should be directed at screening women with recurrent miscarriage associated with placental thrombosis for Factor V Leiden and a policy of targeted thromboprophylaxis during future pregnancies should be assessed in the form of a randomized controlled trial.

Uterine artery Doppler in predicting pregnancy outcome in women with antiphospholipid syndrome.

OBJECTIVE: To assess midtrimester uterine artery Doppler in the prediction of preeclampsia and small for gestational age (SGA) infants in women with primary antiphospholipid syndrome. METHODS: One hundred seventy pregnant women with histories of recurrent miscarriage in association with antiphospholipid antibodies (32 lupus anticoagulant positive, 47 IgG anticardiolipin positive, 78 IgM anticardiolipin positive, and 13 lupus anticoagulant and anticardiolipin antibodies positive) treated with low-dose aspirin and heparin were recruited prospectively. Doppler assessment of the uterine arteries (presence or absence of notches and pulsatility index) were performed at 16-18 and 22-24 weeks. The main outcome measures were the delivery of a SGA infant and the development of preeclampsia. RESULTS: There were 164 live births and six midtrimester losses. The prevalence of preeclampsia and SGA was similar at 10%. In predicting preeclampsia or SGA, uterine artery pulsatility index at either interval was of no value, and the diagnostic accuracy of the Doppler was limited to bilateral uterine artery notches at 22-24 weeks in the subgroup of women with positive lupus anticoagulant. In this subgroup, bilateral uterine artery notches at 22-24 weeks in predicting preeclampsia generated a high likelihood ratio for positive test (12.8, 95% confidence interval 2.2, 75), sensitivity (75%), specificity (94%), positive (75%) and negative (94%) predictive value. In predicting SGA, the corresponding figures were respectively 13.6 (95% confidence interval 1.9, 96), 80%, 94%, 80%, 94%. Uterine artery Doppler was of limited value in pregnancies associated with anticardiolipin antibodies in isolation. CONCLUSION: In pregnancies associated with lupus anticoagulant, uterine artery Doppler at 22-24 weeks is a useful screening test in predicting preeclampsia and SGA infants.

Factor V Leiden and acquired activated protein C resistance among 1000 women with recurrent miscarriage.

Activated protein C (APC) resistance, both in its congenital form, due to the factor V Leiden mutation, and in its acquired form, are important risk factors for systemic venous thrombosis. In view of the suspected thrombotic aetiology of some cases of recurrent miscarriage, the prevalence of APC resistance was determined among 1111 consecutive Caucasian women with a history of either recurrent early miscarriage (three or more consecutive pregnancy losses at <12 weeks gestation; n = 904) or a history of at least one late miscarriage (>12 weeks gestation; n = 207). A control group of 150 parous Caucasian women with no previous history of adverse pregnancy outcome was also studied. Acquired APC resistance was significantly more common among both women with recurrent early miscarriage (8.8%: 80/904; P = 0.02) and those with late miscarriage (8.7%: 18/207; P = 0.04) compared with controls (3.3%: 5/150). In contrast, the frequency of the factor V Leiden allele was similar among (i) women with recurrent early miscarriage (3.3%:60/1808; 58 heterozygotes and one homozygote), (ii) those with late miscarriage (3.9%:16/414; 14 heterozygotes and one homozygote) and (iii) the control group (4.0%:12/300; 12 heterozygotes). Acquired but not congenital APC resistance (due to the factor V Leiden mutation) is associated with both early and late miscarriage.

Recurrent miscarriage--an aspirin a day?

Recurrent miscarriage and later pregnancy complications are in some cases associated with placental thrombosis and infarction. The aim of this study was to assess the value of low dose aspirin (75 mg daily) in improving the subsequent livebirth rate amongst women with either unexplained recurrent early miscarriage (<13 weeks gestation; n = 805) or unexplained late pregnancy loss (n = 250). Amongst women with recurrent early miscarriages, there was no significant difference in the livebirth rate between those who took aspirin (251/367; 68.4%) compared with those who did not take aspirin [278/438; 63.5%; odds ratio (OR) 1.24; 95% confidence interval (CI) 0.93-1.67]. This relationship was independent of the number of previous early miscarriages. In contrast, women with a previous late miscarriage who took aspirin had a significantly higher livebirth rate (122/189; 64.6%) compared with those who did not take aspirin (30/61; 49.2%: OR 1.88; 95% CI 1.04-3.37). The empirical use of low dose aspirin amongst women with unexplained recurrent early miscarriage is not justified. We are currently investigating the role of incremental doses of aspirin in the treatment of women both with early miscarriages associated with thrombophilic abnormalities and in those with late pregnancy losses.

Prospective pregnancy outcome in untreated recurrent miscarriers with thyroid autoantibodies.

