Ce poate ascunde un rezultat NIPT negativ? Mozaicism al trisomiei 18 și anomalii fetale complexe – prezentare de caz

Introduction

An aneuploidy is a numeric chromosomal abnormality occurring due to non-disjunction and resulting in having either extra chromosomes (trisomies) or missing chromosomes (monosomies). Trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome) and monosomy X (Turner syndrome) are some of the most frequent aneuploidies⁽¹⁾.

Trisomy 18 syndrome (Edwards syndrome) is a congenital disorder that was first reported in 1960, when Edwards et al. published the case report of a neonate patient with severe malformations and cognitive deficiency⁽²⁾. Edwards syndrome is the second most frequent trisomy in live-born infants, with a generally poor prognosis. The majority of chromosomal anomalies in fetuses are identified through prenatal ultrasound during the first and second trimesters. The majority of fetuses with trisomy 18 show abnormal findings on ultrasound. Its estimated incidence ranges from 1 in 4000 to 16,000 live births⁽³⁾. The overall prevalence of trisomy 18 was 2.63 per 10,000 total births, including both live births and stillbirths (with or without termination of pregnancy for fetal anomalies), based on data from 21 registers⁽⁴⁾.

Edwards syndrome can occur in three forms: complete trisomy 18 (when there is an extra 18 chromosome), trisomy 18 mosaicism (when a normal cell line and an extra chromosome cell line coexist/appear in the same individual), and partial trisomy 18 (when the triplication occurs in only one fragment of the 18^th chromosome)⁽⁵⁾.

The majority of Edwards syndrome cases are diagnosed antenatally. Among the most consistent presentations, there are increased nuchal translucency, cardiac malformations, omphalocele, fetal growth restriction, excessive amniotic fluid, corpus callosum agenesis, camptodactyly, brachycephaly, and two-vessel umbilical cord⁽⁶⁾. Other frequent anomalies are craniofacial abnormalities, limb anomalies, esophageal atresia, neural tube defects, and diaphragmatic hernia⁽⁷⁾.

Over the last years, prenatal testing developed towards noninvasive testing methods. Accelerated advancements in nowadays modern highly effective molecular technologies and the identification in the maternal plasma of cell-free fetal DNA resulted in significant breakthroughs in fetal aneuploidies screening.  Due to their multiple advantages and diagnostic value, noninvasive prenatal tests (NIPTs) were rapidly integrated in routine clinical practice, becoming standard screening procedures for all pregnant women⁽⁸⁾. Fetal cell-free DNA (cfDNA) found in the maternal plasma seems to be of fetoplacental origin, through apoptosis of cyto- and syncytiotrophoblastic cells. It can be identified in maternal plasma starting from 5 weeks of gestation, and its fraction elevates as the pregnancy progresses⁽⁹⁾.

A number of studies estimated the pool sensitivity of NIPT in high-risk population at 99.3% for Down syndrome, 97.4% for Edwards syndrome, and 97.4% for Patau syndrome, while the specificity reached 99.9% for all three aneuploidies. Nevertheless, considering the general obstetric patients, the positive predictive value of NIPT is much lower due to the lower prevalence of trisomies⁽¹⁰⁾. In spite of its high sensitivity and specificity rates, discordant NIPT results may occur as a result of confines placental mosaicism⁽¹¹⁾. Invasive testing is mandatory in order to confirm a positive NIPT result⁽¹²⁾. These methods include amniocentesis or chorionic villus sampling (CVS), that evaluate fetal chromosomes⁽¹³⁾.

In a study by Gug et al., NIPT proved to have a high detection rate for autosomal aneuploidies in a Romanian population. In our country, NIPT can be a self-funded antenatal screening option for all pregnant wo­men, but an invasive test must be performed in high-risk pregnancies⁽¹⁴⁾.

Case report

We present the case of a 32-year-old patient, 13 weeks pregnant, who came to our clinic for first-trimester ultrasound screening. The patient had a miscarriage in the past, at 6 weeks of pregnancy. She was diagnosed with thrombophilia and received anticoagulant, without other health issues. There were no risk factors for genetic syndromes.

The patient did not have a blood test for PAPP-A and free ß-hCG. She performed a noninvasive prenatal test with an extended panel at 11 weeks of pregnancy, with specificity over 99% for all genetic syndromes tested and with a sensitivity of 99% for trisomy 21, above 99% for trisomy 13 and, respectively, 94.1% for trisomy 18. The results of NIPT were presented in Table 1. NIPT indicated a fetal fraction of 15.43%, a low probability for all anomalies tested, and a female fetus.

The ultrasound scan was performed both transabdominally and transvaginally. Three-dimensional (3D) ultrasound was performed. Doppler and SlowFlow HD technology were used to evaluate blood vessels and the heart anatomy.

