Prenatal Diagnosis and Screening

### **Chapter 9**

## Prenatal Diagnosis: The Main Advances in the Application of Identification of Biomarkers Based on Multi-Omics

*Tong Wang, Jiahe Xu, Lin Wang, Xiumin Cui, Yan Yan, Qiuqin Tang and Wei Wu*

### **Abstract**

Prenatal diagnosis is to make the diagnosis of fetal structural abnormalities, genetic diseases, and pregnancy-related diseases before birth thus could offer evidence for intrauterine treatment or selectively termination of pregnancy. Up to now, researchers have applied multi-omics, including genomics, transcriptomics, and proteomics, in the discovery of prenatal diagnostic biomarkers. They have found some candidate biomarkers for aneuploids, preeclampsia, intrauterine growth retardation, and congenital structural abnormalities. With the momentous progress of biomarkers' identification based on multi-omics for prenatal diagnosis, noninvasive prenatal testing (NIPT) has experienced tremendous progress and is revolutionizing prenatal screening and diagnosis over the past few decades. Extensive studies have also demonstrated the value of biomarkers. In particular, cell-free DNA (cfDNA), allows for a definitive diagnosis in early pregnancy for fetal diseases, including Down syndrome and other common aneuploidies. The cfDNA can be extracted from maternal plasma, posing no risk of miscarriage compared to the traditional invasive diagnosis directly analyzing fetal cells from amniocentesis or chorionic villus sampling. In this review, we would discuss the main advances, strengths, and limitations in the application of biomarkers for prenatal diagnosis along with the analysis of several representative fetal diseases.

**Keywords:** aneuploids, biomarker, cell-free DNA, congenital structural abnormalities, intrauterine growth retardation, preeclampsia, prenatal diagnosis

### **1. Introduction**

Prenatal diagnosis is to make the diagnosis of fetal structural abnormalities, genetic diseases, and pregnancy-related diseases before birth thus could offer evidence for intrauterine treatment or selectively termination of pregnancy [1]. Up to now, the research on noninvasive prenatal screening and diagnosis has undergone

enormous progress. Researchers around the world have applied multi-omics, including genomics, transcriptomics, and proteomics, in the discovery of prenatal diagnostic biomarkers, and found some candidate biomarkers for aneuploids, pre-eclampsia, intrauterine growth retardation, and congenital structural abnormalities. With the momentous progress of biomarkers' identification based on multi-omics for prenatal diagnosis, noninvasive prenatal testing (NIPT) has made great strides over the past few decades and is revolutionizing prenatal screening and diagnosis. Extensive studies have also demonstrated the value of biomarkers. In particular, cell-free DNA (cfDNA), which is widely acknowledged as the main method of NIPT, allows for a definitive diagnosis in early pregnancy for fetal diseases, including Down syndrome and other common aneuploidies, and thus is sought by providers and patients. In this review, we would discuss the main advances, strengths, and limitations in the application of biomarkers for prenatal diagnosis along with the analysis of several representative fetal diseases.

### **2. Prenatal diagnostic techniques**

### **2.1 Noninvasive techniques**

Noninvasive techniques include examining a woman's uterus through maternal serology and ultrasound. Blood tests for selecting trisomies based on detecting placental cfDNA present in maternal blood, which is also known as NIPT, have now become available [2]. However, if a noninvasive screening test indicates an elevated risk of chromosomal or genetic abnormalities, then invasive techniques can be used to gather more information [3]. For example, a detailed ultrasound can provide a definitive diagnosis noninvasively in the case of neural tube defects (NTDs). Biomarkers are involved in some methods, including maternal serum screening and other methods (**Table 1**).

### *2.1.1 Fetal cells in maternal blood*

The inspection of fetal cells in maternal blood requires a maternal blood draw. Because fetal cells contain nearly all of the genetic information of the developing fetus, they could be used for prenatal diagnosis [4].

### *2.1.2 Cell-free fetal DNA (cffDNA) in maternal blood*

Fetal DNA ranges from about 2–10% of the total DNA in maternal blood. The inspection of cffDNA in maternal blood also requires a maternal blood draw. This test can potentially identify fetal aneuploidy [5] and gender. The cffDNA also allows whole genome sequencing of the fetus, thus determining the complete DNA sequence of every gene [6], which is helpful for prenatal diagnosis.

### *2.1.3 Transcervical retrieval of trophoblast cells*

Cervical swabs, cervical mucus aspiration, and cervical or intrauterine lavage could be used to retrieve trophoblast cells for identifying aneuploidies [7]. It has been proven that antibody markers are available to select trophoblast cells for genetic *Prenatal Diagnosis: The Main Advances in the Application of Identification of Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.104981*


### **Table 1.**

*Prenatal diagnostic/screening techniques.*

analysis or to demonstrate that the abundance of recoverable trophoblast cells is reduced in unusual gestations [7].

### *2.1.4 Maternal serum screening*

Maternal serum screening could be used as a routine prenatal test to determine the risk of aneuploidies as well as certain malformations, including NTDs [8, 9]. Maternal serum screening was classically done in the second trimester but now first-trimester screening has also been found equally useful [8]. The related biomarkers contain β-human chorionic gonadotropin (β-hCG), pregnancy-associated plasma protein-A (PAPP-A), alpha-fetoprotein, and inhibin-A.

### **2.2 Invasive techniques**

An invasive method involves probes or needles being inserted into the uterus. The commonly used invasive methods include amniocentesis and chorionic villus sampling (CVS) [8]. One study has compared second-trimester amniocentesis with transabdominal CVS, finding no significant differences in total pregnancy loss between the two procedures [10]. The samples could be used for molecular, cytogenetic, and biochemical tests but especially, the CVS sample is a perfect sample for DNA-based tests when amniotic fluid is desired for cytogenetic analysis [8].

### **3. The research of biomarkers based on multi-omics**

### **3.1 Genomics**

According to the National Cancer Institute, a biomarker is "a biological molecule which is a sign of normal process or disease found in blood, other body fluids or tissues." Liquid biopsy is very promising for noninvasive characteristics and may provide important biomarkers, including cell-free nucleic acids (cf-NAs) [10]. Since the discovery of free fetal DNA, noninvasive prenatal diagnosis (NIPD) has been gaining attention for early pregnancy detection of genetic diseases by analyzing cfDNA or cffDNA in maternal plasma [11].

First detected in cancer patients' sera in 1948 [12], cfDNA, used as a prognostic factor in malignant disease [9, 13], has gradually shown some advantages in the application of prenatal diagnosis since Lo et al. detected circulating fetal DNA in maternal plasma in 1997 [9, 14]. With the rapid advances in molecular biological technologies, it creates a preferable procedure for chromosomal abnormalities and monogenic disorders. The cfDNA refers to a DNA molecule in plasma, typically between 500 and 30,000 bp nucleotides in size. Existing in peripheral blood, synovial fluid, and other body fluids, cfDNA has three forms—free, attached to proteins, or encapsulated in extracellular vesicles [10]. Based on comprehensive studies, nucleosome spacing of cfDNA in healthy individuals suggests its origin—nucleic or mitochondrial from the apoptosis of lymphoid and myeloid cells [15], mainly swallowed by phagocytes for homeostasis. However, there is no hard evidence to support the origin theory, and the mechanism is not clear. Healthy individuals have less cfDNA. Fetal DNA in maternal circulation (3–6%) is reported to be from placental apoptosis [16].

Using cfDNA as a biomarker is advantageous for the accessible sample with little trauma, dynamic monitoring from early pregnancy, sensitive and specific procedure, and reliable outcome. However, the unit cost of cfDNA is relatively more expensive than invasive tests. Screening for trisomies by cfDNA could detect nearly 100% of fetuses with trisomy 21, 98% of trisomy 18, and 99% of trisomy 13, with a combined false-positive rate (FPR) of 0.13% [17]. The application of cfDNA includes quantitative and qualitative methods. The change of cfDNA's quantity may alert us to tumor gene mutations, diseases' progression, and help prognosis prediction [18]. Elevated concentrations of cfDNA are related to cancer, pregnancy, autoimmune disease, or myocardial infarction [18]. The abnormal cffDNA quantity reflects neonatal hemolytic, preeclampsia (PE), and so on. In prenatal diagnosis, rheumatic heart disease (RhD), sex-related diseases, single-gene disorders, such as β-thalassemia, and cartilage dysplasia are in the diagnosable range [14, 17]. Quantitative analysis methods include spectrophotometer, enzyme-linked immunosorbent assay (ELISA), and real-time fluorescence quantitative PCR (qPCR). Qualitative analysis methods can be used to detect activation or inactivation mutations of *Ras*, *P53*, and other tumor suppressor genes, as well as changes in the DNA immunoglobulin heavy chain [18].

Unfortunately, due to the low concentrations of cfDNA, such determination is only feasible by ultra-accurate devices [19]. Including next-generation sequencing (NGS), most methods are still restricted to targeted genomic loci [20]. Until now, only a few noninvasive attempts have been made.

*Prenatal Diagnosis: The Main Advances in the Application of Identification of Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.104981*

### **3.2 Epigenomics**

For genomes, not only do sequences contain genetic information, but modifications can also record genetic information. Epigenomics is the field of studying epigenetic modifications at the level of the genome. Epigenetic modifications act on intracellular DNA and its packaging proteins, histones, and are used to regulate genomic function, as manifested by DNA methylation and post-translational modifications of histones, molecular markers that affect the architecture, integrity, and assembly of chromosomes, as well as the ability of DNA to approach its regulatory elements, and chromatin to interact with functional nuclear complexes. Epigenetic biomarkers, including DNA methylation and histone modifications, are increasingly used for disease diagnosis because of their greater specificity and generalizability.

For prenatal diagnosis, epigenomics has a very extensive application. According to recent research, the level of DNA methylation is related to prenatal alcohol exposure (PAE) [21, 22]. Fetal alcohol spectrum disorders (FASD), however, are a consequence of PAE. Alcohol can affect the phenotype of adult mice by modifying the epigenotype of early mouse embryos. Children with FASD may have unique DNA methylation deficiencies, which suggests the further use of biomarkers in the future. In addition, the translation of non-coding RNA, as microRNAs (miRNAs) into proteins, is part of epigenetic regulation. Differential expression of miRNAs is a potential NIPD of fetal coronary artery disease by abnormal pregnancy-associated miRNAs [23].

### **3.3 Transcriptomics**

Transcriptomics is the field that studies gene transcription and transcriptional regulation in cells at the global level. The transcriptome is the total of all RNAs that living cells can transcribe and is an important tool for studying cellular phenotype and function. Transcriptomics is a diagnostic tool based on providing information about the expression of specific genes under specific conditions, which can infer the function of corresponding unknown genes and reveal the mechanism of action of specific regulatory genes. Therefore, transcriptomics is applied to markers in diagnosis. The technology of microarray, serial analysis of gene expression (SAGE), and massively parallel signature sequencing can be applied to the discovery of biomarkers [24].

In terms of application, microarray technology has become one of the leading techniques for prenatal diagnosis in terms of detection rate and accuracy of results [25]. Chromosome microarray analysis (CMA) is applied to the clinical diagnosis of the genetic cause of congenital heart disease (CHD) [26], which is a pioneered new method to improve the detection rate of CHD in children [27]. Biomarkers relevant for the diagnosis of CHD can be applied using multi-omics techniques [28]. This will be described in detail below.

### **3.4 Proteomics**

Proteins are the main carriers of biological functions. Proteomics refers to the study of proteins, including the dynamic changes in protein composition, the analysis of intracellular expression levels and modification states, the understanding of the interactions and connections between proteins, and the elucidation of the rules of

protein regulation activity. In conclusion, proteomics mainly involves the study of proteomic expression patterns and functional patterns of protein functional groups. Proteomics determines the basic functional properties of proteins through the identification of their species and structures. Based on the relevant studies of proteins, proteomics has a relevant role in biomarkers.

Proteomics has relevant applications in prenatal diagnosis. In the prenatal diagnosis of biomarkers for trisomy 21, proteomics has been applied in large and multiple ways. Differential protein expression can be identified in the urinary proteome by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis to improve the detection of prenatal trisomy 21 [29]. Similarly, mass spectrometry and selective response monitoring (SRM) can be used to screen for differentially expressed proteins in the proteome of maternal serum as biomarkers for trisomy 21.

### **3.5 Metabonomics**

Most of the life activities within a cell occur at the metabolic level, so changes in the metabolites of the cell can more directly reflect the cell's environment. Metabolomics can determine the composition of all small molecules in a cell, map their dynamic patterns of change, create a systematic metabolic map, and determine the link between changes and biological processes. Metabolomics focuses on biological fluids as the object of study, mainly urine and blood. Because of the abundance of endogenous products in blood and the noninvasive nature of urine collection, these body fluids are widely utilized. Compared with genomics and proteomics, metabolomics is more closely related to clinical practice [30]. Based on this advantage, research related to biomarkers is closely affiliated with the application of metabolomics [30].

Non-targeted metabolomics is a powerful tool that can provide a new approach to prenatal diagnosis [31]. It can be utilized for the discovery of affected metabolic pathways and therefore helps to propose potential biomarkers. The search for prenatal biomarkers in preterm birth (PTB) has made full use of metabolomic approaches. Predictive biomarkers of PTB were identified by analysis of prenatal maternal body fluids (amniotic fluid, maternal urine/maternal blood, and cervicovaginal fluid) using nuclear magnetic resonance spectroscopy or mass spectrometry-based methods [32].

### **4. Application of biomarkers for prenatal diagnosis**

### **4.1 Disorder of pregnancy**

### *4.1.1 Preeclampsia (PE)*

PE is a pregnancy-specific syndrome, affecting 3–5% of pregnant women. It is characterized by edemas, proteinuria, and high blood pressure. In women with PE dysfunction of many organs, including liver and kidney, fetus growth restriction is also observed. If untreated, PE may lead to death. In some low-income countries, PE is one of the main causes of maternal and child mortality [33–35].

Hsu et al. identified differentially expressed proteins in serum samples obtained from pregnant women with severe PE and control participants through two-dimensional gel electrophoresis (2-DE) [36]. Then additional serum samples were analyzed by immunoassay for validation. Ten protein spots were discovered to be upregulated in women with PE. Serum α1-antitrypsin, α1-microglobulin, and clusterin levels of

*Prenatal Diagnosis: The Main Advances in the Application of Identification of Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.104981*

PE patients were significantly higher compared to those in the normal participants [36]. Blankley et al. used isobaric tagging to identify certain potential biomarker proteins in plasma obtained at 15 weeks gestation from nulliparous women who later developed PE. The results confirmed the high accuracy of the pregnancy-specific beta-1-glycoprotein 9 (PSG9) as a potential biomarker for the prenatal diagnosis of PE [37]. Kolialexi et al. collected blood samples from pregnant women at 11–13 weeks of gestation and these women were followed up until delivery. Compared to controls, twelve proteins were differentially expressed in the plasma of women who subsequently developed PE [38, 39].

