论文资料：不同妇年纪族群之卵子使用纺锤体影像观察及显微注射

论文资料：不同妇年纪族群之卵子使用纺锤体影像观察及显微注射 O01 不同婦女年紀族群之卵子使用紡錘體影像觀察及顯微注射之相關結果 Meiotic spindle imaging in oocytes and its relationship with ICSI outcomes in different women age groups *1,21141,31,4黃俊嘉 陳秀惠 陳忠義 李宗賢 黃梨香 李茂盛 123 李茂盛婦產科不孕症研究室, 中台科技大學生物技術系, 中山醫學大學醫學 4所, 中山醫學大學附設醫院婦產部 *1,2114Chun-Chia Huang, Hsiu-Hui Chen, Chung-I Chen, Tsung-Hsien Lee, 1,31,4Lii-Sheng Huang, Maw-Sheng Lee 12Division of Infertility, Lee Women’s Hospital, Department of Biotechnology, Central 3 Taiwan University of Science and Technology, Institute of Medicine, Chung-Shan 4Medical University, Department of Obstetrics and Gynecology, Chung-Shan Medical University Hospital Introduction: This study was to analyze the influence of women age on the appearance /location of meiotic spindle deviation from the first PB location and embryo quality. Methods: There 787 mature oocytes were collected from 237 women underwent IVF in this study from Jun 2007 to Dec 2009. According to women age, they were further divided into two groups: group A (female age?38 year, 175 cycles) and group B (female age ?35 years, 62 cycles). All patients underwent ICSI with microscopic digital imaging (ICSI-Guard, OCTAX EyeWare) for meiotic spindle observation. Oocytes after imaging were further divided into 3 groups (?15?, 16-44? and ?45?) according to the angle of spindle deviation from the PB position. The day 2 embryo quality between different groups was also analyzed in this study. Results: The spindle appearance rates of MII oocytes between group A (78.7%, 458/582) and B (74.6% 153/205) were no significant differences. The oocytes of group A showed a significantly higher deviation rate (52.9%, 308/582) from the PB position than group B (40.0%, 82/205, p<0.01). However, women in group A have a significantly lower rate of angle deviation ?15? (33.5%) and a significantly higher rate of angle deviation ?45? (37.8%) than women group B (42.9% vs. 26.3%; p<0.05). There were no significant differences between the group A and B in terms of fertilization rates (75.3% vs. 77.6%) and day 2 good embryo rates (43.8% vs. 41.0%). Conclusions: It was suggested that spindle imaging with ICSI Guard may be an appropriate method to avoid damage of spindle in older women who showed fewer oocytes retrieval and a higher rate of deviation of spindle position. O02 不同培養液在卵子體外成熟過程產生的氨濃度 Ammonium levels in different maturation media during in-vitro maturation of oocytes 林禹宏 黃建榮 謝碧純 陳恆如 新光醫院婦產科 Yu-Hung Lin, Jiann-Loung Hwang, Bih-Chwen Hsieh, Heng-Ju Chen Dept of Ob/Gyn, Shin Kong Wu Ho-Su Memorial Hospital Introduction: It has been shown that some culture media produce embryo-toxic ammonium. Whether ammonium is produced during in-vitro maturation (IVM) of oocytes has never been investigated. The purpose of the study was to investigate whether ammonium is produced during IVM of oocytes, and its possible adverse effects on IVM. Methods: The study used immature oocytes from FVB mice. The immature oocytes were randomly allocated into Quinn’s Advantage Fertilization Medium (Q1) and TCM199 medium, plus 75 mIU/ml hMG, 0.2mM pyruvate, and 10% fetal bovine serum. After 18 hours, ammonium concentrations in the maturation media were measured, and the mature oocytes were fertilized, and cultured to blastocysts. The blastocysts were then stained for inner cell mass (ICM) and trophectoderm (TE). Blank Q1 medium and TCM199 medium without oocytes but with the same composition were used as controls. They were incubated at the same time for 18 hours, and ammonium concentrations in the media were measured. Results: At time 0, ammonium concentrations in Q1 medium and TCM199 medium were 184.9 ? 5.1 and 339.2 ? 7.6 µg/dl, respectively (p = 0.05). After 18 hours of IVM, ammonium concentrations rose slightly in Q1 medium and TCM199 medium (both p = 0.05). Ammonium concentrations in Q1 medium and TCM199 medium were 268.7 ? 13.4 and 443.6 ? 15.5µg/dl, respectively (p = 0.045). Ammonium concentrations in the same medium with and without oocytes were similar. The maturation rate, fertilization rate, and blastocyst rate in Q1 medium were 85.7%, 71.1%, and 18.0% respectively; and those in TCM199 media were 76.6%, 65.2%, and 15.2% respectively. The maturation rate was higher in Q1 medium than in TCM199 medium (p = 0.024), but fertilization and blastocyst rates were similar in the two media. Differential staining of the blastocysts showed no significant differences in total cell numbers and cell numbers of ICM and TE of the blastocysts derived from the two media. But the ICM ratio was slightly higher in the blastocysts derived from Q1 medium. Addition of ammonium to Q1 medium also decreased the maturation rate. Conclusions: The popular TCM199 medium is inferior to Q1 medium for IVM in terms of oocyte maturation. The result may be due to higher ammonium concentration in TCM199 medium. O03 AMH 值多低才算太低? 探討大於40歲不孕症患者中抗血清抗穏勒氏荷爾蒙之 數值.對於 IVF / ICSI 結果之影響 How low is too low? The effects of serum AMH level in patients ? 40 years old on outcomes of ICSI / IVF 吳劭穎* 林時羽 林明輝 胡玉銘 李國光 馬偕紀念醫院 婦產部 Shao-Ying Wu*, Syr-Yu Lin, Ming-Huei Lin, Yuh-Ming Hwu, Robert Kuo-Kuang Lee Department of Obstetrics and Gynecology. MacKay Memorial Hospital Introduction: One of the more subtle, less discussed aspects of modern ART is counseling the patients whom no longer stand a reasonable chance of becoming pregnant. In the past, physician’s assessment of the fecundity was mainly based on patients’ age, which can have great variability. The purpose of this article is to investigate the practicability of using serum levels of anti-mullerian hormone (AMH) combined with biological aging as a simple yet accurate screening method to identify poor candidates for IVF patients for counseling before enrolled into IVF cycles. Methods: Serum AMH were collected from a total of 162 patients who were ? 40 years old when they visited the infertility clinics at MacKay Memorial Hospital from December 2006 to December 2009. The obtained results were stratified into quartiles. We then retrospectively identified the AMH levels of infertility patients ? 40 years old (n = 98) who underwent IVF / ICSI during the same period of time, and respective outcomes were examined. The same analysis was performed for patients ? 35 years old (n = 875), which served as the control group. Results: The range of the calculated AMH quartiles are < 0.32, 0.32~0.82, 0.83~1.79, and >1.79 ng/mL respectively in the ? 40 years old patients. AMH levels in the lowest quartile (<0.32 ng/mL) had a highest cancellation (75%) and zero clinical pregnancy rate. The lowest serum AMH level in ? 40 years old patients who achieved clinical pregnancy was 0.56 ng/mL, which corresponded to the 36.4th percentile of the collected AMH data of the matched age group. For the control group, the lowest serum AMH level achieving clinical pregnancy is 0.26ng/mL, which was equivalent to 0.79th percentile of the matched age group (? 35 years old). Conclusions: Though hundred thousands of IVF cycles are being performed around the globe annually, precise identification of potential poor responder prior to cycle initiation remains a difficult task. We propose using two simple criteria, age ? 40 years old and AMH < 25th percentile (< 0.32 ng/mL) as the markers of extremely poor candidates for IVF/ICSI with high cancellation rate and no viable pregnancy. Table 1. Outcomes of IVF / ICSI cycles in old age women (? 40 years old) Serum AMH Levels Total < 0.32 0.32~0.82 0.83~1.79 > 1.79 Total Cycle 98 8 21 23 46 Cancelled Cycle (%) 17 (17.3) 6 (75) 4 (23.8) 3 (13.0) 3 (6.5) Mean Age 41.6 ? 