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The number of women getting pregnant at a relatively later age is continuously increasing. Because of the increased incidence of Down syndrome (DS) with maternal age, efforts for the prenatal diagnosis of this disorder, as well as some other genetic diseases, are increasing as well. In spite of the intensive studies about fetal DNA isolation from maternal blood for prenatal diagnosis, invasive prenatal tests are still being used extensively in most countries. Amniocentesis (AS) is the most commonly applied invasive diagnostic test to obtain fetal cells for prenatal cytogenetic studies. It is a simple and informative procedure for prenatal genetic diagnosis but can be uninformative in 0.1% of the cases because of the failure of amniocytes to grow in culture.
Major chromosomal abnormalities have an incidence of 0.7%[3, 4] among live births, which is higher at the second trimester. Trisomy 21 is the most common abnormality detected both at delivery[3, 4, 6] and at midtrimester.[7, 8] According to AS data from Southeast Turkey, chromosomal abnormalities were detected in 4.9% of the cases (75% numerical, 25% structural). A larger study from Turkey, which evaluated 6041 genetic AS cases, found a 3% chromosomal abnormality rate (69% numerical, 31% structural).
Frequency of DS increases with maternal age, especially after 35 years of age. On its own, maternal age ≥35 years can detect about 30–50% of DS cases depending on the age distribution of pregnancies in the dealed population. With the use of screening tests, this detection rate can be increased up to 95%. Some ultrasound markers at the second trimester can also increase suspicion of DS and may modify the risk obtained by MSS tests.
In this study, we aimed to analyze the clinical and the cytogenetic properties of 6124 second-trimester AS cases from a center in Turkey dealing with prenatal diagnosis and to lay out our culture success rates, frequency of the common clinical indications and abnormal results. There are several reports from different centers in Turkey about the clinical and cytogenetic results of AS cases,[10, 12] but as far as we know, this is the largest set of AS data that has ever been reported from our country.
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This study includes a retrospective evaluation of the clinical indications and the cytogenetic results of 6124 second-trimester AS cases performed at Suleymaniye Maternity Hospital for Research and Training, Istanbul, Turkey between January 2007 and December 2011. Approval from the local ethics committee was obtained for the use of the hospital data.
Clinical indications for the performance of AS were grouped as: (i) increased risk in MSS tests; (ii) advanced maternal age (AMA) (≥35 years at the expected time of delivery); (iii) pathological ultrasound finding (PUF); (iv) maternal anxiety; (v) history of a fetus or child with a major congenital anomaly; (vi) parental translocation or marker chromosome carriage; and (vii) family history of autosomal recessive Mendelian disorders. PUF included soft markers (hyperechogenic bowel, choroid plexus cysts, pelviectasis, short femur, ventriculomegaly, single umbilical artery), minor malformations (e.g. hypoplasia of the cerebellum, polydactyly, club foot, cleft lip, cleft palate, etc.), major malformations (e.g. cystic hygroma, ventricular septal defect, atrioventricular septal defect, omphalocele, etc.), intrauterine growth retardation and oligohydramnios. All patients except those in the AMA group were <35 years of age at the expected time of delivery. AMA patients with additional findings, such as PUF or increased risk in MSS tests, were included in the AMA group.
All of the patients with any of these indications were given genetic counseling about the possible risk of a chromosomal abnormality in the presence of their associated risk factors. Also, couples with a family history of a Mendelian disorder were informed about the risk of occurrence of the disease in their fetus. Amniocentesis was performed to those patients who decided to undergo this procedure after the genetic counseling.
The amniotic cells were cultured in three separate flasks. The flasks contained CHANG supplement, 1% 200 mM L-glutamine, 100-U/mL penicillin, and 100-μg/mL streptomycin in addition to 2 mL of BIOAMF as the basal medium. When at least 15 colonies were observed, cultures were harvested 10–14 days after seeding. Giemsa banding method banding was performed by conventional cytogenetic methods in all metaphases obtained from all three cultures. In metaphases that were suspicious for polymorphisms, C banding was also performed.
The results of the cytogenetic analysis were examined in three groups as: (i) numerical chromosomal abnormalities; (ii) structural chromosomal abnormalities; and (iii) polymorphisms. The frequency of chromosomal abnormalities according to the clinical indications was also investigated. Patients were informed about the clinical significance of these results by the genetic counselor before deciding on pregnancy termination or continuation.
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Within the indicated time interval, a total of 7657 patients were given genetic counseling due to any of the above indications. Among these, 6124 (79.9%) patients decided to undergo AS. The most common indication for AS in our center was increased risk in MSS tests (56%) followed by AMA (29%) and PUF (11.5%) (Table 1). The other indications (maternal anxiety, history of a fetus or child with a major congenital anomaly, parental translocation or marker chromosome carriage and family history of autosomal recessive Mendelian disorders) were rare and comprised a total of 3.5% of the cases (Table 1).
