Produção Nacional
Revisado por pares
Correlation between semen analysis by motile sperm organelle morphology examination and sperm DNA damage
2010; Elsevier BV; Volume: 94; Issue: 5 Linguagem: Inglês
10.1016/j.fertnstert.2010.01.042
ISSN1556-5653
AutoresJ.B.A. Oliveira, F. C. Massaro, Ricardo Luis Razera Baruffi, Ana L. Mauri, Cláudia G. Petersen, Liliane FI Silva, L.D. Vagnini, J.G. Franco,
Tópico(s)Assisted Reproductive Technology and Twin Pregnancy
ResumoRegression analysis of 538 semen samples demonstrated that percentages of normal nuclear sperm and all spermatozoa with abnormalities of nuclear form at high magnification had significant negative correlation with percentages of DNA fragmentation. On the other hand, there was a positive correlation between percentages of spermatozoa with nuclear vacuoles and those with DNA fragmentation. Regression analysis of 538 semen samples demonstrated that percentages of normal nuclear sperm and all spermatozoa with abnormalities of nuclear form at high magnification had significant negative correlation with percentages of DNA fragmentation. On the other hand, there was a positive correlation between percentages of spermatozoa with nuclear vacuoles and those with DNA fragmentation. As recent studies have demonstrated that intracytoplasmic morphologically selected sperm injection (IMSI), based on sperm normality as defined by the motile sperm organelle morphology examination (MSOME), improves ICSI outcome (1Bartoov B. Berkovitz A. Eltes F. Kogosovsky A. Yagoda A. Lederman H. et al.Pregnancy rate are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection.Fertil Steril. 2003; 80: 1413-1419Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 2Junca A. Cohen-Bacrie M. Hazout P.A. Improvement of fertilization and pregnancy rate after intracytoplasmic fine morphology selected sperm injection.Fertil Steril. 2004; 82: S173Abstract Full Text Full Text PDF PubMed Google Scholar, 3Berkovitz A. Eltes F. Yaari S. Katz N. Barr I. Fishman A. et al.The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm.Hum Reprod. 2005; 20: 185-190Crossref PubMed Scopus (246) Google Scholar, 4Berkovitz A. Eltes F. Lederman H. Peer S. Ellenbogen A. Feldberg B. et al.How to improve IVF-ICSI outcome by sperm selection.Reprod Biomed Online. 2006; 12: 634-638Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 5Berkovitz A. Eltes F. Ellenbogen A. Peer S. Feldberg D. Bartoov B. Does the presence of nuclear vacuoles in human sperm selected for ICSI affect pregnancy outcome?.Hum Reprod. 2006; 21: 1787-1790Crossref PubMed Scopus (164) Google Scholar, 6Hazout A. Dumont-Hassan M. Junca A.M. Cohen Bacrie P. Tesarik J. High-magnification ICSI overcomes paternal effect resistant to conventional ICSI.Reprod Biomed Online. 2006; 12: 19-25Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 7Berkovitz A. Eltes F. Paul M. Adrian E. Benjamin B. The chance of having a healthy normal child following intracytoplasmic morphologically-selected sperm injection (IMSI) treatment is higher compared to conventional IVF-ICSI treatment.Fertil Steril. 2007; 88: S20Abstract Full Text Full Text PDF Google Scholar, 8Vanderzwalmen P. Hiemer A. Rubner P. Bach M. Neyer A. Stecher A. et al.Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles.Reprod Biomed Online. 2008; 17: 5617-5627Abstract Full Text PDF Scopus (165) Google Scholar, 9Antinori M. Licata E. Dani G. Cerusico F. Versaci C. d'Angelo A. et al.Intracytoplasmic morphologically selected sperm injection: a prospective randomized trial.Reprod Biomed Online. 2008; 16: 835-841Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 10Tasaka A. Doshida M. Sato Y. Kyoya T. Nakajo Y. Kyono K. Outcome of IMSI (intracytoplasmic morphologically selected sperm injection) in patients with repeated ICSI failures Fertil Steril. 2009; 92: S76Google Scholar, 11Yazbeck C. Delaroche L. Jacquesson L. Ayel J.-P. Selva J. Rougier N. Intracytoplasmic morphologically selected sperm injection (IMSI): is it a good choice after two or more IVF or ICSI failures?.Fertil Steril. 