1.7 42.3 ? 2.8 42.1 ? 2.0 41.1 ? 1.5 41.4 ? 1.4 1718.6 ? 600 ? 1442.5 ? 1612.8 ? 2287.3 ? Peak E2 level (pg/mL) 1578.9 36.7 2336.7 983.9 1543.1 No. of oocyte retrieved 6.4 ? 5.2 2.5 ? 0.7 5.4 ? 4.9 5.9 ? 2.8 8.8 ? 5.7 No. of mature oocytes 4.9 ? 3.9 1.5 ? 0.7 4.0 ? 2.2 5.0 ? 2.6 6.9 ? 4.3 Embryo obtained 4.5 ? 3.1 1 3.1 ? 2.1 4.4 ? 2.4 5.1 ? 3.4 Embryos transferred 3 ? 1.0 1 2.4 ? 0.9 3.0 ? 0.9 3.1 ? 0.9 Clinical 23 (28.4) 0 (0) 2 (11.7) 7 (35) 14 (32.5) Pregnancy per cycle (%) Abortion per cycle (%) 7 (30.4) 0 (0) 0 (0) 0 (0) 7 (50) Viable Pregnancy Rate per cycle (%) [FHB (?) 16 (19.7) 0 (0) 2 (11.7) 7 (35) 7 (16.2) & > 7 wks.] O04 以子宮鏡治療準備接受人工生殖科技而子宮頸狹窄的病人 Hysteroscopic cervical resection for cervical stenosis in patients undergoing ART 陳恆如 林禹宏 黃建榮 謝碧純 新光醫院婦產科 Yu-Hung Lin, Jiann-Loung Hwang, Bih-Chwen Hsieh, Heng-Ju Chen Dept of Ob/Gyn, Shin Kong Wu Ho-Su Memorial Hospital Introduction: Embryo transfer (ET) is one of the most important parts of ART, and cervical stenosis can be a challenge for physicians performing ET. We propose a technique for correcting cervical stenosis before ART. Methods: All our patients undergoing ART receive mock ET beforehand. We routinely use Wallace Embryo Replacement Catheter (Smith Medical Inc., Kent, U.K.) for ET. If it fails, Sydney IVF Embryo Transfer Set (Limerick, Ireland) or GyneFlex Embryo Transfer Catheter (Gynemed, Lensahn, Denmark) are tried. When all catheters fail to enter the uterine cavity, the patients were referred for hysteroscopic cervical resection. From Dec 2009 to June 2010, 18 patients with the impression of cervical stensosis were referred for the procedure. The procedure was performed as an outpatient surgery under intravenous general anesthesia with Propofol. In 4 patients, the sound could pass the cervical canal after anesthesia, so they were excluded from the study. Cervical stenosis is defined when mock ET fails and the sound can not pass the cervical canal. The procedure proceeded with a 22-F hysteroscope (Karl Storz GmbH, Tuttlingen, Germany) at the cervical os, then the loop-electrode at 80 W cutting current protruded out of the sheath. After resecting small pieces of protrusions at anterior or posterior cervical canal, the hysteroscope could advance a little. Then after resecting protrusions that obstructed the cervical canal, the hysteroscope could advance again. The procedures were repeated until the hysteroscope could enter the uterine cavity, which was inspected for any abnormalities. After the procedures, an 8-F Foley catheter was placed in the uterine cavity to prevent stricture of the cervical canal. Repeat mock ET was performed after the Foley catheter was removed one week later. Results: Among the 18 patients referred for the procedure, 4 were excluded because the sound could pass the cervical canal. Therefore 14 patients were considered to have cervical stenosis. The mean operation time was 19 min (range, 10-40 min). Repeat mock ET was successful in all patients, with slight resistance encountered in 4 patients. Endometrial polyp was found in one patient. Two patients underwent IVF after operation, and both became pregnant. Conclusions: Hysteroscopic cervical resection is an effective treatment for cervical stenosis. O05 促使人類胚胎幹細胞分化為生殖細胞的策略 Strategies to improve the differentiation of human embryonic stem cells into germ cells 陳信孚*1,2, 郭紘志3 吳芳君1 陳佳徽1 楊友仕1 何弘能1,2 Hsin-Fu Chen, Hung-Chih Kuo, Fang-Chun Wu, Chia-Hui Chen, Yu-Shih Yang, Hong-Nerng Ho 1Department of Obstetrics & Gynecology, National Taiwan University Hospital 2Graduate Institute of Clinical Genomics, College of Medicine, National Taiwan University 3Institute of Cellular and Organismic Biology & Genomics Research Center, Academia Sinica Introduction: Human embryonic stem cells (hESCs) can theoretically develop into all cell lineages including germ cells in human. Our recent reports showed that hESCs had the potential to develop into cells expressing female germ cell markers, but with a low efficiency. Since efficiency of germ cells formation need to be high enough for clinical application to treat diseases as infertility and ovarian failure, this study aimed to constitute favorable culture environments to improve differentiating hESCs to germ cells, especially eggs. Methods: NTU1 and transgenic H9:Oct4-eGFP hESCs were cultured on: Laminin-coated dishes, co-culture with mouse ovarian stromal cells (Ova-Co) or its conditioned medium (Ova-CM), co-culture with granulosa cells (Gra-Co) or conditioned medium (Gra-CM) for 7-42 days. BMP4 and retinoid acid were also used. SSEA1(+) and Oct4-eGFP(+)(both germ cell markers) cells were isolated from differentiated NTU1 and H9:Oct4-EGFP hESCs by manual collection or cell sorter, and examined by flowcytometry, immunofluorescence, PCR and Q-PCR for germ cell makers including Oct4, Dazl, c-kit, VASA, GDF9, SCP3, and ZP3. Differentiated H9:Oct4-eGFP(+) cells were also co-aggregated with ovarian cells or granulosa cells and transplanted under kidney capsules or in sterilized ovaries of NOD-SCID mice. The grafts were removed later for histology and immmunocytochemistry. Results: Percentages of SSEA1(+)/Oct4-eGFP(+) cells were significantly increased by treatment with Gra-Co and/or Gra-CM, but these SSEA1(+) cells did not express higher levels of VASA (a specific germ cell marker) and GDF9. Some cells especially those in aggregates remained eGFP(+) after differentiation, suggesting their germ cell nature and they occasionally developed into ovarian follicle-like structures. Gra-CM enriched the number of H9:Oct4-eGFP(+) cells, and these cells expressed higher levels of c-kit, VASA and GDF9, but not SCP3. H9:Oct4-eGFP(+) cells also developed into cells expressing VASA in vivo under mouse renal capsules. Conclusions: Human granulosa cells as an ovarian niche enhance the differentiation of hESCs to germ cells in vivo and in vitro. Transgenic Oct4-eGFP cells improve the enrichment and tracking of these potential germ cells, but SSEA1 is not a good marker for this purpose. However it remains to be tested which system, in vivo or in vitro, is more efficient and whether these germ cells have undergone meiosis and epigenetic modifications. O06 奈米二氧化鈦對於雄性生殖系統之毒性影響 The toxicity effect of nano-titanium dioxide in male reproductive system 1*123 陳惠文 曾啟瑞吳平 李奕旻 12國立陽明大學藥理學研究所 國立台灣大學毒理學研究所 私立台北醫學大學附 3設醫院婦產科 1*123Ping Wu, Yee-Ming Lee, Huei-Wen Chen, Chii-Ruey Tzeng 1Department and Institute of Pharmacology, National Yang-Ming University; Graduate 2Institute of Toxicology, National Taiwan University; Department of Obstetrics and 3Gynecology, Taipei Medical University Introduction: Nano-particles are differing from larger samples of the same material in their chemical or physical properties. However, only limited knowledge of the toxicity of nano-particles in reproductive system has been reported. Previous study demonstrated that the sperm cell viability could be affected by the presence of gold nano-particles and exposure of diesel exhaust particles (DEP) has been shown to induce leydig cell degeneration, increase the number of damaged seminiferous tubules, and reduce daily sperm production. In this study, the toxicity of nano-TiOparticles, in male 2 reproductive system was examined. Materials: Preparation of murine spermatozoa – spermatozoa was collected from cauda epididymis of mice, and purify for motile spermatozoa within centrifugation. The sperm cells were incubated in modified BWW medium for further experiment. Protein tyrosine phosphorylation – purified sperm