In this study, we analysed the distribution of beta tubulins to detect spindle and cytoplasmic microtubules, alpha acetylated tubulins for sperm microtubules and chromatin configuration in oocytes showing fertilization failure after conventional IVF or intracytoplasmic sperm injection (ICSI). A total of 450 human oocytes that failed to fertilize were studied 20-40 h after IVF or ICSI. In all, 287 oocytes were stained for immunofluorescence and chromosomal spreads were performed by Tarkowski's air-drying method in 163 IVF or ICSI oocytes that did not develop pronuclei after the extrusion of a second polar body. Immunofluorescence analysis showed that the main reason of fertilization failure after IVF was no sperm penetration (55.5%). The remaining oocytes showed different abnormal patterns, e.g. oocyte activation failure (15.1%) and defects in pronuclei apposition (19.2%). On the other hand, fertilization failure after ICSI was mainly associated to incomplete oocyte activation (39.9%), and to a lesser extent with defects in pronuclei apposition (22.6%) and failure of sperm penetration (13.3%). A further 13.3% of the ICSI oocytes arrested their development at the metaphase of the first mitotic division. The chromosomal spreads allowed the analysis of abortive activations, in which no pronuclei formed but a second polar body was extruded. Immunofluorescence and cytogenetic analysis provided a useful tool to improve infertility diagnosis and prognosis in each particular case.
ObjectiveThe aim of this study was to investigate the effect of sperm DNA
fragmentation on fertilization rate, embryo development (blastulation rate),
and pregnancy outcomes for ICSI cycles performed in a cohort of couples
using donor eggs and to assess the remaining embryos that were not
transferred or frozen for apoptotic markers.MethodsEighty-two women (egg recipients) were included in the study (2016) were
included in the study. The recipients' mean age was 41.8±5.1 y/o
(36-49), while the egg donors' mean age was 30.8±2.1 y/o (27-33).
Even though donor egg cycles with frozen sperm samples are performed
regularly in our center, 35 cycles were done using fresh sperm samples. The
mean age of the males involved in the procedure was 40.1±5.2 y/o.
Fertilization, blastulation, and pregnancy rates were assessed. The patients
were divided into two groups, TUNEL <15% and ≥15%. In arrested
embryos, ICC was performed to detect cleaved caspase-3, survivin, TUNEL, and
DNA. The Student's t-test was used in between-group
comparisons. The Mann-Whitney U-test was used to assess
homogeneity. Pearson's correlation coefficient was also calculated.
p<0.05 was considered statistically significant.Results This study showed that there is a negative correlation (R=-0.5) between DNA
fragmentation and blastulation rate. High levels of DNA fragmentation were
associated with low blastulation and pregnancy rates (per transfer);
however, fertilization rate was not affected. Samples with higher levels of
DNA fragmentation were associated with higher levels of DNA fragmentation in
blastomeres without activating the apoptotic pathway (9.1% vs. 15.9%)
(p<0.05). Blastomeres from samples with high DNA
fragmentation activated the apoptotic pathway in higher levels than samples
with TUNEL <15% (16.4% vs. 21.9%) (p<0.05).ConclusionSperm DNA fragmentation was negatively correlated with blastulation and
pregnancy rates even in good quality oocytes. High levels of DNA damage
promote embryo arrest and induce the activation of the apoptotic
pathway.
Magnetic activated cell sorting (MACS) with annexin V microbeads recognizes externalized phosphatidylserine (PS) residues on the surface of apoptotic spermatozoa. The successful use of this novel technique applied to a highly apoptotic semen sample before performing intracytoplasmic sperm injection (ICSI) is reported here. The use of annexin V microbeads for selecting non-apoptotic spermatozoa seems to reduce the percentage of altered cells, improving the chance of pregnancy after ICSI.
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