Presence of M540 bodies in human semen: techniques to detect them require attention

20 06 2013

To the Editor:

We read with interest the paper by Gomez-Lopez et al., published in Fertility and Sterility (1), in which the authors investigated the presence of merocyanine 540 bodies (M540 bodies) and their impact on the detection of sperm apoptotic markers, including sperm DNA fragmentation.
In this paper the authors report that the incidence of M540 bodies in the semen of infertile men is very low (about 1%) and that their occurrence does not affect the determination of sperm DNA Fragmentation (sDF) by TUNEL coupled to flow cytometry.

These results contrast with previous data by our group that first described M540 bodies (2) and later on demonstrated that they are apoptotic bodies of testicular origin (3). Indeed, we found that M540 bodies can be present in high amount in semen of sub/infertile men (2, 3) and that they heavily flaw the determination of TUNEL positive sperm by flow cytometry (4). We believe that the cause of such discrepancies is the technique that the authors used to reveal M540 bodies in TUNEL processed samples. They first stained by merocyanine 540 and then washed and processed samples by TUNEL assay. By this procedure, merocyanine labeling is washed away from the bodies, since M540 does not bind in a stable (covalent) manner to bodies. As a consequence, they failed to detect M540+ elements, not because of their absence but because of the loss of merocyanine staining.

Further, Gomez-Lopez et al. give a definition of M540 bodies that is different from that of the studies first reporting these semen elements (2, 3). They defined as M540 bodies, only those elements that are positive for both M540 staining and TUNEL. However, only a small fraction of M540 bodies shows detectable DF, whereas most M540 bodies appear devoid of fragmented chromatin (4). In any case, we have clearly shown that their occurrence heavily interferes with the measures of sDF by flow cytometry (4), since such interference does not depend on the possible presence of fragmented DNA within them, but by the fact that they can mask fractions of DNA fragmented sperm and/or contribute to increase the percentage of global TUNEL negative events (see 4, for a detailed explanation).

Finally, Gomez-Lopez et al. claim that the interference of M540 bodies on the flow cytometric measures of sDF was denied by us in a recent comment (5) about a study comparing the levels of sDF between donors and infertile patients and between neat semen samples and sperm samples selected by density gradient centrifugation. Contrary to what was stated by the authors, in that comment we did stress the importance of excluding M540 bodies from the flow cytometric analyses of spermatozoa, especially in the comparisons between samples with a possible difference in incidence of M540 bodies, like donors and patients or neat semen and selected sperm.

As M540 bodies can virtually affect any flow cytometric analysis of spermatozoa, and they represent a sign of impaired testis function/apoptosis, we believe that the issue of the presence of these elements in semen should not be disregarded.

Monica Muratori, Ph.D., Gianni Forti, M.D. and Elisabetta Baldi, Ph.D.
Department of Experimental and Clinical Biomedical Sciences and Center for Research, Transfer and High Education DeNothe, University of Florence, Firenze, Italy

References

1. Gomez-Lopez N, Estrada-Gutierrez G, Colin A, Flores-Pliego A, Flores-Escobar X, Oehninger S, et al. The apoptotic pathway in fertile and subfertile men: a case-control and prospective study to examine the impact of merocyanine 540 bodies on ejaculated spermatozoa. Fertil Steril 2013;99:1242-8.

2. Muratori M, Porazzi I, Luconi M, Marchiani S, Forti G, Baldi E. AnnexinV binding and merocyanine staining fail to detect human sperm capacitation. J Androl 2004;25:797-810.

3. Marchiani S, Tamburrino L, Maoggi A, Vannelli GB, Forti G, Baldi E, et al. Characterization of M540 bodies in human semen: evidence that they are apoptotic bodies. Mol Human Repro 2007;13:621-31.

4. Muratori M, Forti G., Baldi E. Comparing flow cytometry and fluorescence microscopy for analyzing human sperm DNA fragmentation by TUNEL labelling. Cytometry A 2008;73:785-7.

5. Muratori M, Forti G, Baldi E. M540 bodies interfere with TUNEL analyses in human semen samples. Hum Reprod 2011;26:729.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2013.06.037

The authors respond:

We read with great interest all previous reports about M540 bodies from the group of Dr. Muratori (1-4). As they suggested in previous reports that these structures can heavily affect the determination of TUNEL positive sperm by flow cytometry, we were authentically worried about the underestimation of apoptotic biomarkers in the semen samples that we handled everyday. In our study (5), we reported that M540 bodies did not affect the quantification of apoptotic biomarkers in fertile or subfertile semen samples, and Muratori et al. suggest in their letter to the editor that we failed to detect them because we washed away merocyanine labeling. However, we have shown in the immunofluorescence (5, figure 2, panel D) that we were able to detect the presence of M540 bodies as merocyanine positive cells (red channel), even after they were washed and stained for TUNEL detection. Additionally, it is worth pointing out that all the immunofluorescence results shown in our work were prepared with an aliquot of the same stained cells that were used for flow cytometry, so they could be detected if they were present.

Muratori et al. point out that we defined M540 bodies as positive for both merocyanine and TUNEL, and because of that, we could underappreciate M540 bodies that are devoid of fragmented chromatin, which means cells positive for M540 and negative for TUNEL. However, as we showed in the cytometry plot (5, figure 2, panel E), these cells were barely present (less than 1%), even in subfertile semen samples.

In addition, as we mention in our work, multiple attempts were made to find M540 bodies by light and confocal microscopy due to the low number of these structures in the samples, so we are confident about our results.

In conclusion, we demonstrated that M540 bodies are present in fertile and subfertile samples of men. However, as reported in our study, we believe that the presence of M540 bodies does not interfere in the determination of late apoptotic biomarkers when those are measured by flow cytometry. Finally, we do support the idea that additional studies are needed to assess the real significance of M540 bodies for fertility purposes.

Gerardo Barroso, M.D., M.Sc., Ph.D., Guadalupe Estrada, Ph.D., and Carlos Valdespin, M.D., M.Sc.
Department of Gynecology and Obstetrics, American British Cowdray Medical Center, Mexico City, Mexico
Instituto Nacional de Perinatologia, Mexico City, Mexico

References

1. Muratori M, Porazzi I, Luconi M, Marchiani S, Forti G, Baldi E. AnnexinV binding and merocyanine staining fail to detect human sperm capacitation. J Androl 2004;25:797-810.

2. Marchiani S, Tamburrino L, Maoggi A, Vannelli GB, Forti G, Baldi E, et al. Characterization of M540 bodies in human semen: evidence that they are apoptotic bodies. Mol Hum Reprod 2007;13:621-31.

3. Muratori M, Forti G., Baldi E. Comparing flow cytometry and fluorescence microscopy for analyzing human sperm DNA fragmentation by TUNEL labelling. Cytometry A 2008;73:785-7.

4. Muratori M, Forti G, Baldi E. M540 bodies interfere with TUNEL analyses in human semen samples. Hum Reprod 2011;26:729.

5. Gomez-Lopez N, Estrada-Gutierrez G, Colin A, Flores-Pliego A, Flores-Escobar X, Oehninger S, et al. The apoptotic pathway in fertile and subfertile men: a case-control and prospective study to examine the impact of merocyanine 540 bodies on ejaculated spermatozoa. Fertil Steril 2013; 99:1242-8.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2013.06.038

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