A systematic review had examined 13 studies, 11 of 13 had found an increase in cfDNA among women who subsequently developed PE [39, 40]. Moreover, four studies examining early-onset or severe PE found increased cfDNA levels compared to disease onset [37]. In one study, the median levels and multiples of the median (MoM) values of *HYP2*, a cfDNA marker, were significantly higher in the preeclampsia and hypertensive disorders of pregnancy groups at 6–14 and 15–23 weeks' gestation compared with controls [39, 41]. *HYP2*, located on chromosome 13, is hypermethylated in the placenta and maternal blood cells. *HYP2* has been studied as an epigenetic marker for total cfDNA [42].

### *4.1.2 Intrauterine growth restriction (IUGR)*

Intrauterine growth restriction (i.e., fetal growth restriction) refers to poor growth of the fetus in the uterus during pregnancy. IUGR is defined by evidence of reduced growth and clinical features of malnutrition [43]. IUGR could cause a baby to be small for gestational age (SGA), which is often defined as a weight below the 10th percentile for gestational age, resulting in low birth weight at the end of pregnancy [36].

Current methods of detection commonly include the measurement of symphysis fundal height (SFH) [44], ultrasound biometry, and doppler ultrasonography. Recently, most interest has been put in novel approaches to screening, including the testing of maternal serum biomarkers and nucleic acids, proteins, vesicles, and metabolites [45].

One study showed that pro-angiogenic placenta growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) in the first trimester could increase the sensitivity of detection for early-onset IUGR to 86% and 66% for late-onset IUGR through a larger cohort of 9150 women [46]. Based on a multi-parametric method in the third trimester, Miranda et al. designed a nested case–control cohort study in 1590 pregnant women. Their integrated model contained maternal risk factors, estimated fetal weight (EFW), PlGF, unconjugated estriol, and Uterine artery (Ut) Doppler, achieving a sensitivity of 61% for SGA increasing to 77% for IUGR [47]. In addition, it was also proven that low PlGF (< 5th centile) could indicate IUGR with underlying placental pathology with a specificity of 75% and sensitivity of 98% [48].

Like cfDNA, circulating placental RNA (cpRNA) can also be detected in blood, plasma, serum urine, and amniotic fluid in the first trimester [49, 50]. It has been indicated that compared to those who deliver infants in the normal birth weight range, serum cpRNA, cord blood metabolites, urinary metabolites, and amino acid levels in women who develop IUGR would be changed. Future techniques for the detection of specific analytes may focus on microarrays, digital polymerase chain reaction, and NGS, which could identify some RNA analogs. These novel types of more sophisticated biomarkers are the potential to distinguish certain types of IUGR. However, since placental contributions are more common, chances are that such

biomarkers would have to outperform PlGF. This kind of method is increasingly becoming a more effective screening and diagnostic tool in the diagnosis of IUGR.

### **4.2 Genetic disorders**

### *4.2.1 Down syndrome*

The most common chromosomal disorder is trisomy 21 [51], also known as Down syndrome, with an incidence of 1 per 800 live births [52]. Common biomarkers used for diagnosing Down syndrome include pregnancy-associated plasma protein-A (PAPP-A), β-human chorionic gonadotropin (β-hCG), alpha-fetoprotein (AFP), Estriol (uE3), dimeric inhibin-A (DIA), etc. [52, 53]. These protein measurements are combined with age, race, weight, number of fetuses in the current gestation, diabetes status, and gestational age to provide a risk estimate. For example, PAPP-A and β-hCG levels are higher in Southeast Asian women compared to Caucasian women, and the serum marker levels in twin pregnancies are approximately twice those found in singleton pregnancies [54].

Usually decreased level of PAPP-A is an indicator for Down syndrome and trisomy 18, whereas the increased level of β-hCG suggests a risk of Down syndrome. These are the two most used serum biomarkers for Down syndrome detection and maternal serum screening (MSS) is often performed with nuchal translucency ultrasound screening. The integration of the two methods is known as enhanced first-trimester screening (eFTs). In most cases ultrasound screening can be diagnostic, MSS is intended only to identify women with pregnancies at increased risk [52]. Further diagnostic methods include cell-free fetal DNA screening (cfDNA screening), amniocentesis, CVS, etc. [55].

One thing has to be noticed during the prenatal screening. In the cases of the vanishing-twin syndrome, the PAPP-A level could be affected by the demise of the twin, and thus should not be used as a means of diagnosis except with alternative adjustments [56]. MSS and eFTs can also be done for the detection of other aneuploidies [57], which means aneuploidies can be diagnosed simultaneously.

### *4.2.2 β-Thalassemia*

β-thalassemia is a blood disorder that reduces the production of hemoglobin. Although studies concerned have suggested that several cord blood serum markers have potential diagnostic value, they have not been worked in application [58]. The relatively mature enough technique is the GthapScreen HBB kit, which involves several STR markers. The DNA samples are purified from blood, amniotic fluid, and CVS. Advantages of this technique include perfect accuracy and no need to consider multiple pregnancies [59].

### *4.2.3 Gaucher disease (GD)*

GD is an autosomal recessive lysosomal storage disorder arising predominantly from mutations in the gene *GBA1* [60], which encodes β-glucocerebrosidase. Insufficient amounts of the hydrolase result in glucosyl sphingosine (GluSph) accumulating in the reticuloendothelial cells of the liver, spleen, bone, and lung, as well as the brain in the rarer disease subtypes. Biomarkers applied in prenatal diagnosis include glucocerebrosidase, which is from cultured amniocytes or chorionic villi cells. However molecular analysis of pathogenic GD mutation is still the preferred method of choice in the prenatal diagnosis of Gaucher disease [61].

*Prenatal Diagnosis: The Main Advances in the Application of Identification of Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.104981*

### *4.2.4 Other aneuploidies*

Trisomy 18 and trisomy 13 are the second and third most common autosomal trisomy, respectively, with the incidence being 1 in 7500 and 1 in 15,000 live births [52, 62]. Fetuses with trisomy 18 and 13 often experience intrauterine fetal demise [63, 64]. In the late first trimester, average levels of PAPP-A and free β-hCG tend to be lower in pregnancies with trisomy 18 compared with unaffected pregnancies [63]. However, it is shown in related research that ultrasound findings in the first and second trimester for trisomy 18 seem to be more effective than biochemical screening, thus the combination of sonography, triple test, and amniocentesis makes sense [65]. As for trisomy 13, a decrease in maternal serum-free β-hCG and PAPP-A and an increase in fetal nuchal translucency always come into existence. However, the use of biochemical markers in maternal serum as a screening tool for trisomy 13 seems to be less promising than for other aneuploidies, such as trisomy 21 and trisomy 18 [64]. By using different markers, the hap screen kit technique mentioned in the β-thalassemia part could also be applied in the diagnosis of disorders of chromosomes 21, 18, 13, X, and Y [59].

### **4.3 Congenital structural malformations**

### *4.3.1 Neural tube defects (NTDs)*

NTDs are serious congenital malformation disorders. The neural tube is the central nervous system of the fetus. On the 15th to 17th day of the embryo, the nervous system begins to develop, and by about the 22nd day of the embryo, the two sides of the neural fold begin to close to each other, forming a canal called the neural tube. The embryo closes the anterior foramen and posterior phase on the 24th, 25th, and 26th day. The central neural tube is the part of the embryo that develops into the brain, spinal cord, back of the head, and spine. If the central nervous canal does not develop properly, the above-mentioned parts may be defective when the baby is born. The main manifestations of fetal neural tube malformations are anencephaly, cerebral bulge, cerebrospinal meningeal bulge, and spina bifida [66].

The first biomarker for prenatal testing is related to neural tube defect screening AFP [67]. Maternal serum AFP levels are closely related to the developmental status of the neural tube. Serum AFP screening is generally performed between 15 and 21 weeks of pregnancy. Blood samples can be collected in the form of liquid, whole blood, or dried blood. Studies have shown that anencephalic children have AFP levels that are 6.4 the normal gestation-specific median. In cases associated with spina bifida, AFP levels were 3.8 the normal gestation-specific median. As technology continues to develop and advance, the accuracy of screening for AFP as a biomarker for NTDs has increased and the detection rate of false positives has further decreased.

Recent studies have explored new biomarkers to detect NTDs. AFP-associated maternal serum α-fetoprotein variants L2 and L3 (AFP-L2 and AFP-L3) are more accurate predictors of fetal open neural tube defects (ONTD) with high sensitivity and specificity [68]. In addition, amniotic fluid glial fibrillary acidic protein (AF-GFAP) was shown to be a valid diagnostic biomarker for NTDs by proteomic studies [69]. NTDs were positive in the open stage and negative in the closed stage when the threshold was above 0.2 ng/mL. This confirms that amniotic glial fibrillary acidic protein is a biomarker for the diagnosis of open NTDs and has a negative predictive role in the detection of closed NTDs.

In noninvasive prenatal screening, in addition to conventional methods for AFP level changes, a breakthrough was made in biomarkers of NTDs using isobaric tags for relative and absolute quantitation (iTRAQ ) quantitative proteomics technology [70]. The expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) differed in rat fetuses at different developmental stages [71], with a significant decrease in NTD pregnancy serum and a progressive increase in normal pregnancy and embryonic development serum. Although the possibility of using biomarkers for prenatal testing in humans has not been confirmed, it has a promising prospect.

### *4.3.2 Congenital heart disease (CHD)*

CHD is the most pervasive type of congenital malformation, making up for approximately 28% of congenital malformations. Refers to anatomical abnormalities resulting from abnormal formation or development of the heart and great blood vessels during embryonic development. The heart and great vessels are abnormal at birth, including right-to-right shunt, right-to-left shunt, and no shunt. Tetralogy of Fallot is the most common type of left-to-right shunt CHD [72].

In terms of invasive prenatal testing, the search for suitable biomarkers for prenatal testing is broadly based on two routes—cord blood and amniotic fluid. In the amniotic fluid of fetuses with CHD [73], uric acid and proline were found to be significantly elevated by metabolomic analysis. Among them, uric acid has good specificity and sensitivity and has a promising potential to become a biomarker. Cord blood can be used as a prenatal biological marker for a variety of diseases, including CHD [74]. Analysis of miRNAs reveals significantly elevated expression of miRNA-1, miRNA-208, and miRNA-499, which have the prospect to be biomarkers for CHD.

Noninvasive prenatal testing for CHD is more common. The techniques of proteomics have been used more often in the diagnosis of CHD [75]. In maternal serum [76], proteomic analysis is used to find peptides specifically expressed in fetuses with tetralogy of Fallot. In addition to peptides, it has been shown that maternal serum concentrations of tumor necrosis factor-alpha, vascular endothelial growth factor-d, and heparin-binding epidermal growth factor-like growth factor are associated with CHD with a high degree of specificity [77].

### *4.3.3 Cleft lip and palate (CLP)*

CLP is the most pervasive congenital malformation in the oral and maxillofacial region, mainly due to certain pathogenic factors that cause fetal facial development disorders between the fourth and tenth week of pregnancy [78]. Genetics and maternal conditions are the main causes of CLP. Prenatal diagnosis of fetal CLP is mainly carried out by fetal ultrasound images [78]. However, this technique has many limitations; maternal weight and fetal position can interfere with the diagnostic results. Prenatal diagnosis of CLP is prone to misdiagnosis and underdiagnosis [78].

The discovery of prenatal biomarkers for CLP has made it possible to improve the accuracy of prenatal diagnosis [78, 79]. Three pregnancy-associated PIWIinteracting RNAs (piRNAs) biomarkers (hsa-pri-009228, hsa-pri-016659, and hsa-pri-020496) are reported able to distinguish CLP fetuses from normal fetuses. In CLP fetuses, the expression of piRNAs biomarkers was down-regulated with high accuracy, which is of high clinical value. CLP was first discovered as a related prenatal biomarker, which has high clinical value as a non-invasive detection method.

*Prenatal Diagnosis: The Main Advances in the Application of Identification of Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.104981*

### *4.3.4 Congenital glaucoma*

Congenital glaucoma is a congenital abnormality of the atrial angle structure due to developmental disorders during embryonic life, which blocks the channels for atrial fluid drainage, resulting in increased intraocular pressure and increasing the size of the entire eye. Glaucoma is a disease that causes damage to the optic nerve. When the intraocular pressure increases, it can lead to damage to the optic nerve fibers and cause visual field defects.

The discovery of biomarkers associated with congenital glaucoma offers the possibility of prenatal diagnosis. Human fetuses with cytochrome p4501B1 mutations are more likely to have congenital glaucoma [80]. Detection of cytochrome p4501B1 expression reveals that fetuses with congenital glaucoma have delayed ocular tissue development and decreased cytochrome p4501B1 protein expression, thus increasing oxidative stress biomarkers.

### *4.3.5 Achondroplasia*

Chondrodysplasia is an autosomal dominant disorder with a point mutation in the short arm of chromosome 4, a congenital developmental abnormality due to a defect in endochondral ossification, mainly affecting long bones. A large proportion of cases of chondrodysplasia are stillborn or die in the neonatal period.

The diagnosis of chondrodysplasia is largely dependent on breakthroughs in noninvasive prenatal diagnostic methods. Analysis of cellular free DNA using PCR and restriction endonuclease digestion (PCR-red) is the dominant method for noninvasive prenatal detection of monogenic diseases including chondrodysplasia [81]. A novel NGS assay was found to be more sensitive and specific for chondrodysplasia. In addition, cffDNA may be a useful biomarker for NIPD. Related studies have confirmed the significance of the detection of *FGFR3* gene mutations in cffDNA for the diagnosis of early maternal chondrodysplasia. Based on this biomarker, a novel DNA sensor for detecting disease-causing mutant genes was designed, which is very sensitive for genetic detection in poor cartilage [82].

### **5. Conclusion**

Biomarkers based on multi-omics have a wide variety of applications in prenatal diagnosis, and samples are collected either through invasive or noninvasive ways. Maternal serum biomarkers are ideal diagnostic indexes because of their convenience and security. However, in the present stage, invasive techniques, such as amniocentesis and CVS, are often required to confirm the preliminary result, although they carry a risk of miscarriage and need people with specialty to operate them. Researches on noninvasive techniques are now on the rise. Despite the high cost, noninvasive techniques, such as cfDNA, are quite risk-free and accurate, which is promising for the future.

