Kim, Mi-Young;Lee, Eun-Suk;Lee, Seok-Won;Lee, Yu-Il
Clinical and Experimental Reproductive Medicine
/
v.32
no.2
/
pp.177-185
/
2005
Objective: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. Materials and Methods: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. Results: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. Conclusion: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.
This study was carried out to investigate the survival rates of late mouse molulae frozen in the state of vitrification and then thawed after equilibrating them separately in EFS 40, GFS 40 and DFS 40 at 1$0^{\circ}C$. The results obtained are as follows : 1. Freezing in the state of vitrification and thawing late mouse molulae after equilibrating them at l0$0^{\circ}C$ in EFS 40 for 30 seconds, one minute and two minutes, we obtained survival rates of 76.7%, 96.7% and 100%, respectively. 2. Freezing and thawing them after equilibrating at 1$0^{\circ}C$ in GFS 40 for 30 seconds, one minute and two minutes, we obtained survival rates of 60%, 96.7% and 10%, respectively. These results are as similar as in the case of EFS 40. 3. Freezing and thawing them after equilibrating at l$0^{\circ}C$ in DFS 40 for 30 seconds and one minute, we obtained survival rates of 62.1% and 0%, respectively. These results represent lower survival rates than those obtained with EFS 40 and GFS 40. In conclusion, even equilibrating late mouse molulae in EFS 40 and GFS 40 at 1$0^{\circ}C$ for more than one minute gives a survival rate of more than 97%, while equilibrating them in DFS 40 at 1$0^{\circ}C$ for more than one minute results in a 0% survival rate, which means that DFS 40 has a strong toxicity.
This study was carried out to investigate the in vivo development rates of vitrified-thawed mouse expanding, hatching and hatched blastoc ysts(BL). In vitro fertilization produced blastocysts were vitrified in EFS40(40% ethylene glycol, 30% Ficoll and 0.3 M sucrose in phosphate buffer saline containing 10% FBS). Expanding a and hatching blastocysts were equilibrated in 20% ethylene glyco](EG) for 5 min. before exposure to EFS40 at 25°C for 1 min., they were then vitrified in liquid nitrogen. Hatched blastocysts which cultured in m-CR1 medium supple mented 0.4% bovine serum albumin on day 5. were equilibrated in 10% EG for 5 min. and then vitrified in EFS40 for 30 sec. After thawing, re-expanding blastocysts were transferred to recipients(3 day of pseudopregnant) on one or both uterine horns(6-8 embryos per a horn). Preg¬n nancy rates of recipients and implantation were a assccessed by autopsy on 15 gestation. The res¬u ults obtained in these experiments were summar¬1 ized as follows; 1) The pregnancy and live fetus rates, for vitrified expanding BL(77.8 and 25.0%) and hatching BL(77.8 and 26.4%)n vitro were not significantly difference in those of control BL (66.7 and 42.9%: 83.3 and 40.4%), respectively, 2) in vitro development of vitrified hatched BL was 34.0%. and 3) in vivo developmental rate of vitrified hatched BL was only 33.3%. These results suggested that proposed rapid vitrification p procedures used EFS40 cryoprotectant can be effectively performed in mouse expanding Ihatching blastocysts and that mouse blastocysts a after being hatched from zona pellucida can be successfully cryopreserved.
Kim, E.Y.;Kim, N.H.;Yi, B.K.;Yoon, S.H.;Park, S.P.;Chung, K.S.;Lim, J.H.
Clinical and Experimental Reproductive Medicine
/
v.25
no.1
/
pp.71-76
/
1998
This study was carried out to examine whether the developmental capacity of bovine immature oocytes frozen ultra-rapidly using electron microscope (EM) grids and EFS30 can be obtained. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. As criterior of oocyte viability, the rates of maturation, fertilization and embryonic development were determined. The results obtained in this experiment were summarized as follows: When ultra-rapidly frozen immature oocytes were thawed, 43.2% of them were survived. The rates of maturation (84.1%) and normal 2 pronuclei formation (57.5%) of frozen immature oocytes were not significantly different when compared to those of control (92.5, 65.0%). In addition, the rates of $\geq2$-cell (65.0%) and blastocyst formation (30.8%) of freezing group were not significantly different when compared to those of control (73.7, 35.7%). These results demonstrate that developmental capacity of frozen-thawed bovine immature oocytes can be successfully obtained when survived from the ultra-rapid freezing method using EM grid and EFS30.