cells were incubated with nano-TiO 2 (0.01~500 μg/mL) in modified BWW medium within BSA/Ca2+ activation, then extract cell lysate for electrophorosis analysis and western blotting. Flowcytometry analysis of sperm cell – sperm cell was incubated with nano-TiO, then 2 stain for cell survival assay - propidium iodide staining (PI); ROS production assay - DCFH-DA staining; mitochondria integrity assay - CMXROS staining. Analysis of sperm cell motility via CASA – Sperm cells, pre-incubated with nano-TiO, 2 were detected by CASA. In vitro fertilization (IVF) – Oocytes were collect from super-ovulaton female mice, and then incubated with sperm cells pre-incubated with nano-TiO. The ratio of two cell was 2 further measured. Results: The results showed the level of protein tyrosine phosphorylation (known key factor for capacitation, acrosome reaction, and motility) was significantly decreased in mice sperm treated with different dosage of nano-TiO. CASA analysis also showed that 2 nano-TiO might inhibit sperm motility. IVF data showed the fertilization rate was 2 dose–dependently decreased as sperm exposure to nano-TiO. For animal model, after 2 two weeks of nano-TiO treatment (directly inject single dose of nano-particles into 2 testis), we validate the 2-cell embryo ratio via mating to female mice. We found that there is no 2-cell embryo could be found in nano-TiOtreated group (unfertilized 2 oocytes number = 51, 2-cell embryo=0), as comparing to the control group (unfertilized oocytes number = 23, 2-cell embryo= 42). We also found accumulation of nano-TiO 2 around the seminiferiuous tubules and disruption of spermatogenesis in nano-TiO2 injected testis, as comparing to vehicle control. Conclusion: Our findings indicated that nano-TiO might affect the male fertility via 2 reducing the activities of sperm and disrupt spermatogenesis. These results also support the petition to pay more attention on nano-TiO before introducing into the market. 2 O07 卵泡液與培養液中自由基含量與胚胎品質之關聯性 The association between embryo quality and levels of reactive oxygen species in follicular fluid and culture media 1,2,31 111,2,3李宗賢*曹惠美黃俊嘉 陳忠義李茂盛 123李茂盛婦產科不孕症研究室, 中山醫學大學醫研所,中山醫學大學婦產科 1,2,3111Tsung-Hsien Lee*, Hui-mei Tsao, Chun-Chia Huang, Chung-I Chen, Maw-Sheng 1,2,3Lee 1Division of Infertility Clinic, Lee Women’s Hospital, 2Institute of Medicine, Chung Shan Medical University, 3Deaprtment of Obstetrics and Gynecology, Chung Shan Medical University Hospital Introduction: This study was designed to determine the correlation between the levels of reactive oxygen species (ROS) in follicular fluid and culture media and the early development of human embryos. Methods: Between 2007 and 2008, a total of 174 IVF/ICSI cycles at Lee Women’s Hospital were enrolled in this study. The morphology and good embryo rates in early cleavage stage were recorded. ROS levels in follicular fluid and spent culture media were measured by chemiluminenscence. Pair t test was utilized for embryo quality data during splitting culture and in vitro development. Results: High ROS levels in follicular fluid were associated poor embryo quality on day 3 development, especially in ICSI cycles. The ROS levels in day 3 spent culture media were correlated with fragmentation degree and medium type. Interestingly, ROS levels were different between the two commercial media. Furthermore, better D3 embryo quality were connected with the culture media with low ROS levels. Conclusions: The elevated ROS levels in follicular fluid and culture media in IVF/ICSI cycles were associated with poor development of cleavage stage embryos. The measurement of ROS levels may be a useful parameter for quality assurance process in embryology laboratory. O08 甲型海洋性貧血的著床前胚胎遺傳診斷 Clinical experience of pre-implantation genetic diagnosis of α-thalassaemia 1121111 陳俊凱* 尤星策 Mark Hughes 黃泓淵 王馨世 李奇龍 宋永魁 12 林口長庚醫院婦產部 GGI, USA 1121Chun-Kai Chen*, Hsing-Tse Yu, Mark Hughes, Hong-Yuan Huang, 111 Hsin-Shin-Wang, Chyi-Long Lee, Yung-Kuei Soong 1 Department of Obstetrics and Gynecology, Chang-Gung Memorial Hospital, Chang-Gung University College Of Medicine, Tao-Yuan, Taiwan 2 The Genesis Genetics Institute, USA Introduction: Alpha-thalassaemia of Southeast Asian type (SEA) deletion, the endemic alpha-globin gene cluster abnormality in Taiwanese alpha-thalassemia cases, is caused by deletion of approximately 20.5 kb in length, deleting both alpha-globins, but leaving the embryonic ζ-globin gene on human chromosome 16 intact. A conservative report has estimated at least 3.5% of Taiwanese residents carry this deletion. Thalassemia is an autosomal recessive disorder and carriers of one mutant allele are asymptomatic. SEASEACouples who are both carriers of the - - mutation (- -/αα) run a 25% risk of having a hydropic fetus. Here, we present the experiences of the first four PGD cases of alpha thalassemia in our hospital. SEAMaterials: Between September 2008 and December 2009, four couples with the -- genotype (deletion of 2 α-globin genes in cis) sought for pre-implantation genetic diagnosis. Three couples have previous pregnancies of hydropic fetuses. After counseling and informed consent, the couples start IVF-ET program of long protocol. Embryo biopsy was performed on day 3 post-oocyte retrieval. The aspirated blastomeres were put into small tubes and sent out immediately to another diagnostic laboratory in USA. Mutation and marker data were obtained using nested PCR primers, according to standard PGD practices. The assay directly interrogated the presence or absence of the familial mutation. The test also utilized a chromosome 16 marker genetically linked to the HBA1/HBA2 gene locus, for which allelic phase was known for the family. An ABI 3130 Genetic Analyzer was employed subsequent to PCR to obtain data for both the mutation and marker. These results were then analyzed to determine the genotype of each sample. Results: The average age of these patients were 35.7 years. After stimulation, an average of 15.8 oocytes was retrieved per cycle with a fertilization rate (2PN rate) of 75% from ICSI. The embryo biopsy procedure was successfully performed in all cases. Twenty three (59%) of 39 embryos were biopsied and blastomeres were sent for diagnosis. There was no diagnosis in two embryos (5%) since no DNA signals found after amplification. The data from five embryos (13%) have allele drop-out (ADO) phenomenon. Two were from paternal allele occurring in one couple and three were occurring in another couple (two paternal alleles and one maternal allele). For three couples, three embryos were transferred back each resulting in a twin pregnancy and one singleton. In one couple, the biopsied embryos were frozen due to delay of transportation. After the data available, one unaffected embryo was thawed and transferred back on day 6 resulting one singleton. Conclusions: Pre-implantation genetic diagnosis can help couples get rid of the suffering of abortion. Couples suffered from genetic diseases or carrier should be provided with such treatment choice. Stable blastocyst culture and improved cryopreservation technique both enhance the application of PGD. Since the preimplantation genetic diagnosis (PGD) of alpha thalassemia became mature, it is considered as an alternative for prenatal diagnosis, especially for couples with previous pregnancy of hydropic fetus. O09 使用睪丸切片精蟲或是射精之極嚴重寡少無力畸型精蟲施行顯微受精成果 與出生後兒童的比較研究 Clinical outcome and children born after ICSI with extracted testicular sperm or ejaculated extreme severely oligoasthenoteratzoospermia sperm: a comparative study ,11 11111藍國忠蔡慶璋王儷蓉黃富仁林秉瑤林易奇 龔福財 1 高雄長庚醫院 婦產部 Kuo-Chung Lan, M.D., Ching-Chang Tsai MD., Li-Jung Wang, M.D., Fu-Jen Huang, M.D., Pin-Yao Lin, M.D.,Yi-Chi Lin, B.S., Fu-Tsai Kung, M.D. Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital-Kaohsiung Medical Center Introduction: Infertile men with severe oligoasthenteratzoospermia or azoospermia is uncommon but not rare. Most of these men are healthy, and the cause of impaired spermatogenesis is rarely identified with certainty. Now, even men with no sperm in the ejaculate (azoospermia), whether from obstructions or sperm production problems (non-obstructive azoospermia), may have a chance to father a biological child by ICSI. Although ICSI was initially developed and has been shown to be an effective treatment for male factor infertility, lack of natural selection of sperm in the ICSI procedure, researchers or clinicians have a reason to be concerned about its use and the health of the children born after this technique. Some studies show that children born after assisted reproductive technology (ART) are at increased risk of birth defects, other studies suggest that there is no extra concern about ICSI children conceived with epididymal or testicular sperm. The clinical outcome and children born after ICSI with extracted testicular sperm or ejaculated extreme severe oligoasthenoteratzoospermia sperm has been less thoroughly assessed or comparison. The goal of this study is try to address this issue from ICSI treatment in Taiwan. Materials and Methods: Data were collected from consecutive infertile couples who underwent ICSI and transcervical fresh ET from January 2001 to April 2009 in our . There are 126 ICSI cycles using extracted testicular sperm from azoospermia men and 65 ICSI cycles using fresh ejaculated sperm from extreme severe oligo-astheno-teratospermia men. We defined that infertile men with sperm count < 2 million/ml, with all spermatozoa immotile or only non-progressively motile widely defined as severe oligozoospermia and/or severe asthenozoospermia(severe oligoteratoasthenzoospermia, defined as total sperm count <2 million/ml and/or<5% rapid progressive type A motility in the fresh semen,). Result(s): There was comparable effect in age of female (31.6?5.0 vs. 32.1 ?4.7) and male (36.4?5.6 vs. 35.1?4.9) partners, estradiol (pg/mL) on hCG day (1876.6 ? 1327.1 vs. 2321.6 ? 1747.3), numbers of oocytes retrieved (7.3 ? 4.1 vs. 8.2 ? 3.6), normal fertilization rate (91.3% vs, 90.8 %), Zygote grade 1 score distribution (47.9% vs. 48.0%), numbers of top-quality embryos transferred (1.5?0.8 vs. 1.6?0.9), clinical pregnancy rate per transfer(46.6% vs. 39.7%), chemical pregnancy rate per transfer (4.2% vs.3.2%), implantation rate (26.2% vs. 22.7%),live birth rate per transfer (40.7 % vs. 30.2%) abortion rate per transfer (7.6% vs. 9.5%) between two groups. We further follow up these live birth ICSI children (1-7 years old) and analysis the maternal perinatal characteristics: 60 deliveries from 48 TESE-ICSI cycles and 21 deliveries from 19 severe OAT-ICSI cycles. There are comparable obstetric and perinatal outcomes between two groups. Children conceived by ICSI were healthy without psychomotor or intellectual development retardation. However, one major congenital heart anomalies (TOF) in each groups was observed cannot be dismissed. Conclusion(s): This study shows similar clinical outcome and children development after ICSI with extracted testicular sperm or ejaculated extreme severely oligoasthenoteratzoospermia sperm. O10 對於非男性因素不孕之患者施行單一精蟲注入術會改變胚胎發育之品質嗎? Would ICSI alter embryo quality in non-male factor infertility patients? 易瑜嶠 何彥秉 林宗毅 陳麗宇 何師竹 陳明哲* 谷化芬 陳雅芳 台中榮民總醫院 婦產部 生殖醫學科 Ming-Jer Chen*, Hwa-Fen Guu, Ya-Fang Chen, Yu-Chiao Yi, Jason Yen-Ping Ho, Tsung-I Lin, Li-Yu Chen, Esther Shih-Chu Ho Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Taichung Veterans General Hospital Introduction: The purpose of this study is to investigate whether the performance of ICSI for non-male factor infertility patients could improve the quality of embryos in addition to guaranteeing the fertilization Material and methods: The ART database from Jan 2002 till Jun 2010 was reviewed (total 1265 cycles). We first excluded all male factor patients that ICSI deemed mandatory and all those cycles with total fertilization failure by IVF treatment. Then we chose those repeated cycles with first cycle by IVF and second cycle by ICSI treatment (sequential IVF-ICSI: 88 cycles with 44 sets of patients) and all cycles with half IVF and half ICSI treatment (concomitant IVF-ICSI:55 cycles) for data analysis. Embryo quality (EQ) comparisons between IVF and ICSI treatment were by (1) average embryos score from all resulted embryos (aES), (2) average score from top three embryos (tES), (3) rate of good quality embryos (rGE) and (4) number of good quality embryos(nGE). Paired samples statistics were carried out by SPSS-PC ver.11.5 with p<0.05 as statistical significance. . Results: For those with sequential IVF-ICSI treatment cycles (N=29 sets) with comparable embryo transfer (ET) timing, the aES, tES rGE and nGE in IVF as compared to ICSI cycles were 12.8 vs. 14.2 (p=0.27), 14.9 vs. 18.4 (p=0.04), 0.33 vs. 0.33 (p=0.94) and 1.1 vs. 2.4 (p=0.01). Subgroup analysis revealed that for those IVF cycles (N=9 sets) with fertilization rate (FR) 60% or more, the EQ parameters were not statistically different except aES. (p=0.04, 0.09, 0.32 and 0.10). For those IVF cycles (N=20 sets) with suboptimal FR (less than 60%), the EQ parameters were not statistically different except nGE (p=0.99, 0.20, 0.50 and 0.05). For those with concomitant IVF-ICSI treatment cycles (N=45 sets), the aES, tES rGE and nGE in IVF as compared to ICSI cycles were 15.3 vs. 15.5 (p=0.85), 17.3 vs. 20.0 (p=0.01), 0.45 vs. 0.47 (p=0.65) and 2.0 vs. 2.5 (p=0.14). Subgroup analysis revealed that for those IVF cycles (N=32 sets) with fertilization rate (FR) 60% or more, those EQ parameters were not statistically different. (p=0.98, 0.12, 0.96 and 0.82). However, for those IVF cycles (N=13 sets) with suboptimal FR (less than 60%), the EQ parameters become statistically different (p=0.74, 0.02, 0.44 and 0.05) in tES and nGE. Conclusions: These analyses revealed that in those patients with non-male factor infertility received prior IVF treatment with suboptimal fertilization, subsequent ICSI might not only improve the availability of better quality embryos for transfer (nGE) but also improve the quality of top embryos (tES) (sequential data). These beneficial effects were more obvious in patients with suboptimal IVF fertilization as revealed by patients receiving concomitant IVF and ICSI treatment (concomitant data) O11 比較使用捐贈之冷凍精子給予顯微注射及傳統授精後的受精率 The comparison of fertilization rate of donor cryopreserved sperm with ICSI and convention IVF *11,2141,31,4陳秀惠 黃俊嘉 陳忠義 李忠賢 黃梨香 李茂盛 123 李茂盛婦產科不孕症研究室 中台科技大學生物技術系 中山醫學大學醫學 4所 中山醫學大學附設醫院婦產部 *11,214Hsiu-Hui Chen, Chun-Chia Huang, Chung-I Chen, Tsung-Hsien Lee, 1,31,4Lii-Sheng Huang, Maw-Sheng Lee 12Division of Infertility, Lee Women’s Hospital, Department of Biotechnology, Central 3 Taiwan University of Science and Technology, Institute of Medicine, Chung-Shan 4Medical University, Department of Obstetrics and Gynecology, Chung-Shan Medical University Hospital Introduction: The donated sperm must be cryopreserved for 6 month for HIV screen before IVF and it was suggested the ICSI should be performed in IVF in the past. In this study, we have tentatively named the half-ICSI treatment for combining conventional IVF and ICSI in donated