Archive of Obstetrics Gynecology and Reproductive Medicine (ISSN:2640-2297)
Research Article
Non-Invasive Prenatal Diagnosis
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Non-invasive prenatal diagnosis (NIPD) represents a major advancement in prenatal care, offering pregnant women the ability to detect serious genetic disorders in a fetus with high accuracy using simple blood tests. Unlike traditional invasive methods such as amniocentesis and chorionic villus sampling, which carry a risk of miscarriage, NIPD eliminates procedural risks and enables earlier detection, often as early as 7 to 9 weeks of gestation. This review outlines the main approaches to NIPD, including analysis of intact fetal cells and cell-free fetal nucleic acids in maternal blood and cervical samples. While challenges remain, such as the rarity of fetal cells and the difficulty in distinguishing fetal from maternal DNA, significant clinical applications have already emerged. These include early detection of Rh incompatibility, fetal sex determination, screening for single gene disorders, and identification of chromosomal aneuploidies. Furthermore, elevated levels of fetal DNA in maternal plasma are being explored as markers for obstetric complications like preeclampsia and preterm delivery. Continued technological advances are expected to further integrate NIPD into routine clinical practice, making prenatal genetic diagnosis safer and more accessible.
Someday soon, virtually any pregnant woman 2 will be able to learn -with 98-99% accuracy-whether her fetus has contracted a serious genetic disorder by undergoing nothing more than an in-expensive, non-invasive blood test.
Prenatal genetic diagnosis is offered to women who are at increased risk for carrying a fetus affected with a chromosomal aneuploidy or other genetically inherited diseases. However, the invasive procedures used, amniocentesis and chorionic villus sampling (CVS), can cause miscarriage of pregnancy. Development of non-invasive methods would obviate this risk and change the risk benefit ratio of prenatal diagnosis. Secondly this new technology can attain high level of accuracy and be offered at much earlier (7 to 9 weeks) gestation before CVS or amniocentesis can be offered, minimizing the risks if pregnancy termination is desired.
A. Intact Fetal Cells in Maternal Blood- Intact fetal cells circulate in maternal blood during pregnancy, and their analysis can allow definitive non -invasive prenatal genetic diagnosis. Given the rarity of fetal cells in maternal blood (1 fetal cell per 107 maternal cells); one must enrich the sample for the rare fetal cells6. Analysis for fetal chromosomal complement relies on FISH using chromosome-specific probes. The possible cells that can be isolated from maternal blood are:
a) Trophoblasts - They were the first cells to be identified in maternal circulation because of their large size4, 6. However these are not ideal for prenatal diagnosis.
A. Intact Fetal Cells in Maternal Blood- Intact fetal cells circulate in maternal blood during pregnancy, and their analysis can allow definitive non -invasive prenatal genetic diagnosis. Given the rarity of fetal cells in maternal blood (1 fetal cell per 107 maternal cells); one must enrich the sample for the rare fetal cells6. Analysis for fetal chromosomal complement relies on FISH using chromosome-specific probes. The possible cells that can be isolated from maternal blood are:
a) Trophoblasts - They were the first cells to be identified in maternal circulation because of their large size4, 6. However these are not ideal for prenatal diagnosis.
Disadvantages:
- Very small numbers of these cells are present in maternal blood during the first trimester when it is ideal to perform prenatal diagnosis
- Any trophoblast that is released into the maternal circulation quickly becomes trapped in the lung and rarely remains in peripheral circulation
- They do not share the same chromosome complement as that of the fetus and are either, multinucleated or mosaic
- There occurs development of monoclonal antibodies against trophoblast cell surface antigens
Disadvantages:
- Fetal production of lymphocytes does not begin until the second trimester
- Fetal lymphocytes can persist in maternal circulation for many years after pregnancy and thus may not be indicators of index pregnancy
- Requires paternal HLA typing and specific antisera and hence impossible when paternal and maternal HLA antigens are shared
- There is persistently high number of female bearing pregnancies with Y chromosome positive cells
- They are rare in adult circulation, but common in fetus
- Can be seen in early gestation when hematopoiesis occurs in yolk sac
- Express several unique antigens such as transferring receptor, making it possible to enrich samples of maternal blood for fetal cells
- They produce unique fetal hemoglobin chains such as zeta and gamma, which have the potential to be used as markers to identify these fetal cells
- Have short life span and are thus unlikely to persist from one pregnancy to next
- They are mononuclear and relatively well differentiated
Disadvantages:
- Pregnancy induced erythropoiesis leads to increased number of NRBCs of maternal origin in maternal blood and hence even after enrichment techniques NRBCs of fetal origin are less in number as compared to maternal cells
- Usually difficult to confirm that NRBCs are indeed of fetal origin and not of maternal origin
Advantages:
- Relatively large number of fetal cells is obtained (proportion varying between 4% to 80%)3,6,10
- The higher yield makes feasible the detection of not only trisomies by FISH, but also microarray analysis encompassing microdeletion syndromes and mendelian disorders6
Disadvantages:
- There exists possibility of contamination by sperm in the sample retrieved.
