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Updated: Jul 2, 2026
Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis
Published on: September 7, 2021
Comparative study of noninvasive chromosomal screening using blastocyst culture media: frozen-thawed embryos
Xiaojun Wen1, Junye Huo1, Zhanhui Ou1
1Reproductive Medicine Center, Boai Hospital of Zhongshan, Zhongshan, Guangdong, China.
Background:
Preimplantation genetic testing for aneuploidy (PGT-A) can improve embryo selection in assisted reproductive technology; however, the trophectoderm (TE) biopsy required for testing is invasive and may compromise embryo integrity. While noninvasive chromosomal screening (NICS) using cell-free DNA from spent embryo culture medium (SECM) is a promising alternative, its research in frozen-thawed embryos remains limited. We systematically evaluated and compared the detection performance of SECM-based NICS in fresh and frozen-thawed embryos and explored its clinical application potential. Overall, 104 SECM samples from 27 couples who were undergoing intracytoplasmic sperm injection-PGT-A were analyzed (65 from the fresh group and 39 from the frozen-thawed group). TE biopsy results were used as the gold standard for assessing NICS efficacy. A paired-sample design was employed to conduct synchronous sequencing analyses of TE biopsy cells, fresh SECM, frozen-thawed SECM, and whole-embryo samples from 24 embryos.
Results:
The overall detection success rate of the NICS was 98.08%. When TE biopsy was used as the gold standard, the overall sensitivity, specificity, and clinical consistency of the NICS were 88.46%, 50%, and 79.41%, respectively. Stratified analysis showed that, for SECM-NICS, frozen-thawed embryos demonstrated higher DNA library concentrations (P = 0.046), sensitivity (97.30% vs 78.05%, P = 0.008), positive predictive value (PPV; 100% vs 72.73%, P < 0.01), and clinical consistency (97.37% vs 68.75%, P = 0.001), with a lower rate of complete chromosomal inconsistency (2.63% vs 35.94%, P < 0.01), compared with fresh embryos. The high-concentration group (≥10 ng/µL) exhibited superior PPV and clinical consistency. Paired-sample analysis confirmed a high consistency between the TE biopsy and whole-embryo analysis results (clinical consistency: 100%). When whole-embryo sequencing was used as the absolute gold standard, the frozen-thawed SECM group showed a significantly lower rate of complete chromosomal inconsistency than the fresh group (4.35% vs 33.33%, P = 0.023). Mosaics were a key contributor to diagnostic discordance.
Conclusion:
The detection performance of NICS based on frozen-thawed SECM demonstrated superior sensitivity, PPV, and consistency with the actual chromosomal status compared with that using fresh embryos; this approach, as a noninvasive method, demonstrates potential for genetic assessment of cryopreserved embryos, potentially avoiding the need for secondary, invasive biopsies.