sperm protocol. To evaluate the fertilization ability of cryopreserved sperm in ICSI or convention IVF, the fertilization and day 3 embryo quality between ICSI and convention IVF was compared. Methods: The half-ICSI was carried out 86 cycles of the donated sperm protocol from Mar 2004 to Mar 2009. These donated sperm quality was normal before cryopreservation. Total 1311 mature oocytes (MII) were divided into ICSI (MII=638) and convention IVF (MII=673) after oocytes retrieval. Results: The mean of total fertilization rates were similar between ICSI (80.1?2.0%) and convention IVF (76.1?2.9%, p=0.287). The polyspermic fertilization rate in convention IVF (8.4?1.5%) was significantly higher than that in ICSI (0.6?0.3%, p<0.01). Only one case (1.2%, 1/86) occurred absolute fertilization failure in convention IVF treatment. The day 3 good embryo rates in convention IVF (62.4?3.9%) showed a slightly higher than ICSI group (54.1?3.6%, p=0.044). The pregnancy and implantation rates in this study were 54.7% (47/86) and 26.0% (90/346), respectively. Conclusions: Our data present that ICSI and convention IVF can provide similar higher fertilization rates, and it was suggested that convention IVF was an appropriate choice in IVF with normal donor cryopreserved sperm. O12 ?PICSI的臨床試驗:篩選結合琉璃醣碳基酸精蟲的新方法應用於單一精蟲顯 微注射術 ?A clinical trial evaluating PICSI, a novel method for selection of hyaluronan-bound sperm (HBS) for use in ICSI 1,2111111,2 林正凱* 蘇金琮 林育如 羅筱晴 洪上國 賴宗炫 李發焜 12新竹國泰綜合醫院 婦產科 元培科技大學 醫學檢驗暨生物技術系 1,21111Tseng-Kai Lin*, Jin-Tsung Su, Yu-Ru Lin, Hsiao-Ching Lo, Shang-Gwo Horng, 11,2 Tsung-Hsuan Lai , Fa-Kung Lee 1Department of Obstetrics and Gynecology, Hsinchu Cathy General Hospital, Hsinchu 2Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu Introduction: The technological advances of intracytoplasmic sperm injection (ICSI) enable a single sperm to be introduced into an oocyte. During ICSI, sperm is selected based on sperm morphology and motility. However, this selection procedure is not discriminatory with respect to the identification of spermatozoa with normal chromosome constitution. In addition, in severe male infertility cases, sperm used for ICSI may have an effect in the production of potentially defective embryos, low pregnancy rate and high abortion rate. Hyaluronan (HA), a major constituent of the cumulus matrix, may play a critical role in the selection of functionally competent sperm during in vivo fertilization. The purpose of this study was to evaluate the clinical ?outcomes of the use of hyaluronan-bound sperm in ICSI (PICSI) Materials: Ninety-two ICSI patients were included in this study and randomized to the control (ICSI) and study (PICSI)) group. In the control group, sperm were selected for ICSI via the evaluation of motility and morphology. In the study group, microdrops of hyaluronan were used in the selection of sperm for ICSI. The clinical outcomes of fertilization, good quality embryo, implantation and pregnancy were compared between the two groups. Results: There were no significant differences between each group in terms of the age of patients (33.7?3.4 vs. 33.9?3.6) , fertilization rates (87.7% vs. 84.4%), good quality embryo rates (55.1% vs. 55.1%) and implantation rates (15.4% vs. 22.8%).However, the clinical pregnancy rate in the PICSI group was significantly higher than the control group(54.8% vs. 32.0% , p< 0.05). Conclusions: In this study, the patients who were randomized to the PICSI group demonstrated a statistically significant increase in clinical pregnancy rate over the control group. These results, although preliminary, suggest that the use of immobilized hyaluronan in the in vitro selection of sperm for ICSI may enhance our ability to isolate optimal sperm for injection. In addition, the resulting PICSI embryos may carry a higher level of embryonic potential. O13 多囊性卵巢患者現出較少的內皮前驅細胞 Polycystic ovary syndrome is associated with a decreased level of endothelial progenitor cell count 何信頤* 魏曉瑞 楊文濚 尤三立 郭怡利 台安醫院生殖醫學暨不孕症中心 Hsin-Yi Ho*, Hsiao-Jui Wei, Robert Young, San-Li Yu, I-Li Kuo Center for Reproductive Medicine and Infertility, Taiwan Adventist Hospital Introduction: Endothelial progenitor cells (EPCs) have been reported to be associated with the risk of cardiovascular disease, metabolic syndrome and insulin resistance. However, the relationship of endothelial dysfunction to polycystic ovary syndrome (PCOS) remains unclear. Hence, the purpose of this study was to determine the endothelial progenitor functions of PCOS patients by measuring circulatory EPCs. Methods: Circulatory EPCs were accessed by measuring the percentage of CD146+, CD34+CD133+ and CD34+CD184+ cells using fluorescence activated cell sorting (FACS) analysis in peripheral blood samples of a total of 47 PCOS patients. Results: Compared with normal population, our PCOS patients showed a significant lower level of CD34+CD133+ cells (0.85% vs. 1.34%, p< 0.009), CD34+CD184+ cells (0.019% vs. 0.400%, p< 0.002), showing a reduction in circulatory EPC pool. PCOS <0.006), patients also showed a higher level of CD146+ cell counts (1.000 vs. 0.749%, p which is a marker for mature endothelial cells. Conclusions: EPC levels are reduced in our PCOS patients, suggesting that repair and regeneration potential of endothelial functions are impaired. Insufficient circulating EPCs may be responsible for insulin resistance in the manifestation of PCOS. This finding also provides an additional potential basis for regarding PCOS as a risk factor to cardiovascular disease in women. O14 體外受精與自然懷孕婦女於懷孕前期之妊娠壓力影響因子 Influencing factors of pregnancy stress of IVF and non-IVF pregnant women during early pregnancy 郭碧照*1 黃梨香1 李茂盛2 中山醫學大學 護理學院1 醫研所2 Pi-Chao Kuo*1, Lii-Shung Huang1, Maw-Sheng Lee 2 1 College of Nursing, Chung Shan Medical University, 2 Institute of Medicine, Chung Shan Medical University Introduction: Pregnancy is the potential stress which may cause Pre-eclampsia, premature delivery and physical and mental illness. Many qualitative studies and case reports pointed out that women pregnant by in vitro fertilization (IVF) faced more complex psychological awareness changes. The aim of this study was to explore early pregnant stress and to identify risk factors between IVF women and non-IVF women. Materials: This study was a longitudinal survey from November 2007 to December 2009.Data were collected from 163 IVF women and 94 non-IVF women selected by purposive sampling at 9, 12 and 20 weeks’ gestation (WG) at a ART center in middle Taiwan. The self-report questionnaire included demographic data, the Pregnancy-related Stress Scale, the Symptoms Checklist, the Pregnancy-related Anxiety Scale, the Awareness of the Fetus Scale, the Social Support Apgar and the Chinese Childbearing Attitude Questionnaire. Data was analyzed by Mixed Model by using SPSS v16.0. Results: Results showed that main factors influencing pregnant stress were way of pregnancy, number of fetus, physical and psychological symptoms and social support. Pregnant stress at 20 WG were higher than 9WG and 12WG. In addition, IVF women had higher Pregnant stress than non-IVF women. Conclusions: This study provides useful evidence findings in early pregnant stress those risk factors between IVF women and non-IVF` women. Findings may provide a theoretical basis for nursing intervention with a view to strengthening early pregnant stress in pregnant women and promoting the future quality of care for them. O15 女性之身體質量指數是否影響試管嬰兒之臨床結果? Does the female BMI level influenced the clinical outcome of IVF-ET treatment? 