Genetic Counseling and Prenatal Diagnosis of Triploidy During the Second Trimester of Pregnancy.

INTRODUCTION: Triploidy is a lethal chromosomal numeric abnormality, characterized on extra haploid set of chromosomes. It occurs in 2 to 3% of conceptuses and accounts for approximately 20% of chromosomally abnormal first-trimester miscarriages. As such, triploidy is estimated to occur in 1 of 3,500 pregnancies at 12 weeks', 1 in 30,000 at 16 weeks', and 1 in 250,000 at 20 weeks' gestation. CASE REPORT: We present a case of second-trimester triploidy diagnosed prenataly at our center. 28-years-old gravida with a first spontaneous pregnancy had early gestational hypertension. Ultrasound examination in 146/7 weeks' gestation revealed asymmetric intrauterine growth retardation. We recommended biochemical maternal serum screening during second trimester of pregnancy (AFP, HCG, uE3). Result of biochemical screening was indication for cytogenetic analysis from amniotic fluid cells and we recommended early amniocentesis in 156/7 weeks' gestation. Result showed abnormal karyotype of the fetus (69,XXX triploidy), and DNA analysis confirmed Type-2 Diginy. Parents decided to terminate this pregnancy, and it was done at 22 weeks' gestation.
CONCLUSION: We emphasize the importance of non-invasive prenatal exminationes-biochemical serum screening during second trimester of pregnancy, and ultrasound examinations in prenatal screening of syndroma Down and other chromosomal abnormalities.

1. INTRODUCTION

Triploidy is a rare lethal numeric chromosomal aberation caused by the presence of an extra haploid chromosome set. It occurs in about 17% of all spontaneous abortion during first trimester and occurring in up to 3% of recognized human conceptuses. The majority of fetuses with triploidy are spontaneously miscarried between 7th and 17th week of gestation (1, 2). This extra set of chromosomes is a cause of variety of serious birth defects, placental problems, hydatidiform mollar findings, and severe growth problems in a fetus. Infants with this lethal condition are generally small due to severe intrauterine growth retardation (IUGR) and they have multiple birth defects, including facial abnormalities, micrognatia, cleft lip, heart defects, neural tube defects (spina bifida), and other serious birth defects of limb, kidney and umbilical cord. Anomaly also includes single umbilical artery (3, 4, 5). The low frequency of triploidy among advanced pregnancies has led to discrepancies in prevalence estimates, ranging from as low as 1 in 250,000 at 20 weeks to as high as 1 in 50,000 among live-born infants (6, 7). It can be recurrent, and maternally derived cases appear to live longer than paternally derived cases. There are three different mechanisms that may produce triploidy: an extra haploid chromosome set can be paternal (diandric) or maternal (digynic) in origin (8). The majority of diandric triploids result from simultaneous fertilization by two sperm. Other triploidies of diandric origin arise from normal fertilization of the ovum by a diploid sperm, the result of complete nondisjunction during spermatogenesis. Triploidies of digynic origin result from fertilization of a primary oocyte or from fertilization of a diploid oocyte that is a product of nondisjunction during meiosis I, meiosis II, or retention of a polar body (9, 10).

This can result of two sperm fertilizing one normal egg, it can also occur if a normal sperm fertilizes an egg has an extra set of chromosomes (11).

Cases of triploidy are grouped into two fetal and placental phenotypes that roughly correspond to the parental origin of the extra set of chromosomes (12):

Type I: well formed fetus with a normal or microcephalic head and a large placenta with cystic changes-associated with diandry;

Type II: fetus with growth restriction and a large head and a small, noncystic placenta-associated with digyny.

Genetic counseling and prenatal diagnostics of congenital anomalies is one of the most important improvements in preventive medicine in the last century in the world. Pregnant women were genetically advised and those women who had high risk for chromosomal aberration of the fetus, were recommended for early amniocentesis and chromosome analysis. Second trimester maternal serum alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and unconjugated estriol (uE3) levels are evaluated in pregnancy in order to determine the risk of aneuploidy, trisomy 13,18,21 and poliploidy (13, 14).

Prenatal diagnostics by non-invasive testing (biochemical and ultrasound examinations) during first and second trimester pregnancies, give us most important data about genetic health of fetus (13). Prenatal diagnosis of Sy. Down and other aneuploidies and structural chromosomal aberrations is the most important goal.

