Sperm enrichment from poor semen samples by double density gradient centrifugation in combination with swim-up for IVF cycles

Abstract

Sperm preparation in IVF cycles using density gradient centrifugation (DGC) in combination with swim-up (SU) has been widely adopted in reproductive centres worldwide. It is a fact that the sperm recovery rate following one DGC from poor semen samples (showing liquefaction defects/containing too many unresolvable clots or rare sperm) is relatively low. Our results showed that double DGC (DDGC) is effective at increasing the sperm recovery rate from poor semen samples. However, DDGC may increase the mechanical stress of sperm, thereby potentially impairing embryo development. Therefore, it is necessary to evaluate the safety of using sperm prepared by DDGC/SU for IVF cycles. In this study, we retrospectively analysed the data generated from a total of 529 IVF cycles (from June 2017 to June 2018), and these IVF cycles contributed 622 transfer cycles (from June 2017 to December 2018) in Changzhou Maternal and Child Health Care Hospital. Of them, 306 IVF cycles and the related 355 transfer cycles (normal semen samples prepared by DGC/SU) were set as the normal group, while 223 IVF cycles and the related 267 transfer cycles (poor semen prepared by DDGC/SU) were set as the observation group. The main outcome measures, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, clinical pregnancy rate and live birth rate, birth weight and duration of pregnancy, were compared between the two groups. Compared to semen in the DGC/SU group, semen in the DDGC/SU group showed increased levels of the DNA fragmentation index (DFI) and reduced sperm concentration, percentage of progressive motility (PR) sperm, and percentage of normal morphology sperm. The indicators reflecting in vitro embryo development and clinical outcomes were similar in the DGC/SU group and DDGC/SU group, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, pregnancy rate, implantation rate, spontaneous abortion rate, live birth rate, birth weight and duration of pregnancy. Furthermore, we found that the 1PN zygote formation rate was significantly lower in the DDGC/SU group than that in the DGC/SU group. We concluded that oocytes fertilized by sperm from poor semen samples separated by DDGC/SU achieved the same outcomes as oocytes fertilized by sperm from normal semen separated by DGC/SU, suggesting that DDGC/SU is an effective and safe method of sperm enrichment for poor semen samples in IVF. The main contribution of the present study is the verification of the effectiveness of DDGC/SU in improving sperm recovery from poor semen samples and the safety of using sperm prepared by DDGC/SU for IVF.

Figure 1

A second DGC significantly improves the recovery rate of motile sperm from poor semen samples. Normal or poor semen samples underwent two consecutive DGCs (462 g for 15 min), (A) Representative images of sperm precipitation after the first DGC (left tube) and second DGC (right tube). (B) Ratio of the total sperm count in the second DGC to the first DGC. (C) Ratio of PR sperm in the second DGC to first DGC. Normal semen samples vs. poor semen samples; ***P < 0.001.

Figure 2

Work flow of DDGC. A total of 6 consecutive steps were involved in the entire process. Briefly, semen was placed on the density gradient column in the tube for the first DGC. After the first DGC, sperm precipitation was transferred into IVF medium. The semen in the tube on the column was subject to the second DGC. The sperm precipitation from the second DGC was also transferred to the abovementioned IVF medium. The sperm were then washed by centrifugation and re-suspended in 0.5 ml of IVF medium. Finally, sperm underwent swim-up procedure.