• Proceedings of the KSAR Conference
• /
• 2001.03a
• /
• pp.41-41
• /
• 2001

Chromatin configuration and microtubule assembly were determined in porcine maturing and activated oocytes following intracytoplasmic sperm injection. Microtubule localization was confirmed using a mouse monoclonal antibody to $\alpha$-tubulin and detected using a fluorescent labeled goat anti-mouse secondary antibody. DNA was stained with propidium iodide. The image of microtubules and chromatin was captured using laser scanning confocal microscope. In germinal vesicle stage oocyte, sperm chromatin remained condensation and sperm derived microtubules were not observed at 8 to 12 h after sperm injection. At 24 h after injection, the sperm nucleus developed to the metaphase chromatin along the metaphase structure of female nucleus. In some metaphase I stage oocytes, sperm chromatin decondensed at 8 h to 12 h after injection, sperm aster was seen soon after sperm injection. At 24 h after sperm injection into metaphase I stage oocyte, male chromatin developed to the metaphase chromatin while female chromatin extruded first polar body and formed the metaphase chromatin. At 12 to 15 h after sperm injection into preactivated oocytes, condensed sperm nucleus was located in close proximity of female pronucleus. However, the condensed nucleus did not fuse with female pronucleus. In preactivated ocytes, injected sperm remained condensation, a few sperm organized small microtubular aster. Instead, maternal derived microtubules were organized near the female chromatin, which seem to move condensed male chromatin near to the female pronucleus. These results suggest that sperm nuclear decondensing activity and nucleation activity of centrosome during fertilization are cell cycle dependent. In absence of male functional centrosome, female origin centrosome takes over the role of microtubule nucleation for nuclear movement.

• Clinical and Experimental Reproductive Medicine
• /
• v.26 no.3
• /
• pp.399-405
• /
• 1999

Objective: In the preparation of ICSI, cumulus and corona cells should be removed from the oocytes by using a combination of enzymatic (hyaluronidase) and mechanical (pipetting) methods. But little is known about the effects of different degrees of oocyte denudation and incubation time between denudation and sperm injection on the outcomes of ICSI. The aim of this study was to evaluate the effects of varying the degrees of oocyte denudation and the lengths of incubation time from denudation to sperm injection on the outcomes of ICSI. Methods: In experiment 1, patients (oocytes) were grouped into group A and B according to the degree of denudation, complete and partial, respectively. In experiment 2, patients (oocytes) were grouped into group I, II and III according to the length of incubation time of denuded oocytes until sperm injection as < 1, $1{\sim}2$ and >2 hours, respectively. Results: There was no significant difference between the degree of oocyte denudation on the survival, fertilization and development rates after ICSI procedure. In case of the incubation time of denuded oocytes until ICSI, survival rates was higher in group III (83.1 %) than in group I (61.5%, p<0.05) or group II (64.3%). However no statistically significant differences were found between incubation time and fertilization or development rates. Conclusions: This study reveals that the outcomes of ICSI are not affected by the degree (complete or partial) of oocyte denudation. However, the denuded oocytes with incubation period of more than 2 hours show better outcomes of ICSI than those with the incubation period of less than 2 hours.

• Clinical and Experimental Reproductive Medicine
• /
• v.42 no.2
• /
• pp.45-50
• /
• 2015

