Comparative clinical genetic testing in spontaneous miscarriage: Insights from a study in Southern Chinese women

Abstract Single nucleotide polymorphism (SNP) array and karyotype analyses were conducted on 441 spontaneous miscarriage placental villous tissues collected from women from southern China. Subsequently, the results from these two analyses were compared to evaluate the best diagnostic strategy for subsequent pre‐pregnancy planning. Here, the success rate of genetic testing using karyotyping and SNP array analysis was 78.46% (346/441) and 100.0% (441/441), respectively. The abnormality rate estimated by both methods was 54.9% (242/441). Three hundred and forty‐six cases were successfully detected via both SNP array and karyotype analyses; the rate of consistent detection was 96.24% (333/346), whereas 13 cases were not consistent. There was no substantial positive correlation between age and genetic abnormalities such as Turner syndrome, structural variation or euploidy state in the different age groups studied. However, the aneuploidy rate was significantly different in each age group. Thus, although SNP array has higher success rate and resolution in genetic abnormality detection, supplementary karyotype analysis is needed for a more accurate revelation of the genetic aetiology of miscarriages. Therefore, this study indicates that simultaneous karyotype and SNP array analyses should be performed for spontaneous miscarriages. Furthermore, miscarriages irrespective of maternal age must be genetically analysed.

analysis (CMA), also known as 'molecular karyotype analysis', has been gradually applied to the genetic analysis of abortion villous tissues due to advantages, such as no requirement for culture and high throughput and high resolution. [14][15][16][17][18] CMA can be divided into two categories: array-based comparative genomic hybridization [19][20][21] and single nucleotide polymorphism (SNP) array. 22,23 In addition to detecting copy number variation (CNV), SNP array analysis can also detect loss of heterozygosity (LOH), uniparental disomy, triploidy 15 and a certain level of mosaicism. 24 Here, we investigated the feasibility and superiority of the two methods, traditional karyotyping vs. SNP array analysis, for genetic analysis of abortion villous tissues in 441 cases of spontaneous miscarriages in southern China.

| Study Participants and Samples
Abortion villous tissues were collected from 441 women with spon-

| Villous cell culture and traditional karyotyping
Under aseptic conditions, the villous tissues were rinsed with 0.9% sodium chloride solution and separated from blood clots and non-villi tissue; subsequently, high-quality villi were selected. The selected villi were chopped and divided into two parts, 15-25 mg each for cell culture and DNA extraction, respectively. The villous tissues selected for cell culture were inoculated, cultured and sectioned according to the conventional method. After banding, the slides were placed on a GSL-120 automatic chromosome scanner for scanning. Five karyotypes from 20 mitotic phases were analysed following the ISCN 2013 standards. If mosaicism occurred, 10 karyotypes from 40 the mitotic phases were analysed.

| SNP array analysis
Genomic DNA was extracted using the Qiagen kit (Germany), and the concentration and purity of DNA were determined via ultraspectrophotometry. DNA digestion, amplification, purification, fragmentation, labelling, hybridization, washing, staining and scanning were performed using Affymetrix CytoScan 750K microarray (USA).

| Statistical analysis
SPSS Statistics v20 software (IBM, Armonk, NY) was used. Statistical comparisons with groups were performed using chi-square test, and a P-value of <0.05 was considered statistically significant.

| Traditional karyotyping is effective but plagued by culture success and karyotype quality
Three hundred and forty-six of the 441 abortion villous tissues could be karyotyped successfully, whereas 95 could not be karyotyped.

| SNP array analysis shows higher genetic testing success rate
All 441 villous tissues could be analysed via SNP array. Thus, the success rate was 100% (441/441). SNP array analysis was able to detect 47 cases of chromosomal abnormalities among the 95 karyotyping failure samples. Forty-two of these cases were aneuploidies, whereas five cases were structural abnormalities.
Thus, a total of 235 cases of chromosomal abnormalities were Twenty cases with structure abnormalities were detected via SNP array analysis. However, the parental couples of the two LOH cases detected via SNP array analysis refused peripheral blood verification tests (Table 2). Thus, peripheral blood karyotype analysis for structural abnormalities was carried out in 18 parents of the 20 spontaneous miscarriages; structural abnormalities were detected via villous SNP array. Two maternal carriers of balanced chromosomal translocation and two maternal carriers of inverted chromosomal inversion were found, whereas the remaining 14 parental couples had normal karyotype.

| Genetic testing of spontaneous miscarriage villous tissue with traditional karyotyping and SNP array analysis: Comparison reveals both methods effective but supplement each other
A total of 441 cases were analysed via karyotyping and SNP array analysis. The detection success rate of SNP array analysis (100%, 441/441) was higher than that of karyotyping (78.46%, 346/441).
The difference in the abnormality rates detected by the two meth-  (Table 3).

| D ISCUSS I ON
The aetiology of spontaneous miscarriage is complex, and chromosomal abnormality is the main cause. 26  Approximately half of early spontaneous miscarriage is caused by chromosome abnormality. 35 Here, a total of 441 cases were analysed by karyotyping and SNP array analysis, and the abnormality de- However, the tetraploidy, equilibrium structure abnormality and low proportion mosaicism of X chromosomes (which also comprise the 13 non-overlapping cases) could not be detected by SNP array analysis. Thus, karyotyping has an irreplaceable advantage in the detection of such types of abnormalities.
In summary, the results of this study indicate that a variety of chromosomal abnormalities lead to spontaneous miscarriage.
Therefore, SNP array analysis cannot completely replace karyotyping. Moreover, the vast information output of SNP array analysis requires intensive interpretation abilities.
The risk of miscarriage due to chromosomal abnormalities is increased in women over 35 years of age. [37][38][39] In brief, the older the female is, the more likely the embryo is to have aneuploidy, most probably due to the gradual degeneration of ovarian function with age which may cause the chromosomes to not separate well during the formation of germ cells. 6,13,40 However, the incidence of Turner Various chromosomal abnormalities lead to spontaneous miscarriage. To identify the cause of spontaneous miscarriage and to provide evidence for risk assessment of the next pregnancy, we recommend that patients undergo both tests. Where this is not feasible, karyotyping may be recommended first, followed by SNP array analysis in cases where karyotyping is not possible due to villous cell culture failure or karyotyping results did not reveal any abnormalities. Furthermore, genetic testing is recommended for spontaneous miscarriages irrespective of maternal age. By detecting the chromosomal abnormalities in the aborted villous tissue, pre-pregnancy planning may be strategized for avoiding miscarriage recurrence. Thus, providing a scientific and accurate molecular genetic diagnosis basis is essential for targeted prepregnancy eugenic measures in the next pregnancy.

ACK N OWLED G M ENTS
The present study was supported by the Fujian Provincial Natural Science Foundation, 2017J01238.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated during and/or analysed during the current study are available upon request by contact the corresponding author.