Early Amniotic Fluid Research Articles

Guanosine and Deoxyinosine Structural Analogs Extracted from Chick Early Amniotic Fluid Promote Cutaneous Wound Healing.

Wound healing is a complex, dynamic process supported by a myriad of cellular events that must be tightly coordinated to efficiently repair damaged tissue. These wounds are a significant socioeconomic burden due to their high prevalence and recurrence, which is why the phenomenon of wounds has also been labeled as a "Silent Epidemic". Most of these wounds become "chronic", with around 15% of them remaining unresolved 1-year post incidence, which results in a prolonged yet avoidable burden to patients, families, and the health system. In this experimental study, we tried to purify the potent components in chick early amniotic fluid (ceAF) and applied these components to the wound healing mechanism. We first subjected ceAF to a series of purifications, including an HPLC purification system along with ion-exchange chromatography technology to purify other potential components. Upon narrowing down, we found two structural analogs: guanosine and deoxyinosine. We performed these components' cell scratch and trans-well migration assays to validate the accurate dosage. We also assessed these components via topical administration on the skin of murine model wounds. For this, we randomly divided C57BL/6 (all black, male, 5 weeks old) mice into groups. The wound model was established through excising the skin of mice and treated the wounds with different fractions of guanosine and deoxyinosine continuously for 8-10 day intervals. Once the healing was complete, the skin was excised to determine the inflammatory response and other biochemical parameters of the healed skin, including epidermal thickness, collagen density, macrophages, and neutrophil infiltration at the wounded site. Quantitative real-time PCR and immunoblot assays were performed to determine active gene expression and protein expression of proinflammatory molecules, growth factors, and cytokines. All these findings support our data indicating the promising healing properties of guanosine and deoxyinosine isolated from ceAF.

Chick early amniotic fluid component improves heart function and protects against inflammation after myocardial infarction in mice.

Myocardial infarction (MI) is the major cause of mortality around the world. We recently demonstrated that chick early amniotic fluid (ceAF) can effectively rescue ischemic heart injury, indicating that it has a therapeutic function in MI. However, its functional components and the underlying mechanisms remain to be clarified. Here, we demonstrated that a fraction of ceAF, peak 8 (P8), had a protective effect on acute MI. P8 significantly decreased cardiomyocyte cross-sectional areas and cardiomyocyte apoptosis in MI mice. Using a human embryonic stem cell-derived cardiomyocyte model, which was subjected to hypoxia and reoxygenation, mimicking MI state, we found that P8 treatment reduced apoptosis and reversed myocardial contractility. Mechanistically, P8 improved cardiac function by inhibiting NF-κB signaling and downregulating inflammatory cytokine expression. Using mass spectrometry, we identified that guanosine and deoxynucleoside were the main functional components of P8 that suppressed the inflammatory response in human embryonic stem cell-derived cardiomyocytes. Collectively, our data suggest that specific components from ceAF are promising therapeutic agents for ischemic heart injury and could be a potential supplement to current medications for MI.

Chick Early Amniotic Fluid (ceAF) Deters Tumorigenesis via Cell Cycle Arrest and Apoptosis

In recent years, amniotic fluids have gained attention in cancer research. They have an influential role in protecting embryos against several anomalies. Chick early amniotic fluid (ceAF)-amniotic fluid isolated from growing chicken-has been used in many other studies, including myocardial infarctions and skin regeneration. In this study, we employed ceAF's promising therapeutic applications against tumorigenesis in both in vitro and in vivo studies. We selected three robust proliferating tumor cell lines: BCaP37, MCF7, and RKO. We found that selective dosage is required to obtain maximum impact to deter tumorigenesis. ceAF not only disrupted the uniform colonies of tumor cell lines via disturbing mitochondrial transmembrane potential, but also arrested many cells at growing G1 state via working agonistically with aphidicolin. The significant inhibition of tumor metastasis by ceAF was indicated by in vivo models. This leads to apoptosis analysis as verified by annexin-V staining stays and immunoblotting of critical proteins as cell cycle meditators and apoptosis regulators. Not only on the protein level, but we also tested ceAF's therapeutic potentials on mRNA levels as indicated by quantitative real-time PCR summarizing the promising role of ceAF in deterring tumor progression. In conclusion, our study reveals the potent role of ceAF against tumorigenesis in breast cancer and colon carcinoma. Further studies will be required to determine the critical components present in ceAF and its purification to narrow down this study.

