Miscarriage is a clinical event that termination of pregnancy at less than 28 weeks, and the fetal weight is less than 1000 g, occurs in before 12 gestational weeks is called miscarriage during early pregnancy, occurs in 13–27 gestational weeks is called miscarriage during middle pregnancy (Muttukrishna et al., 2002). The incidence of miscarriage is about 15–20%, and there is a trend of increasing year by year (Quenby et al., 2021). The etiology of miscarriage is closely related to environmental factors (Zhou et al., 2017), genetic factors(Pereza et al., 2017), body immune state (Wang et al., 2021).

Studies have shown that genetic factors play an important role in early miscarriage, with about 50% of the cases caused by chromosomal abnormalities (van den Berg et al., 2012; Ozawa et al., 2019), while the risk factors of late miscarriage (≥28 gestational weeks) are mainly immune and environmental factors (Meng et al., 2020). Types of chromosomal abnormalities including numerical chromosomal abnormality, structural chromosomal abnormality, chimera, and polyploidy. In miscarriage cases, numerical chromosomal abnormality has the highest frequency (up to 90%), structural abnormality accounts for about 6%, and chimeras account for about 12% (Pérez-Durán et al., 2015; Zhang et al., 2019). A study has shown that copy number variations (CNVs) (known as microdeletion/microduplication) accounts for about 2.7% miscarriages, second only to aneuploidy and polyploidy (Wang Y. et al., 2020). CNVs are copy number changes of the genome, which variations can range in size from several dozens of bases (>50 bp) to megabases (Zarrei et al., 2015; Lauer and Gresham, 2019). Regardless of the cause, the etiological analysis of miscarriage is of great significance to the aborted fetuses and the next pregnancy of women with childbearing age.

Low-coverage massively parallel CNV-seq is a method for sequencing analysis of samples, and the sequencing results were compared with the human reference genome, and CNV was found through bioinformatics analysis (Liang et al., 2014). It is a high-resolution and low-cost technology for detecting CNVs in clinical samples. It can detect chromosome aneuploidies, polyploidies, and CNVs, microdeletions and microduplications with a chromosome resolution of 0.1 Mb (Xie and Tammi, 2009; Liang et al., 2014; Dong et al., 2016). In addition, it can detect unknown variations, to find new genetic information related to the disease.

In the current study, chromosomal abnormalities of miscarriages during early and middle pregnancy were evaluated systematically. We further analyzed the genomic regions of detected chromosomal abnormalities to identify potential miscarriage-associated CNVs. Moreover, potential miscarriage candidate genes and gene functions were identified by gene enrichment analysis. This study is expected to provide meaningful data for the genetic etiology of aborted fetuses and for the next pregnancy of women with childbearing age.

The causes of spontaneous miscarriage include endocrine factors (Arredondo and Noble, 2006), reproductive tract malformation (Venetis et al., 2014), infectious factors (Leisher et al., 2021), immune factors (Muyayalo et al., 2018), alcohol exposure (Sundermann et al., 2021), genetic factors (Lan et al., 2021) and some other factors that have not been clarified yet. Despite continuous medical advances, spontaneous abortion during early and middle pregnancy is still an important health problem. Many couples may face the risk of miscarriage when they choose to have children. It places a heavy psychological and financial burden on many families. Genetic factors are one of the main causes, fetal chromosomal abnormality is an important genetic factor of fetal miscarriage (Zhang et al., 2018). CNVs are ubiquitous in the human genome, and although most are benign or VOUS, a considerable number of CNVs are associated with human diseases (Yang X. et al., 2018; Hu et al., 2018).

At present, karyotype analysis, chromosomal microarray analysis (CMA) and CNV-seq were the main methods for detecting chromosomal abnormality (Li Y. et al., 2018). Karyotype analysis is the recognized gold standard for detecting chromosomal abnormality, which can detect the numerical abnormalities and large structural abnormalities of chromosomes. Karyotype analysis requires tissue cell culture and long detection time. The technical requirements for operators are high and the stability is poor. Karyotype analysis has limited resolution and cannot detect CNV. These disadvantages limit its application (Martinez-Portilla et al., 2019). CMA is a fast and effective chromosome analysis technique, which can detect non-equilibrium chromosomal abnormalities in the whole genome through a single experiment. However, its disadvantages are low throughput and high detection cost. In addition, due to its dependence on probe hybridization, variations in areas not covered by the probe and the exact breakpoints cannot be detected (Levy and Wapner, 2018). CNV-seq is a genomic CNV detection technology based on low-depth whole-genome high-throughput sequencing technology (Zhao and Fu, 2019). CNV-seq has several advantages, including low specimen quality requirements, short detection time, high throughput, the ability to detect 100 Kb chromosomal CNV and more accurate detection of breaking points (Ellingford et al., 2017). CNV-seq can achieve the detection efficiency of CMA when CNV-seq with 1×depth (Zhou et al., 2018). Compared with other technologies based on next generation sequencing (NGS), CNV-seq test has a lower cost and can be used as a first-line prenatal diagnosis technology. In addition, a large number of CNVs found by low coverage CNV-seq have undergone secondary verification. It has been gradually applied to the detection of chromosomes in abortive tissues (Shi et al., 2019).

