형광직접보합법을 이용한 착상전 유전진단 기법의 최적화와 경험 축적에 의한 임신율의 향상

Improvement of Pregnancy Rate in Preimplantation Genetic Diagnosis with FISH Procedure by the Laboratory Optimization and Experiences

  • 임천규 (삼성제일병원 생식생물학 및 불임연구실) ;
  • 민동미 (삼성제일병원 생식생물학 및 불임연구실) ;
  • 이형송 (삼성제일병원 생식생물학 및 불임연구실) ;
  • 변혜경 (삼성제일병원 생식생물학 및 불임연구실) ;
  • 박소연 (삼성제일병원 유전학연구실) ;
  • 류현미 (삼성제일병원 산부인과) ;
  • 김진영 (삼성제일병원 산부인과) ;
  • 궁미경 (삼성제일병원 산부인과) ;
  • 강인수 (삼성제일병원 산부인과) ;
  • 전진현 (삼성제일병원 생식생물학 및 불임연구실)
  • Lim, Chun-Kyu (Laboratory of Reproductive Biology & Infertility, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Min, Dong-Mi (Laboratory of Reproductive Biology & Infertility, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Lee, Hyoung-Song (Laboratory of Reproductive Biology & Infertility, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Byun, Hye-Kyung (Laboratory of Reproductive Biology & Infertility, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Park, So-Yeon (Laboratory of Medical Genetics, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Ryu, Hyun-Mee (Department of Obsterics and Gynecology, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Kim, Jin-Young (Department of Obsterics and Gynecology, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Koong, Mi-Kyoung (Department of Obsterics and Gynecology, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Kang, Inn-Soo (Department of Obsterics and Gynecology, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Jun, Jin-Hyun (Laboratory of Reproductive Biology & Infertility, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine)
  • 발행 : 2004.03.30

초록

Objectives: This study was performed to evaluate the laboratory system for successful PGD using fluorescence in situ hybridization (FISH) and the clinical outcome of PGD cycles in five years experiences. Methods: A total of 181 PGD-FISH cycles of 106 couples were performed, and diagnosed chromosome normality in the preimplantation embryos. The laboratory and clinical data were classified by the following optimization steps, and statistically analyzed. Phase I: Blastomere biopsy with two kinds of pipettes, removal of cytoplasmic proteins without treatment of pepsin and culture of biopsied embryos with single medium; Phase II: Blatomere biopsy with single pipette, removal of cytoplasmic proteins with pepsin and culture of biopsied embryos with single medium; Phase III: Blastomere biopsy with single pipette, removal of cytoplasmic proteins with pepsin and culture of biopsied embryos with sequential media. Results: A total of 3, 209 oocytes were collected, and 83.8% (2, 212/2, 640) of fertilization rate was obtained by ICSI procedure. The successful blastomere biopsies were accomplished in 98.6% (2, 043/2, 071) of embryos, and the successful diagnosis rate of FISH was 94.7% (1, 935/ 2, 043) of blastomeres from overall data. Embryo transfers with normal embryos were conducted in 93.9% (170/181) of started cycles. There was no difference in the successful rate of biopsy and diagnosis among Phase I, II and III. However, the pregnancy rate per embryo transfer of Phase III (38.8%, 26/67) was significantly (p<0.05) higher than those of Phase I (13.9%, 5/36) and Phase II (14.9%, 10/67). Conclusions: The laboratory optimization and experience for the PGD with FISH procedure can increase the pregnancy rate to 38.8% in the human IVF-ET program. Our facility of PGD with FISH provides the great possibility to get a normal pregnancy for the concerned couples by chromosomal aberrations.

