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Hum. Reprod. Advance Access published online on June 24, 2009
Human Reproduction, doi:10.1093/humrep/dep088
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© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Selection of sperm based on combined density gradient and Zeta method may improve ICSI outcome

M. Kheirollahi-Kouhestani1, S. Razavi2, M. Tavalaee3, M.R. Deemeh3,4, M. Mardani2, J. Moshtaghian1 and M.H. Nasr-Esfahani3,4,5

1 Department of Biology, Isfahan University, Isfahan, Iran 2 Department of Anatomy, Isfahan Medical University, Isfahan, Iran 3 Department of Andrology and Embryology, Reproductive Medicine Research Center, Royan Institute (Isfahan Campus), ACECR, Tehran, Iran 4 Isfahan Fertility and Infertility Center, Isfahan, Iran

5 Correspondence address: Tel: +98-311-2612900; Fax: +98-311-2605525; E-mail: mh.nasr-esfahani{at}royaninstitute.org

BACKGROUND: Reducing the percentage of sperm anomalies in insemination samples remains a goal to be achieved in the intra-cytoplasmic sperm insemination (ICSI) procedure. The aim of this study was to evaluate the efficiency of density gradient centrifugation (DGC) and Zeta methods to recover sperm with intact chromatin, and to evaluate whether the combined DGC/Zeta procedure improved ICSI outcome.

METHODS: In Experiment 1, DGC and Zeta methods were carried out on 60 unprocessed semen samples. The samples were then assessed by chromomycin A3 staining, acridine orange test, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and the sperm chromatin dispersion test for protamine deficiency and DNA fragmentation. In Experiment 2, sibling oocytes from 30 ICSI candidates were divided into two groups; one group was inseminated with sperm processed by DGC and the second with sperm processed by DGC/Zeta. The outcomes of 30 ICSI cycles were compared between the two groups and also with 34 ICSI candidates whose oocytes were inseminated by DGC-processed sperm.

RESULTS: Both procedures were efficient for the recovery of sperm with normal protamine content and low DNA fragmentation. However, the Zeta method yielded a greater number of sperm with less DNA fragmentation. Fertilization and pregnancy rates were improved following the combined DGC/Zeta procedure. Compared with DGC alone, the pregnancy rate appeared improved but this was not statistically significant (P = 0.091).

CONCLUSIONS: Combining DGC and Zeta procedures improves the quality of semen samples which may increase fertilization rates and possibly pregnancy rates.

Key words: Zeta method/density gradient centrifugation/DNA damage/fertilization rate/pregnancy rate

Submitted on December 4, 2008; resubmitted on March 8, 2009; accepted on March 16, 2009.


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