- This technique is still at least minimally invasive, though the patients who have had this procedure compare it to cervical smear being taken 3, 6.
Disadvantages:
- Pure fetal genetic material cannot be obtained for analysis as there is high preponderance of maternal cell free DNA sequences in maternal plasma
- Half of the genetic code of the fetus will be identical to mother”s genome
Clinical Applications of Non- Invasive Prenatal Diagnosis
a. Rh alloimmunization- Rh-D blood incompatibility between a pregnant woman and her fetus is a significant problem given the potential for maternal alloimmunization and subsequent hemolytic disease of the newborn. The Rh blood group is found among 15% of Caucasians, 3-5% of black Africans, and is rare among Asian population. As such, management of Rh- pregnant women is an important part of prenatal practice. Fetal DNA within maternal plasma has been used to determine fetal Rh-D status in Rh- pregnant women. Unlike more conventional methods such as amniocentesis and CVS, the risk of feto-maternal hemorrhage and further sensitization is removed using this method. Thus it provides a basis for administration of anti-D immunoglobulin as prophylaxis only, in pregnancies with a confirmed Rh+ fetus, thereby preventing unnecessary administration of anti-D immunoglobulin1, 10. Prenatal determination of fetal RhD status thus proves a clinical useful means to limit unnecessary testing or therapeutic intervention, including administration of antenatal anti-D immune-prophylaxis by identifying Rh- D fetuses7.
b. Fetal Sexing- Early determination of fetal gender is useful in the management of X linked genetic disorders like haemophilia and Duchene muscular dystrophy. Male fetus may develop the disease, but female fetus will not. This provides affected women the opportunity to take a decision at right time. Some opt for termination, while others prepare themselves for the birth of a child with abnormality. Defining fetal gender from maternal plasma is possible as early as 14 days post conception, about two months before this information is available through ultrasound scanning 3. Most research groups use sequences of chromosome Y in male embryos as marker of fetal DNA because of the fact that a normal woman/mother (46, XX) does not possess this chromosome in her genome3, 8. Specific sequences in the Y chromosome such as SRY, DYS14, and DAZ loci are generally used to detect fetal gender 5, 9.
Another application of fetal sexing is in pregnancies at risk of congenital adrenal hyperplasia (CAH) where early treatment of pregnancies with an affected female fetus has been shown to reduce the degree of virilisation of the external genitalia. In these situations, NIPD has been shown to reduce the uptake of invasive diagnostic testing by nearly 50% and allowed for early cessation of dexamethasone treatment in pregnancies at risk of CAH where fetus is found to be male or unaffected female 7,9,10.
c. Single Gene Disorders- Non-invasive prenatal diagnosis has been useful in the detection of single gene disorders in families known to be at risk of inherited diseases12. This is currently limited to detecting disease causing mutations that are not present in the maternal genome. So far it has been used to detect autosomal dominant disorders like Hungtington’s disease, Achondroplasia, and Muscular dystrophy10. In cases of autosomal recessive disorders such as cystic fibrosis (CF), prenatal diagnosis currently requires an invasive test as because there are many different mutations that can cause this particular disease and hence detection becomes difficult. Diagnosis of an unaffected fetus may be made by exclusion of the presence of paternally inherited mutation signal in maternal plasma. In that way, invasive prenatal diagnosis could be limited to those pregnancies which have inherited the paternal mutation and so have a potentially affected fetus. Another common autosomal recessive single gene disorder is Beta Thallesemia and this has been excluded by testing for paternally transmitted mutation 9,10.
d. Fetal Aneuploidy- Detection of fetal aneuploidy is the most common indication for prenatal diagnosis and detection using non- invasive means has been technically challenging because of the fact that fetal DNA exists in maternal plasma in a high background of maternal DNA of approximately 1.5 fold that found in normal pregnancies. This difference borders the threshold of real time detection and is not diagnostic 9. Therefore, two effective approaches have been developed that can be used for detection of fetal aneuploidy.
