Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified–warmed ovarian tissue survival

Hye Won Youm,Jung Ryeol Lee,Jaewang Lee,Byung Chul Jee,Chang Suk Suh,Seok Hyun Kim 한국발생생물학회 2014 한국발생생물학회 학술발표대회 Vol.2014 No.9

Study question: What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? Summary answer: The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. What is known already: Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified–.warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. Study design, size, duration: A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. Participants/materials, setting, methods: The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of a-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified–.warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. Main results and the role of chance: The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4–.58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6–.92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. Limitations, reasons for caution: The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. Wider implications of the findings: We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.

Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and <i>in vitro</i> culture on vitrified–warmed ovarian tissue survival

Youm, Hye Won,Lee, Jung Ryeol,Lee, Jaewang,Jee, Byung Chul,Suh, Chang Suk,Kim, Seok Hyun Oxford University Press 2014 Human reproduction Vol.29 No.4

<P><B>STUDY QUESTION</B></P><P>What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation?</P><P><B>SUMMARY ANSWER</B></P><P>The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification.</P><P><B>WHAT IS KNOWN ALREADY</B></P><P>Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of <I>in vitro</I> culture (IVC) on vitrified–warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented.</P><P><B>STUDY DESIGN, SIZE, DURATION</B></P><P>A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and <I>in vitro</I> cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC.</P><P><B>PARTICIPANTS/MATERIALS, SETTING, METHODS</B></P><P>The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of α-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified–warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level.</P><P><B>MAIN RESULTS AND THE ROLE OF CHANCE</B></P><P>The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4–58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6–92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation.</P><P><B>LIMITATIONS, REASONS FOR CAUTION</B></P><P>The limitation of this study was the absence of <I>in vitro</I> fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation.</P><P><B>WIDER IMPLICATIONS OF THE FINDINGS</B></P><P>We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.</P><P><B>STUDY FUNDING/COMPETING INTEREST(S)</B></P><P>This study was supported by a grant (No. A120080) from the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea.</P>

The effects of Angiopoietin-2 on the transplanted mouse ovarian tissue

Hye Won Youm,Jae Wang Lee,Jung Ryeol Lee,Chang Suk Suh,Seok Hyun Kim 한국발생생물학회 2014 한국발생생물학회 학술발표대회 Vol.2014 No.9

The follicle loss of transplanted ovarian tissue (OT) is caused by ischemia and slow revascularization. To shorten the ischemic period and promote angiogenesis, some angiogenic factors have been treated for transplanted tissues. Angiopoietin-2 (ANG-2) is one of the major angiogenic factors and has been reported to promote blood vessels and increase vascular permeability in the ischemic and/or hypoxic environment. So, this study was designed to assess the impact of ANG-2 on follicle integrity and revascularization of mouse OT grafts. The 5-week-old B6D2F1 female mice were divided into 3 groups (a control and 2 ANG-2 groups) followed by ovary collection and vitrification. After warming, the ovaries were autotransplanted into kidney capsules with/without ANG-2 injection (50 or 500 ng/kg), and then killed at day(D)2, 7, 21 and 42 after transplantation. Total 2,437 follicles in OT grafts were assessed for the follicular density, integrity and classification by H&E staining. Apoptosis, revascularization, and serum FSH levels were evaluated by TUNEL assay, CD31 immunohistochemistry, and ELISA respectively. All the ANG-2 groups showed remarkable increase of morphologically intact follicle ratio across all the grafting duration except D21 (no statistical difference). The numbers of CD31(+) vessels (the sum of 3 fields at ×400 magnification) were significantly increased in both ANG-2 groups compared with the control group at all the grafting duration. Especially at D42, the 500ng ANG-2 group showed significantly more vessels than the 50 ng ANG-2 group as well as the control group. However the mean follicle numbers of grafts, apoptosis ratio and serum FSH levels showed no significant difference among the groups. In this study, remarkably well preserved follicles and larger amount of vessels were appeared in ANG-2 treated groups. So we thought that ANG-2 treatment is effective for OT transplantation and improve transplantation outcomes.

