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1 大韓不妊學會誌 : 第 33 卷第 3 號 2006 Kor. J. Fertil. Steril., Vol. 33, No. 3, 2006, 9 염색체이수성과관련된비정상적임신이예상되는환자에서착상전유전진단의결과 성균관대학교의과대학삼성제일병원산부인과 1, 불임연구실 2, 유전학연구실 3 김진영 1 임천규 2 차선화 1 박수현 1 양광문 1 송인옥 1 전진현 2 박소연 3 궁미경 1 강인수 1 Preimplantation Genetic Diagnosis for Aneuploidy Screening in Patients with Poor Reproductive Outcome Jin Yeong Kim 1, Chun Kyu Lim 2, Sun Hwa Cha 1, Soo Hyun Park 1, Kwang Moon Yang 1, In Ok Song 1, Jin Hyun Jun 2, So Yeon Park 3, Mi Kyoung Koong 1, Inn Soo Kang 1 1 Department of Obstetrics and Gynecology, 2 Laboratory of Reproductive Biology and Infertility, 3 Laboratory of Genetics, Cheil General Hospital, Sungkyunkwan University School of Medicine Objectives: The risk of aneuploidies of embryos increases in advanced maternal age or parental karyotype abnormality and it results in poor reproductive outcomes such as recurrent spontaneous abortion (RSA) or repeated implantation failure (RIF). Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) can be applied for better ART outcome by selecting chromosomally normal embryos. The aim of this study is to evaluate the clinical outcome of PGD-AS and which group can get much benefit from PGD-AS among the patients expected to have poor reproductive outcome. Methods: In 42 patients, 77 PGD cycles were performed for aneuploidy screening. Patients were allocated to 3 groups according to the indication of PGD-AS: group I-patients with old age ( 37) and RIF more than 3 times (n=11, mean age=42.2 yrs.), group II-patients with RSA ( 3 times) associated with aneuploid pregnancy (n=19, mean age=38.9 yrs.), group III-parental sex chromosome abnormality or mosaicism (n=18, mean age=29.6 yrs.) including Turner syndrome, Klinefelter syndrome and 47,XYY. PGD was performed by using FISH for chromosome 13, 16, 18, 21, X and Y in group I and II, and chromosome X, Y and 18 (or 17) in group III. Results: Blastomere biopsy was successful in 530 embryos and FISH efficiency was 92.3%. The proportions of transferable embryos in each group were 32.5±17.5%, 23.0±21.7% and 52.6±29.2% (mean ± SD), respectively, showing higher normal rate in group III (group II vs. III, p<0.05). The numbers of transferred embryos in each group were 3.9±1.5, 1.9±1.1 and 3.1±1.4 (mean ± SD), respectively. The clinical pregnancy rates per transfer was 0%, 30.0% and 20.0%, and it was significantly higher in group II (group I vs. group II, p<0.05). The overall pregnancy rate per transfer was 19.6% (10/51) and the spontaneous abortion rate was 20% (2/10) of which karyotypes were euploid. Nine healthy babies (one twin pregnancy) were born with normal karyotype confirmed on amniocentesis. Conclusion: Our data suggests that PGD-AS provides advantages in patients with RSA associated with aneuploidy or sex chromosome