Presented by: Linda Morrison Co-authors: Uli Schmidt, Nico Foley, Rebecca Dorey, Steven McArthur, Genea, Sydney Presented at the World Congress on Human Reproduction 2011, Melbourne

1. Analysis of an Alternative Method for
A l i f Al i M h df
Determining Sperm DNA Integrity and
Comparison to Existing SCSA Methods
and Reference Ranges
and Reference Ranges
Presented by: Linda Morrison
Co‐authors: Uli Schmidt, Nico Foley, Rebecca Dorey, Steven McArthur,
Genea, Sydney
Genea Sydney
Presented at the World Congress on Human Reproduction 2011, Melbourne
Dec 2011 1

2. DNA Fragmentation Index based on DNA staining
DFI = % cells with a red/green fluorescence ratio above
/g
an established threshold
Double‐stranded
Double stranded
Acridine Orange Normal
Single‐stranded
stain bound to DNA Damaged
shifts from green
(normal) to red
(damaged)
Dec 2011 2 © 2011 Genea Limited | genea.com.au

3. Can we Improve Sperm DNA Fragmentation Analysis?
…to assess abnormal chromatin structure
and level of DNA fragmentation
Dec 2011 3 © 2011 Genea Limited | genea.com.au

4. Yes we can!
Counts Through
↑ ‐put ↑
put ↑
Cleaner
p
Sperm outcome
Chromatin
Integrity
I i
Test
Dec 2011
SCIT 4 © 2011 Genea Limited | genea.com.au

5. Analysis after DNA in‐situ acid denaturation
SCSA SCIT
Fluorescense measured by flow Fluorescence measured by High
cytometry (FACS) detector from
y y( ) Content Analyzer (HCA) software
sperm in a sheath fluid from sperm fixed on 96‐well plates
Controls set each day by test run
C t l t hd b t t Semi‐automated controls based
S i t t d t l b d
on standards
Dec 2011 5 © 2011 Genea Limited | genea.com.au

6. Advantages of SCIT over SCSA
8h 2h
Dec 2011 6 © 2011 Genea Limited | genea.com.au

7. Clinical application of SCIT validated step‐by‐step
Change from Reference New limits
SCSA to SCIT limit check established
Dec 2011 7 © 2011 Genea Limited | genea.com.au

8. Current DFI reference limits in relation to fertility
Fertility data in natural conception attempts
Significant lack of fertility >30% DFI
30% DFI
Reasonable fertility
Reasonable fertilit 15‐30% DFI
15 30% DFI
Excellent fertility
Excellent fertility <15% DFI
<15% DFI
Dec 2011 8 © 2011 Genea Limited | genea.com.au

9. IVF conception significantly reduced with increased DFI
Dec 2011 9 © 2011 Genea Limited | genea.com.au

10. Data from 179 treatment cycles – Jan‐Jun 2010
• IVF/ICSI/
Group PICSI
> Correlation and
regression Statistics
• Correlation:
p‐value
analysis
analysis • Scatter plots
Scatter plots
• None / lost
performed • Outliers
Pregnancy
• Internal
> Scatter and range
• None / lost
individual value
individual value Foetal
Foetal • Outliers
plots raised heart • Internal
range
Miscarriage •Rate (%)
Dec 2011 10 © 2011 Genea Limited | genea.com.au

11. Generally no correlation seen between DFI and fertilisation
Dec 2011 11 © 2011 Genea Limited | genea.com.au

12. The only significant difference in fertilisation with IVF 36‐38 y group
Group N Pearson Corr for fert P‐value
ALL 179 ‐0.007 0.916
IVF 57 ‐0.043 0.749
ICSI 105 ‐0.046
0.046 0.639
PICSI 17 0.22 0.396
IVF <36 16 ‐0.004 0.987
IVF 36‐38
IVF 36 38 14 0.55
0 55 0.042*
0 042*
IVF >38 27 ‐0.27 0.173
ICSI <36 39 0.038 0.819
ICSI 36‐38 29 ‐0.19 0.323
ICSI >38 37 ‐0.03 0.862
PICSI <39
PICSI <39 12 0.08
0 08 0.804
0 804
PICSI >38 5 0.718 0.172
Dec 2011 12 © 2011 Genea Limited | genea.com.au

13. Neither high NOR low levels of DFI achieve fetal heart
PICSI
Dec 2011 13 © 2011 Genea Limited | genea.com.au

14. No fetal heart pregnancies in IVF/ICSI treatments when DFI >28% or <3%
Group Fetal hearts DFI Outliers DFI Internal Range
ALL 32 1x33, 1x1 4 to 28
IVF 8 None 3 to 23
ICSI 11 None 4 to 19
4 to 19
PICSI 6 1x33, 1x1 5 to 19
IVF <36 5 none 3 to 17
IVF 36‐38
IVF 36 38 1 none 23
IVF >38 2 none 15 & 17
ICSI <36 5 none 4 to 28
ICSI 36‐38 7 none 4 to 19
ICSI >38 1 none 14
PICSI <39
PICSI <39 6 1x33, 1x1
1x33 1x1 5 to 18
5 to 18
PICSI >38 0 n/a n/a
Dec 2011 14 © 2011 Genea Limited | genea.com.au

15. High DFI group patients who did get pregnant, subsequently miscarried
PICSI
Dec 2011 15 © 2011 Genea Limited | genea.com.au

16. Optimal pregnancy outcomes at DFI level 3‐28%
SCIT
DFI 3‐
3%
DFI <3% 8%
DFI >28%
28%
No foetal heart Good pregnancy and No foetal heart
unless PICSI used
unless PICSI used foetal heart rates
foetal heart rates unless PICSI used
unless PICSI used
Dec 2011 16 © 2011 Genea Limited | genea.com.au

17. SCIT is a viable alternative to SCSA
> 1‐ step reference range
> I
Improved efficiency and reduced
d ffi i d d d
labour time
> Potential second low reference
f f th i ti ti
group for further investigation
> Automation opportunities
Dec 2011 17 © 2011 Genea Limited | genea.com.au

18. Thank you
Thank you
Special thanks to:
Nico Foley
Uli Schmidt
Becc Dorey
Steve McArthur
Teija Peura
Dec 2011 18 © 2011 Genea Limited | genea.com.au