*4.2.2. Genetic analysis of the human embryo and fetus*

generation of 2D and 3D images using synchrotron radiations from appropriate devices [57, 58]. An image of a human embryo at CS 17, obtained by applying a two-crystal X-ray interfer-

**Figure 6.** The results of X-ray CT. A, B: Clinical CT (Toshiba Alexion) in the Laboratory of Physical Anthropology, Graduate School of Science, Kyoto University, Japan. C, D: Phase contrast CT, Photon Factory of the KEK (High Energy Accelerator Research Organization) in Tsukuba, Japan. A, C: Surface reconstruction and B, D: midsagittal section.

Additional imaging modalities can be applied for dead embryos and fetuses. Classically, solid reconstruction and fine drawing were primarily the approaches used; the first 3D morphological imaging technique was the wax plate technique, using serial histological sections of human embryos, which was developed by Born [60]. Recently, the 3D reconstruction of serial sections has been carried out by using computer graphic methods, which has made the 3D reconstruction much easier and quicker than before. The 2D image stacks generated from serial sections have a high resolution, although the issues of section registration and distortion remain unsolved. A solution to this problem is a novel imaging modality for the generation of high-resolution 3D reconstructed images [61], which uses episcopic fluorescence image capture (EFIC). In EFIC imaging, tissue autofluorescence is used to image the block face prior to cutting any section. Although the samples are sliced and some lost during the procedure, the optical resolution of EFIC is reported to reach approximately 5–6 μm [62].

MRI is a useful imaging modality, not only for living prenatal embryos and fetuses, but also for dead embryos and fetuses in autopsy imaging. Despite the longer time taken to capture images, the higher resolution is definitely an advantage; the time required for a high-resolution imaging ranges from several hours to days. MR devices should be selected depending on the sample size; specially, MR microscopy, clinical MRI, and experimental MRI are suitable for small-sized embryos, larger fetuses, and embryos/fetuses with an intermediate size,

X-ray imaging is also used for dead embryos and fetuses. Since there is no need to consider the impact of radiation exposure on the tissue, longer time may be devoted to capture high resolution images. Conventional (absorption-contrast) X-ray CT (cCT) is used for fetal skeletal imaging (**Figure 6A** and **B**). Phase-contrast X-ray CT (pCT) is another method of X-ray imaging [40]. Since X-rays are electromagnetic waves, phase-contrast X-ray imaging is capable of recording the phase-shift of X-rays while passing through the samples and reconstructing

ometer [59], is displayed in **Figure 6C** and **D**.

34 Congenital Anomalies - From the Embryo to the Neonate

*4.2.1. Autopsy imaging of human embryos and fetuses*

respectively [39] (**Figure 5**) [2, 63, 64].

**4.2. New strategies for diagnosis of congenital anomalies**

Amniotic fluid, chorionic villi, and umbilical cord blood are used for genetic analyses of human embryos and fetuses. Recently, a new approach for prenatal testing was proposed in the name of noninvasive prenatal testing (NIPT) that uses DNA fragments derived from maternal villus cells to determine the genetic information of the fetus. In comparison to maternal serum analysis, NIPT has considerably higher sensitivity and specificity for aneuploidy [65]. However, due to the infrequent derivation of cell-free DNA (cfDNA) from multiple sources such as in placental mosaicism, maternal conditions including cancer, or fetal and/or maternal copy number variation (CNV) [66], NIPT has a risk of predicting false-positive and false-negative results.

The cell samples obtained from amniotic fluid and chorionic villi may be used for both screening and diagnostic tests. Traditional karyotype analysis is the most commonly used method to examine cells, obtained from chorionic villus sampling (CVS) and amniocentesis (AC), for the diagnosis of aneuploidies and large rearrangements. The diagnostic accuracy of traditional karyotype analysis is higher than 99% for aneuploidy and for chromosomal abnormalities larger than 5–10 Mb [67]. On the other hand, fluorescence in-situ hybridization (FISH) analysis can detect specific chromosomes or chromosomal regions by using fluorescently labeled probes. The turnaround for FISH results (usually within 2 days) is faster than that of conventional karyotyping results (7–14 days, including the cell culture period). Due to the existence of false-positive and false-negative reports, FISH [68–70] is considered as a mere screening test, although still commonly used to screen chromosomes 13, 18, 21, X, and Y. Therefore, clinical diagnosis using FISH results should be supplemented by other clinical and laboratory analyses such as abnormal ultrasonography, positive screening test using maternal serum and/or soft markers, confirmatory traditional metaphase chromosome analysis, or chromosomal microarray analysis (CMA).

CMA is capable of detecting small chromosomal aneuploidies that cannot otherwise be identified by conventional karyotyping [71]. Since CMA can be performed without cell or tissue culture, the results can be obtained within 3–7 days. Since CMA can also be carried out with nonviable cells, which are not suitable for conventional karyotyping analysis, this technique [71] is applicable to the cases of fetal death or stillbirth. CMA can identify almost all the abnormalities, except for balanced translocations and triploidy. While the results of conventional karyotyping in the detection of structural abnormalities, seen in prenatal ultrasonography, did not show anything notable, approximately 6% of the fetuses were identified for chromosomal defects by CMA [72, 73]; CMA qualifies as the primary test, in case a structural abnormality is detected by fetal ultrasonography, as also recommended by the American Congress of Obstetricians and Gynecologists (ACOG) [71].

