Vitrified-thawed Oocytes Research Articles

MAP kinase phosphorylation in relation to cytoskeletal behavior in human mature oocytes after vitrification

Objective: In previous study, vitrified-thawed oocytes had lower spindle and chromosome nomalities than fresh oocytes. This result suggests that damage in oocyte cytoskeleton system is one of the main cryoinjury. And stabilizing the cytoskeletal system during vitrification is beneficial for improving post-thawed survival and subsequent development of vitrified oocytes (Park et al., 2000). A recent study showed that MAP kinase plays an important role in controlling chromatin and microtubule behavior during cryopreservation which has not been studied in human oocytes. We examined whether cytoskeletal change was correlated with MAP kinase phosphorylation in vitrified-thawed human oocytes.

Microdrop과 Straw 방법으로 초자화 동결한 소 난자의 생존율에 관한 연구

본 연구는 소 난자의 초자화 동결 방법을 설정하기 위하여 체외성숙 소 난자에서 난구세포가 부착된 상태 또는 제거한 상태로 microdrop (MD) 방법과 straw (Straw) 방법을 이용하여 초자화 동결하여 생존율을 검사하였다. 동결 융해난자는 a) 단위발생을 유도하였고 b) 체외수정 후 전핵 형성을 관찰하였으며 c)체외수정 후 수정란 발달을 검사하였다. 초자화 동결 난자의 생존율은 MD 방법을 이용하였을 때가 Straw를 이용하였을 때 보다 높았다 (92.50 vs. 74.19%, p<0.05). MD 방법을 이용하였을 때 대부분의 난자가 생존을 하였다. 단위발생을 유도하였을 때 난할율과 배반포 발달율은 MD (45.05%, 10.81%, p<0.05)가 Straw 방법 보다 높았다 (27.17%, 6.52%, p<0.05). 난구세포의 부착 유무에 따라 동결 융해 후 체외수정 하여 자웅 전핵 형성을 각각 검사하였다. 난구세포 제거 난자에서는 MD와 Straw 방법으로 동결 융해하였을 때 차이가 없었다(80.36% vs. 67.31 %, p<0.05). 정상 수정율 (2PN)에서는 세 처리간에 차이가 없었다 (Fresh; 54.55% vs. MD; 42.22% vs. Straw; 37.14%, p>0.05). 그러나 미수정란 (<1PN)은 Fresh 난자가 동결융해 난자보다 유의적으로 낮았다 (Fresh; 32.47% vs. MD; 57.78% and Straw 62.86%, p<0.05). 다정자 침입은 (3PN) 신선란 (12,99%)에서 발생하였으나 동결 융해 난자에서는 발생하지 않았다. 난구세포가 제거된 난자에서, 정상 수정율 (2PN)은 Fresh와 동결 융해 난자간에 유의적인 차이를 보였다 (Fresh; 59.38% vs. MD; 17.31% and Straw; 30.43%, p<0.05). 또한 미수정율 (<1PN)에 있어서도 신선란과 동결 융해난은 유의적인 차이를 보였다 (Fresh; 23.44% vs. MD; 73.08% and Straw 58.70%, p<0.05). 다정자 침입 (3PN, >4PN)은 신선란과 동결 융해란 모두에서 나타났다. 체외수정 후, 난구세포가 부착된 난자의 2세포기 발달율은 신선란에 비하여 MD 또는 Straw 처리구에서 유의적으로 낮았다 (Fresh; 81.76% vs. MD; 22.22% and Straw; 11.36%, p<0.05). 동결융해난자는 배반포 발달율에 있어서도 신선란에 비하여 유의적으로 낮았다 (Fresh; 28.38 vs. MD; 1.71% and Straw 0%, p<0.05). 난구세포가 제거된 난자에서 2세포기 발달율은 신선란과 MD에서 차이가 없었다 (27.59% vs. 19.25%, p<0.05), 그러나 배반포 발달율에 있어서는 신선란이 MD 또는 Straw 처리구보다 유의적으로 높았다 (4.31% vs. 0.62% and 0%, respectively; p<0.05). 이상의 결과에서, 초자화 동결융해한 소 난자는 체외수정 후 배반포로 발달이 가능함을 나타내주고 있다. Studies on the Viability of In Vitro-Matured Bovine Oocytes Vitrified by Microdrop and Straw Method To establish vitrification method for bovine oocytes, mature bovine oocytes were vitrified by microdrop (MD) or straw (Straw) method and the viability of vitrified oocytes with or without cumulus cells (CC) were examined by several methods; a) parthenogenetic activation; b) pronuclear formation after in vitro fertilization (IVF); and c) embryonic development after IVF. The survival rate of vitrified oocytes by MD was significantly higher than by Straw (92.50 vs. 74.19%, p<0.05). Most of the oocytes survived from vitrification using the MD methods. Cleavage and blastocyst development of parthenogenetically activated oocytes were higher in MD (45.05% and 10.81%, respectively; p<0.05)) than those in Straw method (27.17% and 6.52%, respectively; p<0.05). Male and female pronuclear formation of vitrified-thawed oocytes with or without cumulus cells (CC) after IVF were examined, respectively. The survival rate of vitrified oocytes by MD without CC was no difference between MD and Straw (80.368.14% vs. 67.31%). Normal fertilization (2PN) rates were not different among groups (Fresh; 54.55% vs. MD; 42.22% vs. Straw; 37.14%, p>0.05). While no fertilization (<1PN) rates were significantly different between fresh and vitrified-thawed groups (Fresh; 32.47% vs. MD; 57.78% and Straw 62.86%, p<0.05). The polyspermy (3PN) was appeared in the fresh (12.99%), but no appeared in the vitrified-thawed groups. In the without CC, normal fertilization (2PN) rates were significantly different between fresh and vitrified-thawed oocytes (Fresh; 59.38% vs. MD; 17.31% and Straw; 30.43%, p<0.05). Moreover, no fertilization (<1PN) rates were significantly different between fresh and vitrified-thawed groups (Fresh; 23.44% vs. MD; 73.08% and Straw 58.70%, p<0.05). The polyspermy (3PN, >4PN) was appeared not only fresh but vitrified-thawed groups. After IVF, two-cell developmental rates of vitrified oocytes with CC by MD and Straw were significantly low compared to fresh oocytes (Fresh; 81.76% vs. MD; 22.22% and Straw; 11.36%, p<0.05). Blastocyst developmental rates of vitrified oocytes also were significantly low compared to fresh oocytes (Fresh; 28.38 vs. MD; 1.71% and Straw 0%, p<0.05). In the without CC, two-cell developmental rates were no difference between Fresh and MD (27.59% vs. 19.25%, p<0.05), while blastocyst rates were difference between Fresh and MD or Straw (4.31% vs. 0.62% and 0%, respectively; p<0.05). In conclusion, the results indicate that the vitrified bovine oocytes have the ability to develop to the blastocyst stage after IVF.

