Chromosomal abnormality profiles in abortion tissues from patients with recurrent pregnancy loss vary by etiological subtype: unexplained cases show a higher risk of aneuploidy

The etiology of recurrent spontaneous abortion (RSA) is complex, with embryonic chromosomal abnormalities representing a major contributing factor. This study aims to characterize the embryonic chromosomal abnormalities products of conception (POC) from RSA patients, compare the composition of abnormalities across various etiological subtypes, and analyze the association between maternal clinical factors and different types of chromosomal abnormalities.

A retrospective cohort study was conducted with predefined inclusion and exclusion criteria, a unified etiological evaluation protocol, standardized copy number variation sequencing (CNV-seq) and abnormality interpretation criteria, and systematic clinical variable extraction and statistical analysis to ensure quality control. A total of 534 RSA patients involving 629 pregnancy events who underwent POC CNV-seq in our hospital between January 2021 and December 2024 were recruited. According to systematic etiological evaluation, the patients were classified into a maternal/parental factor-explained recurrent spontaneous abortion (MPF-eRSA) group and a maternal/parental factor-unexplained recurrent spontaneous abortion (MPF-uRSA) group. The distribution of embryonic chromosomal abnormalities was compared between the 2 groups. Mixed-effects logistic regression models were used to evaluate the associations of maternal age, gestational age, number of previous miscarriages, mode of conception, and maternal risk factors with different types of chromosomal abnormalities. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were further performed on genes covered by RSA-related pathogenic CNVs (PCNVs), and the selected candidate genes were preliminarily validated by qRT-PCR.

Among the 629 POC samples, chromosomal abnormalities were detected in 321 cases, with an overall detection rate of 51.0%. Numerical abnormalities were the predominant type, accounting for 75.1% of abnormal cases. Univariate analysis showed that the MPF-uRSA group had higher proportions of aneuploidy and single trisomy than the MPF-eRSA group (P<0.001), whereas mosaicism was more frequent in the MPF-eRSA group (P=0.015). Mixed-effects logistic regression analysis revealed a lower risk of overall chromosomal abnormalities in the MPFeRSA group than the MPF-uRSA group (OR=0.41, 95%CI: 0.21 to 0.82, P=0.011). Maternal age was positively associated with the risk of overall chromosomal abnormalities (OR=1.48, 95%CI: 1.22 to 1.80, P< 0.001), aneuploidy (OR=1.44, 95%CI: 1.17 to 1.77, P<0.001), and single trisomy (OR=1.55, 95%CI: 1.20 to 2.00, P<0.001). The risks of aneuploidy (OR=0.24, 95%CI: 0.11 to 0.51, P<0.001) and single trisomy (OR=0.14, 95%CI: 0.05 to 0.36, P<0.001) were significantly lower in the MPF-eRSA group than the MPF-uRSA group. Maternal endocrine factors were associated with an increased risk of single trisomy (OR=2.87, 95%CI: 1.19 to 6.90, P=0.018), while maternal genetic factors were associated with increased risks of single trisomy (OR=3.75, 95%CI: 1.20 to 11.69, P=0.023) and PCNVs (OR=9.91, 95%CI: 2.87 to 34.21, P< 0.001). GO/KEGG enrichment analyses showed that the genes covered by RSA-related PCNVs were mainly involved in DNA damage repair, extracellular matrix homeostasis, and inflammation-related processes. qRT-PCR showed that RAD21, LIG4, and COL4A1 were downregulated, whereas CTSB and GSDMD were upregulated in the RSA group compared with the control group(P<0.05).

Embryonic chromosomal abnormalities in POC from RSA patients are predominantly numerical abnormalities, and their distribution differs across etiological subtypes. Aneuploidy is more common in MPF-uRSA patients. Maternal age is an important influencing factor for embryonic chromosomal abnormalities, mainly associated with the risk of aneuploidy. POC genetic testing combined with etiological classification may help improve etiological evaluation, risk stratification, and clinical management of RSA.