[4] Goldstein I, Weissman A, Brill-Zamir R, Laevsky I, Drugan A. Ethmocephaly caused by de novo translocation 18;21—Prenatal diagnosis. Prenatal Diagnosis. 2003;**23**:788-790

Congenital Anomalies in Human Embryos http://dx.doi.org/10.5772/intechopen.72628 37

[5] Callahan J, Harmon C, John Aleshire J, Hickey B, Jones B. Alobar holoprosencephaly

[6] Matsunaga E, Shiota K. Holoprosencephaly in human embryos: Epidemiologic studies

[7] Dubourg C, Bendavid C, Pasquier L, Henry C, Odent S, David V. Holoprosencephaly.

[8] Yamada S, Uwabe C, Fujii S, Shiota K. Phenotypic variability in human embryonic holoprosencephaly in the Kyoto collection. Birth Defects Research. Part A, Clinical and

[9] Tonni G, Azzoni D, Pizzi C, Bonasoni MP, Cavalli P, Pattacini P, Ventura A.Anencephalyexencephaly sequence and congenital diaphragmatic hernia in a fetus with 46, XX karyotype: Early prenatal diagnosis, necropsy, and maternal folate pathway genetic analysis.

[11] Copp AJ, Stanier P, Greene ND. Neural tube defects: Recent advances, unsolved ques-

[13] Detrait ER, George TM, Etchevers HC, Gilbert JR, Vekemans M, Speer MC. Human neural tube defects: Developmental biology, epidemiology, and genetics. Neurotoxicology

[14] Mitchell LE, Adzick NS, Melchionne J, Pasquariello PS, Sutton LN, Whitehead AS. Spina

[15] Dixon MJ, Marazita ML, Beaty TH, Murray JC. Cleft lip and palate: Understanding genetic and environmental influences. Nature Reviews. Genetics. 2011;**12**:167-178 [16] Fraser FC. The genetics of cleft lip and cleft palate. American Journal of Human Genetics.

[17] Poswillo D. The aetiology and surgery of cleft palate with micrognathia. Annals of the

[18] Bromley B, Benacerraf BR. Fetal micrognathia: Associated anomalies and outcome.

[19] Mandell DL, Yellon RF, Bradley JP, Izadi K, Gordon CB. Mandibular distraction for micrognathia and severe upper airway obstruction. Archives of Otolaryngology—Head

[20] Collins B, Powitzky R, Robledo C, Rose C, Glade R. Airway management in pierre robin sequence: Patterns of practice. The Cleft Palate-Craniofacial Journal. 2014;**51**:283-289

[12] Cameron AH. The spinal cord lesion in spina bifida cystica. Lancet. 1956;**271**:171-174

[10] Katsanis N. Ciliary proteins and exencephaly. Nature Genetics. 2006;**38**:135-136

tions, and controversies. Lancet Neurology. 2013;**12**:799-810

Royal College of Surgeons of England. 1968;**43**:61-88

Journal of Ultrasound in Medicine. 1994;**13**:529-533

with cebocephaly. Journal of Diagnostic Medical Sonography. 2017;**33**:39-42

of 150 cases. Teratology. 1977;**16**:261-272

Molecular Teratology. 2004;**70**:495-508

and Teratology. 2005;**27**:515-524

bifida. Lancet. 2004;**364**:1885-1895

& Neck Surgery. 2004;**130**:344-348

1970;**22**:336-352

Orphanet Journal of Rare Diseases. 2007;**2**:8

Fetal and Pediatric Pathology. 2010;**29**:69-80

In the late 1980s, single gene disorders were diagnosed from fetal samples. Although only prenatal diagnosis of β-thalassemia was done using amplified fetal DNA [74] initially, the number of diagnosable diseases or genes has increased thereafter. Thus, the whole-genome sequencing, using DNA samples from amniotic fluid, was developed in the next-generation sequencing (NGS) era [75]. In fact, whole-exome sequencing (WES) is more appropriate for fetal genetic analysis, because the coding exons in WES contain 85% of disease-coding mutations, even though it accounts for only 2% of the entire genome. Prenatal WES, using fetal blood samples, has been performed since 2013 [76]. Meanwhile, massive parallel sequencing (MPS) using NGS opened the way to NIPT [77] in the late 2000s. Now, NIPT is widely used for aneuploidy, throughout the world [78], and even some of the fetal single-gene diseases can be detected using cell-free fetal DNA (cffDNA) obtained from maternal blood [79, 80]. Although the number of diseases detectable using cffDNA is gradually increasing, cffDNA analyses are merely screening tests and would not replace the diagnostic testing, as mentioned in the guidelines of professional societies [81–86].
