1. BASIC CYTOGENETICS AND CYTOGENETICS OF INFERTILITY
Basic Genetics for ART Practitioners
BASIC CYTOGENETICS AND CYTOGENETICS OF INFERTILITY
INTRODUCE MYSELF
TALK ABOUT TYPES OF CHROMOSOME ABNORMALITIES SEEN DURING PREGNANCY
THE PHENOTYPES ASSOCIATED WITH THESE ABNORMALITIES
THE WAYS IN WHICH WE DETECT THEM
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.
You will spend the whole of your career examining the most important objects in the living world

3. G-banded karyotype
Groups
Size and position of centromere

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

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 aneuploidy rearrangements 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. Spontaneous abortion products
15% of first trimester pregnancies are lost
46,N
+16
45,X
Triploidy
Other
autosomal
trisomy
50% abnormal
TRISOMY FOR ALL C’SOMES SEEN (EXCEPT 1) – ACCOUNTS FOR 60% OF ALL ABNORMALITIES
MOST COMMON T16 AND 45,X (APPROX 20%)
POLYPLOIDY (MOSTLY TRIPLOIDY) 15%
REMAINING ABNORMALITIES (5%) ARE MOSTLY STRUCTURAL REARRANGEMENTS
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
This is backed up by all major studies. This is a typical one showing a massive increase in the proportion of trisomic pregnancies among older women.

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. Mosaicism Anaphase lag – loss of one X 47,XXY 46,XY 47,XXY 47,XXY
When a chromosome fails to connect to the spindle apparatus or “lags behind” when chromosomes are segregating to the poles this is termed anaphase lag
The chromosome in question fails to be included in the reforming nuclear membrane and instead the single chromosome in cytoplasm forms a micronucleus which is then lost
47,XXY/46,XY

17. Turner syndrome High mortality in first trimester foetuses
Oedema of extremities
Coarctation of the aorta
Webbed neck
NECK WEBBING AND OEDEMA
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 the X-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 Example karyotypes = 47,XXY 47,XXY/46,XY
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
FIRST TALK ABOUT ROBERTSONIAN TRANSLOCATIONS
INVOLVES THE ACROCENTRIC CHROMOSOMES

24. Normal male karyotype: 46,XY
Acrocentric chromosomes
IN BALANCED CARRIERS THE CHROMOSOME NUMBER IS USUALLY 45
LOSS OF P-ARM MATERIAL
CAN BE NON-HOMOLOGOUS OR HOMOLOGOUS

25. Robertsonian translocations der(14;21)(q10;q10)
MOST COMMON ROB IS DER(13;14)
75% OF ALL ROBS
ARROWS INDICATE BREAKPOINTS ON THE NORMAL HOMOLOGUES

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

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
14;21 ACCOUNTS FOR APPROX 10% OF ROBS
HOMOLOGOUS ROBS WILL ALWAYS RESULT IN IMBALANCE
UPD RISK

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

32. Alternate segregation
Normal
Balanced (like carrier parent)

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

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).
In fact the t(2;17) was first detected in an unbalanced form in a fetus that had died in utero. The fetus had inherited the derivative 17 via adjacent 1 segregation and had lissencephally associated with deletion of the LIS1 gene on the short arm of c’some 17

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.
This happens by chance, is more likely to happen as maternal age increases and does not require analysis of parental blood

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