To the Editor:
Although we agree with Dr. Caroppo that the effect of DNA damage of human spermatozoa on fertilization, embryo cleavage, and implantation remains controversial, its detrimental impact on natural conception and miscarriage seems real. Advanced methods for the selection of spermatozoa with healthy DNA will become mandatory, as advocated by Dr. Caroppo (1). As we are now able to significantly reduce the number of spermatozoa with fragmented DNA (2), we will be able to compare prospectively the effect of DNA fragmentation on the outcome of assisted reproductive technology (ART).
Of course, the safety of the process is of concern. However, the safety of the Hoechst dye in combination with fluorescence activated cell sorting (FACS) has been amply documented as much mammalian offspring has been produced with sorting (3). All evidence to date indicates that this dye exerts no detrimental impact. Similarly, the UV laser was described as not having any detectable negative effect on the incidence of DNA damage or mutations in spermatozoa. Only the mechanical aspects of sorting may be detrimental, as the pressure exerted on spermatozoa during their passage through the microfluidic channels has been demonstrated to affect their viability, motility, and velocity in the presence of high pressure (50 psi), but not of lower pressure (30 psi). Here, we used milder pressure (around 20 psi), which can be decreased even further. Due to breakage of the sperm tails, motility is lost during the formation and disruption of little droplets (4). Dr. Caroppo commented that in our study the sperm motility was significantly reduced when compared with the swim-up (3). As mentioned in our report (2), this difference is considerably reduced if we limit the samples to those with lowest quality (concentration ≤10 x 106/ml). In these cases the values are 51.6% +/-14 for sorted spermatozoa and 33% +/-22 in the swim-up.
The second dye used in the study, YO-PRO, does not enter into the viable spermatozoa with intact membranes selected for ART. Four arguments in favor of the safety of YO-PRO are provided (5): first, the authors claimed that 48 hours after adding YO-PRO, myoblasts cell metabolism was not significantly affected, and after 72 hours the difference was approximately 20%. Second, the incubation of viable cells with the dye showed that after 10 hours the nuclei were not stained with YO-PRO. Third, in contrast to metabolically inactive spermatozoa, the myoblasts were metabolically active and proliferating, rendering them much more susceptible to the dye. Fourth, the concentration of the dye used with myoblasts was fivefold higher than the one used in our sorting process. During sorting, staining with YO-PRO lasted 15 minutes, whereas the sorting itself lasted less than 30 minutes. Thereafter the sorted spermatozoa were immediately diluted with fresh medium, leaving them with a much shorter exposure to the dye than the myoblasts.
In our opinion, no isolation procedure is completely harmless to the spermatozoa and neither conventional methods with repeated washing and centrifugation steps nor sorting of spermatozoa with FACS are “physiological.”
Sofia Ribeiro, Ph.D., Gideon Sartorius, M.D., Christian De Geyter, M.D.
Clinic of Gynecological Endocrinology and Reproductive Medicine, University Hospital, University of Basel, Basel, Switzerland
1. Caroppo E. Sperm sorting for selection of healthy sperms: is it safe and useful? Editorial Commentary, Fertil Steril 2013.
2. Ribeiro S, Sartorius G, Pletscher F, De Geyter M, Zhang H, De Geyter C. Isolation of spermatozoa with low levels of fragmented DNA using flow cytometry and sorting (FACS). Fertil Steril 2013.
3. Garner DL. Hoechst 33342: The dye that enabled differentiation of living X-and Y-chromosome bearing mammalian sperm. Theriogenology 2009;71:11-21.
4. Suh TK, Schenk JL, Seidel GE. High pressure flow cytometric sorting damages sperm. Theriogenology 2005;64:1035-48.
5. Gawlitta D, Oomens CWJ, Baaijens FPT, Bouten CVC. Evaluation of a continuous quantification method of apoptosis and necrosis in tissue cultures. Cytotechnology 2004;46:139-50.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2013.06.039