abnormality, decreasing abortion rate and increasing ongoing pregnancy rate. It is not likely to be beneficial in RIF group due to other detrimental factors involved in implantation. Key Words: PGD, Aneuploidy, FISH, RIF, RSA 주관책임자 : 강인수, Department of Obstetrics and Gynecology, Cheil General Hospital, Sungkyunkwan University School of Medicine, Mook Jung-Dong 1-19, Chung-Ku, Seoul, , Korea. Tel: (02) , Fax: (02) , ikang67@yahoo.co.kr * 본논문은 2005 년대한불임학회제 49 차추계학술대회포스터부문우수발표상을수상하였음

2 PGD for Aneuploidy Screening 배아의염색체이수성은착상실패및자연유산의원인이되며, 자연유산의약 50~60% 까지염색체이수성에기인하는것으로보고되고있다. 1,2 배아의이수성의위험은고령의임신에서증가되어, 고령에서의착상실패나자연유산과관련되며, 3~5 습관성유산의원인으로서배아이수성은특발성유산에서보다빈도가낮다고보고되기도하지만, 일정부분습관성유산과관련된다. 6~8 또한터너증후군이나클라인펠터증후군등성염색체이상에서도성선기능부전으로임신확률이매우낮으며임신의경우유산율도매우높은데, 이는대부분태아의염색체이상과관련되는것으로알려져있다. 9,10 클라인펠터증후군의경우특히수술적고환정자채취술 (testicular sperm extraction, TESE) 및세포질내정자주입술 (intracytoplasmic sperm injection, ICSI) 의발전으로보조생식술을이용한임신이보고되고있으나, 11,12 역시채취되는정자및배아의이수성위험이제기되고있다. 13,14 착상전유전진단 (PGD: preimplantation genetic diagnosis) 은착상전의배아에서염색체이상이나유전자이상을진단하여정상적인배아만을선별하여이식할수있는방법으로지금까지다양한단일유전자질환이나염색체구조적이상, 성염색체이상등에서널리시행되고있다. 15,16 나아가고령이나습관성유산등염색체이수성의위험이높은환자들에서정상적인배아를선별이식하면착상율을증가시키고자연유산율을감소시킬수있을것으로생각되어, 배아에서발생하는주요염색체이수성에대한착상전유전진단이시도되고있다. 17,18 이에본연구에서는염색체이수성과관련되어불량한임신예후가예상되는환자들에서염색체이수성에대한착상전유전진단의결과를분석하고, 가장큰효용성을얻을수있는적절한환자군을알아보고자하였다. 연구대상및방법 1. 연구대상 1999년 5월부터 2004년 12월까지, 42명의환자, 77주기에서염색체이수성에대한착상전유전진단을시행하였다. 환자들은착상전유전진단을시행하 게된주적응증에따라 3군으로나누어, 제 1군은 37세이상, 정상염색체핵형을가지며, 체외수정에서 3회이상반복착상실패를한반복착상실패 (repeated implantation failure) 환자군 (n=6, 평균나이 42.2±3.6세 ), 제 2군은 3회이상의습관성유산환자로유산아의염색체이수성이있었던경우 (n=20, 평균나이 39.6±3.6세 ), 제 3군은성염색체이상이나모자이시즘을갖는환자로하였으며이들의염색체핵형은 45,X/46,XX, 47,XXX/45,X/46,XX, 47,XXY, 47,XYY, 47,XXY/46,XY, 48,XXXY/47,XXY/46,XY 등이었다 (n=16, 평균나이 30.8±3.4세 ). 이들에서체외수정시술후발달중인배아로부터 1~2개의할구세포를생검하여 FISH를통한착상전유전진단을시행하고그결과를비교하였다. 통계학적방법으로는 Chi-square test, ANOVA 분석을이용하였다. 2. 난자의채취및수정과배란유도는 Gonadotrophin-releasing hormone (GnRH) agonist (Suprefact, Aventis, France, 또는 Leucrin, Abbott, France) 를이용한황체기중간장기요법을이용하였다. FSH/hMG 제재로는연구초반기에는 highly purified Follicular stimulating hormone (Metrodin, Serono, Switzerland) 및 human menopausal gonadotropin (Pergonal, Serono, Switzerland) 를이용하였고최근에는 recombinant FSH (puregon, Organon, Netherland 또는 Gonal-F, Serono, Switzerland) 를이용하였다. 초음파로난포의크기를관찰하여최소한 18 mm 이상의난포가 2개이상, estradiol 농도가 500 pg/ml 이상되었을때 human chorionic gonadotropin (Pregnyl, Serono, Switzerland) 10,000 IU를주사하였고, hcg 투여 36시간후질식초음파를이용하여난자채취를하였다. 채취된난자는 10% HSA 가첨가된 G-Fert TM 배양액 (Vitrolife, Gothenburg, Sweden) 에넣어 37, 6%CO 2, 94% 공기중배양기에서 3~4시간배양하였다. 배양후성숙된난자에서세포질내정자주입술 (ICSI, Intracytoplasmic Sperm Injection) 을시행하였으며, 16~18시간후에수정을확인하였다. 3. 할구의분리와핵의준비수정이확인된수정란은 G-1 TM version 3 배양액

할구가분리된배아는세척후 G-2 TM version 3 배양액 (Vitrolife, Gothenburg, Sweden) 에서 37, 5%CO 2, 공기중에서배양하였다. 분리해낸할구는배양액으로세척후 hypotonic solution (0.5% sodium citrate in 0.

