• Clinical and Experimental Reproductive Medicine
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• 제28권1호
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• pp.55-63
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• 2001

Objectives: This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Materials and Methods: Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Two-step 1,2-propanediol (PROH), dimethylsulfoxide (DMSO) and 4-step PROH DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. Results: As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in PROH and DMSO was significantly higher than 4-8 cell (64.5% versus 62.1 %,79.7% versus 73.2%) (p<0.01, p<0.01), but the development rates of 4-8 cell embryos in PROH and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4-8 cell embryos in PROH were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in PROH (74.4% versus 64.5%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The developmen1 rate from morular to hatching blastocyst, however, was significantly higher in PROH than in DMSO during 48 hr (p<0.01). The survival rate of 4-8 cell embryo was 62.1% in PROH and 73.2% in DMSO. The development rates of embryo in PROH were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. Conclusions: The survival rate of embryo in 2 cell stage was higher than in 4-8 cell stage, and PROH appears more effective cryoprotectant than DMSO because PROH showed better development rates of embryos in 2 and 4-8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

• 한국수정란이식학회지
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• 제30권3호
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• pp.195-200
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• 2015

본 연구에서 배아의 생식세포 동결에 가장 흔히 쓰이고 있는 침투성 동결보호제(DMSO, EG, Glycerol, 1,2-PROH)의 독성을 비교하고자 생쥐 수정란 모델을 이용한 실험을 하였다. 생후 6주령의 암컷 생쥐 F1 hybrid mice에 10 IU의 PMSG를 복강 주사하여 과배란을 유도하고, 2-세포기 배아를 획득하고 침투성 동결보호제(DMSO, EG, Glycerol와 1,2-PROH) 각각 실온에서 60분간 노출시킨 후, 배양을 하였다. 배반포의 전체 세포수는 2-세포기 단계에서 DMSO($68.1{\pm}24.1$), EG($68.1{\pm}24.1$), Glycerol($81.2{\pm}27.0$), 1,2-PROH($68.1{\pm}24.1$) 침투성 동결보호제 처리군은 대조군에 비해 유의적으로($99.0{\pm}18.3$)(p<0.001) 낮았다. DMSO와 Glycerol 처리구가 EG와 1,2-PROH 처리구에 비해 세포수가 적었다. DMSO($15.4{\pm}1.5$), EG($10.2{\pm}1.4$), Glycerol ($10.2{\pm}1.4$), 1,2-PROH($10.2{\pm}1.4$) 네 처리구는 대조구($6.1{\pm}0.9$, p<0.0001)와 비교해서 배반포에서 세포사 비율이 더 높음을 확인했다. 또한, DMSO와 Glycerol 처리구는 EG와 1,2-PROH 처리구(p<0.001)보다 더 많은 세포사멸된 세포가 각각 확인되었다. DMSO, EG, Glycerol과 1,2-PROH 처리군과 대조군 사이에는 배아 부화율에 있어서 차이가 있었으며 이는 배아에 대한 동결보호제의 잠재적인 독성을 확인한 결과였다. 이번 연구에서 장기간 처리했을 때 EG와 1,2-PROH 처리군보다 DMSO와 Glycerol 처리군에서 배아발달과 세포수가 저하된 것은 DMSO와 Glycerol의 독성이 더 높을 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제31권1호
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• pp.59-65
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• 2004

Objectives: The aim of this study was to assess toxicities of cryoprotectants. Methods: Toxicities of two cryoprotectants, dimethyl sulfoxide (DMSO) and 1, 2-propanediol (PROH), were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to either DMSO or PROH. Embryo development was evaluated up to the blastocyst stage. Blastocysts were stained with bis-benzimide to evaluate the cell count and with terminal deoxynucleotidyl transferase mediated dUTP nick labeling (TUNEL) to assess apoptosis. Results: The total cell count of blastocysts that were treated with DMSO at the 2-cell stage was significantly lower than that were treated with PROH ($75.9{\pm}27.0$) or the control ($99.0{\pm}18.3$) (p<0.001). On comparison of two cryoprotectant treated groups, the DMSO treated group showed a decreased cell count compared with the PROH treated group (p<0.05). Both DMSO ($14.2{\pm}1.5$) and PROH ($11.2{\pm}1.4$) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control ($6.2{\pm}0.9$, p<0.0001). In addition, the DMSO treated group showed more apoptotic cells than the PROH treated group (p<0.001). Conclusions: The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to either DMSO or PROH at room temperature. When comparing two cryoprotective agents, PROH appeared to be less toxic than DMSO at least in a murine embryo model.

