Embryo development after intracytoplasmic sperm injection can be predicted by assessment of sperm nuclear chromatin.

PURPOSE: To assess the influence of structural differences in sperm nuclei on embryo development in intracytoplasmic sperm injection (ICSI). METHODS: Semen obtained from forty-four infertile patients who underwent ICSI was examined. In assessing blastocyst development, only those patients who had successfully obtained over five fertilized eggs were included to exclude any oocyte factors (n = 22). Spermatozoa were assessed using excitation fluorescence flow cytometry with acridine orange and the sperm chromatin dispersion (SCD) test. RESULTS: There was a significant positive correlation between the COMP values obtained from flow cytometry and blastocyst formation. (r = 0.477, p = 0.025). There was a significant negative correlation between the SCD values representing DNA fragmentation and blastocyst formation. (r = 0.796, p < 0.001). COMP values and SCD values were independent parameters to assess sperm nuclear quality regarding embryo development in vitro (r = 0.224, p = 0.080). CONCLUSION: Results suggest that injection of spermatozoa with fewer disulfide bonds and less nuclear DNA fragmentation could achieve better blastocyst formation in human ICSI. Assessment of sperm chromatin should help to predict embryo development after ICSI.

The present status of artificial oocyte activation in assisted reproductive technology.

Intracytoplasmic sperm injection (ICSI) is the most effective treatment for achieving fertilization in assisted reproductive technology (ART). However, fertilization failure occurs. The incidence of fertilization failure after ICSI is 1-5%. Approximately 50% of fertilization failure cases could be attributed to the abnormality of sperm factor. As the fertilization fails after ICSI using mature sperm, round spermatids and globozoospermia, artificial oocyte activation may provide a means of improving fertilization rates in such cases. The oocyte activation treatments used in clinical research include calcium (Ca) ionophore treatment, electrostimulation and strontium treatment. In terms of the efficiency of oocyte activation, electrostimulation and Ca ionophore gave better outcomes than strontium treatment. Strontium treatment causes Ca2+ oscillations in mice, so it has been viewed favorably. However, in human oocytes calcium oscillation has not been observed. The fertilization rate after ICSI was low in the case of globozoospermia and wiht round spermatids. Some cases of pregnancy were achieved by ICSI alone and oocyte activation methods were not essential in these cases. Among the various oocyte activation methods currently used, it should be noted that issues of genetic safety have not been addressed for the combined use of these oocyte activation methods. (Reprod Med Biol 2008; 7: 133-142).

Role of mammalian sperm nuclear structure in fertilization and embryo development.

Although intact spermatozoon is successfully collected from infertile patients, repeated implantation failure or pregnancy loss are often experienced. Sperm nuclear defects have been thought to be one of the most important reasons for repeated assisted reproductive technology failure. In comparison with other mammalians, characteristic heterogeneity has been found in each mature human sperm nuclei, therefore it is necessary to investigate the significance between fertilization failure, developmental disability and structural abnormality of human sperm nuclei. Furthermore, if close relationships between the heterogeneity of human ejaculated sperm nuclei and DNA fragmentation are defined by analyzing sperm nucleoproteins, it would be clearly shown that impaired sperm chromatin leads to failure of embryo development in vitro or in vivo, so called late paternal effect on embryo development. It will be necessary in the near future to study the strategy for more novel methodology than those previously reported in terms of sperm selection. The present report reviews the roles of mammalian sperm nuclear structure, especially in humans, in fertilization and embryo development after the insemination procedure. (Reprod Med Biol 2006; 5: 161-168).

Use of diamide-acridine orange fluorescence staining to detect aberrant protamination of human-ejaculated sperm nuclei.

OBJECTIVE: To investigate the influence of human sperm nuclear chromatin on fertilization. DESIGN: Prospective study. SETTING: Assisted reproductive technology unit at a university teaching hospital. PATIENT(S): Fifty men starting an IVF-ET program. INTERVENTION(S): Epifluorescent microscopic observation of human-ejaculated sperm nuclei stained with diamide-acridine orange. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of extracted sperm nucleoproteins. MAIN OUTCOME MEASURE(S): Usefulness of diamide-acridine orange in analysis of human sperm nuclear chromatin and fertilization ability. RESULT(S): There was no correlation between the semen parameters and the diamide-acridine orange observation. A positive correlation was observed between the fertilization rate after conventional IVF and the green-type increase ratio (percentage of green-pattern sperm after diamide-acridine orange staining/percentage of green-pattern sperm after acridine orange staining). Furthermore, it was suggested by SDS-PAGE that structural differences were noticed between the fertile men and the men with sperm immaturity diagnosed after diamide-acridine orange staining. CONCLUSION(S): Diamide-acridine orange staining was a more precise method for detecting chromatin abnormalities in human-ejaculated sperm and evaluating fertilization ability than acridine orange staining alone. This method can be used as a diagnostic tool to assess the fertilization ability of human-ejaculated spermatozoa before IVF procedures.

