It's presented in the 7th International Congress of the Turkish-German Gynecological Association, Antalya, Turkey, May 16-20, 2007.

Introduction

Chromosome diseases are genetic diseases where a large part of the genetic code has been disrupted. Most cases of simple aneuploidy (monosomy or trisomy) are likely due to meiotic non-disjunctions. Down syndrome is the most common chromosomal problem accounting for significant morbidity and mortality. Down syndrome occurs in approximately one in 1250 children born to women in their 20s. The chances increase to one in 400 by age 35, and one in 100 at age 40. Other syndromes include trisomy 13, 18, Turner and Cri du Chat syndromes, etc. First trimester screening test (nuchal translucency, serum-free beta-hCG and PAPP-A), and second trimester triple test (alpha-fetoprotein, unconjugated oestriol and hCG with maternal age) are the most common methods used for chromosomal anomaly screening. Generally, the standard care in prenatal diagnosis is to offer invasive genetic diagnosis to determine fetal karyotype in pregnant women 35 years and older, whereas resources for noninvasive screening tests (such as ultrasound or maternal serum biochemistry) are usually used in younger women (₁). Definitive diagnosis requires chorion villus sampling (CVS) or amniocentesis, and current maternal serum-based testing requires a false-positive rate of about 5% to detect 85% of affected pregnancies.

Second-trimester amniocentesis is the most common invasive technique according to its safety, cytogenetic accuracy and low cost than some other screening methods. For patients attending after the 14 weeks of pregnancy and at the clinics where CVS experience is not enough; still anomaly screening by ultrasound and triple screen combination seems to be a reliable, safe method for prenatal diagnosis. In the literature amniocentesis complication rates range from 0.5 to 2.2% (₂,₃,₄). The aim of this study was to evaluate the indications, karyotype results, and maternal-fetal complications of amniocentesis performed in our clinic.

Materials and Methods

We retrospectively analysed the results of 189 amniocentesis cases performed in our clinic between 2002-2007 for different indications including advanced maternal age (≥35 years), high risk in triple screening test performed, advanced maternal age and high risk in triple screening test, high risk in first trimester screening test, multiple fetal anomalies detected by obstetric ultrasonography, anxiety and other indications. First trimester screening test was performed between 11-13 weeks of pregnancy by measuring nuchal translucency, freeBeta-hCG and PAPP-A. Triple screening test was performed by measuring alpha-fetoprotein, unconjugated estriol and hCG with maternal age in maternal blood between 16 and 20 weeks of pregnancy. Interferential process with the purpose of karyotype determination related with gestational week was applied to pregnants who had >1/250 risk of giving birth with Down syndrome. All the patients signed a written consent and were checked in terms of general blood biochemistry, hepatitis porter, Rh incompatibility. Abnormal karyotypes were and the structural chromosomal anomalies were recorded. The success of cell culture and neonatal outcome had been also evaluated. All women were in singleton pregnancy. Toshiba SSA-240A 3.75 MHz (Toshiba Corporation, Japan) transabdominal convex probe was used at interferences. After a detailed systematic ultrasonography determination and placenta localization, iodine solution was swabbed onto her belly in order to cleanse the area and sterile drapes were placed around the area. The amniocentesis was performed between 16 and 22 weeks of gestational age with free hand technique by 22 G spinal needle and 1 ml of amniotic fluid was taken for every week of pregnancy. The first 2 cc of amniotic fluid was discarded in order to minimize the contamination with the mother's blood or cells, then 18-20 cc of the amniotic fluid was removed.

Results

The maternal age varied between 19 and 41 (median 32). The tissue cell culture was unsuccesful in 3 (1 %) of cases and procedure was repeated. The most common indication for genetic amniocentesis was maternal age ≥ 35 years in 75 (40 %). The other indications were: high risk in triple screening test 51 (27 %), advanced maternal age and high risk in triple screening test 11 (6 %), high risk in first trimester screening test 11 (6 %), multiple fetal anomalies detected by obstetric ultrasonography in 14 (7 %), anxiety in cases 5 (3 %) and other indications in 22 cases (12 %), (Table 1). Other indications included obstetric history for giving birth with chromosomal anomalies in 11 cases (8 Down syndrome, 2 spinal muscular atrophy, 1 Turner syndrome) and with mental retardation in one case; advanced maternal age and obstetric history with Down syndome in one case, advanced maternal age + fetal anomalies (with cystic hygroma in 5 cases, with cockayne syndrome in one case), and advanced maternal age + recurrent abortion history in 2 cases. The karyotype was not available in three samples (1%) due to culture failure.

Figure 1

Table 1: Chromosomal abnormalities, indications of amniocentesis, and outcome of the pregnancy after amniocentesis.

Abnormal karyotypes were detected in 11 of 189 cases (6 %): two trisomy 13 (1 %), two trisomy 18 (1 %), one turner (1 %), three trisomy 21 (2 %), and three structural chromosomal anomalies (2 %). All pregnancies were terminated after the diagnosis of genetic anomalies, (Table 1). Three pregnancies were terminated because of corpus callosum agenesis, holoprosencephaly, ventriculomegaly, encephalocele, omphalocele, cleft lip and palate detected on ultrasound. The chromosomal analyses of these patients were already normal. No more than two needle insertions were done on the same day. There was no fetal losses and maternal complication due to the procedure.