키워드

참고문헌

  1. Gardner RL, Edwards RG. Control of the sex ratio at full term in the rabbit by transferring sexed blastocysts. Nature 1968; 218: 346-8 https://doi.org/10.1038/218346a0
  2. McLaren A. Prenatal diagnosis before implantation: opportunities and problems. Prenat Diagn 1985; 5: 85-90 https://doi.org/10.1002/pd.1970050115
  3. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990; 344: 768-70. https://doi.org/10.1038/344768a0
  4. Munne S, Dailey T, Sultan KM, Grifo J, Cohen J. The use of the first polar bodies for preimplantation genetic diagnosis of aneuploidy. Hum Reprod 1995; 10: 1014-20 https://doi.org/10.1093/oxfordjournals.humrep.a136027
  5. Munne S, Sandalinas M, Escudero T, Fung J, Gianaloi L, Cohen J. Outcome of preimplantation genetic diagnosis of translocations. Feril Steril 2000; 73: 1209-18 https://doi.org/10.1016/S0015-0282(00)00495-7
  6. Scriven PN, Flinter FA, Braude PR, Ogilvie M. Robertsonian translocations-reproductive risks and indications for preimplantion genetic diagnosis. Hum Reprod 2001; 16: 2267-73 https://doi.org/10.1093/humrep/16.11.2267
  7. 임천규, 한미현, 전진현, 송견지, 김정욱, 박소연 등. 균형 전좌 또는 Robertsonian 전좌 보인자의 체외수정 및 배아이식술에서 형광직접보합법을 이용한 착상전 유전자 진단의 임상적 적용. 대한산부학회지 2000; 43: 1147-53
  8. 김진영, 임천규, 송인옥, 유근재, 양광문, 한국선 등. 유전질환 및 염색체 이상의 예방을 위한 착상전 유전진단의 결과. 대한불임학회지 2002; 29: 269-78
  9. 임천규, 민동미, 이형송, 김진영, 궁미경, 강인수 등. 착상전 배아의 분리된 할구에서 중기염색체 상을 획득하기 위한 효율적인 방법의 개발에 대한 연구: 미세소관 형성 저해제의 효과. 대한불임학회지 2003; 30: 111-8
  10. Inzunza J, Iwarsson E, Fridstorm M, Rosenlund B, Hilensjo T, Blennow E, et al. Application of single-needle blastomere biopsy in human preimplantation genetic diagnosis. Prenat Diagn 1998; 18: 1381-8 https://doi.org/10.1002/(SICI)1097-0223(199812)18:13<1381::AID-PD495>3.0.CO;2-N
  11. Conn CM, Harper JC, Winston RML, Delhanty JD. Infertile couples with Robersonian translocations: preimplantation genetic analysis of embryos reveals chaotic divisions. Hum Genet 1998; 102: 117-23 https://doi.org/10.1007/s004390050663
  12. Van Assche E, Staessen C, Vegotti W, Bonduelle M, Vandervorst M, Van Steirteghem A, et al. Preimplantation genetic diagnosis and sperm analysis by fluorescencein-situ hybridization for the most common reciprocal translocation t(11;22). Mol Hum Reprod 1999; 5: 682-90 https://doi.org/10.1093/molehr/5.7.682
  13. Escudero T, Michael L, Carrel D, Blanco J, Munne S. Analysis of chromosome abnormalities in sperm and embryos from two 45,XY,t(13;14) (q10;q10) carriers. Prenat Diagn 2000; 20: 599-602 https://doi.org/10.1002/1097-0223(200007)20:7<599::AID-PD883>3.3.CO;2-H
  14. Conn CM, Cozzi J, Harper JC, Winston RML, Delhanty JD. Preimplantation genetic diagnosis for couples at high risk of Down syndromepregnancy due to parental translocation or mosaicism. J Med Genet 1999; 36: 45-50
  15. Fraccaro M, Lindsten J, Ford C, Iselius L. The 11q;22q translocation: A European collaborative analysis of 43 cases. Hum Genet 1980; 56: 21-51
  16. Iselius L, Lindsten J, Aurias A, Fraccaro M, Bastard C, Bottelli AM, et al. The 11q;22q translocation: A collaborative study of 20 new cases and analysis of 110 families. Hum Genet 1983; 64: 343-55 https://doi.org/10.1007/BF00292366
  17. Iwarsson E, Malmgren H, Inzunza J, Ahrlund-Richter L, Sjoblom P, Rosenlund B, et al. Highly abnormal cleavage divisions in preim plantation embryos from translocation carriers. Prenat Diagn 2000; 20: 1038-47 https://doi.org/10.1002/1097-0223(200012)20:13<1038::AID-PD976>3.0.CO;2-8
  18. Scriven PN, Mahony FO, Bickerstaff H, Yeong CT, Braude P, Ogilvie M. Clinical pregnancy following balstomere biopsy and PGD for areciprocal translocation carrier: analysis of meiotic outcomes and embryo quality in two IVF cycles. Prenat Diag 2000; 20: 587-92 https://doi.org/10.1002/1097-0223(200007)20:7<587::AID-PD873>3.0.CO;2-W
  19. Wells D, Escudero T, Levy B, Hirschhorn K, Delhanty JDA, Munne S. First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy. Fertil Steril 2002; 78: 543-9 https://doi.org/10.1016/S0015-0282(02)03271-5
  20. Malmgren H, Sahlen S, Inzunza J, Aho M, Rosenlund B, Fridstorm M, et al. Single cell CGH analysis reveals a high degree of mosaicism in human embryos from patients with balanced structural chromosome aberrations. Mol Hum Reprod 2002; 8: 502-10 https://doi.org/10.1093/molehr/8.5.502