Abstract Study question What are the pregnancy and perinatal outcomes of twice-cryopreserved embryos compared to once-cryopreserved embryos? Summary answer Transfers of twice-cryopreserved embryos result in similar live birth rates (LBR) and perinatal outcomes compared to transfers of once-cryopreserved embryos. What is known already Repeated cryopreservation of viable surplus embryos in frozen embryo transfer (FET) cycles is a potential method to increase the cumulative pregnancy rate and reduce the risks related to multiple pregnancies. Currently, evidence on the safety and success of repeated cryopreservation is limited. Existing data from a few studies indicate that the vitrification of previously slow-frozen or vitrified embryos does not negatively impact pregnancy outcome, and no long-term health consequences in neonates have been reported. However, due to the limited number of reported pregnancies and children, more studies are needed. Study design, size, duration This retrospective register-based case-control study included FETs (n = 2834) performed at the University Hospital of Turku and the Central Hospital of Central Finland, Finland, between January 2012 and December 2019. The case group consisted of twice-cryopreserved FETs (n = 89), and the control group consisted of once-cryopreserved FETs (n = 304). The matching criteria were embryonic age at transfer and female age category of less or over 35 years. Participants/materials, setting, methods All the FETs in the case group and 86% in the control group were single-embryo transfers (p = <0.001). The first cryopreservation was performed by slow freezing or vitrification (cases 58% vs 42% and controls 40% vs 60%, p = 0.002, respectively). The re-cryopreservation method was vitrification. Mixed effects logistic regression was used to analyse the pregnancy outcomes, and a linear mixed model was used to analyse neonatal weight, adjusting for gestational age, neonatal sex, parity and BMI. Main results and the role of chance The survival rate of the twice-cryopreserved embryos was 92.2% (94/102), and 93.7% (89/95) of the planned FETs could be carried out. FET was performed with D3–4 embryos in 17 cases and 68 controls and with D5–6 embryos in 72 cases and 238 controls. The rates of live birth, clinical pregnancy and miscarriage in the case and the control groups were comparable (27.0% vs 31.9%, p = 0.35; 31.4% vs 36.8%, p = 0.35 and 4.5% vs. 3.9%, p = 0.77, respectively). No difference was seen in the preterm delivery rate (cases 4.2% vs controls 10.3%, p = 0.69). Twenty-five children were born in the case group and 100 in the control group. No differences in birth weights were detected between the groups (3730 g, upper and lower quartiles 3500 g and 4050 g, vs 3490g, upper and lower quartiles 3150 g and 3900 g, p = 0.28), and, in the case group, all the newborns’ birth weights were appropriate for gestational age. There were no congenital malformations among the newborns in the case group. In the control group, there was one pregnancy termination due to aneuploidy, one case of undescended testicles, one child with a hypoplastic aortic valve without stenosis and one child with craniosynostosis. Limitations, reasons for caution This study was retrospective, and the small sample size limits interpretation of the results. FET has been demonstrated to increase the risk for fetal macrosomia and gestational hypertension/pre-eclampsia. Whether repeated cryopreservation enhances these effects or influences neonatal health in the long term needs further investigation. Wider implications of the findings: Acceptable LBR and neonatal outcomes may be expected after transfer of twice-cryopreserved embryos. Also, the survival rate is high. To avoid embryo wastage or transfer of multiple embryos, good quality surplus embryos from FET cycles may be considered for repeated cryopreservation by vitrification. Trial registration number Not applicable