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 previous studies (2,3) and our recent report (4). The authors found the percentage of non-necrotic sperm (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 of PI positive (necrotic) sperm. Thus, assuming that necrotic sperm are immotile, the mean motility after semen processing was less than 67%. This is a surprising result, since the efficiency of density-gradient separation is usually much higher. In fact, the authors report that the sperm motility was significantly improved in all 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 non-significant difference in the incidence of late apoptosis between prepared and non-prepared 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 author gated sperm population using forward-angle light scatter, while 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. In order to overcome this problem, we have suggested performing a preliminary FC using 6-carboxyfluoresceindiacetate (6-CFDA) to identify the live sperm and using propidium iodide (PI) to identify dead cells (5). This preliminary analysis allows performing the classical bivariate Annexin V/PI analysis on the sperm population rightly 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 (PS) on the inner leaflet of the plasma membrane (6). Thus, AV/PI combination cannot reliably distinguish between late apoptotic and necrotic sperm. Staining with AV, therefore, can be used as a specific marker only for apoptosis in the early phase, where the cell membrane is still intact. Recently, we have shown that the combination of Syto 16 /7-AAD 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.a
Sandra Perticarari, B. Sc.b
Rita Boscolo, B. Sc.a
Martinelli Monica, B. Sc.a
a Assisted Reproduction Unit
Department of Obstetrics and Gynaecology
bClinical Analysis Unit
Department of Laboratory Medicine Institute for Maternal and Child Health IRCCS Burlo Garofolo and University of Trieste
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 2008 May 5. [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 2008 Jan 16. [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 and 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.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2008.12.109
The Authors Respond:
We are very happy that Dr Ricci and his colleagues found our article interesting. Our aim was to examine the relationship between sperm chromatin integrity and maturity with sperm membrane changes detected by annexin V assay. We evaluated DNA damage by sperm chromatin structure assay (SCSA), Toluidine blue staining, annexin V/ PI assay and sperm DNA cytometry for detecting the percentage of apoptotic cells (PI positive cells). We agree with Dr. Ricci that each assay has some limitations; for example PI negative population may contain some necrotic cells that may fail to stain with PI. All viability markers for staining the DNA may not be permeable to cell membrane except at certain stages of membrane integrity. In such instances early or dead cells may not allow the PI and other DNA probes to reach the cell nucleus.
Significant improvement in sperm motility after preparation of sperm by density gradient does not indicate the absence of membrane or nuclear damage that may subsequently affect the sperm quality. Objective parameters such as percentage of late apoptotic sperm in Annexin/ PI staining assay and %DFI in SCSA or by objective parameters such as the Toluidine blue assay failed to show significant improvement. These observations confirm that there may be something more than the methodology. Also there may be patients to patient variations in sperm preparation as well as in the sperm preparation obtained from healthy donors.
We agree with Dr. Ricci that cell debris must be taken into consideration when running FCM analysis. Advancement in flowcytometry technology allows the dual Annexin V/ PI staining for flowcytometry with greater ability to exclude cell debris. Adequate instrument settings, acquisition setting, compensation, negative and single dye control as well as an experienced analyzer using sophisticated software for correct data analysis and interpretations are critical factors for FCM analysis. Certain dyes such as 6-carboxyfluoresceindiacetate (CFDA) or SYBR green may be used to identify the viable fraction along with PI staining.
Finally, we agree with Dr. Ricci that the differentiation between late apoptotic (PI positive/AV positive) and necrotic (PI positive/AV negative) cells is not practical since late apoptosis is an irreversible stage of apoptosis. Annexin V is not permeable to the plasma membrane; it can only bind to externalized phosphatidylserine (PS) portion. Cell debris may occur as cell damage progresses and plasma membrane fragments. Many assays have been recently introduced for assessing viability such as Annexin V/ PI, Yo-Pro-1/ PI, SYBR green/ PI, Syto 16/ PI, or Syto/ 7-AAD. Syto16 is superior when combined with 7-AAD than PI because of the narrower emission spectrum of 7-AAD compared with PI. In our lab we use combination of Yo-Pro1 and PI. Finally we agree with Dr. Ricci’s conclusion and suggest that the best reliable method is the one that has been standardized by the lab based on available equipment with correct interpretation of its results and the limitations.
Again we would like to thank Dr. Ricci and his colleagues for affording us the opportunity to address these important points.Reda Mahfouz, M.D.a
Rakesh Sharma, Ph.D.a
Jakob Lackner, M.D.b
Nabil Aziz, M.D.c
Ashok Agarwal, Ph.D., HCLDa aCenter for Reproductive Medicine Glickman Urological and Kidney Institute and Obstetrics and Gynecology and Women’s Health Institute Cleveland Clinic Cleveland, Ohio bDepartment of Urology Medical University of Vienna Vienna, Austria cLiverpool Women’s Hospital Liverpool, United Kingdom
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2009.03.054