Therapeutic application of chick early amniotic fluid: effective rescue of acute myocardial ischemic injury by intravenous administration

Myocardial regeneration has been considered a promising option for the treatment of adult myocardial injuries. Previously, a chick early amniotic fluid (ceAF) preparation was shown to contain growth-related factors that promoted embryonic growth and cellular proliferation, though the nature of the components within ceAF were not fully defined. Here we tested whether this ceAF preparation is similarly effective in the promotion of myocardial regeneration, which could provide an alternative therapeutic for intervening myocardial injury. In this study, a myocardial ischemic injury model was established in adult mice and pigs by multiple research entities, and we were able to show that ceAF can efficiently rescue damaged cardiac tissues and markedly improve cardiac function in both experimental models through intravenous administration. ceAF administration increased cell proliferation and improved angiogenesis, likely via down-regulation of Hippo-YAP signaling. Our data suggest that ceAF administration can effectively rescue ischemic heart injury, providing the key functional information for the further development of ceAF for use in attenuating myocardial injury.

Therapeutic Application of Chick Early Amniotic Fluid: Effective Rescue of Acute Myocardial Ischemic Injury by Intravenous Administration

Background: Stem cell therapy has been reported to be effective and promising for the treatment of adult myocardial ischemic injury. However, few stem cells survive and differentiate into cardiomyocytes after transplantation and the mechanisms of repair by stem cells are multiplex and undetermined. Furthermore, different types of stem cells elicit various degrees of immunogenicity and tumorigenicity, which poses a high risk for their future clinical applications. Chick early amniotic fluid (ceAF) administration could be a valuable alternative or addition to current stem cell therapy. Methods: Mice myocardial infarction and swine ischemic reperfusion injury models were established and received ceAF by intravenous injection after surgery. Findings: ceAF efficiently rescued damaged cardiac tissue, stimulated cardiac repair, and markedly improved heart function in adult mice and swine models of myocardial ischemic injury. Notably, no noticeable abnormalities were observed in the treated animals and no systemic inflammation or immune reactions were perceived after recurring intravenous infusion of ceAF in rats and monkeys. We further show that ceAF promoted heart repair through multiple mechanisms, including, down-regulation of the Hippo-YAP signaling pathway, increased cell proliferation, improved angiogenesis, and recruitment of pro-healing macrophages after myocardial damage in adult mice and pigs. Interpretation: Together, our data indicate that intravenous injection of ceAF effectively rescued ischemic heart injury through multiplex mechanisms and could be a novel and safe non-invasive therapy for ischemic heart disease in substitution of stem cell therapy. Funding Information: This work was supported by ZheJiang HygeianCells Biomedical Co. Ltd, Hangzhou, Zhejiang, 310019, China. Declaration of Interests: None declared. Ethics Approval Statement: All animal protocols were reviewed and approved by the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Publication No. 85-23, Revised), by the U.S. Department of Agriculture (USDA) in the Animal Welfare Act (Public Law 99-198), and were carried out under the supervision of the Fudan University Institutional Animal Care and Use Committee (IACUC), Shanghai Mincal Medical Research Co. IACUC, WuXi AppTec (Shanghai, China) IACUC, or the JOINN Laboratories IACUC (Suzhou, China).

Metabolomic profiles of mid-trimester amniotic fluid are not associated with subsequent spontaneous preterm delivery or gestational duration at delivery