2008; 90: S416Abstract Full Text Full Text PDF Google Scholar), attention has been given to the existence of a correlation between sperm morphologic abnormalities observed at high magnification (>×6,000), particularly as to the presence of nuclear vacuoles (12Franco Junior J.G. Baruffi R.L.R. Mauri A.L. Petersen C.G. Oliveira J.B.A. Vagnini L. Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI.Reprod Biomed Online. 2008; 17: 42-45Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 13Garolla A. Fortini D. Menegazzo M. De Toni L. Nicoletti V. Moretti A. et al.High power magnification microscopy and functional status analysis of sperm in the evaluation and selection before ICSI.Reprod Biomed Online. 2008; 17: 610-616Abstract Full Text PDF PubMed Scopus (120) Google Scholar), and DNA damage. Nevertheless, the significance of each particular nuclear sperm form observed at MSOME in relation to DNA alteration has yet to be determined. To better comprehend the diagnostic/prognostic value of morphologic analysis of semen by high magnification, the present study aimed to evaluate the correlation between the MSOME classification and sperm DNA damage. Semen samples were obtained from 538 men from an unselected group of infertile couples. This study received internal Institutional Review Board approval. A portion of each semen sample was processed for MSOME and the remainder analyzed for DNA damage. Determination of morphology by MSOME was carried out as previous described (12Franco Junior J.G. Baruffi R.L.R. Mauri A.L. Petersen C.G. Oliveira J.B.A. Vagnini L. Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI.Reprod Biomed Online. 2008; 17: 42-45Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 14Oliveira J.B. Massaro F.C. Mauri A.L. Petersen C.G. Nicoletti A.P. Baruffi R.L. Franco Junior J.G. Motile sperm organelle morphology examination is stricter than Tygerberg criteria.Reprod Biomed Online. 2009; 18: 320-326Abstract Full Text PDF PubMed Scopus (39) Google Scholar). At least 200 motile spermatozoa per patient were evaluated at ≥×8,400 magnification by inverted microscope equipped with Nomarski differential interference contrast optics, and the percentages of the following spermatozoa forms were determined: normal nuclear spermatozoa (smooth, symmetric, and oval nucleus measuring 3.28 ± 0.20 μm in width and 4.75 ± 0.20 μm in length, with absence of vacuoles occupying >4% of nuclear area) (7Berkovitz A. Eltes F. Paul M. Adrian E. Benjamin B. The chance of having a healthy normal child following intracytoplasmic morphologically-selected sperm injection (IMSI) treatment is higher compared to conventional IVF-ICSI treatment.Fertil Steril. 2007; 88: S20Abstract Full Text Full Text PDF Google Scholar); abnormalities of nuclear form (spermatozoa with small or large oval nuclear forms [length ≤4.19 μm or ≥5.31 μm] (15Bartoov B. Berkovitz A. Eltes F. Kogosowski A. Menezo Y. Barak Y. Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome.J Androl. 2002; 23: 1-8PubMed Google Scholar), spermatozoa with wide or narrow nuclear forms [width >3.7 μm or 50% of the nuclear area]). Sperm cells with any severe abnormality (e.g., pin, amorphous, tapered, round, multinucleated head, double tail) easily identified at low magnification (×200–×400) were not assessed in this study. Spermatozoids that presented more than one alteration were classified as being the most severely altered (3Berkovitz A. Eltes F. Yaari S. Katz N. Barr I. Fishman A. et al.The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm.Hum Reprod. 2005; 20: 185-190Crossref PubMed Scopus (246) Google Scholar, 4Berkovitz A. Eltes F. Lederman H. Peer S. Ellenbogen A. Feldberg B. et al.How to improve IVF-ICSI outcome by sperm selection.Reprod Biomed Online. 2006; 12: 634-638Abstract Full Text PDF PubMed Scopus (171) Google Scholar) (small/large < wide/narrow < regional shape abnormality < with vacuoles occupying >4% of the nuclear area). DNA damage was measured by DNA fragmentation analysis using the terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay as previous described (12Franco Junior J.G. Baruffi R.L.R. Mauri A.L. Petersen C.G. Oliveira J.B.A. Vagnini L. Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI.Reprod Biomed Online. 