• Reproductive and Developmental Biology
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• 제32권2호
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• pp.111-115
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• 2008

The aim of this study was to examine more effective cryoprotectant for the cryopreservation of mouse preantral follicles. Enzymetically isolated preantral follicles from 12-day-old mice were cryopreserved by a slow freezing protocol with 1.5 M propanediol (PROH), dimethyl sulphoxide (DMSO) or glycerol (GLY) and then grown and matured in vitro for 11 days after thawing. The survival of preantral follicles immediately after freezing and thawing was not different among the PROH (68.2%), DMSO (72.4%) and GLY (72.1%). After grown and matured in vitro, the rates of survival and metaphase II oocytes were 54.9% and 36.6% for PROH which was significantly higher rates (p<0.05) compared with the rates obtained from DMSO (16.9% and 9.0%) and GLY (16.3% and 7.5%). The diameter of metaphase II oocytes from pre antral follicles frozen in PROH ($67.4{\pm}1.8\;{\mu}m$) was significantly (p<0.05) smaller than that of the fresh preantral follicles ($69.1{\pm}2.3\;{\mu}m$). The results from the present study revealed that PROH is more suitable cryoprotectant for the cryopreservation of mouse preantral follicles.

• 한국수정란이식학회지
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• 제18권3호
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• pp.179-186
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• 2003

Human embryonic stem (hES) cell lines have been derived from human blastocysts and are expected to have far-reaching applications in regenerative medicine. The objective of this study is to improve freezing method with less cryo-injuries and best survival rates in hES cells by comparing various vitrification conditions. For the vitrifications, ES cells are exposed to the 4 different cryoprotectants, ethylene glycol (EG), 1,2-propanediol (PROH), EG with dime-thylsulfoxide (DMSO) and EG with PROH. We compared to types of vehicles, such as open pulled straw (OPS) or electron microscopic cooper grids (EM grids). Thawed hES cells were dipped into sequentially holding media with 0.2 M sucrose for 1 min, 0.1 M sucrose for 5 min and holding media for 5 min twice and plated onto a fresh feeder layer. Survival rates of vitrified hES cells were assessed by counting of undifferentiated colonies. It shows high survival rates of hES cells frozen with EG and DMSO (60.8％), or EG and PROH(65.8％) on EM grids better than those of OPS, compared to those frozen with EG alone (2.4％) or PROH alone (0％) alone. The hES cells vitrified with EM grid showed relatively constant colony forming efficiency and survival rates, compared to those of unverified hES cells. The vitrified hES cells retained the normal morphology, alkaline phosphates activity, and the expression of SSEA-3 and 4. Through RT-PCR analysis showed Oct-4 gene expression was down-regulated and embryonic germ layer markers were up-regulated in the vitrified hES cells during spontaneous differentiation. These results show that vitrification method by using EM grid supplemented with EG and PROH in hES cells may be most efficient at present to minimize cyto-toxicity and cellular damage derived by ice crystal formation and furthermore may be employed for clinical application.

• Clinical and Experimental Reproductive Medicine
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• 제31권1호
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• pp.9-17
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• 2004

Objective: The aim of this study were to compare the effects of EG and PROH on cryopreservation of mouse and human embryos, and to find the optimal protocol for embryo freezing. Methods: Human embryos derived from fertilized eggs showing 3 pronuclei (PN) and mouse embryos were divided into two groups respectively: dehydrated with 1.5 M EG + 0.2 M sucrose or 1.5 M PROH + 0.2 M sucrose using the slow freezing method. Moreover mouse embryos were controlled the exposure time of cryoprotectant during dehydration or rehydration steps. Results: The survival rates of human embryos were 79.2% (84/106) in EG group and 77.9% (88/113) in PROH group. In mouse embryos, the survival and development rates up to blastocyst were 70.6% (245/347), 44.1% (123/279) in EG group and 62.1% (198/319), 45.1% (123/279) in PROH group, respectively. However, in EG group, partially damaged embryos after thawing were decreased compared to PROH group. In combination group, when the exposure time during dehydration and rehydration were reduced, the survival and embryonic developments were increased slightly, but not significant. Conclusion: Cryopreservation of mouse and human embryos at cleavage stage by using EG or PROH exhibited no statistical difference in the survival rate and/or developmental rate to blastocyst. However, the use of EG for cryopreservation of embryos might reduce the exposure time of the cryoprotectant because of a high permeation of EG and result in lessen its toxic effects.

