自首例試管嬰兒寶寶Louise Joy Brown於1978年在英國誕生，體外受精-胚胎移植 (In Vitro Fertilization-Embryo Transfer, IVF-ET；俗稱試管嬰兒) 發展至今已有三十五年之久，全球利用試管嬰兒技術誕生的嬰兒數量迄今也已超過五百萬名。在IVF-ET技術中，使用的培養液也由早期的實驗室自行泡製，發展至目前所研發出兩種類型的商業化培養液：單步驟培養液（Single step medium；Global Medium）和連續性培養液（Sequential medium；Sage Medium），也因為有此新的研發產品出現，提升了胚胎培養的品質和懷孕率。本實驗收集210個進行試管嬰兒療程的個案，將取得之卵子以單一精蟲顯微注射方式授精後，隨機分配到兩組商業化培養液中，皆由第一天培養至第三天並觀察與記錄每天胚胎的品質，做為二種培養液對於胚胎發展的比較。本研究結果顯示，在胚胎培養的第一天，Global Medium和Sage Medium受精率各為75.5%和77.5%。第二、三天Global Medium在胚胎型態分級中的Grade 1和胚胎的細胞分裂數目皆比Sage Medium多，但兩組的懷孕率並無差異；另外年齡在≧38歲的個案中，在Global Medium的胚胎的細胞分裂數目也比Sage Medium多，但在胚胎型態分級中的Grade 1之比較，兩者則是無差異的，懷孕率亦無差異。由於商業化培養液的發展，可將培養天數拉長，但有些個案因受其他因素影響，無法培養至第五天囊胚期。若於胚胎培養的第三天，胚胎細胞數＞6 cells、胚胎的細胞碎片百分比也是最低，如此就可以藉由胚胎形態學的挑選，降低胚胎培養的時間及胚胎植入的數目，而得到與第五天胚胎植入相同的懷孕率。經由本實驗得知，單步驟培養液第三天培養出來的細胞數>6 cells，Grade 1的胚胎較多，細胞碎片亦較少，等級好的胚胎會比連續步驟培養液還來得多。因此，就第三天的胚胎形態學觀點，若因有其他因素，致無法使胚胎培養到第五天囊胚期的情形時，利用單步驟培養液培養到第三天的做法，是一個值得施行的選擇。

Since Louise Joy Brown, the first baby of In Vitro Fertilization-Embryo Transfer (IVF-ET), was born in the United Kingdom in 1978, there had been over five million successful cases over the course of 35 years. There are two commercially available culture media in the market: Single Step Medium (Global Medium) and Sequential Medium (Sage Medium). The development of these two media had enhanced the quality of embryo and increased the pregnancy rate in IVF-ET. In this collection of 210 cases of IVF-ET, oocytes were fertilized by intracytoplasmic sperm injection（ICSI）and randomly assigned to the two commercially-available culture media. The quantity and quality of embryos were observed and recorded for three consecutive days in order to compare the development of embryos in the two commercial culture medium. A fertilized rate of 75.5% in Global Medium and 77.5% in Sage Medium were observed on the first day. There was a higher number of cleaved cells in Global Medium and embryos of Grade 1 cells than those in Sage Medium both on the 2nd and the 3rd day, though no significant differences were noted in the pregnancy rates between the two samples. In the group of those over 38 years old, there was a higher number of cells in Global Medium, but exhibited no difference in either embryonic pattern or in the pregnancy rate. Though the development of commercial culture medium had enabled a longer cultivation, some cases still failed be cultured to the blastocytic stage due to other limitations. It had been attempted with morphologic selection in embryos by the 3rd day of culture, with a criteria of over six cells per embryo and minimal cell fragmentation, to reach compatible pregnancy rate of embryo implantation of the 5th day by conventional method. Our study indicated that the there was higher number of Grade 1 embryos with more than 6 cells and lower embryo fragmentation in Single Step Medium, thus yielded a better overall quality of embryo than Sequential Medium. It can be speculated, therefore, that the Single Step Medium would offer a valuable choice in patients with difficulty in reaching blastocytic stage by conventional method due to various factors.

