Effect of blastocyst morphology on the incidence of monozygotic twinning pregnancy after single blastocyst transfer: a retrospective cohort study

Background: Visual morphology-based selection for transfer is a currently common method with evaluation of blastocyst quality and prediction of pregnancy and live birth. The aim of this study is to explore whether blastocyst morphology [blastocyst stage, inner cell mass (ICM) and trophectoderm (TE) grading] has an impact on the occurrence of monozygotic twinning (MZT) after single blastocyst transfer (SBT). Methods: : All patients who obtained a clinical pregnancy after single blastocyst transfer between January 2015 and September 2021 were retrospectively included. Blastocyst morphology scores were assessed using Gardner grading system. Monozygotic twinning was defined as more than 1 gestational sac (GS), or 2 or more fetal heartbeats (FHB) in a single GS via ultrasound at 5-6 gestational weeks. MZT incidence associated with blastocyst morphological parameters was analyzed by multivariable logistic regression modeling. Results: : The overall MZT rate was 2.46% (227 of 9229 cases), of which was the highest in blastocysts of grade A TE and lowest in those with grade C TE (grade A: B: C= 3.40%: 2.67%: 1.58%, P =0.002). The higher risk of MZT pregnancy was associated with higher trophectoderm grading [grade A vs C: aOR, 1.883, 95% CI 1.069-3.315, P =0.028; grade B vs C: aOR, 1.559, 95% CI 1.066-2.279, P =0.022], but not with extended culture in vitro (day 5 vs day 6), vitrification (fresh vs frozen-thawed ET), assisted hatching (AH), blastocyst stage (stage 1-6) or ICM grading (A vs B). Conclusions: : TE grade is an independent risk factor of MZT after single blastocyst transfer. The effects of extended culture, AH, frozen-thawed procedures, blastocyst stage and ICM grade were not demonstrated.