2008; 17: 42-45Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 16Vagnini L. Baruffi R.L.R. Mauri A.L. Petersen C.G. Massaro F.C. Pontes A. et al.The effects of male age on sperm DNA damage in an infertile population.Reprod Biomed Online. 2007; 15: 514-519Abstract Full Text PDF PubMed Scopus (77) Google Scholar). At least 200 spermatozoa in randomly selected areas on microscope slides were evaluated using a fluorescent microscope, and the percentage of spermatozoa with fragmented DNA (TUNEL-positive cells) was determined. Correlations were performed using the Spearman rank correlation test. The level of significance was set at P<.05. The average age of the men was 37.4 ± 6.2 years, 34.6% had fathered at least one child (or a pregnancy that had ended in miscarriage), 14.1% had varicocele; 12.5% were smokers, 62.8% regularly used alcohol, and 13.6% regularly took vitamin supplements. According to MSOME, in samples examined, the mean incidence of morphologically normal nuclear spermatozoa was 1.8 ± 2.5% and the incidence of each abnormal form was: large/small spermatozoa 1.4 ± 1.7%; wide/narrow spermatozoa 1.8 ± 1.8%; spermatozoa with regional disorder 3.2 ± 2.7%; spermatozoa with vacuoles occupying 5%–50% of the nuclear area 65.7 ± 16.3%; and spermatozoa with vacuoles occupying >50% of the nuclear area 25.9 ± 19.2%. The mean DNA fragmentation was 18.4 ± 10%. Regression analysis demonstrated different results depending on the sperm form considered. First, there was a significant negative correlation between percentage of DNA fragmentation and percentage of normal nuclear sperm forms (P<.05; r = −0.16). In the same manner, percentages of sperm with abnormal nuclear form also presented significant negative correlations with percentages of DNA fragmentation, as follows: spermatozoa with small or large oval nuclear forms (P<.05, r = −0.13); spermatozoa with wide or narrow nuclear forms (P<.05, r = −0.15); and spermatozoa with regional shape abnormality of nuclear form (P<.05, r = −0.14). On the other hand, in relation to sperm with abnormalities of nuclear chromatin content, there was not a significant correlation between percentage of DNA fragmentation and percentage of spermatozoa with vacuoles occupying 5%–50% of the nuclear area (P>.05; r = −0.05). However, there was a significant positive correlation between the percentage of DNA fragmentation and the percentage of spermatozoa with large nuclear vacuoles (P<.05, r = 0.10). Figure 1 summarizes these results. Success in human reproduction depends on, among other factors, the integrity of sperm DNA. Clinical evidence now shows that sperm DNA damage is detrimental to reproductive outcomes and that the spermatozoa from infertile men possess substantially more DNA damage than do those of fertile men (17Zini A. Libman J. Sperm DNA damage: clinical significance in the era of assisted reproduction.CMAJ. 2006; 175: 495-500Crossref PubMed Scopus (147) Google Scholar). The present study evidenced a significant negative correlation between normal nuclear sperm levels at MSOME evaluation and DNA fragmentation levels. This finding corroborates the result of a previous study by our group (12Franco Junior J.G. Baruffi R.L.R. Mauri A.L. Petersen C.G. Oliveira J.B.A. Vagnini L. Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI.Reprod Biomed Online. 2008; 17: 42-45Abstract Full Text PDF PubMed Scopus (134) Google Scholar), in which a relatively low DNA fragmentation percentage (15.9%) was found in normal nuclear spermatozoa selected by high magnification. However, negative correlation with DNA fragmentation was not exclusive of normal nuclear sperm forms. The forms with alterations in nuclear dimensions (small/large or wide/narrow) presented a significant negative correlation with DNA fragmentation levels very close to those presented by normal nuclear forms. These findings indicate safety, from the perspective of DNA fragmentation, in using spermatozoa with these nuclear alterations. In addition, the sperm form with regional disorders (extrusion and/or invagination of chromatin), that theoretically would have a greater possibility of DNA damage, also presented negative correlation with DNA fragmentation level near that observed with normal nuclear form. In a study comparing normal nuclear sperm forms with spermatozoa exclusively with chromatin extrusion (18Mauri A.L. Oliveira J.B.A. Baruffi R.L.R. Petersen C.G. Vagnini L.D. Massaro F.C. et al.Significance of extruded nuclear chromatin mass (regional nuclear shape malformation) in human spermatozoa: implications for ICSI.Hum Reprod. 