論文審定書 i
中文摘要 ii
英文摘要 iii
第一章 前言 1
第一節 試管嬰兒的起源 1
第二節 何謂試管嬰兒 1
第三節 試管嬰兒誘導療程 1
第四節 人工協助生殖技 2
第五節 胚胎培養液 5
第二章 研究動機 9
第三章 研究方法 10
第四章 實驗結果 17
第五章 討論 20
第六章 結論 25
參考文獻 26
圖次 36
表次 47

一、中文部分
衛福部台灣地區人工生殖施行結果分析報告，2011，頁37。
二、英文部分
Alikani M, Cohen J, Tomkin G. Human embryo fragmentation in vitro and
its implications for pregnancy and implantation. Fertil Steril 1999; 71:
836–842.
Anderson AR, Wiemer KE, Weikert ML, Kyslinger ML. Fertilization, embryonic development and pregnancy losses with intracytoplasmic sperm injection for surgically-retrieved spermatozoa. Biol Reprod Online 2002; 5: 142-7.
Angus S, Grunert GM, Dunn RC, Valdes CT, Schenk LM, Mangal RK. No advantage of using the sequential GIII media versus the single media Global. Fertil Steril 2006; 86 Suppl 2: S229.
Balakier H, MacLusky N, Casper R. Characterization of the first cell cycle in human
zygotes: implications for cryopreservation. Fertil Steril 1993; 59: 359–65.
Behr B, Pool TB, Milki AA, Moore D, Gebhardt J, Dasig D. Preliminary clinical
experience with human blastocyst development in vitro without co-culture. Hum
Reprod 1999; 14: 454-457.
Biggers JD, McGinnis LK, Raffin M. Amino acids and preimplantation development of
the mouse in protein-free potassium simplex optimized medium. Biol Reprod
Online 2000; 63: 281-293.
Biggers JD, McGinnis LK, Lawitts JA. Enhanced effect of glycyl-glutamine on mouse
preimplantation embryos in vitro. Biol Reprod Online 2004; 9: 59-69.
Biggers JD, Racowsky C. The development of fertilized human ova to the blastocysy
stage in KSOM(AA) medium: is a two-step protocol necessary? Biol Reprod
Online 2002; 5: 133-140.
Bodri D, Vernaeve V, Guillen JJ. Comparison between a GnRH antagonist and a GnRH
agonist flare-up protocol in oocyte donors: a randomized clinical trial. Hum. Reprod
2006; 21: 2246-2251.
Brison DR, Houghton FD, Falconer D, Roberts SA, Hawkhead J, Humpherson PG, et
al.Identification of viable embryos in IVF by noninvasive measurement of amino
acid turnover. Hum Reprod 2004; 19: 2319-2324.
Bruce S., Kevin S., Dee C., Said T. Influence of patient age on the growth and transfer
of blastocyst-stage embryos. Fertil Steril 2002; 77: 700-705.
Bungum M, Humaidan P, Bungum L. Recombinant human albumin as protein source in
culture media used for IVF: a prospective randomized study. Reprod Biomed Online 2002; 4: 233-236.
Capmany G, Taylor A, Braude P, Bolton V. The timing of pronuclear formation, DNA
synthesis and cleavage in the human 1-cell embryo. Mol Hum Reprod 1996; 2: 299
-306.
Carillo A, Lane B, Pridham D, Risch P, Pool T, Silverman I. Improved clinical
outcomes for in vitro fertilization with delay of embryo transfer from 48 to 72
hours after oocyte retrieval: use of glucose- and phosphate-free media. Fertil Steril
1998; 69: 329-334.
Carrillo AJ, Yalcinkaya TM. Improved embryo development using a single medium(SIN) vs. a sequential media (SEQ) system from the day of egg retrieval to day 6: comparison of sibling oocytes. Fertil Steril 2008; 90: Suppl O-83: S30.
Carrillo AJ, Yalcinkaya TM. Improved embryo development and comparable pregnancy rates using single vs. sequential culture media from the day of oocyte retrieval to the blastocysts stage of development. J. Clin. Embryol 2010; 13(4): 35-42.
Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J. of Reprod and Fertil 1989; 86: 679-688.
Chen C. Pregnancy after human Oocyte cryopreservation, thawing and transfer of an eight-cell embryo. Lancet 1986; 327: 884-886.
Christine C.S., Jackson KV, Racowsky C. Early compaction on day 3 may be associated with increased implantation potential. Fertil Steril 2006; 86: 1386-1391.
