Sperm DNA fragmentation: new tool in the management of the male from infertile couples.

Impact of lifestyle and environment

Aleksander Giwercman

Dept. of Clinical Sciences & Reproductive Medicine CentreLund University

MalmöSweden

Lund University / Faculty / Department / Unit / Document / Date

Conflict of interest declaration

• I declare no commercial relationships or other activities that may be perceived as a potential conflict of interest.

Lund University / Faculty / Department / Unit / Document / Date

Infertility is a result of dysfunction in two subjects

Therefore• It will not be possible to develop

a sperm test predicting fertility, aswe have no good way of assessingfemale fertility

• The best we can achieve is topredict in/subfertility

50% 50%

Lund University / Faculty / Department / Unit / Document / Date

?

?

? ?

??

?

Lund University / Faculty / Department / Unit / Document / Date

Clinical questions to be answered by semen analysis

• Spontaneous pregnancy – no/very low chance• Intrauterine insemination - no/very low chance• Standard IVF - no/very low chance• Fertilisation, good embryo quality, pregnancy - no/very low

chance• Offspring health – risk• Can it be improved by changes in environment/lifestyle or

medical intervention?

Lund University / Faculty / Department / Unit / Document / Date

How does it work with standard semen parameters?

• Even very low concentration, motility, morphology does not exclude spontaneous pregnancy

• Poor criteria for selection of type of assisted reproduction

• No association with offspring health

• No clue about specific treatment

Bonde et al, Lancet 1998

Lund University / Faculty / Department / Unit / Document / Date

Increased sperm DNA damage in infertile men

• Several studies have shown that men from infertile couples have increased proportion of spermatozoa with DNA damage

• Is testing for sperm DNA- breaks a valuable clinical tool?

Zini et al, Urology 2001

Lund University / Faculty / Department / Unit / Document / Date

A two-step hypothesis for the origin of DNA damage in human spermatozoa

Aitken R , and De Iuliis G Mol. Hum. Reprod. 2010;16:3-13

Lund University / Faculty / Department / Unit / Document / Date

Most commonly used tests

Test What is

measuredAdvantages Disadvantages

SCSA • DNA strand breaks

• Protamination• ?

• Quick analysis• Many cells analysed• Standardised • Methodology (repeatable)• Clinical thresholds

• Need of expensive equipment

COMET • Single and double strand breaks

• Altered bases

• Allows analysis of few cells• Can detect DNA damage at

cell level• High repeatability• Clinical thresholds

• Lack of standardisation

TUNEL • Single and double strand breaks

• Microscopy or flow cytometry

• No clinical threshold• Poor standardisation

Lund University / Faculty / Department / Unit / Document / Date

Sperm Chromatin Structure Assay COMET(SCSA)

Sperm with native DNA

Evenson et al. 1980; 2002 Östling & Johansson, 1984

Singh et al. in 1988

Lund University / Faculty / Department / Unit / Document / Date

Low inter-laboratory (SCSA) and intra-laboratory (SCSA; COMET) variation

• Intra-laboratory CV:– COMET: 2-4%– SCSA: 2-5%

Giwercman et al, Fertil Steril, 2004

On average: 1% difference between Rome and Malmö

Lund University / Faculty / Department / Unit / Document / Date

?

?

? ?

??

?

Lund University / Faculty / Department / Unit / Document / Date

SCSA and natural conception

No pregnancies for DFI>30%

OR 6.54 (1.71-24.91)

Evenson et al, Hum Reprod 1999

TTP inversely associated with DFI:

starts decreasing DFI>20%

negligible at >40%

OR 7.59 (2.54-22.67)

Spanò et al, Fertil Steril 2000

DFI

Lund University / Faculty / Department / Unit / Document / Date

Prediction of fertility• Simon et al, Fertil Steril 2011:

COMET: DNA fragmentation >25%, OR for infertility – 180 (21-1600)

Giwercman et al, Int J Androl 2010

Lund University / Faculty / Department / Unit / Document / Date

Conclusion 1

• Sperm DNA integrity is a good tool for prediction of ”low chance for spontaneous pregnancy”

• SCSA can, therefore, be used for selection of couples who should be referred for assisted reproduction (ART)

Lund University / Faculty / Department / Unit / Document / Date

?

