BASIC CYTOGENETICS AND CYTOGENETICS OF INFERTILITY

1. Basic Genetics for ART Practitioners BASIC CYTOGENETICSAND CYTOGENETICS OF INFERTILITY Richard Hall BSc SRCS Cytogenetics Department, Guy's & St Thomas' NHS Foundation Trust

2. Chromosomes? • The most important objects in the living world, for the genes they carry determine the existence and form of organisms.

3. G-banded karyotype

4. Cytogenetics? • The study of the genetic constitution of cells through the visualisation and analysis of chromosomes. • G-banding (and other traditional techniques) • Fluorescence in situ hybridization (FISH) • Molecular techniques (QF-PCR, MLPA)

5. MICROARRAYS FISH 4p dup 8p del CGH MLPA 10p del QF-PCR Chromosome analysis techniques

6. Preparation of metaphases CULTURE SYNCHRONISE HARVEST 72 hours to 14 days ANALYSE CHROMOSOMES STAIN SLIDES PREPARE SLIDES

7. Traditional microscopy METAPHASE Low power x100 High power x1000

8. Traditional microscopy High power (1000x) view. Next stage of analysis involves locating each chromosome pair and comparing them band for band. Random distribution of chromosomes can hinder the accuracy and efficiency of the band comparison. Typically 1000 bands per cell.

9. Chromosome abnormalities • Aneuploidy • too many chromosomes • too few chromosomes • Rearrangements • translocations • balanced • unbalanced • inversions

10. Chromosome abnormalities Chromosome abnormalities seen in adults referred for: • infertility mostly sex chromosome aneuploidyrearrangements involving sex chromosomes • recurrent miscarriage balanced chromosome rearrangements e.g. translocations and inversions 2.5% 6% However, up to 50% of first trimester loss is due to foetal chromosome abnormality – mostly de novo

11. +16 Other 45,X autosomal trisomy Triploidy Other 46,N Spontaneous abortion products 15% of first trimester pregnancies are lost 50% abnormal 50% normal

12. Aneuploidy • Mostly from meiotic non-disjunction. • Meiosis is the specialised cell division that generates haploid gametes. • Errors in meiotic segregation occur frequently in human females, especially in MI.

13. Chromosome abnormalities and maternal age

14. Meiosis I non-disjunction Meiosis I Meiosis II Disomic Nullisomic

15. Mosaicism • The presence of two or more cell lines that are genetically identical, except for the chromosomal difference between them, in a single zygote. • Frequently seen in patients with sex chromosome aneuploidy. • Abnormal cell line may be in the minority.

16. Anaphase lag – loss of one X Mosaicism 47,XXY 46,XY 47,XXY 47,XXY 47,XXY 46,XY 46,XY 47,XXY/46,XY

17. Turner syndrome • High mortality in first trimester foetuses • Oedema of extremities • Coarctation of the aorta • Webbed neck Classical karyotype = 45,X (45%)

18. Turner syndrome Phenotype very variable, often mild and dependant on karyotype • Short stature • Increased carrying angle • Infertility 7% mosaic, eg 45,X/46,XX 45% structural abnormality, eg 46,X,i(X)(q10)

19. Structural abnormalities of theX-chromosome Monosomy for short arm is associated with features of Turner syndrome or primary ovarian failure The location of the breakpoint in the X may influence gonadal function Partial monosomy for, or balanced rearrangements with, breakpoint in long arm more likely to be associated with premature ovarian failure

20. Structure of the X chromosome • Xp11.2-p22.1 • Ovarian failure (gonadal dysgenesis) • Xq13 • X inactivation centre (XIST) • Xq13-q26 • ‘Critical region’ for ovarian function • Breakpoints within this region are associated with gonadal insufficiency • Except breakpoints in Xq22

21. Klinefelter syndrome • Incidence = 1/1000 • Usually taller than average • Disproportionately long limbs • 30–50% gynaecomastia • Infertility/azoospermia • IQ may be reduced relative to siblings Example karyotypes = 47,XXY 47,XXY/46,XY

22. Klinefelter syndrome • Phenotype very variable – some patients are not diagnosed until they try for a family. • Mosaics 47,XXY/46,XY may have milder phenotype and may be fertile. • Therefore always carry out mosaicism check as infertility is the main clinical problem.

23. Chromosome translocations • Exchange of material between chromosomes • Two types • Robertsonian • reciprocal

24. Normal male karyotype: 46,XY Acrocentric chromosomes

25. Robertsonian translocationsder(14;21)(q10;q10)

26. Robertsonians and infertility • Some male carriers are infertile as they have spermatogenic arrest. • Thought to be due to failure of pairing of the translocation in meiosis, which allows it to interfere with the X-Y bivalent. • The more often this occurs the greater the effect on the sperm count. • Prevalence of 1 in 1000. • 10x excess in infertile men.

27. Behaviour at meiosis Robertsonians and miscarriage Female carriers of der(14;21) have 10% risk of Down’s syndrome child Alternate segregation Adjacent segregation

29. Robertsonian translocations Summary • Result from fusion of two acrocentric chromosomes (13, 14, 15, 21, 22) • Prevalence of 1 in 1000 • Balanced carriers have reproductive risks present as: • recurrent miscarriage • Patau syndrome • Down’s syndrome • male infertility

30. Reciprocal translocations • Exchange of material between two non-homologous chromosomes • Prevalence of 1 in 500 • Balanced carriers are generally phenotypically normal • Reproductive consequences because of behaviour at meiosis

31. Behaviour at meiosis • The homologous chromosomes cannot pair properly • Instead they must form a quadrivalent

32. Alternate segregation Balanced (like carrier parent) Normal

33. Unbalanced trisomy and monosomy Unbalanced monosomy and trisomy Adjacent-1 segregation

34. Reciprocal translocations:reproductive risks • For most translocations, ~50% of conceptions will have either normal chromosomes or the balanced translocation. • Unbalanced products result in: • miscarriage (large segments) • dysmorphic delayed child (small segments).

35. Reciprocal translocations Summary • Chromosome rearrangements are rare, but chromosome analysis is indicated if a couple have had three or more miscarriages of unknown aetiology. • Essential that both partners are investigated as either the male or the female could carry a balanced rearrangement. • Aneuploidy is the most common chromosomal cause of early miscarriage and requires no follow-up.

36. Suggested reading • Gardner, RJM & Sutherland GR (2004). Chromosome abnormalities and genetic counseling. 3rd edn. Oxford University Press, New York.