Non-invasive chromosome screening of embryos fertilized in vitro : A single center, retrospective study

Abstract

Objective: To evaluate the center’s experience with non-invasive preimplantation genetic testing for aneuploidy (NIPGT-A), focusing on embryo aneuploidy assessment, euploidy-linked pregnancy outcomes, and the relationship between embryo morphology and chromosomal status.

Methods: A retrospective study was conducted on couples undergoing in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles that yielded day-5 blastocysts eligible for NIPGT-A. Spent culture media from 159 blastocysts were analyzed using multiple annealing and looping-based amplification cycles (MALBAC)-based whole genome amplification followed by next- generation sequencing with a 10 Mb resolution threshold for copy- number variation detection. Embryo ploidy was categorized as euploid, low mosaic, high mosaic, or aneuploid based on cell- free DNA (cfDNA) abnormality levels. Euploid embryos were transferred in subsequent frozen embryo transfer (FET) cycles. Clinical outcomes included biochemical pregnancy, clinical pregnancy, ongoing pregnancy, and live birth rates.

Results: Fifty couples were included in this study. Among 213 blastocysts formed, 159 (74.7%) underwent NIPGT-A. Of these, 43.4% were euploid, 19.5% aneuploid, 23.3% mosaic, and 13.8% uninterpretable. A total of 82 embryos were transferred across 45 FET cycles, including euploid (69), mosaic (7), and uninterpretable (6) embryos. The clinical pregnancy rate was 53.3%, with a biochemical loss rate of 6.7% and early pregnancy failure of 4.4%. Ongoing pregnancy occurred in nearly half of the participants, and several successful outcomes were documented even among mosaic and uninterpretable embryos. Embryo morphology did not correlate significantly with euploidy, reaffirming the limited predictive value of morphologic grading alone.

Conclusion: NIPGT-A is a feasible, safe, and clinically meaningful alternative to invasive PGT-A, offering favorable pregnancy outcomes while avoiding embryo manipulation. Although technical limitations such as cfDNA variability and uninterpretable results persist, NIPGT-A enhances embryo selection, reduces miscarriage risk, and is particularly beneficial in low-yield or high-risk IVF populations. Continued refinement of laboratory protocols and long-term outcome studies are essential for broader clinical adoption.

Keywords

NIPGT-A / Spent culture media / Non-invasive genetic testing / IVF / Embryo selection / Aneuploidy / cfDNA / MALBAC / Next-generation sequencing

Conflict of interest statement

The authors declare that there are no conflict of interest.

Acknowledgements

The authors wish to thank the Institution for lending support.

Funding

No funding was obtained for this study.

Data availability statement

The raw data files will be available on request to the corresponding author.

Authors’ contributions

Dr. Gayathri K, consultant gynaecologist designed the study, analysed clinical and experimental data, and wrote the manuscript. Dr. Manipriya R, senior consultant gynaecologist evaluated the experimental data and revised the manuscript. All authors approved the final manuscript.

Publisher’ s Note

The Publisher of the Journal remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

[1] Franco JG Jr, Vagnini LD, Petersen CG, Renzi A, Canas MCT, Petersen B, et al. Noninvasive preimplantation genetic testing for aneuploidy (niPGT-A): The first Brazilian baby. JBRA Assist Reprod 2020; 24(4): 517-520.

[2] Forman EJ, Hong KH, Ferry KM, Tao X, Taylor D, Levy B, et al. In vitro fertilization with single euploid blastocyst transfer: A randomized controlled trial. Fertil Steril 2013; 100(1): 100-107.

[3] Rubio C, Rienzi L, Navarro-Sánchez L, Cimadomo D, García-Pascual CM, Albricci L, et al. Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: Concordance rate and clinical implications. Fertil Steril 2019; 112(3): 510-519.

[4] Xu J, Fang R, Chen L, Chen D, Xiao JP, Yang W, et al. Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization. Proc Natl Acad Sci U S A 2016; 113(42): 11907-11912.

