Abstract Study question Is the incidence of tripronuclear zygotes (3PN) in IVF cycles influenced by clinical, patient and treatment parameters? Summary answer Tripronuclear zygotes incidence in IVF cycles was influenced by Antral Follicle Count, number of oocytes retrieved, sperm progressive motility, and the use of fresh semen. What is known already Tripronuclear zygotes are indicative of abnormal fertilisation and are not routinely considered suitable for transfer. It is therefore important to optimise their detection and minimise their occurrence. Tripronuclear zygotes can originate as a result of the combination of one maternal and two paternal sets of chromosomes (fertilisation of a haploid oocyte by two haploid spermatozoa or by one diploid spermatozoon) or the combination of one paternal and two maternal sets of chromosomes (fertilisation of a diploid oocyte by one haploid spermatozoon) (1). Study design, size, duration This is a retrospective study of 550 IVF cycles performed between April 2018- April 2021, in a single UK clinic. IVF cycles either within (group 1) or exceeding (group 2) the key performance indicator (KPI) for the incidence of 3PN (<6%) (2), were assessed for associations with quantitative cycle variables. Proportion of 3PN per cycle was assessed for associations with qualitative variables. Cycles using donor oocytes (n = 73) were excluded for analyses assessing patient (maternal) parameters. Participants/materials, setting, methods Continuous variables including age, body mass index, Antral Follicle Count (AFC), anti-Mullerian hormone (AMH), oocytes recovered, timing from sperm production to insemination or oocyte recovery to insemination, sperm concentration, progressive motility and morphology were analysed by Mann-Whitney U Test. Qualitative variables including patient ethnicity, own/donor oocyte source, fresh/frozen sperm, stimulation regimen and oocyte recovery needle were analysed by logistic regression assuming binomial errors. Significance was assumed at p < 0.05. Main results and the role of chance There was no difference between groups where 3PN incidence exceeded or fell within the KPI when procedure timings and clinical parameters were assessed. There was also no relationship between patient characteristics (age, body mass index, AMH levels, ethnicity, oocyte source (own/donor)), semen concentration or semen morphology and the proportion of 3PN per cycle. However, number of oocytes retrieved in group 2 was significantly higher than group 1 (group 1, n cycles = 307, median oocytes retrieved = 8; group 2, n cycles = 170, median oocytes retrieved = 12 [95% CI -4, -1]) (p < 0.001). Similarly, AFC was significantly increased in group 2 (group 1, n cycles = 254, median AFC = 11; group 2, cycles n = 150, median AFC = 13 [95% CI -4, -1]) (p = 0.009). Likewise, the mean sperm motility parameters were higher for cycles exceeding the 6% 3PN KPI, with 64.6 ± 0.91% vs 68.8 ± 1.11% total motility (p = 0.004), and 57.1 ± 0.97% vs 61.7 ± 1.2% progressive motility (p = 0.003) for group 1 and 2 respectively. Proportion of 3PN per cycle was also significantly higher with fresh semen compared to frozen (6.6 ± 0.39% vs 4.7 ± 0.51%) (p = 0.006). Limitations, reasons for caution This is a retrospective study of a single UK clinic. Further analysis, expanding sample size, are required to confirm these findings. Wider implications of the findings Our finding that number of oocytes recovered influences 3PN incidence is in agreement with previous studies (3, 4). Fresh and high progressively motile sperm may be more capable of zona pellucida penetration leading to polyspermy and 3PN. Adjustment of sperm concentration at insemination could be considered in higher risk circumstances. Trial registration number na