- The first approach involves the measuring of the allelic ratio of single nucleotide polymorphism (SNP) in the mRNA rather than the DNA coding region of placenta. The main advantage of cell free RNA (cff-RNA) over cell free DNA (cff-DNA) is that it is highly expressed in placental tissues but barely or no contribution in maternal tissues, i.e., it is fetal specific. By extracting cell-free RNA rather than cell-free DNA from maternal plasma and testing a SNP located in the PLAC4 fetal mRNA sequence, the maternal: paternal chromosome allelic ratio can be determined. A ratio of 1:1 indicates that the fetus has two copies of chromosome 21 (normal) and a ratio of 2:1 indicates that the fetus has three copies of chromosome 21 (Down syndrome)7,9. The major disadvantage of this approach is the need to have informative polymorphic region.9
- The next approach involves analysing both maternal and fetal DNA and looking for differences in the DNA methylation pattern. SERPINB5 and RASSF1A are examples of such epigenetic markers. SERPINB5 coding for maspin is hypomethylated in placental tissues and hypermethylated in maternal blood cells. Through the targeting of such fetal specific markers the detected signal is virtually completely fetal in origin, thus allowing fetal chromosomal dosage via the analysis of the allelic ratio of the genetic variations at the detected loci9.
- Preeclamsia - Elevated levels of fetal DNA is seen in plasma of pregnant women with pre- eclampsia and their levels might be a useful as a marker for diagnosing and/or monitoring this condition. Plasma DNA concentration can also be used for early prediction of pre-eclampsia. However, the exact mechanism by which quantitative increase in plasma DNA occurs is still under research1.
- Hyperemesis gravidarum - Increased fetal DNA levels are detected in maternal plasma in cases of hyperemesis-gravidarum (HG) since hyperactivation of the maternal immune system is thought to destroy trophoblast cells. Levels of fetal DNA vary with the clinical severity of the condition.
- Invasive placenta (abnormal adherence of the placenta) - Antenatal detection of invasive placenta might be achievable through analysis of fetal DNA concentrations in maternal plasma. Trophoblasts are destroyed by the maternal immune system during the process of invasion of myometrium, thus resulting in increased concentrations of fetal DNA. They are a useful marker to follow patients with retained placental tissue after delivery.
- Preterm delivery - Fetal DNA is present in greater concentrations in the plasma of pregnant women who undergo preterm labor, as compared to those who deliver at term.
CONCLUSION
Detection of fetal cells in the maternal circulation is a rapidly expanding field of investigation with important implications for both prenatal diagnosis and for a better understanding of the physiology of feto-maternal interactions. It is hoped that further new concepts and technological advances will now hasten the development of this field and lead to the introduction of non-invasive prenatal diagnosis into routine clinical practice.
- Recent advances in non –invasive prenatal DNA diagnosis through analysis of maternal blood-J. (2007) Obstet. Gynaecol. Res. 33, No 6:747-764.
- Recent progress in non-invasive prenatal diagnosis-Seminars in Fetal & Neonatal Medicine (2008) 13: 57-62.
- Non-Invasive Prenatal Diagnosis-ASJOG. 2006..
- Prenatal Diagnosis Using Fetal Cells Isolated From Maternal Peripheral Blood: A Review-Clinical Obstetrics and Gynaecology (1996) 39: 4:801-813.
- Noninvasive prenatal diagnosis: Current practice and future prospective - Current Opinion in Obstetrics and Gynaecology (2008) 20:146-151
- Obstetrics‐Normal and Problem Pregnancies, Fifth Edition, Gabbe, Niebyl and Simpson, Prenatal Genetic Diagnosis, Pages 152‐179.
- Non-Invasive Prenatal Diagnosis Using Cell –Free DNA in Maternal Blood, Scientific Advisory Committee Opinion Paper-15, 2009.
- Ramos ES. Cell-free fetal DNA in maternal plasma and noninvasive prenatal diagnosis. Rev Latino- am Enfermagem 2006 Novembro-Dezembro; 14(6):964-967.
- Non-Invasive Prenatal Diagnosis: From Dream to Reality,JPOG, 129
- Review Non –invasive prenatal Diagnosis- Illanes S, Abdel-Fatah S, Soothill P: The Obstetrician & Gynaecologist 2006;8:91-95
- The SAFE project: towards non- invasive prenatal diagnosis: Biochem. Soc.Trans (2009) Volume 37;460-465.
- Cell –free fetal DNA and RNA in maternal blood: implications for safer antenatal testing; Caroline F Wright, Lyn S Chitty. 2009.
- Cell -free fetal DNA and Non-invasive Prenatal Diagnosis; Imran Rafi, Br. J. Gen. Pract. 2009 ;59(562): e146-e148.
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