大韓不妊學會誌 : 第 32 卷第 4 號 2005 Kor. J. Fertil. Steril., Vol. 32, No. 4, 2005, 12 체외수정시술시획득한미성숙난자의환자연령에따른체외성숙률및수정률비교 분당서울대학교병원산부인과 1, 서울대학교의과대학산부인과학교실 2, 서울대학교의학연구원인구의학연구소 3 한상훈 1 이정렬 2 김현준 1 문정희 1 지병철 1 구승엽 2 서창석 1,2,3 김석현 2,3 최영민 2,3 김정구 2 문신용 2,3 Effects of Age on in vitro Maturation and Fertilization of Immature Oocytes from Stimulated Cycles in Human IVF-ET Program Sang Hoon Han 1, Jung Ryeol Lee 2, Hyun Jun Kim 1, Jung Hee Moon 1, Byung Chul Jee 1, Seung-Yup Ku 2, Chang Suk Suh 1,2,3, Seok Hyun Kim 2,3, Young Min Choi 2,3, Jung-Gu Kim 2, Shin Yong Moon 2,3 1 Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam, Gyeonggi-do, Korea, 2 Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul, Korea, 3 Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, Korea Objective: To investigate the effects of female age on in vitro maturation and fertilization of immature oocytes from controlled ovarian hyperstimulation (COH) in human IVF-ET program. Method: A total of 96 immature oocytes (GV & metaphase I) obtained from 40 cycles of IVF-ET (29 patients). The mean age of female patients was 31.8±3.1 years. Ovulation was triggered by urinary or recombinant hcg. Immature oocytes were cultured with YS medium containing 30% of patients' human follicular fluids, LH (1 IU/mL), FSH (1 IU/mL) and EGF (10 ng/ml), and then matured oocytes were fertilized by ICSI. In vitro maturation and fertilization of immature oocytes were analyzed according to age of female (< 34 or 34 years). Results: The maturation rate was similar between two groups (68% vs 64%). The fertilization rate of in?vitro-matured oocytes was higher in patients < 34 years old, but there was no statistical significance (64% vs 50%, p=0.347). The fertilization rate of in-vitro-matured oocytes was significantly lower compared with those of in-vivo-matured oocytes in both age groups (64% vs 79%, p=0.035, 50% vs 86%, p=0.007). Conclusion: In older female group, fertilization rate of in-vitro-matured oocytes seems to be decreased. Further investigations should be warranted to increase fertilization potential of in-vitro-matured oocytes. Key Words: Immature oocyte, Female age, In vitro maturation, Controlled ovarian hyperstimulation, Fertilization 주관책임자 : 지병철, 우 ) 463-707, 경기도성남시분당구구미동, 분당서울대학교병원산부인과 Tel: (031) 787-7254, Fax: (031) 787-4054, e-mail: blasto@snubh.org - 331 -