Introduction Spontaneous preterm delivery (<37 gestational weeks) has a multifactorial etiology with still incompletely identified pathways. Amniotic fluid is a biofluid with great potential for insights into the feto-maternal milieu. It is rich in metabolites, and metabolic consequences of inflammation is yet researched only to a limited extent. Metabolomic profiling provides opportunities to identify potential biomarkers of inflammatory conditioned pregnancy complications such as spontaneous preterm delivery. Objective The aim of this study was to perform metabolomic profiling of amniotic fluid from uncomplicated singleton pregnancies in the mid-trimester to identify potential biomarkers associated with spontaneous preterm delivery and gestational duration at delivery. A secondary aim was to replicate previously reported mid-trimester amniotic fluid metabolic biomarkers of spontaneous preterm delivery in asymptomatic women. Method A nested case-control study was performed within a larger cohort study of asymptomatic pregnant women undergoing mid-trimester genetic amniocentesis at 14–19 gestational weeks in Gothenburg, Sweden. Medical records were used to obtain clinical data and delivery outcome variables. Amniotic fluid samples from women with a subsequent spontaneous preterm delivery (n = 37) were matched with amniotic fluid samples from women with a subsequent spontaneous delivery at term (n = 37). Amniotic fluid samples underwent untargeted metabolomic analyses using liquid chromatography-mass spectrometry. Multivariate random forest analyses were used for data processing. A secondary targeted analysis was performed, aiming to replicate previously reported mid-trimester amniotic fluid metabolic biomarkers in women with a subsequent spontaneous preterm delivery. Results Multivariate analysis did not distinguish the samples from women with a subsequent spontaneous preterm delivery from those with a subsequent term delivery. Neither was the metabolic profile associated with gestational duration at delivery. Potential metabolic biomarker candidates were identified from four publications by two different research groups relating mid-trimester amniotic fluid metabolomes to spontaneous PTD, of which fifteen markers were included in the secondary analysis. None of these were replicated. Conclusions Metabolomic profiles of early mid-trimester amniotic fluid were not associated with spontaneous preterm delivery or gestational duration at delivery in this cohort.

Elevated placenta growth factor levels in the early second-trimester amniotic fluid are associated with preterm delivery

Objective: The purpose of this study was to determine how angiogenesis-related factors correlate with preterm delivery.Methods: A cohort of 382 pregnant women undergoing early second-trimester genetic amniocentesis was enrolled and followed-up until delivery, and the amniotic fluid was collected and stored as a nested case-control study. Cases with preterm delivery (n = 31) were compared with matched controls with term delivery (n = 62). The amniotic fluid concentrations of placenta growth factor (PlGF), angiogenins, angiopoietin-2, soluble fms-like tyrosine kinase and soluble endoglin were determined using enzyme-linked immunosorbent assays.Results: Women who delivered preterm had a higher amniotic PlGF concentration compared with the control group (median 12.6 pg/ml versus 6.1 pg/ml; p=0.027). Other angiogenesis-related factors did not show any differences between case and control groups. The odds ratio for preterm delivery based on amniotic fluid PlGF was 1.031 (95% confidence interval: 1.002–1.061; p=0.035). Additionally, when the cases were subdivided into early preterm, late preterm and term groups, PlGF values between the early preterm and term delivery groups were significantly different (median 16.6 pg/ml versus 6.1 pg/ml; Bonferroni-adjusted p=0.018).Conclusion: Amniotic fluid PlGF levels in the early second trimester of pregnancy are associated with preterm delivery.

Soluble Fas and Fas-ligand levels in mid-trimester amniotic fluid and their associations with severe small for gestational age fetuses: a prospective observational study

We aimed to determine the second-trimester amniotic fluid (AF) levels of soluble Fas (sFas) and Fas-ligand (FasL) and investigate their association with fetal growth. Therefore, sFas and FasL levels were measured by enzyme immunoassay in the AF of 21 small for gestational age (SGA), 13 large for gestational age (LGA), and 44 appropriate for gestational age (AGA) fetuses of pregnant women who underwent amniocentesis at between 15 and 22 weeks gestation. Our study results showed that sFas and FasL levels were detectable in AF. sFAS median (25th–75th centile) levels were 3.8 (2.8–4.6)ng/ml in SGA, 3.6 (3.1–4.5)ng/ml in AGA, and 4.0 (3.1–4.4)ng/ml in LGA. FasL median (25th–75th centile) levels were 26.0 (20.3–32.7)pg/ml in SGA, 22.7 (18.4–28.5)pg/ml in AGA, and 21.5 (15.8–30.9)pg/ml in LGA. The differences were not statistically significant. Nevertheless, statistically significant differentiation of FasL levels existed when SGA fetuses in the extremes of distribution (≤5th, ≤2.5th centile) were considered. This is the first study presenting sFas and FasL concentrations in early second-trimester amniotic fluid in AGA, SGA, and LGA fetuses. We found indications that severe and very severe SGA fetuses (≤5th and ≤2.5th centile) have high levels of FasL in the amniotic fluid. This finding probably reflects the increased rate of apoptosis that is assumed to exist in cases of extreme growth restriction.