鍾明廷 蔡永杰 康介乙 盧道權 唐訓翰 林亮吟 宋豔香 蔡婷芳 陳姵君 財團法人奇美醫院 婦產部生殖醫學中心 Ming-Ting Chung, Yung-Chieh Tsai, Chieh-Yi Kang, Tao-Chuan Loo, Hsun-Han Tang, Liang-Yin Lin, Yen-Hsiang Sung, Ting-Fang Tsai, Irene Chen. Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Chi-Mei Medical Center Objective: The BMI level of the female will influence the ovulation had been report. In here we try to compare the clinical outcome of IVF-ET between those groups with different BMI level. Material & Method: From Dec. 2004 to Dec 2009, patients who received IVF-ET cycles in our hospital were retrospectively classified into three groups according to the BMI level of femlae. The group one included 510 cycles those females with BMI < 23 22 cm/m. The group 2 included 119cycles those females with BMI between 23 cm/mand 2227 cm/m. The group three included 37 cycles those females with BMI > 27 cm/m. Only patients younger than 40 years old were included. All patients received oral pills and standard long protocol down regulation. Ovulation induction was individualized according to the hormone datas at day 2 of cycle after down regulation.10000 unit of hCG was administrated when at least 2 follicles ?16 mm. The progesterone was given immediately after oocytes retrieval in both groups. Clinical pregnancy, which was defined as visible gestation sac in the uterine cavity. And the ongoing pregnancy means the pregnancy beyond the twenty gestational weeks. Clinical outcome was compared between two groups. ,2 test was used for statistical analyses. P < 0.05 was considered statistically significant. Results: The mean age (y/o) of patient in group one was 33.3,3.4 compared to group two 34.0,3.3,and group three 34.6,2.9 p,=0.017 (Group1 vs. Group 3). The FSH level on the day 3 was 8.4,3.1 mIU/mL in group one compared to 7.6,2.7 mIU/Ml in group two and 6.9,1.8 mIU/mL in group 3, P. 0.012** (Group1 vs. Group 3) The dosage of gonadotropines (unit) used in group one was 2368,1111 compared to 2359,1172 in group 2 and 2475,934 in group3 with P>0.05. The average number of oocyte per retriveal was 7.2,4.4 in group one and 6.7,3.9 in group two and 7.1,4.1 in group 3, with P > 0.05.The number of embryos per transfer was 2.6,1.0 in group one compared to 2.7,0.9 in group two, and 2.9,0.9 in group 3 with P> 0.05. There was no statistically significant difference in clinical pregnancy rate between three groups 47.3%(241/510) in group one and 41.2%(49/119) in group two and 51.4%(19/37) in group 3 with P> 0.05 .The implantation rate in group one was 27.2%(366/1348) compared to 20.6%(66/320) in Group 2 and 24.3%(26/107) in group 3 with P>0.05. The ongoing pregnancy rate in group one was 40.2%(205/510) compared to 31.9%(38/119) in Group 2 and 29.7%(11/37) in group 3 with P>0.05. Conclusion: There was no statistically significant difference in clinical outcome of IVF-ET treatment in these data. However, in the group 3 the on going pregnancy rate was lower than the other groups although there were no statistically significant. O16 子宮內膜異位瘤及腹腔鏡腫瘤切除手術對卵巢之影響 The Impact of Endomtrioma and Laparoscopic Cystectomy on Ovarian Reserve 111112胡玉銘吳劭穎 李國光 林明輝 林時羽 孫芳如 12台北馬偕紀念醫院 婦產部不孕症學科 醫研部 1 111Yuh-Ming Hwu,Frank Shao-Ying Wu, Robert Kuo-Kuang Lee, Shyr-Yeu Lin, 2Fang-Ju Sun 1 2Department of Obstetrics and Gynecology,Department of Medical Research, Mackay Memorial Hospital Introduction: Currently, laparoscopic ovarian cystectomy is recommended as the treatment of choice for ovarian endometrioma. However, lots of studies reported that ovarian reserve is damaged after excision of ovarian endometriomas. Regarding the degree of ovarian reserve damage due to the endometrioma or ovarian cystectomy, AMH may also be a very informative marker. The aim of this study was to evaluate the impact of endometrioma and laparoscopic cystectomy on ovarian reserves by serial AMH level measurements. In addition, the differences in ovarian reserve impairment between unilateral and bilateral endometrioma or cystectomy were also investigated. Methods: A total of 1596 subjects were enrolled in this study and were divided into four groups according to the status of endometrioma and cystectomy. Group 1 (control group) (n = 1323): No evidence of endometrioma; Group 2 (endometrioma group) (n= 121): endometrioma diagnosed by clinical and image finding and without surgical intervention; Group 3 (endometrioma with previous cystectomy group) (n =122): endometrioma with previous surgical intervention; Group 4 (current cystectomy group) (n = 30): endometrioma diagnosed pre-operatively and confirmed by pathology obtained from laparoscopic cystectomy (n = 30). For group 1, 2, and 3, serum AMH levels are measured at the initial visit. For group 4, AMH are measured both pre-operatively and 3 months post-operatively. Results: Serum AMH level was significantly negatively correlated with increasing age in group 1, 2, and 3. Bonferroni t-test showed that mean serum AMH level of control group (mean ?SEM: 2.99?0.06) was statistically significantly higher than endometrioma group (2.99?0.06 vs. 2.48?0.16, P<0.05) or previous cystectomy group (2.99?0.06 vs.1.62?0.14, P< 0.001). Mean serum AMH level of endometrioma group was also statistically significantly higher than previous cystectomy group (2.48?0.16 vs.1.62?0.14, P< 0.01). Mean serum AMH level was statistically significantly decreased in patients with bilateral endometrioma compared with unilateral endometrioma (mean?SEM: 2.65?0.18 vs.1.80?0.31, P< 0.05). In group 4, the mean serum levels significantly decreased from 3.87?0.43 pre-operation to 1.91?0.21 -month post-operation (P <0.001). 3 Conclusions: Laparoscopic cystectomy can inflict significant short and long term damage to ovarian reserve. Presence of endometrioma in short term may not have a continuing damage to ovarian reserve other than the initial insult, but can still have significant negative impacts in long term. Bilateral endometriomas have a more profound impacts on ovarian reserve than unilateral endometrioma, regardless of surgical or conservative management. O17 在採用GnRH拮抗劑療程的新鮮胚胎植入和下次冷凍胚胎植入週期,注射 HCG當天過高的E2濃度是否會影響IVF的成功率, Dose high serum E2 concentration impair the success of IVF treatment with GnRH antagonist protocol in fresh ET cycles and in subsequent frozen-thawed ET cycles? 林明輝 胡玉銘 吳劭穎 黃夢婷 林時羽 李國光 馬偕紀念醫院 婦產部不孕症學科 Ming-Huei Lin, Yuh-Ming Hwu, Shao-Ying Wu, Meng-Ting Huang, Shyr-Yeu Lin, Robert Kuo-Kuang Lee Department of Obstetrics and Gynecology, Mackay Memorial Hospital Introduction: Several studies have shown high serum E2 concentrations have an impact on pregnancy achievement in IVF cycles treated with GnRH agonist protocol, whereas others have found no adverse effects. Assessment of the association between E2 levels and success of IVF treatment with GnRH antagonist protocol remains scanty. The purpose of this study was to examine the effects of high serum E2 concentrations on the day of HCG administration on pregnancy rates in fresh IVF cycles with GnRH antagonist protocol and in subsequent frozen-thawed embryo transfer cycles in those who did not become pregnant in the fresh cycles. Materials and Methods: There were 379 patients treated by IVF/ICSI with rec-FSH/GnRH antagonist protocol at our center between 2007-2009, included in this study. Exclusion criteria were the presence of the following conditions: 1) age > 40 years; 2) infertility attributed to endocrine abnormalities, such as hyperprolactinemia, thyroid dysfunction, and Cushing syndrome; 3) previous COH with documented poor response resulting in a mature oocyte yield < 3; 4) occult ovarian failure