### **Author details**

Tong Wang1,2,3, Jiahe Xu1,2,4, Lin Wang1,2,4, Xiumin Cui1,2,4, Yan Yan5 , Qiuqin Tang6 \* and Wei Wu1,2\*

1 State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing, China

2 Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China

3 The Stomatological College of Nanjing Medical University, Nanjing, China

4 The First Clinical Medical College, Nanjing Medical University, Nanjing, China

5 Nanjing Hanwei Public Health Research Institute, Nanjing, China

6 Department of Obstetrics, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, China

\*Address all correspondence to: t19871004@sina.com and wwu@njmu.edu.cn

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Prenatal Diagnosis: The Main Advances in the Application of Identification of Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.104981*

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## Prenatal Screening: A Tool to Predict, Prevent, and Prepare

*Brinda Sabu and Vidyalekshmy Ranganayaki*

### **Abstract**

There has been a considerable reduction in maternal mortality from 6 to 9/1000 live births and infant mortality from 100/1000 live births in the 1900s to less than 0.1/ 1000 live births and 7/1000 live births, respectively, in the 2000s. This is mostly due to nutritional improvement and obstetric and fetal medicine advancements. However, in the current era, prevention of mortality is not the only goal but also the prevention of morbidity. Thus comes the importance of prenatal screening, which would help us to predict and prevent maternal-fetal complications and in non-preventable conditions to prepare ourselves for optimal care of the mother and fetus. Prenatal screening is thus a test to detect potential health disorders in pregnant mothers or the fetus and to identify a subset who may need additional testing to determine the presence or absence of disease. It is done to categorize mothers into high-risk and low-risk pregnancies to prevent maternal complications, screen the fetus for aneuploidies, anomalies, and growth abnormalities, and decide on any indicated interventions and the time and mode of safe delivery so that an optimal perinatal outcome is achieved. Prenatal screening not only caters to identify fetal complications but also attempts to identify maternal complications early.

**Keywords:** prenatal screening, aneuploidy, preeclampsia, preterm labor, small for gestational age, fetal anomalies, adverse pregnancy outcomes, preventive strategies, screening models

### **1. Introduction**

In 1929, the Ministry of Health in the UK set forth guidelines advocating for pregnant women to be first seen in the antenatal clinics at 16 weeks, followed by 24 and 28 weeks visits, fortnightly till 36 weeks, and weekly until delivery [1, 2] (**Figure 1a**). However, in 2011, Prof. Nicolaides inverted this pyramid of prenatal care by introducing a new model where a comprehensive assessment of the mother and the fetus is done at 11–13 weeks. According to this "inverted pyramid model", combining the data from maternal characteristics and history along with biophysical and biochemical tests performed on the mother can define the patient-specific risk for a variety of pregnancy complications, namely aneuploidies, preeclampsia, preterm delivery, gestational diabetes, fetal growth restriction, and macrosomia [5]. (**Figure 1b**). In 2017, Ljubic proposed an "extended inverted pyramid of care" based

### **Figure 1.**

*(a) Pyramid of care in 1929, (b) inverted pyramid of prenatal care (adapted from [3]), (c) three floor model which includes prepregnancy and postpregnancy care (adapted from [4]).*

on the concept that the roots of these disorders are dysfunctional placentation and thus must be sought in the earlier period of pregnancy and in the deeper, subcellular level [6]. This means that the changes that lead to insufficient implantation should be sought in the preimplantation period, in the relation between the embryo and endometrium. Prepregnancy approaches such as optimizing maternal comorbidities, adequate weight management, blood pressure and glycemic control, smoking cessation, and spacing pregnancies may improve the placentation leading to an optimal pregnancy outcome [7]. Year 2016 saw the emergence of another model put forward by Moshe and Nicolaides called the "Three-floor model" where the care is extended to prepregnancy and postnatal periods [4] (**Figure 1c**). This model was proposed based on the concept that the health effects of women and their offspring are mediated by epigenetic and genetic pathways contributing to the increased risk of developing noncommunicable diseases (NCD) that are passed onto the next generations, which is a vicious cycle. Thus, this model of care helps in the assessment of NCDs in the prepregnancy period (first floor) and optimizing the disease state followed by the inverted pyramid of care (second floor) starting at 11–14 weeks till delivery, to the postnatal period (third floor) where appropriate management can minimize the long

term harmful effects in both mother and her offspring. Hence, this model of prenatal screening, which starts from the prepregnancy period and continues through pregnancy into the postnatal period, would be an ideal screening model, and this would reduce the harmful effects of the epigenetic/genetic factors on the fetus and the mother, thereby reducing the long term development of NCDs.

In this chapter we shall discuss the different screening methods which can be applied to these three floors of prenatal care:


### **2. Prepregnancy period**

Prepregnancy care aims to identify the women with NCDs and treat them and optimize their disease state. This is done by the family physician or primary obstetrician by gathering patient information either through personal interviews or from electronic records regarding medical, pregnancy, and family history, drug intake, and smoking. A physical examination is done to calculate the BMI and BP and investigations like HbA1C and total cholesterol are conducted. Seven cardiovascular health (CVH) metrics proposed by the American heart association (AHA) are assessed which include four health behaviors (weight, physical activity, smoking, and diet) and three health risk factors (blood pressure, fasting blood glucose, and total cholesterol). Based on these metrics patients are stratified into different risk categories, namely ideal [8–12], intermediate [4, 7, 13–15], and poor (0–4) categories [13]. Two points are awarded for ideal, one point for intermediate, and zero points for poor, ranging from 0 to 14 [13].

Women who score ideal risk are reassured and advised to plan their families. Those women who score intermediate risk should be referred to either dieticians or physical trainers to optimize their pregnancy issues at hand. The poor score women are referred to a maternal-fetal medicine (MFM) specialist to optimize comorbidities like anemia/hypertension/diabetes, evaluation of the end organs in chronic morbidities, conversion to pregnancy-safe medications, screen for infections, and immunization of varicella/rubella and hepatitis B. Carriers of inherited genetic disorders should be offered counseling and workup by geneticist including index child workup, genetic evaluation for carrier status, and preimplantation genetic diagnosis (PIGD). Periconceptional folic acid should be advised as and where indicated.

### **3. Inverted pyramid of care**

In the 11–14 weeks period, a comprehensive evaluation of the mother is done based on the demographics, medical/obstetric, and family history along with biophysical markers like mean arterial pressure (MAP), biochemical markers like human chorionic gonadotrophin (HCG), pregnancy-associated placental plasma protein A (PAPP-A) and placental growth factor (PLGF), and ultrasound (USG) parameters like nuchal translucency (NT) and uterine artery Doppler pulsatility index (UTPI) thus quantifying the

woman's risk for developing any chromosomal aberrations, preeclampsia, spontaneous preterm birth, fetal growth restriction, and gestational diabetes. Thus, they are stratified as low-risk and high-risk pregnancies. Low-risk mothers enter the routine care regimen and high-risk mothers enter the specialist care regimen.

The inverted pyramid of care thus includes:


### **3.1 Aneuploidy screening**

Aneuploidy screen has come a long way since its inception in the 1970s. Early detection of Down syndrome is the main objective of prenatal aneuploidy screening since this syndrome is the most common autosomal trisomy among live births which is compatible with life. Trisomy 21 affects 1 per 500 pregnancies with a live birth prevalence of 1 per 740 while trisomy 18 occurs in 1 per 2000 pregnancies and 1 per 6600 live borns, and trisomy 13 is identified in 1 per 5000 pregnancies and 1 per 12,000 live borns [14, 15]. As detection of aneuploidies is also observed in younger age groups, screening is universal in the current era and all pregnant women should be offered screening for aneuploidies. There are two different types of aneuploidy screening:


Conventional screening is the established method of screening using NT performed by USG along with biochemical screening in the first trimester and only biochemical screening in the second trimester. It is further divided into three types:


Cell-free DNA screening which was implemented in 2011, identifies circulating DNA fragments that are primarily placental in origin, from apoptotic trophoblasts [8, 9] and is considered to be the best available screening test with a good positive predictive value, and a very low false positive rate especially when applied appropriately.

*Prenatal Screening: A Tool to Predict, Prevent, and Prepare DOI: http://dx.doi.org/10.5772/intechopen.105598*


Components of FTS are as follows:


### *3.1.1.1.1 Pretest counseling*

Every pregnant woman is counseled regarding the options of undergoing a screening test, diagnostic test, or no test at all, their detection rates and it is completely her choice to proceed with any testing. She is counseled that the purpose of the screening test is to provide information and if the test results come positive, it does not mean that the fetus is affected (false positive) and there is the option of diagnostic testing to confirm the same. Decisions cannot solely be taken based on screening tests. Similarly, it does not mean the fetus is unaffected if the test results come negative (false negative). The benefits of diagnosis, early intervention if affected and the costs of the screening and diagnostic tests are also explained. Additional evaluation and counseling are suggested if a patient has had a previous fetus or neonate with autosomal trisomy, Robertsonian translocation, or other chromosomal abnormality.

### *3.1.1.1.2 USG evaluation*

There has been a paradigm shift in utilizing USG for the detection of aneuploidy markers to early identification of structural and genetic abnormalities. The salient applications of USG in the first trimester are:


### *3.1.1.1.2.1 Calculation of GA*

Dating is of paramount importance before aneuploidy testing because each screening test is valid only within a specific gestational age window, 11–14 weeks for first trimester screening and 15–21 weeks for second trimester screening. Moreover, when risk assessment is done each component of a screening test should be adjusted for gestational age when calculating multiples of the median, and false positive rates are reduced when gestational age is assessed by USG [10]. Crown-rump length (CRL) is the length of the embryo or fetus from the top of its head to the bottom of the torso (**Figure 2a**). Popularly called the Robinson's CRL curve, it is the most accurate estimation of gestational age in early pregnancy, owing to the little biological variability at that time. Thus, CRL measurement has become the universal pregnancy dating tool to avoid the last menstrual date recall error [11, 12]. If the GA is >14 weeks then head circumference (HC) is used for dating the pregnancy (**Figure 2b**).

### *3.1.1.1.2.2 Diagnose multiple pregnancies and determination of chorionicity*

Overall, twin pregnancies are at higher risk than singleton pregnancies for aneuploidy. This is mostly due to advanced maternal age in twin pregnancies. Determination of chorionicity of a twin pregnancy is of paramount importance, and the first trimester assessment has a better accuracy rate of 96–100% versus approximately 80% in the second trimester [16–19]. Chorionicity, rather than zygosity, has a major impact on the outcome of twin pregnancies mainly because of specific complications secondary to placental anastomoses, such as twin-to-twin transfusion syndrome (TTTS), selective fetal growth restriction (sFGR), twin anemia polycythemia sequence (TAPS), twin reversed arterial perfusion (TRAP) [20, 21]. The presence of the lambda sign (due to the interposed chorionic tissue) (**Figure 3a**) is suggestive of DCDA twins and the presence of the T sign is suggestive of MCDA twins (**Figure 3b**).

### **Figure 2.**

*Dating parameters. (a) Midsagittal plane of a fetus showing measurement of -rump length (CRL)-dating parameter <14 weeks GA, (b) measurement of head circumference (HC)-dating parameter >14 weeks GA.*

**Figure 3.** *Assessment of chorionicity. (a) Lambda sign-DCDA twins, (b) T sign-MCDA twins.*

*Prenatal Screening: A Tool to Predict, Prevent, and Prepare DOI: http://dx.doi.org/10.5772/intechopen.105598*

### *3.1.1.1.2.3 Identification of aneuploidy markers*

Nuchal translucency (NT) is a subcutaneous collection of fluid between the skin and soft tissue overlying the fetal spine at the back of the neck in the sagittal plane. It is the most important marker used in the first trimester for aneuploidy risk calculation. This was demonstrated by Nicolaides in the early 1990s and was found to be strongly associated with fetal aneuploidy [22, 23]. Increased NT is associated with trisomy 21/13/18, Turner syndrome and other chromosomal defects, fetal structural malformations, and genetic syndromes. Though NT tends to resolve, it can evolve into increased nuchal fold thickness or cystic hygromas with or without hydrops. **Figure 4a** and **b** is representative of normal and increased nuchal translucency, respectively.

NT must be accurately imaged and measured in a reproducible way following the standards put forth by the Fetal Medicine Foundation and Perinatal Quality Foundation for aneuploidy detection to be accurate. The optimal gestational age for measurement of fetal NT is 11–13 + 6 weeks when the fetal CRL is between 45 and 84 mm. Measurement is done in the sagittal plane with the neck in a neutral position, and the image is magnified so that the screen is filled with the fetal head, neck, and upper thorax. The calipers are placed on the inner borders of the widest aspect of the nuchal space, perpendicular to the long axis of the fetus, with the horizontal crossbar within the space [24]. Though there is no clarity in the definition of increased NT beyond the cutoff of 3.5 mm, NT is said to be increased when it is >99th centile for the CRL or > 1.9 MOM for the measured CRL [25]. The causes of increased NT [23, 26] are cardiac defects and dysfunction, venous congestion in the head and neck, the altered composition of the extracellular matrix, failure of lymphatic drainage, fetal anemia, fetal hypoproteinemia, and fetal infection [26].

### *3.1.1.1.2.4 Other aneuploidy markers*

### *3.1.1.1.2.4.1 Heart rate*

In normal pregnancy, the fetal heart rate (FHR) increases from about 100 bpm at 5 weeks of gestation to 170 bpm at 10 weeks and then decreases to 155 bpm by 14 weeks. Between 11 and 14 weeks, trisomy 13 and Turner syndrome are associated with tachycardia, whereas bradycardia is noted in trisomy 18 and triploidy. Inclusion

### **Figure 4.**

*Measurement of nuchal translucency (NT). (a) Sagittal section of fetus showing normal nuchal translucency— 1.8 mm, (b) sagittal section of fetus showing increased nuchal translucency—4.1 mm.*

of FHR is important in differentiating trisomy 18 and 13, which in other respects show common features like increased fetal NT and decreased maternal serum free B hCG and PAPP-A [26].

### *3.1.1.1.2.4.2 Nasal bone*

Nasal bone (NB) assessment is done between 11 and 13 + 6 weeks when the CRL is between 45 and 84 mm. In the midsagittal view of the fetal profile, NB is imaged as three distinct lines. The top line represents the skin and the bottom one, which is thicker and more echogenic than the overlying skin, represents the NB. A third line, almost in continuity with the skin, but at a higher level, represents the tip of the nose. At 11–13 + 6 weeks the NB is absent in 1–3% of euploid fetuses, 60% of fetuses with trisomy 21, 50% of fetuses with trisomy18, and 40% of fetuses with trisomy 13. Assessment of the NB improves the performance of combined screening, increasing the detection rate from 90% to 93% and decreasing the false positive rate from 5% to 3% [26]. **Figure 5a** and **b** shows the ossified and unossified nasal bone, respectively.