The purpose of this study was to determine the prevalence of thyroid antibodies in women with recurrent miscarriage and to observe whether their presence was predictive of future pregnancy outcome. A total of 870 consecutive, non-pregnant women with a history of three or more pregnancy losses and normal parental karyotypes were investigated for the presence of thyroglobulin antibodies (TgAb) and for thyroid microsomal antibodies (TmAb). Thyroid antibodies were found in 162 (19%) women. TgAb only were found in eight women (5%); TmAb only in 98 (60%) and both TgAb and TmAb were found in 56 (35%). Thirteen women had a history of thyroid disease and a further 15 women were found to have abnormal thyroid function. All 28 were excluded from the pregnancy outcome study. Among the remaining 134 thyroid antibody positive women, 36 women were not tested and normal thyroid stimulating hormone results were obtained for 98. In the group proven euthyroid, 14 of 24 untreated pregnancies resulted in live births (58%). Among the 710 thyroid antibody negative women, 47 of 81 untreated pregnancies resulted in live births (58%). The future risk of pregnancy loss in women with unexplained recurrent miscarriage is not affected by their thyroid antibody status.

Polycystic ovaries and recurrent miscarriage--a reappraisal.

The prevalence of polycystic ovaries (PCO) was established amongst 2199 consecutive women (median age 33 years; range 19-46) with a history of recurrent miscarriage (median 3; 3-14). A diagnosis of PCO was made if the ovarian volume was enlarged (>9 ml), there were >/=10 cysts of 2-8 mm in diameter in one plane and there was increased density of the stroma. In a cohort study, the prospective pregnancy outcome of 486 of the women scanned who were antiphospholipid antibody negative and who received no pharmacological treatment during their next pregnancy was studied. The prevalence of PCO was 40.7% (895/2199). The livebirth rate was similar amongst women with PCO (60.9%; 142/233) compared to that amongst women with normal ovarian morphology (58.5%; 148/253; not significant). Neither an elevated serum luteinizing hormone concentration (>10 IU/l) nor an elevated serum testosterone concentration (>3 nmol/l) was associated with an increased miscarriage rate. Polycystic ovarian morphology is not predictive of pregnancy loss amongst ovulatory women with recurrent miscarriage conceiving spontaneously. The search for a specific endocrine abnormality that can divide women with PCO into those with a good and those with a poorer prognosis for a future successful pregnancy continues.

Bone density changes in pregnant women treated with heparin: a prospective, longitudinal study.

Heparin plus aspirin significantly improves the live birth rate of women with primary antiphospholipid syndrome. Osteopenia is a major concern of long-term heparin therapy. We studied prospectively the bone mineral density (BMD) changes during pregnancy and the puerperium in 123 women with primary antiphospholipid syndrome treated with low-dose aspirin and subcutaneous low-dose heparin (46 women took unfractionated heparin and 77 took low-molecular-weight heparin). Lumbar spine, neck of femur and forearm BMD were measured, using dual energy X-ray absorptiometry, at 12 weeks gestation, immediately postpartum and 12 weeks postpartum. The mean heparin duration was 27 weeks (range 22-29). During pregnancy, BMD decreased by 3.7% (P < 0.001) at the lumbar spine and by 0.9% (P < 0.05) at the neck of femur with no significant change at the forearm. Lactation was associated with a significant decrease in the lumbar spine and neck of femur BMD. There was no significant difference in BMD changes between the two heparin preparations. No woman suffered a symptomatic fracture. Long-term heparin treatment during pregnancy is associated with a small but significant decrease in BMD at the lumbar spine and neck of femur. This decrease is similar to that previously reported to occur in untreated pregnancies.

Pregnancy complications in women with recurrent miscarriage associated with antiphospholipid antibodies treated with low dose aspirin and heparin.

OBJECTIVE: To study the obstetric course of women with a history of recurrent miscarriage associated with antiphospholipid antibodies, lupus anticoagulant and anticardiolipin antibodies, treated with low dose aspirin and low dose heparin. DESIGN: Prospective observational study. SETTING: University based tertiary referral clinic. POPULATION: One hundred and fifty pregnant women with a history of recurrent miscarriage associated with persistently positive tests for antiphospholipid antibodies. METHODS: Lupus anticoagulant was detected using the dilute Russell's viper venom time together with a platelet neutralisation procedure. IgG and IgM anticardiolipin antibodies were detected using a standardised enzyme linked immunosorbent assay. An IgG anticardiolipin level > or = 5 per litre units and an IgM anticardiolipin level > or = 3 per litre units was considered positive. Aspirin (75 mg daily) was commenced at the time of a positive pregnancy test and heparin (5000 units subcutaneously 12 hourly, or enoxaparin 20 mg daily) was started when fetal heart activity was demonstrated on ultrasound. Treatment was stopped at the time of miscarriage or at 34 weeks of gestation. RESULTS: One hundred and seven pregnancies (71%) resulted in a live birth. Forty-one pregnancies (27%) miscarried, the majority in the first trimester. One woman had a stillbirth, and one a premature baby who died in the neonatal period. One pregnancy was terminated for a fetal anomaly. Gestational hypertension complicated 17% (18/108) of ongoing pregnancies and antepartum haemorrhage 7% (8/108). Twenty-six babies (24%) were delivered before 37 weeks of gestation. Fifty women (46%) were delivered by caesarean section. The median birthweight of all live born infants was 3069 g (range 531-4300); however 15% (16/108) of the infants were small for gestational age. CONCLUSION: Combination treatment with aspirin and heparin leads to a high live birth rate among women with recurrent miscarriage and antiphospholipid antibodies. However, successful pregnancies are prone to a high risk of complications during all trimesters. Close antenatal surveillance and planned delivery of these pregnancies in a unit with specialist obstetric and neonatal intensive care facilities are indicated.