with day 3 basal FSH concentration > 10 IU/L, or AMH < 1.0 . All women were determined to have normal uterine cavities by HSG or hysteroscopy. According to serum E2 level on the day of HCG administration, patients were categorized into two groups: group A 1000-3500 pg/mL, and group B >3500 pg/mL. Results: In fresh cycles, group A had significantly lower good embryo rates, when compared with group B (55.4% versus 64.8% respectively, P=0.0126). The clinical pregnancy rates in group A and group B were similar (42.4% versus 42.9% respectively, P=0.2496). In frozen-thawed embryo transfer cycles, no significant difference in clinical pregnancy rates between group A and group B were found (46.5% versus 60.0% respectively, P=0.5487). Conclusions: Although higher good embryo rate has been observed in the presence of high E2 levels, no subsequent significant difference in pregnancy rate was noticed. In addition, similar pregnancy rates were present in the frozen-thawed ET cycles that no ovulation stimulation was performed. This observation suggests that extremely high E2 concentrations could have no potential deleterious effect on endometrial receptivity in IVF treatment with GnRH antagonist protocol. O18 鐵質沉積與重度地中海型貧血婦女之低抗穏勒式荷爾蒙值 Iron overload is associated with low anti-Müllerian hormone in women with transfusion-dependent beta-thalassemia *12 2222221陳美州 張修豪盧孟佑 呂俊毅 楊永立 周憲堂 林東燦 林凱信 楊友仕 12台大醫院 婦產部 小兒部 1 2222Mei-Jou Chen*,Hsiu-Hao Chang, Meng-Yao Lu, Chun-Yi Lu, Yung-Li Yang, 2221 Shiann-Tarng Jou, Dong-Tsamn Lin, Kai-Hsin Lin, Yu-Shih Yang 1 2 Departments of Obstetrics and Gynecology and Pediatrics, National Taiwan University Hospital, Taipei Introduction: The ovarian function of women with transfusion-dependent β-thalassemia is controversial. In this study, we investigated the anti-Müllerian hormone (AMH), as a surrogate marker of ovarian reserve in women with transfusion-dependent β-thalassemia, and the relationship between AMH and iron overload. Methods: Twenty-nine women with transfusion-dependent β-thalassemia and 29 healthy controls of a similar age were recruited. The history of iron overload-related morbidities, hematologic phenotypes, and serum levels of AMH, ferritin, hormonal profiles were recorded, measured, and analyzed. Results: The serum levels of AMH, LH, and E2 were significantly lower in women with transfusion-dependent β-thalassemia than normal controls. In women with transfusion-dependent β-thalassemia, the serum AMH level was significantly related to the ferritin level, but not to the presence of hypogonadotrophic hypogonadism, hepatitis C, diabetes, and hematologic phenotypes. In addition, with the exception of AMH, the serum ferritin level was also significantly and positively associated with advanced age, the presence of hypogonadotropic hypogonadism and hepatitis C infection in the study subjects. The inverse relationship between AMH and ferritin still exist after further adjustment for advanced age. Conclusions: Though the present study comprises small study population, the results still indicate that the serum AMH levels in women with transfusion-dependent β-thalassemia are significantly lower when compared to normal healthy women of a similar age, and are also negatively correlated with serum ferritin levels. This implies that ovarian function might be impaired by the chronic iron overload status in women with transfusion-dependent β-thalassemia. O19 胚胎著床與Notch 1基因表現量之相關性-小鼠胚胎體外模式 The relationship between Notch 1 gene expression and embryo implantation - A model of ex vivo murine embryo 朱伯威* 王宜珮 陳逸菁 武國璋 三軍總醫院婦產部 Po-Wei Chu, Yi-Pay Wang, I-Ching Chen, Gwo-Jang Wu Department of Obstetrics and Gynecology, Tri-service General Hospital Introduction: To improve the implantation rate is an important issue in the assisted reproductive technology. In our study, the evolution of the murine embryo was taken as a model for understanding the correlation between embryo implantation and the Notch 1 gene of Notch signaling pathway. Methods: To detect Notch1 gene expression during embryo implantation, we used real-time quantitative PCR analysis, which revealed Notch 1 mRNA relative expression levels in pre-and post-implantation stage of embryos, both were compared to the oocyte stage. For advanced understanding of the role Notch 1 plays during implantation, we used an ex vivo model system with Matrigel. The invasion and spreading assays of murine embryos were also performed for understanding and evaluation of embryo implantation. To see if the effect of attenuated Notch signaling could be rescue by the support of endometrial cells efficiently, we placed endometrial cells on the base of the Matrigel system. Collected murine embryos were placed in the inner cup of Matrigel. Results: 1. The result of the qPCR analysis showed higher expression level of Notch 1 mRNA at the stage post-implantation than pre-implantation stage (3.26 fold and 1.95 fold). 2. When supplemented with 10 nM DAPT, a specific Notch 1 inhibitor, lower implantation rate could be observed (66.7% vs. 87.2%, p<0.05). Furthermore, treat with 30 nM DAPT also reduced the ability of implantation (53.3% vs. 87.2%, p<0.05). We found a dose-dependent effect and eliminate the possibility of completely block of blastocyst survival when add the Notch 1 inhibitor. 3. When the group applied DAPT was cultured with endometrial cells, the result of spreading and invasion area seemed wider than without endometrial cells Conclusions: The inhibition of Notch 1 reduced the implantation rate of murine embryo on matrigel. Supplement of DAPT (a Notch 1 inhibitor) and cultured with endometrial cells further confirmed attenuated Notch signaling could be rescue by the support of endometrial cells efficiently. Further and ongoing studies are focused on explore the detailed mechanism underlying the effect of Notch 1 on embryo during the implantation window, which means the candidate genes regulated by upstream Notch 1 expression, would be identified by microarray analysis. O20 Follistatin isoforms 在調控小鼠胚胎幹細胞「自我更新」時的角色 Follistatin isoforms regulate mouse embryonic stem cell self-renewal ,1 21111122林時羽* 邱志勇 魏承紘 邱于恬 彭佳琪 汪世崇 胡玉銘 李國光 林2明輝 12馬偕紀念醫院 醫研部幹細胞實驗室 婦產部不孕症學科 1, 2111Shyr-Yeu Lin*, Chih-Yung Chiou, Cheng-Hung Wei, Yu-Tien Chiu, Jia-Chi 11222Peng, Shih-Chung Wang, Yuh-Ming Hwu, Kuo-Kuang Lee, Ming-Huei Lin 1. Stem Cell Lab, Department of Medical Research, 2. Department of Obstetrics and Gynecology, Mackay Memorial Hospital. Introduction: Follistatin (fst) is a protein that is able to bind and antagonize members of TGF-β superfamily, including activins and bone morphogenetic proteins (BMP). As activins and BMPs play an important role in embryonic stem (ES) cell self-renewal and pluripotency, it is likely that follistatin is also involved in these important characteristics of ES cells. Follistatin isoforms arise from alternative pre-mRNA splicing and proteolytic cleavage and exist as three isoforms (i.e., fst288, fst303 and fst315) differing in length at the C terminus. In this work, we try to delineate the roles of follistatin isoforms in mouse ES cell self-renewal and pluripotency. Methods: We constructed four fst isoform-specific transgenes (fst288, fst303, fst315 and fst-myc), which are drived by the EF1 promoter and can consistently overexpress follistatin isoform 288, 303 and 315, respectively. The fst-myc was fst315 with a myc-tag linked at its C terminus, which design may be able to prevent fst315 from proteolysis into fst303 in vivo. Mouse ES cells were transfected with these four transgenes individually