Double Density Gradient Centrifugation as a Routine Sperm Preparation Method for In Vitro Fertilization in Males With Extremely Severe Oligospermia

Background: In males with extremely severe oligospermia (MESO), single density gradient centrifugation (SDGC) has low sperm enrichment efficiency, making intracytoplasmic sperm injection (ICSI) challenging. This study aimed to determine whether double density gradient centrifugation (DDGC) can efficiently enrich sperm from MESO samples and whether these sperm are safe for clinical use. Methods: MESO was defined as having ≤2000 motile sperm/mL of semen, whereas males with severe oligospermia (MSO) were defined as having 2000–10,000 motile sperm/mL. We compared sperm recovery between SDGC and DDGC in MESO samples and retrospectively analyzed in vitro fertilization (IVF) data from 39 MESO cases (sperm prepared using DDGC) and 78 MSO cases (sperm prepared using SDGC) collected from 2017 to 2023. The SDGC group served as the control group. Results: The results showed that the sperm recovery rate of DDGC was approximately three-fold higher than that of SDGC in MESO samples. We hypothesized that in normal semen samples, sperm aggregate into a pellet during centrifugation, enabling efficient enrichment by SDGC. In MESO, where sperm count is extremely low, sperm fail to form a pellet, leading to slower sedimentation and lower recovery rates with SDGC, thereby necessitating additional centrifugation. Importantly, sperm prepared by DDGC from MESO semen samples showed comparable in vitro and in vivo embryo developmental parameters to sperm prepared by SDGC. Interestingly, the DDGC group showed a significantly higher usable blastocyst formation rate compared to SDGC group (73.48% vs. 62.63%, p = 0.009). Conclusions: In conclusion, DDGC can effectively enrich sperm from MESO samples, and no obvious adverse clinical outcomes were observed. The method is simple, requires no additional equipment, and may be considered as a routine sperm preparation technique for MESO in clinical use. However, the long-term safety of using DDGC for sperm preparation from MESO for ICSI still requires further confirmation, and more effective methods for sperm enrichment from MESO are needed.