3 (Vitrolife, Gothenburg, Sweden) 에서 3일간배양한후배아를 mineral oil로덮은 biopsy media (MediCult, Denmark) 에이전하였다. 할구세포의생검을위해 30 µm의 pipette을이용하여 acidified Tyrode 용액 (MediCult, Denmark) 으로투명대에작은구멍을만들고, 같은 glass needle을이용하여조심스럽게할구세포를흡인하여생검하였다. 할구가분리된배아는세척후 G-2 TM version 3 배양액 (Vitrolife, Gothenburg, Sweden) 에서 37, 5%CO 2, 공기중에서배양하였다. 분리해낸할구는배양액으로세척후 hypotonic solution (0.5% sodium citrate in 0.4 mg/ml bovine serum albumin) 에이전하여 10초간배양후, Tween 20 용액내에 Superfrost plus 슬라이드에옮겨 Tween 20 용액을소량씩계속첨가하여세포막이터지도록하였다. 슬라이드를공기중에서완전히건조시킨후 phosphate-buffered saline (PBS) 으로세척하고건조시켰다. 슬라이드를 0.01% pepsin과 0.01 N HCl 로 37 에서 10분간배양한후, 실온에서 H 2 O 및 1 PBS 용액으로세척하였다. 그후 1% paraformaldehyde에 4 에서 10분간두었다가 PBS및 H 2 O로세척하고, 70, 85, 100% ethyl alcohol을각각 2분씩처리하여탈수시켰다. 4. 할구세포에서 FISH 의시행과결과확인염색체 13 (LSI 13 green), 16 (CEP 16 green, orange), 18 (CEP 18 aqua), 21 (LSI 21 orange), X (CEP X orange, aqua), Y (CEP Y green, aqua) 에대한 FISH probe (Vysis, Inc, Illinois, USA) 를이용하였고, 성염색체이상을가진군에서는 X, Y와 18 또는 17번염색체에대한 probe를이용하였다. 10 microliter의 probe 혼합액을 12 mm 원형 cover glass에넣고, 할구의핵이준비된 slide glass에올려놓고 rubber cement로밀봉하였다. DNA의 denaturation은 75 hot plate에서 10분간시행한후 humidified chamber에서 37 에서 overnight으로 hybridization 시켰다. Hybridization 후 50% formamide/2x SSC, 2X SSC로 42 에서 10분간세척하여, 잔여 probe를제거하였다. 세척후 125 ng/ml DAPI가첨가된 Vectashield mounting medium (Vysis, USA) 을각슬라이드에첨가하고 mm cover slip을덮었다. 10분후형광현미경 (Optiphot-2, Nikon) 으로 1,000 배율로 FISH signal을확인하였다. 결과확인시 2개이상의 signal이근접해있을경우에 2 signal의거리가 signal 크기의 2배이상이면 2 개의 signal로, 2배이하이면한개의 signal로판정하였다. 19 정상으로판정된배아를난자채취 4일째 A B Figure 1. FISH signals in the blastomeres in preimplantation genetic diagnosis for aneuploidy screening. A) Euploidy embryo showing two green signals for chromosome 13 (LSI 13), two aqua signals for 18 (CEP 18), two orange signals for 21(LSI 21), two green/orange signals for 16 (CEP 16 green/orange), one orange/aqua signal for X (CEP X orange/aqua), and one green/aqua signal for Y (CEP Y green/aqua). B) Abnormal embryo showing FISH signals of one green signal for chromosome 13, one aqua signal for 18, one orange signal for 21, one green/orange signal for 16, and one orange/aqua signal for X, indicating monoploidy

4 PGD for Aneuploidy Screening Table 1. Results of preimplantation genetic diagnosis for aneuploidy screening Group I Group II Group III No. of patients Age 42.2± ± ±3.4 * No. of cycles No. of transfer cycles Transferable embryo rate (%) 32.5± ± ±29.2 * No. of transferred embryo 3.9± ± ±1.4 Clinical PR (%) per transfer 0 (0/11) 30.0 * (6/20) 20.0 (4/20) Implantation rate (%) 0 (0/43) 21.6 * (8/37) 6.0 (4/62) Abortion rate (%) (2/ 6) 0 Delivery rate (%) per transfer 0 (0/11) 20.0 (4/20) 20.0 (4/20) Values are mean ± SD. RP: pregnancy rate. *: p<0.05 에자궁내이식하였다. 