• 한국수정란이식학회지
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• 제10권3호
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• pp.219-227
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• 1995

This study was investigated to test in vitro-maturation rate of bovine follicular oocytes freezability of in vitro-matured bovine follicular oocytes with different stock solution in Glycerol and Propanediol, freezability of in vitro-rnatured bovine follicular oocytes on cryoprotectants, the viability of in vitro-rnatured bovine follicular oocytes by morphologically normal and FDA staining method. 1. The maturation rates of bovine follicular oocytes classified as grade A, B and C was 88, 63 and 21%, respectively. 2. Freezability of in vitro-matured bovine follicular oocytes on stock solution, TCM-199+5% FCS and m-PBS + 5% FCS was 61%(n=105), 48%(n=62) in $_1$M Glycerol and freeability of in vitro-matured bovine follicular oocytes on stock solution, TCM-199 +5% FCS and m-PBS + 5% FCS was 68%(n=112), 42%(n=57) in 1~2 Propanediol. The results indicate that freezability of in vitro-matured bovine follicular oocytes with different stock solution is important. 3. Freezability of in vitro-matured bovine follicular oocytes on cryoprotectants was Glycerol and PROH was 56%(n=167), 57%(n=169). The results indicate that PROH was superior to Glycerol. 4. The rates of morphologically normal IVM oocytes after thawing of cryopreserved oocytes with Glycerol and PROH were 39%(n=$_1$8), 65%(n=39), respectively. The results indicate that PROH was superior to Glycerol. 5. The fluorescent light intensity after thawing of cryopreserved oocytes classified with Positive, Partial-I, Partial-II, Negative with Glycerol and PROH. The results of FDA-positive 24%, 42%, Partial-I 17%, 10%, Partial- H 20%, 12%, FDA-negative 39%, 37%, and Partial-I, II, respectively.

• Clinical and Experimental Reproductive Medicine
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• 제25권3호
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• pp.261-268
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• 1998

The present study was to assess the effect of ultrarapid freezing on the development of 1-cell mouse zygote using cryoprotectants, DMSO (dimethyl sulfoxide) or PROH (1,2-propanediol). We investigated the effect of the type and concentration of cryoprotectant, and of the temperature and time of prefreezing equilibration on their capacity to develop to the blastocyst stage in vitro. The concenration, the equilibration temperature, and the exposure time seemed to serve as an important factor in ultrarapid freezing of 1-cell mouse zygotes. In addition to the exposure time and the concentration of cryoprotectant appeared to playa key role in the development of the embryo. In general, the development of the embryo was more effective at $3^{\circ}C$ than $23^{\circ}C$ and 4.5 M than 3 M for 3 to 5 minutes. At $23^{\circ}C$ the development of the embryo was stimulated by DMSO while at $3^{\circ}C$ it was stimulated by PROH. Thus it has been suggested that there exists a correlation between the concentration of cryoprotectants and exposure time in the development of the embryo. In conclusion, we found that for ultrarapid freezing of mouse 1-cell embryos in DMSO, or PROH-based solution, viability shown optimum depending on the cryoprotectant, the concentration of the cryoprotectant and on the temperature and the duration of equilibration.

• Clinical and Experimental Reproductive Medicine
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• 제18권1호
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• pp.55-61
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• 1991

In order to increase the pregnancy rate by means of cryopreservation of the excess oocytes in IVF-ET program, the survival rate of the frozen-thawed oocytes of mouse was examined according to the stages of maturation, cryoprotectants and their treatment. The results were summarized as follows. First, during the continuous treatment with cryoprotectant media, the survival rate of oocytes was higher in DMSO than in PROH, and higher at low temperature($4^{\circ}C$) than at room temperature($25^{\circ}C$). Second, as regard with the maturation of immature(GV-intact) oocytes after treatment with cryoprotectant media, the rate of maturation in DMSO-treated group(52%) was higher than in PROH-treated group(35%). Third, according to the treatment of cryoprotectant media, the survival rate of frozen-thawed oocytes in DMSO-treated group (45%) was higher than in PROH-treated group(29%), and that of oocytes in DMSO 4-step treated group was higher than any other groups. Finally, in the post-thaw oocytes frozen at various stage of maturation, the survival rate of immature oocytes with GV was the highest in all groups. These results suggest that in the cryopreservation of mouse oocytes, DMSO was better than PROH as cryoprotectant, in treatment of cryprotectant the multi-step treatment was better than single-step, and the post-thaw survival rate of oocytes was closely related to the maturity of oocytes. It is assumed that the highest survival rate of mouse oocytes with GV is due to the stability of the structures in nucleus and intracelluar organelles, and of physiological function.

• Clinical and Experimental Reproductive Medicine
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• 제21권3호
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• pp.325-330
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• 1994

The study has been carried out in order to evaluate the effects of embryonic stage, and cryopreservation method on the rates of viability and development of the cryopreserved mouse early embryos. The results were as following:In the treatment steps of cryoprotectant, for the fertilized oocyte with pronucleus(PN), 2-step was better than the others. And for the other embryos, 4-step was better than 2- or 3-step. In respect to the embryonic stage, as the embryos developed from fertilized oocytes to 8-cell embryos, the rates of viability and development were increased higher. Therefore, 8-cell embryo was better stage than the others. In respect to the kind of cryoprotectants, PROH was better than DMSO for the fertilized oocyte, as a cryoprotectant. DMSO, for the 2-cell embryos and PROH and DMSO for the 4- and 8-cell embryos were suitable for cryopreservation.