?

? ?

??

?

Lund University / Faculty / Department / Unit / Document / Date

SCSA and Intrauterine Insemination (IUI) (387 cycles)

• These results are well-matched with those for spontaneous pregnancy

• 20-30% of men fulfilling IUI criteria – DFI>30%

ORs for BP and D significantly lower in the group with DFI>30%

- 0.19 (95% CI: 0.07-0.48) for biochemical pregnancy- 0.15 (95% CI: 0.04-0.48) for delivery (adjusted for sperm conc, motility, female age and BMI)

Bungum et al, Hum Reprod, 2007

Lund University / Faculty / Department / Unit / Document / Date

ICSI vs. IVF (611 cycles)

DFI < 30% NSDFI > 30% Biochemical pregnancy OR (95% CI) 3.0 (1.4-6.2)

Delivery OR (95% CI) 2.0 (1.0-4.5)

Bungum et al, Hum Reprod, 2007

Lund University / Faculty / Department / Unit / Document / Date

Meta-analysis of sperm DNA fragmentation, IVF

Sperm DNA damage associated with lower IVF pregnancy rates Combined OR 1.57 (95% CI, 1.18 - 2.07)

Ref. Assay N OR (95% CI)

Høst et al., 2000 M-TUNEL 175 1.92 (0.92-4.04)

Henkel et al., 2003 F-TUNEL 208 2.24 (1.09-4.58)

Huang et al., 2005 F-TUNEL 217 1.30 (0.66-2.56)

Boe-Hansen et al., 2006

SCSA 139 2.43 (0.28-20.83)

Borini et al., 2006 F-TUNEL 82 1.66 (0.33-8.28)

Lin et al., 2007 SCSA 137 0.88 (0.35-2.19)

Benchaib et al., 2007

M-TUNEL 84 0.46 (0.11-2.00)

Bungum et al., 2007

SCSA 388 1.24 (0.69-2.26)

Frydman et al., 2007

FCM-TUNEL 117 2.97 (1.39-6.32)

Zini & Sigman, J Androl, 2008

Lund University / Faculty / Department / Unit / Document / Date

Meta-analysis of sperm DNA fragmentation, ICSI

Sperm DNA damage not associated with ICSI pregnancy rates Combined OR 1.14 (95% CI, 0.86 - 1.54)

Ref. Assay N OR (95% CI)

Høst et al., 2000 M-TUNEL 61 0.79 (0.28-2.25)

Henkel et al., 2003

F-TUNEL 54 3.67 (1.12-12.0)

Gandini et al., 2004

SCSA 22 0.36 (0.06-2.08)

Huang et al., 2005

F-TUNEL 86 1.80 (0.76-4.27)

Zini et al., 2005 SCSA 60 0.87 (0.23-3.22)

Check et al., 2005

SCSA 104 1.34 (0.52-3.43)

Boe-Hansen et al., 2006

SCSA 47 0.76 (0.21-2.72)

Borini et al., 2006 F-TUNEL 50 7.36 (1.67-32.4)

Lin et al., 2007 SCSA 86 1.21 (0.45-3.23)

Benchaib et al., 2007

M-TUNEL 218 1.55 (0.70-3.41)

Bungum et al., 2007

SCSA 223 0.65 (0.37-1.14)

Zini & Sigman, J Androl, 2008

Lund University / Faculty / Department / Unit / Document / Date

COMET and IVF/ICSI

• DNA Fragmentation vs. motility in predicting IVF fertilisation rate (>70%)

– Specificity 93% vs. 78%– OR 24 vs. 4.8

• No predictive value in relation to ICSI outcome

DNA fragmentation

(%)

Couples(%)

Life births(%)

0-24 16 26

25-50 43 19

>50 41 12Simon et al, RBM Online, 2013

Lund University / Faculty / Department / Unit / Document / Date

Conclusion 2

• Sperm DNA testing has good predictive value in relation to IUI-failure

• Sperm DNA testing may be valuable in relation to choice of IVF vs. ICSI

Lund University / Faculty / Department / Unit / Document / Date

?

?

? ?

??

?

