{"title":"无创植入前基因检测的作用-利用废培养基非整倍体及其与滋养外胚层活检的一致性：概念证明和验证研究。","authors":"Neeta Singh, Ankita Sethi, Lata Rani, Monika Saini, Ritu Gupta, Neena Malhotra, Reeta Mahey","doi":"10.4103/jhrs.jhrs_192_24","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>While trophectoderm (TE) biopsy with next-generation sequencing (NGS) remains the gold standard for preimplantation genetic testing for aneuploidy (PGT-A), the discovery of cell-free DNA (cfDNA) in spent culture media (SCM) has sparked interest in non-invasive PGT-A (NiPGT-A) as a potential alternative.</p><p><strong>Aim: </strong>The study was conducted to assess the feasibility of cell cfDNA from SCM as a tool for NiPGT-A in patients undergoing IVF for advanced age, repeated implantation failure or severe male factor infertility.</p><p><strong>Settings and design: </strong>This is a prospective study where a total of 44 embryos having TE biopsy for aneuploidy testing and their respective SCM collected at day 5/6 were analysed.</p><p><strong>Materials and methods: </strong>All aneuploid blastocysts (WB) were subjected to DNA extraction and amplification using Sureplex DNA amplification system followed by library preparation using VeriSeq™ PGS Library Prep kit and sequencing on MiSeq (Illumina, California, USA).</p><p><strong>Statistical analysis used: </strong>Copy Number Variation visualisation and analysis were carried out using BlueFuse Multi Software (Illumina). The statistical data were analysed by STATA version 14.</p><p><strong>Results: </strong>Informative results were obtained in 36/44 (81.2%) SCM samples. The reads were analysable in 26 paired (SCM and TE biopsy) samples. Concordant NGS results for both TE biopsy and SCM sample were obtained in 17/26 (65.38%) embryos. The per chromosome concordance rate was 85.13% (487/572) and the sex chromosome concordance rate was 73% (19/26). The sensitivity and specificity of NiPGT-A were 66.6% and 60%, respectively. On comparing the ploidy concordance rate, poor morphology embryos had better, but not statistically significant concordance rate (83.33%) as compared to good morphology embryos (50%, <i>P</i> = 0.16). Although not significant, day 6 embryos had better per chromosome as well as sex chromosome concordance rate as compared to day 5 embryos.</p><p><strong>Conclusion: </strong>Aneuploidy testing using cf DNA in SCM is a promising technique but needs more research on larger cohort size to improve the sensitivity, specificity and concordance rate.</p>","PeriodicalId":15975,"journal":{"name":"Journal of Human Reproductive Sciences","volume":"18 2","pages":"70-79"},"PeriodicalIF":1.1000,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12306720/pdf/","citationCount":"0","resultStr":"{\"title\":\"Role of Non-invasive Preimplantation Genetic Testing-Aneuploidy Using Spent Culture Media and its Concordance with Trophectoderm Biopsy: A Proof of Concept and Validation Study.\",\"authors\":\"Neeta Singh, Ankita Sethi, Lata Rani, Monika Saini, Ritu Gupta, Neena Malhotra, Reeta Mahey\",\"doi\":\"10.4103/jhrs.jhrs_192_24\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>While trophectoderm (TE) biopsy with next-generation sequencing (NGS) remains the gold standard for preimplantation genetic testing for aneuploidy (PGT-A), the discovery of cell-free DNA (cfDNA) in spent culture media (SCM) has sparked interest in non-invasive PGT-A (NiPGT-A) as a potential alternative.</p><p><strong>Aim: </strong>The study was conducted to assess the feasibility of cell cfDNA from SCM as a tool for NiPGT-A in patients undergoing IVF for advanced age, repeated implantation failure or severe male factor infertility.</p><p><strong>Settings and design: </strong>This is a prospective study where a total of 44 embryos having TE biopsy for aneuploidy testing and their respective SCM collected at day 5/6 were analysed.</p><p><strong>Materials and methods: </strong>All aneuploid blastocysts (WB) were subjected to DNA extraction and amplification using Sureplex DNA amplification system followed by library preparation using VeriSeq™ PGS Library Prep kit and sequencing on MiSeq (Illumina, California, USA).</p><p><strong>Statistical analysis used: </strong>Copy Number Variation visualisation and analysis were carried out using BlueFuse Multi Software (Illumina). The statistical data were analysed by STATA version 14.