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The prenatal screening modalities, recent trends in India:

Dr. Ashish Fauzdar, Ph.D

Background: Chromosome abnormalities manifest at an approximate rate of 1 in 150 live births, predominantly comprising aneuploidies in chromosomes 13, 18, 21, X, and Y, accounting for a substantial 80-95% of clinically significant cytogenetic anomalies linked to congenital malformations. The occurrence of fetal chromosomal abnormalities, known as aneuploidy, tends to increase as the mother’s age goes up. This trend does not show a preference based on a person’s race or ethnicity.

Notably, chromosome abnormalities occur in approximately 2% of pregnancies involving women aged over 35 years.(1) Within the Indian context, a staggering number of nearly 500,000 neonates are born annually afflicted with genetic disorders, congenital malformations, or birth defects. It is pertinent to acknowledge that more than half of spontaneous abortions stem from chromosomal anomalies (2), among which Down syndrome stands as the most frequent etiological agent, manifesting an incidence rate of 1 in every 1150 births in India. (3)

It is noteworthy that chromosome abnormalities exhibit a greater prevalence during the early phases of gestation, thereby contributing significantly to the incidence of pregnancy loss. The primary imperative for prenatal screening lies in women demonstrating an augmented susceptibility to chromosome anomalies. This susceptibility is observed among individuals with advanced maternal age (typically exceeding 35 years), those having a familial history of parental chromosome translocations or other chromosomal anomalies, those with discernible fetal gross structural abnormalities via ultrasound, and individuals who return positive results in biochemical maternal serum tests—such as double, triple, or quadruple markers—conducted during the first and second trimesters of gestation.

Symptoms:

Down Syndrome is a common genetic condition in which individuals have an extra copy of chromosome 21. It is the most prevalent autosomal chromosomal aneuploidy in live-born babies, occurring in approximately 1 in 700 births worldwide. In India, the high birth rate results in nearly 500,000 babies born each year with genetic abnormalities. Of these, around 21,000 babies are born with Down syndrome, which means that approximately 1 in every 1,150 births in India is affected by this condition. (4)

People with Down syndrome typically exhibit intellectual disabilities and physical features that are different from the general population. They may also have birth defects affecting the heart and other organs, as well as hearing and vision problems. This condition can happen when a person has an additional, full or partial copy of chromosome 21. Down syndrome comes in three main types: trisomy 21 (non-disjunction), which makes up 95% of cases, translocation (around 4%), and mosaicism (about 1%).

The presence of this additional genetic material disrupts normal development and leads to the characteristic features associated with Down syndrome. Individuals with Down syndrome are at an increased risk of certain medical conditions, including congenital heart defects, respiratory and hearing issues, Alzheimer’s disease, childhood leukemia, and thyroid problems. Fortunately, many of these conditions can be treated, allowing most people with Down syndrome to lead healthy lives.

Early intervention programs, such as speech, physical, occupational, and educational therapy, can help individuals with Down syndrome develop their skills and abilities. With the right support and treatment, many people with Down syndrome can live happy and productive lives. (NIH: National Institute of Child Health and Human Development) (5)

Patau’s Syndrome (Trisomy 13), condition occurs in approximately 1 in 5,000 live births. Sadly, there is a very high fetal loss rate of 97% for pregnancies affected by trisomy 13, and most babies born with this condition do not survive beyond four months. (7) Trisomy 13 is characterized by babies being smaller than expected for their gestational age, with central nervous system abnormalities, facial defects in the midline of the face, and urogenital malformations. In trisomy 13, the main central nervous system malformation observed is holoprosencephaly, but spina bifida and agenesis of the corpus callosum can also occur. (8)

Trisomy 18, also called Edwards syndrome, was initially described by John Hilton Edwards in 1960. (7) It affects around 1 in 5,000 births, but the incidence is higher during the prenatal period due to a significant number of fetal losses. After birth, about 60% of children with trisomy 18 do not survive beyond two months, and more than 95% do not make it past their first year.

Most cases of trisomy 18 are the result of a maternal meiotic nondisjunction, with only 5% being caused by a balanced reciprocal translocation in the parents. The characteristic features of trisomy 18 include facial abnormalities like a small jaw, low-set ears, and excessive body hair. Along with this, multiple organ system malformations are common, like spina bifida, omphalocele (a type of abdominal wall defect), heart defects, clubfeet, and radial aplasia (underdevelopment of the forearm bone). (8-10)

It’s important to understand the terms “monosomy” and “trisomy.” Monosomy refers to having only one copy of a particular chromosome when two are expected, while trisomy means having three copies of a chromosome when two are expected.

X and Y chromosomal aneuploidies, which mean having an abnormal number of sex chromosomes, are among the most common changes in human chromosome counts. These variations happen quite often, with an estimated occurrence in the overall population ranging from 1 in 400 to 1 in 1,000 for each of these sex chromosome syndromes. However, complex aneuploidies are much less common. (11-13)

Mosaic sex chromosome aneuploidies are also common. This means that a person may have a mixture of cells with different chromosomal arrangements, often in the presence of normal cell lines. Some common mosaic cell lines include 45,X/46,XX, 46,XX/47,XXX, 46,XY/47,XXY, and 46,XY/47,XYY. More complex sex chromosome aneuploidies, involving gaining more than one extra copy of the X or Y chromosome, or structural rearrangements of these chromosomes, occur much less frequently. These complex variations can be seen in either the sole cell line or in a mosaic form. (14,15)

Diagnostics:

In the past 25 years, there has been significant progress in the field of prenatal screening for chromosome abnormalities, especially in the detection of Down syndrome (trisomy 21). The most commonly used approach is to perform screening tests at a single point in time during pregnancy.