결과총 548개의배아에서할구생검을시도하여 530 개배아에서성공적으로할구의생검이가능했으며 (96.7%), 이중 FISH 분석으로진단이가능하였던경우가 489개로 FISH 효율은약 92.3% 였다. 각각의 FISH signal을분석하여배아의이수성을진단하여정상으로진단된배아를이식하였다 (Figure 1). FISH 상염색체이상으로진단된배아들의경우를보면, 하나의염색체이상을보인경우가 46.5%, 두개의염색체에대해이상을보인경우가 37.6%, 세개의염색체이상을보인경우가 10.9%, 네개의염색체이상을보인경우가 4.0%, 진단한다섯개의염색체에대해동시에모두이상을보인경우도 1.0% 였다. 착상전유전진단의임상적결과는 Table 1과같다. 전체배아중 FISH 진단상정상염색체를가진경우로진단된배아의비율은각각제 1군에서 32.5 ±17.5 (%), 제 2군에서 23.0±21.7 (%), 제 3군에서 52.6±29.2 (%) 로습관성유산환자군에서는의미있게낮게나타났으며, 성염색체이상환자들에서는정상배아율이의미있게높았다. 총 10주기에서임상적임신이이루어져, 전체적으로이식주기당임상적임신율은 19.6% (10/51) 였으며착상율은 8.5% 였다. 각군별로는제 1군에서는임신된예가없었 Table 2. Confirmation of pregnancy outcome of preimplantation genetic diagnosis for aneuploidy screening Case Age Indication Outcome Karyotyping 1 36 RSA Term delivery 46,XY 2 42 RSA Term delivery 46,XY 3 38 RSA Miscarriage 46,XX * 4 38 RSA Miscarriage 46,XX * 5 38 RSA Term delivery 46,XX 6 36 RSA Term delivery, twin 46,XX and 46,XY ,XXY Term delivery 46,XY ,XXY Term delivery 46,XX ,XXY Term delivery Not done Turner mosaic Term delivery 46,XX RSA: recurrent spontaneous abortion. *: karyotyping of abortus, 2 miscarriages in the same patient. 고, 제 2군에서 30%, 제 3군에서 20% 로나타났다. 습관성유산이군중한환자에서두번의임신이모두유산이되어유산율은 20% (2/10) 를나타냈으며, 유산아의염색체핵형은정상이었다. 임신이진행된 8예에서모두임신 16주경양수검사를시행하여착상전유전진단결과를확인하였으며, 모두정상으로판명되어만삭분만을하였고, 이중 1예는쌍

5 태임신으로총 9명의건강한정상아가출생하였다 (Table 2). 고찰배아의염색체이수성은배아의착상실패나유산, 기형아출생을초래하며고령이나반복적인착상실패, 성염색체모자이시즘이나염색체전좌에서위험이증가된다. 체외수정전극체생검을통한난자의염색체검사상 35세이상의고령에서는난자의염색체이수성비율이 33~43% 정도로높아유전적진단이필요하며, 20 비정상적인발달양상을보이는배아에서는약 70% 에서염색체이상이있으며, 정상적으로발달하는배아라도고령이나반복착상실패환자의배아에서는 57% 정도에서, 특히 40세이상에서는대부분염색체이상을갖는것으로보고되고있다. 21,22 착상전유전진단은착상전의배아에서염색체이상이나유전자이상을진단하여정상적인배아만을선별하여이식할수있는방법이다. 유전병에이환된배아의착상예방을위해시행된이래, 23 지금까지다양한단일유전자질환이나염색체구조적이상, 성염색체이상등에서유전병을예방과자연유산율감소의효과를얻는방법으로그시행이증가되고있다. 24 최근에는그적용이점차확대되어, 습관성유산이나염색체이수성의위험이높은고령임신, 반복착상실패등에서염색체이수성에기인한착상실패와자연유산을감소시켜임신유지율을향상시키기위해염색체이수성에대한착상전유전진단이시행되고있다. 17,18 특히 35세이상의고령에서체외수정시착상전유전진단으로임신율의유의한증가는없으나자연유산율을유의하게감소시키는효과가있다고보고되었다. 25,26 본연구에서는고령이나, 염색체이수성과관련된습관성유산, 반복착상실패등염색체이수성과관련되어불량한임신예후가예상되는환자들에서염색체이수성에대한착상전유전진단의결과를살펴보고어떤경우에서가장큰효용성을얻을수있는지알아보고자하였다. 그결과착상전유전진단으로정상적인임신을얻을수있었고, 유산율도 20% 정도로일반집단의수준으로낮출수있었다. 특히이러한효과는염색 체이수성과관련된습관성유산과성염색체이상에서큰효용성이있는것으로생각된다. 습관성유산군에서는환자의나이가고령 ( 평균 39.6세 ) 임을감안하더라도특히정상배아율이 23.0% 로다른군에비해낮아, 이러한환자들에서염색체이수성과관련되어정상적인임신확률이낮을것을예상할수있다. 따라서이러한환자군에서염색체이수성을스크리닝함으로써정상적인임신결과를얻는데효용성이클것으로생각된다. 그러나 1예에서정상염색체핵형을갖는자연유산이 2회연속발생하여, 이들에서염색체이수성에대한착상전유전진단으로유산의예방에한계점이있으며, 유전적요인이외의인자들에의한영향도고려되어야할것으로생각된다. 