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Chromosome Analysis of Spermatozoa using FISH

Chromosome Analysis of Spermatozoa using FISH. = FISH 를 이용한 정자의 염색체 분석 =. Jee Byung Chul, M.D. Department of OBGY, College of Medicine, Cheju National University. History of Sperm Chromosome Analysis. - Heterochromatic staning : 1, 9, Y(‘70)

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Chromosome Analysis of Spermatozoa using FISH

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  1. Chromosome Analysis of Spermatozoa using FISH = FISH를 이용한 정자의 염색체 분석 = Jee Byung Chul, M.D. Department of OBGY, College of Medicine, Cheju National University

  2. History of Sperm Chromosome Analysis - Heterochromatic staning : 1, 9, Y(‘70) - Zona-free hamster oocytes(Rudak et al., 1978) : mataphase, conventional cytogenetics - In situ hybridization, Fluorescence in situ hybridization(FISH), Dual color, Multi-color FISH : interphase,

  3. FISH Procedures Probe DNA Target DNA within cells Denaturation Hybridization Immunocytochemistry Conuterstaning

  4. FISH Procedures Direct method 형광 물질을 직접 probe DNA에 결합시킨 후 target DNA와 hybridization Indirect method biotin 또는 digoxigenin-labelled probe를 이용하여 target DNA와 hybridization 시키고 peroxidase + DAB 또는 FITC(green), rhodamine(red)로 발색 반응을 유도

  5. (4) Telomeric Probes (1) Centromeric Probes (2) Whole Chromosome Painting Probes (3) Chromosome-specific Unique Sequence Probes

  6. 1. Centromeric probe : centromere 또는 pericentromere에 존재하는 repetitive DNA 서열로서 염색체 수적 이상을 진단하는데 주로 이용 2. Whole chromosome painting probe : 단일 염색체내 특정부위에 대한 각각의 서열들을 모은 많은 probe의 집합체로 중기 염색체에서 염색체 수적 이상, marker 염색체의 기원, translocation을 분석하는데 이용

  7. 3. Unique sequence probe : Locus-specific DNA segment를 가지며 유전자 특이 서열을 검색하는데 이용, 특히 특정 염색체 내의 microduplication 혹은 microdeletion을 검색하는데 이용 4. Telomeric, subtelomeric probe : 가장 최근에 개발되기 시작한 probe로 reciprocal translocation, hidden tranlocation 등을 검색하고자 할 때 이용

  8. FISH Signal Interpretation • Nullisomy : 대상 염색체에 대한 signal은 없고 다른 염색체에 대한 signal은 한 개가 관찰될 때 • Disomy : 대상 염색체에 대한 signal은 두 개면서 다른 염색체에 대한 signal은 한 개가 관찰될 때 • Diploidy : 대상 염색체에 대한 signal은 두 개면서 다른 염색체에 대한 signal도 두 개가 관찰될 때

  9. FISH Interpretation Disomy Diploidy Normal Nullisomy

  10. X X Y FISH Interpretation X Y Y X Disomy Y X X Y Normal X Y Y Nullisomy Diploidy

  11. FISH Adavntages higher efficiency interphase relatively short time ~1x104 per patients Limits decondensation method nullisomy ~ DDx with hybridization failure disomy ~ DDx with splitted signals d/t over-decondensation a few chromosomes

  12. Technical Aspects 1. Decondensation of sperm nucleus 2. Number of spermatozoa to score 3. Use of internal controls 3. Scoring criteria 4. Specificity of DNA probes

  13. = Decondensation of sperm nucleus ~sperm chromatin is highly compacted ~decompacting agent (DTT) is needed to make DNA sequences accessible to probes ~decondensation method is an important step to increase hybridization efficiency

  14. = Number of spermatozoa to score ~the size of the sample will affect the validity of the result (1x104 per probe) ~the higher the number of spermatozoa scored, the closer the obtained values will become to mean of the population, ~especially in low frequency of aneuploidy

  15. = Use of internal control ~to differentiate between disomy and diploidy autosomal aneuploidy -> at least two probes; one specific probe + one control probe gonosomal aneuploidy -> at least three probes; X probe + Y probe + one control probe

  16. = Scoring criteria “strict scoring criteria”(Williams et al., 1993) sperm nuclei are considered to be disomic if hybridization yielded two compact distinct signals of equal size that were separated from each other by a distance of at least one signal domain within that cell

  17. Sperm chromosome abnormalities in sperms from normal men using hamster technique hyperhaploid 1.00% aneuploid 3.69% consevative estimate of aneuploidy 2.00% structural abnormalities 8.44% sex ratio(X:Y) 52:48 Kearns et al., 1996