MAP kinase phosphorylation in relation to cytoskeletal behavior in mouse mature oocytes after vitrification

Objective: In a previous study, vitrified-thawed oocytes had fewer spindles and more chromosomal abnormalities than fresh oocytes. However, with the addition of a cytoskeleton stabilizer, Taxol™ to vitrification solution, the post-thawed preimplantation development was improved. The result suggests that damage in oocyte cytoskeleton system may be one of the main cryoinjuries so that stabilizing the cytoskeleton system during vitrification is beneficial for the post-thawed survival and the subsequent development of vitrified oocytes (Park et al., 2000). A recent study showed that MAP kinase plays an important role in controlling chromatin and microtubule behavior. However, the relationship of MAP kinase phosphorylation to cytoskeletal behavior during cryopreservation has never been studied. The objective of the present study was to determine whether a relationship exists between cytoskeletal changes and MAP kinase phosphorylation in vitrified-thawed oocytes.

Mitochondrial ATPase 6 gene expression in mouse oocytes matured in vitro after vitrification at the germinal vesicle stage

Objective: Deficiencies in mitochondrial ATP production may be associated with various cellular and chromosomal disorders. In previous study, vitrified-thawed oocytes had lower spindle and chromosome normalities than fresh oocytes. This study was subsequently conducted to examine whether level of mitochondrial ATPase 6 expression changed after vitrification. This gene was chosen because of its central role in ATP production.

Pregnancies derived from vitrified human zygotes

Pregnancies derived from vitrified human zygotes

Improved post-thawed preimplantation development after vitrification using Taxol™, a cytoskeleton stabilizer.

Objective: We previously reported (Park et al., 2000 ASRM meeting) that mouse mature oocytes can survive and develop to the blastocyst stage after vitrification and thawing using ethylene glycol (EG) and an electron microscope (EM) grid. Since preimplantation development of vitrified-thawed oocytes was lower than that of fresh oocytes, this study was subsequently conducted to examine whether the addition of Taxol™, a cytoskeleton stabilizer, to vitrified solution could improve post-thaw development of vitrified mouse oocytes.

Cryopreservation of ICR mouse oocytes: improved post-thawed preimplantation development after vitrification using Taxol™, a cytoskeleton stabilizer

Objective: To establish an effective cryopreservation method. Design: In vitro model study. Setting: Infertility Medical Center, Pochon CHA University. Animal(s): Four-week-old ICR mice superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin. Intervention(s): Vitrified-thawed oocytes were fertilized and subsequently cultured in vitro. Main Outcome Measure(s): Post-thawed development, chromosome/spindle normalities, and blastocyst quality. Result(s): More cumulus-enclosed oocytes were fertilized and developed to the 8-cell stage after vitrification and thawing than denuded oocytes. However, cryopreserved oocytes of both types had lower spindle and chromosome normalities than fresh oocytes, which resulted in reduced developmental competence after thawing. The addition of 1 μM of Taxol™, a cytoskeleton stabilizer, to vitrification solution greatly promoted the blastocyst formation of vitrified-thawed oocytes, compared with no addition (24.0% vs. 58.6%). No difference in blastocyst quality, which was evaluated by blastomere and inner cell mass cell numbers and inner cell mass cell per trophoblast ratio, was found between fresh oocytes and oocytes vitrified with Taxol™. Conclusion(s): A vitrification solution consisting of 5.5 M ethylene glycol, 1.0 M sucrose, 10% fetal bovine serum, and 1 μM Taxol™ greatly improved post-thawed development of vitrified oocytes.