Cooke S, Quinn P, Kime L, Ayres C, Tyler JP, Driscoll GL. Improvement in early
human embryo development using new formulation sequential stage-specific culture media. Fertil Steril 2002; 78: 1254-1260.
Da Motta EL, Alegretti JR, Baracat EC, Olive D, Serafini PC. High implantation and
pregnancy rates with transfer of human blastocysts developed in preimplantation
stage one and blastocyst media. Fertil Steril 1998; 70: 659-663.
Di Falco Cossiello R, Aggelis A, Faundes D, Petta CA. Morphological differences in human zygotes and embryos cultured in different media. Zygote 2011; 1-7.
Edwards RG, Donahue RP, Baram TA, Jones HW. Preliminary attempts to fertilize human oocytes matured in vitro. Am J Obstet Gynecol 1966; 96: 192-200.
Erbach GT, Lawitts JA, Papaioannou VE, Biggers JD. Differential growth of the mouse preimplantation embryo in chemically defined media. Biol Reprod Online 1994; 50: 1027-1033.
Fleming R, Adam AH, Barlow DH, et al. A new systematic treatment for infertile women with abnormal hormone profiles. Br J Obstet Gynaecol 1982; 89: 80-83.
Friedler S, Schachter M, Strassburger D, Esther K, Ron El R, Raziel A. A randomized clinical trial comparing recombinant hyaluronan/recombinant albumin versus human tubal fluid for cleavage stage embryo transfer in patients with multiple IVF-embryo transfer failure. Hum Reprod 2007; 22: 2444-2448.
Gardner, D.K. Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology 1998a; 49: 83-102.
Gardner DK, Lane M. Development of viable mammalian embryos in vitro: evolution
of sequential media. Principles of cloning. Amsterdam: Academic Press, 2002:
187-213.
Gardner, D.K. Embryo development and culture techniques. In Clark, J. (ed.), Animal Breeding: Technology for the 21st Century. Harwood Academic, Berkshire, UK 1998b; pp. 13-46.
Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A
prospective randomized trial of blastocyst culture and transfer in in-vitro
fertilization. Hum Reprod 1998c; 13: 3434-3440.
Gardner DK, Phil D, Vella P, Lane M, Wagley L, Schlenker T, et al. Culture and
transfer of human blastocysts increases implantation rates and reduces the need for
multiple embryo transfers. Fertil Steril 1998d; 69: 84-88.
Gardner, D.K. and Lane, M. Embryo culture. In Gardner, D.K., Weissmann, A.,
Howles, C.M. and Shoham, Z. (eds), extbook of Assisted Reproductive
Technologies. Laboratory and Clinical Perspectives 2001. Martin Dunitz, London,
pp. 203-222.
Gemzell CA, Diczfalusye E, Tillinger G, Human pituitary follicle stimulating hormone
(FSH). Ciba Found Colloq Endocrinol 1960; 13: 191.
Gordts S, Roziers P, Campo R, Notov. Survival and pregnancy outcome after ultrarapid
freezing of human embryos. Fertil Steril 1990; 53: 469-472.
Greenglatt E, Di Berardino T, Chronis-Brown P, Holt D, Lains A. Comparison of
Global medium and G1/G2 cleavage/blastocyst sequential medium for culture of
human embryos after IVF. Hum Reprod 2005; 20, Suppl 1: i21.
Herrero L, Bou C, Aragones M, Mifsud Y, Minguez Y. Comparacion de dos medios de
cultivo embrionario hasta ei estadio de blastoclato: IVF-CCM y GLOBAL. In
Asociacion para el Estudio de la Biologia de la Reproduccion 2007; Bilbao, Spain
pp. Poster P-043.
Hill G, Freeman M, Bastias M. The influence of oocyte maturity and embryo quality on
pregnancy rate in a program for in vitro fertilization embryo transfer. Fertil Steril
1989; 52: 801-806.
Ho Y, Wigglesworth K, Eppig JJ, Schultz RM. Preimplantation development of mouse
embryos in KSOM: augmentation by amino acids and analysis of gene expression.
Mol Reprod Dev 1995; 41: 232-238.
Houghton FD, Hawkhead JA, Humpherson PG, Hogg JE, Balen AH, Rutherford AJ, et
al. Non-invasive amino acid turnover predicts human embryo developmental
capacity. Hum Reprod 2002; 17: 999-1005.