The use of hormonal stimulation in human in vitro fertilization and embryo transfer (IVF-ET) leads to increased production of embryos for ET. So to avoid high pregnancies and to allow conception in future, unstimulated cycles, cryopreservation of spare embryos is desirable. One of the improvement of cryopreservation methods is vitrification. We cryopreserved mouse day 3 embryos by vitrification using the three different vitrification solution (EFS40, VS11 and VS3a). EFS40 solution is consisted of 40% (v/v) ethylene glycol, Ficol170 30% (w/v) and 0.5M sucrose and VS11 is 6.0M ethylene glycol and 1.8M glycerol. And VS3a is 6.5M glycerol and 6% (w/v) BSA (bovine serum albumin). First we tested the toxicity of three vitrification solution by exposure to these solution during 3 min. After washing by thawing solution, the survival rates of each groups are 95.5%, 90.9% and 84.4% (EFS40, VS11 and VS3a). High percentages of them developed to expanded blastocyst and hatching embryos in culture 48hrs 94.2%, 97.7%, 100% and 97.4% (no treatment group, EFS40, VS11 and VS3a). So there is no significant differences among the each group. Second, after thawing of vitirfied embryos, the survival rates of each groups are 96.8% (slow freeze), 94.1% (EFS40), 85.5% (VS11) and 80.0% (VS3a, P vs. no freeze or EFS40 is 0.01). Vitrified embryos exhibited a high rate of development in vitro after 48hrs culture. The percentages of each group to blastocyst and hatching embryos are 88.7% (no freeze), 91.8% (slow freeze), 93.4% (EFS40), 87.7% (VS11) and 73.0% (VS3a, P vs. other group is 0.01). The results suggest that there is no significant differences in exposure of various vitrification solution and day 3 mouse embryos can be vitrified in solution EFS40 and VS11 by simple procedure.
Kim, M.K.;Kim, E.Y.;Yi, B.K.;Yoon, S.H.;Park, S.P.;Chung, K.S.;Lim, J.H.
Clinical and Experimental Reproductive Medicine
/
v.25
no.1
/
pp.87-92
/
1998
This study was carried out to investigate in vitro/in vivo development of vitrified mouse oocytes. Mouse oocytes were vitrified using EFS30, 35 and 40 (30, 35 and 40% ethylene glycol, 18% ficoll and 0.5 M sucrose in M2 medium). After being exposed or vitrified-thawed, oocytes of normal morphology were inseminated in vitro by $1-2\times10^6/ml$ of epididymal sperm. The rates of fertilization, in vitro/in vivo development and cell number (inner cell mass and tropectoderm cell) of blastocysts in each treatment group were examined. The results obtained in these experiments were summarized as follows: The cleavage rates were obtained in EFS35 containing 35% ethylene glycol higher than in EFS30 and EFS40. The development rate of vitrified-thawed oocytes to two-cell stage after in vitro fertilization (51.1%) was significantly different compared to that of exposed to vitrification solution without cooling (60.0%) and control (68.2%) (p<0.05). However, there were no differences in the blastocyst formation from the cleaved embryos among groups (75.0, 73.3 and 80.0%). Also, the mean number of cells per blastocysts of vitrified group $(92.5{\pm}2.9)$ was similar to that of the exposed $(98.5{\pm}5.3)$ and control $(100.9{\pm}4.8)$. In vivo development of the blastocysts derived from vitrified-thawed oocytes resulted in fetal development (50.7%) and implantation rates (80.0%) which are very similar to those of control (58.2, 78.2%). These results suggest that mouse oocytes could be cryopreserved using vitrification solution (EFS35) based on ethylene glycol.
