Gene expression analysis and e-karyotyping of human blastocysts using whole transcriptome sequencing

Introduction. The study of early human embryogenesis involves the analysis of a limited amount of biological material. Most methodological approaches do not allow for the simultaneous analysis of several blastocyst characteristics on the same material. This paper presents the results of a pilot study on RNA sequencing of the inner cell mass (ICM) and trophectoderm (TE) cells of human blastocysts.

Aim: evaluation of the feasibility of gene expression analysis and blastocyst karyotype reconstruction using whole transcriptome sequencing data.

Methods. Material – 16 human blastocysts, which were obtained as part of in vitro fertilization (IVF) cycles at the Regional Perinatal Center named after I.D. Yevtushenko. Blastocysts were mechanically divided into the ICM (2 fragments) and TE (3 fragments). Sample preparation of libraries for RNA sequencing was carried out using the commercial QIAseq FX Single Cell RNA Library Kit (Qiagen, Germany). Sequencing was performed using a NextSeq 550 instrument and commercial NextSeq 500/550 High Output Kit v2.5 sequencing kit and NextSeq 500/550 High Output Kit v2 kit (Illumina, USA), as well as a NextSeq 2000 instrument (Illumina, USA) and a commercial NextSeq 2000 P3 reagents (300 cycles) kit (Illumina, USA). Bioinformatics data processing was performed using FastQC, multiQC, STAR. RStudio. superFreq. Embryoid bodies (EBs) obtained from induced pluripotent stem cell (iPSC) lines were used as reference samples with confirmed normal karyotype to reconstruct molecular karyotypes of the studied samples.

Results. In blastocysts on day 7 of development, in comparison with blastocysts on day 5 (7vs5), a group of genes responsible for blastocyst growth (CTR9, GINS1 and ZNF830) was identified. When comparing 7vs5 and 6vs5, several groups of genes responsible for protein localization and translation were identified. A total of 286 signs of chromosomal instability (CIN) were identified. Amplifications, deletions, partial amplifications and deletions of autosomes occurred with a frequency of 48.3%, 34.3%, 11.2% and 6.3%, respectively. The most frequently detected copy number abnormalities were chromosomes 3, 5, 9, 10, 12, 15, 16, 17, 19, 22. We found an inverse statistically significant correlation between the quality of TE and the number of CIN in the ICM, as well as a tendency to decrease CIN in the trophectoderm with a change in the quality of TE from “C” to “A”.

Conclusions. Reconstruction of blastocyst karyotypes based on whole-transcriptome analysis data is possible and is of interest from the point of view of studying the relationship between the expression profiles of blastocyst cells and their chromosomal constitution.

1. Shaw L., Sneddon S.F., Zeef L., et al. Global gene expression profiling of individual human oocytes and embryos demonstrates heterogeneity in early development. Plos One. 2013;8(5):e64192.

2. Rossant J., Tam P.P. Early human embryonic development: Blastocyst formation to gastrulation. Dev Cell. 2022;57(2):152-165.

3. Alfarawati S., Fragouli E., Colls P., et al. The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender. Fertil Steril. 2011;95(2):520-524.

4. Schoolcraft W.B., Gardner D.K., Lane M., et al. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril. 1999;72(4):604-609.

5. Andrews S. FastQC: a quality control tool for high throughput sequence data. 2010. [Electronic resource] Available at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc. Accessed: 20.10.2024.

6. Ewels P., Magnusson M., Lundin S., et al. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32(19):3047-3048.

7. Dobin A., Davis C.A., Schlesinger F., et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29(1):15-21.

8. Liao Y., Smyth G.K., Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923-930.

9. Groff A.F., Resetkova N., DiDomenico F., et al. RNA-seq as a tool for evaluating human embryo competence. Genome Res. 2019;29(10):1705-1718.

10. Love M., Anders S., Huber W. Differential analysis of count data– the DESeq2 package. Genome Biol. 2014;15(550):10-1186.

11. Zhu A. Statistical methods for sequencing count data and integrative functional genomics. 2019. [Electronic resource] Available at: https://doi.org/10.17615/y2et-dh46. Accessed: 20.10.2024.

12. Flensburg C., Sargeant T., Oshlack A., et al. SuperFreq: Integrated mutation detection and clonal tracking in cancer. PLoS Comput Biol. 2020;16(2):e1007603.

13. Malakhova A.A., Grigor’eva E.V., Pavlova S.V., et al. Generation of induced pluripotent stem cell lines ICGi021-A and ICGi022-A from peripheral blood mononuclear cells of two healthy individuals from Siberian population. Stem Cell Res. 2020a;48:101952.

14. Malakhova A.A., Grigor’eva E.V., Vasilyeva O.Y., et al. Generation of two induced pluripotent stem cell lines from peripheral blood mononuclear cells of a patient with Wilson’s disease. Stem Cell Res. 2020b;47:101922.

15. Zhigalina D.I., Malakhova A.A., Vasilyeva O.Y., et al. Generation of an induced pluripotent stem cell line ICGi030-A from a Wilson’s disease patient carrying a frameshift mutation p. Lys1013fs and missense mutation p. H1069Q in the ATP7B gene. Stem Cell Res. 2021;57:102556.

16. Zhang K., Haversat J.M., Mager J. CTR9/PAF1c regulates molecular lineage identity, histone H3K36 trimethylation and genomic imprinting during preimplantation development. Dev Biol. 2013;383(1):15-27.

17. Genuth N.R., Shi Z., Kunimoto K., et al. A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production. Nat Commun. 2022;13(1):5491.

18. Tranguch S., Chakrabarty A., Guo Y., et al. Maternal pentraxin 3 deficiency compromises implantation in mice. Biol Reprod. 2007;77(3):425-432.

19. Huang K., Maruyama T., Fan G. The naive state of human pluripotent stem cells: a synthesis of stem cell and preimplantation embryo transcriptome analyses. Cell Stem Cell. 2014;15(4):410-415.

20. Franasiak J.M., Forman E.J., Hong K.H., et al. Aneuploidy across individual chromosomes at the embryonic level in trophectoderm biopsies: changes with patient age and chromosome structure. J Assist Reprod Genet. 2014;31:1501-1509.