During the prenatal period, several prenatal diagnosis approaches, such as visualization and genetic tests, can be used to determine diagnostic prompts. Due to recent progress in imaging techniques, physicians can now represent smaller embryos and fetuses with a higher resolution. Here we describe the prenatal diagnosis methods for visualizing live or dead embryos and fetuses. In addition, we provide information on the latest prenatal diagnosis methods of genetic testing, with special emphasis on technical and ethical aspects.
Imaging of the Human Embryo and Fetus
Ultrasonography is usually performed throughout pregnancy. Gestational sac for about 5 weeks, yolk sac for 5.5 weeks, flickering cardiac motion after 6 weeks, etc. are observed with transvaginal ultrasound as part of prenatal diagnosis. Embryos and early fetuses within the 12 weeks of pregnancy are usually examined by transvaginal ultrasonography, and fetuses more than 12 weeks are studied using transabdominal ultrasound as part of prenatal diagnosis too.
Ultrasonography plays several roles in the study of embryos and fetuses. One of its roles is the measurement of embryos and fetuses to determine gestational age and assess the weight of the fetus. Detection (including evaluation) of congenital anomalies of the fetus is also another role of ultrasonography. The first application of ultrasound for the diagnosis of congenital disease is the evaluation of anencephaly, and at present, various anomalies can be determined using ultrasound. For effective screening of morphological anomalies, the definition of optimal fetal anatomy surveys was published in the guidelines of the ISUOG. Soft markers in ultrasonography, which in themselves are not harmful, indicate an increased risk of chromosomal abnormalities. Currently, soft markers in combination with maternal serum can achieve a high detection rate of aneuploidy.
When an abnormality is detected during prenatal ultrasound examination or when there is an increased risk of developing neuro-structural disorders, MRI is the next image step for further examination. Fetal MRI can reveal anomalies of the central nervous system that do not appear with prenatal ultrasound in about 20% of cases. If at the glomus of the choroid plexus on the axial ultrasound image at the level of the thalamus is found the width of the atrium ≥ 10 mm, it is considered that it is ventriculomegaly; thus, an MRI scan will be performed for further diagnosis. Fetal MRI reveals additional anomalies in up to 50% of cases of ventriculomegaly detected in ultrasound images.
Usually associated anomalies include agenesis of the corpus callosum, migrational abnormalities, and ventricular hemorrhage. Ultrasound and MRI can also detect Holoprosencephaly (HPE); however, milder cases of lobar EPS may be difficult to detect, taking into account a wide range of HPV symptoms. Cortical malformations are characteristics of target diseases in fetal MRI, although the normal smoothness of the cortex in the second trimester makes it difficult to distinguish it from that of migratory disorders. Intracranial tumors are rare and can be detected with embryonic MRI, mainly as a supratentorial lesion with mixed/high signal intensity on T2-weighted sequences.
MRI is also effective for other congenital anomalies. For congenital diaphragmatic hernia (CDH), which occurs in one of the 4,000 live births, embryonic MRI surpasses ultrasound when recognizing subtypes of CDH. The analysis of the volume of embryonic lung is important in CDH to estimate the prognosis, and MRI is usually used to calculate volume during the evaluation. This function of MRI can also be applied to other diseases associated with pulmonary masses, such as lung sequestration, congenital malformations of the respiratory tract of the lungs and, very rarely, neoplasms. In addition, MRI is used to diagnose abdominal wall abdominal wall defects, such as gastroschisis and omphalocele.
Axial T2-weighted sequences are most useful for identifying abdominal wall defects and umbilical cord position, whereas T1-weighted sequences are useful for tracking the intestine. Evaluation of the musculoskeletal system is still one of the complex aspects of the use of fetal MRI. Recently, clinical imaging of diffusion tensor imaging (DTI) has been applied in clinical MRI, and muscle fibers can be visualized by DTI in adults.
