Prenatal diagnosis of fetuses conceived by assisted reproductive technology by karyotyping and chromosomal microarray analysis

Study design and participants

A retrospective cross-sectional study was carried out to assess fetuses conceived by assisted reproductive technologies delivered between January 2015 and December 2021 at the Affiliated Hospital of Jining Medical University, a tertiary hospital, in Shandong, China. The study population included singleton pregnant women who conceived via ART procedures with invasive genetic testing by both karyotyping and CMA. All genetic samples were obtained by amniocentesis. Inclusion criteria were pregnancy conceived via ART procedures in women, with or without other abnormal medical indications, single fetus and invasive genetic tests (both karyotyping and CMA). Exclusion criteria were natural conception in women, multiple pregnancy, no first-trimester ultrasound, no invasive genetic tests (both karyotyping and CMA) only karyotype analysis or CMA.

Based on ultrasound findings and other medical indications, the enrolled pregnant women were classified into two groups: ART only (isolated ART group) and ART accompanied by soft ultrasound markers, ultrasound malformations and other medical indications (non-isolated ART group). In the non-isolated ART group, the most common medical indications were abnormal ultrasound findings (AUS), including structural abnormalities, increased nuchal translucency, intrauterine growth restriction, intestinal hyperechogenicity and amniotic fluid abnormalities, etc, followed by elevated odds of maternal serum Down syndrome screening, confirmation of a known anomalous fetal NIPT, a family history of a genetic disease or chromosomal alteration and adverse pregnancy history including trisomy 21 reproductive history and a reproductive history of a genetic condition or chromosomal abnormality.The control population was a group of individuals with naturally conceived fetuses and no medical indications. Pregnancies without medical indications underwent CMA and karyotyping by maternal request and elevated maternal age in Affiliated Hospital of Jining Medical University. There were no other indications for performing invasive diagnostics at the subgroup that underwent solely ART and at the control group.

Follow-up of pregnancy outcomes

Clinical follow-up assessments of pregnancy outcome, prenatal and postnatal development were performed regularly by telephone.

Karyotype analysis

Ultrasound-guided amniocentesis was carried out at pregnancy weeks 17–28, collecting amniotic fluid samples (30 ml each). Then, 20 ml amniotic fluid samples were assessed based on the amniotic fluid karyotyping procedure of the prenatal diagnosis department of our hospital (320 to 400 bands). All prenatal samples were routinely cultured, mounted on slides and subjected to G-banding (additional C-banding and N-banding if required). Karyotype analysis was performed on a GSL-120 Streamlines Cytogenetic Analysis System (Leica Microsystems; Mannheim, Germany). At least 40 karyotypes were counted for each case, and five karyotypes were randomly selected for analysis.

CMA analysis

The remaining 10 ml amniotic fluid is used for chromosomal microarray analysis. DNA extraction was performed with the QIAamp DNA Blood Kit (QIAGEN, Hilden, Germany) following the kit’s handbook (http://www.qiagen.com). CMA was carried out by high-resolution genotyping single nucleotide polymorphism microarray with Affymetrix CytoScan 750k Array (Affymetrix, USA). CNV analysis was based on findings reported for the human reference genome 37 (NCBI37hg19) by the National Centre for Biotechnology Information. CNV was assessed by reviewing multiple databases, including DGV (http://dgv.tcag.ca/dgv), OMIM (https://omim.org/), DECIPHER (https://decipher.sanger.ac.uk/), PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) and others.

Next, CMA findings were categorized as follows: (1) clinically significant, i.e., pathogenic/likely pathogenic; (2) variants of unknown significance (VOUS) (findings of unknown significance or CNV with clinical penetrance below 10%, e.g., duplications at 15q13.3, 16p11.2 and 16p11.13 loci) (Maya et al., 2018; Rosenfeld et al., 2013); (3) normal findings, i.e., no CNV, benign/likely benign CNVs, or VOUS findings below the reported cutoffs of 1 and 2 Mb for deletions and duplications, respectively.

Statistical analysis

SPSS 20.0 (IBM, Armonk, NY, USA) was utilized for data analysis. Continuous and categorical data were expressed as mean ± standard deviation and frequency or percentage, respectively; they were compared by the Student’s t test and the chi-square test or Fisher’s exact test, respectively. P < 0.05 indicated statistical significance.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Affiliated Hospital of Jining Medical University, and the ethics approval number is 2020c052. The date of approval was 2 December 2020. All subjects signed an informed consent form.

Prenatal diagnostic karyotyping data

Karyotyping in all ART groups revealed chromosomal anomalies in eight fetuses, with seven aneuploidy cases and one unbalanced translocation. The clinico-genetic features of fetuses with abnormal karyotypes are shown in Table 2. Precisely, chromosomal alterations were found in 3.57% of individuals (8/224). The commonest chromosomal alterations included trisomy 21 (1.79%, 4/224) and sex chromosome abnormalities (1.79%, 4/224). Sex chromosome abnormalities included Klinefelter’s syndrome, hyperestrogenism, hyperandrogenism and unbalanced sex chromosome translocation. Compared with the control population, there was elevated rate of chromosomal anomalies in the total ART group (0.49 vs. 3.57%, χ² = 11.990; p = 0.001, Chi-square test) (Table 1).

