Meiotic Errors and Spermatogenesis Mexico City, 2007

Rene Martin, Ph.D., FCCMG Professor, Dept. of Medical Genetics University of Calgary Canada Research Chair in Genetics

Chromosome Abnormalities

very common in humans

.6%

newborns

6% stillborns spontaneous abortions

60%

estimates

at conception: 20 - 50%

Animal Models
Frequency of chromosome abnormalities in gametes and zygotes:
laboratory rodents domestic animals primates

0 - 5% 1 - 8% 12%

Cause of Chromosomal Abnormalities

very little information produced in eggs and sperm, but most die as embryos - information lost

need to study chromosome abnormalities in human eggs and sperm

Methods to Study Chromosomes in Human Spermatozoa

sperm karyotypes

hamster oocytes fused with human sperm

fluorescence in situ hybridization (FISH)

chromosome-specific DNA probes
single sperm analysis for research analysis of single genes

polymerase chain reaction (PCR)

cytoimmunofluorescence of meiotic chromosomes

analysis of recombination

Distribution of Aneuploidy Among Chromosome Groups

clues about etiology of aneuploidy

all chromosomes equal frequency? certain chromosomes predisposed

Aneuploidy in Humans
13 18 21 sex chromosomes susceptible to nondisjunction or compatible with survival

newborns:

trisomy

11,615 Sperm Karyotypes

hyperhaploidy in all chromosome groups all chromosomes susceptible to nondisjunction significant increase for chromosome 21 and sex chromosomes (p=.0001)

Sperm Karyotyping

plus
precise

sperm karyotypes numerical and structural abnormalities information on all chromosomes

minus
technically

difficult, slow, expensive available in only a few labs worldwide requires sperm with fertilization capability

FISH Studies in Human Sperm

study interphase sperm

no

fertilization

rapid, inexpensive increase sample size

from

11,000 to >10,000,000

Mean Disomy Frequency in Sperm

Chromosome 1 2 4 9 12 13 15 16 18 20 21 22 sex % Disomy .09 .08 .11 .14 .16 .19 .11 .11 .11 .12 .29* p<.001 1.21* p<.001 .43* p<.001

* = significant

Nondisjunction in Individual Chromosomes

FISH results corroborate results from sperm karyotypes increased frequency of aneuploidy for G group chromosomes (21 & 22) and sex chromosomes in human sperm

Other FISH Studies

increased frequency of disomy for sex chromosomes

Williams et al., Spriggs et al., Scarpato et al., Kunathikom et al.,

1993 1995 1998 2002

increased frequency of disomy 21

Spriggs et al., Blanco et al., Oliver-Bonet et al.,

1996 1996 2001

Increased Susceptibility to Nondisjunction

G-group (21 and 22) and X-Y bivalent have only one crossover

if recombination absent or reduced, may increase the chances of nondisjunction

Most Sex Chromosomal Aneuploidies Result from Paternal Nondisjunction

paternal: 47,XYY 45,X 47,XXY 47,XXX

100% 80% (Hassold et al., 1988) 50% (Jacobs et al., 1988) 7% (May et al., 1990)

Meiotic Pairing of Sex Chromosomes

recombination required in pseudoautosomal region

necessary

for proper segregation of sex chromosomes

decreased recombination in 47,XXY of paternal origin (Hassold et al., 1991)

Single Sperm Typing

to determine if there is a relationship between recombination in the pseudoautosomal region and nondisjunction compared frequency of recombination between STS/STS pseudogene (sex specific locus) and DXYS15 (pseudoautosomal locus) in unisomic vs disomic sperm

Heterozygous Male

Methods

sperm sonicated to remove tails

flow cytometer separates individual sperm
single

sperm into 96-well plates (+ve and -ve controls)

disomic sperm (24,XY) identified by FISH, scraped off slide & micromanipulated into individual tubes PCR: DXYS15 and STS/STS pseudogene

Results of Single Sperm Typing

329 unisomic sperm - 38% recombination 150 disomic (24,XY) - 25% recombination significant decrease in recombination in XY sperm (p=.001)

lack of recombination directly linked to nondisjunction

Chromosome Abnormalities in Male Infertility

somatic chromosomal abnormalities

consequences

in sperm

infertile men with normal karyotypes

chromosomal

abnormalities in sperm

Sperm Chromosomal Abnormalities in Infertile Men

oligo-, aestheno- or teratozoospermia 46,XY somatic karyotype

518 sperm karyotypes

significant

increase in aneuploidy (4X) 1,12, X, Y

FISH on 103,130 sperm

chromosome significant

increase in disomy 1 and XY (3X)

Infertile Men

FISH analysis for chromosomes 13 & 21

90,809 sperm from 9 infertile men

significant increase in the frequency of disomy for chromosomes 13 (p<.0001) and 21 (p<.0001), and of diploidy (p<.0001) may not have as much clinical significance as XY disomy since greater embryonic lethality

Infertile Men: Other 3-Colour FISH Studies

>30 multicolour FISH studies

most

show an increased frequency of disomy for autosomes and sex chromosomes

severe infertility (OAT) or mixed infertility

mainly

Increased Frequency of Chromosome Disorders After ICSI

Van Opstal et al., 1997

6/71 prenatal diagnosis: 8% abnormal sex chrom. abnormalities: all paternal origin

Aboulghar et al., 2001

15/430 consecutive babies abnormal: 3.5% sex chromosomes and autosomes

Devroey & van Steirteghem, 2004

2622 fetal karyotypes: 3% abnormal 1.6% de novo: sex chromosomes and autosomes 1.4% inherited

Immunofluorescence Methods to Study Meiosis

allows study of recombination in all chromosomes analysis of chromosome pairing by visualization of synaptonemal complex antibodies to:

synaptonemal

complex (SCP1/SCP3) recombination foci (MLH1) centromeres (CREST)

Lepto-diplotene meiosis diagram

8-1-20

Analysis of Synaptonemal Complexes 27 Normal men

testicular samples from vasectomy reversals (15) and cancer patients (12) recombination foci - mean 48.5/cell

90% cells in pachytene

5% cells have at least 1 bivalent with no

recombination foci

no significant difference in vasectomy reversals vs cancer patients

Infertile Men with Pachytene Cells

6/6 men with obstructive azoospermia (OA) had pachytene cells

mainly

congenital absence of the vas deferens (CF)

14/29 men with nonobstructive azoospermia (NOA) had pachytene cells

14

men with no meiotic cells 1 man with a block at zygotene

SC 12-3-2

Mean Frequency of Recombination

49 47 43

Control

OA*

NOA*

*p<0.0001, nested ANOVA

% Cells with at Least 1 Bivalent with no Recombination

29

9 5

Control A

OA

NOA*

*p=0.0005, Z-test

noa sc h7-3-5

cenM-FISH / Karyotyped SC

Summary

in both NOA and OA, abnormalities in:

chromosome pairing decreased frequency of recombination increased frequency of bivalents with no recombination foci

could lead to meiotic arrest or increased frequency of aneuploid sperm

Acknowledgements

Qinghua Shi Maria Oliver-Bonet Fei Sun Nafisa Moosani Monica Mikhaail-Philips Jena Smith Brenda McInnes

Evelyn Ko Paul Turek Cal Greene

Peter Moens Marv Fritzler Terry Ashley Fred Rademaker

Canadian Institutes of Health Research Canada Research Chair in Genetics