Proteomic Biomarkers in Second Trimester Amniotic Fluid That Identify Women Who Are Destined to Develop Preeclampsia

This study investigated whether proteomic analysis of amniotic fluid (AF) in the early second trimester can be used to predict the development of preeclampsia. Amniotic fluid samples were collected at the time of genetic amniocentesis (15-19 weeks of gestation) from women who subsequently developed preeclampsia and from gestational age-matched normotensive controls (n = 10 for each). Amniotic fluid samples were subjected to proteomic analysis using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, sodium dodecyl sulfate polyacrylamide gel coupled with in-gel tryptic digestion, electrospray ionization tandem mass spectrometry (MS/MS), immunodepletion assays, and enzyme-linke immunosorbent assay. Five proteomic biomarkers were identified, which were differentially expressed in women who subsequently developed preeclampsia compared with those women who did not; four of these peaks were significantly upregulated (mass-to-charge ratio of 9080 [P = .006], 14 045 [P = .010], 14 345 [P = .049], and 28 087 [P = .006]) and one was significantly downregulated (mass-to-charge ratio of 4679 [P = .014]) in women who subsequently developed preeclampsia. Using electrospray ionization MS/MS and immunodepletion assays, two protein peaks were identified as albumin fragment and apolipoprotein A-I. Using proteomic technology, this study identified protein biomarkers that are differentially expressed in the early second trimester AF from women who subsequently develop preeclampsia compared with women who remained normotensive. Early identification of women at risk of developing preeclampsia will allow clinicians to better optimize maternal and perinatal outcomes.

Fetal overgrowth (FOG) is associated with early perturbations in amniotic fluid (AF) glucose, insulin and insulin like growth factors (IGF) in mothers with and without gestational diabetes (GDM)

FOG occurs in mothers with and without GDM. Few biochemical markers have been identified. We investigated the possibility that perturbations in 2nd trimester AF glucose, insulin, IGF 2 or its binding proteins (BP) 1 or 3 might underscore FOG and that these early AF disturbances might differ in mothers with or without GDM. Our study population (n=701) was stratified as follows: mothers were classified as GDM or NON GDM and their offspring as appropriate (AGA) or large for gestational age (LGA). ANCOVA, multiple linear and logistic regressions were performed and, using pairs of AF constituents, 2D Bayesian probability plots were created to predict LGA. In the GDM population AF insulin was positively and IGF 2 was negatively associated with growth centile, while elevated AF glucose increased the odds of neonates being born LGA. In the NON GDM population AF insulin was positively and BP 3 negatively associated with growth centile, but the likelihood of LGA was not increased by AF glucose. Bayesian probability plots showed that specific AF constituents separated LGA from AGA infants in GDM mothers but were less discriminating in NON GDM mothers. In conclusion, fetal exposure to increased AF glucose in the first trimester increases the risk of delivery of an LGA infant in GDM mothers. Lower BP 3 in NON GDM mothers and IGF 2 in GDM mothers provide further evidence that perturbations in growth factors may underscore FOG. [Funded by CIHR]Grant Funding Source: CIHR

Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation

Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.

