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Glucose와 Phosphate가 제거된 M-TALP 배지에서의 난구세포 공배양에 의한 임신율 향상에 관한 연구
정범식,장우현,이문희,김지연,방지호,김규현,서태광,Chung, B.S.,Chang, W.H.,Lee, M.H.,Kim, J.Y.,Bang, J.H.,Kim, K.H.,Suh, T.K. 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.1
The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.
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박영식,서태광,이택후,전상식 경북대학교 병원 1998 경북대학교병원의학연구소논문집 Vol.2 No.1
본 연구는 난자의 동결보존에 초급속동결법을 효율적으롤 이용하기 위하여, 이의 효율성에 영향을 주는 여러 요인 중에서 난자의 탈수방법, 난자의 생존활력, 및 저온조절방법이 동결융해후 난자의 생존성에 미치는 효과를 조사하기 위하여 실시하였다. 생쥐난자를 3.5M DMSO와 0.25M sucrose가 함유된 mPBS에서 탈수한 다음 액체질소에 직접 침적하여 초급속동결하였다. 이어 동결된 난자를 37℃에서 융해하여 0.25M sucrose가 함유되어 있는 mPBS에서 복수시킨 다음, HAM's F10에 반복 세척하여 형태의 정상성을 조사하였다. 초급속동결난자의 생존성은 동결전 탈수방법, 난자의 질, 및 탈수와 복수시 0℃ 조절방법에 따라서 차이가 있었다. 1) 다단계로 탈수한 난자의 융해후 생존율은 48.4±13.8%로서 2단계 탈수한 난자 의 생존율 40.9±14.0% 보다 높았다. 2) 수정란의 융해후 생존율은 87.0±14.0%로서 미수정란의 융해후 생존율 5.4±5.4%보다 유의하게 높았다(p<0.01) 3) 냉각장치를 이용하여 탈수와 복수처리한 난자의 융해후 생존율은 95.8±4.2% 로서 얼음위에서 처리한 난자의 84.1±9.9% 보다 유의하게 높았다(p<0.05) 결론적으로 초급속동결방법을 이용하여 난자를 체외에서 효율적으로 보존하기 위해서는 생존활력이 높은 난자를 선별하고 탈수하는 동안 삼투충격을 적게 받도록 하며, 고삼투환경에 있을 때 온도를 일정하게 유지하여야 한다. This study was carried out to efficiently use the ultrarapid freezing method in the cryopreservation of mouse ova. For this, the effects of dehydration method, oval vigour and 0℃ controlling method on post-thawing viability were investigated. Fresh mouse ova were dehydrated in mPBS with 3.5M DMSO and / or 0.25M sucrose, and directly immersed in LN₂for ultrarapidly freezing. The frozen ova were thawed at 37℃, rehydrated in mPBS with 0.25M sucrose, and then repeatedly washed in HAM's F10 before evaluating the morphological normality of frozen-thawed ova. The results obtained showed that there was difference between treatments in a experiment. 1) The post-thawing viability of ova dehydrated in multi-step ((48.4±13.8%) was higher than that of ova in two-step (40.9±14.0%). 2) The post-thawing viability of fertilized ova (87±14.0%) was significantly(p<0.01) higher than that of unfertilized ova (5.4±5.4%). 3) The post-thawing viability of ova dehydrated and rehydrated using a cooling machine (95.8±4.2%) was significantly(p<0.05) higher than that on ice(84.1±9.9). In conclusion, in order to efficiently cryopreserve ova in vitro with ultrarapidly freezing method, highly viable embryos should be selected, heavy osmotic shock to the dehydrating ova should be avoided, and embryos in high osmotic condition were dehydrated and rehydrated in a constantly low temperature.
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