The questionable effectiveness of routine semen examination in establishing male infertility issues has led to the implementation of new techniques allowing the evaluation of sperm DNA integrity and the prediction of fertilization capacity aiming to improve ART outcomes. In fact, evidence suggests that sperm DNA quality adversely affects ART results [21] and fertilization capacity depends on a substantial amount of sperm with chromatin anomalies.

Firstly, our results indicated a significant improvement in the proportion of motile progressive sperm recovered from the 90% layer after the DGC technique which is about 3.5 folds compared with unprocessed semen. This observation consists with those published by Ricci et al. [22], Hashimoto et al. [23], and Noguchi et al. [24] who reported that the DGC technique leads to a higher progressive motile sperm rate than other sperm processing techniques.

Besides, this method revealed its efficiency in the improvement of the proportion of spermatozoa with normal morphology after density gradient sperm preparation as reported in previous studies [21, 25, 26].

In fact, the DGC technique consists of separating the whole semen on layers of particular solutions with different densities. It is a time-saving technique as it only needs around 20 min of centrifugation compared with 1 h of incubation for the swim-up technique. It is simple to perform within sterile conditions which makes it easy to implement in routine clinical work.

Additionally, in the case of a sperm head deformity, the synchronization between head rotation and flagellar motion is altered. Some authors suggest that this desynchronization could explain, among other things, the low fertility potential during conventional in vitro fertilization [27]. This abnormal progression, resulting in variable degrees of asthenospermia, would therefore explain the difficulty faced by these spermatozoa in crossing the two layers of the gradient so that they are not selected. Acrosomal status is a major factor involved in the fertilizing capacity of the sperm as it contains enzymes that are essential to permeate and fertilize the oocyte and take part in capacitation and acrosome reaction [28]. The results of the current study revealed that the number of spermatozoa with normal acrosome was also remarkably increased by 1.4 folds after DGC preparation.

Secondly, using the SCD test, we revealed that spermatozoa retained in the 90% layer after the DGC technique present a significant decrease in the chromatin decondensation rate corresponding to ½ compared to untreated semen. Interestingly, these results are in accordance with those obtained by the AO assay regarding the DNA denaturation levels which decreased from 49.76% in the initial sperm sample to reach a mean value of 15.71% in the 90% layer after centrifugation (p < 0.001). Hence, one of the strengthen points of the current study is that it provides complementary data allowing DNA integrity appreciation: decondensation and denaturation status, especially since these two parameters were evaluated on each semen sample which makes the results more reliable than those where two different techniques were tested but on different semen samples [29].

To go further in selecting the best male germ cells, other reports have proven the efficiency of DGC on isolating the sperm population with longer telomeres [30]. This criterion seems to be predictive on fertilizing sperm ability but currently available data is still controversial [30, 31].

Our results are consistent with other studies focusing on the impact of DGC in reducing the proportion of sperm DNA fragmentation originally present in the neat semen [32, 33].

In accordance with these data, Sakkas et al. [34] noted a significant decrease in the percentage of damaged sperm DNA after DGC, but they did not find any significant amelioration when they used the swim-up method. Moreover, Hammadeh et al. [25] proved that density gradient centrifugation not only ameliorates sperm motility but also leads to the selection of sperm with normal morphology and chromatin integrity.

In a study conducted by Malvezzi et al. [35], three commercially available gradient media (among which SpermGrad that we used in the present study) were employed to evaluate sperm selection quality. Even if conducted on only 20 semen samples, the study has shown that all three media improved sperm parameters as well as DNA quality, which favor previous results that density gradient can recuperate high-quality sperm with little DNA damage. Despite all the previously described trials, the effects of DGC on sperm DNA integrity are still controversial; in fact, other studies have not found DGC to be useful for the selection of sperm with high DNA integrity. A study conducted by Muratori et al. [14], using the pure sperm density gradient, demonstrated that sperm preparation with DGC may even induce sperm DNA fragmentation. They found that after DGC, about 50% of subjects had higher levels of sperm DNA fragmentation when compared to pre-DGC values, proposing the induction of DNA damage over the manipulation. The authors argue for a potential contamination of commercially available colloidal silicon gradients by transition metals which may be the principal reason of sperm DNA breakage during DGC.

This hypothesis was supported by the findings of Aitken et al. [36] who revealed an oxidative DNA damage in sperm following the use of PureSperm® discontinuous colloidal silicon gradients suggesting that metal transition present in the medium particularly Fe, Al, and Cu are responsible to promote free radical generation in the immediate vicinity of DNA. According to these authors, this damage can be significantly accentuated by reducing agents, such as ascorbate (p < 0.001), and inhibited by selective chelation (p < 0.001).

Taken together, it is important that we avow the potential damage that may be induced by certain metals on sperm DNA integrity during sperm preparation and so we must seek to counteract their effects by adding in the medium antioxidants that block the initiation or propagation of chain oxidation. It is well reported that zinc may act by competition with the ferrous ions to the oxygen ligands in the oxidized polyunsaturated fatty acids in the sperm membrane and so it prevents the binding of these redox-active metals [37].

Another study reported by Zini et al. [38] showed a 2-fold augmentation in denatured sperm DNA when compared with raw semen, after processing with two and four layers of Percoll gradients.

The authors attributed this discrepancy to the nature of the gradient itself and the centrifugal force utilized which could be responsible for ROS production and iatrogenic DNA damage induction. The centrifugal force employed when processing the sperm was considerably higher than that used in the current study.

Furthermore, a previous study reported that an excessive amount of ROS produced by seminal leucocytes or immature spermatozoa during sperm incubation has been confirmed to potentially harm sperm function via oxidized DNA adducts and DNA fragmentation [8, 14]. The use of antioxidants in sperm preparation media (albumin was used in this study) can save spermatozoa from oxidative attacks, which could explain the significant amelioration of sperm motility, normal morphology, and especially better DNA quality.

Selecting spermatozoa on the basis of chromatin integrity is a crucial point in the process of fertilization. Indeed, both the lack and the excess of sperm chromatin compaction could be deleterious. On the one hand, a supernormal sperm chromatin compaction could prevent the delivery of the paternal genome in the oocyte [39]. On the other hand, the oocyte has an important DNA repair capacity but is limited to DNA strand breaks and the capacity to manage with an abnormal chromatin structure is very poor [40].

To our knowledge, we herein describe for the first time the relationship between sperm deformity rate and DNA integrity using the SCD and AO assay after DGC preparation.

In fact, we demonstrated a positive correlation between the results provided by these two tests giving us an idea about the sperm DNA integrity by measuring the rate of sperm chromatin dispersion and DNA denaturation.

To conclude, the classic procedures might be ameliorated by conducting in-depth more sophisticated methods, so as to find efficient semen processing that can be used in ART. Therefore, the use of the DGC technique for sperm preparation before ART treatment as reported in this study will be possibly an attractive new strategy that can ameliorate DNA integrity and overcome the shortcomings of routine semen examination to point male infertility issues and to improve ART results.

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