Proceedings of the Korean Society of Developmental Biology Conference
/
2003.10a
/
pp.113-113
/
2003
This study was conducted to investigate the effect of vitrification solution(VS) on in vitro developmental competence of immature porcine oocytes. The immature porcine oocytes were exposed to the following vitrification solution, at RT. 1) EFS sol. : 20% ethylene glycol (EG) 3 min, 40% EG + 18%(w/v) Ficoll(MV70, 000) + 0.3 M sucrose 30 sec, 2) GE sol. : 10% glycerol 5 min, 10% G + 20% EG 5 min, 25% G +25% EG 30 sec, 3) EG sol : 1.5M EG 2.5 min, 5.5 M EG + 1.0 M sucrose 30 sec. Oocytes were immediately transferred into 1.0 M, 0.5 M, 0.25 M, 0125 M, 0 M sucrose solution for 2.5 min each at RT. After removal of VS, immature oocytes were matured in vitro and subsequently all oocytes were subjected to IVF followed in vitro culture for 7 days. Maturation rates of oocytes were 38.8%, 44.5%, 22.4% and 57.6%, in EFS, EG, GE and Control, respectively, maturation rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.01). Fertilization rates of oocytes in Control was significantly higher than other treated groups(P<0.05), but no difference were observed among treated groups. Polyspermic rates were no significant difference among four groups. Cleavage rates of oocytes were 21.9%, 47.1%, 19.0% and 65.9%, in EFS, EG, GE and Control, respectively, cleavage rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.05), but blastocyst formation rates were no significant difference among four groups. These results suggested that the use of EG solution could be a great challenge for reaching a successful vitrification of immature porcine oocytes.
This study was carried out to confirm whether the in vivo developmental potential of mouse hatched blastocysts (HBs) can be obtained by vitrification method using the cryoprotectant EFS35. The HBs ($\theta$ 130$\mu\textrm{m}$) were cultured in vitro until day 5 from zygotes produced in vivo and were equilibrated in 10% ethylene glycol(EG) for 5 min, and then were exposed or vitrified in EFS35 (35% EG, 18% Ficoll and 0.3M sucrose). After 30 min thawing, re-expanding HBs were transferred into one or both uterine horns of pseudopregnant recipients on day 3 (4~6 embryos /horn). Pregnancy rates of recipients and implantation were assessed by autopsy on day 15 of gestation. The results obtained in these experiments were summarized as follows : After thaw-ing, in vitro survival of HBs was not significantly different between exposed (65.5%) and vitrified(54.5%) group. Also, when the in vivo development potential was examined, total implantation was not different between control (58.5% ) and vitrified (41.0%) group, although the live fetus formation of vitrified group (24.0%) was significantly lower than that of control (58.3%) group (p< 0.05). These results suggested that vitrification freezing method of mouse HBs using EFS35 can be used to make wide the utility of embryo transfer of the more embryos produced in vitro.
Park, Yong-Deuk;Park, Hyung-Seo;Cui, Zheng-Yun;Park, Hyoung-Jin
The Korean Journal of Physiology and Pharmacology
/
v.7
no.3
/
pp.169-174
/
2003
${\gamma}$-Aminobutyric acid (GABA) has been reported to enhance exocrine secretion evoked not only by secretagogues but also by intrinsic neuronal excitation in the pancreas. The pancreas contains cholinergic neurons abundantly that exert a stimulatory role in exocrine secretion. This study was undertaken to examine effects of GABA on an action of cholinergic neurons in exocrine secretion of the pancreas. Intrinsic neurons were excited by electrical field stimulation (EFS; 15 V, 2 msec, 8 Hz, 45 min) in the isolated, perfused rat pancreas. Tetrodotoxin or atropine was used to block neuronal or cholinergic action. Acetylcholine was infused to mimic cholinergic excitation. GABA $(30{\mu}M)$ and muscimol $(10{\mu}M)$, given intra-arterially, did not change spontaneous secretion but enhanced cholecystokinin (CCK; 10 pM)-induced secretions of fluid and amylase. GABA (3, 10, $30{\mu}M$) further elevated EFS-evoked secretions of fluid and amylase dose-dependently. GABA (10, 30, $100{\mu}M$) also further increased acetylcholine $(5{\mu}M)$-induced secretions of fluid and amylase in a dose-dependent manner. Bicuculline $(10{\mu}M)$ effectively blocked the enhancing effects of GABA $(30{\mu}M)$ on the pancreatic secretions evoked by either EFS or CCK. Both atropine $(2{\mu}M)$ and tetrodotoxin $(1{\mu}M)$ markedly reduced the GABA $(10{\mu}M)$-enhanced EFS- or CCK-induced pancreatic secretions. The results indicate that GABA enhances intrinsic cholinergic neuronal action on exocrine secretion via the $GABA_A$ receptors in the rat pancreas.