비정상으로진단된배아들의염색체이상의양상에있어서는두개이상의염색체에대해동시에이상을갖는배아들도많이나타났으며특히 5개모두이상을나타낸경우도 1% 정도로, 복합적인비정상을나타내는배아들이많음을알수있었다. 반복착상실패군에서는임신된예가없었는데, 환자군이매우고령이었으며반복적인착상실패에관련되는다양한요인들이복합적으로관련되어염색체이수성스크리닝만으로효과를얻지못한것으로생각된다. 그러나시행된예가너무적고환자의나이가매우고령 ( 평균 42.2±3.6세 ) 이었으므로, 추후적절한연령기준을설정하여더많은예에서연구가필요할것으로생각된다. 염색체이수성에대한착상전유전진단에대해서아직명확한전향적무작위연구가없고, 체외수정시행시염색체이수성에대한스크리닝으로착상율과임신율이기대만큼현저히증가되지않는다는논란도있다. 27 그제한점으로는 FISH로스크리닝할수있는염색체의수가제한적이며, 배아의할구생검이나생검된배아의배양에서배아의발달에영향을받을수있고, 배아의모자이시즘이있을수있어 FISH 진단의정확성에영향을줄수있다는점들이제기되고있다. 27 또한원인불명의습관성유산환자들에서염색체이수성에대한착상전유전진단을시행하여유의한효과가없었으며, 원인이매우이질적인환자군을대상으로시행하는것은큰효과가없을것으로생각된다. 28 습관성유산에서염색체이수성과의관련성을살펴보면, 산발적유산과

6 PGD for Aneuploidy Screening 습관성유산에서다음임신시염색체이수성을비교한결과산발적유산에서는 42.1% 에서, 습관성유산에서는약 25.4% 에서염색체이수성이나타나, 산발적인유산에서염색체이수성발생율이더높고습관성유산에서반복적인염색체이수성이반복적으로나타나는경우는일부에국한된다고보고되기도하며, 8 2~4회의습관성유산에서는약 60% 에서염색체이수성을보이고 4회이상의습관성유산에서는염색체이수성이 29% 정도로낮으며, 35세이하에서는정상염색체를갖는유산이많고 35세이상에서는이수성과관련된유산이증가되는것으로보고되고있다. 29 따라서염색체이수성이원인이되는일부군에서착상전유전진단이큰효과를얻을수있을것이다. Staessen 등 30 은 37세이상의고령에서포배기배아이식만시행한경우와염색체이수성에대한착상전유전진단과함께포배기배아이식을시행한경우를전향적으로비교한결과, 정상배아율은 36.8% 였으며착상전유전진단을한경우에서이식된배아의수는적었지만포배기배아이식의경우와착상율이나임신율은차이가없었다. 따라서염색체이수성에대한착상전유전진단은이식하는배아의수가제한되거나, 다태임신을피하기위해배아를선별해야할경우에서특히유용하다고보고하였다. 또한반복적인태아의염색체이수성이나고령에서는염색체이수성에대한착상전유전진단이효과적인것으로알려지고있으며, 31 특히 38세이상이나비정상적염색체핵형을갖는경우, 3회이상체외수정실패시착상전유전진단으로착상율을증가시킬수있다고보고되기도하였다. 32 또한고령이아닌 35세이전에서도염색체이수성을가진임신의과거력이있었던경우에서는배아의염색체이수성비율이 37.4%, 반복착상실패군에서는약 27% 정도로증가된다고보고된바있어, 33 염색체이수성과관련되어습관성유산이예상되거나고령등적절한환자군을대상으로시행한다면착상전유전진단으로큰효과를얻을수있을것으로생각된다. 착상전유전진단에서는단일세포에서 FISH를이용하므로진단하는염색체가제한적인데, 일반적으로자연유산에서발견되는염색체이수성의약 70% 는 13, 14, 15, 16, 18, 21, 22, X, Y 등으로알려지고 있다. 이들에대한착상전유전진단시고령이나습관성유산, 반복착상실패등의환자에서정상배아율이약 42% 정도였으며, 유산율을일반군에서와유사하게감소시킬수있다. 34 또한나이증가에따라현저히이수성이증가되는염색체는 1, 15, 17, 21, 22번으로보고된바있어, 35 35세이상에서 9개의염색체, 즉 13, 15, 16, 18, 21, 22, X, Y외에 1, 7, 14 또는 17를대상으로착상전유전진단을시행하여유의하게착상율을증가시킬수있으며, 이때수정란의개수가 8개이상이고반복착상실패가아닌경우에서특히착상율의향상을보이며유산율도감소될수있어, 36 진단염색체수를최대한증가시키면그효과를더욱증대시킬수있을것으로생각된다. 그러나 Kahraman 등 37 은남성요인으로 ICSI 를시행하는경우착상전유전진단으로고령과반복착상실패군에서유사한임신율을얻을수있어두군에서모두효과적으로적용될수있다고하였다. 한편, 성염색체모자이시즘에서의배아이수성증가위험이제기되고있으며, 본연구에서는정상배아율이다른군에비해비교적높게나타났지만, 착상전유전진단시염색체이수성이증가되며성염색체뿐아니라오히려상염색체의이수성도증가되는것으로알려져있고, 38 클라인펠터증후군에서나타난비정상적인배아들은동시에여러염색체에대해이수성을갖거나 chaotic aneuploidy 가나타나는경우가많다고보고되었다. 39 이외에도착상전유전진단을통해수술적고환내정자채취술이나미성숙난자의체외성숙시염색체이수성이증가됨을알수있고, 이전주기의정상배아의수에따라다음주기임신의가능성을예측할수있는방법으로서도효용성이제시된바있다. 40 결론적으로염색체이수성에대한착상전유전진단은염색체이수성에기인하는착상실패나자연유산을예방할수있는방법으로서, 염색체이수성과관련되어불량한임신예후가예상되는적정한환자군에서시행하여착상율을증가시키고자연유산을감소시켜보조생식술의효율을높일수있다. 특히, 이수성과관련된습관성유산, 성염색체모자이시즘환자군에서효용성이클것으로생각된다. 추후더욱포괄적으로염색체진단을할수있는기법을적