Analysis of Participant Factors That Affect the Diagnostic Performance of Screening Mammography: A Report of the Alliance for Breast Cancer Screening in Korea

Kim, Young Joong,Lee, Eun Hye,Jun, Jae Kwan,Shin, Dong-Rock,Park, Young Mi,Kim, Hye-Won,Kim, Youme,Kim, Keum Won,Lim, Hyo Soon,Park, Jeong Seon,Kim, Hye Jung,Jo, Hye-Mi unknown 2017 KOREAN JOURNAL OF RADIOLOGY Vol.18 No.4

<P><B>Objective</B></P><P>To analyze participant factors that affect the diagnostic performance of screening mammography.</P><P><B>Materials and Methods</B></P><P>We enrolled 128756 cases from 10 hospitals between 2005 and 2010. We analyzed recall rate, cancer detection rate (CDR) per 1000 examinations, positive predictive value (PPV), sensitivity, specificity, false positive rate (FPR), and interval cancer rate (ICR) per 1000 negative examinations according to participant factors including age, breast density, and number of visit to the same institution, and adjusted for confounding variables.</P><P><B>Results</B></P><P>Increasing age improved recall rates (27.4% in 40's, 17.5% in 50's, 11.1% in 60's, and 8.6% in 70's), CDR (2.7, 3.2, 2.0, and 2.4), PPV (1.0, 1.8, 1.8, and 2.8%), sensitivity (81.3, 88.8, 90.3, and 94.7%), specificity (72.7, 82.7, 89.0, and 91.7%), and FPR (27.3, 17.3, 11.0, and 8.4%) (<I>p</I> < 0.05). Higher breast density impaired recall rates (4.0% in P1, 9.0% in P2, 28.9% in P3, and 27.8% in P4), PPV (3.3, 2.3, 1.2, and 1.3%), specificity (96.1, 91.2, 71.4, and 72.5%), and FPR (3.9, 8.9, 28.6, and 27.6%) (<I>p</I> < 0.001). It also increased CDR (1.3, 2.1, 3.3, and 3.6) and ICR (0.2, 0.3, 0.6, and 1.6) (<I>p</I> < 0.05). Successive visits to the same institution improved recall rates (20.9% for one visit, 10.7% for two visits, 7.7% for more than three visits), PPV (1.6, 2.8, and 2.7%), specificity (79.4, 89.6, and 92.5%), and FPR (20.6, 10.4, and 7.5%) (<I>p</I> < 0.001).</P><P><B>Conclusion</B></P><P>Young age and dense breasts negatively affected diagnostic performance in mammography screening, whereas successive visits to the same institution had a positive effect. Examinee education for successive visits to the same institution would improve the diagnostic performance.</P>

Transgenic potato expressing Aβ reduce Aβ burden in Alzheimer’s disease mouse model

Youm, Jung Won,Kim, Hee,Han, Jee Hye Lo,Jang, Chang Hwan,Ha, Hee Jin,Mook-Jung, Inhee,Jeon, Jae Heung,Choi, Cheol Yong,Kim, Young Ho,Kim, Hyun Soon,Joung, Hyouk Elsevier 2005 FEBS letters Vol.579 No.30

<P><B>Abstract</B></P><P>Beta amyloid (Aβ) is believed one of the major pathogens of Alzheimer’s disease (AD), and the reduction of Aβ is considered a primary therapeutic target. Immunization with Aβ can reduce Aβ burden and pathological features in transgenic AD model mice. Transgenic potato plants were made using genes encoding 5 tandem repeats of Aβ1–42 peptides with an ER retention signal. Amyloid precursor protein transgenic mice (Tg2576) fed with transgenic potato tubers with adjuvant showed a primary immune response and a partial reduction of Aβ burden in the brain. Thus, Aβ tandem repeats can be expressed in transgenic potato plants to form immunologically functional Aβ, and these potatoes has a potential to be used for the prevention and treatment of AD.</P>

「건축서비스산업진흥법」시행 이후의 공공건축 설계공모제도 운영 실태에 관한 연구

염철호(Youm, Chirl-Ho),박석환(Park, Seok-Hwan),이혜원(Lee, Hye-Won) 대한건축학회 2021 대한건축학회논문집 Vol.37 No.12

The purpose of this study is to understand how the architectural design competition has been operated since the enforcement of the act on the promotion of building service industry and to draw implications for future system improvement. In this study, 2,083 projects announced from January 2015 to June 2019 were analyzed for the application characteristics of the design competition method and the compliance status of the design competition operation guidelines. In order to analyze the application characteristics of the design competition method, the order time, the size of the project, the use of the building, the design competition method, and the type of ordering agency were set as units of analysis, and the correlation was investigated. In addition, the status and characteristics of compliance with the design competition operation guidelines were investigated for four items: design competition period, format and specification of submissions, list of judges, and compensation for public offering costs. As a result of the analysis, the proposal competition is continuously increasing, but school facilities often do not present clear proposal request tasks. It was also confirmed that it was very low that the compliance rate with the regulations on disclosure of the list of judges and compensation for public offering costs. Based on the above, we proposed the necessity of inducing the adoption of appropriate proposal request tasks consistent with the purpose of introducing the proposal competition method and the need to strengthen procedures and monitoring to improve the compliance ratio of design competition guidelines.

