Evaluation of the efficiency of full-genome sequencing for karyotyping of spontaneous abortus cells with no proliferative activity

Background. Determination of chromosomal imbalance has an important clinical and biological significance in cases of spontaneous abortion. It is necessary to establish the cause of non-pregnancy, to exclude hereditary factors, to provide further genetic counselling, and to understand the mechanisms of chromosomal anomalies. The gold standard for karyotype analysis is microscopic analysis of chromosomes after GTG staining. However, this method has limitations as it depends on the mitotic activity of the cells and the presence of well-visualised chromosomes. An alternative cytogenomic approach to detect chromosomal imbalance may be low-coverage and ultra-low coverage whole-genome sequencing, used to detect aneuploidies and large copy number variations.

Objective: to perform whole-genome sequencing with ultra-low coverage for molecular karyotyping of spontaneous and medical abortuses.

Methods. Uncultured material of extraembryonic mesoderm of spontaneous and induced abortions was used. Molecular karyotyping was performed using ultra-low coverage whole genome sequencing. The results were verified using fluorescence in situ hybridization (FISH) and real-time PCR.

Results. Among spontaneous abortuses, aneuploidies were detected in 25 out of 71 (35.2%) cases, among which autosomal trisomies were the most frequent abnormality (92%), while sex chromosome number abnormalities were detected in 8% of cases. Polyploidy was detected in 4 out of 71 cases (5.6%), giving a cumulative incidence of chromosomal abnormalities of 40.8%.

Conclusions. Determination of the efficacy and specificity of ultra-low coverage whole-genome sequencing for the detection of aneuploidy showed values of 100% in both measures on the control groups. Confirmation of karyotype abnormalities in spontaneous abortions by reference methods showed that ultra-low coverage whole-genome sequencing is effective in the diagnosis of aneuploidy in the absence of alternative methods.

1. American College of Obstetricians and Gynecologists’ Committee on Practice Bulletins—Gynecology. ACOG Practice Bulletin No. 200: Early Pregnancy Loss. Obstet Gynecol. 2018;132(5):e197-e207. doi:10.1097/AOG.0000000000002899

2. Essers R., Lebedev I.N., Kurg A., et al. Prevalence of chromosomal alterations in first-trimester spontaneous pregnancy loss. Nat Med. 2023;29(12):3233-3242. doi:10.1038/s41591-023-02645-5

3. Smolander J., Khan S., Singaravelu K., et al. Evaluation of tools for identifying large copy number variations from ultra-low-coverage whole-genome sequencing data. BMC Genomics. 2021;22(1):357. doi:10.1186/s12864-021-07686-z

4. Lund R.J., Nikula T., Rahkonen N., et al. High-throughput karyo- typing of human pluripotent stem cells. Stem Cell Res. 2012;9(3):192-195. doi:10.1016/j.scr.2012.06.008 8

5. Lund R.J., Närvä E., Lahesmaa R. Genetic and epigenetic stability of human pluripotent stem cells. Nat Rev Genet. 2012;13(10):732-744. doi:10.1038/nrg3271

6. Kader T., Goode D.L., Wong S.Q., et al. Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue. Genome Med. 2016;8(1):121. doi:10.1186/s13073-016-0375-z

7. Chin S.F., Santonja A., Grzelak M., et al. Shallow whole genome se- quencing for robust copy number profiling of formalin-fixed paraf- fin-embedded breast cancers. Exp Mol Pathol. 2018;104(3):161-169. doi:10.1016/j.yexmp.2018.03.006

8. Benjamini Y., Speed T.P. Summarizing and correcting the GC con- tent bias in high-throughput sequencing. Nucleic Acids Res. 2012;40(10):e72. doi:10.1093/nar/gks001

9. Quinlan A.R., Hall I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26(6):841-842. doi:10.1093/bioinformatics/btq033

10. Yakut S., Toru H.S., Çetin Z., et al. Chromosome abnormalities iden- tified in 457 spontaneous abortions and their histopathological find- ings. Turk Patoloji Derg. 2015;31(2):111-118. doi:10.5146/tjpath.2015.01303

11. Vlachadis N., Papadopoulou T., Vrachnis D., et al. Incidence and Types of Chromosomal Abnormalities in First Trimester Sponta - neous Miscarriages: a Greek Single-Center Prospective Study. Maedica (Bucur). 2023;18(1):35-41. doi:10.26574/maedica.2023.18.1.35

12. Zhou W., Dinh H.Q., Ramjan Z., et al. DNA methylation loss in late-replicating domains is linked to mitotic cell division. Nat Gen- et. 2018;50(4):591-602. doi:10.1038/s41588-018-0073-4

13. Tisato V., Silva J.A., Scarpellini F., et al. Epigenetic role of LINE-1 methylation and key genes in pregnancy maintenance. Sci Rep. 2024;14(1):3275. doi:10.1038/s41598-024-53737-2

14. Zhou Q., Xiong Y., Qu B., Bao A., Zhang Y. DNA Methylation and Recurrent Pregnancy Loss: A Mysterious Compass?. Front Immunol. 2021;12:738962. doi:10.3389/fimmu.2021.738962

15. Matsumoto Y., Shinjo K., Mase S., et al. Characteristic DNA meth- ylation profiles of chorionic villi in recurrent miscarriage. Sci Rep. 2022;12(1):11673. doi:10.1038/s41598-022-15656-y