Triploidy may be prenatally diagnosed through cytogenetic analysis of cells obtained through procedures such as amniocentesis. Fetal nuchal translucency in the first trimester is frequently increased in fetuses with triploidy (15, 16). It is also associated with elevated maternal serum alpha fetoprotein (AFP) and total and beta-human chorionic gonadotropin (hCG) levels and low maternal serum pregnancy-assisted plasma protein-A (PAPP-A) levels in the first trimester (17,18). Fetal ultrasound examination during first and second trimester of gestation is most important, and gives support in discovery of chromosomal aberration of the fetus and congenital anomalies (19). Screening for Sy. Down includes measurement of nuchal translucency and nuchal fold skin thickens, identification of asymmetric IUGR and congenital anomaly of the fetus. (20, 21).

Here we present a case of prenatally diagnosed triploidy of fetus undergoing routinely amniocentesis, where indications for prenatal diagnosis were ultrasound findings and biochemical markers screened in the 146/7 weeks’ gestation. We discovered only one case of triploidy during 12 years.

2. PATIENT AND METHODS

We present prenatally diagnosed abnormal karyotype of fetus. It was poliploidy - 69, XXX triploidy Type 2 Diginy, discovered during second trimester of pregnancy. The patient was a 28-years-old woman with a spontaneous pregnancy referred to our institution at 146/7 weeks’ gestation after anatomy ultrasound examination revealed severe intrauterine growth restriction. Pregnancy complications included gestational hypertension.

Ultrasound examination revealed asymmetric intrauterine growth restriction. Ultrasound screen and result maternal biochemical serum screening (Triple test) during second trimester of pregnancy showed high risk of chromosomopathies and it was indication for cytogenetic analyses (early amniocentesis). Result of citogetic analysis of amniotic cells showed abnormal karyotype-triploidy and DNA (PCR) analysis discovered Type II (Diginy), with extra set of haploid chromosomes of maternal origin.

Ultrasound examination

Ultrasound examination revealed asymmetric intrauterine growth restriction (Figure 1). Gestational age according to last menstrual period was 146/7, but according to ultrasound finding (USGA) it was 116/7 (CRL 47,0 mm; Nuchal translucency 1,7 mm–1,32 Mom, and IUGR 3 gw; Interorbital distance-11mm/18gw). Other abnormal ultrasound findings included micrognatio and single umbilical artery. Placental tissue had normal morphology and did not have trofoblastic tissue-molla (characteristics for triploidy Type 1, with extra set of haploid chromosomes of paternal origin). Amniotic fluid was normal and no other pathological ultrasound findings were found (Figure 2).

Figure 1

Ultrasoud examination: asimetric IUGR of the fetus and micrognatio

Figure 2

Ultrasound examination: gestational age 116/7 gw; Fl/AC 0.30; asimetric IUGR (3 weeks); normal ultrasound morfology of placental tissue and amniotic fluid

Biochemical findings

Result of biochemical analysis during the second trimester of pregnancy (alpha-fetoprotein 18,1-0,61 MoM low; human chorion gonadotropin-hCG 4630,0–0,14 MoM low; unconjugated estriol-uE3, 3,36 – 0,51 MoM low), did not show high risk of Syndroma Down (1:6241) but showed high risk of Syndroma Edwards (1:101) and triploidy 69 XXX.

Genetic counseling, cytogenetic analysis and DNA (PCR) analysis

Genetic counselor advised that cytogenetic analyses should be done. Results of cytogenetic analysis revealed numeric chromosomal aberrations (69, XXX-tripoidy) (Figure 3) and PCR-DNA method showed type II-poliploidy (diginy), with extra haploid set of chromosomes of maternal origin (Figure 4).

Figure 3

Karyotype of the fetus: Poliploidy-triploidy 69,XXX (karyotype from amniotic fluid)

Figure 4

PCR - DNA analysis: Triploidy 69, XXX, Type-2 (Diginy)

Pathoanatomical examination of fetus after termination of pregnancy confirmed ultrasound pathological findings. No other pathological findings were found.

Chromosomal findings from amniotic fluid: Amniocytes were grown using standard method for harvesting of amniocytes: amniotic cell pellet was seed into tissue flask containing Amniomax Complete II (Invitrogen-Gibco). After passaging cells into duplicate flask, colchicines were added for 3 hours afterwards pellet was treated with hypotonic solution (0.56% KCl) for 15 minutes at 37°C, cells were collected by centrifugation, and fixed using Karnoy’s fixative (methanol: acetic acid in the ratio 3:1). Fixed cells were sped onto clean slides, and stained with Giemsa. Metaphase analyses were carried out using microscope Zeiss, magnification 1400. Metaphases were recorded by CCD camera Axiocam 5. Classic and GTG banded chromosomes were analyzed. Analysis revealed karyotype 69,XXX.