Objective: Artificial oocyte activation (AOA) is an effective method to avoid total fertilization failure in human in vitro fertilization-embryo transfer (IVF-ET) cycles. AOA performed using a calcium ionophore can induce calcium oscillation in oocytes and initiate the fertilization process. We evaluated the usefulness of AOA with a calcium ionophore in cases of total fertilization failure in previous cycles and in cases of severe male factor infertility patients with non-motile spermatozoa after pentoxifylline (PF) treatment. Methods: The present study describes 29 intracytoplasmic sperm injection (ICSI)-AOA cycles involving male factor infertility at Cheil General Hospital from January 2006 to June 2013. Patients were divided into two groups (control, n=480; AOA, n=29) depending on whether or not AOA using a calcium ionophore (A23187) was performed after testicular sperm extraction-ICSI (TESE-ICSI). The AOA group was further split into subgroups according to sperm motility after PF treatment: i.e., motile sperm-injected (n=12) and non-motile sperm-injected (n=17) groups (total n=29 cycles). Results: The good embryo rate (52.3% vs. 66.9%), pregnancy rate (20.7% vs. 52.1%), and delivery rate (10.3% vs. 40.8%) were lower in the PF/AOA group than in the control group. When evaluating the effects of restoration of sperm motility after PF treatment on clinical outcomes there was no difference in fertilization rate (66.6% vs. 64.7% in non-motile and motile sperm, respectively), pregnancy rate (17.6% vs. 33.3%), or delivery rate (5.9% vs. 16.7%) between the two groups. Conclusion: We suggest that oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles regardless of restoration of sperm motility after PF treatment. AOA may be useful in selected patients who have a low fertilization rate or total fertilization failure.

• Clinical and Experimental Reproductive Medicine
• /
• v.43 no.3
• /
• pp.157-163
• /
• 2016

Objective: The decision to use in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or split insemination (IVF-ICSI) in the first cycle is based on the number of motile sperm. Hence, total fertilization failure (TFF) often occurs during IVF cycles, despite normozoospermia. To investigate whether the cumulative motile swim-up spermatozoa percentage at 22 hours post-insemination (MSPPI) is an indicator for ICSI, we analyzed TFF, fertilization, blastocyst development, chemical pregnancy, clinical pregnancy, and live birth rates. Methods: This prospective study was performed using data obtained from 260 IVF cycles. At 22 hours after insemination, the remaining swim-up spermatozoa were observed and divided into six groups according to MSPPI (<10%, 10% to <30%, 30% to <50%, 50% to <70%, 70% to <90%, and 90% to 100%). Results: Regardless of the ejaculated motile sperm concentration ($0.6-280{\times}10^6/mL$ motile spermatozoa), the incidence of TFF significantly increased when MSPPI was <10%, and the fertilization rate significantly decreased when MSPPI was <30%. We found that cumulative MSPPI correlated with the cumulative fertilization rate (Spearman correlation, 0.508, p<0.001). Regarding embryo development, we observed no significant differences in the rates of blastocyst development, chemical pregnancy, clinical pregnancy, or live birth among all groups. Conclusion: Our findings suggest that MSPPI is a viable indicator for split IVF-ICSI and ICSI. Taken together, by employing the MSPPI test in advance before IVF, ICSI, or split IVF-ICSI cycles, unnecessary split IVF-ICSI and ICSI may be avoided.

• Clinical and Experimental Reproductive Medicine
• /
• v.22 no.3
• /
• pp.273-278
• /
• 1995

Mammalian, including human, spermatozoa undergo morphological and physiological changes during sperm maturation. There were, these changes may affect the fertilization and embryonic development. In this study, we examined the pronucleus formation, pronucleus disappearance and embryonic development in the human oocytes fertilized by intracytoplasmic sperm injection (ICSI). The injected spermatozoa were grouped into ejaculated, epididymal and testicular by the collecting region. Among 363 metaphase II injected oocytes, 287(79.1%) oocytes were normally fertilized and displayed two pronuclei. There were no difference in the fertilization rates and in the pronucleus formation and pronucleus disappearance at 16, 20 and 24 hr after ICSI, among the each spermatozoa group. Also, at 64 hr, the appearance of embryonic development was similar. From these results it can be concluded that there was no difference of maturity among the sperm collected from ejaculated, epididymis and testis in the pronucleus formation and embryonic development. Therefore, testicular spermatozoa are successfully used with ICSI in IVF-ET program.