  18. Sperm chromosome abnormalities in sperms from normal men using FISHtechnique Disomy autosome 0.06 ~ 0.26% gonosome 0.4% estimated total aneuploidy 7.5% Kearns et al., 1996

  19. 46,XY 46,XY Meiosis I NDJ 22,-- 24,XY 23,X 23,Y Meiosis II NDJ 22,-- 22,-- 24,XY 24,XY 22,-- 24,XX 22,-- 24,YY 22,-- = 24,XX + 24,XY + 24,YY 24,XX : 24,XY : 24,YY = 1 : 2 : 1

  20. Sex chromosome disomy rate = 0.4% 24,XX 24,XY 24,YY 0.1% 0.2% 0.1% x2 x1/2 47,XXY conceptus 47,XXY liveborn 24,XY 0.2% ~0.4% ~0.2%

  21. Genetic Abnormalities in Infertile Males Cytogenetic abnormality -> Somatic cell abnormality -> Gamete (Sperm) abnormality Y microdeletion

  22. Chromosomal abnormality after ICSI Liebaers et al.(1995) ~ Belgian Group 0.85% sex chromosomal abnormality In't Veld et al.(1995) 5/15 sex chromosomal abnormality Wisanto et al.(1996) ~ 1.2% overall chromosomal abnormality in prenatal Dx ESHRE(1996) ~ 2.2% overall chromosomal abnormality in prenatal Dx 1.1% overall chromosomal abnormality in postnatal Dx Bonduelle et al.(1998) 2.5% overall chromosomal abnormality in prenatal Dx(n=1082) de novo chromosomal aberration : 1.66% sex chromosomal aberration : 0.83% autosomal chromosomal aberration : 0.83% inherited chromosomal aberration : 0.92%

  23. FISH results in sperms of infertile males with normal karyotypes -Ushijima1 et al.(2000) -X/Y, 13, 18, 21 -OAT(n=8), control(n=10) -Aneuploidy(Disomy) 13;0.13% vs 0.09%(sig) 18;0.12% vs 0.13%(ns) 21;0.24% vs 0.19%(sig) X/Y;0.59% vs 0.38%(sig) -Diploidy 0.29% vs 0.16%(sig) -Nishikawa et al.(2000) -X/Y, 18 -OAT(n=10), control(n=5) -Aneuploidy(Disomy) XY;0.36% vs 0.14%(sig) XX;0.10% vs 0.15%(ns) YY;0.13% vs 0.14%(ns) 18;0.15% vs 0.14%(ns) -Diploidy 0.14% vs 0.10%(ns)

  24. FISH results in sperms of infertile males with normal karyotypes -Damri et al.(2000) -X/Y, 18 -ICSI candidates(n=12) control(n=3) -Aneuploidy(Disomy) sex;0.28 vs 0.18%(ns) 18;0.11 vs 0.06%(ns) -Diploidy 0.11% vs 0.05%(ns) -Schultz et al.(2000) -X/Y, 13, 18, 21 -infertile male(n=5) control(n=7) -Aneuploidy(Disomy) sex;0.44 vs 0.43%(ns) -Diploidy 0.22% vs 0.20%(ns)

  25. Defective spermatogenesis systemic disease, malnutrition endocrinological disorders anatomical obstruction infection, environmental toxins genetic defects ~ meiotic abnormalities, non-disjunction within testes

  26. ~Incidence of aneuploidies were significantly higher in peripheral lymphocytes and spermatozoa of infertile men compared with donors. ~There was a positive correlation between the incidence of chromosome aneuploidies in the somatic cells and sperm. ~mitotic instability in idiopathic oligozoospermia. Gazvani et al., 2000

  27. FISH results in sperms of infertile males with abnormal karyotypes -Guttenbach et al.(1997) -47,XXY (n=2,206) -normal : 82.2% 23,X : 43.4% 23,Y : 48.8% -disomy : 2.68% XX : 1.22%(sig) XY : 1.36%(sig) YY : 0.10%(ns) -diploidy : 0.23%(sig)

  28. FISH results in sperms of infertile males with abnormal karyotypes -47,XXY/46,XY -increased incidence of 24,XY 0.92%(Cozzi et al., 1994) 1.3%(Martini et al., 1996) 2.09%(Chevret et al., 1996) -Roland et al.(1998) -XXY/XXXY/XY -hyperhaploidy :7.5% 24,XX : 2.0% 24,XY : 5.0% 25,XXY : 0.5%

  29. How can we select chromosomally normal sperms? - Pfeffer et al.(1999) The type and percentage of aneuploid sperm for all patients with OAT found in both swim-up and pellet fractions were not different, with the exception of diploid sperm - Van Dyk Q et al.(2000) : Hemi-zona assay The zona-bound spermatozoa had a significantly lower frequency of aneuploidy than the swim-up motile fraction or the pellet fraction

  30. Thank You!

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