Despite the risk to the fetus (exposure to ionizing radiation), X-ray computed tomography (CT) plays a vital diagnostic role during the prenatal period, especially for fetal skeletal diseases (SDs). Sensitivity to SDs in ultrasonography screening is limited from 40 to 60%, and diagnostic three-dimensional ultrasonography provides a better sensitivity of about 80%. MRI is no more effective than ultrasound for further diagnosis of SDs. In recent years, the skeletal CT of the fetus has been used to visualize the fetal skeleton. A low-dose CT protocol with 3D reconstruction was proposed to reduce the adverse effects of X-rays, and fine images can be obtained for accurate diagnosis.
Autopsy Imaging of Human Embryos and Fetuses
For dead human embryos and fetuses, additional imaging techniques can be used. The classically solid reconstruction and thin pattern were the basic approaches used, for example, the method of the wax plate using successive histological sections of human embryos was the first method of 3D morphological imaging. Recently 3D-reconstruction of serial partitions is carried out using computer graphic methods; so, 3D-reconstruction becomes easier and faster than before. Stacks of 2D images created from consecutive sections have a high resolution; however, they have problems registering and distorting the partition.
The solution to this problem is the use of episcopic fluorescence image capture (EFIC), a new way of image processing to create 3D-reconstructed high-resolution images. In the EFIC image, tissue autofluorescence is used to represent the face of the block before cutting each section. Although the samples are cut and lost during the procedure, the optical resolution of the EFIC is reported to reach about 5-6 μm. MRI is a useful method of visualization for not only live embryos and fetuses of the prenatal period, but also for dead embryos, and fetuses as an autopsy. Since it takes more time to capture images, higher resolution images can be obtained.
The processing time for high-resolution images ranges from several hours to several days. MR-devices should be selected depending on the sample size; MR-microscopy, clinical MRI and experimental MRI are suitable for small embryos, large embryos and embryos or fetuses with intermediate size, respectively.
X-ray imaging is also used for dead embryos and fetuses. Since there is no need to consider the effect of radiation exposure, you can devote more time to visualization. For embryonic skeletal imaging, a conventional (absorption-contrast) X-ray CT (cCT) is used. Phase contrast X-ray CT (pCT) is another method of radiography. Due to the characteristics of X-rays as electromagnetic waves, the phase-contrast X-ray image can visualize the phase shift of X-rays passing through the samples and restore 2D or 3D images of the samples in combination with CT. The embryo or early fetus is mostly composed of soft tissues due to a lack of bone structure and, therefore, is suitable for pCT.
Ultrasonography of live embryos and fetuses is currently usually performed, and many developmental defects can be diagnosed in the early prenatal period. In cases of termination of pregnancy, not all aborted fetuses are dismembered, and pathologically diagnosed. There is technically difficult to dissect small fetuses. The visualization methods presented here can be used to visualize the opening of embryos and fetuses in the future. If the clues about the diseases that could bear fetus can be identified by visualization, you can conduct the appropriate genetic tests and get the final accurate diagnosis. After the final diagnosis, parents will have sufficient information about their lost pregnancy and can receive appropriate genetic counseling for the next pregnancy.
Depending on the period of pregnancy, appropriate methods should be used to visualize the dead embryo or fetus.
Genetic Analysis of the Human Embryos and Fetuses
Amniotic fluid, chorionic villus and umbilical cord blood were used for the genetic analysis of human embryos and fetuses. Recently, DNA fragments derived from villus cells have been identified in maternal blood, and genetic information about the fetus could be determined from a mother’s blood test. This approach is called non-invasive prenatal genetic testing (NIPT). Compared to mother serum analysis, NIPT for aneuploidy has significantly higher sensitivity and specificity. Nevertheless, NIPT is a screening test with the potential for false positive and false negative results, since cell-free DNA (cfDNA) can also be obtained from several sources, such as placental mosaicism, maternal conditions, including cancer, or an embryonic and/or maternal copy number variation (CNV).
Cell samples derived from amniotic fluid and chorionic villus are used for both screening and diagnostic tests. For prenatal genetic diagnosis, several laboratory methods can be used. Traditional karyotype analysis is most often used to study cells obtained by sampling of chorionic villus (CVS) and amniocentesis (AC). This method is suitable for the diagnosis of aneuploidy and large rearrangements.