Kim, E.Y.;Yi, B.K.;Nam, H.K.;Lee, K.S.;Yoon, S.H.;Park, S.P.;Chung, K.S.;Lim, J.H.
Clinical and Experimental Reproductive Medicine
/
v.25
no.2
/
pp.129-134
/
1998
The objective of this study was to test whether the developmental capacity of human multi-pronuclear (PN) zygotes after ultra-rapid freezing using EM grid can be maintained. For this experiment, multi-PN zygotes which produced in human IVF program were used as an alternatives of normal 2PN zygotes, and they were separated into 3PN or $\geq4PN$ zygotes to compare their in vitro development and cryoinjury according to PN number. As freezing solution, EFS30 which consisted of 30% ethylene glycol, 18% bcoll, 0.5 M sucrose and 10% FBS added D-PBS was used. The result obtained in this experiment was summarized as follows; When the multi..PN zygotes were ultrarapidly frozen and thawed, the high mean percentages (85.5%) were survived. Also when the cleavage rates between control and freezing group were compared with PN number, there were not significantly different in each group (3PN; 81.3% & 85.4% and $\geq4PN$; 90.0% & 95.7%). When the in vitro development rates after thawing were examined, freezing 3PN group (22.0%) was not differed to control 3PN group (38.5%), although the development result of freezing $\geq4PN$ group (45%) was significantly lower than that of control $\geq4PN$ group (44.4%) (p<0.05). These results demonstrate that developmental capacity of human zygote can be obtained by ultra-rapid freezing method using EM grid and EFS30.
본 웹사이트에 게시된 이메일 주소가 전자우편 수집 프로그램이나
그 밖의 기술적 장치를 이용하여 무단으로 수집되는 것을 거부하며,
이를 위반시 정보통신망법에 의해 형사 처벌됨을 유념하시기 바랍니다.
[게시일 2004년 10월 1일]
이용약관
제 1 장 총칙
제 1 조 (목적)
이 이용약관은 KoreaScience 홈페이지(이하 “당 사이트”)에서 제공하는 인터넷 서비스(이하 '서비스')의 가입조건 및 이용에 관한 제반 사항과 기타 필요한 사항을 구체적으로 규정함을 목적으로 합니다.
제 2 조 (용어의 정의)
① "이용자"라 함은 당 사이트에 접속하여 이 약관에 따라 당 사이트가 제공하는 서비스를 받는 회원 및 비회원을
말합니다.
② "회원"이라 함은 서비스를 이용하기 위하여 당 사이트에 개인정보를 제공하여 아이디(ID)와 비밀번호를 부여
받은 자를 말합니다.
③ "회원 아이디(ID)"라 함은 회원의 식별 및 서비스 이용을 위하여 자신이 선정한 문자 및 숫자의 조합을
말합니다.
④ "비밀번호(패스워드)"라 함은 회원이 자신의 비밀보호를 위하여 선정한 문자 및 숫자의 조합을 말합니다.
제 3 조 (이용약관의 효력 및 변경)
① 이 약관은 당 사이트에 게시하거나 기타의 방법으로 회원에게 공지함으로써 효력이 발생합니다.
