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X - 정자와 Y - 정자의 분리에 관한 연구 1 . Sephadex Gel 여과에 의한 인간정자의 분리
엄기붕,정길생,정병현 ( K . B . Oum,K . S . Chung,B . H . Chung ) 한국축산학회 1986 한국축산학회지 Vol.28 No.12
These experiments were carried out to develop new techniques useful for in vitro separation of X- and Y-bearing spermatozoa. One ㎖ of human semen was applied to the column (14 × 1.06 ㎝) filled with swellen Sephadex G-25 fine or G-50 fine gel and then elutriated with Locke solution. Elutriated solution was fractionated into 1 ㎖ by automatic fraction collector and spermatozoa included in each fraction were subjected to the estimation of viability and recovery rate and to F-body test. The results obtained in these experiments were summarized as follows: 1. Few spermatozoa were visible in the 1st to 3rd fractions and the highest concentration of spermatozoa was obtained from 7th to 9th fractions. 2. In case of Sephadex G-25 fine, the viable rate of spermatozoa before and after chromatography was 81.1 and 83.3%, respectively and in case of Sephadex G-50 tine, it was 79.9 and 82.35%, respectively. 3. The recovery rates of sperm applied to Sephadex G-25 and 50 fine were 40.8% and 52.3%, respectively. 4. The percentage of spermatozoa without F-body was increased from 53.0 to 67.1% and 52.6 to 68.2, respectively by the filtration through Sephadex G-25 and 50 column.
정자직접주입법시술시 동결보존된 인간정소정자에 의한 수정 및 임신성공
엄기붕(Ki Boong Oum),윤태기(Tae Ki Yoon),차광열(Kwang Yul Cha),김현주(Hyun Joo Kim),남윤성(Yoon Sung Nam),김현규(Hyun Kyoo Kim),곽인평(In Pyung Kwak),한세열(Sei Yul Han) 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.1
N/A There are not much reports concerning with clinical results using frozen-thawed testivular sperm in ICSI program. It is speculated that the necessity of cryopreservation of testicular sperm to avoid repeating surgical procedure for obtaining sperm for ICSI. This study was carried out to confirm whether frozen-thawed testicular sperm could be fertilized and pregnancy could be achieved using embryos fertilized with frozen-thawed testicular sperm in ICSI program or not. Testicular sperm obtained from obstructive- or non-obstructive azoospermia patients were co-cultured for 3 days with Vero cells to improve sperm motility. By co-culturing with Vero cells for 3 days, O-ll% of sperm motility after thawing increased up to 8-42% after co-culturing. ICSI was performed using frozen-thawed, and co-cultured sperm with 66 oocytes obtained from 8 patients and 62 oocytes were survived and 49(79.0%) oocytes were fertilized normally. Embryo transfer was possible in 7 out of 8 patients, and pregnancy was achieved in 6 patients(85.7%). These results indicated that not only fresh testicular sperm but frozen-thawed testicular sperm can be used in ICSI program.
X- 정자와 Y- 정자의 분리에 관한 연구 2 . Percoll 중층원심분리법에 의한 인간정자의 분리
엄기붕(K . B . Oum),이주영(J . Y . Lee),고대환(D . H . Ko),정길생(K . S . Chung) 한국축산학회 1988 한국축산학회지 Vol.30 No.5
These experiments were carried out to develop new techniques for in vitro separation of X-and Y-bearing spermatozoa. One ㎖ of washed human sperm suspension was loaded on the isotonic discontinuous Percoll density gradient, and then it was centrifuged at 250×G for 25 min. After centrifugation, spermatozoa were fractionated according to Percoll density gradient. Spermatozoa included in each fraction were subjected to the estimation of motility, morphological abnormality, F-body test, and recovery rate of spermatozoa was also investigated. The results obtained in these experiments were summarized as follows: 1. Following centrifugation of discontinuous Percoll density gradient, population of spermatozoa increased progressively from low density to high density. The highest concentration of spermatozoa was observed in 7th fraction which included 20% of spermatozoa. 2. High percentage of motile spermatozoa was observed at high Percoll concentration and the highest percentage was obtained at 6th fraction. 3. Following Percoll centrifugation, percentage of X-sperm increased from 53.2% (control) to 74.1% (7th fraction). 4. Following centrifugation, sperm abnormality was increased at low Percoll gradient and decreased at high Percoll gradient. The lowest abnormality (15.0%) and the highest abnormality (46.0%) were observed at 7th fraction and 1st fraction, respectively.