Jones G, Trounson A, Lolatgis N, Wood C. Factors affecting the success of human
blastocyst development and pregnancy following in vitro fertilization and embryo
transfer. Fertil Steril 1998; 70: 1022-1029.
Jones HW, Jones GS, Andrews MC, et al. The program for in vitro fertilization at
Norfolk. Fertil Steril 1982; 38: 14-21.
Khoury C, Coffler M, Potter D, Frederick J, Batagglia D. Improved blastocyst development using a single step medium versus a sequential medium. Fertil Steril 2012; 97: S4-S5.
Kiltz R. J., Woodhouse D. J., Polanski V., Miller D. B. Comparison of clinical
pregnancy rates and incidence of high order multiple gestations in day 3 vs. day 5
embryo transfers (ET). Fertil Steril 2000; 74, Suppl 1: S97.
Kuleshova L, Gianaroli L, Magli C. Birth following vitrification of a small number of
human oocytes: case report. Hum Repord 1999; 14: 3077-3079.
Lambalk CB, Leader A, Olivennes F. Treatment with the GnRH antagonist ganirelix
prevents premature LH rises and luteinization in stimulated intrauterine
insemination: results of a double-blind, placebo-controlled, multicentre trial. Hum
Reprod 2006; 21: 632-639.
Lawitts JS, Biggers JD. Optimization of mouse embryo culture media using simplex methods. J. Reprod 1991; 91: 543-556.
Lawitts JS, Biggers JD. Joint effects of sodium chloride, and glutamine, and glucose in mouse preimplantation embryo culture media. Mol Reprod Devt 1992; 31: 189-194.
Lee TH, Lin YH, Seow KM. Effectiveness of cetrorelix for the prevention of premature luteinizing hormone surge during controlled ovarian stimulation using letrozole and gonadotropins: a randomized trial. Fertil Steril 2008; 90: 113-120.
Liebermann J., Pelts E. J., Matthews J. M., Sanchez S. R., Uhler M., Beltsos A.
Single-step vs. tow-step media: Are there any differences in embryonic
development? Fertil Steril 2012; 98: P-193, S168.
Matsubara T, Nakamura S, Hashimoto C, Mukaida T, Takahashi K. Examination of blastocyst culture systems (sequential media system vs. single-step media system). In Proceedings of the 24th Annual Meeting of the Japan Society for Fertilization and Implantation, Karuizawa, Japan 2006; p 206.
Michael L., Amanda Hamic, Douglas J., Charles L. Continuous uninterrupted single
medium culture without medium renewal versus sequential mediaculture: a sibling
embryo study. Fertil Steril 2009; 92: 1783-1786.
Milki A, Fisch J, Behr B. Two-blastocyst transfer has similar pregnancy rates and a
decreased multiple gestation rate compared with threeblastocyst transfer. Fertil
Steril 1999; 72: 225-228.
Milki, A.A., Hinkley, M.D., Fisch, J.D., Dasig, D. and Behr, B. Comparison of
blastocyst transfer with day 3 embryo transfer in similar patient populations.
Fertil Steril 2000; 73: 126-129.
Nikas G, Ao A, Winston R. Compaction and surface polarity in the human embryo
in-vitro. Biol Reprod Online 1996; 55: 32-37.
Palermo, G., Joris, H., Devroey, P. et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340: 17-18.
Pomeroy KO, Foley S, Faber B, Johnson MD, Moffitt DV. A comparison of sequential medium to non-sequential medium: do some patient`s embryos culture better in one over the other? Reprod Fert Dev 2009; 21: 162-163.
Porter RN, Smith W, Craft IL, et al. Induction of ovulation for in vitro fertilization using buserelin and gonadotropins. Lancet 1984; 324: 1284-1285.
Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985; 44: 493-498.
Quinn P, Warnes GM, Kerin JF, Kirby C. Culture factors in relation to the success of human in vitro fertilization and embryo transfer. Fertil Steril 1984; 41: 202-209.
Racowsky C, Combelles C, Nureddin A, Pan Y, Finn A, Miles L, et al. Day 3 and day 5
morphological predictors of embryo viability. Bio Reprod Online
2003; 6: 323-331.
Racowsky C., Jackson K. V., Cekleniak N. A., Fox J. H., Hornstein M. D., Ginsburg E.
