DHEA administration in poor responders.

may have been influenced by the presence of M540 bodies. M540 bodies are indeed mysterious structures. They stain positively with merocyanine, a fluorescent membrane probe normally associated with the measurement of membrane potential and disorder, and also generate signals in the TUNEL assay, even though these structures do not stain with DNA sensitive fluorochromes such as YOPRO-1 (Marchiani et al., 2007, Fig 2A). We are certainly intrigued by M540 bodies but doubt that their presence had a significant impact on the results of our paper for the following reasons: (1) Percoll centrifugation effectively removes M540 bodies from human sperm suspensions so, for all of the results reported in our paper that involved sperm preparation by discontinuous gradient centrifugation, M540 body contamination would not have been an issue. (2) While it remains theoretically possible that the presence of M540 bodies affected our analysis of 'unfractionated' semen samples, this does to seem to have been the case for our cohort of subjects. Thus, while we have recently been able to confirm the presence of M540 bodies in semen samples from our patients and donors, we found that these structures were present in insufficient numbers to influence our patient versus donor comparison. (3) This difference with Muratori's analyses may be due to the fact that our patient population comprised unselected subjects attending our local IVF clinical who possessed semen quality that was essentially normal (See Fig. 1 of our original paper). If, like Marchiani et al. (2007), we had selected patients with severe defects in their semen profile, including oligoasthenotera-tozoospermia, then M540 bodies might well have had a more significant influence on our results. Further studies with more seriously compromised semen samples will be needed to establish this point. (4) In our unfractionated samples, the threshold for discriminating abnormally high levels of TUNEL and 8-hydroxy-2 ′-deoxyguano-sine (8-OHdG) reactivity was around 40% for both criteria, in keeping with the fact that levels of TUNEL positivity and 8-OHdG formation were highly correlated (Aitken et al., 2010, Fig. 2). This correlation would not have been impacted by the presence of M540 bodies because the latter contain no detectable DNA and so should not have stained with an antibody against 8-OHdG. Again, further studies will be needed to establish this point. (5) Muratori et al. suggest that 'The percentages of TUNEL in unse-lected semen are expected to be greater than those reported by Aitken et al., (2010)'. …