Comparison of zeta potential and physiological intracytoplasmic sperm injection in obtaining sperms with a lower DNA fragmentation index: A cross-sectional study

Abstract Background The sperm DNA fragmentation index (DFI) is one of the men's reproductive health criteria that affects assisted reproductive technique outcomes. Efforts in obtaining high-quality mature sperms seem to be necessary. Advanced sperm selection techniques (including physiological intracytoplasmic sperm injection [PICSI], zeta potential, microfluidic, etc.) have gained popularity in this regard. Objective The study aimed to compare the efficacy of zeta potential and PICSI sperm selection in obtaining sperms with better DNA integrity. Materials and Methods In this cross-sectional study, 48 couples were enrolled where the male partner had increased sperm DFI in his ejaculated sample and the female was in normal reproductive health. For each male partner, the semen sample was processed with zeta potential and PICSI techniques, then the sperm DFI of neat semen was compared to zeta and PICSI samples by the sperm chromatin dispersion test. Results Data showed that both the zeta potential and PICSI technique decreased sperm DFI in comparison with the neat semen sample (p < 0.001 for both). In addition, there was a statistically significant difference in sperm DFI between the PICSI and zeta potential samples (p < 0.01). Conclusion The current study showed that both zeta potential and PICSI could result in sperm with a lower DFI. However, PICSI seems to be superior to zeta potential in this regard.


Introduction
According to the World Health Organization definition, about 15% of couples face infertility and its related problems (1). Male fertility potential is traditionally assessed with a semen analysis, which is considered a cornerstone in the andrology clinic. However, about 15% of infertile men may have normal semen analysis (2).
Assessment of sperm DNA damage seems to be necessary for such patients. Previous studies have suggested that an increased sperm DNA fragmentation index (DFI) may lead to lower natural and intrauterine insemination pregnancy rates as well as compromised assisted reproduction technique (ART) outcomes (3,4).
There are reports about the temporal decline in semen quality and also increased use of ART techniques, worldwide (1,5 (14).
In the current study, we therefore aimed to compare the efficacy of PICSI sperm selection over zeta potential in obtaining sperms with better DNA integrity and chromatin compaction.

Sample collection
Semen samples were collected after 3 days of abstinence from intercourse by masturbation.
Each semen sample was divided into 3 parts: the 1 st part (group 1) included the unprocessed semen sample considered as the control group, part 2 and 3 (group 2 and 3) were processed by the density gradient method and then sperm selection was made with PICSI and zeta potential, respectively.

Semen analysis was performed based on
World Health Organization guidelines (15).
Sperm morphology was analyzed based on strict criteria. Motility was reported as a percentage of progressive, non-progressive, and immotile sperms. Also, sperm DNA integrity was analyzed by SCD.

SCD
The SCD test was used to assess sperm DNA

Zeta potential
The technique was performed similar to the protocol previously described else where

Ethical considerations
The

Results
A total of 48 men were enrolled in the current    Our results are not in agreement with another similar study that stated the superiority of zeta potential over hyaluronic sperm selection in terms of sperm DNA integrity. This difference may be partly due to differences in HA-selection tools and the role of an embryologist in sperm selection. In our study, by using a standard PICSI dish (PICSI dish; Biocoat, Inc., Horsham, PA, USA), seemingly suitable sperm were selected for injection by an embryologist. They were collected in a tiny dot of sperm wash (on an agarose-coated slide), and their DNA integrity was assessed with an SCD test. Meanwhile, the aforementioned study evaluated the DNA integrity of unselected HA-bound sperms on a handmade slide coated with HA. We believe that the role of an embryologist in sperm selection and the use of a standard PICSI dish may have led to the aforementioned difference in findings. However, this difference may also be due to our small sample size or the small number of selected sperms. Another recent study compared PICSI with MACS techniques in selecting spermatozoa with lower DNA fragmentation. This study showed both of these techniques were effective in sperm selection, but intimated that MACS was preferential in cases with younger women and PICSI was more effective in the cases with older ones (19).
Since advanced sperm selection methods were developed comparatively recently, assessment of their efficacy seems to be necessary. It has been proposed that PVP, used in ICSI procedures, may have a toxic effect on spermatozoa (11,20). PVP may result in sperm membrane injury and embryonal maldevelopment (20). It has been shown that this negative effect may be dosedependent (21). Whereas PVP is widely used in zeta potential to reduce sperm motility, such a goal is achieved by a more physiologic substitute (HA) in the PICSI technique (9).
The hyaluronic-based sperm selection technique was developed to obtain mature sperm. As mentioned before, some authors suggest that only mature functional sperms might gain the ability to bind to the HA receptors. These sperm usually show a high degree of nuclear and cytoplasmic maturity (22). However, the effect of HA-bound sperms on ART outcome remains the subject of debate. A prospective study on 232 ICSI cycles randomized to PVP-ICSI (107 cycles) and HA-selected sperms (125 cycles), showed better embryo development in the latter group (23). However, British scientists performed a welldesigned randomized trial on 2772 couples and their study indicated no statistically significant difference between PICSI and conventional ICSI in terms of clinical pregnancy and live birth rate (24).
Recently, another study stated that PICSI might significantly increase the fertilization rate and embryo transfer in couples with previous ART failures compared to routine ICSI. The authors concluded that this technique should be performed with infertile couples with a history of unsuccessful IVF cycles (25). The current study showed statistically significant differences between PICSI and zeta potential in obtaining sperms with better DNA integrity. However, the clinical significance of this finding in terms of ART outcome is yet to be defined and should be evaluated in upcoming studies.
Sperm chromatin is well-organized and has high nuclear condensation, and any alteration of it during spermatogenesis could have harmful effects on sperm functions (26). As shown in table III, there is a positive correlation between male age and neat sperm DFI. This means that advanced paternal age may negatively affect sperm DNA integrity (increased sperm DFI). This result is in line with recent studies which showed that increased male age might result in decreased sperm DNA quality (27,28). This effect may be due to increased reactive oxygen species production, defective sperm chromatin compaction, disordered apoptosis, and changes in telomer length (29). Also, it is widely accepted that sperm DNA fragmentation may influence the semen quality (26,30). Among the sperm characteristics, sperm morphology may have a crucial role in the diagnosis of male fertility potential. It means that abnormally shaped spermatozoa may have increased rate of chromosomal aneuploidy, a higher DFI, and an increased chance of mitochondrial dysfunction (31). In this regard, this study's data showed a significant negative correlation between normal morphology spermatozoa and sperm DFI. This result aligns with earlier studies suggesting that normal morphology sperm selected with motile sperm organelle morphology examination and intracytoplasmic morphologically selected sperm injection had significantly lower DFI than those from ICSI (32).
There are other limitations in the current study. Since the number of embryologist-selected sperms were limited, only sperm DNA integrity could be assessed, and the status of sperm chromatin compaction remained unclear. Further attempts to select a larger number of sperms may result in increased sperm DFI due to prolongation of the process.
Finally, given the small number of participants included in the current study, the results should be evaluated with larger samples in future studies.

Conclusion
The current study showed that advanced sperm selection techniques may lead to obtaining sperms with better DNA integrity. In this regard. PICSI may result in sperms with higher quality in comparison with zeta potential. However, further studies are needed to determine the clinical significance of this finding.