In this study, 505 fetal aborted tissues were examined by CNV-seq. The chromosomal abnormality rate was 54.3%, and the normal rate was 45.7%. The results are similar to those of other studies (Dai et al., 2019; Sheng et al., 2021). A total of 202 cases with numerical chromosomal abnormality, and the trisomy variation mainly occurred on chromosomes 16, 22, 21, 15, 13, and 9. The results in this study are similar to those of other studies (Liu et al., 2015; Wang M.-z. et al., 2017; Li F. X. et al., 2020). Trisomy 16 is one of the common genetic causes of early miscarriage, accounting for about 6% of early miscarriages (Martin et al., 2004). Some genes on chromosome 16 were associated with abnormal fetal head circumference (Pasternak et al., 2020), and CNVs on chromosome 16 have relationship with prenatal growth retardation (Redaelli et al., 2019). In this study, numerical chromosomal abnormalities were not detected on chromosomes 1, 11 and 19. Several studies have found that numerical abnormalities of chromosomes 1 and 19 were associated with miscarriage (Banzai et al., 2004; Vicić et al., 2008; Li H. et al., 2018). A study using CMA to detect chromosomal abnormalities in products of conception (POC) showed that the rate of abnormalities in POC specimens was 44.6%, with the most common were aneuploidies, including trisomy 16, triploidy, monosomy X, trisomy 22, trisomy 21 and trisomy 15, while the least encountered aneuploidies were trisomy one and trisomy 19 (Wang et al., 2014). The results showed that CMA and CNV-seq were similar in the detection of numerical chromosomal abnormalities in pregnancy products. In Wang BT's study, the proportion of triploidy was high (14%), while this study did not detect triploidy. In addition, one fetus with chromosome 7 tetrasomy (48,XN,+7,+7) was found in this study. Study has shown that partial tetrasomy of chromosome 7 can cause dysmorphic signs, congenital heart defect, and developmental delay (von Beust et al., 2005).

Structural chromosomal abnormality is also one of genetic factors in miscarriage except numerical chromosomal abnormality. Because the number of CNVs in the genome is so large and contains a large number of genes, identifying specific genes associated with miscarriage is a challenge. In this study, VOUS CNVs were detected in 48 fetal tissues, pCNVs and likely pCNVs were detected in 18 fetal tissues. 168 genes are involved in the VOUS CNVs and pCNVs. 41 terms and 12 pathways were enriched by GO analysis and KEGG pathway analysis in miscarriages during early and middle pregnancy, respectively. Some biological processes were enriched such as organ differentiation and development, nervous system formation and development, transmembrane transport, and cellular functions. KEGG analysis results showed that the most enriched pathways of these genes were adherens junction, amyotrophic lateral sclerosis, tight junction, and so on. The functional pathways, such as “Adherens junction” and “Tight junction” pathways may control monolayer barrier function and may be paralleled by altered cytoskeletal organization. These pathways can control barrier function by altered cytoskeletal organization (Cvitic et al., 2020). Study has also shown that “Adherens junction” was associated with repeated implantation failure (Bastu et al., 2019). ‘Tight junction’ pathway was associated with fetal neural tube defects (Wang L. et al., 2017). Amyotrophic lateral sclerosis (ALS) pathway is considered have relationship with fetuses of isolated agenesis of the corpus callosum (Chia et al., 2018; She et al., 2021). Study has shown that fetuses with isolated agenesis may be associated with amyotrophic lateral sclerosis pathway (She et al., 2021). ‘Pathways in cancer’ include Hedgehog (Hh), Wnt, phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), Janus kinase (Jak)/signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK), hypoxia-inducible factor-1 (HIF-1), transforming growth factor-beta (TGF-β), vascular endothelial-derived growth factor (VEGF), and peroxisome proliferator-activated receptor (PPAR) pathways. The hedgehog pathway in the uterine stroma causes deferred implantation and embryonic loss (Harman et al., 2011). Wnt signaling pathway plays an important role in embryonic development by regulating cell differentiation, proliferation and apoptosis (Tepekoy et al., 2015; Nayeem et al., 2016). The Treg/Th17 balance serves a vital role in maintaining the steady state of the maternal-fetal interface (Qian et al., 2018). The differentiation of Treg and Th17 cells is controlled by PI3K/Akt signaling pathway (Yang Y. et al., 2018). In the early pregnancy decidua, the immunoregulation system must work to prevent fetus rejection. NK cells play very important roles in the maintenance of pregnancy (Saito et al., 2007). The JAK-STAT pathway may involve in the regulation of NK function, and it may contribute to the maintenance of immune tolerance at the maternal-fetal interface (Fu et al., 2017). Early oxygen exposure can cause oxidative damage leading to pregnancy disorders. The source of NADPH oxidase in early pregnancy may be related to the activation of MAPK pathway (Hernandez et al., 2019). Study has showed that HIF-1α/VEGF pathway may regulate villous angiogenesis in early pregnancy and HIF-1α/VEGF may be a novel biomarker for missed abortion (Zhi et al., 2018). The TGF-β signaling has been shown to regulate cell growth, immune response, and inflammation. Moreover, TGF-β is able to induce immature lymphocytes to maintain the homeostasis of the immune system. Thus, TGF-β signaling may play a role during pregnancy by regulating immune system homeostasis (Li X. et al., 2020). The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that contribute to the developmental plasticity of the placenta by regulating lipid and glucose metabolism pathways, and placental signaling pathways (Lendvai et al., 2016). Moreover, calcium signaling pathway and leukocyte transendothelial migration pathway were associated with promoting endothelial cell contraction and increased permeability, mediating inflammatory responses (Dalal et al., 2020). If there is a disorder of fetal inflammatory response, it may lead to fetal multiple organ dysfunction, and even fetal death (Jung et al., 2020).