In the isolated ART group, there was no chromosomal abnormality. In the non-isolated ART group, chromosomal anomalies were found in 8 fetuses. In comparison with the isolated ART group, the non-isolated ART group had starkly elevated rate of chromosomal anomalies (0 vs. 5.71%, χ² = 4.978; p = 0.026, Chi-square test). In comparison with the control population, chromosomal abnormalities had no starkly elevated rate in the isolated ART group (0.49 vs. 0%, χ² = 0.414; p = 0.520, Chi-square test). In comparison with the control population, there was markedly elevated rate of chromosomal alterations in the non-isolated ART group (0.49 vs. 5.71%, χ² = 21.573; p = 0.000, Chi-square test) (Table 1).

Prenatal diagnostic findings by CMA

Pathogenic/likely pathogenic CNVs were detected in 13/224 fetuses, indicating a total detection rate in CMA for genetic alterations of 5.80%, including seven aneuploidies and one unbalanced sex translocation. Of the 216 fetuses with normal karyotype, five showed pathogenic/likely pathogenic CNVs. Totally seven fetuses were detected with VOUS (Table 3).

In case 1, a nulliparous 31-year-old woman with a low-risk pregnancy showed unremarkable first- and second-trimester ultrasound findings. The karyotype was normal, but CMA revealed a pathogenic CNV, showing a duplication in 16q24.1q24.3 that may cause intellectual disability, generalized hypotonia and global developmental delay. In case 2, CMA showed a microdeletion in 17p12, which may be involved in hereditary motor neuropathy with liability to pressure palsies (HNPP). In case 3, the 1q21.1q21.2 recurrent microdeletion was found and could lead to microcephaly, moderate cognitive impairment, moderate dysmorphic facial traits, eye abnormalities and cardiac defect. In case 4, CMA showed a duplication in 17q12 that may cause microcephaly, short stature, developmental delays, and renal and cardiac abnormalities. In case 5 (a male fetus with high risk of trisomy 18), CMA revealed a duplication in Xp22.31 that might induce ichthyosis, intellectual disability and seizure.

In the total ART group, CMA anomalies were detected in 20 fetuses, including 13 pathogenic/likely pathogenic CNVs and seven VOUS. In the isolated ART group, CMA anomalies were detected in six fetuses, including four pathogenic/likely pathogenic CNVs and two VOUS. In the non-isolated ART group, CMA anomalies were detected in 14 fetuses, including nine pathogenic/likely pathogenic CNVs and five VOUS. Compared with the control population, the total ART group had no starkly elevated rate of CMA anomalies (6.70 vs. 8.93%, χ² = 1.205; p = 0.272, Chi-square test). Compared with control individuals, no starkly elevated rate of CMA anomalies was detected in the isolated ART group (6.70 vs. 7.14%, χ² = 0.023; p = 0.879, Chi-square test). Compared with control individuals, CMA anomalies had remarkably higher rate in the non-isolated ART group (6.70 vs. 10%, χ² = 1.831; p = 0.176, Chi-square test). However, compared with the control group, the prevalence of pathogenic/likely pathogenic CNVs showed a significant increase in the isolated ART group (1.47 vs. 4.76%, χ² = 4.365; p = 0.037, Chi-square test). In comparison with control individuals, the non-isolated ART group had markedly elevated rate of pathogenic/likely pathogenic CNVs (1.47 vs. 6.43%, χ² = 11.988; p = 0.001, Chi-square test). In comparison with the isolated ART group, pathogenic/likely pathogenic CNVs had non-significantly elevated rate in the non-isolated ART group (4.76 vs. 6.43%, χ² = 0.267; p = 0.606, Chi-square test). The prevalence rates of VOUS were similar among groups (P > 0.05). Using pairwise comparison methods to assess VOUS, nonsignificant differences were found between the total ART and control groups and among isolated ART, non-isolated ART, and control groups (p > 0.05) (Table 1).

Comparing the detection rate of abnormalities by karyotyping, the detection rate of genetic abnormalities detected by CMA was increased in the non-isolated ART group, but with a nonsignificant difference (5.71 vs.10%, χ² = 1.776; p = 0.183, Chi-square test); however, the rate of genetic alterations detected by CMA was significantly increased in the isolated ART group (0 vs. 7.14%, χ² = 6.222; p = 0.013, Chi-square test) .CMA markedly elevated the diagnostic yield of pathogenic/likely pathogenic anomalies in fetuses conceived by assisted reproductive technologies compared to karyotyping(0 vs. 4.76%, χ² = 4.098; p = 0.043, Chi-square test) (Table 1).