일반적인체외수정및배아이식술에서과배란유도후약 20~30% 의난자는미성숙난자상태로얻어진다. 1,2 이러한미성숙난자를체외배양하면일부에서성숙난자로진행하는데특히전체얻어진난자수가적은저반응군 (poor responder) 환자의경우미성숙난자의체외성숙성공은중요한의미를가진다. 현재까지이러한미성숙난자를성숙시켜임신에까지이른보고는많지않다. 2~4 미성숙난자의체외성숙률 (in vitro maturation rate) 은 germinal vesicle (GV) 의경우약 75%, 제1 감수분열중기 (metaphase I) 난자의경우약 74% 정도이며, 이들중 51~68% 정도가수정능이있는것으로알려져있다. 1~3 체내성숙난자의수정률및배아발달률에비하여차이가없다는보고도있지만대개는미성숙난자를체외성숙시켜얻은난자의수정률및배아발달률은약간낮은것으로보고되고있다. 2 미성숙난자가이렇게낮은수정률및배아발달률을보이는것은난자자체의결함이나배양조건때문으로여겨지고있다. 즉과배란유도를하였는데도미성숙난자상태로나오는난자는내재적인결함이있을수있고또한, 현재사용하는배양조건이불완전해서일수도있다. 최근다낭성난소증후군환자에서과배란유도를하지않고미성숙난자를채취하여체외성숙시키려는시도가이루어지고있는데, 5 이에대한연구들에서미성숙난자의체외성숙률을높이는가장중요한요소는배양조건이라고알려져있다. 그러나미성숙난자의성숙률과수정률에있어배양조건이외의다른인자들의영향에대해서는아직까지잘알려져있지않다. 이에저자들은환자의연령이채취된미성숙난자의성숙률과수정률에어떠한영향을주는지를알아보고자하였다. 연구대상및방법 1. 연구대상 2003년 7월부터 2004년 12월까지분당서울대학교병원에서성선자극호르몬 (gonadotropin) 으로과배란유도를하고체외수정시술을받았던환자중미성숙난자를얻었던 40주기 (29명, ICSI 17주기포함 ) 를대상으로하였다. 무정자증으로수술적정자채취 (surgical sperm retrieval) 를하였던경우는제외하 였다. 한사람이여러번을시행하였을경우각각독립된것으로간주하였다. 얻어진 96개의미성숙난자에서성숙과미성숙여부에따라서연구대상군에서 Receiver Operator Characteristic (ROC) 도표를이용하여 34세를기준으로하여연령에따라 34세미만과 34세이상의두군으로나누고체외성숙률과체외수정률을비교하여보았다. 미성숙난자에는 MI과 GV 단계의난자가모두포함되며이둘을따로구분하지는않았다. 2. 연구방법 1) 과배란유도및난자채취월경주기제2~3 일부터성선자극호르몬을투여하였는데 17주기에서는 HMG (Pergonal, Serono, Switzerland), 8주기에서는 pure FSH (Metrodin, Serono), 7주기에서는 recombinant FSH (Gonal-F, Serono), 3주기에서는 recombinant FSH + hmg, 3주기에서는 HMG + pure FSH, 2주기에서는 letrozole (Femara, Norvatis) + pure FSH가이용되었다. 난포의크기가 14 mm에도달하면조기 LH surge의예방목적으로 GnRH antagonist (Cetrotide, Serono) 0.25 mg을매일투여하였으며 (flexible protocol), 우성난포의크기가 19 mm 에도달하면 urinary hcg (Profasi, Serono) 5,000~ 10,000 IU 또는 recombinant hcg (Ovidrel, Serono) 250 µg을투여하여배란촉발을유도한후 35~36시간뒤에질식초음파유도하에난자를채취하였다. 2) 미성숙난자의배양및수정채취한난자는고식적인 (conventional) IVF의경우난구세포 (cumulus cell) 의분포를보고난자의성숙여부를판단하였다. 세포질내정자주입법 (ICSI) 을시행할환자에서얻어진난자는 0.1% hyaluronidase 농도의 PBS 용액에서피펫으로난구세포를제거한후에입체현미경 (stereomicroscope) 하에서 MII 단계에나타나는제1극체 (polar body) 의유무로난자의성숙여부를판단하였다. 성숙난자로판명되면 4시간후에수정을시행하였고, GV 또는 MI 단계의미성숙난자는체외성숙용배양액이준비될때까지 G-Fert (Vitrolife, Sweden) 배양액에보관하였다. 체외성숙용배양액은 YS 배양액 6 70% 와환자의난포액 30% 을섞고 LH 1 IU/ml, FSH 1 IU/ ml, EGF 10ng/ml를첨가하여사용하였다. 난자채 - 332 -