Table 1. Distribution of chromosomal abnormalities according to the clinical indications for amniocentesis cases
|Indication for amniocentesis||Number (%)||Number of chromosomal abnormalities (Positive predictive value)|
|Increased risk in maternal serum screening tests||3429(56%)||83(2.4%)|
|Increased risk in the triple test||1990(58%)||19(1%)|
|Increased risk in combined test||1130(33%)||58(5.1%)|
|Increased risk in quadruple test||309(9%)||6(1.9%)|
|Advanced maternal age(>35 years of age at the expected time of delivery)||1777(29%)||92(5.2%)|
|Pathological ultrasound finding||706(11.5%)||28(4%)|
|History of a fetus or child with a major congenital anomaly||90(1.5%)||5(5.6%)|
|Parental translocation or marker chromosome carriage||7(0.1%)||7(100%)|
|Thalassemia major(only molecular analysis was performed)||4(0.07%)||0|
|Congenital muscular dystrophy(only molecular analysis was performed)||2(0.03%)||0|
The mean gestational age ± SD of the patients undergoing AS was 17.6 ± 1.5. The mean age ± SD of the patients was 33.3 ± 6.6; 29% of all cases were ≥35 and 71% of the cases were <35 years of age at the expected time of delivery.
From the AS material, karyotype analysis was performed in 6118 cases, but molecular analysis was only performed in six cases (four cases for the prenatal diagnosis of thalassemia major and two cases for prenatal diagnosis of congenital muscular dystrophy). In 72 (1.2%) cases, cell proliferation did not occur in the culture, so no cytogenetic result could be obtained. The success rate of cell culture as a total was 98.8%.
Increased risk in MSS tests comprised the most common (56%) indication for AS and this indication had 2.4% positive predictive value (PPV) for the detection of chromosomal abnormalities. Abnormal triple test result was the most frequent MSS abnormality (58%), followed by the abnormal combined test (33%) and abnormal quadruple test (9%). Among the MSS tests, the combined test had the highest PPV (5.2%) followed by quadruple test (1.9%) and triple test (1%). Parental translocation or marker chromosome carriage had a PPV of 100%, history of a fetus or child with major congenital anomaly had 5.6% PPV, AMA had 5.2% PPV, and PUF had 4% PPV. Increased risk in MSS tests was followed by AMA (29%), PUF (11.5%), maternal anxiety (1.8%) and a few other indications (1.7%) in decreasing frequency (Table 1).
The group that was most commonly associated with chromosomal abnormalities was the AMA group; of 1777 cases, chromosomal abnormality was detected in 92. Therefore, AMA had the highest PPV (5.2%) among all indications. In patients <35 years of age, abnormal MSS result was the most common indication, but this indication had the lowest PPV (2.4%) among all other indications in this age group.
Chromosomal abnormality was detected in 215 (3.6%) of the 6052 cases in whom cytogenetic studies for prenatal diagnosis were performed. Among these, 74.9% (161/215) were numerical and 25.1% (54/215) were structural chromosomal abnormalities. The most frequent numerical chromosomal abnormality was trisomy 21 (61.9%; 99/161) (Tables 2, 3). Polymorphisms (clinically insignificant structural chromosomal aberrations that are not expected to have any phenotypic effects) were not included in the structural abnormality group (Table 4). These were the most frequent structural chromosomal aberrations and were found in 571 cases (Table 5). The most frequently observed polymorphism was increase in heterochromatin region in the 1st chromosome (n = 158).