② 당 사이트는 이 약관을 개정할 경우에 적용일자 및 개정사유를 명시하여 현행 약관과 함께 당 사이트의
초기화면에 그 적용일자 7일 이전부터 적용일자 전일까지 공지합니다. 다만, 회원에게 불리하게 약관내용을
변경하는 경우에는 최소한 30일 이상의 사전 유예기간을 두고 공지합니다. 이 경우 당 사이트는 개정 전
내용과 개정 후 내용을 명확하게 비교하여 이용자가 알기 쉽도록 표시합니다.
제 4 조(약관 외 준칙)
① 이 약관은 당 사이트가 제공하는 서비스에 관한 이용안내와 함께 적용됩니다.
② 이 약관에 명시되지 아니한 사항은 관계법령의 규정이 적용됩니다.
제 2 장 이용계약의 체결
제 5 조 (이용계약의 성립 등)
① 이용계약은 이용고객이 당 사이트가 정한 약관에 「동의합니다」를 선택하고, 당 사이트가 정한
온라인신청양식을 작성하여 서비스 이용을 신청한 후, 당 사이트가 이를 승낙함으로써 성립합니다.
② 제1항의 승낙은 당 사이트가 제공하는 과학기술정보검색, 맞춤정보, 서지정보 등 다른 서비스의 이용승낙을
포함합니다.
제 6 조 (회원가입)
서비스를 이용하고자 하는 고객은 당 사이트에서 정한 회원가입양식에 개인정보를 기재하여 가입을 하여야 합니다.
제 7 조 (개인정보의 보호 및 사용)
당 사이트는 관계법령이 정하는 바에 따라 회원 등록정보를 포함한 회원의 개인정보를 보호하기 위해 노력합니다. 회원 개인정보의 보호 및 사용에 대해서는 관련법령 및 당 사이트의 개인정보 보호정책이 적용됩니다.
제 8 조 (이용 신청의 승낙과 제한)
① 당 사이트는 제6조의 규정에 의한 이용신청고객에 대하여 서비스 이용을 승낙합니다.
② 당 사이트는 아래사항에 해당하는 경우에 대해서 승낙하지 아니 합니다.
- 이용계약 신청서의 내용을 허위로 기재한 경우
- 기타 규정한 제반사항을 위반하며 신청하는 경우
제 9 조 (회원 ID 부여 및 변경 등)
① 당 사이트는 이용고객에 대하여 약관에 정하는 바에 따라 자신이 선정한 회원 ID를 부여합니다.
② 회원 ID는 원칙적으로 변경이 불가하며 부득이한 사유로 인하여 변경 하고자 하는 경우에는 해당 ID를
해지하고 재가입해야 합니다.
③ 기타 회원 개인정보 관리 및 변경 등에 관한 사항은 서비스별 안내에 정하는 바에 의합니다.
제 3 장 계약 당사자의 의무
제 10 조 (KISTI의 의무)
① 당 사이트는 이용고객이 희망한 서비스 제공 개시일에 특별한 사정이 없는 한 서비스를 이용할 수 있도록
하여야 합니다.
② 당 사이트는 개인정보 보호를 위해 보안시스템을 구축하며 개인정보 보호정책을 공시하고 준수합니다.
③ 당 사이트는 회원으로부터 제기되는 의견이나 불만이 정당하다고 객관적으로 인정될 경우에는 적절한 절차를
거쳐 즉시 처리하여야 합니다. 다만, 즉시 처리가 곤란한 경우는 회원에게 그 사유와 처리일정을 통보하여야
합니다.
제 11 조 (회원의 의무)
① 이용자는 회원가입 신청 또는 회원정보 변경 시 실명으로 모든 사항을 사실에 근거하여 작성하여야 하며,
허위 또는 타인의 정보를 등록할 경우 일체의 권리를 주장할 수 없습니다.
② 당 사이트가 관계법령 및 개인정보 보호정책에 의거하여 그 책임을 지는 경우를 제외하고 회원에게 부여된
ID의 비밀번호 관리소홀, 부정사용에 의하여 발생하는 모든 결과에 대한 책임은 회원에게 있습니다.
