STRATEGIES TO REDUCE SPERM DNA DAMAGE Edited by: Talebi AR. Ph.D

Sperm chromatin/DNA remodeling  Testicular phase (histone-protamine substitution)  Epididymal phase (disulfide bonds formation)  Ejaculation phase (zinc addition)

CHROMATIN / DNA ABNORMALITIES  Excessive histone  Absence or deficiency of protamines (P1, P2 )  Reduction in disulfide bonds formation  Hypostabilized chromatin due to the zinc deficiency  DNA fragmentation  DNA denaturation

MECHANISMS OF CHROMATIN / DNA ABNORMALITIES 1 - Impairement in DNA nicking / ligation and Topoisomerase II 2 - Programmed cell death via apoptosis 3- Oxidative stress (Reactive Oxygen Species) most important in ejaculated spermatozoa

Etiologies of sperm DNA damages Age Smoking Varicocele Inflammation Hyperthermia Febrile illness Spinal cord injury Testicular cancer Environmental toxins Drugs, chemotherapy and radiation Hormonal factors like testosterone

STRATEGIES TO REDUCE SPERM DNA DAMAGE 8 chief strategies regarding the problem of raised sperm DNA damage have been recommended recently: Using surgically-retrieved testicular spermatozoa instead of ejaculated ones [219], Using ejaculated spermatozoa after at least two months of oral antioxidant therapy [249], Micro-injection (ICSI) with spermatozoa selected with the use of a high-magnification optical system (high-magnification ICSI) [481], Suitable preparation of the semen samples [18,426, 482], In-vitro culture conditions [483] Selected type of ART programs [364] Changing the lifestyle Repeated ejaculation during one week before ART programs

1. Testicular Spermatozoa Spermatozoa spend a long period of time in the epididymis and so, it has been suggested that oxidative stress can probably damage male germ cells in this organ due to long exposure of ROS. It was found by Greco et al (2005) that DNA breakages in ejaculated spermatozoa assessed by the TUNEL are significantly higher than those in testicular spermatozoa (23% versus 4.5%). They also reported that there is a higher pregnancy rate with using testicular spermatozoa compared to ejaculated ones [219]. However, it should be considered that if the origin of sperm DNA damage is abnormalities in topoisomerase II activity or abortive apoptosis (two other causes of DNA damage), there will not be any advantages for testicular spermatozoa.

2. Antioxidant Therapy… Because spermatozoal DNA damages are supposed to be mainly due to high levels of ROS, an antioxidant therapy can markedly diminish the percentage of DNA-fragmented spermatozoa in the ejaculate. Several in-vivo and in-vitro studies demonstrated that antioxidants have positive effects on oxidative-induced sperm DNA damage The patients can be given oral antioxidant during 3 months (at least one spermatogenesis cycle) before an ICSI attempt. The subsequent ICSI cycle led to a significant increase in implantation rates and clinical pregnancy in comparison to the pretreatment ICSI outcomes despite of the absence of differences in fertilization and cleavage rates or in embryo quality.

Roles of Antioxidants  Protect normal sperm from ROS-producing sperm  Protect normal sperm from WBC-derived ROS  Suppress premature sperm maturation

In classification reviewed by Agarwal et al (2008), antioxidants can be placed into two broad categories, enzymatic and non- enzymatic. Seminal plasma contains three important enzymatic antioxidants; Superoxide dismutase (SOD), catalase and Glutathione peroxidase/glutathione reductase (GPX/GRD) system. Non-eazymadc antioxidants are including, Vitamin C, Vitamin E and Vitamin A (carotenoids), Coenzyme Q-10, proteins like Albumin, Transferrin, hepatoglobulin, Ceruloplasmin, Glutathione (GSH), Pyruvate, Ubiquinol and bilirubin Types of Antioxidant

Antioxidant Therapy Greco et al (2005) showed that daily oral adminstration of vitamin C and vitamin E for two months reduced the number of TUNEL positive sperm cells from 22.1% to 9.1% The results of an in vitro study suggested that addition of vitamin E or vitamin C to the sperm preparation media during density gradient sperm preparation protected spermatozoa from DNA damage. It is also shown that albumin helps to neutralize lipid peroxide-mediated damage to the sperm plasma membrane and DNA. It is reported that zinc therapy in men with asthenozoospermia resulted in significant improvement in sperm quality with increases in sperm concentration, progressive motility, sperm nuclear integrity and improved conception and pregnancy rates.

