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

Capsule:
The presence of merocyanine 540 staining bodies does not affect quantification of early and late apoptotic biomarkers in spermatozoa from fertile and subfertile men.

Authors:
Nardhy Gomez Lopez, Ph.D., Guadalupe Estrada-Gutierrez, Ph.D., Alinne Colin M.D., M.Sc., Arturo Flores-Pliego M.Sc., Xochitl Flores-Escobar B.Sc., Sergio Oehninger, M.D., Ph.D., Gerardo Barroso M.D., M.Sc.

Volume 99, Issue 5, Pages 1242-1248, April 2013

Abstract:

Objective:
To evaluate the presence of merocyanine 540 (M540) bodies and their impact on the measurement of apoptotic biomarkers in human spermatozoa.

Design:
Case-control, prospective study.

Setting:
Academic centers.

Patient(s):
Fertile and subfertile subjects.

Interventions:
Semen samples from subfertile and fertile men, eleven per group, were analyzed for basic semen parameters, and early (Annexin-V binding) and late (TUNEL) sperm apoptotic biomarkers by flow cytometry. Samples were also stained with Merocyanine 540 (M540) to assess the presence of M540 apoptotic bodies.

Main Outcome Measure:
Presence of M540 apoptotic bodies.

Results:
Groups differed significantly in the expression of early and late apoptosis biomarkers. The percentage of M540 bodies between groups was not different. The exclusion of M540 bodies from TUNEL results did not have a significant impact on measurement in either fertile or subfertile groups.

Conclusions:
This study confirmed (i) the occurrence of M540 bodies in semen, and (ii) that male infertility is associated with an increased expression of apoptosis biomarkers. Moreover, we demonstrated that presence of M540 bodies did not affect the quantification of apoptotic biomarkers in either group.

  • jim hotaling

    This article examines M540 bodies and their impact on the assessment of apoptotic biomarkers in human spermatozoa. There appears to be controversy over the methodology employed in the detection of these bodies. If these bodies do exist, it would be very interesting to examine whether their presence, absence or number correlates with microTESE outcome. Have either of the authors who have worked in this area considered such a study?

  • Monica Muratori, PhD

    In this study the Authors failed to reveal
    both the presence of M540 bodies and (hence) the effect of such bodies on the
    measures of sperm DNA fragmentation (sDF), in contrast with a previous paper by
    my group (Hum Reprod, 23, 2008: 1035-43). The reason of such a discrepancy
    appears to be 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. By this procedure, merocyanine labeling is
    washed away from the bodies, since M540 is not bound 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 the Authors gave a definition of M540 bodies that is different from
    that of the studies first reporting these semen elements (J Androl, 2004, 25:797-810
    and Mol Hum Reprod, 2007, 13:621-31 ). They considered as M540 bodies, only
    those elements that are positive for both M540 staining and TUNEL. However,
    bodies with detectable DNA fragmentation are present in low quantity in semen
    and not in all the infertile patients, whereas most M540 bodies appear devoid
    of chromatin (Cytometry, 2008, 73:785-7). In any case, their occurrence heavily
    interferes with the measures of sDF by flow cytometry, since such interference
    does not depend by the possible presence of fragmented DNA within them, as
    extensively explained in Cytometry, 2008, 73:785-7.

  • Monica Muratori, PhD

    In this study the Authors failed to reveal both the presence of M540 bodies and (hence) the effect of such bodies on the measures of sperm DNA fragmentation (sDF), in contrast with a previous paper by my group (Hum Reprod, 23, 2008: 1035-43). The reason of such a discrepancy appears to be 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. By this procedure, merocyanine labeling is washed away from the bodies, since M540 is not bound 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 the Authors gave a definition of M540 bodies that is different from that of the studies first reporting these semen elements (J Androl, 2004, 25:797-810 and Mol Hum Reprod, 2007, 13:621-31 ). They considered as M540 bodies, only those elements that are positive for both M540 staining and TUNEL. However, bodies with detectable DNA fragmentation are present in low quantity in semen and not in all the infertile patients, whereas most M540 bodies appear devoid of chromatin (Cytometry, 2008, 73:785-7). In any case, their occurrence heavily interferes with the measures of sDF by flow cytometry, since such interference does not depend by the possible presence of fragmented DNA within them, as extensively explained in Cytometry, 2008, 73:785-7.

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