### *3.1.1.1.2.4.3 Tricuspid regurgitation (TR)*

TR assessment is done between 11 and 13 + 6 weeks when the CRL measures 45– 84 mm. No regurgitation should be noted across the fetal tricuspid valve during

**Figure 5.** *Assessment of NB. (a) NB—seen as the bottom line, (b) unossified nasal bone—absence of bottom line.*

### **Figure 6.**

*Assessment of tricuspid flow. (a) Axial section of the fetal thorax with spectral wave Doppler imaging showing normal flow pattern across the tricuspid valve, (b) axial section of the fetal thorax with spectral wave Doppler flow showing tricuspid regurgitation.*

### *Prenatal Screening: A Tool to Predict, Prevent, and Prepare DOI: http://dx.doi.org/10.5772/intechopen.105598*

systole. Regurgitation is significant if it occurs in more than half of systole with a velocity of more than 60 cm/s and is noted in about 1% of euploid fetuses, 55% of fetuses with trisomy 21, and 30% in fetuses with trisomy 18 and trisomy 13. **Figure 6a** and **b** shows normal tricuspid flow pattern and tricuspid regurgitation, respectively. Assessment of the tricuspid flow improves the detection rate from 90% to 95% and decreases the false positive rate from 3% to 2.5%. If there is tricuspid regurgitation, it is pertinent that a detailed cardiac evaluation is done to diagnose or exclude major cardiac defects [26].

### *3.1.1.1.2.4.4 Ductus venosus flow*

The ductus venosus is a short trumpet-shaped vessel that shunts oxygenated blood preferentially to the fetal heart. It has a triphasic pattern and the forward 'a wave corresponds to the atrial systole. Reversed a wave in ductus venosus is associated with an increased risk for chromosomal abnormalities, cardiac defects, and fetal death. At 11–13 + 6 weeks, abnormal ductal flow is observed in 5% of chromosomally normal fetuses and about 80% of fetuses with trisomy 21. Assessment of ductus venosus improves the performance of combined screening, increasing the detection rate from 90% to 95% and decreasing the false positive rate from 3% to 2.5%. Though in 80% of cases with isolated reversed a wave the pregnancy outcome is normal, a detailed cardiac evaluation should be done to rule out cardiac defects in fetuses with an a-wave reversal in ductus venosus (**Figure 7a** and **b**) [26]'.

### *3.1.1.1.2.5 Identification of structural anomalies*

There has been a paradigm shift from the identification of fetal aneuploidies in the 1980s to the early identification of structural/genetic abnormalities due to innovation in USG imaging technology. The commonly identified anomalies are given below: Acrania, alobar holoprosencephaly, body stalk anomaly, omphalocele, megacystis, and early hydrops. **Figure 8a–f** depicts these commonly diagnosed first trimester anomalies.

### *3.1.1.1.3 Biochemical screening/risk assessment.*

Every pregnant woman has a background or apriori risk to bear a fetus with aneuploidy. This is based on her age and history of aneuploidy. The risk for trisomies increases with maternal age, but Turner syndrome and triploidy do not change with maternal age. The patient-specific risk is calculated by multiplying the apriori risk

### **Figure 7.**

*Assessment of ductus venosus (DV) flow. (a) Axial section of the fetal abdomen with spectral wave Doppler flow showing normal ductus venosus flow with normal 'a' wave, (b) axial section of the fetal abdomen with spectral wave Doppler flow showing abnormal ductus venosus flow with reversed 'a' wave.*

**Figure 8.** *Commonly identified anomalies in the first trimester. (a) Acrania, (b) alobar holoprosencephaly, (c) body stalk anomaly, (d) omphalocele, (e) megacystis, (f) early hydrops.*

with a composite likelihood ratio, which is obtained from the screening tests performed during the pregnancy in the first and second trimesters. Each time a test is carried out the apriori risk is multiplied by the likelihood ratio of the test to calculate a new risk, which subsequently becomes the apriori risk for the next test.

Aneuploid pregnancies are associated with altered maternal serum concentrations of various fetoplacental products, namely HCG and PAPPA. First trimester combined screening integrates nuchal translucency with maternal serum HCG and PAPPA. HCG assay can be either intact HCG or free beta unit of HCG depending on the local laboratory guidelines and clinical practice, albeit the two are considered comparable [23]. Individual analytes are converted to multiples of the median (MOM) adjusting for maternal age, maternal weight, smoking status, ethnicity, and gestational age. The pattern of increase or decrease in analyte levels affects the risk for trisomies 21, 18, and 13 and at a predetermined value the test is deemed positive or abnormal. For


**Table 1.**

*Indications of 11–14 weeks assessment.*

trisomy 21, this is often presented as a first trimester risk of 1:250 and for trisomy 18 and trisomy 13, a cut-off of 1:150 is used (**Table 1**).

In euploid pregnancies, the average maternal serum HCG is 1.0 MOM and PAPP-A is 1.0 MOM. In trisomy 21 pregnancies, maternal serum HCG is increased by twofold and PAPP-A is reduced to half compared to normal pregnancies. In trisomies 18 and 13, maternal serum HCG and PAPP-A are decreased. In cases of sex chromosomal anomalies, maternal serum HCG is normal and PAPP-A is low. In paternally derived triploidy, maternal serum free HCG is greatly increased, whereas PAPP-A is mildly decreased. Maternally derived triploidy is associated with markedly decreased maternal serum HCG and PAPP-A [26, 27]. **Table 2** depicts the analyte levels in various fetal trisomies in the first trimester and screening by a combination of fetal NT and maternal serum PAPP-A and HCG can identify about 90% of all these chromosomal abnormalities for a false positive rate of 5% [26, 27]. **Table 3** depicts the detection rates and false positive rates of various combinations of screening tools and markers used in the first trimester [26].


### **Table 2.**

*Multiple marker levels associated with trisomies in the first trimester.*


### **Table 3.**

*Detection and false positive rates of different combinations of screening tools and markers used in the first trimester. Modified from [26].*

As the biochemical analytes (HCG and PAPPA) are increased twofold in twin pregnancies, the performance of combined screening is 15% lower when compared to singleton pregnancies [28].

### *3.1.1.1.4 Posttest counseling*

Posttest counseling is mandatory for any screening program. When a positive or negative screening test result is obtained, the patient should be counseled regarding the adjusted likelihood of carrying a fetus with the aneuploidies evaluated and a diagnostic test is offered. The possibility for the fetus to be affected by genetic disorders which are not evaluated by the screening or diagnostic test should also be reviewed. In the event of a prenatal diagnosis of fetal aneuploidy, the patient and family should be counseled appropriately so that she can make informed decisions regarding further pregnancy management.

### *3.1.1.2 Second trimester screening*

The triple screen which includes the analytes alpha feto protein (AFP), HCG, and serum estriol has a poor detection rate of 60% and was used in the 1980s. The quadruple test which was formulated by the addition of dimeric inhibin A to the triple screen in the 1990s replaced it and is the only current second trimester multiple marker screening tests widely used. A quadruple screen is performed between 15 and 21 weeks when the Biparietal diameter (BPD) is between 34 and 52 mm, the measurement varying between labs. The pattern of change in analytes is depicted in **Table 4** [29].

Since the early 2000s, the quadruple test detection rate is 81–83% at a 5% false positive rate in two large prospective trials- the Serum, Urine, and Ultrasound Screening Study (SURUSS) [24] and the First and Second Trimester Evaluation of Risk (FASTER) [10] trials.

As the second trimester quadruple marker screening offers no advantage over first trimester screening, it is used only if first trimester screening is unavailable in certain settings or if the antenatal woman books too late to receive first trimester screening.

### *3.1.1.3 Combination of first and second trimester screening*

This is based on the concept that aneuploidy detection will be significantly increased if first trimester biochemistry and nuchal translucency screening is combined with a second trimester quadruple marker test. However, these two tests should not be done as independent tests as it increases the false positive rate thus making counseling difficult. There are two different methods of screening—integrated test


**Table 4.**

*Multiple marker levels associated with trisomies in the second trimester.*

and sequential screening, which are further subdivided into stepwise and contingent sequential screening.

Integrated screening involves testing the first trimester serum analytes (HCG and PAPPA) and NT at 11–14 weeks followed by quadruple screening between 15 and 21 weeks of GA and a single risk is calculated. This integrated approach has a 94% sensitivity in detecting T21 and 93% detection of T18 [30] and the result was abnormal in 93% of cases with trisomy 13, in 91% with triploidy, and 80% with monosomy. Serum integrated screening is done when NT is unavailable and only the biochemistry is taken into consideration. This has a detection rate of 85–88% for T21 at a 5% false positive rate [24].

In sequential screening, first trimester screening with nuchal translucency and serum analytes is performed and the patient is informed about the results based on the understanding that, if the risk exceeds a predetermined cut-off (≥1 in 30), she will be offered diagnostic testing. There are two types of sequential screening. With stepwise sequential screening, women at high risk in the first trimester are offered diagnostic testing and the rest go on to complete the quadruple marker screening in the second trimester, after which the women who are screen positive are offered diagnostic testing and the rest are reassured and no further testing is indicated. Based on data from the FASTER trial, stepwise sequential screening using a first trimester risk cutoff of 1:30 and an overall cut-off of 1:270 yielded a 92% trisomy 21 detection rate at a 5% false positive rate [24]. In contingent sequential screening, after women at high risk are offered a diagnostic test, the remaining women are divided into two groups. The lowest risk women (<1:1500) are reassured and receive no further screening, whereas those at intermediate risk (1 in 270 to 1 in 1500) are followed up with quadruple marker screening. Based on data from the FASTER trial, the trisomy 21 detection rate was 91% with the contingent screening at a 5% false positive rate [24].

### *3.1.2 Cell-free–based DNA screening*

Noninvasive prenatal screening (NIPS) or cell-free–based DNA screening was introduced into the armamentarium of aneuploidy screening in 2011. This test identifies circulating DNA fragments that are primarily placental in origin, from apoptotic trophoblasts. Assaying of cell-free DNA is done in three ways for aneuploidy screening: whole-genome sequencing (massive parallel shotgun sequencing-MPSS); chromosome selective sequencing (targeted); and single nucleotide polymorphism analysis (SNP). It can be performed after 9–10 weeks of gestation, and the turnaround time of the results is within 7–10 days. The detection rate is 99% for trisomy 21, 96% for trisomy 18, and approximately 90% for trisomy 13 and monosomy X [8, 9]. According to a meta-analysis of 37 studies of cell-free DNA screening in high-risk pregnancies, the pooled sensitivity to detect trisomy 21 was 99.2% (95% confidence interval 98.5–99.6%), and the specificity was 99.9%, and the false positive rate was only 0.1% [29, 31]. Detection rates of trisomies 18 and 13 are 96% and 91%, respectively, each with a specificity of 99.9%. For detection of monosomy X (Turner syndrome), the sensitivity of cell-free DNA was approximately 90% with a specificity of 99.8% [29, 31–35]. **Table 5** depicts the different characteristics, the detection rates, the false positive rates, and the positive predictive values of the different screening tests to detect T21 [29].

The high PPV of cell-free DNA screening is dependent on maternal age at delivery which means in younger women, a positive screening test result is more likely to be falsely positive regardless of the aneuploidy. For a woman in her early 20s, the PPV


### **Table 5.**

*Characteristics and performance of different screening tests [29].*

may be close to 50% for fetal trisomy 21, but this percentage is considerably higher in older women, which is clinically relevant while counseling before cell-free DNA screening considering the expensive nature of the test [29].

As the placenta and the fetus do not share the same chromosomal content, false positives can occur especially when there is confined placental mosaicism (CPM) and a vanishing co-twin with an identifiable fetal pole. Hence cell-free DNA screening is not recommended in such conditions [36, 37]. Moreover, as this screening examines the maternal DNA, rare cases of maternal mosaicism and malignancy have also been identified [38, 39] by the presence of more than one aneuploidies in the test. Another disadvantage of this screening method is the 'No Call' result which is seen in 4–5% of screened pregnancies. This is due to a reduced fetal fraction of less than 4% which is seen in lower gestational age, obese women, small placentation, and aneuploidies [40–42]. If a no-call result is reported the patient should be counseled by a geneticist in detail regarding the possible cause and is offered a repeat test or invasive amniocentesis keeping in mind the high chance of no-call in the repeat test which is as high as 40%.


### **Table 6.**

*Comparison of traditional and cell-free DNA screening [29].*

*Prenatal Screening: A Tool to Predict, Prevent, and Prepare DOI: http://dx.doi.org/10.5772/intechopen.105598*

### **Figure 9.**

*Commonly noted second trimester USG soft markers. (a) Profile view of the fetal face showing unossified nasal bone (UNB), (b) transcerebellar plane showing increased nuchal fold thickness (NFT), (c) fetal transventricular plane showing ventriculomegaly, (d) axial section of fetal thorax showing aberrant right subclavian artery (ARSA), (e) axial section of the fetal abdomen showing bilateral renal pelviectasis, (f) axial section of fetal thorax showing echogenic cardiac focus in the left ventricle, (g) sagittal view of abdomen showing fetal bowel as echogenic as surrounding bone, (h) short femur length corresponding to 17 weeks in a fetus at 19 weeks of gestation.*

Because of its high detection and low false positive rate, cell-free DNA screening may be offered as either a primary screen or secondary screening test to women who test positive on a traditional screening test before proceeding with a diagnostic test. If an abnormal traditional screening result is followed by a normal cell-free DNA screen, the risk for a chromosomal abnormality is approximately 2% [43]. However, the time required for cell-free DNA screening (7–10 days) may delay aneuploidy diagnosis to the point that pregnancy termination may no longer be an option for those who choose it. Because of the above-mentioned limitations and the reduced costeffectiveness in low-risk pregnancies, traditional screening tests are still considered the choice of first-line screening for low-risk pregnancies [8]. However, cell-free DNA screening is recommended as a screening option in advanced maternal age (maternal age > 35 years at delivery), high/intermediate-risk in traditional screening, presence of an ultrasonographic soft marker, prior pregnancy with h/o trisomy, or known carrier of a balanced Robertsonian translocation involving chromosomes 21, 13, and 14 [9]. Currently, cell-free DNA screening detects specific chromosomal abnormalities, namely trisomy 21, 18, and 13; 45, X; and 47 XXX, XXY, and XYY [44]. It should be noted that prenatal diagnosis is recommended whenever an aneuploidy screening test is abnormal and pregnancy termination should not be based on the results of any screening test. A comparison of traditional and cell-free DNA screening is given in **Table 6**. Cell-free DNA is not offered if the first trimester scan reveals any structural abnormalities.