김현규,엄기붕,김현주,고정재,이숙환,윤태기,차광열,Kim, Hyun-Kyoo,Oum, Ki-Boong,Kim, Hyun-Joo,Ko, Jung-Jae,Lee, Sook-Hwan,Yoon, Tae-Ki,Cha, Kwang-Yul The Korean Society for Reproductive Medicine 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.2
폐색성 혹은 비폐색성 무정자증에서 부정소 정자채취법 등이 부적절하다고 여겨질때는 정소 조직을 일부 절제하여 그 조직으로부터 정자를 직접 채취하게 되는데 일반적으로 이렇게 정소로부터 추출한 정소정자는 운동성이 전혀 없거나 매우 약한 운동성을 보이는 경우가 많다. 본 연구의 목적은 이러한 정소정자를 Vero cell과 공배양을 시킴으로써 운동성을 획득시키거나 향상시키고 이를 수정시키는 시기까지 지속시킴으로써 정소정자추출술 (TESE)을 시행하는 환자나 의료진들에게 보다 편안하고 융통성있는 시간대를 부여하고, 아울러 정자직접주입술 (ICSI)을 보다 용이하게 하여 성공적인 수정률과 임신율을 얻음에 있다. 또한 ICSI를 시행한 후, 운동성이 향상된 잉여의 정소정자를 냉동보존함으로써 차후에 TESE을 다시 시행치않고도 시험관 아기 시술을 시도할 수 있는 부가적인 잇점도 있다고 할 수 있다. 대상환자군은 정관폐색증(n=11) 혹은 비정관폐색증(n=2)을 보이는 13명의 무정자증의 남성불임환자였으며 난자회수예정일 3일전에 TESE를 시행하여 정소정자를 얻은 후 이를 정자직접주입술이 시행되는 당일까지 Vero cell과 공배양을 실시하였다. Vero cell과의 공배양에 의하여 운동성이 있는 정소정자의 수는 공배양전과 비교하여 평균 3.3배가 증가하였으며, 특히 공배양전에 운동성이 있는 정소정자의 수가 50,000/ml이하의 미약한 운동성만을 보였던 경우 (n=5)에는 공배양 후에 운동성이 있는 정소정자 수의 평균증가율이 7.7배였다. 공배양전 정자운동성이 전혀 없었던 2례의 비정관폐색증환자중 3일간의 공배양을 통하여 1례에서 운동성을 획득한 정소정자를 얻을 수 있었으며 (14,300/ml), 정자직접주입술을 통하여 성공적인 수정 및 임신에 도달할 수 있었다. Vero cell과 공배양을 하고 ICSI했던 결과, 평균 수정률은 75.0% 이었으며 임신율은 61.5%였다.
Swim-up, Percoll, Sil-Select를 이용한 정자처리법에 의한 정자회수율, 운동성 및 체외수정율의 비교분석
하정희,엄기붕,정형민,정미경,김현규,고정재,윤태기,차광열,Ha, J.H.,Oum, K.B.,Chung, H.M.,Chung, M.K.,Kim, H.K.,Ko, J.J.,Yoon, T.K.,Cha, K.Y. 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.2
It is well known that discard of seminal plasma from the semen and separation of motile sperm should be preceded before insemination for IUI or IVF. Till now, more than ten kinds of semen treatment methods have been developed. Of those, swim-up and Percoll methods have been used widely in ART laboratories as a routine semen treatment methods because of its advantages. However, there are reports that Percoll can make a genetic trouble because of its chemical structure and therefore the necessity has been arisen to substitute Percoll for other equivalent materials. This study was performed to evaluate the effects of three different sperm preparation methods (swim-up, Percoll and Sil-Select) on sperm motility, sperm recovery rate and fertilization rate. Also, the feasibility of using Sil-Select instead of Percoll in ART was evaluated. Each semen samples were divided into three fractions and motile sperm were recovered by swim-up, Percoll and Sil-Select gradient centrifugation methods. Normal and sub-normal criteria of fifteen semen samples and seventeen IVF cycles were included in these study. As results, no significant difference was found in sperm recovery rate in normal semen treated by a Swim-up, Percoll and Sil-Select method ($13.2{\times}10^6,\;17.5{\times}10^6\;and\;17.7{\times}10^6$ respectively). The initial sperm motility was 61.9% and this increased to 87.1%, 92.6% and 89.5% through Swim-up, Percoll and Sil-Select treatment, respectively. Higher motility was observed in Percoll and Sil-Select treated groups (81.5%, 79.2%, respectively) than swim-up group (66.8%) after incubation for 24hrs. In sub-normal group, sperm recovery rates were higher in Sil-Select group $(2.9{\times}10^6)$ than Percoll gradients group $(1.8{\times}10^6)$. In IVF cycles, the outcomes of fertilization using sperm treated by swim-up and Sil-Select group were similar (82.2%, 79.7% respectively). In conclusion, our results indicate that Sil-Select can be used as a substitute material for sperm preparation instead of Percoll.
항정자항체가 일반적 체외수정 방법 및 정자직접 주입법(ICSI)에 미치는 영향에 관한 연구
오종훈,엄기붕,최동희,정미경,한세열,차광열,정길생,Oh, Jong-Hoon,Oum, Ki-Boong,Choi, Dong-Hee,Chung, Mi-Kyung,Han, Sei-Yul,Cha, Kwang-Yul,Chung, Kil-Saeng 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.3
The purpose of this study was to examine the effects of anti-sperm antibody (ASA) on the fertilization processes using conventional IVF and ICSI procedure in human and hamster oocytes. In human IVF, we have observed restricted fertilization with sperm testing positive for ASA. ($23{\sim}90%$ IgA, 60-97 % IgG). However, if ICSI was perform in the next IVF cycle with the same patients, we could successfully fertilize the oocytes (37%; p<0.001), thus achieving pregnancy and delivery. When the sperm were cocultured in medium containing ASA, there were binding of ASA to sperm surface. In addition, the mean rate of the acrosomal reaction in an in vitro acrosome reaction test was lower for Ab-bound sperm (43.5%) than for Ab-free sperm group (51.3%, p<0.05). We used human sperm and hamster oocytes to confirm the negative effects of the ASA on fertilization. The sperm and/or oocytes have been expose to medium containing ASA before IVF and ICSI. In this experiment, the ASA was bound to the oocyte and sperm surface. The following results were obtain by using various combinations of ASA free or ASA bound sperm with ASA free or ASA bound oocytes for IVF. When ASA free sperm were inseminate with ASA free and ASA bound hamster oocytes, the fertilization rates are 89.6% and 74.3% respectively. However, when ASA bound human sperm were use the results were 62.5% and 55.6% respectively. These shows the fertilization rate was significantly decreased in both ASA bound and ASA free oocytes when using ASA bound sperm. No difference found when ASA are present on the oocyte surface. When the hamster oocytes was treated by ICSI with ASA free or ASA bound human spermatozoa, no significant difference was found. These results showed that ICSI is the most promising method for couples who fertilization was not possible by conventional IVF because of ASA.