Table 2. Distribution of numerical chromosomal abnormalities detected among 6124 second-trimester amniocentesis cases
|Numerical chromosomal abnormalities||Number (%)|
|Sex chromosome abnormality||22(13.6%)|
|Rare autosomal trisomies||4(2.5%)|
Table 3. Number and spectrum of numerical chromosomal abnormalities given in relation to the indication of amniocentesis and the pathologic ultrasound finding if present
|Indication for amniocentesis||Numerical chromosomal abnormality||Abnormal ultrasound finding||Number of patients|
|Advanced maternal age||—||—||76|
|+ TTR. PUF||45,X||ND||9|
|+ PUF||47,…+21||Hyperechogenic bowel. Increased NT. Aberrant subclavian artery. Cystic hygroma. Short femur. Omphalocele. Ventricular septal defect||15|
|+ CTR. PUF||47,XXY||Increased NT||6|
|Increased risk in maternal serum screening tests||—||—||65|
|+ HxMCA||46,XY,rob(14;21)(q10;q10),+21 (15cenh>)||ND||1|
| ||48,XXY,+21 [22 ps >]||ND||1|
|+ PUF||45,X/46XY||Bilateral pyelectasis||2|
|Pathologic ultrasound finding|| ||ND||19|
| ||47,XX,+18||Bilateral choroid plexus cysts. Bilateral talipes. Omphalocele. AVSD. Regurgitation in the atrioventricular valves||6|
| ||47,XX,+13||Polydactyly. Cerebellar hypoplasia. Omphalocele. Cleft lip and palate. AVSD. Dandy Walker malformation. Pyelectasis. Cardiac abnormality||6|
| ||69,XXY||Intrauterine growth retardation. Oligohydramnios||3|
| ||46,XY/47,XY,+15||Left pyelectasis. Increased NT. Ventricular septal defect||1|
| ||Marker chromosome||ND||3|
Table 4. Number and spectrum of structural chromosomal abnormalities given in relation to the indication of amniocentesis and the pathologic ultrasound finding if present
|Indication for amniocentesis||Structural chromosomal abnormality||Pathologic ultrasound finding||Number of patients (%)|
|+ PUF||46,XY,t(1;19)(q34.1;p13.3)||Increased NT||1|
|+ PUF||46,XY,inv(12)(p11;q13)||Agenesis of the ductus venosus||1|
| ||46,XX, t(1;11)(p2;p12.4)||ND||1|
| ||46,XY,t(5;16)(q23;p12) 15cen+||ND||1|
|+ PUF||46,XX,t(6;19)(q10;q10)||Bilateral pyelectasis||1|
| ||der(18)add18p11.3||AVSD. Intracardiac hyperechogenic focus. Bilateral pyelectasis||1|
| ||46,XX,t(2;12)(q22;q21)||Right sided ventriculomegaly||1|
| ||46,XX,t(6;9)(p21;p23)||Single umbilical artery. Mega cisterna magna. Hypoplasia of the vermis||1|
| ||5p del||ND||1|
Table 5. Spectrum of polymorphisms detected among 6124 amniocentesis cases
In total, there were 161 numerical chromosomal abnormalities. Among these, there were 97 trisomy 21 cases, 22 sex chromosomal abnormalities, 19 trisomy 18 cases, seven trisomy 13 cases, four rare autosomal trisomies, two Double aneuploidies and four marker chromosomes (Table 2). The indication for AS was carriage of a marker chromosome in one and PUF in three of the cases in which marker chromosomes were detected.
Amniocentesis-related total pregnancy loss rate was 0.26% (16/6124). Most of the mothers in our series elected termination of pregnancy after the baby was diagnosed with a major chromosomal abnormality. As some of the patients coming from different centers turned back to their primary physicians with the AS result, we do not have the exact number of terminated pregnancies. However, we know that all of the cases with a diagnosis of fetal trisomy 13 and 18, 74 of those with a diagnosis of fetal trisomy 21 and all but three of those that were diagnosed with unbalanced structural chromosomal abnormalities of the fetus elected pregnancy termination.
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Prenatal diagnosis of fetal chromosomal abnormalities is an important issue in routine prenatal care. DS is the most common chromosomal abnormality among live births[3, 4, 6] and the frequency of this disorder necessitates screening and, in high-risk cases, invasive testing for diagnosis. Amniocentesis is the most frequently used method of invasive prenatal testing for the diagnosis of DS and other chromosomal abnormalities. Reports about the amniocyte culture success rates from Turkey include data from İzmir, which were found to have improved from 97.97% in 1998 to 99.74% in 2001. A collaborative study from four Turkish centers later reported an overall culture success rate of 99.7%. Our culture success rate was 98.8% and was closer to the upper range of the previous results from our country.
Several studies on AS results and indications have been reported to date from Turkey and from other countries. For example, as reported from the results of 1068 AS cases from Southeast Turkey, abnormal MSS result was the most common indication for AS (37.6%). In contrast, a larger study from Turkey, which included 6041 cases, found that AMA was the most common indication followed by positive serum screening. In an even larger study from Korea, including 31 615 cases of mid-trimester AS, abnormal MSS result was the most common indication for AS since 1994, followed by AMA and PUF. In another study, which included 13 648 cases, the AS indications in decreasing frequency were high-risk serum screening, AMA, PUF, paternal/maternal chromosomal abnormality and other factors. The same was true for our study, and the group with abnormal MSS result included 56% of all indications; this was followed by AMA (29% of our patients were above the age cut-off) and PUF and other indications. Before the development of MSS tests, DS screening was solely based upon maternal age, and the group with AMA was directly offered invasive prenatal testing. In the 1970s this approach could detect only 30% of the DS cases, but in recent years, it has detected about 50% of the DS cases, especially in developed countries, as the proportion of pregnant women at older ages has increased. With the development of MSS with high detection rates, abnormal MSS results have become a more frequent indication for invasive testing. Our results are in accordance with this trend.
