CNVs can occur anywhere in the human genome, and although most CNVS are benign or VOUS, a significant number of CNVs are associated with human diseases(YANG et al., 2018). In addition, with the rapid advancement of genomics, the OMIM database adds an average of 1.5 genes per day, but the design of probes used in Chromosome Microarray Analysis (CMA) cannot keep up with this rate of updates, leading to missed detection of some known pathogenic regions (LAN et al., 2020). The high cost and low throughput of CMA limit its widespread use in prenatal diagnosis. Since the first report in 2009 that CNV-seq can be used for accurate analysis of chromosomal CNVs, more and more studies have confirmed its reliability in detecting them, however.
CNV-seq is a low-depth whole-genome sequencing technique based on next-generation sequencing technology that uses bioinformatics analysis to discover the CNVs of the tested samples. This technique has the advantages of high throughput, low sample requirements, and broad detection range. However, it has limitations such as the inability to detect triploidy, polyploidy, uniparental disomy, balanced translocations, or inversions. Therefore, it is necessary to combine STR to solve the problems of triploidy, uniparental disomy, and maternal contamination. In this study, the combined use of STR and CNV-seq analysis revealed six cases of maternal contamination, three cases of triploidy, and one case of absence of heterozygosity (AOH).
There are many causes of spontaneous abortion, including embryo chromosomal abnormalities, genetic factors, reproductive tract abnormalities, hormonal imbalances, maternal systemic diseases, immune factors, infectious diseases, toxins, environmental factors, poor lifestyle habits, as well as other unknown factors(QUINTERO-RONDEROS et al., 2020; KARIM et al., 2017; ZWIERZCHOWSKA et al., 2018; TOSIC-PAJIC et al., 2017), and despite advances in medicine, spontaneous abortion remains a significant health issue. Many couples may face the risk of miscarriage when planning to have children. Genetic factors are one of the main causes, and fetal chromosomal abnormalities are an important genetic factor in fetal miscarriage (ZHANG et al., 2018). Among them, abnormal embryo chromosomal numbers and CNVs are the main causes of spontaneous abortion. In this study, chromosomal structural abnormalities were detected in 12 cases out of 97 cases successful fetal tissue samples (13.9%), with a total of 17 CNVs identified. Among the 17 CNVs detected, 14 were pathogenic CNVs. These pathogenic CNVs can lead to severe developmental delays, malformations, intellectual disabilities, delayed language development, distinctive facial features, hypotonia, and congenital heart disease in embryos or fetuses. One case of 4p16.3 loss was found involving Wolf-Hirschhorn syndrome. Patients with this syndrome typically have low birth weight, failure to thrive after birth, microcephaly, developmental delays, and hypotonia. Facial features include downward-slanting eyelids, a ‘Greek warrior helmet’ appearance, a short nose, and a very short midsection. They may also suffer from conditions such as iris tumors and epilepsy (YIN et al., 2019). In this case, both parents underwent peripheral blood karyotype testing, and the father was identified as a carrier of a balanced translocation [46,XX, (t 4;9)]. It was recommended that they undergo preimplantation genetic diagnosis (PGD) for genetic disease screening when planning to have children again.
Additionally, one case involving a 17p13.2-p13.3 deletion associated with Miller-Dieker syndrome was also identified in this study. The main characteristic of this syndrome is lissencephaly (“smooth brain” lacking normal brain gyri and sulci). Clinical manifestations also include a high and prominent forehead, vertical furrows, narrowed temples, widely spaced eyes, upward-slanting eyelids, short and upward-turned nose, inverted red lips, long, wide and thick upper lip, congenital heart disease, umbilical hernia, and joint contractures. MDS patients typically have severe intellectual disabilities, epilepsy, and shortened lifespan. Both parents in this case underwent peripheral blood karyotype testing, and it was found that the mother was a carrier of a balanced translocation [46,XX, t(2;17)(p23;p13), 21ph+]. Given that there is only a 2/18 chance of having a normal fetus in future pregnancies, it was recommended that they undergo preimplantation genetic diagnosis (PGD) to assess for genetic diseases during reproductive planning.
CNV-seq technology can efficiently and rapidly detect chromosomal structural abnormalities that may lead to abortion, and analysis of the peripheral blood karyotypes of both spouses can guide assessment for future pregnancies. Under normal circumstances, offspring inherit one homologous chromosome from the mother and one from the father. However, cases where a pair of homologous chromosomes in the offspring is entirely or partly derived from either the father or the mother are referred to as absence of heterozygosity (AOH). Although the incidence of AOH is low, it can increase the risk of recessive disease-causing genes that can lead to complex clinical symptoms. In this study, combined STR and CNV-seq testing detected a homozygous phenomenon in the 2p25.3q37.3 region, spanning 242Mb, which may have led to an increased risk of recessive genetic disorders in that specific region. Currently, there is no conclusive evidence that the occurrence of uniparental diploid on chromosome 2 is directly related to embryo termination. Research have found that after ovulation in vitro, oocyte quality decreases with time, and the expression of spindle assembly checkpoint (SAC) in oocytes is negatively correlated with oocyte senescence. Decreased SAC expression also leads to errors in sister chromatid separation during the second meiotic division (MII) of oocytes, thereby increasing the incidence of early embryonic aneuploidy (SHIMOI et al., 2019). In this study, the rate of chromosomal abnormalities in aborted tissues in the ≥ 35 age group accounted for 58.8%, and the rate of chromosomal abnormalities in the < 35 age group accounted for 48.7%, Although the difference was not statistically significant (P = 0.451), the abnormality rate in the ≥ 35 group was 10.1% higher than that in the < 35 group, indicating a positive correlation between the frequency of abnormal karyotypes and maternal age.
Chromosomal abnormalities in fetuses aborted in the first trimester may involve any one or multiple chromosomes, although chromosomal abnormalities in fetuses aborted in the second trimester may be trisomy 13, 18, and 21, sex chromosome aneuploidy, or CNV, which are similar to the types of chromosomal aberrations detected in live birth (ZHANG et al., 2021; LAN et al., 2021). With the development of molecular biology technology, CNV-seq technology has been gradually applied to chromosomal testing of fetuses aborted, mostly used in the detection of chromosome number abnormalities, large fragment deletion/duplication, and copy number variation of pathogenic genomes[19]. Although chromosomal karyotyping is considered the gold standard for detecting chromosomal abnormalities, it cannot detect deletions/duplications smaller than 5-10Mb. However, CNV-seq technology can efficiently detect routine chromosomal number abnormalities and identify pathogenic microdeletions/microduplications that cannot be detected by karyotype analysis. It can also detect unknown variations and discover new genetic information associated with diseases, thus improving the detection rate. CNV-seq technology can provide genetic diagnostic evidence for the etiology of miscarriage, guide future pregnancies with reliable laboratory evidence, and offer valuable reference data for the prevention, diagnosis, and treatment of abortion.