The main purpose of this study was to determine the possible relationship between chromatin condensation (Aniline Blue staining), the morphology of spermatozoa according to strict criteria, and the fertilization, cleavage and pregnancy rate in an intracytoplasmic sperm injection (ICSI) programme. A total of 60 patients were divided into two groups (27 versus 34) according to sperm stainability by Aniline Blue. The first group involved patients having a positive Aniline Blue staining test with 0-29% stained. The fertilization rate in this group was 60.8%, cleavage rate 54.4% and pregnancy rate 18.5%. In the second group in which > 29% spermatozoa were positively stained, the fertilization rate was 62.1%, cleavage rate 62.0% and pregnancy rate 35.3%. There was no statistically significant difference between the two groups. Furthermore, the influence of morphology according to strict criteria after Papanicolaou staining on successful fertilization, cleavage and pregnancy was studied in 85 patients who were divided into two groups according to the percentage of morphologically normal sperm. The fertilization, cleavage and pregnancy rates were 44.21, 63.37, and 39.47% respectively in the first group (< 4%), the corresponding values for the second group (> 4%) were 56.50, 46.04 and 21.21%. There was no significant correlation between the fertilization (P = 0.722), cleavage (P = 0.519) and pregnancy (P = 0.096) rates in either group. This study demonstrates that neither chromatin condensation (Aniline Blue staining) nor morphology could assess the fertilization potential, cleavage and pregnancy rate in an ICSI programme.
The purpose of this study was to determine the negative effects (cryodamage) on human spermatozoa after freeze-thawing and to determine whether freeze-thawing of spermatozoa with a programmed slow freezer is better than freezing with liquid nitrogen vapour (rapid freezing) with regard to alterations in sperm chromatin and morphology in semen from fertile (donor) and subfertile, IVF/ICSI, patients. Ninety-five semen samples were obtained either from patients attending our IVF unit for treatment (n=34) or from donors (n=25) with proven fertility and normal sperm quality according to WHO guidelines. Each semen sample was divided into two parts after liquefaction and addition of the cryoprotectant. The first part was frozen using a programmed biological freezer and the second part was frozen by means of liquid nitrogen vapour. Smears were made before the freezing and after the thawing procedure to assess morphology (strict criteria) and chromatin condensation (Acridine Orange test). The mean percentage of chromatin condensed spermatozoa in the samples from donors (control group) was 92.4 +/- 8.4% before freezing and this decreased significantly (p < 0.0001) to 88.7 +/- 11.2% after freeze-thawing with the computerized slow-stage freezer and to 87.2 +/- 12.3% after using static liquid nitrogen vapour (p < 0.001). The corresponding values for semen obtained from patients was 78.9 +/- 10.3% before freezing which decreased to 70.7 +/- 10.8 and 68.5 +/- 14.8%, respectively (p < 0.001). On the other hand, the mean percentage of normal sperm morphology in the control group decreased from 26.3 +/- 7.5% before freezing to 22.1 +/- 6.4% (p < 0.0001) after thawing with the computerized slow-stage freezer and to 22.2 +/- 6.6% (p < 0.0001) after the use of static liquid nitrogen vapour. In the patient group, the mean percentage of normal morphology decreased from 11.7 +/- 6.1% after freezing with the biological freezer to 9.3 +/- 5.6% and to 8.0 +/- 4.9% after freezing with static liquid nitrogen vapour. This study demonstrates that chromatin packaging and morphology of human spermatozoa decrease significantly after the freeze-thawing procedure, not only after the use of static liquid nitrogen vapour but also after the use of a computerized slow-stage freezer. However, the chromatin of semen samples with normal semen parameters (donor sperm) withstand the freeze-thaw injury better than those with low quality semen samples. Therefore, the computerized slow stage freezer could be recommended for freezing of human spermatozoa, especially for subnormal semen samples, for example, ICSI and ICSI/TESE candidates and from patients with testicular tumours or Hodgkin's disease, in order to avoid further damage to the sperm chromatin structure.
In this study, a total of 95 ejaculates from infertile patients were investigated morphologically according to Kruger's strict criteria and 78 of the 95 ejaculates were stained for chromatin condensation with acidic aniline blue. Patients were divided into two groups based on the percentage of morphologically normal spermatozoa as follows: Men with normal sperm morphology < 14% (Group I), and men with normal morphology > 14% (Group 2). The relationship between percentage of normal sperm morphology and fertilization, cleavage and pregnancy rate was analysed. The rates were 33.7%, 57.1% and 0.0% respectively, in the first group. The corresponding values for the second group were 76.1%) 68.2% and 32.1 YO. The fertilization and pregnancy rates correlate significantly with morphologically normal spermatozoa.In regard to the percentage of morphologically normal spermatozoa stained with aniline blue, patients were divided into two groups: patients with 0-20°/0 stained spermatozoa (Group I) and those with >20% (Group 2). Fertilization and pregnancy rates were higher in the first group than in the second group (79.9%, 52.8% vs. 58.8%, 29.5%).The results demonstrate that chromatin condensation visualized by aniline blue staining is a good predictor for IVF outcome and should be considered besides morphology by sperm assessment for patients undergoing IVF treatment.
A significant association between male subfertility, imperfect spermiation and abnormal nuclear condensation has been suggested. The DNA content of spermatozoa might be responsible for inducing alterations in sperm morphology. The final nuclear shape, which is species-specific, depends on chromatin condensation during spermatogenesis as well as a precise organization of DNA within the nucleus. Many reports have described the association between disturbances in sperm chromatin condensation, morphology and male infertility. Chromatin condensation is achieved by gradual substitution of lysinerich somatic histones by testis-specific histone and finally by protamine. In this study two groups of patients were compared: the first consisted of 63 patients who had undergone intracytoplasmic sperm injection (ICSI) with freshly ejaculated spermatozoa whereas the second included 47 patients assigned to ICSI with testes biopsy-extracted spermatozoa. In both groups chromatin condensation was assessed by aniline blue staining and morphology evaluated according to strict criteria. The condensed chromatin and morphology of spermatozoa were significantly (P < 0.0001) less in the second group compared to the first. However the fertilization, cleavage, implantation and pregnancy rates were almost the same in both investigated groups. There was no significant difference between the two groups with respect to ICSI outcome. The percentage of chromatin condensation (nuclear maturity) and morphologically-normal spermatozoa were significantly higher (P < 0.0001) in the ejaculated spermatozoa than in those from testis biopsy but the ICSI outcome (fertilization, cleavage, implantation and pregnancy rates) was the same. In view of these results the fertilization capability and the embryo quality obtained using testis biopsy extracted spermatozoa is not influenced by chromatin condensation and sperm morphology in testicular sperm extraction (TESE)-ICSI programmes. Therefore, it could be said that neither chromatin condensation nor morphology of testis extracted sperm could predict the fertilization, implantation and pregnancy rate in TESE-ICSI programmes.
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