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

- 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, History of Sperm Chromosome Analysis

FISH Procedures Probe DNATarget DNA within cells Denaturation Hybridization Immunocytochemistry Conuterstaning

Direct method 형광 물질을 직접 probe DNA 에 결합시킨 후 target DNA 와 hybridization Indirect method biotin 또는 digoxigenin-labelled probe 를 이용하여 target DNA 와 hybridization 시키고 peroxidase + DAB 또는 FITC(green), rhodamine(red) 로 발색 반응을 유도 FISH Procedures

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

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

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

l Nullisomy : 대상 염색체에 대한 signal 은 없고 다른 염색체에 대한 signal 은 한 개가 관찰될 때 l Disomy : 대상 염색체에 대한 signal 은 두 개면서 다른 염색체에 대한 signal 은 한 개가 관찰될 때 l Diploidy : 대상 염색체에 대한 signal 은 두 개면서 다른 염색체에 대한 signal 도 두 개가 관찰될 때 FISH Signal Interpretation

Normal Disomy Nullisomy Diploidy FISH Interpretation

Normal Disomy Nullisomy Diploidy FISH Interpretation X X X X Y Y Y Y X X X YY Y

FISH Adavntageshigher efficiency interphase relatively short time ~1x10 4 per patients Limits decondensation method nullisomy ~ DDx with hybridization failure disomy ~ DDx with splitted signals d/t over-decondensation a few chromosomes

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

= 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

= Number of spermatozoa to score ~the size of the sample will affect the validity of the result (1x10 4 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

= 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

= 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

Sperm chromosome abnormalities in sperms from normal men using hamster technique hyperhaploid1.00% aneuploid3.69% consevative estimate of aneuploidy2.00% structural abnormalities8.44% sex ratio(X:Y)52:48 Kearns et al., 1996

Sperm chromosome abnormalities in sperms from normal men using FISH technique Disomyautosome0.06 ~ 0.26% gonosome0.4% estimated total aneuploidy7.5% Kearns et al., 1996

46,XY Meiosis I NDJ 22,--24,XY 22,-- 24,XY 46,XY Meiosis II NDJ 23,X23,Y 22,--24,XX22,--24,YY 22,-- = 24,XX + 24,XY + 24,YY 24,XX : 24,XY : 24,YY = 1 : 2 : 1

24,XY24,XX24,YY Sex chromosome disomy rate = 0.4% 47,XXY conceptus 24,XY 47,XXY liveborn 0.2% ~0.4% ~0.2% x2x1/2 0.1% 0.2% 0.1%

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

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% Chromosomal abnormality after ICSI

-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) FISH results in sperms of infertile males with normal karyotypes

-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) -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) FISH results in sperms of infertile males with normal karyotypes

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

~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

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)

-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) FISH results in sperms of infertile males with abnormal karyotypes -Roland et al.(1998) -XXY/XXXY/XY -hyperhaploidy :7.5% 24,XX : 2.0% 24,XY : 5.0% 25,XXY : 0.5%

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

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