Relevance of Sperm DNA Fragmentation - eshre

Relevance of Sperm DNAFragmentationMarianne MoserLandes-Frauen- und KinderklinikLinz, Austria

Relevance of Sperm DNAFragmentation1. Sperm DNA peculiarity2. Etiology of DNA fragmentation (DF)3. Test methods4. Influence of laboratory techniques5. DF and outcome in infertility treatments6. Conclusions

1. Sperm DNA peculiarityDuring spermiogenesisspermatids repackagetheir DNA withprotamines, a smallresidue of histone-boundDNA is retained (15%).

protamines• Are proteins with a high content of positively chargedamino acids (48% arginine)• Form a highly condensed complex with the sperm DNA(DNA has a strong negative charge)• Incorporate cysteins• Cysteins allow the formation of disulphide bondsbetween the protamines• Therefore strongly stabilize the nucleoprotaminecomplex

In mature sperm the DNA is• 85% protamine bound• 15 % remains histone boundprotamine deficiency: more susceptible for ROSInfertile men have a higher histone:protamine ratio than fertile men(Oliva 2006, Zhang 2006)5-10% of infertile men have a complete protamine deficiency

Fig. 1: The human spermZini, A. et al. CMAJ 2006;175:495-500Copyright ©2006 Canadian Medical Association or its licensors

2. Etiology of DNA Damagethe etiology of sperm DNA damage ismultifactorial• In the testis during the process ofspermatogenesis:– Apoptosis: screening mechanisms,that mark individual apoptoticsperms which causes phagocytosisof these cells, failed (double sb)– during remodeling of spermchromatin the DNA is unwoundthrough the induction of strandbreaks (single sb), sperms withunrepaired strand breaks →semen.

Etiology of Sperm DNA Damage• post-testicular:– during sperm transport through theseminiferous tubules and the epididymis– Varicocele– Genital tract infections (leukocytes)– Immature sperms (cytoplasmic droplet)– radio- and/or chemotherapy– Lifestyle factors (obesity, cell phones,nicotine), environmental toxicants– Laboratory factors• Majority of DNA damage is associated withROS (Aitken et al., 2010)

ROS• Small levels are essential for normal spermfunctions (capacitation, acrosome reaction,sperm-oocyte fusion (Sikka et al, 1995)• Balance between ROS production andscavenging system is important• in 25% of infertile men high ROS levels havebeen detected in their semen.

levels of ROS may fluctuatwithin a fertile man but do not affectsperm concentration and motility.This may be possible due to thepresence of adequate antioxidantdefense mechanismsin the presenthealthy individual.this variations may have physiologic,seasonal, or lifestyle-related causes

3. DNA Fragmentation TestsDFI: DNA Fragmentation Index %

DNA fragmentation - Tests• DIRECT– TUNEL (terminal deoxynucleotydil dil transferasemediateddeoxyuridin triphosphate- nick-end labelingassay ) (s&dSB)– ISNT (in situ-nick translation)– Comet Assay at neutral pH (sdSB) (single cell gelelectrophoresis• INDIRECT (need denaturation of DNA)– SCSA (sperm chromatin structure assay)– SCD (sperm chromatin dispersion test, HalospermAssay)– Comet Assay at acid or basic pH (sdSB)

TUNEL assayLabels single and double strand breaks, quantifies the incorporation of flourescent dUTP

SCSA Testsperm chromatin structure assayacid-induced denaturation of DNA followed by staining with AO.is a flow-cytometric method, measures the metachromatic shift ofAO fluorescence from green to red– green (native DNA)– red (denatured DNA).

SCD or Halo TestThe SCD is based on the principlethat sperm with fragmented DNA failto produce the dispersed DNA loopsafter acidic denaturation (halos)Normal sperm produce a haloEbner et al, 2011

Observed a strong relationship between the tests.AO only coincided on values >30%, (technical problems of instinct colours, rapid fading,heterogenous staining of slides, also reported in other studies)

4. Influence of lab procedures

storage and sperm DFIUnselected groupOur data indicate that anysample that will not beanalyzed within 4 hours ofcollection should be frozen toprevent increasing i DNAdamage.Among different methods offreezing, there was no statisticallysignificant difference in theresultant amount of DNAfragmentation

The potential detrimental effect of density gradient centrifugationon sperm DNA integritiy is related to initial semen quality (Zini)Sperms of infertile patients are more susceptible to externalSperms of infertile patients are more susceptible to externalinfluences

Sperm processing and DFI• DFI Levels immediately followingprocessing were significantly lower forswim-up, density gradient and DG&SU than for fresh and washed semensamples.