Factors determining outcome in gastroschisis: clinical experience over 18 years

Although the survival in gastroschisis has improved to more than 85%, serious morbidity with consequent long hospital stay is still very common. Alternative strategies such as planned early delivery and amniotic fluid exchange are being explored. In order to evaluate these approaches, adequate risk stratification of patients with gastroschisis is required. The aim of this study was to identify the risk factors associated with increased morbidity and mortality. A retrospective analysis of all patients with gastroschisis treated at our institution between 1988 and 2005 was conducted. Patients' data were collected from the files and compared for statistical significance with three outcome measures: (1) complicated post-operative course; (2) time to full enteral feeding (TFE); (3) length of hospital stay (LOS). SPSS was used for statistical analysis. Fifty-nine newborns with gastroschisis were admitted during the study period. Antenatal diagnosis was made in 40 cases (68%). Associated malformations (other than intestinal anomalies) were present in eight patients. One patient with trisomy 13 was excluded from the analysis. Ten patients (17%) had compromised bowel (atresia, severe ischaemia or a combination). Primary repair was successful in 47 (81%) and in 11 patients a silo was applied. Overall survival was 54/58 children (93%), four patients died. Complications associated with closure occurred in eight patients. Post-operative course was uncomplicated in 45 patients and complicated in 13. The RR for patients with compromised bowel to have a complicated post-operative course is 9 (95% CI 2-39) and the RR to die is 20 (95% CI 2-222) for these patients. Risk stratification of patients with gastroschisis appears possible based on the presence of compromised bowel before closure.

Prenatal diagnosis of some metabolic diseases using early amniotic fluid samples: report of a 15 years, experience

In the present study, we report the results of 132 prenatal diagnoses performed on chorionic villi and cell-free amniotic fluid obtained simultaneously at 12-13 weeks of gestation. In addition, we report the result of 59 prenatal diagnoses performed at 12-13th week using amniotic fluid only. A total of one fetal loss (1/191) was observed when a sample of amniotic fluid was obtained at around 12-13 weeks, whereas three losses (3/82) were observed after midtrimester amniocentesis. We attribute this finding to the fact that only a very small volume of amniotic fluid was sampled using a very small needle. From these data it appears that when a couple is facing a high risk of recurrence of some metabolic diseases, the study of chorionic villus and amniotic fluid sampled simultaneously offers a safe and reliable method of early prenatal diagnosis.

Xenobiotic phenols in early pregnancy amniotic fluid

We found detectable levels of three phytoestrogens (enterolactone, daidzein and genistein) and bisphenol A (BPA) in 21 residual amniotic fluid specimens that were collected before 20 weeks gestation. Samples were obtained by amniocentesis from women who were referred to the Mount Sinai Medical center because of advanced maternal age. Phytoestrogens were present in higher concentrations than BPA. Enterolactone was detected at the highest concentration (median 95.9 μg/L), followed by daidzein and genistein (9.5 and 1.4 μg/L, respectively). BPA was present at very low concentrations (10% > LOD of 0.5 μg/L). The relative concentration of the chemicals measured in amniotic fluid were identical to those in urine reported by other studies, i.e. enterolactone > daidzein > genistein ≫ BPA. Amniotic fluid is a source of fetal exposure to polar xenobiotics that come from the mother.

Echocardiographically detected mass “in transit” in early amniotic fluid embolism

Amniotic fluid embolism is a catastrophic illness related to the passage of fetal material into the pulmonary circulation causing cardiovascular collapse. A 29-year-old female sustained cardiopulmonary arrest during delivery presumably due to amniotic fluid embolism. A right atrial mass "in transit" was detected by echocardiography. It had an appearance and pattern of motion that was suggestive of a gelatinous consistency and is likely to have been an amniotic fluid embolus. There was also evidence of acute right ventricular overload. We recommend that echocardiography be considered early on such conditions to gain more insight into the pathogenesis of this complication.

Early mid-trimester amniotic fluid leptin concentration: indicator of fetal growth?

Early mid-trimester amniotic fluid leptin concentration: indicator of fetal growth?

Adrenomedullin Concentrations in Early 2nd-Trimester Amniotic Fluid: Relation to Preterm Delivery and Fetal Growth at Birth