### *3.1.2.1 Role of USG in the second trimester (to rule out anomalies and evaluate for soft markers)*

We have already elaborately learned about the role of USG in the first trimester. Targeted imaging of fetus for anomalies (TIFFA) is a level 2 USG done at 18–24 weeks depending on the local protocols. There is a role for targeted scan after a positive aneuploidy testing as the presence of an abnormality or multiple soft markers increases the risk of aneuploidy by 50–60% [45]. It is also noted that 25–30% of fetuses with Down syndrome and almost all fetuses with T18/13 will have major abnormalities [46, 47]. Soft markers are normal USG variants with no/trivial clinical sequelae, are transient and resolve with advancing gestation or after birth, and are noted in 10% of euploid pregnancies. The most commonly noted second trimester soft


### **Table 7.**

*Various second trimester soft markers and their likelihood ratios.*

### *Prenatal Screening: A Tool to Predict, Prevent, and Prepare DOI: http://dx.doi.org/10.5772/intechopen.105598*

markers are unossified/hypoplastic NB, ventriculomegaly, increased nuchal skinfold thickness, aberrant right subclavian artery, echogenic intracardiac focus, echogenic bowel, pelviectasis, and short femur or humerus length. When a marker has been identified, the posttest odds for trisomy 21 are derived by multiplying the pretest odds (obtained by first/second trimester screening) by the positive LR for each detected marker. The images of various second trimester soft markers are depicted in **Figure 9a–h**. Metaanalysis by Agathakleous et al. in 2013 suggested that when the targeted anomaly scan reveals no abnormalities and soft markers then the aneuploidy risk is reduced by 7.7-fold. **Table 7** shows the various markers and their likelihood ratios [48].

### *3.1.2.2 Diagnostic tests of aneuploidy*

Diagnostic testing allows patients to know with certainty whether the pregnancy is affected by a particular genetic condition. Abnormal screening tests in the first or second trimester must be followed up by diagnostic tests before any final decisions are made. Commonly performed diagnostic tests include chorionic villus sampling (**Figure 10**), and amniocentesis (**Figure 11**). Preimplantation genetic diagnosis is considered in known cases of familial syndromes or previously affected children with

**Figure 10.** *Transabdominal chorionic villus sampling.*

parents being carriers. Rapid aneuploidy testing using either quantitative fluorescent polymerase chain reaction (qfPCR) or fluorescent in-situ hybridization (FISH), will detect the major trisomies (13, 18, and 21) and Turners syndrome (45XO) and the results are issued in 1–3 working days. Full karyotyping is then performed following culturing of the cells. This takes 10–14 days and involves microscopic examination of cells and can detect other chromosomal rearrangements. However, as this approach will not detect very small submicroscopic changes, known as copy number variations (CNVs), chromosomal microarray (CMA) has replaced conventional karyotyping in identifying the CNVs.

The advantage of prenatal diagnosis is that when an anomaly or a genetic disease is diagnosed prenatally, it helps the obstetrician and neonatologist to counsel the family, discuss the available options, and to initiate a neonatal management plan even before delivery of the fetus. In certain cases, treatment may be instituted in utero. Although diagnostic testing is recommended to be available to all women, regardless of maternal age, patients should be counseled regarding types of invasive procedures, including the expected benefits, risks, and technical aspects of the test.

The indications of diagnostic testing are as follows:


4.A desire to have the most reliable information about the fetal karyotype.

5.A desire to have a comprehensive genetic analysis that will detect both autosomal and sex chromosome aneuploidy and pathological copy number variants.

### *3.1.2.3 Chorionic villus sampling (CVS)*

CVS is the only diagnostic test available in the first trimester and allows for diagnostic analyses, including quantitative Fluorescent Polymerase Chain Reaction (qFPCR), karyotype, microarray, molecular testing, and gene sequencing. CVS is performed between 10 and 14 weeks of gestation. Early CVS which was performed before 9 weeks in the past is no longer recommended as it is shown to increase the risk of limb deformities and oromandibular malformations.

Under ultrasonographic guidance, a sample of placental tissue is collected for genetic evaluation through a catheter placed through either the transcervical or transabdominal route without entering the sac (**Figure 10**). CVS allows for earlier prenatal diagnosis and earlier pregnancy termination if desired. A disadvantage of CVS is confined placental mosaicism (CPM) which is noted in 1–2% of CVS results. Pregnancy loss attributed to CVS is approximately 1 in 450 according to recent data [49–51].

### *3.1.2.4 Amniocentesis*

Amniocentesis is a technique by which amniotic fluid is withdrawn from the amniotic sac using a needle under continuous ultrasound guidance via a transabdominal approach to obtain a sample of fetal exfoliated cells, transudates, urine, or secretions. It can be performed from 16 weeks of pregnancy onwards (**Figure 11**). The various tests which can be performed in the amniotic sample are chromosomal, biochemical, molecular, and microbial studies, the most common being prenatal diagnosis of chromosomal abnormalities, single-gene disorders, and fetal infection The procedure has a risk of fetal loss of approximately 0.5% (range, 0.06–1%) [50, 51].

### *3.1.2.5 Preimplantation genetic diagnosis*

Preimplantation genetic diagnosis (PGD) is a test to detect the abnormality before embryo transfer so that only unaffected embryos are transferred to the patient. This helps in the earlier detection of chromosomal and genetic abnormalities. After in vitro fertilization (IVF) a polar body or a single cell from the blastocyst is removed and examined for aneuploidies/genetic disorders (**Figure 12**). However, it is recommended that all pregnancies conceived with IVF/PGD should be offered confirmatory testing with CVS or amniocentesis as false negative reports are possible [52, 53].

### **3.2 Preeclampsia/SGA screening**

Prediction of PE and SGA can be done in the first trimester by a combination of maternal demographic characteristics, uterine artery pulsatility index (Ut art PI), mean arterial pressure (MAP), and maternal serum biochemical markers serum PAPP-A and PlGF [54].

**Figure 12.** *Preimplantation genetic diagnosis.*

SGA is defined as birth weight below the 10th centile for the gestational age though there are cutoffs varying between the 3rd and 10th centile. The prevalence of SGA is estimated to be 8–11%. The SGA babies are prone to develop complications like prematurity, neonatal asphyxia, hypothermia, hypoglycemia, hyperbilirubinemia, hypocalcemia, polycythemia, sepsis, and death [54]; and long-term morbidities like learning difficulties, cognitive, and behavioral defects.

Preeclampsia (PE) is a multisystem disorder of pregnancy [55, 56] and develops in 2–5% of pregnant women and is one of the leading causes of maternal and perinatal morbidity and mortality [57, 58]. The International Society for the Study of Hypertension in Pregnancy (ISSHP) definition is the accepted one by international bodies, [59] which defines gestational hypertension as systolic blood pressure (SBP) at ≥140 mm Hg and/or diastolic blood pressure (DBP) at ≥90 mm Hg on at least two occasions measured 4 h apart developing after 20 weeks of gestation in previously normotensive women. PE is defined as gestational hypertension accompanied by ≥1 of the following conditions at or after 20 weeks of gestation: (a) Proteinuria (≥30 mg/ mol protein: creatinine ratio; ≥300 mg/24 h; or ≥ 2 + dipstick) (b) Maternal organ dysfunction, including acute kidney injury (creatinine ≥90 μmol/L; 1 mg/dL) liver involvement (elevated transaminases, e.g. alanine aminotransferase or aspartate aminotransferase >40 IU/L) with or without right upper quadrant or epigastric abdominal pain, neurological complications (e.g. eclampsia, altered mental status, blindness, stroke, clonus, severe headaches, and persistent visual scotomata) or hematological complications (thrombocytopenia—platelet count <150,000/μL) or c) uteroplacental dysfunction (fetal growth restriction, abnormal umbilical artery Doppler waveform analysis, or stillbirth).

PE can be further subclassified into [59]:


3.Late-onset PE (with delivery ≥34 weeks GA)

4.Term PE (with delivery ≥37 weeks GA)

Most common maternal complications include placental abruption, HELLP syndrome, acute pulmonary edema, respiratory distress syndrome, acute renal failure, intracranial hemorrhage, and death [60, 61]. The early perinatal complications are fetal growth restriction, nonreassuring FHR during labor, oligohydramnios, intrauterine fetal death (IUFD) preterm birth, low Apgar scores, need for NICU admission, and long-term complications are cerebral palsy, hearing loss, visual impairment, insulin resistance, diabetes mellitus, coronary artery disease, and hypertension.

Thus, the occurrence of PE and SGA contributes significantly to adverse pregnancy outcomes. Hence screening at 11–14 weeks GA, is of paramount importance as one can identify the patients prone to develop these disorders, prevent them to a considerable extent by starting on prophylactic Aspirin and be prepared to tackle the maternal and perinatal morbidity associated with it.

Maternal risk factors for PE and SGA prediction are nulliparity, age ≥ 40 years, BMI ≥35 kg/ m<sup>2</sup> , family history of PE, interpregnancy interval > 10 years, hypertensive disease in a previous pregnancy, chronic hypertension, chronic renal disease, diabetes mellitus, or autoimmune disease [62]. Based on the history and presence of risk factors, the detection rate is only 39% for preterm PE and 34% for term PE at a 10.3% false positive rate. Thus, though history-based screening is useful in identifying at-risk women in clinical practice, it is not a sufficient tool for the effective prediction of PE.

Combined risk assessment for both early PE and preterm SGA is based on maternal characteristics, assessment of biophysical markers like MAP, uterine artery pulsatility index (UTPI), and biochemical markers, namely placental growth factor (PLGF) and PAPPA.

### *3.2.1 Measurement of mean arterial pressure (MAP)*

MAP should be measured by validated automated and semiautomated devices. Women should be seated, with their arms well supported at the level of their heart and an appropriate-sized cuff should be used according to the mid-arm circumference (small, medium, or large). After resting for 5 min, blood pressure is recorded in both arms simultaneously and two sets of similar recordings are made at 1-minute intervals (**Figure 13**). The four sets of SBP and DBP measurements are included in the risk calculator and the final average MAP measurement is used for the calculation of patientspecific risk. The formula for the calculation of MAP is DBP + (SBP DBP)/3 [63].

### **Figure 13.**

*Measurement of mean arterial pressure. Courtesy: Perkin Elmer life and analytical sciences (Wileyonline library.com).*

### *3.2.2 Measurement of Uterine artery pulsatility Index (UTPI)*

UTPI is measured along with an NT scan when the fetal CRL is between 45 and 84 mm and the GA between 11 and 13 + 6 weeks according to the criteria put forward by Fetal Medicine Foundation. For this measurement, a sagittal section of the uterus is obtained identifying the cervical canal and internal cervical os by transabdominal USG. Keeping the transducer in the midline and gentle tilting to both sides will identify each uterine artery in color flow mapping alongside the cervix at the level of the internal os. Pulsed-wave Doppler is then applied with the sampling gate at 2 mm to cover the whole vessel and the angle of insonation should be less than 30° (**Figure 14**). When three to five consecutive waveforms are obtained, the UTPI is measured and the mean UTPI of the left and right arteries is calculated [64, 65]. The first trimester abnormal UTPI is defined as greater than the 90th percentile, achieving a detection rate of 48%, at an 8% false positive rate, for the identification of early-onset PE. However, the detection rate for predicting late-onset PE reduces to 26% at a 7% false positive rate [64].

### *3.2.3 PLGF and PAPPA*

PLGF and PAPPA are glycoproteins secreted by trophoblastic cells and changes in their levels have been implicated in the development of PE [66, 67]. Women who are prone to develop PE have significantly lower maternal PLGF and PAPPA concentrations in the first trimester than those with normal pregnancies. These biomarkers alone have a detection rate of 55% and 33%, respectively, at a 10% false positive rate, for the identification of both early and late-onset PE [68–71]. However, the detection rate with the combined approach is 90% for early PE, 75% for preterm PE, and 45% for term PE with a false positive rate of 10%. The detection rate of preterm SGA is 55% and term SGA is 44% with a false positive rate of 10%.

The ASPRE trial concluded that administration of low-dose aspirin, resulted in a 62% reduction in the incidence of preterm PE, when compared to placebo but did not have a significant reduction in the incidence of term PE [72]. However, it is pertinent to note that this combined first trimester screening of PE is less effective at predicting and preventing preeclampsia developing >37 weeks of gestation and hence the need for second trimester screening methods for PE.

### **Figure 14.**

*Identification of the uterine artery at the level of the internal os and the demonstration of typical waveforms of the uterine artery Doppler in the first trimester of pregnancy.*

### *3.2.4 Second trimester PE prediction*

This is based on the concept that uteroplacental dysfunction occurs due to an imbalance in angiogenic and antiangiogenic factors. Circulating levels of the antiangiogenic protein, soluble fms-like tyrosine kinase-1 (sFlt-1) is increased, proangiogenic factor, PlGF is decreased and the sFlt-1/PlGF ratio is elevated before the onset of PE. Therefore, measurement of angiogenic markers, either alone or combined as part of the sFlt-1/PlGF ratio, has a significant value in preeclampsia prediction [73–75].

The prospective PROGNOSIS study [76], aimed to investigate the value of using the sFlt-1/PlGF ratio to predict the absence of PE within 1 week and to predict the presence of PE within 4 weeks in women with clinical suspicion of PE. sFlt-1/PlGF ratio cutoff of ≤38 was shown to have an NPV of 99.3% for ruling out development of PE within 1 week and a ratio > 38 demonstrated a PPV of 36.7% for ruling in preeclampsia within 4 weeks in a cohort of 700 women. The PPV for the occurrence of a combined endpoint of preeclampsia/eclampsia/HELLP syndrome, maternal and/or fetal adverse outcomes within 4 weeks was 65.5% [76]. Similar results were obtained in a separate study involving Asian women [77]. Thus, sFlt-1 and PlGF can be valuable biomarkers for the short-term prediction and detection of evolving preeclampsia in women with clinical signs and symptoms of the disorder, demonstrating a high NPV for ruling out preeclampsia, although the PPV remains relatively low. However, more research is needed to elucidate the benefits of the second trimester PE screening considering perinatal and maternal risk reduction and resource optimization.

Thus, the guideline to prevent PE is following the first trimester screening and assessment for preterm PE, women identified at high risk should receive aspirin prophylaxis commencing at 11–16 weeks of gestation at a dose of 150 mg to be taken every night until either 36 weeks of gestation, when delivery occurs, or when PE is diagnosed [78].

### **3.3 Screening for preterm labor**

Approximately 11% of infants worldwide are born preterm, and the majority of cases occur in low-income countries [79]. Preterm birth (PTB) continues to be one of the leading causes of perinatal morbidity and mortality worldwide [80, 81]. Twothirds of PTB cases are attributed to spontaneous PTB (SPTB) and the remaining onethird are medically indicated, due to maternal or fetal complications [82]. SPTB is defined as birth between 20 and 37 weeks of gestation following the spontaneous onset of labor, preterm prelabor rupture of membranes, or premature dilation of the cervix [83].