취시얻어진환자의난포액은원심분리하여상층액만취해서 56 water bath에 30분동안두었다가세포파편등을제거하기위하여여과 (filtration) 한후 5% CO 2, 37 하에서 1시간정도두었다. 이렇게준비된체외성숙용배양액에미성숙난자를넣고 37, 5% CO 2 상태의배양기에서배양하였다. GV 상태였던경우는 20시간후에, 제1 감수분열중기 (MI) 의경우는 4시간내에성숙상태를확인하였다. 성숙이확인되면준비된정자로세포질내정자주입법 (ICSI) 을시행하였으며, 18시간후도립현미경 (inverted microscope) 으로두개의전핵이관찰되는경우수정된것으로간주하였다. 세포질내정자주입법 (ICSI) 및이후의과정은이미본교실에서발표한바와같은통상적인방법으로진행하였다. 7 3. 통계처리통계프로그램으로윈도우용 SPSS (ver 12.0) 를이용하였고비율비교시 Chi-square test를, 평균치비교시에는 Student's t-test를시행하여 p 값이 0.05 미만인경우통계학적유의성이있는것으로간주하였다. 결과전체적으로 34세미만과 34세이상의두군간에남편의연령, 불임의기간, 세포질내정자주입법 (ICSI) 의비율, 채취된난자중미성숙난자의비율은차이가없었다. 34세미만군에서난관요인이더많 은경향이있었다 (Table 1). 두군에서미성숙난자의성숙률및수정률은통계학적으로유의한차이는없었으나, 34세이상에서수정률이더낮은경향을나타냈다 (Table 2). 미성숙난자로부터얻어진성숙난자의전체적인수정률 (61%) 은동시에채취된성숙난자의수정률 (81%) 에비하여유의하게낮게나타났으며또한, 두연령군 Table 1. Characteristics of subjects according to female age Group of Age < 34 34 No. of patients 18 11 No. of cycles 28 12 No. of immature oocytes 74 22 Immature oocytes/total aspirated oocytes 74/210 (35%) 22/72 (31%) Husband's age (years) 34.0±1.9 40.7±5.5 Duration of Infertility (years) 4.8±2.1 5.5±2.3 No. of ICSI cycle 12 (43%) 5 (42%) Diagnosis Tubal factor 15 (54%) 3 (25%) Endometriosis 6 (21%) 2 (17%) Unexplained 4 (14%) 2 (17%) Male 3 (11%) 2 (17%) Uterine factor 0 1 ( 8%) Age factor 0 1 ( 8%) High FSH 0 1 ( 8%) Table 2. Maturation rate of immature oocytes and their fertilization rate according to female age Group of age < 34 34 Total No. of patients 18 11 29 No. of cycles 28 12 40 No. of immature oocytes 74 22 96 No. of in-vitro-matured (IVM) oocytes 50 (68%) 14 (64%) 64 (67%) No. of fertilized IVM oocytes 32 (64%) 7 (50%) 39 (61%) No. of in-vivo-matured oocytes 136 50 186 No. of fertilized in-vivo-matured oocytes 108 (79%) 43 (86%) 151 (81%) ; p<0.05 when compared with fertilization rate of in-vitro-matured oocytes - 333 -
모두에서각각유의하게낮게나타났다 (p<0.05). 체내성숙난자의경우에서도 34세미만과 34세이상의연령군의수정률은통계학적으로유의한차이를나타내지않았다 (Table 2). 고찰일반적으로과배란유도를통한난자채취시미성숙난자는약 20~30% 정도에서얻어지며, 본연구에서도전체회수난자중 34% 가미성숙난자였다. 미성숙난자의체외성숙은여분의성숙난자를부가적으로얻을수있는장점이있어, 전체난자의수가적은저반응군 (poor responder) 의경우매우유용하다. 이러한여분의미성숙난자의임상적응용을최적화하면시술횟수와소요되는비용을줄일수있다. 그러나, 일반적으로고식적인체외수정시술 (conventional IVF) 시체외성숙된난자 (in-vitromatured oocyte) 의수정률은체내에서성숙되는난자 (in-vivo-matured oocyte), 즉채취당시부터이미성숙난자의경우보다낮다고보고되고있다 8. 