90% of patients showed no strandbreaks after processing

Ebner et al, 2010

abSwim-up for maximum 2 hoursEbner et al, 2010

39 patientswith male subfertility(51% isolatedteratozoospermia(WHO 1999)).Halo testRaw semeneAfterdensity gradientAfter Zech-selectorEbner et al, 2011

DFImotilityRaw semen 15.8 ± 7.8% 36.6 ± 19.4 %a and bDensity gradient 14.2 ± 7.0% notinvestigatedZech Selector 0.4 ± 1.1% 100% a

• Motility seems to be the utmost t factor, since theselector separates spermatozoa according totheir motilty and not to their morphology.• This is supported by the literature Ramos andWetzels, 2001, Van den Berg et al 1998) and byour own observation, that the sperm swimmingclose to the surface of the supernatant showsignificantly reduced rates of DF after densitycentrifugation and swim-up.• Simply overlaying a sperm sample with medium,(w/o centrifugation) could lead to similar results.

Why do fast progressive sperms show no sign of DF?• Both nuclear and mitochondrial DNA can be harmed by strandbreaks• Mitochondrial DNA could cause alterations in ATP production,which is a prerequisite for optimal sperm motility• Deletions within mitochondrial DNA have been associated withreduced sperm motility (Ozmen et al, 2007)• It could be possible, that grade a spermatozoa could be neitherharmed by nuclear or mitochondrial DNA damage• In the selector grade b spermatozoa cannot overcome thecapillary gap between the outer and the inner ring in 2 hoursdue to their reduced energy and forward movement.

Correlation of sperm parametersand DFIYesNoOligozoospermia Burallo et al 2004Host et al 1999Tomlinson 2001Gandini et al. 2000Teratozoospermia Host et al 1999Muratori et al 2003Trisini et al 2004Tomlinson 2001Asthenozoospermia Giwercman et al 2003Irvine et al 2000Mahfouz 2010Varghese ez al 2009Chan et al. 2001Donnely et al 2001??

5. Is DFI testing relevant forfertility assessment

DFI and IUI• withaDFI< 27% the chances forpregnancy are significantly higher than inpatients with a DFI > 27%

Fertility potential, threshold levelsEvenson et al (1999), Spano (2000):If >30% sperm have abnormal chromatin: fertility ishampered independent of sperm number, morphologyand motility.Evenson et al (2002):Fertility potential according to DFI fraction:Excellent 30%

Ahi Achievement of pregnancy:– 84% of men with DFI of

• in the general population:1-4% of men have a DFI > 30%.•Infertility patients– a DFI > 30% have• IUI patients: 7%•IVF patients: 16%• ICSI patients: 33%

Retrospective study on 282 patients

23 papers that investigated the influence of DNA damage onFertilization Embryo Pregnancy PregnancyqualitylossTotal nr.of19 18 21 12papersYES 8 8 12 3NO 11 10 9 9

DFI and pregnancy loss

CONCLUSIONSsperm DNA damage is associated with a significantly increasedrisk of pregnancy loss after IVF and ICSI.The data provide a clinical indication for the evaluation of sperm DNAThe data provide a clinical indication for the evaluation of sperm DNAdamage prior to IVF or ICSI and a rationale for further investigating theassociation between sperm DNA damage and pregnancy loss.

Let´s not forget the oocyte

When oocytes from infertile patients were employed, DF had a statisticallysignificant negative impact on chance of pregnancy.For every 10% increase in DF, the probability bilit of not achieving i pregnancyincreased by 1.31.Wh d d l d DF did h i i llWhen donated oocytes were employed, DF did not have a statisticallysignificant effect

Conclusions I• Infertile men have higher DF than fertile men• Sperms of infertile men are more susceptible todamage age (ROS)• sperm with damaged DNA can successfullyfertilize - but may cause de novo mutations inthe offspring. (despite the ability of the oocyteand embryo to repair some damage)→ causes concern about the safety of ICSI• Et Extensive DF might ihtnot tbe overcome by oocyterepair mechanisms.• Oocyte repair capacity cannot be measured.

Conclusions II• Small but significant ifi association between DFand pregnancy – but- no indication for routineuse in male evaluation (Collins)• ? Indication in failed IVF, several pregnancylosses, several failed IUI´s• More studies needed to identify subgroups thatwould merit from DF tests• Apply careful sperm processing method, so notto enhance DFI• ICSI: use methods to pick out sperms withreduced risk for DF

ThomasMeManuelaRenateThank you for your attention

(infertile patients!)Infertile PatientsHDS: high DNA stainability: a measure of nuclear chromatin compactionSperm head abnormalities may in part be due to incomplete sperm chromatincondensation

Is sperm dna damage associated with IVF embryo quality?A systematic review.Zini A, Jamal W, Cowan L, Al-Hathal N.Division of Urology, Department of Surgery, McGill University, Montreal, Quebec, Canada,ziniarmand@yahoo.com.28 studies (8 IVF, 12 ICSI and 8 mixed IVF-ICSI studies) that evaluated therelationship between sperm DNA damage and embryo quality.3226 treatment cycles (1033 IVF and 873 ICSI, 1320 mixed IVF-ICSI cycles)CONCLUSIONS: This systematic review indicates that the evaluable studiesare heterogeneous and that overall, there is no consistent relationshipbetween sperm DNA damage and embryo quality and/or development.