Preterm babies require prolonged hospitalization and are at high risk of adverse outcomes, including respiratory difficulty, necrotizing enterocolitis, feeding difficulties, blindness, deafness, intraventricular hemorrhage, higher risk of death at the age of 5 years, and neurodevelopmental sequelae when compared to their term counterparts [80, 84]. Thus, they need immense and prolonged health care, and hence for both the family and society PTB constitutes a major public health problem. Considering these issues, screening and early detection of pregnancies at the highest risk for SPTB will guide us in the implementation of management options and secondary prevention of morbidities associated with SPTB.

### *3.3.1 Identification of maternal risk factors*

Demographic risk factors like African race, low socioeconomic status, and maternal characteristics like low BMI have been identified as poor risk factors with a relative risk (RR)—<2 in identifying women who are destined to develop SPTB. Other maternal risk factors are further subclassified into prior risk factors and pregnancy-specific risk factors. The prior risk factors are previous h/o preterm birth, a short interpregnancy interval of <6 months, family h/o preterm labor, congenital uterine malformations, infections of urinary and genital tracts, maternal smoking, and drug abuse. Pregnancyspecific risk factors are mid trimester short cervix <2.5 cm and bleeding per vaginum in the first or second trimester [82, 85]. Though the greatest risk factor for SPTB is a history of the previous SPTB, prediction of SPTB beyond that is very challenging considering the heterogeneous nature of risk factors and etiology.

### *3.3.2 USG screening*

Universal cervical length screening is controversial due to its concern about costeffectiveness and the possibility of unnecessary interventions. The most important risk factor for SPTB is a combination of short cervical length in a woman with previous h/o SPTB, which contributes to a relative risk of 3.3 [86, 87]. Cervical assessment is done by transvaginal ultrasound measurement of cervical length which is a safe, reliable, and highly reproducible tool when performed by trained providers [88]. In a mid trimester (16–24 weeks of gestation) scan, a cervical length of 2.5 cm corresponds to the 10th centile for the period of gestation, and hence if the transvaginal cervical length is <2.5 cm, it is considered to be short [89] (**Figure 15**). According to the guidelines put forth by the Society for Maternal-Fetal Medicine and the American College of Obstetricians and Gynecologists, serial cervical length surveillance is indicated for pregnant women with prior h/o SPTB from 16 to 24 weeks gestation though studies have shown that 82% of women who developed SPTB did not have a short cervical length during screening by transvaginal ultrasound [90].

### *3.3.3 Fetal fibronectin measurement*

Fetal fibronectin (fFN) is an extracellular matrix glycoprotein that is present at the maternal-fetal interface of the amniotic membrane and is found in minimal quantity (<50 ng/ml) in the cervicovaginal secretions between 22 and 35 weeks of GA [91] and

**Figure 15.** *Transvaginal cervical length measurement showing (a) normal cervix (3.8 cm) and (b) short cervix (1.6 cm).*

hence levels >50 ng/mL at >22 weeks gestation is associated with an increased risk of SPTB [92]. However, one should keep in mind that false positive test results can be noted in sexual intercourse, vaginal bleeding, and vaginal lubrication or douching [93].

A qualitative assay involves doing a swab test that detects whether fetal fibronectin is present in the cervicovaginal secretion. A positive fFN test (≥50 ng/mL) has low sensitivity and a positive predictive value [94]. However, a negative fFN test has a high negative predictive value up to 35 weeks gestation and strongly suggests that SPTB will not occur within the following 2 weeks [95]. Due to its limited predictive ability, the American College of Obstetricians and Gynecologists (ACOG) discourages the use of this test as a screening strategy in asymptomatic women, as there is a lack of evidence for better perinatal outcomes.

Quantitative assays are tests that will measure the amount of fetal fibronectin in the cervicovaginal secretions and studies have demonstrated that increasing concentration of qfFN is directly proportional to the rate of SPTB. A threshold of 10 ng/ml has high sensitivity (96%) and negative predictive value (98%) to detect those women unlikely to deliver preterm. The higher the qfFN concentration, the greater the need for surveillance and intervention. It is predicted that quantitative fetal fibronectin measurements enhance the accuracy in the identification of women at risk of preterm delivery [87]. However, studies have shown that combined fFN assay and cervical length screening had low sensitivity to predict SPTB before 35 weeks gestation which was ratified in a systematic review by Berghella et al. [91].

### *3.3.4 Role of Insulin-like growth factor binding protein (IGFBP-1)*

Insulin-like growth factor binding protein-1 (IGFBP-1) is one of the major secretory proteins of the decidualized endometrium and is present in large amounts in the amniotic fluid. Decidua contains more phosphorylated IGFBP-1 (phIGFBP-1) and amniotic fluid contains more nonphosphorylated IGFBP-1. So, when there is a detachment of the fetal membrane, phIGFBP-1 may leak into cervical secretions and trigger the cascade of SPTB. A strong phIGFBP-1-positive result which is an immunochromatography-based dipstick test predicted delivery before 35 completed weeks with a sensitivity of 72.7%, a specificity of 83%, a PPV of 47%, and a negative predictive value of 93.6% [96]. The advantage of this test over fFN is that IGFBP-1 is less prone to influence by sexual intercourse [97].

### *3.3.5 Role of placental alpha microglobulin 1 (PAMG-1)*

PAMG-1 is another glycoprotein synthesized by the decidua and is present in the amniotic fluid in high concentrations. There is a transudation of PAMG-1 through chorioamniotic pores in fetal membranes during uterine contractions due to the inflammatory process of labor or infection. An immunoassay bedside 'dipstick test' is done by a vaginal swab between 20 and 37 weeks to obtain the result within 5 min. This test has a high specificity of 97.5% and NPV of 97.5% and the advantage is that the test results will not be affected by vaginal examination, and thus can be used shortly after the vaginal examination [98].

### *3.3.6 Role of biomarkers*

Certain pro-inflammatory cytokines, such as interleukins, tumor necrosis factoralpha (TNF-α), C-reactive protein (CRP), granulocyte colony-stimulating factor (G- CSF), soluble intercellular adhesion molecule-1 (sICAM-1), alkaline phosphatase, stromal cell-derived factor-1a (SDF-1a), interferon-c, and matrix metalloproteinase-8 (MMP-8) are hypothesized to respond to infection at the maternal-fetal interface and stimulate the release of prostaglandins thereby causing uterine contractility and subsequent cervical change triggering SPTB. Based on this concept, an assay of these biomarkers should predict spontaneous preterm birth in women with singleton pregnancies with no symptoms of preterm labor [99]. However, multiple studies and a subsequent meta-analysis by Agudelo et al. have proven that none of the novel biomarkers are clinically useful for predicting SPTB [95, 98] and more research is needed to clarify their efficacy as predictors.

### *3.3.7 Preventive strategies for SPTB*


*Prenatal Screening: A Tool to Predict, Prevent, and Prepare DOI: http://dx.doi.org/10.5772/intechopen.105598*

The different types of cervical stitch are McDonald cerclage which involves placing a transvaginal purse-string suture at the cervical isthmus junction, without bladder mobilization [116]. High transvaginal or Shirodkar cerclage involves placing a transvaginal purse-string suture above the level of the cardinal ligaments following bladder mobilization, [117] and transabdominal cerclage which involves placing the suture at the cervicoisthmic junction by laparotomy or laparoscopy [118]. Transabdominal cerclage can be performed in women with previous unsuccessful transvaginal cerclage and is done in the preconception period or early pregnancy.

### **3.4 Screening for diabetes**

Screening and prediction of diabetes in pregnancy are advisable as it causes increased morbidity, namely fetal macrosomia, trauma during birth, induction of labor, increased chance of cesarean section, shoulder dystocia, neonatal hypoglycemia, and perinatal death. Early diagnosis will ensure the patient follows medical nutritional therapy along with exercise and if glycemic control is not achieved, early recourse to oral hypoglycemic agents or Insulin can be undertaken thereby preventing the abovementioned morbidity.

The risk factors for the development of diabetes in pregnancy are:


Screening is done by the 75-g 2-h oral glucose tolerance test (OGTT) to test for gestational diabetes in women with risk factors. Women who had gestational diabetes in a previous pregnancy, can either be offered early self-monitoring of blood glucose or a 75-g 2-h OGTT as soon as possible after booking (whether in the first or second trimester) and a further 75-g 2-h OGTT at 24–28 weeks if the results of the first OGTT are normal. In women with no risk factors, OGTT is offered at 24–28 weeks. Gestational diabetes mellitus (GDM) is diagnosed if the fasting plasma glucose level is 5.6 mmol/L or above or a 2-h plasma glucose level is 7.8 mmol/L or above and the woman is advised a dietician consultation and medical nutrition therapy (MNT-Diet/exercise) is initiated. If her glycemic control is inadequate within 2 weeks, she should be referred to a diabetologist for the start of oral hypoglycemic agents (OHA)/Insulin. In a woman with preexisting DM, multidisciplinary team care should be offered for optimal glycemic control and adequate end-organ assessment from preconception to delivery [119].

### **3.5 USG to screen for anomalies**

USG has been established as an essential modality in the prenatal assessment of the fetus and thus obtain an optimal outcome for the mother and fetus. As a majority of fetal abnormalities occur in the low-risk group, targeted imaging of fetal anomalies is offered to all pregnant women. The mid trimester USG is done between 18 and

24 weeks of gestation according to the local protocol regarding the legal limit of termination of pregnancy. The sensitivity in detecting anomalies improves when done in close to 24 weeks. The request for the scan should originate from the primary obstetrician and the pregnant woman should be counseled regarding the potential benefits and limitations of a second trimester fetal ultrasound scan and a consent form should be signed before the evaluation. Mid trimester USG should be performed by trained professionals, and it includes a detailed and systematic evaluation of the external and internal anatomy of the fetus. The established accuracy in diagnosing fetal anomalies according to the EUROFETUS study is 55–60% [120].

For the mid trimester scans, a USG machine with the following capabilities should be used: real-time, grayscale transabdominal/transvaginal transducers, necessary software applications, color Doppler, power Doppler, adjustable acoustic power output controls with output display standards, freeze-frame capabilities, electronic calipers, and capacity to print/store images. High-end machines with elaborate software settings and the use of 3D/4D probes will hasten the diagnosis and reporting process in certain circumstances.

Though the safety of USG has been established in many studies [121–123], the evaluation time should be minimized, using the lowest possible power output needed to obtain diagnostic information, following the as low as reasonably achievable (ALARA) principle [124]. Apart from evaluation of cardiac activity, fetal number, fetal environment, placental appearance and location, evaluation of biometry to assess fetal growth is recommended in the mid trimester USG. Biometry includes biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), and femur length (FL). Details of evaluation and images of mandatory biometry are given in **Table 8**. The minimum evaluation and checklist which is recommended in the mid trimester USG are described in **Table 9** [125].


### **Table 8.**

*Planes of evaluation and images of mandatory biometry in mid trimester scan.*


### **Table 9.**

*Minimum requirements recommended in 18–24 weeks USG (modified from ISUOG practice guideline for midtrimester USG, 2011).*

Evaluation of the cervix, uterine pathology like fibroids and adnexa also should be done to look for any pathology. A proper referral mechanism should be in place once a diagnosis of an anomaly is made and a detailed report including the name**,** date of USG, any relevant medical or obstetric conditions, the scan indication, the best estimate of gestational age, estimated delivery date, amniotic fluid assessment, BPD, HC, AC, and FL (in centiles), EFW in grams with centile graphs, Dopplers, diagnostic impression, and recommendations for follow up examination or management.

Thus, the inverted care pyramid model helps in the identification of low, intermediate, and high-risk antenatal mothers. The low-risk mothers continue their antenatal care in the general obstetrician's clinic, the high-risk group is treated by a multidisciplinary team including perinatologists, genetic counselors, dieticians, endocrinologists, and USG experts. The intermediate group in their further visits is stratified as either high or low-risk groups and managed accordingly.

### **4. Postnatal period**

Care during this period is based on the concept that the prevalence of various NCD, namely metabolic syndromes and premature cardiovascular diseases is increased in women with uteroplacental dysfunction. These women are referred to specialist care, namely diabetologists, nephrologists, endocrinologists, genetic counselors, cardiologists, and nutritionists thereby preventing future occurrence of NCDs. This can be achieved using lifestyle changes, exercise, and medication. Furthermore, the women who have preexisting medical morbidities like connective tissue disorders, renal disease, and neurological disease are referred to appropriate specialist physicians for a reevaluation of their medical condition and alteration of medication if indicated.

### **5. Conclusion**

Incorporation of the above-mentioned protocols in the prenatal screening process helps in the standardization of antenatal care from the preconception period, into the pregnancy till delivery, and through the postnatal period. This structured care will help in the substantial reduction of adverse outcomes in pregnancy thus achieving an optimal perinatal outcome. Thus, prenatal screening helps us to predict the at-risk mother and fetus, and prevent the problem from occurring by means of prophylactic measures and timely interventions. Nevertheless, in unpreventable conditions, a multidisciplinary team-based approach is considered and relevant care is given to both mother and fetus to make the process of delivery and the postnatal period a less stressful and more pleasant one.

### **Acknowledgement**

This chapter is dedicated to "Roshan Jethro Rollands, my son, my guardian angel."

### **Author details**

Brinda Sabu\* and Vidyalekshmy Ranganayaki Department of High-Risk Pregnancy and Perinatology, KIMSHEALTH, Trivandrum, Kerala, India

\*Address all correspondence to: brinda\_sabu@yahoo.co.in

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Prenatal Screening: A Tool to Predict, Prevent, and Prepare DOI: http://dx.doi.org/10.5772/intechopen.105598*

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### **Chapter 11**

## Common Indications and Techniques in Prenatal MRI

*Ryan Holman*

### **Abstract**

Fetal and perinatal diagnostic imaging with MRI has evolved and expanded during recent times, allowing more widespread use and availability. Common indications are for neurodevelopmental conditions that are inconclusive with ultrasonography. The modality is pivotal in treatment planning for *in utero* interventions, such as repair of neural tube defects, and for particular obstetrical complications. The technique is also useful for identifying neurological sequelae from conditions like congenital heart defects and maternal viral infections. Many other applications are not indicated for routine use, particularly due to the high cost, but show much promise in research applications. Recently, complications associated with COVID-19 have been an area of interest, with prenatal MRI cohorts and case studies reporting obstetrical complications and neurodevelopmental effects. This review is aimed at highlighting common indications for the use of MRI in maternal-fetal medicine, including the MRI sequences and physics often implemented. Also, an in-depth analysis of the SARS-CoV-2 virus is discussed; in addition to pregnancy-related complications and the role of prenatal MRI in diagnosis and treatment.