체외에서성숙되는제1 감수분열중기 (MI) 난자의경우도성숙난자의수정률보다낮다 (52.7% vs 70.8%). 2 본연구결과에서도체외에서성숙시킨미성숙난자의수정률과성숙난자의수정률을비교하면체외성숙난자의수정률이낮게 (61% vs 81%) 나타났다. 이처럼체외성숙난자의수정률이감소하는이유로는미성숙난자가성숙난자로진행하기위한충분한준비가되지않은상태에서채취된다는점, 미성숙난자의체외배양시핵성숙에비하여세포질성숙이상대적으로느리게진행된다는점, 체외성숙난자의세포질구성이체내성숙난자에비하여안정되지못한점, 환자의나이가증가함에따라서난자의발달능 (competency) 이감소하는점, 체외의배양환경이체내의배양환경보다좋지못한점등을고려해볼수있다. 그러나아직까지도과배란유도후에채취된미성숙난자의적합한체외성숙법은정립되지못한상태이며체외성숙률에영향을주는인자에대해서도명확하게알려져있지않다. Combelles 등은채취된성숙난자의경우는이미어느정도의안정된방추사 (spindle) 가있는반면체외에서성숙된인간의미성숙난자는 MII의방추 사가빨리퇴화되고불안정하여, 체외성숙난자에서핵의질 (quality) 이더저하될것이라는주장을하였고, 9 미성숙난자의체외성숙은장시간체외배양조건에노출되기때문에투명대 (zona pellucida) 경화를유발하여고식적인체외수정법을이용하면수정률이감소한다고알려져있다. 10 또한나이가증가함에따라서난자의발달능 (competency) 이저하되므로체내성숙난자의수정률뿐만아니라체외성숙난자의수정률또한나이가증가함에따라감소하는것으로보고되고있다. 11 본연구에서도체외성숙난자의수정률은나이가증가함에따라감소하는경향을나타냈다. 체외성숙된난자의발달능이감소하는기전은아직잘알려져있지않지만일단난자가체내에서성숙이되면어느정도수정능이획득되어수정률이나이의증가에영향을받지않지만, 미성숙난자는채취당시에성숙및배란촉발을위한 hcg의자극에둔화될정도로난포내의환경이혈관공급등의내부적인장애가존재하거나국소적으로호르몬작용 (paracrine) 환경이체내성숙난자에비하여좋지않은상태의난포에서채취하여체외에서성숙시키므로수정능및발달잠재력이체내에서채취된성숙난자보다낮을것으로추측해볼수있다. 또한체외에서의성숙환경이체내에서보다미성숙난자의성숙에상대적으로빈약하다고생각되기때문에나이의증가에따른영향을더많이받을것으로추측해볼수있다. 12,13 과배란유도에서질초음파를이용한난자를채취하였을경우가장작은난포에서채취되는난자의경우는정상적인착상전 (preimplantation) 발달에필요한물질들을다생산하지못하여서, 핵과세포질의발달이동시에이루어지지못하고, 이로인해정상배아발달을위한난자의발달능이감소한다는보고가있다. 이처럼핵의성숙에비해세포질의성숙이지연되면세포내소기관의배열에이상이생겨서수정에장애를받는다고알려져있다. 14 본연구의제한점으로는, 대상수가적고, 후향적연구라는점과 GV와 MI 미성숙난자는그특성과능력면에서다소차이가있는데 GV와 MI을구분하지않은점, ICSI 주기와고식적체외수정시술주기를모두를포함하여서 ICSI 주기에서난자의난 - 334 -
구세포제거 (denudation) 에의한미성숙난자의판별및체외성숙도의영향이변수로작용할수있다는점을들수있다. 또한, 수정률뿐만아니라배아발달에따른진행양상을비교해보면난소의발달잠재능을평가하는데더도움이되었을것이다. 실제로난소의발달능이저하되면수정률은비슷하더라도, 임상적인임신율은낮다고보고되었다. 15,16 대상환자에서 40세이상의환자수가너무적은것도나이와수정률의관계를나타내는데문제점이될수있다. 그러므로, 추후의연구에서는 40세이상을포함하는많은수의환자를대상으로 GV와 MI을구분한연구가필요하다고할수있다. 결론적으로본연구결과에따르면과배란유도시채취된미성숙난자의성숙률은환자의연령에영향을받지않지만수정률은연령이증가함에따라감소하는경향을보였다. 체내성숙된난자의수정률이연령에영향을받지않았지만, 체외성숙된난자의수정률은연령에다소영향을받는것같다. 이는아마도체외성숙된난자의경우여성의나이가증가할수록세포질성숙이더욱느려지는것에기인하는것으로사료된다. 그러므로세포질성숙에적절한배양조건을개발하고체외성숙난자의수정률을증진시키기위한연구가지속적으로필요하다. 참고문헌 1. Mandelbaum J, Junca AM, Belaisch-Allart J, Salat- Baroux J, Plachot M, Antoine JM, et al. Oocyte maturation and intracytoplasmic sperm injection. Contracept Fertil Sex 1996; 24: 534-8. 