Chromosomal findings in cordocentesis: 0.5ml of cord blood sample was put into medium PBmax karyotyping medium and harvested up to 72 hours. Metaphases were collected by adding colchicine for 3 hours. After standard preparation and fixation metaphase spreads were analyzed on Giemsa stained slides. In parallel to classical chromosomal analysis chromosomes were analyzed employing Giemsa-trypsin-Giemsa banding technique. Analysis revealed karyotype 69,XXX.

DNA analysis: DNA was isolated from amniocytes (duplicate culture), whereas sample of mother DNA was isolated from buccal cells. 14 markers were compared (TPOX, D18S51, D3S1358, THO1 vWA, D135317, D16S539, D2S13382, D2S1334, D165519, D138317, D138317, THO1-2, D351358). Results have shown that 2 markers were noninformative, whereas in other 12 doubled copies of maternal genetic material have been found in fetal tissue. The result of the DNA analysis have shown that triploidy is of maternal origin. This finding was in accordance to ultrasound observation and autopsy finding of placenta.

3. DISCUSSION

Triploidy is the third most frequent chromosomal anomaly and is responsible for 15-18% of spontaneous abortions. Only 1 in 1,200 triploid fetuses live after birth. The frequency of triploidy in live births is 1:10,000, and males represent 51-69 % of all cases (1, 6).

Two different mechanisms have been described that are responsible for triploidy. The extra-haploid set of chromosomes may be maternal (digynic triploids) or paternal (diandric triploids). Digynic triploidy may result from the fertilization of normal oocyte produced through an error at either the first or second meiotic division. Diandric triploidy may occur through fertilization of normal oocyte by a diploid sperm as a result of an error at either the first or second meiotic division or through fertilization of an oocyte by two sperms (dispermy) (8, 9). According to previous reports, 66.4% of triploid infants are due to dispermy, 23.6% due to s diploid sperm and 10% due to a diploid oocyte. Usually, the extra-haploid set of chromosomes has a paternal origin. Dyginic embrios have a better chance to reach the second trimester of gestation or even to be born (10, 22).

The sex chromosomes in triploid individuals of maternal origin should be in 50% of the cases XXX and 50% XXY, and in those of paternal origin, 25% XXX, 50% XXY and 25% XYY. According to previous studies, the frequency of triploids is the following: 31-49% 69,XXX, 49-69% 69,XXY and 0,3% 69,XYY. The low frequency of the 69,XYY chromosomal aberration suggests that this karyotype leads to low viability and early abortion of the zygote, otherwise the mechanism through which it occurs is very rare (7, 11, 12).

Many pregnancies were terminated by spontaneous abortion during the first trimester pregnancy, because had chromosomal aberration or congenital anomaly.

Some affected fetuses had chromosomal aberration and congenital anomaly but were not terminated by spontaneous abortion and we have to submit this pregnancies to prenatal diagnostics. Syndroma Edwards and triploidy have many similar biochemical and ultrasound findings and characteristics (20, 21, 22). We have to introduce cytogenetic and PCR-DNA analysis during diagnosis and these results give us more information about genetic health of the fetus and chromosomal aberration.

4. CONCLUSION

Frequency of chromosomopaties diagnosed by prenatal diagnostics in Brcko District is about 2% during 8 year period (2003-2011) and all pregnancies were terminated on request of pregnant women and approval of Ethical committee before 24 weeks’ gestational age. We emphasize the importance of prenatal ultrasound and biochemical screening and testing during the first and second trimester. This case report highlights the importance of cytogenetic and PCR–DNA analysis in discovered congenital anomaly and chromosomal aberration of the fetus during the second trimester of pregnancy.

There are no risk factors. Triploidy is not more common in older mothers, like some other chromosomal abnormalities such as Sy. Down. Couples who have one pregnancy with triploidy do not have an increased risk in future pregnancies. Multidisciplinary approach of different specialists (gynecologist, genetic counselor, biologist, psychologist, social worker) is important for good results in early prenatal detection of chromosomopathies.