In the first trimester, a common screening method involves measuring the nuchal translucency (NT) of the fetus, along with analyzing the levels of two serum analytes, β-human chorionic gonadotropin (hCG) and pregnancy-associated plasma protein (PAPP-A). This is known as the double marker test. In the second trimester, other tests such as the triple and quadruple marker tests are often used.

If a pregnant woman receives a “High Risk” or positive result in one of these biochemical hormone screening tests, she is typically required to undergo further, more invasive prenatal diagnostic procedures. However, these invasive procedures are associated with a small risk, around 0.5-1%, of causing fetal loss as a result of the procedure.

The most reliable test for confirming the presence of chromosomal abnormalities in a developing fetus is chromosome analysis, which is often called a karyotype test. This test requires growing amniotic fluid cells (amniocytes) or placental cells (chorionic villus sample) in a lab to examine the chromosomes.

Although maternal serum screening is generally considered safe, it has its advantages and disadvantages. One drawback is that it can result in lower sensitivity and relatively high false positive results. When these results are obtained, it is necessary to follow up with further invasive testing procedures to confirm or rule out chromosomal abnormalities.

These invasive procedures involve collecting tissue samples from the placenta or amniotic fluid from the pregnant woman. It’s important to note that they carry a risk of miscarriage or fetal loss related to the procedure. Therefore, these prenatal sampling procedures should only be performed by experienced and trained fetal medicine doctors.

In recent years, non-invasive prenatal screening methods, also known as Cell-Free Fetal DNA Screening (NIPS/NIPT), have become increasingly popular worldwide, including in India. This is because the cost of these tests has come down, and they have shown a high success rate in detecting major chromosome abnormalities like Down syndrome.

This groundbreaking technique was first developed in 1997 by Dennis Lo and his team. They made a remarkable discovery that the blood of pregnant women contains cell-free DNA (cfDNA). It turns out that a significant amount of this cfDNA in the mother’s blood comes from the placenta and contains genetic material from the developing fetus. This makes it a suitable source for non-invasive prenatal testing.

Further research into sequencing the maternal and fetal cell-free DNA has revealed the tremendous potential of this method as a screening test. It offers a new and non-invasive way to detect fetal chromosomal abnormalities, providing an alternative to more traditional and invasive testing method

Prevention and Treatment:

NIPS, or Non-Invasive Prenatal Screening, is a test that helps identify significant chromosome abnormalities in a developing fetus by examining cell-free fetal DNA from the mother’s blood. The process involves extracting fetal DNA, amplifying it, aligning the sequences, and then identifying the origin of each chromosome through bioinformatics analysis to interpret the results.

There are currently two commercially validated methods for detecting fetal aneuploidy (chromosome abnormalities) using next-generation sequencing techniques. One method involves counting DNA sequences across the entire genome, while the other targets specific segments known as SNPs (single nucleotide polymorphisms).

The procedure begins with the laboratory receiving a blood sample from the pregnant woman, which is then centrifuged to separate it into three layers. The top layer contains cell-free DNA from both the mother and the fetus. In the SNP method, the top two layers are sequenced using next-generation sequencing, targeting 19,488 SNPs covering major chromosome abnormalities for chromosomes 13, 18, 21, X, and Y. Bioinformatics algorithms are used to process the maternal genotype from the white blood cells in the middle layer, which provides insight into the fetal genotype. Bayesian statistical methods are applied to analyse the sequencing results, determining the copy number of the chromosomes in question. The results are reported with an individualized risk score, categorizing them as high risk, low risk, or suggesting a repeat sampling if necessary.

Current non-invasive prenatal screening tests are validated to detect core trisomies, including common autosomal abnormalities like trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), and common sex chromosome abnormalities like monosomy X (Turner syndrome), Triple X (Triple X syndrome), (XXY) Klinefelter syndrome, and XYY syndrome. More advanced versions of NIPS tests are also available after validation to detect common microdeletion syndromes, such as DiGeorge syndrome, 1p36 syndrome, Angelman syndrome, Prader-Willi syndrome, Cri-du-chat syndrome, Wolf-Hirschhorn syndrome, Jacobsen syndrome, and Langer-Giedion syndrome.

NIPS, or NIPT, is an extremely sensitive test, with an accuracy of over 99% in detecting trisomy 21 (Down Syndrome) in fetuses. This makes it a highly reliable screening test, alleviating the anxiety of pregnant women concerned about the possibility of having a baby with Down syndrome and helping to avoid invasive procedures that carry a risk of miscarriage. NIPS is also valuable for women who have undergone assisted reproductive technologies, including those with twin or triplet pregnancies, egg donors, or surrogate mothers.

NIPS is considered one of the safest and most convenient prenatal screening methods because it only requires a simple blood draw from the pregnant woman. It has the potential to spare women from invasive fetal testing procedures like amniocentesis or CVS and rule out chromosome abnormalities due to its improved sensitivity. In clinical settings, NIPS appears to be the most effective prenatal screening test for detecting Down syndrome, especially in women with positive results from maternal serum screening, women aged 35 and older at delivery, those with abnormal ultrasonography findings, those with a previous history of a trisomy-affected baby, or those with a family history of chromosomal abnormalities or balanced translocations.

Recent trends have shown that Non-Invasive Prenatal Screening (NIPS) holds great promise as a highly accurate prenatal screening method to rule out major chromosome abnormalities and reduce the anxiety and stress in pregnant women with positive maternal serum screening results, thus avoiding unnecessary invasive procedures and the risk of having a baby with chromosomal abnormalities.

Author:

Dr. Ashish Fauzdar, Ph.D (Department of Reproductive Biology,  All India Institute of Medical Sciences (AIIMS), New Delhi).

November 6, 2023

Edited by:

Dr. Dhanlaxmi Shetty, Ph.D and Dr. Saima Naz Khan, Ph.D

References:

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