[5] Vera-Rodriguez M, Diez-Juan A, Jimenez-Almazan J, Martinez S, Navarro R, Peinado V, et al. Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development. Hum Reprod 2018; 33(4): 745-756.

[6] Chen L, Sun Q, Xu J, Fu H, Liu Y, Yao Y, et al. A non-invasive chromosome screening strategy for prioritizing in vitro fertilization embryos for implantation. Front Cell Dev Biol 2021; 9: 708322.

[7] Brouillet S, Martinez G, Coutton C, Hamamah S. Is cell-free DNA in spent embryo culture medium an accurate source for preimplantation genetic testing? Reprod Biomed Online 2020; 40(6): 779-796.

[8] Yeung QSY, Zhang YX, Chung JPW, Lui WT, Kwok YKY, Gui B, et al. A prospective study of non-invasive preimplantation genetic testing for aneuploidies (NiPGT-A) using next-generation sequencing on spent culture media. J Assist Reprod Genet 2019; 36(8): 1609-1621.

[9] Sun B, Wang Y, Wen S, Zhou L, Zhang C, Wu Z, et al. Effectiveness of non-invasive chromosomal screening: Normal chromosomes and chromosomal rearrangements. Front Genet 2023; 14: 1036467.

[10] Yan J, Qin Y, Zhao H, Sun Y, Gong F, Li R, et al. Live birth with or without preimplantation genetic testing for aneuploidy. N Engl J Med 2021; 385(22): 2047-2058.

[11] Yang S, Xu B, Zhuang Y, Zhang Q, Li J, Fu X. Current research status and clinical applications of noninvasive preimplantation genetic testing: A review. Medicine 2024; 103: 40(e39964).

[12] Kim MK, Park JK, Jeon Y, Choe SA, Lee HJ, Kim J, et al. Correlation between morphologic grading and euploidy rates of blastocysts, and clinical outcomes in in vitro fertilization preimplantation genetic screening. J Korean Med Sci 2019; 34(4): e27.

[13] Bolton H, Graham SJL, van der Aa N, Kumar P, Theunis K, Fernandez Gallardo E, et al. Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential. Nat Commun 2016; 7: 11165.

[14] Rubio C, Rienzi L, Navarro-Sánchez L, Cimadomo D, García-Pascual CM, Albricci L, et al. Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: Concordance rate and clinical implications. Fertil Steril 2019; 112(3): 510-519.

[15] Fang R, Yang W, Zhao X, Xiong F, Guo C, Xiao J, et al. Chromosome screening using culture medium of embryos fertilised in vitro: A pilot clinical study. J Transl Med 2019; 17: 73.

[16] Sialakouma A, Karakasiliotis I, Ntala V, Nikolettos N, Asimakopoulos B. Embryonic cell-free DNA in spent culture medium: A non-invasive tool for aneuploidy screening of the corresponding embryos. In Vivo 2021; 35(6): 3449-3457.

[17] Kakourou G, Mamas T, Vrettou C, Traeger-Synodinos J. An update on non-invasive approaches for genetic testing of the preimplantation embryo. Curr Genomics 2022; 23(5): 337-352.

[18] Huang L, Bogale B, Tang Y, Lu S, Xie XS, Racowsky C. Noninvasive preimplantation genetic testing for aneuploidy in the spent medium may be more reliable than trophectoderm biopsy. Proc Natl Acad Sci USA 2019; 116(28): 14105-14112.

[19] He F, Yao YX, Wang J, Zhao DM, Wan AQ, Ren J, et al. Non-invasive chromosome screening for embryo preimplantation using cell-free DNA. Reprod and Dev Med 2022; 6(2): 113-120.

[20] Li X, Hao Y, Chen D, Ji D, Zhu W, Zhu X, et al. Non-invasive preimplantation genetic testing for putative mosaic blastocysts: A pilot study. Hum Reprod 2021; 36(7): 2020-2034.