S. The number of eight-cell embryos is a key determinant for selecting day 3 or
day 5 transfer. Fertil Steril 2000; 73: 558-564.
Reed ML, Hamic A, Thompson DJ, Caperton CL. Continuous uninterrupted single
medium culture without medium renewal versus sequential media culture: a sibling
embryo study. Fertil Steril 2009; 92: 1783-1786.
Scholtes MCW, Zeilmaker GH. Blastocyst transfer in day-5 embryo transfer depends
primarily on the number of oocytes retrieved and not on age. Fertil Steril
1998; 69: 78-83.
Sepulveda S, Garcia J, Arriaga E, Diaz J, Noriega-Portella L, Noriega-Hoces L. In vitro development and pregnancy outcomes for human embryos cultured in either a single medium or in a sequential media system. Fertil Steril 2009; 91: 1765-1770.
Skiadas CC, Jackson KV, Racowsky C. Early compaction on day 3 may be associated
with increased implantation potential. Fertil Steril 2006; 86: 1386-1391.
Steptoe PC, Edwards RG. Birth after the reimplantation of a human embryo. Lancet 1978; 2: 366.
Summers MC, Bhatnagar PR, Lawitts JA, Biggers JD. Fertilizatio in vitro of mouse ova from inbred and outbred strains: complete preimplantation embryo development in glucose-supplemented KSOM. Biol Reprod Online 1995; 53: 431-437.
Summers MC, McGinnis LK, Lawitts JA, Raffin M, Biggers JD. IVF of mouse ova in a simplex optimized medium supplemented with amino acids. Hum Repord 2000; 15: 1791-1801.
Summers MC, McGinnis LK, Lawitts JA, Biggers JD. Mouse embryo development following IVF in media containing either L-glutamine or glycyl-L-glutamine. Hum Reprod 2005; 20: 1364-1371.
Trounson AO, Leeton JF, Wood C, et al. Pregnancies in humans by fertilization in vitro and embryo transfer in the controlled ovulatory cycle. Science 1981; 212:
681-682.
Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eigth-cell embryo. Nature 1983; 305: 707-709.
Tsai Y, Chung M, Sung Y, Tsai T, Lin L. Clinical value of early cleavage embryo. Int J
Gynaecol Obstet 2002; 76: 293-297.
Tsirigotis M. Blastocyst stage transfer: pitfalls and benefits. Too soon to abandon
current practice? Hum Reprod 1998; 13: 3285-3289.
Whittingham DG. Culture of mouse ova. J. of Reprod and Fertil 1971; Suppl 14: 7-21.
Wiemer KE, Anderson AR, Kyslinger ML, Weikert ML. embryonic development and pregnancy following sequential culture in human tubal fluid and a modified simplex optimized medium containing amino acids. Biol Reprod Online 2002; 5: 323-327.
Wirleitner B, Vanderzwalmen P, Stecher A, Zech MH, Zintz M, Zech NH. Individual demands of human embryos on IVF culture medium: influence on blastocyst development and pregnancy outcome. Biol Reprod Online 2010; 21: 776-782.
Vansteenbrugge, C. Vastersaegher, P. Fontaine, C. Klay, D. Rodez, P. Comparison of a single medium with sequential media for the development of human zygotes to the blastocyst stage. Fertil Steril 2007; 88: Suppl P-640: S319.
Volpes A, Sammartano F, Coffaro F, Mistretta V, Scaglione P, Allegra A. Number of
good quality embryos on day 3 is predictive for both pregnancy and implantation
rates in in vitro fertilization/ intracytoplasmic sperm injection cycles. Fertil Steril
2004; 82: 1330-1336.
Zech N, Stecher A, Zech H, Uher P, Vanderzwalmen P. Prospective analysis of embryo
development to day 5 and transfer outcomes in sequential medium (G 1.3-G2.3) vs
a one step protocol (Global medium). Hum Repro2006; 21 Suppl 1: i162.
Zeilmaker GH, Alberda AT, van Gent I. Two pregnancies following transfer of
intact frozen-thawed embryos. Fertil Steril 1984; 42: 293-296.
Ziebe S, Petersen K, Lindenberg S, Andersen A, Gabrielsen A, Andersen A. Embryo
morphology or cleavage stage: how to select the best embryos for transfer after
in-vitro fertilization. Hum Reprod 1997; 12: 1545-1549.
三、網路部份
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