2009; 24: i217-i224Crossref Google Scholar), similar levels were also observed between groups as to DNA fragmentation, although those with extrusion presented more DNA denaturation than normal ones. In this context, the spermatozoa with altered nuclear form appear to present a prognosis as favorable as that of normal nuclear sperm in relation to the possibility of DNA fragmentation. On the other hand, the identification of abnormal chromatin content represented a change in the correlation with DNA fragmentation, in that this parameter was not significantly correlated with sperm showing vacuoles occupying 5%–50% of the nuclear area but presented a significant positive correlation with those presenting large nuclear vacuoles (>50% of the nuclear area). These results, besides indicating the detrimental effect of nuclear vacuole presence on sperm quality, demonstrate that the extent to which the nucleus is compromised (by vacuoles) reflects the extent of sperm DNA damage. These data corroborate the findings of other studies. Berkovitz et al. (3Berkovitz A. Eltes F. Yaari S. Katz N. Barr I. Fishman A. et al.The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm.Hum Reprod. 2005; 20: 185-190Crossref PubMed Scopus (246) Google Scholar, 4Berkovitz A. Eltes F. Lederman H. Peer S. Ellenbogen A. Feldberg B. et al.How to improve IVF-ICSI outcome by sperm selection.Reprod Biomed Online. 2006; 12: 634-638Abstract Full Text PDF PubMed Scopus (171) Google Scholar), who graded the severity of nuclear morphologic alterations while highlighting the presence of large vacuoles, suggested that vacuolization of the sperm nucleus reflects some underlying chromosomal or DNA defects. Berkovitz et al. (5Berkovitz A. Eltes F. Ellenbogen A. Peer S. Feldberg D. Bartoov B. Does the presence of nuclear vacuoles in human sperm selected for ICSI affect pregnancy outcome?.Hum Reprod. 2006; 21: 1787-1790Crossref PubMed Scopus (164) Google Scholar) and Bach et al. (19Bach M. Neyer A. Stecher A. Uher P. Vanderzwalmen P. Zintz M. et al.Morphological integrity of human sperm nuclei and blastocyst formation after intracytoplasmic morphologically selected sperm injection (IMSI).Hum Reprod. 2007; 22: i108-i109Google Scholar) reported that the presence of vacuoles provokes harm to embryo development, reduction in the pregnancy rate and increase in the miscarriage rate. Vanderzwalmen et al. (8Vanderzwalmen P. Hiemer A. Rubner P. Bach M. Neyer A. Stecher A. et al.Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles.Reprod Biomed Online. 2008; 17: 5617-5627Abstract Full Text PDF Scopus (165) Google Scholar) demonstrated that nuclear vacuoles negatively affect the percentage of embryos that reach the blastocyst stage in ICSI cycles. Franco et al. (12Franco Junior J.G. Baruffi R.L.R. Mauri A.L. Petersen C.G. Oliveira J.B.A. Vagnini L. Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI.Reprod Biomed Online. 2008; 17: 42-45Abstract Full Text PDF PubMed Scopus (134) Google Scholar) showed an association between large nuclear vacuoles and both the presence of DNA fragmentation and denaturation in the spermatozoa. In addition, Garolla et al. (13Garolla A. Fortini D. Menegazzo M. De Toni L. Nicoletti V. Moretti A. et al.High power magnification microscopy and functional status analysis of sperm in the evaluation and selection before ICSI.Reprod Biomed Online. 2008; 17: 610-616Abstract Full Text PDF PubMed Scopus (120) Google Scholar) showed that the presence of nuclear vacuoles affects mitochondrial function, chromatin status, and aneuploidy rate. The present data support recent studies that propose classifications for defining semen quality based on analysis at high magnification, with special emphasis on the number and extension of nuclear vacuoles (8Vanderzwalmen P. Hiemer A. Rubner P. Bach M. Neyer A. Stecher A. et al.Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles.Reprod Biomed Online. 2008; 17: 5617-5627Abstract Full Text PDF Scopus (165) Google Scholar, 20Cassuto N.G. Bouret D. Plouchart J.M. Jellad S. Vanderzwalmen P. Balet R. et al.A new real-time morphology classification for human spermatozoa: a link for fertilization and improved embryo quality.Fertil Steril. 2009; 92: 1616-1625Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 21Saïdi R. Rives N. Gruel E. Mazurier S. Mousset-Simeon N. Mace B. Nouvelle classification du spermogramme à fort grossissement.Med Reprod Gyn Endo. 2008; 10: 315-324Google Scholar). But despite the diagnostic and prognostic advantages of these classifications, concerns are raised from the clinical point of view regarding individuals that present nuclear vacuoles in 100% of their spermatozoids (in our sampling, ∼10% of men). In this situation, obtaining success in the reproduction processes should depend on the possibility (but not certainty) of correction, by oocytes, of probable damage in sperm DNA (20Cassuto N.G. Bouret D. Plouchart J.M. Jellad S. Vanderzwalmen P. Balet R. et al.A new real-time morphology classification for human spermatozoa: a link for fertilization and improved embryo quality.Fertil Steril. 2009; 92: 1616-1625Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 22Tesarik J. Mendoza-Tesarik R. Mendoza C. Sperm nuclear DNA damage: update on the mechanism, diagnosis and treatment.Reprod Biomed Online. 2006; 12: 715-721Abstract Full Text PDF PubMed Scopus (62) Google Scholar). Furthermore, incomplete or incorrect repair of sperm DNA damage also can lead to impairment in the reproductive process and, in turn, in the offspring. On the other hand, the existence of a correlation would imply that strategies to reduce sperm DNA damage would serve as alternatives for diminishing the percentage of vacuolated spermatozoa (23Aitken R.J. De Iuliis G.N. Origins and consequences of DNA.damage in male germ cell.s Reprod Biomed Online. 2007; 14: 727-733Abstract Full Text PDF PubMed Scopus (300) Google Scholar, 24Zini A. San Gabriel M. Baazeem A. Antioxidants and sperm DNA damage: a clinical perspective.J Assist Reprod Genet. 2009; 26: 427-432Crossref PubMed Scopus (134) Google Scholar, 25Tunc O. Thompson J. Tremellen K. Improvement in sperm DNA quality using an oral antioxidant therapy.Reprod Biomed Online. 2009; 18: 761-768Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 26Ménézo Y.J.R. Hazout A. Panteix G. Robert F. Rollet J. Cohen-Bacrie P. et al.Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect.Reprod Biomed Online. 2007; 14: 418-421Abstract Full Text PDF PubMed Scopus (225) Google Scholar). In conclusion, the results of the present study show that both normal and abnormal nuclear forms, under high-magnification analysis, appear to be equally favorable from a DNA fragmentation point of view. The only sperm type that correlates with a high rate of DNA fragmentation is the category of sperm with >50% vacuolated nucleus. Based on clinical/laboratory findings on the repercussions of possible DNA damage in offspring (27Carrell D. Symposium: genetic and epigenetic aspects of assisted reproduction. Contributions of spermatozoa to embryogenesis: assays to evaluate their genetic and epigenetic fitness.Reprod Biomed Online. 2008; 16: 474-484Abstract Full Text PDF PubMed Scopus (62) Google Scholar), and given that sperm nuclear vacuoles are evaluated more precisely at high magnification by MSOME (15Bartoov B. Berkovitz A. Eltes F. Kogosowski A. Menezo Y. Barak Y. Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome.J Androl. 2002; 23: 1-8PubMed Google Scholar), the present results support the routine use of MSOME for ICSI and as a criterion for semen analysis with potential clinical repercussions. The authors thank the Research Support Group, Sao Paulo State University for revising the English text.
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