Abstract Study question Do the anti-centromere antibodies (ACA) affect the multi pronuclear formation (MPF) rate? Summary answer MPF rate was higher in patients with ACA. The MPF rate of ACA patients with antibody titers ≥160-fold was significantly higher than in titers 40-fold. What is known already ACA is an anti-nuclear antibody (ANA), and specifically recognizes the centromere. Recently, several studies have reported that the rate of oocyte maturation is lower in patients with ACA. Moreover, in MI oocytes collected from ACA patients, female chromosomes were frequently dispersed in the cytoplasm. Study design, size, duration A total of 4756 patients from our clinic were tested for ANA before oocyte retrieval from January 2014 to December 2021. After retrospective ANA testing, patients were classified according to 3 groups; 62 ACA patients (with only ACA), 1134 non-ACA patients (with ANA but not with included ACA) and 3560 non-ANA patients (without ACA and any ANA).We considered ACA-positive levels at titers ≥40. ACA patients were further classified into 6 groups by antibody titer. Participants/materials, setting, methods The MPF rate after ICSI was compared in the 3 groups (ACA patients, non-ACA patients, non-ANA patients) and at 6 ACA titers in each group (titer of ACA; 40-fold, 80-fold, 160-fold, 320-fold, 640-fold, ≥1280-fold).MPF rate was calculated by dividing the number of embryos that formed three or more pronuclei by the number of embryos inseminated by ICSI.Ryan's method was used for multiple comparisons of ratios. Main results and the role of chance MPF rate was 3.8% (1997/53240) in non-ANA patients, 4.3% (733/17003) in non-ACA patients, and 32.1% (351/1092) in ACA patients, being significantly higher in ACA vs other groups (P<.01). In comparisons between ACA titers, MPN rate was 8.7% (4/46) at 40-fold , 13.0% (3/23) at 80-fold , 36.1% (56/155) at 160-fold , 32.4% (48/148) at 320-fold , 36.0% (111/308) at 640-fold , and 31.3% (129/412) at ≥ 1280-fold, respectively. MPN rate of patients with titers ≥160-fold was significantly higher than with 40-fold. Limitations, reasons for caution A potential limitation of the present study is the small sample size. This is because ACA patients account for only 1% of patients who underwent ART treatments. Wider implications of the findings MPF rate in ACA patients was significantly higher than in non-ANA and non-ACA patients and was also significantly higher in ACA patients with titers of ≥ 160-fold vs 40-fold. The dispersion of the female chromosome in the cytoplasm of MI oocytes may be a cause of MPN formation. Trial registration number None