Objective: The purpose of this study was to evaluate whether adrenomedullin concentrations in the early 2nd-trimester amniotic fluid predict preterm delivery or fetal growth at birth. Methods: The adrenomedullin concentrations in early 2nd-trimester amniotic fluid were measured in 70 pregnancies with term delivery and in 3 pregnancies with preterm delivery. Total and free adrenomedullin concentrations were measured from early 2nd-trimester amniotic fluid samples using an immunoradiometric assay. Results: The amniotic fluid total adrenomedullin concentrations in women with preterm delivery were significantly higher (129.7 ± 19.6 fmol/ml) than those in women with term delivery (92.5 ± 28.2 fmol/ml; p < 0.05). There were no significant differences for amniotic fluid free adrenomedullin concentrations and free/total adrenomedullin ratios between the two groups. Total or free adrenomedullin concentrations in the early 2nd-trimester amniotic fluid showed an inverse correlation both with birth weight (r = 0.27, p < 0.05, and r = 0.21, p < 0.05) and height (r = 0.30, p < 0.05, and r = 0.28, p < 0.05). There were no correlations between placental weight and total or free adrenomedullin concentrations in the early 2nd-trimester amniotic fluid. Conclusions: These results suggest that adrenomedullin concentrations in the early 2nd-trimester amniotic fluid might be related to further in utero fetal growth and that high levels of adrenomedullin in the early 2nd-trimester amniotic fluid may be involved in the occurrence of preterm delivery.

Improving the prenatal diagnosis of citrullinemia using citrulline/ornithine+arginine ratio in amniotic fluid.

Prenatal diagnosis of citrullinemia is performed using a direct argininosuccinate synthetase (ASS) assay on chorionic villi (CV) and citrulline concentration measurement in early amniotic fluid (AF). Here we report the results of 40 prenatal diagnoses performed using this method, discuss the difficulties encountered in interpreting the results, and propose the use of the citrulline/ornithine+arginine ratio (which is more discriminatory than citrulline concentration alone) when performing prenatal diagnosis of citrullinemia.

Amniotic fluid insulin at 14-20 weeks' gestation: association with later maternal glucose intolerance and birth macrosomia.

To examine the hypothesis that early second trimester amniotic fluid (AF) insulin concentration is elevated and later fetal growth is augmented in gravidas demonstrating later oral glucose intolerance. In this prospective observational cohort study, AF was sampled at 14-20 weeks' gestation in 247 subjects, and 1-h 50-g oral glucose challenge tests (GCTs) were performed at > or = 24 weeks. AF insulin was assayed by an automated immuno-chemiluminometric assay (8). Macrosomia was defined as birth weight above the 90th centile. AF insulin concentration (range 1.4-44.5 pmol/l) correlated positively with gestational age and maternal weight. A logistic regression analysis, adjusted for maternal age and midpregnancy weight, showed increased AF insulin multiples of gestational age-specific medians to be associated with subsequently diagnosed gestational diabetes mellitus (GDM) (OR 1.9, CI 1.3-2.4, P = 0.029). Among 60 subjects with GCT values > 7.2 mmol/l, each unit increase in AF insulin multiple of median (MOM) was associated with a threefold increase in fetal macrosomia incidence (3.1, 1.3-4.9, P = 0.048). An elevated AF insulin concentration at 14-20 weeks' gestation is associated with subsequently documented maternal glucose intolerance. Among gravidas with GCT values > 7.2 mmol/l, elevated early AF insulin concentration is associated with fetal macrosomia. Maternal glucose intolerance may affect fetal insulin production before 20 weeks' gestation.

Cytogenetic aspects of the Canadian early and mid-trimester amniotic fluid trial (CEMAT)

Cytogenetic results from a large multicentre randomized controlled study of 2108 amniotic fluids obtained at 11+0-12+6 weeks (EA) and 1999 fluids at 15+0-16+6 weeks (MA) were compared. There was no statistically significant difference in the rate of chromosome abnormalities (EA =1.9 per cent; MA=1.7 per cent) or level III mosaicism (EA=0.2 per cent; MA= 0.2 per cent) between the groups. Level I and Level II mosaicism occurred more frequently in MA. Maternal cell contamination was not significantly different between the groups, but maternal cells only were analysed from one bloody EA fluid. The number of repeat amniocenteses because of cytogenetic problems was 2.2 per cent in the EA group compared with only 0.3 per cent in the MA group. On average, culture of EA fluids required one day more than MA fluids. Although both culture success (97.7 per cent) and accuracy (99.8 per cent) were high for patients randomized to the EA group, routine amniocentesis prior to 13 weeks' gestation is not recommended for clinical reasons including an increased risk of fetal loss and talipes equinovarus.