The ultrasound scan revealed a live intrauterine fetus with a crown-rump length (CRL) of 6.11 cm, corresponding to a gestational age of 12 weeks and 4 days. Several complex fetal anomalies were found. Generalized skin edema (Figure 1) and nuchal translucency (NT) with a thickness of 3.3 mm were observed (Figure 2).

It was observed that the lateral ventricles were almost completely occupied by the choroid plexuses in transventricular axial view, a suggestive image for the “dry brain” sign (Figure 3). Also, in the axial plane, the posterior abnormal displacement of the mesencephalon was observed, which suggested the “crash sign” (Figure 4). These two images from fetal brain were associated with the presence of spina bifida aperta (Figures 5 and 6).

The evaluation of the fetal heart began with the estimation of heart axis which was greater than 90 degrees (Figure 7). Using Doppler and SlowFlow HD technology, we obtained a four-chamber view of the heart with an enlargement of the right ventricle (Figure 8) associated with tricuspid regurgitation (Figure 9). A severe cardiac anomaly, with common arterial trunk type I (Figure 10) and right aortic arch (Figure 11), was observed.

The ductus venosus was identified, with a positive a wave, and the pulsatility index (PI) was 1.24. In the axial (Figure 12) and sagittal (Figure 13) planes, the presence of the omphalocele with herniation of the liver and intestine was confirmed.

Left renal agenesis was observed, with the absence of the left renal artery (Figure 14) and the presence of a duplex collecting the system of the right kidney (Figure 15). Another anomaly observed was the single umbilical artery.

All aforementioned fetal anomalies were explained to the patient, and she was carefully advised regarding the diagnostic and therapeutic options. After understanding the risks and possible complications of continuing the pregnancy, she and her partner decided to terminate the pregnancy and undergo genetic testing of the fetus.

With the patient’s consent, medical abortion was induced at 13 weeks of pregnancy. All the fetal anomalies described before were confirmed following the pathological examination. The fetal molecular karyotype analysis was performed, using the single-nucleotide polymorphism (SNP) microarray method, and an abnormal female profile was identified. A gain of genetic material with a size of 77.87 Mb in the chromosomal region 18p11.32 -> 18q23 was observed, a region that represents an entire chromosome 18. The duplication of chromosome 18 was identified in approximately 30% of the cells. This result corresponds to trisomy 18 mosaicism. According to the analyzed databases, the genomic imbalance described has pathogenic significance, the duplicated region having an important genetic content associated with the development of pathological conditions. Genetic counseling of the partners was recommended.

Discussion

We presented the case of a 32-year-old woman, thirteen weeks pregnant, who performed NIPT as the first screening test in the first trimester, that indicated a reduced probability for the tested genetic anomalies. After this, multiple fetal anomalies were revealed by screening ultrasound scan: spina bifida aperta, tricuspid regurgitation, common arterial trunk type I, right aortic arch, omphalocele and left renal agenesis. In this case, the molecular analysis of the karyotype resulted in trisomy 18 mosaicism. We highlighted the importance of performing a screening ultrasound scan before genetic tests for chromosomal abnormalities, in order to have an effective prenatal diagnosis and a correct management of the case. NIPT should not be used as the only screening test in the first trimester of pregnancy. It must always be associated with an ultrasound scan, because there are several anomalies that can be detected in the first trimester and are not associated with trisomies 13, 18 and 21.

Prenatal diagnosis refers to a medical procedure that uses different methods to assess the health and well-being of a developing fetus. The information obtained helps avoid negative outcomes for both the fetus and the mother, especially since congenital abnormalities are responsible for 20-25% of perinatal fatalities⁽¹⁵⁾.

The International Society of Ultrasound in Obstetrics and Gynecology (ISUOG) has published practice guidelines for an ultrasound scan in the first trimester of pregnancy between 11 and 14 weeks of gestation, whose objectives are the accurately pregnancy dating, verification of viability and plurality, identification of major structural anomalies, and screening for aneuploidies and preterm preeclampsia⁽¹⁶⁾.

Over the past years, many studies have investigated the clinical use of NIPT for detecting common trisomies and sex chromosome aneuploidies. While the detection rate (DR) for trisomy 21 consistently exceeds 95% across all studies, concerns have been raised about the accuracy of the DR for trisomy 18 and trisomy 13, especially when focusing on specific subgroups rather than on high-risk populations⁽¹⁷⁾. In a meta-analysis conducted by Taylor-Phillips for the National Institute of Clinical Excellence, the detection rate for Down syndrome was 96%, while it was 87% for trisomy 18, and 77% for trisomy 13 in the general population⁽¹⁰⁾.

Although numerous professional societies currently advocate for NIPT to be utilized as a screening tool rather than a diagnostic test, its high sensitivity (true positive rate) and specificity (true negative rate) make it a compelling alternative to traditional serum screening and invasive procedures. Additionally, these societies advise that NIPT should be paired with genetic counseling to enable families to make well-informed reproductive decisions⁽¹⁸⁾.