by electroporation, and then stable clones were screened. To test the influence of follistatin isoforms on self-renewal, mouse ES cell clones were cultivated in various conditions with different growth factors. During cultivation, the percentage of Oct4 positive ES cells was determined by flow cytometry. In the meanwhile, expression levels of self-renewal marker genes (Oct4, Nanog, Rex1, Sox2 and alkaline phosphatase) were evaluated by qRT-PCR. Passage survival of different ES cell clones under various conditions was also observed. Assessments of cell proliferation and apoptosis were performed by flow cytometry for cell counting, BrdU incorporation, Annexin V-7 ADD staining and TUNEL. To determine the effect of follistatin isoforms on pluripotency, embryoid body (EB) differentiation (day 0, 3, 9, 15 21 and 27) was made. The samples were then used for analyses of early and late endodermal, mesodermal and ectodermal markers by qRT-PCR and immunostaining. Studies into the potential interaction of follistatin isoforms with specific signalings (LIF, BMP, Act, PI3K/Akt and Wnt) were conducted by Western blots. Results: In the presence of LIF but without feeders, fst288, fst303, fst315 and fst315myc can be propagated for 4 passages, compared with mES that can be propagated for at least 6 passages. Follistatin isoforms assume different effects on proliferation. Follistatin isoforms appear to reduce apoptosis of mouse ES cells in absence of LIF and serum. Follistatin isoforms did not block the endodermal, mesodermal, or ectodermal commitments, however, with different extents of diverse gene expressions during EB differentiation. Conclusions: Follistatin isoforms regulate mouse ES cell self-renewal and pluripotency with distinct responses to different culture conditions. O21 第十八型介白質系統於人類子宮肌腺症子宮內膜肌肉層介面之表現模式:子 宮肌腺症之致病機轉研究 Interleukin-18 (IL-18), IL-18 receptor (IL-18R) and IL-18 binding protein (IL-18BP) expression at the level of endometrial-myometrial interface (EMI) in human uterine adenomyosis 1, 221111, 21, 21, 2黃泓淵* 黃鴻儒 李金蓉 尤星策 吳憲銘 李奇龍 王馨世 宋永魁 12 林口長庚醫院 婦產部 長庚大學醫學院 婦產科 1, 2211Hong-Yuan Huang*, Hung-Ru Huang, Chin-Jung Li, Hsing-Tse Yu, Hsien-Ming 11, 21, 21, 2Wu, Chyi-Long Lee, Hsin-Shih Wang, Yung-Kuei Soong 1Department of Obstetrics and Gynecology, Lin-Kou Medical Center, Chang Gung 2Memorial Hospital; Department of Obstetrics and Gynecology, Chang Gung University and School of Medicine, Tao-Yuan Introduction: Adenomyosis is defined by the presence of endometrial mucosa within the myometrium as well as by a diffuse enlargement of the uterus, thereby disrupting the EMI. IL-18 system is one of the major cytokine involved in human endometrium of different menstrual cycle and might perform a defensive role against maternal immune response. In this study, we investigate the human IL-18 system expression at the level of EMI in patients with uterine adenomyosis. The purpose of this study is to investigate IL-18 and its corresponding receptor and antagonist expression in human uterine adenomyosis. Methods: A total of 10 paired samples of human uterine eutopic, ectopic endometrium and corresponding normal myometrium tissues were obtained individually from surgical specimens of women undergoing hysterectomy for uterine adenomyosis after informed consent and IRB approval. Real-time quantitative PCR was used to quantitative IL-18 system mRNA expression in paired human uterine samples. To determine the presence of IL-18 system proteins, tissues were fixed and processed for immunohistochemical study. IL-18 system florescence was quantified by confocal microscope. Data analysis was done with ANOVA and Pearson's correlation. Results: IL-18, IL-18R as well as IL-18BP mRNA were expressed in human eutopic, ectopic endometrium and normal myometrium. According to Real-time quantitative PCR with C value quantification, IL-18 was not significantly different in eutopic, T ectopic endometrium and normal myometrium, but IL-18R expression was significantly lower in ectopic endometrium and normal myometrium in comparison to eutopic endometrium (p<0.05). IL-18BP was also significantly lower in normal myometrium. Immunoreactive protein levels and quantified florescence of IL-18 system was also present in paired tissue samples. Conclusions: We have shown that a complete IL-18 system including the agonist, receptor and antagonist expressed in the eutopic, ectopic endometrium and corresponding myometrium at the level of EMI in uterine adenomyosis. According to our results, IL-18 may implicate as a local immune regulator and modulating cytokine networks at the level of EMI of uterine adenomyosis and also provide indirect molecular evidence of transplantation theory of adenomyosis. O22 高解析度溶解曲線法快速鑑定分析多囊性卵巢症候群與CAPN10基因 UCSNP- 19及UCSNP-63多型性變異關聯性 The association of CAPN10 alleles UCSNP-19 and UCSNP-63 polymorphisms and polycystic ovary syndrome rapidly identified by high-resolution melting curve analysis 123341 林大欽顏瑞美倪靜華王郁菁李文琮郭宗正 1234臺南郭綜合醫院 婦產部 小兒部 醫研部 檢驗部 12334Ta-Chin Lin, Jui-mei Yen, Jing-Hua Ni, Yuh-Jing Wang, Wen-Tsung Lee, 1Tsung-Cheng Kuo 1Departments of Obstetrics and Gynecology, 2Department of Pediatrics, 3Department of Research, 4Department of Clinical Laboratory, Kuo General Hospital Introduction: PCOS, in which insulin resistance is considered as a key feature, has been independently associated with CAPN10 (calpain-10 gene) allelic variants. This study investigates whether UCSNP-19, UCSNP-63 polymorphism in the CAPN10 gene is related with PCOS etiology. We purposely study the allelic frequencies and genotypes of CAPN10 UCSNP-19, UCSNP-63 polymorphism in PCOS-affected women. We use high-resolution melting (HRM) analysis for mutation detection and real-time PCR to investigate whether UCSNP- 19, UCSNP-63 polymorphism of CAPN10 allele is associated with PCOS-affected women in the Taiwan population Materials and Methods: Forty-three women with Rotterdam definition of PCOS and 50 healthy women were included in this study. Informed consents were obtained from all patients. Genomic DNA was taken from all patients and isolated from whole blood. The primers were designed for the detection of UCSNP-19, UCSNP-63 polymorphism. The CAPN10 UCSNP-19, UCSNP-63 genotyping were analyzed with real time PCR and HRM. The real time PCR condition is a final volume of 10ul using 10 ng of genomic DNA,1 mM each amplification primer, 4 ul of LCGreen Light Scanner Master Mix. The HRM condition is start temperature 75?, final temperature 95?, and rapid cooling to 60? with a slope of 0.3?/sec.The HRM analysis of polymorphism were confirmed with sequence analysis. Results: The HRM analysis had classified the UCSNP-19 and UCSNP-63 into L, N, and M types. These were corresponded to ins/ins, del/ins, del/del in UCSNP-19 polymorphism and CC, CT, TT in UCSNP-63 polymorphism. The association studies in PCOS revealed: The proportions of ins/ins, del/ins, del/del in UCSNP-19 were 28%, 56%, 16% in PCOS group and 48%, 40%, 12% in control group. While, CC, CT, TT in UCSNP-63 were 49%, 40%, 11% in PCOS women and 41%, 41%, 18% in control group respectively. In the analysis of polymorphic allele SNP-19, we obtained a positive correlation: 48 of 86 (55.8%) PCOS alleles have the deleted allele of this polymorphic marker, compared with 32 of 100 (32%) control chromosomes (P=0.048). Conclusions: HRM is a sensitive, robust technique to detect polymorphism of CAPN10 in PCOS women. In this study, there is clear evidence of UCSNP-19 (del) polymorphism of genome CAPN10 has a relationship with PCOS susceptibility and its phenotype.