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Abstract Study question Is follicular flushing associated with oocyte maturity, fertilisation and embryo cleavage, in low responders? Summary answer In low responders, follicular flushing is negatively associated with oocyte maturity, but is not associated with oocyte fertilisation and embryo cleavage. What is known already Follicular flushing has been proposed to increase the number of cumulus-oocyte complexes (COCs) retrieved during oocyte collection. The number of times follicular flushing is performed is an arbitrary choice, ranging from 1-10 in the published literature. However, its association with oocyte maturity, fertilisation and cleavage has not been adequately evaluated. This is important in light of recent RCTs that used identical flow rates for both the follicular flushing and the single aspiration groups, showing an increase in the number of COCs retrieved with follicular flushing, across low, normal and hyper responders, as well as in patients with mono-follicular development. Study design, size, duration A single-center observational cohort study, in 209 low responders was performed between 12/2021 and 12/2023. Patients underwent ICSI after treated with recombinant-FSH and GnRH-antagonist and were included in the study only once. The primary outcome was oocyte maturity, whereas the secondary outcomes were oocyte fertilisation and embryo cleavage. Multivariable analysis was performed adjusting for key confounding factors. Statistical significance was set at a p-level of 0.05. Participants/materials, setting, methods Eligible patients had ≤5 follicles ≥ 11mm on the day of triggering final oocyte maturation, were <43 years, with BMI 18–35kg/m². Oocyte retrieval was performed 36h after triggering, using a 16G double-lumen needle set at 190mmHg and a flow rate of 0.42mL/sec. Retrieved COCs were categorized into groups based on whether they were retrieved in the initial-aspirate (IA), 1st, 2nd, 3rd, 4th, 5th flush. Each oocyte was individually cultured to evaluate its developmental potential. Main results and the role of chance In this study, 522 COCs from 209 low responders were analyzed. Of the collected COCs, 91% were retrieved by the second flush (IA: 39.46%, 1st :39.66%, 2nd: 11.88%, 3rd: 5.36%, 4th: 3.64%). Multivariable analysis indicated that the probability of oocyte maturity decreased from 1st to 4th flush compared to the IA (1st flush OR: 0.65, 95% CI: 0.42-1.01; 2nd flush OR: 0.63, 95% CI: 0.33- 1.19; 3rd flush OR: 0.37, 95% CI: 0.16- 0.84; 4th flush OR: 0.18, 95% CI: 0.07- 0.46). However, this reduction became statistically significant in the 3rd and 4th flushes compared to the IA (pvalue = 0.017; pvalue < 0.001 respectively). The rates of fertilization did not show statistically significant correlation with follicular flushing (1st flush OR: 1.10, 95% CI: 0.68–1.77; 2nd flush OR: 1.18, 95% CI: 0.57–2.44; 3rd flush OR: 2.87, 95% CI: 0.80–10.24; 4th flush OR: 1.77, 95% CI: 0.37–8.43), neither the embryo cleavage on day 2 (1st flush OR: 0.68, 95% CI: 0.15–3.02; 3rd flush OR: 0.34, 95% CI: 0.03–3.82). No ORs could be calculated for the 2nd and 4th flush regarding embryo cleavage. Limitations, reasons for caution The current study allows the evaluation of the effect of the number of times follicular flushing is performed on oocyte maturity, fertilisation and embryo cleavage. However, it does not provide data on the blastulation rates or pregnancy outcomes. Wider implications of the findings The current study suggests that in low responders, COCs retrieved after repeated flushing are less likely to be mature. However, once mature, they exhibit an equivalent likelihood of fertilization and cleavage compared to those retrieved during the initial aspiration. Trial registration number No