**Keywords:** prenatal MRI, fetal MRI, birth defects, obstetrics, radiology, COVID-19

### **1. Introduction**

Infection, preterm birth, and perinatal complications including asphyxia are among the leading causes of neonatal deaths worldwide [1, 2]. Neonatal and antenatal mortality and morbidity is most often associated with preterm birth that can result in respiratory complications, developmental abnormalities, and high-risk of infections [3, 4]. Infection has been reported in approximately 23% of worldwide neonatal deaths with an estimated 84% of instances being preventable with proper medical treatment [1, 5]. Preterm birth results in the majority of neonatal morbidity and mortality, is the direct cause of approximately 35% of neonatal deaths worldwide, and is the major risk factor for all types of neonatal deaths [1, 3, 4, 6]. Hypoxic birth asphyxia is expected to cause approximately 30% of worldwide neonatal mortality, identified by the inability to perform voluntary breathing at birth, can be observed intrapartum with techniques including Doppler ultrasound

and auscultation, and can be diagnosed by an arterial pH in the umbilical cord less than 7.2 [7].

Some of the most common birth defects include congenital heart disease (CHD), down syndrome, and neural tube defects. Congenital cardiac complications are the most common form of congenital abnormalities, with an estimated worldwide prevalence in about 0.8% of all live births, resulting in approximately 1/3 of all congenital abnormalities that cause significant medical and social consequences [8, 9]. Down syndrome is expected in about 1 in 400–1500 births, is the most common chromosomal abnormality, can be diagnosed early in pregnancy with chorionic villus sampling or amniocenteses, and predominately results from trisomy of chromosome 21 [10]. Global neural tube defect prevalence is estimated at 0.05–1% of live births, are characterized by improper closure of the neural tube during fetal development, are commonly asymptomatic, with spina bifida being the most common type, of which the most severe is myelomeningocele [11–13].

The understanding of normal *in utero* fetal brain development is still largely unknown, with techniques like magnetic resonance imaging (MRI) being uncommon in absence of disease [14]. Fetal MRI has allowed better understanding of the physiological processes involved with normal neurodevelopmental maturation, *in utero* and *ex utero* comparison, the underpinnings of congenital disease mechanisms, and longterm outcomes for specific conditions [14–17]. In the clinic, fetal MRI is often undertaken after referral from a maternal-fetal medicine specialist and indicated to help in diagnosis of particular conditions, management of known conditions, and to provide additional information for pregnancies considered for termination [9]. MRI is indicated after inconclusive results with ultrasonography, for a variety of structural abnormalities related to fetal development, particularly for imaging and identification of anomalies of the central nervous system, prior to fetal surgery, and for particularly difficult deliveries [18–21]. Fetal MRI is generally used in addition to ultrasound, primarily due to the relative cost, and can be complicated by fetal motion, wraparound artifacts limiting the field-of-view, and from multi-slice magnetization transfer from off-resonance artifacts between adjacent slices [22]. Fetal MRI is often performed at a 20-week ultrasound scan [9]. Recently, the MERIDIAN study found that ultrasound provided accurate diagnosis of fetal brain abnormalities at 70% and 64% above and between 18 and 24 weeks, respectively; while fetal MRI in combination with ultrasound increased the accuracy to 92% and 94%, respectively [23]. Clinical radiologists report common referrals to include neurological diagnosis, treatment planning for *in utero* surgery, imaging of congenital masses, and imaging of congenital cardiac defects [24].

### **2. Fetal MRI sequences and safety for imaging neurodevelopmental and cardiac anomalies**

MRI sequence for fetal brain analysis include functional imaging, structural imaging, and diffusion imaging [25]. The predominant sequences used in fetal MRI are single-shot T2W (SST2W) sequences, such as rapid acquisition with relaxation enhancement (RARE) sequences on Bruker, Single-Shot half-Fourier Turbo Spin Echo (SShTSE) on Philips, Single-shot Fast Spin Echo (SSFSE) on General Electric, and half-Fourier acquisition single-shot turbo spin echo (HASTE) sequences on Siemens, with protocols provided by the MRI vendor [22, 26]. These T2W sequences are quick enough to be acquired without sedation and are common for neuroanatomical fetal

### *Common Indications and Techniques in Prenatal MRI DOI: http://dx.doi.org/10.5772/intechopen.105361*

imaging; [9] with other common sequences being T1W to view hemorrhaging, perfusion MRI, diffusion MRI, and spectroscopy [9, 22]. Default SST2W sequences are generally capable of good image generation with 1x1x4 mm voxel size; using half-Fourier acquisitions, with refocusing pulses with flip angles between 120°-150° [22]. Though difficult to implement, diffusion-weighted imaging (DWI) allows identification of ischemic brain lesions, while T1W images can provide improvement over T2W for detection of calcifications, fat, and hemorrhaging [26].

Fetal cardiac sequences are often balanced steady state free precession (bSSFP) and HASTE to encompass small voxel size and reduce acquisition times needed to avoid motion artifacts, with bSSFP being particularly beneficial for imaging blood vessels and cavities containing fluid [26, 27]. Fetal cardiac MRI can be used to view structure, function, vasculature; in addition to performing quantitative MRI measurements including blood flow velocity and oxygen saturation [27]. Blood oxygen level-dependent (BOLD) functional MRI sequences have shown useful for illustrating the improvement of fetal oxygenation during maternal respiratory oxygen therapy for fetuses with impaired cerebral oxygenation resulting from certain types of CHD [28]. Abnormal placenta pathology has been linked with high rates of CHD and is a possible compounding factor for higher severity brain lesions [29]. Neurological implications are not distinct from CHD. Impaired cardiac development is linked with mild brain injury, delayed maturation, shorter gestational age, and smaller brain volumes [30, 31]. Fetal cardiac MRI complications include the smaller size of the fetal heart, lack of gating technologies, and higher heart rate [27].

The primary safety concerns in fetal MRI involve radiofrequency exposure in terms of specific absorption rate (SAR), high acoustic noise, and possibility of peripheral nerve stimulation [22]. MRI is generally considered safe during pregnancy with no evidence of harming the fetus, but is typically not recommended when the fetus is less than about 13 weeks gestational age, and gives best information after completion of organogenesis [22]. The United States Food and Drug Administration (FDA) fetal MRI SAR limit is set at 4 W.kg<sup>1</sup> [22, 32]. Fetal MRI scans are usually recommended to be performed at 1.5 T, and as a "golden rule", remain below 25 seconds [20, 22]. 3 T fetal MRI is often used only within research settings because the SAR is four times higher than at 1.5 T; with the upper limit generally at 4 T for research applications [9]. Although, some institutions perform routine 3 T fetal imaging during the late second trimester and throughout the third trimester [33]. Contrast enhancement is not recommended in fetal MRI, thought to enter into the fetal vasculature, passing through the renal system, before emptying into the amniotic fluid [9, 34].

### **3. Common neurodevelopmental indications for Fetal MRI**

Prenatal MRI is most routine for neural abnormalities because of the improved capability for fetal brain scans. In addition to treatment planning of delivery complications, a variety of conditions have high diagnostic rates with fetal MRI, including diagnosis for mild to moderate ventriculomegaly, a variety of neural tube defects, posterior fossa malformations, and twin-to-twin transfusion syndrome [9, 35]. A USA retrospective study for fetal neurology consultations (n = 94) with diagnostic MRI over 14 months reported the most common conditions were posterior fossa malformations, agenesis or dysgenesis of the corpus callosum, congenital acqueductal stenosis, ventriculomegaly, isolated malformations of cortical development, and holoprosencephaly at 19%, 15%, 14%, 11%, 8.5%, and 6%, respectively [36].

Malformations of cortical development are a collection of developmental malformations resulting from disruption during one of the stages of cerebral cortex formation, often causing cognitive impairment, cerebral palsy, and epilepsy. The cortical development occurs in three major stages, including neuronal stem cell proliferation, neuronal migration along radial glial fibers or axons to the developing cerebral cortex, and neuronal organization [37]. Malformations due to abnormal neuronal stem cell proliferation include microcephaly, megalencephaly, and cortical dysplasia. Malformations during neuronal migration and failure for proper cessation of neuronal migration, include: periventricular heterotopia, subcortical band heterotopia, classic lissencephaly, and cobblestone lissencephaly. While, neuronal organization abnormalities include polymicrogyria and schizencephaly [37, 38]. Historically, autopsy or surgical tissue samples were used for diagnosis of these conditions, being difficult to diagnose with ultrasound. MRI has greatly improved the ability to diagnose these conditions during development, rather than in childhood [39]. Retrospective assessment of cortical development malformations has shown high diagnostic accuracy of fetal MRI when compared to postnatal MRI [40].

Ventrigulomegaly is characterized by dilation of the cerebral lateral ventricles during fetal development. Congenital hydrocephalus is a type of ventrigulomegaly that results specifically from increased cerebrospinal fluid pressure, which causes birth defects resulting in abnormally large head size and many other anomalies, and most frequently results from aqueductal stenosis from outlet obstruction in the third ventricle [41, 42]. An illustration of hydrocephalus is shown in **Figure 1**. Characteristic findings seen postnatally are not often observed prenatally, such as aqueduct funneling or obstruction. Fetal MRI diagnostic indicators, for disease severity from aqueductal stenosis, include the extent of enlargement in the lateral and third ventricle, increased size of the third ventricle of inferior recesses, and observance of diverticulum outpouching in the lateral ventricles [43]. A cohort at the national maternity hospital in the Republic of Ireland reported suspected ventriculomegaly as the most common indication for fetal MRI at the facility, with severe ventriculomegaly (exluding termination) showing a 72% survival rate (n = 74) and a 65% rate for cesarean delivery (n = 72) [44].

**Figure 1.** *Illustration of hydrocephalus with MRI. Rumruay/shutterstock.com*

### *Common Indications and Techniques in Prenatal MRI DOI: http://dx.doi.org/10.5772/intechopen.105361*

Failure of neural tube closure during development results in a variety of neural tube defects, causing spinal anomalies in cases of spinal dysraphism like spina bifida; or cranial anomalies like with anencephaly, characterized by absence of a major portion of the cranium. Though, anencephaly is less indicated for MRI [45]. Distinguishing characteristics of common types of spina bifida are shown in **Figure 2**. Worldwide incidence varies geographically, but estimated on average about 0.1–1% of live births, with anticonvulsants correlating with increased risk, and folic acid associated with reduced risk of neural tube defects [45]. Spinal dysraphism occurs from improper closure of the spinal cord and surrounding membranes during fetal development, and can be classified by open or closed. Closed spina bifida accounts for about 15% of instances, with spina bifida occulta as the most common form, and is usually asymptomatic [33]. Open spina bifida accounts for about 85% of open spinal dysraphisms with myelomenengocele (MMC) and myelocele being predominant, and nearly always presents with Chiari type II malformation [33]. The randomized MOMS trial compared spina bifida outcomes from fetal surgery compared to surgery after delivery, with fetal MRI playing a pivotal role in treatment planning. Outcomes showed fetal surgery for MMC allowed less need for cerebrospinal fluid shunt placement, improved cognitive function in early childhood, though higher risk of preterm birth was observed in the fetal surgery group [33, 46, 47].

Posterior fossa anomalies are characterized by neurodevelopmental malformations in the posterior fossa of the skull cranial cavity. Posterior fossa anomalies are some of the most frequent indications for fetal MRI, occurring in approximately 1 in 5000 live births, encompass a broad spectrum of conditions, and can be categorized as developmental disruptions and malformations [48, 49]. Posterior fossa anomalies include: mega cisterna magna, Blake's pouch cyst, Dandy-Walker malformation, arachnoid cyst, Joubert syndrome, rhombencephalosynapsis, and Chiari malformation [50]. The malformations can present with either an enlarged cyst appearing with abnormally

**Figure 2.** *Comparison of spina bifida subtypes. Rumruay/shutterstock.com*

high retrocerebellar fluid, such as in Dandy-Walker malformation, mega cisterna magna, and Blake's pouch cyst. Or the malformations cause an unusually small posterior fossa such as in Dandy-Walker variant [51, 52]. The most common reported malformation is generally Dandy-Walker malformation, presenting with macrocephaly in 90–100% of children within months of delivery [49]. Comparison of fetal MRI and fetal ultrasound images in the diagnosis of Dandy-Walker malformation is shown in **Figure 3**. Prognosis of these conditions is highly influenced by concomitant anomalies, with co-occurring conditions like agenesis and cerebral hypoplasia often resulting in cognitive impairment. Other conditions like mega cisterna magna without hydrocephalus typically result in normal development [50]. In a USA retrospective cohort for ultrasonography referrals for fetal MRI involving posterior fossa anomalies (n = 180), the most common indications for fetal MRI were Dandy-Walker continuum (Dandy-Walker malformation in addition to Dandy-Walker variant) at 42%, mega cisterna magna at 22%, with a change in diagnosis in 70% of cases, and 60% agreement between fetal MRI and postnatal MRI [54].

### **Figure 3.**

*Dandy-Walker malformation in a 26 week fetus, first suspected as Dandy-Walker variant with ultrasonography, and confirmed as Dandy-Walker malformation with T2W HASTE MRI. A) Ultrasonography illustrating mild ventriculomegaly B) ultrasonography image illustrating cisterna magna that is abnormally large. C) MRI image illustrating direct connection between the cisterna magna and 4th ventricle. D) Sagittal MRI of abnormally large posterior fossa. Reprint Sohn et al., 2008 under CC BY-NC 3.0 [53].*

### *Common Indications and Techniques in Prenatal MRI DOI: http://dx.doi.org/10.5772/intechopen.105361*

The corpus callosum is a white matter commissural nerve tract, connecting cortical regions of left and right hemispheres, and composed of myelinated axons that allow action potential propagation [55]. The corpus callosum forms between gestational weeks 11–22, is composed of five distinct regions, and hyperplasia or hypoplasia of these regions is termed callosal dysgenesis, while total absence is deemed callosal agenesis [56]. Agenesis of the corpus callosum rarely occurs in complete isolation, and generally occurs in combination with other disorders. MRI can provide more detail for the extent of the condition than ultrasonography alone [55]. This allows confirmation that the corpus callosum is intact and visualization of co-occurring and associated malformations [9]. Diffusion tensor imaging and fiber tractography in developing research applications has greatly improved the understanding of the neuronal tracts of the corpus callosum, and complications associated with different degrees of agenesis [55]. Tractography has allowed characterization of normal developmental patterns for the nerve bundles of the corpus callosum with increasing gestational age, showing an increase in volume and fractional anisotropy, with a decrease in apparent diffusion coefficient [57].

