BACKGROUND To prevent the transfer of embryos affected by monogenic conditions and/or chromosomal defects, preimplantation genetic testing (PGT) requires trophectoderm biopsy and cryopreservation. In 2–6% of biopsies, the diagnosis may be inconclusive due to DNA amplification failure or low-quality results. In these cases, a round of re-warming, re-biopsy, and re-cryopreservation is required to obtain a genetic diagnosis. In other cases, when the IVF centre starts providing PGT and/or when the patients develop an indication because of multiple failures, miscarriages or the birth of an affected child after IVF, cryopreserved untested embryos may be warmed, biopsied, and then re-vitrified. However, it is still unclear whether multiple manipulations may reduce reproductive outcomes after PGT. OBJECTIVE AND RATIONALE This study aimed at conducting a systematic review to investigate the available evidence on the safety of double biopsy and/or double cryopreservation–warming and provide recommendations in this regard. We performed meta-analyses of the differences in the reproductive outcomes (live birth per embryo transfer [LBR per ET], clinical pregnancy rate per ET [CPR per ET], and miscarriage rate per clinical pregnancy [MR per CP]) in double cryopreservation and single biopsy (CBC) or double biopsy and double cryopreservation (BCBC) flows vs the control single biopsy and single cryopreservation (BC) flow. Cryo-survival rates before ET and gestational and perinatal outcomes were also reported. SEARCH METHODS PRISMA guidelines were followed to gather all available information from the literature (PubMed, Scopus, and Embase). We used Medical Subject Headings (MeSH) terms and a list of specific keywords relevant for the study question. We searched for original studies in humans, published in peer-reviewed journals in English up to April 2024. Four independent authors assessed the articles for inclusion. One included paper was retrieved from another source. OUTCOMES A total of 4219 records were identified, and 10 studies were included in the meta-analysis. Certainty of evidence level ranged from low to moderate. Both the CBC and BCBC groups showed reduced reproductive outcomes compared to the control (BC). Specifically, live birth rates per embryo transfer were lower in the CBC group (OR: 0.56, 95% CI: 0.38–0.81, I2 = 58%; six studies) and the BCBC group (OR: 0.51, 95% CI: 0.34–0.77, I2 = 24%; six studies). CPR per ET were also lower in the CBC group (OR: 0.68, 95% CI: 0.51–0.92, I2 = 57%; seven studies) and the BCBC group (OR: 0.60, 95% CI: 0.46–0.78, I2 = 0%; seven studies). Additionally, MR per CPs were higher in both the CBC group (OR: 1.68, 95% CI: 1.02–2.77, I2 = 50%; seven studies) and the BCBC group (OR: 2.08, 95% CI: 1.13–3.83, I2 = 28%; seven studies). Cryo-survival as well as gestational and perinatal outcomes were within the expected norms in the studies reporting them. WIDER IMPLICATIONS Improved genetic technologies, standardization of laboratory protocols, operators’ proficiency with biopsy and cryopreservation, and continuous monitoring of the performance are essential to minimize inconclusive diagnoses and the putative impact of additional embryo manipulations. Although poorer reproductive outcomes might result from double biopsy and/or double cryopreservations, these practices may still be worthwhile to avoid transferring affected/aneuploid blastocysts. Therefore, the risks must be weighed against the potential benefits for each specific couple. REGISTRATION NUMBER PROSPERO (ID: CRD42024503678)

中文翻译：

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PGT 中双滋养外胚层活检和/或冷冻保存的系统评价和荟萃分析：平衡诊断需求与伤害风险


背景 为了防止受单基因条件和/或染色体缺陷影响的胚胎移植，植入前基因检测 （PGT） 需要滋养外胚层活检和冷冻保存。在 2-6% 的活检中，由于 DNA 扩增失败或结果质量低，诊断可能不确定。在这些情况下，需要进行一轮重新保温、重新活检和重新冷冻保存才能获得基因诊断。在其他情况下，当 IVF 中心开始提供 PGT 和/或当患者因多次失败、流产或 IVF 后受影响儿童出生而出现适应症时，冷冻保存的未经测试的胚胎可能会被加热、活检，然后重新玻璃化。然而，目前尚不清楚多次操作是否会降低 PGT 后的生殖结局。目的和基本原理 本研究旨在进行系统评价，以调查关于双重活检和/或双重冷冻保存-加温安全性的现有证据，并在这方面提供建议。我们对双重冷冻保存和单次活检 （CBC） 或双重活检和双重冷冻保存 （BCBC） 流与对照单次活检 （BCC） 流中生殖结局 （每次胚胎移植的活产率 [LBR per ET] 、每次 ET 的临床妊娠率 [CPR 每次 ET] 和每次临床妊娠的流产率 [MR per CP] 的差异进行了荟萃分析。还报告了 ET 前的冷冻存活率以及妊娠和围生儿结局。检索方法 遵循 PRISMA 指南从文献 （PubMed、Scopus 和 Embase） 中收集所有可用信息。我们使用了医学主题词 （MeSH） 术语和与研究问题相关的特定关键词列表。 我们检索了截至 2024 年 4 月发表在同行评审英文期刊上的人类原始研究。四位独立作者评估了这些文章的纳入情况。其中一篇论文是从另一个来源检索到的。结果 共确定 4219 条记录，其中 10 项研究纳入荟萃分析。证据质量从低到中等不等。与对照组 （BC） 相比，CBC 和 BCBC 组均显示生殖结局降低。具体而言，CBC 组（OR：0.56,95% CI：0.38-0.81，I2 = 58%;6 项研究）和 BCBC 组（OR：0.51,95% CI：0.34-0.77，I2 = 24%;6 项研究）的每次胚胎移植活产率较低。CBC 组（OR：0.68,95% CI：0.51-0.92，I2 = 57%;7 项研究）和 BCBC 组（OR：0.60,95% CI：0.46-0.78，I2 = 0%;7 项研究）每 ET 的 CPR 也较低。此外，CBC 组（OR：1.68,95% CI：1.02-2.77，I2 = 50%;7 项研究）和 BCBC 组（OR：2.08,95% CI：1.13-3.83，I2 = 28%;7 项研究）的每 CP MR 较高。冷冻生存率以及妊娠期和围生期结局在报告它们的研究中处于预期正常范围内。更广泛的影响 改进的遗传技术、实验室方案的标准化、操作者对活检和冷冻保存的熟练程度以及对性能的持续监测对于最大限度地减少不确定的诊断和额外胚胎操作的假定影响至关重要。尽管双重活检和/或双重冷冻保存可能会导致较差的生殖结果，但这些做法可能仍然值得避免转移受影响的/非整倍体囊胚。因此，必须权衡风险与每对特定夫妇的潜在好处。注册号 PROSPERO （ID： CRD42024503678）