Methods for defecting sperm apoptosis

LETTER TO THE EDITOR Methods for defecting sperm apoptosis To the Editor: We read with interest the recent article by Mahfouz et al. (1), and we have some observations. First, the authors concluded that after semen processing, ‘‘the incidence of late apoptotic sperm remains unchanged.’’ This finding is not consistent with earlier studies (2, 3) and our recent report (4). The authors found the percentage of nonnecrotic sperm (propidium iodide fluorescence (PI)– negative sperm) to be 63.1% in neat semen and 66.9% after density-gradient separation. In other words, the semen processing provided a sample with one-third PI-positive (necrotic) sperm. Therefore, assuming that necrotic sperm are immotile, the mean motility after semen processing was <67%. This is a surprising result, because the efficiency of density-gradient separation is usually much higher. In fact, the authors report that the sperm motility was significantly improved in all of the samples after density-gradient separation. In our recent study, we found a significantly lower percentage of necrotic sperm in the capacitated fraction (8.4%) compared with neat semen (13.1%). Second, the authors suggest that the nonsignificant difference in the incidence of late apoptosis between prepared and nonprepared sperm might be due to the small sample size in their study. We believe, on the contrary, that the problem was the methodology used to detect sperm apoptosis. The authors gated sperm population using forward-angle light scatter, and side-angle light scatter was used to exclude debris. However, some particles and cell debris present in the ejaculate may have frontal and side light scatter parameters similar to those of the sperm. This makes it difficult to discriminate by flow cytometry (FC) between sperm and particles or debris on the sole basis of scatter parameters. To overcome this problem, we have suggested performing a preliminary FC using 6-carboxyfluoresceindiacetate to identify the live sperm and using PI to identify dead cells (5). This preliminary analysis allows performing the classic bivariate annexin V (AV)/PI analysis on the sperm population correctly identified, regardless of the amount of particles and cell debris present in the ejaculate. Third, the authors stated that AV combined with PI can distinguish among viable, necrotic, early, and late apoptotic cells. This is a very controversial issue. There is no agreement on how to classify PI-positive/AV-positive and PI-positive/ AV-negative cells. When the membrane loses its integrity, the cell becomes PI positive, indicating that the cell is necrotic. However, disruption of membrane integrity during necrosis make the cell interior accessible to AV that can bind to phosphatidylserine on the inner leaflet of the plasma

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membrane (6). Therefore, AV/PI combination cannot reliably distinguish between late apoptotic and necrotic sperm. Staining with AV can be used as a specific marker for apoptosis only in the early phase, when the cell membrane is still intact. Recently, we have shown that the combination of Syto 16/7amino-actinomycin D provides a more reliable assay for detection of sperm apoptosis, in both neat and prepared semen (4, 7). In conclusion, we agree with the authors that ‘‘simultaneous evaluation of apoptosis and sperm chromatin status is important for processing sperm in assisted reproductive procedures,’’ but we suggest using a more reliable method to detect sperm apoptosis. Giuseppe Ricci, M.D. Rita Boscolo, B.Sc. Monica Martinelli, B.Sc. Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy Sandra Perticarari, B.Sc. Clinical Analysis Unit, Department of Laboratory Medicine, Institute for Maternal and Child Health, IRCCS Burlo Garofolo and University of Trieste, Trieste, Italy September 5, 2008 REFERENCES 1. Mahfouz RZ, Sharma RK, Said TM, Erenpreiss J, Agarwal A. Association of sperm apoptosis and DNA ploidy with sperm chromatin quality in human spermatozoa. Fertil Steril. Published online May 5, 2008 [Epub ahead of print]. 2. Paasch U, Grunewald S, Fitzl G, Glander HJ. Deterioration of plasma membrane is associated with activated caspases in human spermatozoa. J Androl 2003;24:246–52. 3. Marchetti C, Gallego MA, Deffosez A, Formstecher P, Marchetti P. Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters. Hum Reprod 2004;19:1127–34. 4. Ricci G, Perticarari S, Boscolo R, Montico M, Guaschino S, Presani G. Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique. Fertil Steril. Published online January 16, 2008 [Epub ahead of print]. 5. Ricci G, Perticarari S, Fragonas E, Giolo E, Canova S, Pozzobon C, et al. Apoptosis in human sperm: its correlation with semen quality and the presence of leukocytes. Hum Reprod 2002;17:2665–72. 6. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994;84: 1415–20. 7. Perticarari S, Ricci G, Granzotto M, Boscolo R, Pozzobon C, Guarnieri S, et al. A new multiparameter flow cytometric method for human semen analysis. Hum Reprod 2007;22:485–94.

doi:10.1016/j.fertnstert.2008.12.109

Fertility and Sterility Vol. 92, No. 2, August 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

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