Lund University / Faculty / Department / Unit / Document / Date

Sperm DNA integrity and pregnancy loss after ART

11 studies, 1549 IVF/ICSI cycles, 640 pregnancies, 122 pregnancy losses Combined OR 2.48 (95% CI, 1.52 - 4.04)

Zini et al, Hum Reprod 2008

Lund University / Faculty / Department / Unit / Document / Date

Sperm DNA-fragmentation and fertilisation rate, embryo quality and miscarriage

Unpublished data (SCSA)

853 IVF and ICSI cycles

DFI had no predictive value in relation to:

• Fertilisation rate• Embryo quality• Miscarriage (early/late)

Generally – conflicting data

0

5

10

15

20

25

10 20 30 40 50 60 70

DFI (%)

OR

for m

isca

rria

ge

Bungum et al, Hum Reprod, 2007

998 cycles, 333 pregnancies, 77 pregn.loss

Lund University / Faculty / Department / Unit / Document / Date

Conclusion 3

• Some studies have indicated negative impact of high DNA-fragmentation in relation to fertilisation rate, embryo quality and miscarriage rate

• The available data are inconclusive

Lund University / Faculty / Department / Unit / Document / Date

?

?

? ?

??

?

Lund University / Faculty / Department / Unit / Document / Date

Sperm DNA-damage and offspring health

• Animal data indicating morbidity in the offspring

• Paternal smoking – increased cancer (Lee et al, Leuk Res, 2009) and cryptorchidism (Kurahashi et al, Med Sci Monit, 2005)

• Sperm DNA-damage excluding fertilisation in vivo can be overcame by IVF/ICSI

• No impact of sperm DFI (SCSA) on birth weight or gestational age (Bungum et al, Int J Androl 2011)

Lund University / Faculty / Department / Unit / Document / Date

Unexplained infertility - no longer unexplained

In 15-20% of couples with ”unexplained infertility”

– DFI 20-30% (18% of couples)– DFI >30% (8.4% of couples)

(Oleszczuk et al, Andrology, 2013)

Lund University / Faculty / Department / Unit / Document / Date

Sperm DNA damage – a treatable condition?

Lund University / Faculty / Department / Unit / Document / Date

Antioxidants and birth rate

(Showell et al, Cochrane Database Syst Rev. 2011)

Lund University / Faculty / Department / Unit / Document / Date

Smoking and sperm DNA damage

Assay used No participants Effect Reference8-OHdG 60 ↑ Shen et al. Reprod Toxicol 1997

SCSA 25 = Rubes et al. Fertil Steril 1998

SCSA 277 = Spano et al. Human Reprod 1998

TUNEL 70 ↑ Potts et al. Mutat Res 1999

SCSA 70 ↑ Potts et al. Mutat Res 1999

TUNEL 97 = Sergerie et al. Human Reprod 2000

SCSA 65 = Saleh et al. Fertil Steril 2002

COMET (alkaline) 40 = Belcheva et al. Int J Androl 2004

COMET (neutral) 257 = Trisini et al. Fertil Steril 2004

TUNEL 108 ↑ Sepaniak et al. Toxicology 2006

OxyDNA Assay 55 = Viloria et al. Fertil Steril 2009

Lund University / Faculty / Department / Unit / Document / Date

PCB exposure and sperm DNA breaks

RignellHydbom et al. EHP 2005

Lund University / Faculty / Department / Unit / Document / Date

Some urgent issues to be addressed

• Results should be repeated by other research groups

• Standardisation of methodology – common clinical cut off values

• Which methods to be applied for what

• Antioxidant treatment – Yes or No (in principle no other efficient medical treatments of non-hypogonadotropic male subfertility)

• Preventive measures (smoking; weight loss; environment) –effect on sperm DNA

Lund University / Faculty / Department / Unit / Document / Date

Acknowledgements

• Mona Bungum• Marcello Spanó• Katarina Jepson• Leif Bungum• Krzysztof Oleszczuk• Jens Peter Bonde• Gunnar Toft• Irene Leijonhufvud

Numerous medical students• Lars Lindstedt• Mattias Larsson• Adam Gustafsson • Anders Langegård• Linn Augustinson• Najia Bayat