Discussion

Amniocentesis is the most common invasive prenatal diagnostic procedure undertaken in the world and was first included to clinical practice by making karyotype determination from fetal cells (₅). It can be used to diagnose a large number of genetic and chromosomal abnormalities in the fetus. Amniocentesis is usually done between the 14th and 20th weeks of pregnancy, most commonly at weeks 15-16. As was seen in the presented study, the most common reason of amniocentesis is for maternal age of 35 or over (₆).

Complications of amniocentesis can include cramping and bleeding, amniotic fluid leakage, and miscarriage/fetal death (₂,₃,₄). More unusual complications include fetal respiratory problems, birth defects, uterine infection (₇). Because of the increased rate of miscarriage, talipes equinovarus, culture failure, multiple-needle insertion, amniotic fluid leakage, and respiratory problems, early amniocentesis, at 11–13 weeks of gestation, has largely been discredited for prenatal testing at this time (₈). It was shown that amniocentesis applied in this period and rate of fetal loss related with interference brought 1% additional risk as to group which was not applied amniocentesis (₇). The rate of amniotic fluid leakage after amniocentesis as approximately 1-2 % (₃,₄). Most of the time, the fluid loss is minimal. Because the body is constantly making and replacing amniotic fluid, lost fluid is thought to be replaced within twelve hours to a few days of the procedure. Bed rest is the recommended management in those patients. Normally, spontaneous occlusion and repair of the penetration tract is expected by conservative management. Some authors propose the maternal blood clot patch procedure in postamniocentesis amniorrhea that does not respond to expectant management in 5 days (₉). Annapoorna V et al. found that children exposed to second trimester amniocentesis had an increased incidence of respiratory illness compared with controls (₇,₁₀). This was an unexpected finding, and they called for further study on the issue. In contrast to the study of Annapoorna V, Finegan JA et al. did find a higher rate of ear infections in children who had been exposed to amniocentesis as babies (₁₁). If amniotic fluid plays a role in inner ear and mid-ear development, amniotic fluid leakage might indeed result in long-term damage to the ears. However, little corroborating information can be found for this risk.

The risk of miscarriage ranges from 1 in 400 to 1 in 200 (₇,₈). The miscarriage rate depends greatly on the skill of the provider and can occur because of infection in the uterus, the water breaks or labor is induced prematurely. No fetal losses due to the procedure were detected in our cases.

Every needle insertion raises the risk for problems so it is best to avoid multiple insertions whenever possible. If success does not come after two needle insertions, the amniocentesis should probably be abandoned that day and, if desired, tried again later. Of the 189 cases, in two cases, a second needle insertion was required. However, no maternal or fetal complications were detected. In addition, no membrane rupture was recorded.

Sometimes amniotic membrane “tenting” occurs, where the membranes resist penetration by the needle and are pushed back but not penetrated by the needle. This may be more associated with fetuses with chromosomal abnormalities like Down Syndrome, but also is more common in earlier amniocentesis procedures (₈,₁₂). However, in the presented study, tenting occurred in 2 cases with advanced maternal age and high risk in triple screening test but no relation has been found between tenting and chromosomal abnormalities leading to that once again, the size of this study was limited.

When all amniocentesis cases were evaluated, only 3 of 189 cases had culture failure. Culture success from fetal cells we obtained was 98%. Our success rate was similar to the result of Leung WC et al. (₁₃). Contamination of samples was the reason for cases having no reproduction within our series. When laboratory workers are unable to culture enough cells from the amniotic fluid to do karyotyping, a second amniocentesis or fetal blood sampling would be necessitate. A second amniocentesis was performed in our three cases which was found to be normal in the second fetal cell culture.

Chromosome anomaly rate of 6% (n:11) obtained from 189 cases. No chromosome anomaly was found in 73 cases within our series which were applied interferential process due to only advanced maternal age. In this group, Down syndrome and a structural chromosomal anomaly (46,XY, inv 9 (p11q13) were detected in one case. In total, 3 of all had structural anomalies as inversions. The neonatal outcome in these three cases was found to be normal. Additionally, karyotype of 11 cases was normal which had high risk for Down syndrome in triple test and had age risk. Two trisomy 18 and one Turner syndrome were also found. Trisomy 18 was found in two cases with advanced maternal age + cystic hygroma. In the ultrasonographic examinations, the fetus with Turner syndrome had cystic hygroma; omphalocele, cleft lip and palate, and ventriculomegaly were found in one of the fetus with trisomy 13 and ventriculomegaly, omphalocele were found in the second fetus with trisomy 13. Although triple-marker screening has been advocated as a more efficient method of diagnosing Down syndrome among fetuses of women older than 35 years of age, its value as a replacement for amniocentesis in these groups has not been assessed properly. We perform routinely amniocentesis in these pregnant women with advanced maternal age. In the present study, the reason for not finding chromosome abnormalities in cases with advanced maternal age + high risk in the triple screening test which were in only advanced maternal age group may be explained by the limited population size.

In conclusion, although the size of this study was limited, amniocentesis being the most common prenatal diagnostic method, continues to be a reliable and safe method with low fetal loss rate.

Correspondence to

Dr. Mehmet A. OSMANAĞAOĞLU Karadeniz Technical University Faculty of Medicine, Department Obstetrics and Gynecology 61080 Trabzon- Turkey Tel : 0 (462) 377 54 19 Fax : 0 (462) 325 05 18 e-mail : osmanaga@meds.ktu.edu.tr