Abstract Study question Are there more advantages in performing frozen embryo transfer (FET) immediately after ovarian stimulation or in delaying it by at least one menstruation? Summary answer In natural cycles, timing of frozen embryo transfer does not affect clinical pregnancy rates, but immediate transfer is associated with higher rates of cycle cancellation. What is known already In order to achieve a reduced time to pregnancy and avoid additional stress for the couple, some Centers initiate FETs immediately, i.e. on the first menstrual cycle after ovarian stimulation. However, the detrimental effect of hormonal stimulation on ovarian function and endometrial receptivity may persist in the following cycle and immediate FETs may increase the possibility of cycle cancellation and reduce pregnancy rate. Available evidence on this subject, mainly focused on endometrial preparation with hormonal replacement therapy (HRT), showed contrasting results. Moreover, studies on FETs performed on natural cycles are lacking. Study design, size, duration This is a retrospective study conducted at the Infertility Unit of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico in Milan, Italy. A large amount of single FETs, performed between 2014 and 2021, was considered and was then divided in two groups according to their timing (immediate or delayed). The primary outcome was to assess the clinical pregnancy rate (CPR); secondary outcomes were live birth rate (LBR) and the cycle cancellation rate. Participants/materials, setting, methods True natural cycles (T-NC) and modified cycles with the use of triggered ovulation (M-NC) protocols for endometrial preparation were pursued in women with regular menses; otherwise, HRT was used. Immediate FET was defined when performed on the first menstruation following ovarian stimulation (i.e. withdrawal bleeding occurring after ovarian stimulation or after a failed fresh embryo transfer). Delayed FETs included those performed after at least two menstruations. Only the first transfer after ovarian stimulation was considered. Main results and the role of chance A total of 5,765 FET cycles were included in the study. FET cycles were achieved through T-NC in 4,477 cases (78% of all FETs), of which 364 were immediate and 4,113 were delayed. M-NC cycles accounted for 1% of the total amount of FETs and HRT protocols were pursued in 11% of cases. The analysis showed no impact of FET timing on CPR both in the whole cohort and in the subgroups based on different endometrial preparation protocols. Focusing on T-NC cycles, the CPR in the immediate group was comparable to that of the postponed group (29 vs. 32% respectively, OR 0.90 [95% CI 0.71 - 1.14]). On the other hand, LBR in T-NC cycles was significantly lower in the immediate group than in the delayed group (20% vs. 25% respectively, OR 0.74 [95% CI 0.56 - 0.96]). Consequently, immediate FETs resulted to be associated with a significantly higher rate of pregnancy loss (31% vs. 21% respectively, p = 0.03). Furthermore, cycle cancellation occurred more frequently in the immediate T-NC group, compared with the delayed group (18% vs. 12% respectively, p = 0.003). Similar results were observed in HRT cycles (15% in immediate FETs vs. 7% in delayed FETs, p = 0.08). Limitations, reasons for caution The retrospective nature of this study involves a risk of selection bias. Moreover, the reason for cycle cancellation was not always available; thus, these data were not included in the analysis. Lastly, these outcomes are only applicable to a similar cohort, that is Centers following a non-elective freeze-all policy. Wider implications of the findings This is the largest investigation on T-NC FETs and is among the very few studies providing separate results on different endometrial preparation protocols. Evidence from this study is in line with some previous retrospective analyses and in contrast with others. Thus, randomized controlled trials are needed to confirm these results. Trial registration number not applicable