철도교통 트랜스폰더 태그의 선로변 설치를 위한 내환경성 연구

김혜윤(Hye Yun Kim),박성수(Sungsoo Park),양영구(Youngoo Yang),이승원(Seung Won Lee),염기중(Ki Jung Youm),이재호(Jae-Ho Lee) 대한전기학회 2015 전기학회논문지 Vol.64 No.6

In this paper, we studied the environmental requirements for the railway transponder system. The transponder system is comprised of a reader installed beneath the train, a tag installed on the track, and a portable programmer. Among them, the transponder tag should be designed to withstand harsh environments, such as wide operating temperature range, mechanical shock and vibration, etc. To validate stable and reliable service under railway conditions, we carried out environmental test for transponder tag. We then installed the tags on the Honam high-speed test line.

구리이온을 함유한 PCB 폐에칭액의 Cross-flow 나노여과

박혜리 ( Hye Ri Park ),남상원 ( Sang Won Nam ),염경호 ( Kyung Ho Youm ) 한국화학공학회 2014 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.52 No.2

In this study the nano filtration (NF) membrane treatment of a sulfuric acid waste solutions containing copperion (Cu+2) discharging from the etching processes of the printed circuit board (PCB) manufacturing industry has been studied for the recycling of acid etching solution. SelRO MPS-34 4040 NF membrane from Koch company was tested to obtain the basic NF data for recycling of etching solution and separation efficiency (total rejection) of copper ion. NF experiments were carried out with a cross-flow membrane filtration laboratory system. The permeate flux was decreased with the increasing copper ion concentration in sulfuric acid solution and lowering pH of acid solution, and its value was the range of 4.5~23 L/m2·h. Total rejection of copper ion was decreased with the increasing copper ion concentration, lowering pH of acid solution and decreasing cross-flow rate. The total rejection of copper ion was more than 70% at the experimental condition. The SelRO MPS-34 4040 NF membrane was represented the stable flux and rejection for 1 year operation.

Isolation of a Tryptophan-Overproducing Strain Generated by EMS Mutagenesis of Candida rugosa

Kim, Sun-Hye,Ha, Yu-Mi,Youm, Hyoung-Joon,An, Gil-Hwan,Lee, Bong-Duk,Won, Mi-Sun,Song, Kyung-Bin The Korean Society of Food Science and Nutrition 2004 Preventive Nutrition and Food Science Vol.9 No.2

To isolate a mutant strain which overproduces tryptophan, mutants of Candida rugosa were screened after EMS (ethyl methane sulfonate) mutagenesis. Fluorotryptophan, a tryptophan analogue, was used for selection of a tryptophan-overproducing mutant after mutagenesis. Among 50 mutants, several candidates were selected based on intracellular tryptophan content. Amino acid analysis results showed that C3 was the best strain because it had the highest amount of tryptophan among the mutants.

Effects of supplementation with antifreeze proteins on the follicular integrity of vitrified-warmed mouse ovaries: Comparison of two types of antifreeze proteins alone and in combination

Kim, Min Kyung,Kong, Hyun Sun,Youm, Hye Won,Jee, Byung Chul The Korean Society for Reproductive Medicine 2017 Clinical and Experimental Reproductive Medicine Vol.44 No.1

Objective: The aim of this study was to analyze the effect of supplementing vitrification and warming solutions with two types of antifreeze proteins (AFPs) and the combination thereof on the follicular integrity of vitrified-warmed mouse ovaries. Methods: Ovaries (n=154) were obtained from 5-week-old BDF1 female mice (n=77) and vitrified using ethylene glycol and dimethyl sulfoxide with the supplementation of 10 mg/mL of Flavobacterium frigoris ice-binding protein (FfIBP), 10 mg/mL of type III AFP, or the combination thereof. Ovarian sections were examined by light microscopy after hematoxylin and eosin staining, and follicular intactness was assessed as a whole and according to the type of follicle. Apoptosis within the follicles as a whole was detected by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. Results: The proportion of overall intact follicles was significantly higher in the type III AFP-supplemented group (60.5%) and the combination group (62.9%) than in the non-supplemented controls (43.8%, p<0.05 for each). The proportion of intact primordial follicles was significantly higher in the FfIBP-supplemented (90.0%), type III AFP-supplemented (92.3%), and combination (89.7%) groups than in the non-supplemented control group (46.2%, p<0.05 for each). The proportions of non-apoptotic follicles were similar across the four groups. Conclusion: Supplementation of the vitrification and warming solutions with FfIBP, type III AFP, or the combination thereof was equally beneficial for the preservation of primordial follicles in vitrified mouse ovaries.