BASIC CYTOGENETICS AND CYTOGENETICS OF INFERTILITY

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BASIC CYTOGENETICSAND CYTOGENETICS OF
INFERTILITY
Basic Genetics for ART Practitioners

• Richard Hall BSc SRCS
• Cytogenetics Department, Guy's St. Thomas' NHS
  Foundation Trust.

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CHROMOSOMES ?

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

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G-banded Karyotype
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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)

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CHROMOSOME ANALYSIS TECHNIQUES
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Preparation of Metaphases
CULTURE
SYNCHRONISE
HARVEST
72 HOURS TO 14 DAYS
ANALYSE CHROMOSOMES
STAIN SLIDES
PREPARE SLIDES
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TRADITIONAL MICROSCOPY
METAPHASE
LOW POWER x100
HIGH POWER x1000
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TRADITIONAL MICROSCOPY
High power (1000x) view. Next stage of analysis
involves locating each chromosomes 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.
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CHROMOSOME ABNORMALITIES

• ANEUPLOIDY
• too many chromosomes
• too few chromosomes
• REARRANGEMENTS
• translocations
• balanced
• unbalanced
• Inversions

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CHROMOSOME ABNORMALITIES

• Chromosome abnormalities seen in adults referred
  for
• Infertility -
• MOSTLY SEX CHROMOSOME ANEUPLOIDY
• REARRANGEMENTS INVOLVING SEX CHROMOSOMES
• Recurrent Miscarriage
• BLANCED CHROMOSOME REARRANGEMENTS
• E.g. translocations and inversions

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HOWEVER UPTO 50 OF FIRST TRIMESTER LOSS IS DUE
TO FETAL CHROMOSOME ABNORMALITY MOSTLY DE NOVO
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SPONTANEOUS ABORTION PRODUCTS
15 first trimester pregnancies are lost
50 ABNORMAL
50 NORMAL
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Aneuploidy

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

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Chromosome abnormalities and maternal age
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MEIOSIS I NON-DISJUNCTION
MEIOSIS I
MEIOSIS II
DISOMIC
NULLISOMIC
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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

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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
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TURNER SYNDROME
High mortality in first trimester fetuses oedema
of extremities coarctation of the aorta webbed
neck
Classical karyotype 45,X (45)
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TURNER SYNDROME

• Phenotype very variable, often mild and dependant
  on karyotype
• short stature
• increased carrying angle
• Infertility

7 mosaic e.g. 45,X/46,XX 45 structural
abnormality e.g.46,X,i(X)(q10)
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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
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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

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Klinefelter Syndrome

• Incidence 1/1000
• Usually taller than average
• Disproportionately long limbs
• 30-50 gynaecomastia
• Infertility / Azoospermia
• I.Q may be reduced relative to sibs

Example karyotypes 47,XXY 47,XXY/46,XY
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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

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CHROMOSOME TRANSLOCATIONS

• Exchange of material between chromosomes
• 2 types
• Robertsonian
• Reciprocal

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NORMAL MALE KARYOTYPE 46,XY
Acrocentric chromosomes
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ROBERTSONIAN TRANSLOCATIONSder(1421)(q10q10)
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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 of Robs in infertile men

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Robertsonians and Miscarriage
Behaviour at meiosis
Female carriers of der(1421) have 10 risk of
Down syndrome child
alternate segregation
adjacent segregation
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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 miscarriages
• Patau syndrome
• Down syndrome
• male infertility

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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

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Behaviour at meiosis

• The homologous chromosomes cannot pair properly
• Instead they must form a quadrivalent

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Alternate segregation
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Adjacent-1 segregation
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RECIPROCAL TRANSLOCATIONSREPRODUCTIVE 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)

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RECIPROCAL TRANSLOCATIONS

• Summary
• Chromosome rearrangements are rare, but
  chromosome analysis is indicated if a couple have
  had 3 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

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Suggested reading

• Gardner, RJM. Sutherland GR. (2004). Chromosome
  abnormalities and genetic counseling. 3rd Ed.
  Oxford University Press, New York.