3. High-Magnification ICSI Presence of sperm head defects and intranuclear vacuoles has been shown to be a signal for abnormalities of sperm chromatin packaging and incomplete nuclear remodeling during late stages of spermiogenesis. Sperm cells with normal morphology at standard ICSI magnification, may show a variety of structural abnormalities including intranuclear vacuoles, at high- magnification ICSI system. The ICSI cycles which were carried out with this system have been shown to significantly increase pregnancy rates compared to conventional ICSI procedure in patients with high DNA fragmentation.

4. Semen Preparation Techniques… Several new techniques have been introduced to select the best spermatozoa from ejaculate. Glass wool filtration, density gradient centrifugation and swim-up are examples of techniques used to prepare the semen. These methods can significantly improve sperm DNA integrity compared to that of native semen. It was reported that DNA damaged spermatozoa dropped from 12 to 5.5 % after using swim-up and 28 to 14 % after density gradient preparation.

Semen Preparation Techniques … A new technique based on the electrophoretic separation of sperm has recently been developed for the selection of male germ cells for use in ART programs. The method is based on the fact that the negatively charged glycocalyx which has a high level of sialic acid residues, causes mature spermatozoa to be more electronegative. Spermatozoa are separated by moving toward the positively charged cathode and away from the negatively charged anode. It was shown that electrophoretically isolated spermatozoa have low DNA damage assessed by TUNEL examine. First pregnancy and normal birth were reported after ICSI using electrophoretically isolated spermatozoa by Ainsworth and colleagues (2007).

Semen Preparation Techniques The other new method is the separation of sperm cells by magnetic-activated cell sorting (MACS). The technique is based on the ability of spermatozoa to express the apoptotic marker phosphatidylserine, which binds to Annexin-V-conjugated micro-beads. According to Said et al (2005), spermatozoa with signs of apoptosis including DNA fragmentation and phosphotidylserine externalization could be separated by a magnetic field to Annexin-V positive and negative fractions and therefore, could be used in ART programs.

5. In Vitro Culture Condition… In-vitro culture for hrs at 37°C has been shown to improve the motility, post-thaw recovery rate and DNA integrity of testicular spermatozoa, because: 1)The degeneration of single-stranded DNA- damaged spermatozoa 2)Development of immature spermatids with double-stranded DNA may provide an explanation for this occurrence

In Vitro Culture Condition Studies have shown that repeated cycles of centrifugation involved in conventional sperm preparation techniques used for ART, increase significantly the levels of ROS production due to elimination of seminal antioxidant capacity!! Oxidative stress may also damage sperm during cryopreservation. A study conducted by Bilodeau et al (2000) revealed that ROS generated during freeze-thaw cycles are detrimental to sperm function and that levels of antioxidants were decreased during each cycle.

6. Kinds of ART Programs Bungum et al 2004 and 2007 showed that when the DFI value was above 30 percent, the result of ICSI treatment was more better than the result in IVF, because: 1) One possible explanation could be that ICSI procedure eliminates the oxidative stress to which the spermatozoa are exposed during hyperactivation and acrosome reaction as well as zona pellucida penetration. Therefore, ICSI cycles will lead to a better chance of pregnancy than after IUI and/or IVF. 2) Other possible explanation could be that the zygote has a better capability to repair sperm DNA damages after ICSI than IVF treatment. In conclusion, if the DFI value is below 25%, the IUI and IVF are recommended. But if the DFI value raises above 25 percent, the chance to get pregnant by intrauterine insemination (IUI) is approximately zero, IVF very low and it is therefore highly recommended that the couple register for treatment by intra cytoplasmic sperm injection.