Relationship between Fertilizing Ability of Ejaculated Human Spermatozoa and Its Chromatin Heterogeneity.

OBJECTIVE: The aim of this study was to examine the relationship between the fertilizing ability of ejaculated human sperm and its chromatin heterogeneity. METHOD: We used the D-AO staining method (acridine orange epifluorescence accompanied by diamide, a thiol oxidizing agent) to analyze the sperm chromatin structure of infertile patients with IVF-ET treatment. SDS-PAGE was performed to analyze the sperm nuclear proteins collected from patients with immature sperm (stained red with D-AO staining) and proven fertile men. RESULTS: 1. It was suggested that D-AO staining allowed the immature sperm to be divided into two groups. One was immature sperm, which had disturbance of S-S formation in the epididymides, and the other was sperm that which had an abnormal exchange process of nuclear proteins in the testes. 2. The stainability after D-AO staining showed no correlation with the findings of semen analysis. 3. The fertilization rate of IVF-ET was significantly correlated to the percentage of green sperm staining with D-AO staining. 4. In the pregnant group after IVF-ET, it was noticed that sperm of the green type with D-AO staining was increased in comparison with the non-pregnancy group. 5. The fertilization rate in the group of the sperm stained red with D-AO staining was increased to 73.5% by ICSI. 6. Definite differences were noticed between the protein components of the patients with immature sperm and those of the proven fertile men by analysing with SDS-PAGE. CONCLUSION: D-AO staining was an efficient method for evaluating the fertilizing ability of human ejaculated sperm, and to determine an appropriate ART tool such as ICSI.

Fertilization failure from a sperm chromatin defect in couples with unexplained infertility.

OBJECTIVE: To investigate the relationship between unexplained infertility and fertilization failure from nucleoprotein defects in ejaculated human sperm and to study the usefulness of sperm chromatin assays, using AO fluorescence dye, to evaluate patients with unexplained infertility before treatment. STUDY DESIGN: From January 1999 to January 2000, 513 infertile couples had the clinical causes of their infertility assessed. During the next investigative period (February 2000-February 2001), 137 cases of unexplained infertility (n = 80) were chosen for this study, as were cases of tubal factor infertility (n = 57) as controls. The status of nuclear chromatin in ejaculated sperm was examined using acridine orange staining, followed by a conventional in vitro fertilization procedure. RESULTS: The number of patients with immature ejaculated sperm was 16 of 30 (53.3%) unexplained infertility cases involving fertilization failure, 8 of 50 (16.0%) unexplained infertility cases without fertilization failure and 5 of 57 (8.8%) tubal factor infertility cases. A significant difference was observed between unexplained infertility cases with fertilization failure and the other groups (P < .0001). CONCLUSION: These results suggest that the nuclear immaturity of ejaculated human sperm may be 1 of the primary factors underlying unexplained infertility.

Chromosomal analysis of mouse spermatozoa following physical and chemical treatments that are effective in inactivating HIV.

Human immunodeficiency virus (HIV) can be inactivated by heating at 56 degrees C for 30 min, treating with 50% ethanol at room temperature for 10 min, or treating with 2% sodium hypochlorite solution (NaClO) at room temperature for 60 min. Using a mouse model, we evaluated the risk of generating chromosome damage in spermatozoa following these treatments. The spermatozoa were all dead after the treatments. Although 41.3% of oocytes injected with ethanol-treated spermatozoa successfully activated, none of the oocytes injected with heated or NaClO-treated spermatozoa activated. When artificial stimulation with strontium was used, the fertilization of oocytes with heated or ethanol-treated spermatozoa was completely rescued. Sperm nuclei treated with NaClO neither decondensed nor developed to a male pronucleus. The incidences of structural chromosome aberrations in 1-cell zygotes derived from the heated spermatozoa (45.6%) and ethanol-treated spermatozoa (91.2%) were significantly higher than those in the matched controls (5.5% and 10.5%, respectively). Further study is needed to develop a methodology for the protection of spermatozoa against chromosome damage or the separation of damaged spermatozoa before intracytoplasmic sperm injection.