Day 5/6 sequential hatching/biopsy versus day 3 hatching before blastocyst biopsy: systematic review and meta-analysis

This systematic review and meta-analysis evaluated whether day 5/6 sequential hatching/biopsy immediately before trophectoderm biopsy leads to different preimplantation genetic testing (PGT) and embryo transfer outcomes compared with day 3 artificial hatching. Following PRISMA guidelines, PubMed, EMBASE and the Cochrane Library were searched on 15 January 2026. Two randomised controlled trials (RCTs) and three cohort studies (one a conference abstract) met the inclusion criteria; study types were analysed separately due to methodological heterogeneity. Compared with day 3 artificial hatching, day 5/6 sequential hatching/biopsy was associated with higher probability of post-warming survival (1 cohort study, OR=5.50, 95%CI=2.79-10.85, P<0.001), clinical pregnancy (2 RCTs, pooled OR=1.92, 95%CI=1.02-3.64, P=0.04; 2 cohort studies, pooled OR=1.51, 95%CI=1.25-1.83, P<0.001) and live birth (2 RCTs, pooled OR=1.86, 95%CI=1.30-2.67, P<0.001; 1 cohort study, OR=1.44, 95%CI=1.19-1.75, P<0.001) following embryo transfers. No significant differences were observed in ongoing pregnancy or miscarriage rates. However, day 5/6 sequential hatching/biopsy was associated with increased mosaicism (2 RCTs, pooled OR=2.50, 95%CI=1.97–3.17, P<0.001) and no-PGT-result reporting (2 RCTs, pooled OR=5.84, 95%CI=3.09-11.01, P<0.001), despite comparable euploidy and aneuploidy rates. Our findings suggest day 5/6 sequential hatching/biopsy may be associated with improved embryo transfer outcomes compared with day 3 artificial hatching, while the increased reporting of mosaicism and no-PGT-result may have limited clinical implications given their low absolute incidence.