This is not my research specialty.

Thanks

Pradman

I would postulate that the likelihood is very small, but remember that (i) the oocyte can repair DNA and (ii) depending on the way DNA damage was measured it implies that 95% of sperm has  damaged DNA while 5% of spermatozoa has normal DNA - these can more than likely still generate fertilization and embryo development. However these cells might also be compromised to a certain extent.

with regards to sperm DNA fragmentation, is it possible for it to be happening due to an oxidative stress if there is a high number of leukocytes in the ejaculate?

Yes, it is very possible that it is due to increased ROS produced by granulocytes that can lead to OS once an imbalance exist between ROS (oxidants) and antioxidants. Leukocytes present in semen produce large quantities of ROS and is generally accepted to cause DNA damage

A DNA fragmentation index of 95% is really very high but the meaning depends on the methodology you are using. If it is COMET, having a normal cut-off at 45%, it is a relatively normal finding. If it is rather TUNEL or SCSA or SCD, with a cut-off around 20%, 95% is too high to be true. In this case an inflammatory origin is the very first you have to rule out. As a second step, you should check the global oxi-redox state of the patient because such a high level of oxidative damage could parallel a similarly high systemic oxidative stress. This is of high prognostic value for the patient independently of the fertility issues (a serious disease might be in place).

Back to the fertility issues, if the 95% is coming from COMET there are chances to isolate a relatively good sperm for the ICSI (try with HA binding). If rather it refers to the other tests there are no real chances to obtain a viable embryo: DNA fragmentation is mainly due to apoptosis and if the damage was so high to make it evident in a so high rate of sperms it is sure that hidden defects (not yet developed enough to trigger evident apoptosis) are present in all cells.

Sperm DNA oxidative damage is largely repaired into the zygote, nevertheless the repair capacity is finite, sometime it is too much. Moreover, these damages associate to damages to the chromatin structure that may hamper the ability of the sperm to activate the embryo independently of the possible repair of molecular damages. Thus, even if you get fertilization by ICSI, you are then likely to suffer no activation or late activation or chaotic activation of the embryo contributing to implantation failure or early pregnancy loss. Finally, pregnancies obtained as such are at risk for pregnancy complications and  for newborn safety.

Consider testing this patient for sperm nuclear decondensation/maturity by blue aniline staining. It is simple and cheap and in our experience it is far better predictive then fragmentation, especially if you want to check the effect of a treatment. The decrease of fragmentation by antioxidants may be just a cosmetic effect by blocking apoptotic removal of damaged cells. On the other side, an improvement of chromatin maturity directly reflects on the fertility potential.

Thank you very much for your valuable comments, I think we will have to also screen this patient for possible infection as his level of DFI is abnormally high. I already tested his sample on two different days to role out sampling, processing error.

hmm for increase the exit you must have stonger  slection sytem, if you have that big number in DNA aberration.

the classic swin-up help you, but you used centrifugation in special médium. Anyway, good luck with the cells selection

I am absolutely agree with Dr. Dattilo. Anyway, although it could be obvious, I might add that if inflammatory causes occur you should take into account spermatogenic cycle duration before semen evaluation and FIV.

Good luck!!

This is still a open question for andrology researchers and there is much debate regarding the effect of sperm DNA damage on embryo quality. Theoretically, 95% DNA damaged sperm should not be allowed (by zygotic machinery) to proceed further with embryo development. The activation of the embryonic genome in human begins between 4 and 8 cell stages. It is this stage after which the compromised sperm DNA quality (sperm DNA damage) could affect the further embryo development. Since mature sperm is deficient in DNA repair mechanism, certain level of DNA damage can be repaired by oocyte upon fertilization. In  my study I didn't find any significant association of DNA damage with embryo quality (using comet assay, for ICSI patients). 

U can use COMET or TUNEL assay for identification of DNA fragmentation.

DNA fragmentation are due to oxidative stress or increased leukocyte.

If it is due to oxidative stress the outcome may b improved by supplementing the patient with anti oxidant rich diet according to some previous research report.l

Dear Katarzyna,

there is a new product for the magnetic depletion of apoptotic sperm cells:  MACS ART Annexin V Complete Kit (200-070-507). The protocol is fast and simple and should be combined with DGC.

In the case of extremely high percentages of apoptotic cells the protocol might have to be adjusted.

If you send me an email ([email protected]) I'll be happy to send you a visual overview of the protocol as well as references.

Kind regards, Sandra

Our experience suggests there is a direct correlation between DNA fragmentation and fertilization failure.  I think Don Evenson's work also suggests the same correlation.  Furthermore keep in mind that fragmentation may not be random and may disrupt the genome in key regions that are required for normal fertilization and development. Hope this helps,

When using a semen sample with a high level of DNA damage, as 95%, we assume that we have a 95% probability of using a sperm with damaged DNA. But how damaged is the DNA of the sperm individually?, repair capacity of oocytes depends on the percentage of sperm with damaged DNA in the semen sample? or it depends on the extent of DNA damage in the sperm used. I do not know if the latter is possible to determine

Are you determining DNA.chromatin fragmentation based on SCSA? If you did, you can assume that the germline of this patient has germline genomic instability which we determine by small pool PCR.We have found that a subset of these patients are at a high risk of developing testicular cancer as they age. Most of this genomic instability is due to

oxidative stress.