The rate of numerical chromosomal abnormalities in fetuses of ≥35 year-old age pregnant women was higher than that in fetuses of <35 year-old age pregnant women, while the rate of structural chromosomal abnormalities in fetuses of ≥35 year-old age pregnant women was lower than that in fetuses of <35 year-old age pregnant women. It is known that reduced fertility in women over 35 years of age because maternal age is associated with the quality of oocytes. This is due to the rate of chromosome segregation errors during meiotic divisions are increasing with maternal age, and lead to the production of numerical chromosomal abnormalities (Mikwar et al., 2020). A study showed that the frequency of structural chromosomal abnormality seemed to be independent of maternal age (Xanthopoulou et al., 2012). We may need more cases to study this. The rate of chromosomal abnormalities of fetuses in early pregnancy was higher than that of fetuses in middle pregnancy. Chromosomal abnormality is the main causes of early miscarriage (Qu et al., 2019).

In addition to the above CNV detection methods, there is a new method called Optical Genome Mapping (OGM). OGM is considered as a disruptive, novel, and high resolution genome analysis technology. It has been suggested that it can be used to detect all types of genomic variations in many diseases (Pastor et al., 2020). In reproductive medicine and prenatal diagnosis, clinically significant structural variations can be detected in a single analysis by OGM. One study showed that Bianano optical mapping (BOM) can determine the number of D4Z4 repeats and exclude interference of the 10q26.3 homologous region, and in combination with karyomapping, can be used for rapid and accurate prenatal diagnosis of FSHD1 (Zheng et al., 2020). The single-molecule optical mapping (SMOM) has potential clinical application as a rapid tool to screen patients with balanced reciprocal translocations (BRTs) for underlying genetic causes of infertility and other diseases (Wang H. et al., 2020). OGM is a method that can compensate for the limitations of karyotype analysis, fluorescence in situ hybridization (FISH), and CMA by detecting all types of structural variations, including CNVs (Sahajpal et al., 2021).

There were some limitations in this study. First, CNV-seq technology has obvious advantages in detecting chromosomal abnormalities, but it is unable to detect chromosome structural rearrangements such as translocation, inversion, and loss of heterozygosity (LOH) such as uniparental disomy (UPD). Second, the sample size was not large enough to detect all CNVs in miscarriages during early and middle pregnancy. Third, the enrichment analysis of gene function conducted in this study was not in-depth enough. The detected genes were enriched in some functions and signal pathways, but this is just part of the functions of genes in the human body that are associated with miscarriages during early and middle pregnancy. Therefore, we need to analyze larger cohorts to screen out candidate genes related to miscarriage, and conduct basic experiments to study the mechanism of this phenomenon.

The results of this study showed that CNVs were one of the genetic factors of miscarriage during early and middle pregnancy. Some meaningful genetic etiology information such as genes and pathways has been obtained, it may provide useful genetic guidance for pregnancy and prenatal diagnosis. It may provide valuable reference data for the prevention, diagnosis and treatment of miscarriage.