7- Changing the lifestyle…. Cigarette smoking causes oxidative stress either by producing high levels of free radicals or by decreasing the antioxidant capacity of seminal plasma. Sperm DNA fragmentation, axonemal damage and decreased sperm count show significant positive relationship with heavy smoking.

Changing the lifestyle…. Ethanol causes a significant decrease in the percentage of motility, concentration and normal morphology of spermatozoa in human semen and also in ethanol-consuming animals. It is believed that ROS has a critical role in alcohol- induced fertility reduction Talebi et al (2010) showed that alcohol causes the production of spermatozoa with less condensed chromatin and high DNA damage which they can be the results of oxidative stress.

Changing the lifestyle…. Different kinds of ROS such as hydrogen peroxide (H2O2), superoxide anion and hydroxyl radical can be produced by radiation and environmental pollution. However, Rosa and colleagues (2003) demonstrated that traffic pollution has detrimental effects on sperm cells and may decrease fertility potential in young and also middle-aged men.

Radiation & sperm DNA X-irradiation can affect the fertility potential of spermatozoa and cleavage rate of embryo due to DNA damage (Hendricks, 2010) The type of DNA damage that is observed depends on the dose of irradiation and the stage of development of the exposed germ cells (reviewed in Kamiguchi and Tateno, 2002). It is likely that irradiation induces oxidative stress; (Ishii et al., 2005).

EXPOSURE TO XENOBIOTICS It is demonstrated that exposure to pesticides and insecticides has been associated with increased levels of DNA damages in spermatozoa (Xia et al. 2005). Exposure to xenobiotics can alter chromatin in human and animal sperm. Abnormalities such as deficiency in protamines (Cho et al., 2001) or in histone modification (Baarends et al., 2007) are related to xenobiotics. Insecticides: Pyrethroid, Carbaryl and Chlorpyrifos cause sperm DNA damage (Meeker JD, 2004 and 2008).

Metals Workers with high blood lead levels have elevated sperm DNA damage (Hsu et al., 2009). Lead interacts with protamine 2 to decrease its binding to DNA, altering chromatin stability (Quintanilla-Vega et al., 2000). On the other hand, lead and other cations (mercury, copper) may replace zinc in chromatin structure: => Failure or delay in sperm chromatin decondensation in fertilitzation process => Susceptibility to DNA damage agents There is evidence that acute iron or cadmium exposure causes oxidative DNA damage in sperm (Wellejus et al., 2000; Manna et al., 2008).

Mobile phone radiation induces reactive oxygen species production and DNA damage in human spermatozoa in vitro (Geoffry N, 2009) Purified human spermatozoa which were exposed to electromagnetic radiation (as the same as mobile) have shown high levels of oxidative DNA damage bio- marker, 8-OH-dG, and DNA fragmentation index. Also, Aitken (2008) identified high levels of sperm DNA fragmentation following16 hours exposure to mobile.

Chronic exposure to MDMA (ecstasy) increases DNA damage in sperm (Barenys, 2009 and 2010). Cocaine can affect sperm DNA integrity and cause apoptosis (Li, 1999). Acetaminophen and hydroxyurea alter sperm chromatin structure in laboratory mice (Wiger, 1995) Antidepressants like paroxetine can induce DNA damage in spermatozoa (New Scientist. 2008) Drugs

8. Repeated ejaculation during one week before ART programs Elimination of destroyed spermatozoa Decrease in exposure time of spermatozoa to ROS

Indications of sperm DNA damage tests 1.Counselling people who are planning their first pregnancy: 2. Counselling people planning to undergo intrauterine insemination 3.Counseling people planning to undergo IVF or ICSI specially IVF failures 4.All idiopathic couples 5.Men older than 40 years even if prior fertility 6.Men with known exposure to toxicans 7.Men who are at risk of DNA damages like cancer, smoking, varicocele etc. 8.Repeated pregnancy loses 9.Spinal cord injured men

Dr. Talebi AR. Andrology lab. Director Research & Clinical Center For Infertility Dr. Talebi AR. Andrology lab. Director Research & Clinical Center For Infertility