The use of NIPT in clinical settings to screen high-risk patients for fetal aneuploidy is growing more widespread. The high sensitivity and specificity of noninvasive prenatal tests increased hopes that these tests may lead to a decrease in the number of invasive diagnostic procedures and the risks they entail. According to research by Chiu et al., basing referrals for amniocentesis or CVS on NIPT results could potentially avoid around 98% of invasive diagnostic procedures⁽¹⁹⁾. Furthermore, these tests have been extensively validated in high-risk groups. In fact, the American College of Obstetricians and Gynecologists (ACOG) advises that high-risk women undergo testing, while also highlighting the need for further research to assess the performance of these tests in low-risk populations⁽²⁰⁾.

Noninvasive prenatal tests cannot differentiate between the various types of aneuploidy. For instance, it is unable to specify whether Down syndrome is caused by an additional chromosome (trisomy 21), a Robertsonian translocation involving chromosome 21, or high-level mosaicism. When mosaicism is present, including confined placental mosaicism, the results may be unreliable⁽²¹⁾. Identifying the mechanism behind aneuploidy is crucial for advising on recurrence risks, and it highlights the need for confirmatory testing after NIPT, such as chorionic villus sampling, commonly used. However, since CVS examines a small sample of placental tissue, there is a possibility that placental mosaicism may occur at a higher rate⁽²²⁾.

The occurrence of aneuploidy is closely linked to maternal age and the developmental stage. While only 0.6% of newborns have aneuploidy, its incidence rises to 45% when examining miscarriages. Among newborns and stillbirths, trisomies 13, 18 and 21, as well as sex chromosome aneuploidies (such as 45X, 47XXX, 47XXY and 47XYY) are the most frequently observed abnormalities. The only significant monosomy seen after implantation is 45,X. In miscarriages, trisomies of all chromosomes have been reported, with trisomies 15, 16, 21 and 22 being the most common. Except for trisomy 21, these abnormalities are typically lethal early in pregnancy, with fetal survival beyond the first trimester only possible in cases of mosaicism⁽²³⁾.

Around 2-3% of all NIPT tests result in inconclusive findings. Maternal and fetal factors appear to influence cell-free fetal DNA (cffDNA) fraction. A negative correlation has been suggested between fetal fraction and factors such as maternal age, BMI (Body Mass Index) or body weight, LDL (low-density lipoprotein) cholesterol, triglyceride levels, therapies like metformin, heparin and enoxaparin, hemoglobinopathies, and physical activity. Additionally, patients from South and East Asia seem to have a lower fetal fraction compared to Caucasians. There are several reasons for this, including issues with sample collection, transportation, storage, technical errors, maternal or fetal mosaicism, and maternal malignancies. Some laboratories do not assess the fetal fraction, although past research has shown that a fetal fraction under 3-4% raises the likelihood of receiving no result. By failing to measure the fetal fraction, these labs increase the risk of providing inaccurate results⁽²⁴⁾. On average, the fetal fraction is approximately 10%, but it varies between individuals. It tends to increase with gestational age and decrease as maternal weight rises⁽²⁵⁾. Getting a no-result report raises the likelihood of negative outcomes, including miscarriage, chromosomal abnormalities, preeclampsia, and gestational diabetes mellitus⁽²⁶⁾. Conversely, a positive correlation has been found with gestational age, free b-hCG (beta-human chorionic gonadotropin), PAPP-A (pregnancy-associated plasma protein A) levels, living at high altitudes, and twin pregnancies. However, further research is needed, particularly focusing on controversial factors, using populations adjusted for BMI and gestational age⁽²⁷⁾. Research has shown that, in the case of patients with small placentas, the likelihood of receiving a negative result is higher, which is particularly evident in cases of trisomy 13 and trisomy 18, where PAPP-A and b-hCG levels are also notably low. The fetal fraction is influenced by ethnicity, being lower in women of Afro-Caribbean descents compared to Caucasians. It is also lower in IVF (in vitro fertilization) pregnancies and increases with fetal crown-rump length, the levels of PAPP-A, serum b-hCG, smoking, and the presence of trisomy 21⁽²⁸⁾.

Conclusions

Noninvasive prenatal testing should not be used as the only screening method for fetal anomalies, even though an extended panel of tests is used. Ultrasound scanning has a primary role in the detection of fetal anomalies. The remarkable progress of ultrasound technologies and devices contributes to the early diagnosis of numerous anomalies and to a better management of pregnancies. Analyzing the information obtained by ultrasound scan is essential for guidance to a specific genetic test and for choosing the therapeutic options during pregnancy.

Autor corespondent: Romina-Marina Sima E-mail: romina.sima@umfcd.ro

CONFLICT OF INTEREST: none declared.

FINANCIAL SUPPORT: none declared.

This work is permanently accessible online free of charge and published under the CC-BY.