Background and Aim: To compare the fertilization, blastocyst utilization, clinical pregnancy rate (cPR), implantation rate (IR) and on-going clinical pregnancy rate between fresh versus frozen-thawed micro-epididymal aspirated sperm (MESA) in intra-cytoplasmic sperm injection (ICSI) cycle. Methods: A retrospective study of a total of 27 MESA-ICSI cycles done from the year 2019 to 2022 was carried out. Patients were categorized into fresh MESA-ICSI group (n=16) and frozen-thawed MESA-ICSI group (n=11). The maternal mean age of both groups was 33.0 and 32.5 respectively. For fresh MESA-ICSI group, retrieved sperm were being injected on the same day while frozen-thawed MESA-ICSI group, sperm sample were being frozen after the MESA procedure and utilized in the future. Sample was processed using sperm medium (COOK MEDICALⓇ). All embryos were cultured in time-lapse monitoring incubator and assessed according to modified Gardner’s grading system. On day 5/6, blastocyst range from grade A to C will be vitrified for frozen embryo transfer (FET). A total of 22 FET procedures were done, and statistical analysis was performed using Fisher’s exact test, p<0.05. The mean number of embryos transferred for MESA-ICSI group and frozen-thawed MESA-ICSI group are 1.43 and 1.13 respectively. Results: There is no significant difference in the fertilization rate between fresh MESA-ICSI, 71.1% (106/149) and frozen-thawed MESA-ICSI group, 65.4% (70/107). The blastocyst utilization rate between fresh and frozen-thawed MESA-ICSI group are also similar, 45.3% (48/106) and 51.4% (36/70) respectively. Besides, the cPR shown higher in the fresh MESA-ICSI group, 64.3% (9/14) compared to frozen-thawed MESA-ICSI group, 37.5% (3/8). The IR, 50.0% (10/20) and on-going pregnancy rate, 57.1% (8/14) were higher in the fresh MESA-ICSI group compared to frozen-thawed MESA-ICSI group, 33.3% (3/9) and 12.5% (1/8) respectively, p<0.05. Conclusion: The results show the trend of higher cPR, IR and on-going pregnancy rate in fresh MESA-ICSI group compared to frozen-thawed MESA-ICSI group. However, further study is required to conduct in a larger population in order to obtain a significant result.