Detection of Sperm DNA Fragmentation in IVF-ICSI Patients and Correlation with Sperm Quality and Treatment Outcome

Med. J. Cairo Univ., Vol. 77, No. 3, June: 109-113, 2009 www.medicaljournalofcairouniversity.com Detection of Sperm DNA Fragmentation in IVF-ICSI Pat...
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Med. J. Cairo Univ., Vol. 77, No. 3, June: 109-113, 2009 www.medicaljournalofcairouniversity.com

Detection of Sperm DNA Fragmentation in IVF-ICSI Patients and Correlation with Sperm Quality and Treatment Outcome SHERIF A. GHAZI, M.D.*; HOWIDA K. ABDULFATTAH, M.D.**; AMANY A. SHALTOUT, M.D.***; AMAL M. SHOHEIB, M.D.***; MONA M. MOSTAFA, M.D.*** and EMAN K. SHOEIR, M.D.*** The Departments of Andrology*, Genetic Laboratories** and Obstetrics & Gynaecology***, Faculty of Medicine, Cairo University.

Abstract

cause is obscured. Several putative mechanisms for DNA damage was suggested including apoptosis, abnormal DNA condensation during spermiogenesis and deficiencies in paternal DNA repair mechanism [2-4] .

The incidence of DNA fragmentation in the sperm head (DFI) is higher in infertile male population compared to the fertile one. Several methods can be used to detect sperm DNA fragmentation. Among this method TUNEL assay is reported to be the most related to the male fertility potential. The aim of this study is to examine the relationship between sperm DNA fragmentation and the outcome of intracytoplasmic sperm injection (ICSI). Eighty-five couples undergoing ICSI procedure were recruited to this study. Sperm from the row ejaculate was examined for sperm DFI was using TUNEL assay. There was no relation between the sperm DFI and the conventional WHO semen parameter. There was also no significant difference in the fertilization and cleavage rate between couples with low, moderate or high DFI (30% respectively). Pregnancy rate was significantly lower when sperm DFI was higher than 15% and no pregnancy was achieved when the DFI was higher than 30%. These results demonstrate the negative effect of the DNA fragmentation on the ICSI outcome. This deleterious effect seems to affect later stages of embryonic development.

Several tests are used to detect sperm DNA damage. The most commonly used tests are TUNEL (Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labelling) and SCSA (sperm chromatin structure assay) techniques. TUNEL technique identifies the DNA breaks by labelling 3'OH terminal using exogenous terminal deoxynucleotidyl transferase, while in sperm chromatin structure assay (SCSA) the acid extracted sperm nuclei are stained with acridine orange. Intracytoplasmic sperm injection (ICSI) is increasingly used to help infertile couples wishing to have a child. However pregnancy rate is still relatively low. Classic semen parameters like sperm concentration, motility and abnormal forms can not predict ICSI outcome.

Key Words: Sperm DNA – FISH – ICSI.

Introduction THE main function of the sperm is the transmission of the paternal genomic information to the developing embryo. In order to protect this message during the sperm transit through the genital tract, sperm undergo a series of changes in the later stages of spermatogenesis including the replacement of histones with protamines. These changes condense the sperm DNA tightly and turn it into tough crystalline-like state which is bio-chemically almost inaccessible [1] . Nevertheless, sperm DNA remains susceptible to damage. All ejaculates contain variable percentage of sperm with damaged DNA. Such damage can be attributed to factors like exposure to irradiation, gonado-toxins, varicocele and increased level of seminal reactive oxygen species. However in most of the cases the

There are several reports of possible correlation between the incidence of sperm DNA damage and the fertility potential both in vivo and in vitro [510] . The aim of this study is to explore the possible correlation between DNA fragmentation in sperm as evaluated by the TUNEL technique and the outcome of ICSI procedure in terms of fertilization and pregnancy rates. Patients and Methods The study group included 85 consecutive couples undergoing ICSI procedure using ejaculated spermatozoa. Patient with sperm count less than 100000 sperm/ml were excluded to allow for easy

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Detection of Sperm DNA Fragmentation in IVF-ICSI Patients

counting of sperm in tunnel technique. Couples using testicular sperm or frozen thawed sperm were also excluded. Semen analysis and sperm preparation: On day of ICSI semen samples were collected by masturbation in sterile plastic jars. After liquifaction a small aliquot was used for semen assessment according to WHO criteria. Semen volume, sperm density, motility and the percentage of normal shaped sperm were recorded. Sperm were prepared for ICSI using discontinuous puresperm gradient. 0.5ml of semen was layered on the 40, 80, 100% gradient and centrifuged for 25 minutes at 300G. Sperm from the 100% layer were obtained and washed in HEPES IVF media before being used for ICSI. ICSI procedure and embryo assessment: After down regulation of the pituitary gland using GnRH analogue (Decapeptyl, Ferring) controlled ovarian stimulation was achieved using personalized dosage of recombinant FSH (Gonal F, Serono or Purigon, Organon) guided by follicular maturation monitoring using ultrasonography and plasma estradiol. When ovarian follicles reach satisfactory level of maturation, 10000 IU of hCG was administered 36 hours before schedules oocytes' collection which is done through ultra sound guided vaginal aspiration. At 16-18 hours after microinjection, the oocytes were checked for the development of the two pronuclei as a sign of fertilization. Embryos were examined three days following microinjection before transfer and they are graded according to quality to grade A: less than 10% fragments and blastomeres are regular, grade B:

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