③ 회원은 당 사이트 및 제 3자의 지적 재산권을 침해해서는 안 됩니다.
제 4 장 서비스의 이용
제 12 조 (서비스 이용 시간)
① 서비스 이용은 당 사이트의 업무상 또는 기술상 특별한 지장이 없는 한 연중무휴, 1일 24시간 운영을
원칙으로 합니다. 단, 당 사이트는 시스템 정기점검, 증설 및 교체를 위해 당 사이트가 정한 날이나 시간에
서비스를 일시 중단할 수 있으며, 예정되어 있는 작업으로 인한 서비스 일시중단은 당 사이트 홈페이지를
통해 사전에 공지합니다.
② 당 사이트는 서비스를 특정범위로 분할하여 각 범위별로 이용가능시간을 별도로 지정할 수 있습니다. 다만
이 경우 그 내용을 공지합니다.
제 13 조 (홈페이지 저작권)
① NDSL에서 제공하는 모든 저작물의 저작권은 원저작자에게 있으며, KISTI는 복제/배포/전송권을 확보하고
있습니다.
② NDSL에서 제공하는 콘텐츠를 상업적 및 기타 영리목적으로 복제/배포/전송할 경우 사전에 KISTI의 허락을
받아야 합니다.
③ NDSL에서 제공하는 콘텐츠를 보도, 비평, 교육, 연구 등을 위하여 정당한 범위 안에서 공정한 관행에
합치되게 인용할 수 있습니다.
④ NDSL에서 제공하는 콘텐츠를 무단 복제, 전송, 배포 기타 저작권법에 위반되는 방법으로 이용할 경우
저작권법 제136조에 따라 5년 이하의 징역 또는 5천만 원 이하의 벌금에 처해질 수 있습니다.
제 14 조 (유료서비스)
① 당 사이트 및 협력기관이 정한 유료서비스(원문복사 등)는 별도로 정해진 바에 따르며, 변경사항은 시행 전에
당 사이트 홈페이지를 통하여 회원에게 공지합니다.
② 유료서비스를 이용하려는 회원은 정해진 요금체계에 따라 요금을 납부해야 합니다.
제 5 장 계약 해지 및 이용 제한
제 15 조 (계약 해지)
회원이 이용계약을 해지하고자 하는 때에는 [가입해지] 메뉴를 이용해 직접 해지해야 합니다.
제 16 조 (서비스 이용제한)
① 당 사이트는 회원이 서비스 이용내용에 있어서 본 약관 제 11조 내용을 위반하거나, 다음 각 호에 해당하는
경우 서비스 이용을 제한할 수 있습니다.
- 2년 이상 서비스를 이용한 적이 없는 경우
- 기타 정상적인 서비스 운영에 방해가 될 경우
② 상기 이용제한 규정에 따라 서비스를 이용하는 회원에게 서비스 이용에 대하여 별도 공지 없이 서비스 이용의
일시정지, 이용계약 해지 할 수 있습니다.
제 17 조 (전자우편주소 수집 금지)
회원은 전자우편주소 추출기 등을 이용하여 전자우편주소를 수집 또는 제3자에게 제공할 수 없습니다.
제 6 장 손해배상 및 기타사항
제 18 조 (손해배상)
당 사이트는 무료로 제공되는 서비스와 관련하여 회원에게 어떠한 손해가 발생하더라도 당 사이트가 고의 또는 과실로 인한 손해발생을 제외하고는 이에 대하여 책임을 부담하지 아니합니다.
제 19 조 (관할 법원)
서비스 이용으로 발생한 분쟁에 대해 소송이 제기되는 경우 민사 소송법상의 관할 법원에 제기합니다.
[부 칙]
1. (시행일) 이 약관은 2016년 9월 5일부터 적용되며, 종전 약관은 본 약관으로 대체되며, 개정된 약관의 적용일 이전 가입자도 개정된 약관의 적용을 받습니다.