The diagnostic accuracy of the traditional karyotype analysis is above 99% for aneuploidy and chromosomal abnormalities of more than 5-10 MB. Fluorescence in situ hybridization (FISH) analysis can detect specific chromosomes or chromosomal regions using fluorescently labeled probes. The turnover for FISH results (usually within 2 days) is faster than for usual results of karyotyping (7-14 days, including the period of cell culture). FISH is commonly used as a screen panel for chromosomes 13, 18, 21, X and Y. This is considered a screening test, as false positive and false-negative results were reported with FISH. Thus, clinical diagnosis using FISH results should be supported by other clinical and laboratory analyzes, such as abnormal ultrasonography, a positive screening test using serum and/or soft markers in the mother or confirming traditional metaphase chromosomal analysis or chromosome microarray analysis (CMA), as described below.
CMA can detect small chromosomal aneuploidies that cannot be identified by conventional karyotyping. Duplicated or deleted regions of DNA are called CNV. CMA can be carried out without a cell or tissue culture. Thus, the results are obtained in about 3-7 days. Because CMA can also be performed with nonviable cells that are unsuitable for routine karyotype analysis, such fetal deaths or stillbirths can be studied using this technique. CMA can identify almost all anomalies, except for balanced translocations and triploidy. When CMA is compared with conventional karyotyping in the detection of structural disorders by prenatal ultrasonography, approximately 6% of the fetuses were identified with chromosomal abnormalities with CMA. However, a normal karyotype analysis showed normal results. Therefore, CMA should be the main criterion if a structural anomaly is detected using fetal ultrasonography, as recommended by ACOG.
In the late 1980s, disorders with one gene were diagnosed using fetal samples. First, prenatal diagnosis of β-thalassemia was performed using amplified embryonic DNA, and then the number of diagnosed diseases or genes increased. The sequence of the whole genome using DNA samples from the amniotic fluid was performed in the next generation sequencing (NGS) era. Whole-exome sequencing (WES) is also an option for genetic analysis of the fetus, since the coding of exons sequenced in WES is only 2% of the genome, but contains 85% of the mutations coding for the disease. Prenatal WES using embryonic blood samples has been conducted since 2013. In the late 2000s massive parallel sequencing (MPS) using NGS opened the way to NIPT. Now, NIPT for aneuploidy is widely used in the world, and some fetal diseases of one gene can be detected using a cell-free fetal DNA (cDNA) derived from maternal blood. Although the number of diseases that can be detected using cDNA is gradually expanding, cffDNA analysis is screening tests and does not replace diagnostic testing, as indicated in the guidelines of professional societies.
Ethical Issue of Prenatal Diagnosis
Prenatal diagnosis involves certain ethical issues, and recent progress in fetal genetic testing also poses additional ethical challenges. Four ethical principles based on principles are: a) respect for autonomy, b) beneficence, c) non-maleficence, and d) justice. All women have reproductive rights to make a final reproductive decision after appropriate counseling or consultation based on these principles. Respect for autonomy requires that parents receive accurate information so that they can make a well-informed decision. For example, specific steps in the field of genetic counseling for pre-NIPT include pre-training, counseling and informed consent; a screening or testing procedure; a laboratory component that includes a test interpretation; and finally, disclosure to the patient in a context that includes appropriate education, counseling and follow-up.
Moreover, since termination of fetal abnormalities can have long-term psychological consequences, identifying women vulnerable to poor psychological adaptation and promoting strategies to overcome problems associated with a lower level of grief can be beneficial. In Ukraine, prenatal genetic screening and testing are offered to women whose children are at risk of serious illnesses in the early stages of the disease and/or chromosomal abnormalities, according to the criteria provided by Ukrainian professional societies. Some people argue that identifying congenital anomalies should reduce incidence of disability. The WHO response to this statement is that the presence of genetic tests should not allow the illusion that most disabled people can be prevented and therefore unacceptable to society.
In the near future, we will easily discover complete genomic information, the epigenetic state and the molecular structure of the fetus. In the context of prenatal diagnosis, we must consider our ethical responsibilities to two patients: mother and fetus. The more we possess, the harder quandaries lead.