In twin-to-twin transfusion syndrome, unequal blood supply to the fetuses leads to demise of one twin. Untreated cases have dismal survival rates [58]. The condition indicates diagnostic fetal MRI due to improved capabilities over ultrasonography for identifying ischemic lesions and neurodevelopmental abnormalities. The condition often warrants intervention including serial amniocentesis or *in utero* fetoscopic laser ablation of the blood supply of the surviving twin [18]. This condition is hypothesized to be the cause of death for two fetuses found in the tomb of King Tutankhamen, whom are believed to be his two stillborn twin daughters [59, 60].

### **4. Common cardiac indications for Fetal MRI**

Ultrasonography is the primary imaging modality for monitoring and diagnosis in both congenital and acquired pediatric heart disease and antenatal complications [61]. Ultrasonography and MRI have been determined safe for fetal imaging, but suggested to be used prudently, with common concerns and power limits due to potential tissue heating and acoustic damage [62]. Fetal cardiac MRI can improve outcomes by allowing earlier preparation of treatment procedures [63]. The American Heart Association (AHA) and British Association of Perinatal Medicine (BAPM) suggest neonatal MRI for newborn patients with high-risk CHD in combination of evidence for intracranial hemorrhaging or parenchymal brain trauma, though not recommended for routine use for CHD [9].

CHD is the most common form of congenital abnormalities, occurring in about 0.6–0.8% of live births, with as much as half of the patients requiring open-heart surgery, and is associated with high rates of neurodevelopmental problems [9]. CHD is associated with high neonatal morbidity, particularly in preterm infants [64]. Some of the most common congenital heart abnormalities include atrial septal defects, ventricular septal defects, Tetralogy of Fallot, patent ductus arteriosus, and pulmonary stenosis [65, 66]. A depiction of several types of congenital heart defects is shown in **Figure 4**. Ventricular septal defects are the most common congenital cardiac anomaly, often requiring surgical repair, though a high percentage will also spontaneously close with age [66–68].

Prenatal cardiac MRI for CHD has generally been limited to a research setting [69]. This has been due to factors including inability to perform electrocardiogram gating,

**Figure 4.**

*Illustration of common congenital heart defects. N.Style/shutterstock.com*

fetal motion, insufficient safety data, and the relatively small size of the features of the fetal heart [70, 71]. Prenatal cardiac MRI allows evaluation of cardiac anatomy, cardiac function, vascular anatomy, flow quantification, and oxygen content [69].

Recent advances has allowed image reconstruction techniques to obtain highresolution 3D MRI of the fetal heart to assess for congenital heart defects. 3D MRI with motion-corrected image registration was shown in a cohort study to significantly increase visualization and diagnosis of major fetal vascular heart defects in lategestational age fetuses, when compared to 2D MRI [72]. Additionally, Doppler ultrasonography has shown capable of performing cardiac gating of the fetal heart to generate high-quality bSSFP cine images [73].

A cohort study reported the use of a non-contrast velocity-selective arterial spin labelling (VSASL) sequence to assess placental perfusion in fetuses with CHD compared to fetuses without CHD [74]. The study found decreased global perfusion and increased variation of regional perfusion were linked to increasing gestational age in CHD fetuses. The results also suggest that early placental perfusion may increase to compensate for the heart defect.

A Chinese retrospective study reported findings in 1379 confirmed cases for fetal cardiac MRI from 2005 to 2019, referred after echocardiography could not show the four cardiac chambers in addition to ventricular outflow [75]. Imaging sequences were SSFP, real-time cine SSFP, non-gated phase contrast sequences, and SSTSE. The findings were normal in 92.5% of cases, 5.1% presented with CHD, and 2.4% were diagnosed with an alternative heart condition. In the CHD cases, 56% received correct diagnosis with MRI, which was similar to other studies, as prenatal detection rates for CHD for patients that eventually underwent congenital heart surgery, have tended to be low and less than 50% [76].

### **5. MRI in Fetal surgery**

Most conditions are best treated when the fetus is delivered at term; however, certain instances warrant the use of *in utero* fetal surgery [77]. Traditionally, this has *Common Indications and Techniques in Prenatal MRI DOI: http://dx.doi.org/10.5772/intechopen.105361*

been limited to cases of high likelihood of mortality for the fetus without intervention, as the technique is high-risk of morbidity and mortality to the mother. More recently, fetal surgery has allowed interventions for improved life quality [78]. MRI has proved beneficial for fetal surgery planning when indicated for conditions, including fetal tracheolaryngeal airway obstruction, congenital diaphragmatic hernia, congenital pulmonary airway malformation, myelomeningocele spina bifida, congenital heart defects, and lower urinary tract obstruction [77, 78]. Additionally, fetal MRI has shown useful to assess effects of fetal myelomeningocele repair, by comparison of before and after MRI images to uncomplicated fetuses of the same gestational age [79]. Also, MRI has shown beneficial in patient selection for fetal intervention prior to EXIT delivery in congenital high airway obstructive syndrome [80].

### **6. Fetal MRI for pregnancy complications**

Again, ultrasonography is recommended as the first imaging modality, but MRI is often indicated in a variety of maternal obstetric and non-obstetric complications during pregnancy, including placental adhesive disorders, placental abruption, prognosis of uterine rupture, restricted circulation in placental bed disorders, placental insufficiency, acute appendicitis during pregnancy, prediction of preterm labor, ovarian cysts, and urolithiasis [18, 81]. Additionally, MRI is indicated in treatment planning for difficult deliveries, such as those that require the EXIT procedure due to fetal airway obstruction [9]. Moreover, the technique has proved useful in risk scoring for massive intraoperative hemorrhage in patients with previous cesarean sections and exhibiting placenta previa and accreta [82]. Fetal MRI was recently used in a randomized control trial to assess fetal neurodevelopmental improvement for supplemental pomegranate juice in pregnancies with intrauterine growth restriction [83].

### **7. Fetal MRI for viral infections**

Prenatal MRI is useful for diagnosis of complications associated with maternal viral infections, including the more recent complications associated with SARS-CoV-2 infection.

### **7.1 Prenatal MRI for complications from viral infections other than SARS-CoV-2**

A variety of fetal complications arising from viral infection can be imaged with MRI, particularly for identifying neurological sequelae, but also for conditions including fetal ascites, hydrops, cardiomegaly, and pericardial effusion [84]. Fetal MRI can be indicated for diagnosis of suspected neurotropic pathogens, such as cytomegalovirus, Zika virus, and toxoplasmosis [85–88]. Cytomegalovirus is a member of the Herpesviridae family, the most common vertically transmitted congenital viral infection, and the most common infection that results in deafness and intellectual disability in children [89, 90]. MRI and ultrasonography can identify fetal brain lesions resulting from cytomegalovirus infection. MRI diagnosis of infection-related complications allows the possibility of treatment planning for investigational therapies, including antiviral therapy such as Valaciclovir or hyperimmunoglobulin therapy, in the neonates and in fetuses [18, 91, 92].

### **7.2 Prenatal MRI for complications involving the SARS-CoV-2 virus**

SARS-CoV-2 is a positive sense, lipid-enveloped, single-stranded, RNA coronavirus that causes both upper and lower respiratory tract infection, which can result in severe pulmonary inflammation and pneumonia, in a condition denoted human coronavirus disease or more recently COVID-19 [93–95].

SARS-CoV-2 relies upon two types of entry pathways to enter cells through the interaction of the virion spike (S) protein with angiotensin-converting enzyme 2 (ACE2), with release of internal RNA within the cell occurring after cleavage of the S-protein subunits [95]. After binding to ACE2, if transmembrane protease serine 2 (TMPRSS2) is present on the cell surface, the cleavage event occurs through TMPRSS2 and furin, initiating membrane fusion and fusion pore formation on the cell membrane, and release of viral RNA into the cellular cytoplasm [95]. Alternatively, if little or no TMPRSS2 is present on the surface, the clathrin-mediated endocytosis occurs and the virus is internalized intracellularly within endolysosomes, followed by a cathepsin-cleavage event within the endosome, resulting in membrane fusion and release of the viral RNA into the cell cytoplasm [95].

The BNT162b2 (Pfizer, BioNTech) and Spikevax (Moderna, NIAID) are both mRNA-based vaccines that encompass an mRNA strand encoding the spike protein for the original Wuhan-Hu-1 strain, in a liposomal mRNA-lipid nanoparticle, which has a notable ability for large-scale production [95, 96]. The vaccine causes cells to encode the vaccine mRNA to produce spike proteins that are then expressed into the cell membrane. This causes an antibody response that identify these spike protein antigens as a foreign body, stimulating a B-cell and T-cell lymphocyte response to produce antibodies that will tag future spike proteins from SARS-CoV-2 viremia [97]. The viral mutations of these spike protein antigens result in reduced efficacy of the vaccines to induce a immunogenic response. Because mRNA vaccines require antibody neutralization of viremia, mutations in the spike proteins can allow variants to exhibit resistance to the vaccines, potentially causing more severe infections, higher transmissibility, and the possibility of re-infection in vaccinated individuals [98, 99].

A prospective U.K. cohort found 0.5% incidence of SARS-CoV-2 infection during pregnancy that required hospital admission (n = 427) [100]. Of the patients that delivered or experienced pregnancy loss at the time of the article (n = 262), 10% required intensive care unit (ICU) admission and death occurred in 1.2%. From the SARS-CoV-2 positive pregnancies with live born births, 59% had cesarean deliveries and 25% of neonates were admitted to the neonatal intensive care unit (NICU). Preterm delivery occurred in 25% of cases, most of which were induced labor due to COVID-19 complications, and 5% of neonates were COVID-19 positive within 12 hours of birth.

Pregnant women are at high risk of developing severe COVID-19 compared to nonpregnant women, in terms of adjusted risk. Comparing COVID-19 positive pregnancies with non-COVID-19 pregnancies, studies have observed a factor of 3 increase in ICU admissions and invasive intubation with mechanical ventilation, a factor of 2.4 increase in odds for extracorporeal membrane oxygenation, and 70% increase in death [101]. Severe COVID-19 complications are linked with increased rates of preterm birth, hypertensive disorders, and cesarean births [101]. Studies have linked COVID-19 with significant increased mortality for mothers post-delivery and in neonates; particularly for symptomatic patients and those with underlying comorbidities [102, 103]. Neonatal outcomes have been reported as generally favorable, with about half of cases being asymptomatic; though, neonates and children less than one year of age are thought to possibly exhibit higher risk of acute respiratory failure than other children [104].

Risk of vertical transmission of SARS-CoV-2 from mother to fetus is considered low, with the primary transmission to the neonate being through horizontal transmission [101, 105]. Although, at least one case study has confirmed vertical transplacental transmission [106]. There is little evidence for transmission of SARS-CoV-2 through breast milk to the neonate, but pasteurization has been shown to inactivate the SARS-CoV-2 virus and might be considered in specific cases for positive SARS-CoV-2 mothers [101, 105, 107]. Transmission between members of the same family cluster is the primary means of infection from SARS-CoV-2 in children [108]. Infection in children and adolescents has tended to result in milder symptoms and good prognosis, in general [109].

The American College of Radiology (ACR) has suggested limiting the use of MRI to only cases that are absolutely necessary, for COVID-19 positive patients and those suspected of infection [110]. The use of fetal MRI for COVID-19 positive mothers does not have a common indication for routine use and has mostly been reported as case studies or small cohorts. Fetal MRI has been used in cohorts to assess possible neurodevelopmental damage in the fetuses of mothers with SARS-CoV-2 infection during early pregnancy, with results showing no abnormal findings [111]. However, a case study of *in utero* transplacental transmission did reveal white matter damage in a neonate, causing placental inflammation in the mother, and ill-effects in the neonate, including bilateral gliosis and white cortical matter damage on MRI from which the infant slowly recovered [106]. A cohort of 34 pregnant patients assessed lung volume with fetal MRI for complications associated with infection in mildly symptomatic SARS-CoV-2 positive mothers. The study found that the fetal lung volume to body weight ratio was noticeably reduced, particularly when the infection occurred during the third trimester; though neonates did not exhibit respiratory distress [112]. Many cases studies are reported for MRI diagnosis of non-obstetric complications of pregnant COVID-19 patients for a variety of common complications, such as stroke [113] and appendicitis [114].

A significant increase in obstetrical complications in COVID-19 has been observed, compared to non-COVID-19 pregnancies. Studies have shown higher rates of fetal deaths, maternal deaths, ICU admissions, preterm births, and cesarean deliveries. These outcomes highlight the benefit of vaccination during pregnancy, to reduce the risk of maternal and fetal complications [101].

### **8. Conclusions**

Prenatal MRI offers useful complementary diagnostic information to ultrasonography, particularly for neurodevelopmental complications. The technique can be used for diagnosis, for guiding treatment decisions, and to counsel parents for scenarios like potential termination. MRI has been determined safe for fetal health, though low field strengths and non-contrast imaging are generally used, as these scenarios are lower risk to the fetus. MRI can improve diagnostic accuracy for neurodevelopmental and cardiac anomalies when used in conjunction with ultrasonography, but factors like additional cost limits the number of indications for prenatal diagnosis. Studies have shown increased rates of pregnancy-related complications in patients infected with SARS-CoV-2 during pregnancy. Although, studies with fetal MRI for assessing fetal developmental complications due to maternal COVID-19 has been limited, but results have been reported in case studies and small cohorts.

### **Acknowledgements**

Thanks to the Center for Biomedical Imaging and the Image-guided Interventions Laboratory for supporting the development of this manuscript. Thanks to Dr. Daniela Dumitriu LaGrange for reviewing the manuscript and suggesting improvements.

### **Author details**

Ryan Holman Image Guided Interventions Laboratory, Department of Radiology, University of Geneva, Switzerland

\*Address all correspondence to: ryan.holman@unige.ch

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Common Indications and Techniques in Prenatal MRI DOI: http://dx.doi.org/10.5772/intechopen.105361*

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*Edited by Wei Wu, Qiuqin Tang, Panagiotis Tsikouras, Werner Rath, Georg-Friedrich Von Tempelhoff and Nikolaos Nikolettos*

Ectopic pregnancy is an implantation occurring elsewhere than in the cavity of the uterus. It is the leading cause of maternal morbidity and mortality during the first trimester. While the incidence of extrauterine pregnancy has increased in recent years, the rapid development of multi-omics has also provided an effective method of prenatal diagnosis. This book focuses on the diagnosis and treatment of ectopic pregnancy, fetal malformation, and the different screening methods for prenatal diagnosis.

Published in London, UK © 2022 IntechOpen © gueritos / iStock

Ectopic Pregnancy and Prenatal Diagnosis

Ectopic Pregnancy and

Prenatal Diagnosis

*Edited by Wei Wu, Qiuqin Tang, Panagiotis Tsikouras, Werner Rath, Georg-Friedrich Von Tempelhoff* 

*and Nikolaos Nikolettos*