The efficacy of assisted hatching has been widely debated. The variable results reported for AH are confounded by the numerous methods of performing AH, including mechanical partial zona dissection, acid tyrode’s, and more recently by laser assistance. Assisted hatching has most commonly been performed on Day 3 embryos. This study assesses if Day 2 laser assisted hatching can improve implantation rates for fresh blastocyst transfers. Prospective observational cohort. On the morning of Day 2 of culture, all embryos were sorted into groups (<4-cell, 4-cell and >4-cells). Those in the AH group underwent laser assisted hatching using an Octax laser (4.0ms) on the morning of Day 2 at the time of embryo check. All embryos were then cultured undisturbed until assessment on the morning of Day 5 when the highest quality embryo(s), based on morphology, were selected for embryo transfer. A total of 446 fresh Day 5 transfers between Jan 2016 - Mar 2019 were analyzed (244 transfers with AH and 202 transfers without AH). A total of 682 embryos were transferred in the 446 cycles (363 embryos in the AH group and 319 embryos in the non-AH group). Because 206 of the 446 transfers involved transfer of more than 1 embryo, a mixed model accounting for both fixed and random effects (i.e. repeated measurements) was used, with SAC modeled as a function of the fixed effects assisted hatching (AH), age, and body mass index (BMI). Day 2 assisted hatching is associated with successful implantation (p=0.036). As expected, age was negatively associated with implantation rate (p<0.0001). BMI was not. The R-square for the model was 0.52, and the variance component of the random effects (owing to multiple embryo transfers) was 0.082 (95% CI [0.046-0.11]).Tabled 1No AHWith AHTotal# Fresh Day 5 Transfers202244446Mean Maternal Age35.634.435.0Mean BMI26.827.026.9Mean # Embryos Transferred1.581.491.53# Embryos319363682# Sacs137194331Implantation Rate (%)42.9%53.4%48.5% Open table in a new tab Large data sets such as the SART database do not allow for evaluation of the impact of specific AH techniques. Certainly, any embryo handling and exposure has the potential to be detrimental to blastocyst development and implantation rates. Here, we show that Day 2 laser AH can lead to a significant increase in implantation rates from 42.9% without AH to 53.4% with AH. Day 2 embryos generally have larger perivitelline space which may allow for reduced peripheral laser exposure to the blastomeres. Further studies are needed to confirm if Day 2 laser AH confers benefit over Day 3 laser AH.