2. De Vos A, Van de Velde H, Joris H, Van Steirteghem A. In-vitro matured metaphase-i oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-ii oocytes after intracytoplasmic sperm injection. Hum Reprod 1999; 14: 1859-63. 3. Coetzee K, Windt ML. Fertilization and pregnancy using metaphase I oocytes in an intracytoplasmic sperm injection program. J Assist Reprod Genet 1996; 13: 768-71. 4. Edirisinghe WR, Junk SM, Matson PL, Yovich JL. Birth from cryopreserved embryos following in-vitro maturation of oocytes and intracytoplasmic sperm injection. Hum Reprod 1997; 12: 1056-8. 5. Child TJ, Phillips SJ, Abdul-Jalil AK, Gulekli B, Tan SL. A comparison of in vitro maturation and in vitro fertilization for women with polycystic ovaries. Obstet Gynecol 2002; 100: 665-70. 6. Yoon HG, Yoon SH, Son WY, Kim JG, Im KS, Lim JH. Alternative embryo transfer on day 3 or day 5 for reducing the risk of multiple gestations. J Assist Reprod Genet 2001; 18: 262-7. 7. Jee BC, Ku SY, Suh CS, Choi YM, Kim JG, Moon SY, et al. Cumulative ongoing pregnancy rate in intracytoplasmic sperm injection cycles. J Obstet Gynaecol Res 2004; 30: 372-6. 8. De Vos A, Van de Velde H, Joris H, Van Steirteghem A. In-vitro matured metaphase-i oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-ii oocytes after intracytoplasmic sperm injection. Hum Reprod 1999; 14: 1859-63. 9. Combelles CM, Cekleniak NA, Racowsky C, Albertini DF. Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes. Hum Reprod 2002; 17: 1006-16. 10. De Vos A, Van Steirteghem A. Zona hardening, zona drilling and assisted hatching: new achievements in assisted reproduction. Cells Tissues Organs 2000; 166: 220-7. 11. Sauer MV. The impact of age on reproductive potential: lessons learned from oocyte donation. Maturitas 1998; 30: 221-5. 12. Armstrong DT. Effects of maternal age on oocyte developmental competence. Theriogenology 2001; 55: 1303-22. 13. Sutton ML, Cetica PD, Beconi MT, Kind KL, Gilchrist RB, Thompson JG. Influence of oocytesecreted factors and culture duration on the metabolic activity of bovine cumulus cell complexes. Reproduction 2003; 126: 27-34. 14. Sutton ML, Gilchrist RB, Thompson JG. Effects of in-vivo and in-vitro environments on the metabolism of the cumulus-oocyte complex and its influ- - 335 -
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