</p><p><strong>Results: </strong>Informative results were obtained in 36/44 (81.2%) SCM samples. The reads were analysable in 26 paired (SCM and TE biopsy) samples. Concordant NGS results for both TE biopsy and SCM sample were obtained in 17/26 (65.38%) embryos. The per chromosome concordance rate was 85.13% (487/572) and the sex chromosome concordance rate was 73% (19/26). The sensitivity and specificity of NiPGT-A were 66.6% and 60%, respectively. On comparing the ploidy concordance rate, poor morphology embryos had better, but not statistically significant concordance rate (83.33%) as compared to good morphology embryos (50%, <i>P</i> = 0.16). 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引用次数: 0
摘要
背景：虽然用下一代测序（NGS）进行滋养外胚层（TE）活检仍然是非整倍体（PGT-A）着床前基因检测的金标准，但在废培养基（SCM）中发现无细胞DNA （cfDNA）引发了人们对非侵入性PGT-A （NiPGT-A）作为潜在替代方法的兴趣。目的：本研究旨在评估SCM细胞cfDNA在高龄、反复着床失败或严重男性因素不育的IVF患者中作为NiPGT-A工具的可行性。背景和设计：这是一项前瞻性研究，共44个胚胎进行TE活检进行非整倍体检测，并在第5/6天收集各自的SCM进行分析。材料和方法：所有非整倍体囊胚（WB）采用Sureplex DNA扩增系统进行DNA提取和扩增，随后使用VeriSeq™PGS library Prep试剂盒制备文库，并在MiSeq （Illumina, California， USA）上测序。采用统计分析：使用BlueFuse Multi Software （Illumina）进行拷贝数变异可视化和分析。统计数据采用STATA version 14进行分析。结果：在36/44例（81.2%）SCM样本中获得了信息丰富的结果。在26对（SCM和TE活检）样本中可分析读数。17/26（65.38%）胚胎TE活检和SCM样本的NGS结果一致。每条染色体的一致性率为85.13%(487/572)，性染色体的一致性率为73%（19/26）。NiPGT-A的敏感性和特异性分别为66.6%和60%。形态差胚的倍性一致性率为83.33%，而形态好胚的倍性一致性率为50%，P = 0.16，但差异无统计学意义。虽然不显著，但第6天胚胎的每条染色体和性染色体的一致性率高于第5天胚胎。结论：利用cf DNA检测SCM非整倍体是一种很有前景的技术，但需要更多的研究来提高灵敏度、特异性和一致性。
Role of Non-invasive Preimplantation Genetic Testing-Aneuploidy Using Spent Culture Media and its Concordance with Trophectoderm Biopsy: A Proof of Concept and Validation Study.
Background: While trophectoderm (TE) biopsy with next-generation sequencing (NGS) remains the gold standard for preimplantation genetic testing for aneuploidy (PGT-A), the discovery of cell-free DNA (cfDNA) in spent culture media (SCM) has sparked interest in non-invasive PGT-A (NiPGT-A) as a potential alternative.
Aim: The study was conducted to assess the feasibility of cell cfDNA from SCM as a tool for NiPGT-A in patients undergoing IVF for advanced age, repeated implantation failure or severe male factor infertility.
Settings and design: This is a prospective study where a total of 44 embryos having TE biopsy for aneuploidy testing and their respective SCM collected at day 5/6 were analysed.
Materials and methods: All aneuploid blastocysts (WB) were subjected to DNA extraction and amplification using Sureplex DNA amplification system followed by library preparation using VeriSeq™ PGS Library Prep kit and sequencing on MiSeq (Illumina, California, USA).
Statistical analysis used: Copy Number Variation visualisation and analysis were carried out using BlueFuse Multi Software (Illumina). The statistical data were analysed by STATA version 14.
Results: Informative results were obtained in 36/44 (81.2%) SCM samples. The reads were analysable in 26 paired (SCM and TE biopsy) samples. Concordant NGS results for both TE biopsy and SCM sample were obtained in 17/26 (65.38%) embryos. The per chromosome concordance rate was 85.13% (487/572) and the sex chromosome concordance rate was 73% (19/26). The sensitivity and specificity of NiPGT-A were 66.6% and 60%, respectively. On comparing the ploidy concordance rate, poor morphology embryos had better, but not statistically significant concordance rate (83.33%) as compared to good morphology embryos (50%, P = 0.16). Although not significant, day 6 embryos had better per chromosome as well as sex chromosome concordance rate as compared to day 5 embryos.
Conclusion: Aneuploidy testing using cf DNA in SCM is a promising technique but needs more research on larger cohort size to improve the sensitivity, specificity and concordance rate.
期刊介绍：
The Journal of Human Reproductive Sciences (JHRS) (ISSN:0974-1208) a Quarterly peer-reviewed international journal is being launched in January 2008 under the auspices of Indian Society of Assisted Reproduction. The journal will cover all aspects human reproduction including Andrology, Assisted conception, Endocrinology, Physiology and Pathology, Implantation, Preimplantation Diagnosis, Preimplantation Genetic Diagnosis, Embryology as well as Ethical, Legal and Social issues. The journal will publish peer-reviewed original research papers, case reports, systematic reviews, meta-analysis, and debates.
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