In the study by Balkan et al., chromosomal abnormality was detected in 4.9% of the cases; 75% of the chromosomal abnormalities were numeric, and 25% were structural. In our study the results were very similar: 74.9% of the chromosomal abnormalities were numeric and 25.1% were structural, but chromosomal abnormality was detected in 3.6% of the cases. Chromosomal abnormality was detected in 3% of the cases in the study by Karaoğuz et al., 69% of which were numeric and 31% of which were structural.
The most common chromosomal abnormality among live births is trisomy 21 and the incidence of trisomy 21 is even higher in the second trimester, because some of the fetuses with the disorder are spontaneously lost in utero before delivery. In our study, 46% of all (and 61.5% of numerical chromosomal abnormalities) were trisomy 21. The study by Zhang et al., which examined the amniotic fluid of 13 795 cases, also reported that trisomy 21 was the most common abnormality, with an incidence of 35.6% among abnormal karyotypes. Similarly, Han et al., who examined the amniotic fluid of 31 615 cases, also reported that DS was the most frequent chromosomal abnormality, which comprised 36.9% (359/973) of all chromosomal abnormalities. Our study supports all these and similar reports that DS is still the most common chromosomal abnormality detected in the second trimester.
Families in whom the fetus was detected to have a cytogenetic abnormality were given genetic counseling. They were informed about the possible clinical implications of the associated disorder, possibility and options of treatment and the recurrence risk. In our country, elective abortions can be performed up to the 10th gestational week legally. After this gestational age, pregnancy termination is legal only in the presence of severe fetal chromosomal or congenital abnormalities of if the pregnancy severely threatens the maternal health. There is no legally determined upper gestational age limit for termination, however, because of ethical reasons, most of the centers and obstetricians in our country do not agree to perform termination beyond 23 weeks of gestation, which is accepted to be the limit of viability.
The mid-trimester AS-related procedure loss rate was reported to vary between 0.6% and 1% (1/175–1/100), but the authors mentioned that it was unique to the individual depending on many different variables. It was also mentioned that this rate could be as low as 0.19% or as high as 1.53%. The procedure-related loss rate in our center (0.26%) was also consistent with these numbers.
AMA alone had a PPV of 5.2% for the detection of chromosomal abnormalities. In patients that are <35 years of age, parental chromosomal abnormality had 100% PPV, PUF had 4% PPV, and abnormal serum screening had 2.4% PPV. Among the abnormal serum screening results, the abnormal combined test had the highest PPV (5.2%).
Major abnormalities in the chromosome structure can include translocations, deletions, gene inversions and gene duplications. The spectrum of major structural changes detected in our cytogenetic studies is listed in Table 4. It should be kept in mind that parental carriage of a balanced autosomal abnormality causes a decrease in pregnancy success rate and an increase in the risk of a miscarriage. Besides, in these couples, it should be remembered that the fetus can be affected by multiple congenital anomalies, mental retardation and a chromosomal abnormality that may result in decreased survival. Parents that carry a balanced translocation are the most suitable group of candidates for the application of prenatal genetic diagnosis (PGD). Studies show that the pregnancy rate per oocyte can increase from 29% to 38% and the abortion rate in the natural pregnancies of carrier parents can decrease from 92% to 12.5% after the application of PGD. In addition to couples who carry balanced chromosomal translocations, PGD can also be considered for couples who are carriers of other familial or non-familial genetic diseases. In genetic screening, which is performed for optimal reproductive outcome, detection of patients at high risk of having a baby affected by a disease with fatal and/or severe abnormalities should be the basic aim.
Polymorphisms are small abnormalities in the structure of the chromosome. These may include short tandem repeats, single nucleotide polymorphisms and copy number variants. These are responsible from most of the genetic variations in populations and are generally not associated with clinical diseases. But, during recent years, possible clinical importance of polymorphisms in the heterochromatin region of chromosomes has been reported. Their possible association with reproductive failure and recurrent spontaneous miscarriages has been reported. We detected polymorphisms in 571 of the 6052 cases (Table 5).
In conclusion, an overview of the indications and results of a limited number of AS cases from a center dealing with prenatal diagnosis in Turkey in our study shows that abnormal MSS result is the most frequent indication and DS is the most commonly detected abnormality, which are findings that support the already present literature. Even if it is the largest series of AS cases from our country as far as we know, further larger studies will of course more clearly compare the situation in the Turkish population with other populations.