7 용하여더많은예에서의전향적연구가필요할것으로사료된다. 참고문헌 1. Simpson JL. Genes, chromosomes and reproductive failure. Fertil Steril 1980; 33(2): Sanchez JM, Franzi L, Collia F, De Diaz SL, Panal M, Cubner M. Cytogenetic study of spontaneous abortions by transabdominal villus sampling and direct analysis of villi. Prenat Diag 1999; 19: Munne S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod 1993; 8: Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates and maternal age are correlated with chromosome abnormalities. Fertil Steril 1995; 64: Kuliev A, Cieslak J, Ikewitch Y, Verlinsky Y. Chromosomal abnormalities in a series of 6733 oocytes in preimplantation diagnosis of age-related aneuploidies. Reprod Biomed Online 2003; 6: Stern JJ, Dofrman AD, Gutierez-Najar MD. Frequency of abnormal karyotype among abortuses from women with and without a history of recurrent spontaneous abortion. Fertil Steril 1996; 65: Carp H, Toder V, Aviram A, Daneily M, Mashiach S, Barkai G. Karyotype of the abortus in recurrent miscarriage. Fertil Steril 2001; 75: Sullivan AE, Silver RM, LaCoursiere DY, Proter TF, Branch DW. Recurrent fetal aneuploidy and recurrent miscarriage. Obstet Gynecol 2004; 104: Tarani L, Lampariello G, Raguso F, Colloridi F, Pucarrelli I, Pasquino AM, et al. Pregnancy in patients with Turner's syndrome: six new cases with review of literature. Gynecol Endocrinol 1998; 12: Swapp GH, Johnston AW, Watt JL, Couzin DA, Stephen GS, et al. A fertile woman with non-mosaic Turner's syndrome. Br J Obstet Gynecol 1989; 96: Ron-El R, Friedler S, Strassburger D, et al. Birth of a healthy neonate following the intracytoplasmic injection of testicular spermatozoa from a patient with Klinefelter's syndrome. Hum Reprod 1999; 14: Friedler S, Raziel A, Strassburger D, Schachter M, Bern O, Ron-El R. Outcome of ICSI using fresh and cryopreserved-thawed testicular spermatozoa in patients with non-mosaic Klinefelter's syndrome. Hum Reprod 2001; 16 (12): Denschlag D, Tempfer C, Kunze M, Wolff G, Keck C. Assisted reproductive techniques in patients with Klinefelter syndrome: a critical review. Fertil Steril 2004; 82(4): Silber S, Escudero T, Lenahan K, Abdelhadi I, Kilani Z, Munne S. Chromosomal abnormalities in embryos derived from testicular sperm extraction. Fertil Steril 2003; 79(1): Harper JC, Boelaert K, GeraedtsJ, Harton G, Kearns WG, Moutou C, et al. ESHRE PGD consortium data collection V: Cycles from January to December 2002 with pregnancy follow-up to October Hum Reprod 2006; 21(1): 김진영, 임천규, 송인옥, 유근재, 양광문, 한국선, 등. 유전질환및염색체이상의예방을위한착상전유전진단의결과. 대한불임학회지 2002; 29(4): Munne S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome aneuploidies in human preimiplantation embryos. Hum Reprod 1993; 8: Munne S, Magli C, Cohen J, Morton P, Sadowy S, Gianaroli L, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod 1999; 14(9): Munne S, Marquez C, Magli C, Morton P, Morrison L. Scoring criteria for preimplantation genetic diagnosis of numerical abnormalities for chromosomes X, Y, 13, 16, 18 and 21. Mol Hum Reprod 1998; 4:

8 PGD for Aneuploidy Screening 20. Munne S, Sepulveda S, Balmaceda J, Fernandez E, Fabres C, Mackenna A, et al. Selection of the most common chromosome abnormalities in oocytes prior to ICSI. Prenat Diagn 2000; 20: Munne S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod 1993; 8(12): Magli M, Gianaroli L, Munne S, Ferraretti AP. Incidence of chromosomal abnormalities from a morphologically normal cohort of embryos in poor prognostic patients. J Assist Reprod Genet 1998; 15(5): Handyside AH, Pattinson JK, Penketh RJ, Edlhanty JD, Winston RM, Tuddenham EG. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1989; 18: Kuliev A, Verlinsky. Thirteen years' experience of preimplantation diagnosis: report of the Fifth international symposium on preimplantation genetics. Reprod Biomed Online 2003; 8(2): Munne S, Magli C, Cohen J, Morton P, Sadowy S, Gianaroli L. Positive outcome after preimplantation genetic diagnosis of aneuploidy in human embryos. Hum Reprod 1999; 14(9): Munne S, Fischer J, Warner A, Chen S, Zouves C, Cohen J, et al. Preimplantation genetic diagnosis significantly reduces pregnancy loss in infertile couples: a multicenter study. Fertil Steril 2006; 85: Hill David H. Ten years of preimplantation genetic diagnosis-aneuploidy screening: review of a multicenter report. Fertil Steril 2004; 82(2): Plateau P, Staessen C, Michiels A, Steirteghem AV, Liebaers I, Devroey P. Preimplantation genetic diagnosis for aneuploidy screening in patients with unexplained recurrent miscarriages. Fertil Steril 2005; 83(2): Stephenson MD, Awartani KA, Robinson WP. Cytogenetic analysis of miscarriages from couples with recurrent miscarriage: a case-control study. Hum Reprod 2002; 17: Staessen C, Platteau P, Assche EV, Michiels A, Tournaye H, Camus M, et al. Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial. Hum Reprod 2004; 19(12): Obasaju M, Kadam A, Biancardi T, Sultan K, Fateh M, Munne S. Pregnancies from single normal embryo transfer in women older than 40 years. RBMonline 2001; 2: Gianaroli L, Magli MC, Ferraretti AP, Munne S. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999; 72(5): Munne S, Sandalinas M, Magli C, Gianaroli L, Cohen Jwarburton D. Increased rate of aneuploid embryos in young women with previous aneuploid conceptions. Prenat Diagn 2004; 24: Munne S, Magli C, Bahce M, Fung J, legator M, Morrison L. Preimplantation diagnosis of the aneuploies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22. Prenat Diangn 1998; 18: Gianaroli L, Magli MC, Ferraretti AP, Tabanelli C, Trombeta C, Boudjema E. The role of preimplantation genetic diagnosis for aneuploidies. RBMonline 2001; 4.suppl.3: Munne S, Sandalina M, Escudero T, Velilla E, Walmsley R, Sadowy S. Improved implantation after preimplantation genetic diagnosis of aneuploidy. RBMonline 2003; 7: Kahraman S, Bahce M, Samli H, Imirzalioglu N, Yakism K, Cengiz G, et al. Healthy births and ongoing pregnancies obtained by preimplantation genetic diagnosis in patients with advanced maternal age and recurrent implantation failure. Hum Reprod 2000; 15(9):

9 38. Magli MC, Gianaroli L, Ferraretti AP, Gordts S, Feliciani E. Impact of parental gonosomal mosaicism detected in peripheral blood on preimplantation embryos. RBMonline 2002; 5(3): 김진영, 임천규, 전진현, 박소연, 서주태, 차선화, 등. 클라인펠터증후군환자에서착상전유 전진단의결과. 대한불임학회지 2004; 31(4): Ferraretti AP, Magli MC, Kopcow L, Gianaroli L. Prognostic role of preimplantation genetic diagnosis for aneuploidy in assisted reproductive technology outcome. Hum Reprod 2004; 19(3): = 국문초록 = 목적 : 배아의염색체이수성은착상실패나자연유산과관련되며이는고령이나부부의염색체이상이있는경우그위험이증가되어습관성유산이나반복착상실패등불량한임신결과를초래한다. 염색체이수성에대한착상전유전진단은정상염색체를갖는배아를선별적으로이식할수있으므로이러한환자들에서효과적으로적용될수있다. 이에염색체이수성에대한착상전유전진단의임상적결과를알아보고어떠한환자군에서가장큰효용성을얻을수있는지알아보고자하였다. 연구방법 : 총 42명의환자에서 77주기의염색체이수성에대한착상전유전진단을시행하였다. 환자군을적응증에따라세군으로나누어제 1군은 37세이상이며 3회이상반복착상실패를보인경우 (11예, 평균나이 42.2세 ), 제 2군은 3회이상의습관성유산및이중 1회이상염색체이수성을보인경우 (19예, 평균나이 38.9세 ), 제 3군은터너증후군이나클라인펠터증후군, 47,XYY 등성염색체이상이나모자이시즘을가진환자였다 (18예, 평균나이 29.6세 ). 착상전유전진단은제1군과 2군에서는 13, 16, 18, 21, X, Y염색체에대한 FISH를, 제 3군에서는 X, Y 및 18 또는 17번염색체에대한 FISH를시행하였다. 결과 : 총 530개의배아에서할구생검이가능하였고 FISH 진단효율은 92.3% 였다. 정상배아의비율은각군에서 32.5±17.5%, 23.0±21.7%, 및 52.6±29.2% (mean ± SD) 였으며제 3군에서유의하게높았다 (group II vs. III, p<0.05). 배아이식은 51주기에서시행하였으며이식된배아의수는각각 3.9±1.5, 1.9±1.1 및 3.1±1.4개 (mean ± SD) 였다. 임상적임신율은각군에서 0%, 30.0% 및 20.0% 로제 2군에서유의하게높았다 (p<0.05). 전체적인임신율은 19.6% (10/51) 였고자연유산율은 20% (2/10) 였으며유산된경우의염색체는정상이었다. 쌍태아 1예를포함하여총 9명의정상아가출생되었으며, 양수검사로정상염색체핵형을확인하였다. 결론 : 염색체이수성에대한착상전유전진단은염색체이수성과관련된불량한임신예후가예상되는환자에서유용하게이용될수있으며, 특히이수성과관련된습관성유산이나성염색체이상에서효용성이있으며, 반복착상실패에서는착상에관련되는다른원인들의복합적인영향으로큰효용성이없는것으로생각된다. 중심단어 : 착상전유전진단, 염색체이수성, 착상실패, 습관성유산

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歯 PDF * * The Effects of Oocyte Oocytes Intracytoplasmic ICSI) Kee TaekHoo Hai * SikChun Department of Obstetrics and Gynecology, School of Medicine, Kyungpook Taegu, *Department of Animal Science, Taegu Kyungbuk,