• Clinical and Experimental Reproductive Medicine
• /
• v.41 no.3
• /
• pp.137-139
• /
• 2014

The effect of artificial oocyte activation (AOA) with a calcium ionophore on intracytoplasmic morphologically selected sperm injection (IMSI) was examined in patients with histories of repeated failed implantation attempts. Four singleton pregnancies and one twin pregnancy were obtained after embryos transfer (5/14, 35.7%). Therefore, AOA combined with IMSI can be considered an option for cycles with a fertilization defect and recurrent implantation failures.

• Journal of Embryo Transfer
• /
• v.17 no.1
• /
• pp.13-21
• /
• 2002

Intracytoplasmic Sperm Injection (ICSI) has been widely used fur both human infertility and basic research. However, the high incidence of chromosomal abnormality is severe problem in cattle. Various oocyte activation stimuli, therefore, were compared by assessment of developmental capacity and chromosome analysis. Motile sperm selected by Percoll-density gradient were treated with 5 mM dithiothreitol (DTT) and injected into an oocyte matured fur 24 h. Eggs were then allocated into 5 treatment groups. Group 1 (control), sperm injection was performed without any further activation stimuli to the oocytes. Group 2 (handled control), sham injection was performed without sperm. In Group 3, oocytes exposed to 5 (M ionomycin for 5 min at 39(C. Group 4. ionomycine + 1.9 mM demethylaminopurine (DMAP, 3 h) and Group 5, ionomycine + 3 h culture in Ml99 + DMAP. Cleavage and the later development rate in Groups 1, 2 and 3 were significantly (P<0.05) lower than those in Groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycine was relatively higher than in the embryo of Group 3 h, delayed DMAP treatment. From this results DMAP caused to be arrested the release of the 2nd polar body, resulting in changes of chromosomal pattern. Therefore, the time interval between ionomycin and DMAP is a crucial role in bovine ICSI.

• 대한생식의학회:학술대회논문집
• /
• 1996.11a
• /
• pp.66.1-66.1
• /
• 1996

• Korean Journal of Animal Reproduction
• /
• v.26 no.4
• /
• pp.361-368
• /
• 2002

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilization. After 44h in vitro maturation, spermatozoa was injected into the cytoplasm of oocytes. After injection, all oocytes were transferred to NCSU23 medium and cultured at 39'E under 5% CO2 in air. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8 to 9 h following the injection of porcine sperm, and 6 to 8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte center. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. These results suggested that DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, and the sperm nuclear decondensing activity and microtubule nucleation abilities of the male centrosome are cell cycle dependent.

• Clinical and Experimental Reproductive Medicine
• /
• v.44 no.2
• /
• pp.90-95
• /
• 2017

Objective: We investigated whether the insemination method (in vitro fertilization [IVF] or intracytoplasmic sperm injection [ICSI]) affected morphokinetic events and abnormal cleavage events in embryonic development. Methods: A total of 1,830 normal fertilized embryos were obtained from 272 IVF and ICSI cycles that underwent ovum retrieval culture using a time-lapse system (Embryoscope) from June 2013 to March 2015. All embryos were investigated by a detailed time-lapse analysis that measured the developmental events in the hours after IVF or ICSI insemination. Results: No significant differences were observed between the two groups regarding clinical outcomes (p>0.05). ICSI-derived embryos showed significantly faster morphokinetics than those derived from conventional IVF, from the time to pronuclear fading to the time to 6 cells (p<0.05). However, no significant differences were found from the time to 7 cells to the time to expanded blastocyst (p>0.05). There were no differences in abnormal cleavage events between the two groups (p>0.05); they showed the same rates of direct cleavage from 1 to 3 cells, 2 multinucleated cells, 2 uneven cells, and reverse cleavage. Conclusion: The morphokinetics of embryo development was found to vary between IVF- and ICSI-fertilized oocytes, at least until the 6-cell stage. However, these differences did not affect the clinical outcomes of the embryo. Additionally, no significant differences in abnormal cleavage events were found according to the fertilization method.