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

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.

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).

i. Noninvasive techniques:

Preimplantation genetic diagnosis (PGD)

External examination

Ultrasound detection

Fetal heartbeat

Non-stress test

Maternal blood pressure

Maternal weighing

Fetal cells in maternal blood (FCMB)

Cell-free DNA in maternal blood (cfDNA)

Glucose tolerance testing

Transcervical retrieval of trophoblast cells

Maternal serum screening

ii. Invasive techniques:

Chorionic villus sampling (CVS)

Amniocentesis

Embryoscopy and fetoscopy

Percutaneous umbilical cord blood sampling

Table 1.

Prenatal diagnostic/screening techniques.

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 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.

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, 34, 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 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 pre-eclampsia 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].

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 PIWI-interacting 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.

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.