Effect of Alcohol Consumption on the Sperm DNA Integrity : A Systematic Review

There is no solid conclusion on the conventional sperm parameters in association with alcohol consumption, evaluation of sperm DNA integrity thus become a more reliable parameter. Hereby, this literature search was performed to summarize alcohol consumption on the sperm DNA integrity. A computerized database search was done through MEDLINE via Ovid (since 1946 until August 2017) and Cochrane was used. The following set of keywords: ‘alcohol consumption OR alcohol intake OR alcohol diet OR drinking alcohol OR ethanol diet’ AND ‘sperm DNA OR sperm chromatin OR sperm genome OR sperm histone OR sperm protamine’ were utilised. 24 articles were retrieved where only five studies conform to the inclusion criteria All studies demonstrated a negative effect of alcohol consumption on sperm DNA integrity, regardless of various range of alcohol doses and duration of alcohol consumption. Out of five studies reviewed, four studies were using a different approach to measure the sperm DNA damage. Hereby, this review identified a need to use a single approach of DNA damage test by having various method of alcohol administration and/or vice versa so that the extension of sperm DNA damage to alcohol consumption will have a better conclusion. On the same note, a few studies have reported the reversibility on conventional semen parameters, none has been done on the sperm DNA damage upon alcohol withdrawal. Therefore, the role of alcohol withdrawal on the reversibility of sperm DNA damage needs to be as well investigated further.

Due to this variation, the conventional sperm parameters may therefore do not reflect the true quality of the spermatozoa in association of alcohol intake.Agarwal and Allamaneni (2005) supported this fact where they found approximately 15% of male with fertility problem presented with normal sperm parameters (Agarwal & Allamaneni, 2005).In this case, assessment of sperm DNA damage seems to be a more precise tool in diagnosis infertility in male.
Hereby, this systematic review was done in order to summarize the available information regarding the effect of alcohol consumption on the sperm DNA integrity based on preclinical and clinical evidences.Subsequently, we would like to point out where is needed for further research in the future.

Search Strategy
A computerized literature search was conducted on the reported article regarding the effect of alcohol consumption on the status of sperm DNA integrity in particular.The literature research was conducted through MEDLINE via Ovid (1946until August 2017) and COCHRANE databases with the following keywords: 'alcohol consumption OR alcohol intake OR alcohol diet OR drinking alcohol OR ethanol diet' AND 'sperm DNA OR sperm chromatin OR sperm genome OR sperm histone OR sperm protamine'.

Study Inclusion and Exclusion Criteria
The eligible articles were reviewed independently by two authors (FHF and SFI) based on preclinical and clinical evidences.The following criteria were considered: original article published full-length in English; there is a group of sample that consumed solely alcohol regardless of any fertility factors measured as confounding; measurement of sperm DNA integrity, chromatin packaging and/or DNA breakage as the dependent variables.

Data Extraction and Management
Selection of the study involved two phases.During the first phase, the titles and abstract were scrutinized thoroughly.Following this, the study that did not meet the inclusion criteria were immediately excluded.Full articles were obtained for the remaining list that were likely to meet the selection criteria.Inclusion and exclusion criteria were finalized upon examination of the full articles.Any opinion opposition regarding the inclusion and exclusion criteria were resolved by third reviewer (KO) consensus.
For this review, we recorded the sample size that was related only to alcohol consumption, dose and duration of alcohol consumed, the age of animal sample or human subject, sperm DNA assay type, cut off point of DNA damage and sperm DNA test results in relation to alcohol consumption.

Preclinical Evidence
The first animal study that will be reviewed used Long Evans and Sprague Dawley male rats.Its objectives were to assess the effect of strain, paternal and maternal alcohol consumption.The treatment group received a variation of ethanol concentration; 35%, 17% and 0%-ethanol derived calories (EDC).The study found out that paternal alcohol consumption resulted in decreased litter size and testosterone levels.Fortunately, it did not affect postnatal mortality and passive avoidance learning of offspring.On the same note, the rats strain affected their offspring activity.Ethanol 35% consumption showed a decrement in offspring activity in Long Evans but increment in Sprague Dawley rats.In contrast, maternal ethanol consumption did not affect offspring activity but it was associated with lower birth rate, lower offspring weight at weaning, poorer passive avoidance learning and increased in postnatal mortality.Despite of the main design of the study, the study had selected sperm from Long Evans for their DNA study.Instead of using three doses tested in the main study design, only two doses were used for the sperm DNA damage evaluation, 0% and 35%.They found out that sperm DNA from that strain migrated further compared to its control following ethanol consumption (Abel, 1989).However, this study failed to report the statistical analysis.Thus, the significance of 50 kilobase pairs between the tested group could not be concluded.
In another experimental study, alcohol abuse model used Wistar rats.This study is interesting because it took spermatogenesis cycle duration (50 days) into consideration.It also documented for the first time the effect of alcohol towards nuclear sperm DNA.They measured sperm motility and chromatin integrity from cauda epididymal aspiration.The study introduced a "cut-off" value for the cytochemical test done.Their findings showed that ethanol consumption did not affect sperm morphology but decreased its progressive and non-progressive motility.Even though they did not have a positive and negative control on the sperm DNA evaluation, this study did test the efficacy and quality of the stain by using acid denaturation method in a normal sample.This study found out that ethanol caused DNA damage and affected its chromatin integrity (Talebi et al., 2011).
Generally, many risk factors for infertility.Apart from alcohol, chronic diseases like diabetes has been associated with infertility too.Another experimental study that involved ethanol consumption in diabetes animal model has been established (Pourentezari et al., 2016).This study used male mice with streptozotocin (200 mg/kg, single dose, intraperitoneal) for diabetes induction.While considering diabetes a cofounding factor, this study used 35 days of ethanol liquid diet.This duration won't be able to complete a spermatogenesis cycle.However, the dose of ethanol liquid diet in supplemented in this study is higher (10 mg/kg compare to 5% v/v) compared to the study done by Talebi, Sarcheshmeh, Khalili, and Tabibnejad (2011).Despite of this situation, the findings suggested that ethanol consumption with or without hyperglycemic state decreased all sperm parameters and affected sperm DNA integrity.Through AO staining, the study showed that alcoholism with or without diabetes as cofounding factor would increase denaturation of sperm single stranded DNA.While, TB and CMA3 stain confirmed that alcohol has a profound detrimental effect on sperm chromatin condensation.

Clinical Evidence
We reviewed two studies on human sample in this paper.The first study involved the evaluation of alcohol consumption with cigarette smoking as a cofounding factor (Anifandis et al., 2014).This study chosen sperm DNA fragmentation index (SDF) as their indicator and this were measured by using sperm chromatin dispersion (SCD) assay kit, Halosperm®.Study concluded that smoking causes an irreversible damage to the DNA while sperm morphology was evidently suggested to be reversible upon discontinuation of alcohol consumption (Kuller et al., 1978).Yet, the reversibility of DNA damage upon alcohol discontinuation was not evaluated.The percentage of no halo which represent high percentage of DNA fragmentation was not significant between no alcoholics, moderate and heavy alcoholics group.On the other hand, there was significant difference of small halo percentage between moderate and heavy alcoholics group.Thus, the consumption of more than 7 units (1 alcohol unit was considered for 100 ml wine/200 ml beer/30 ml whisky/30 ml vodka) of alcohol per week will be resulting in low percent of DNA damage.Even though, smoking and alcohol act independently, this study has concluded that both factors acted synergistically when combined.
In a second study of human sample, the paternal effect in the etiology of fetal alcohol spectrum disorder (FASD) was investigated.An irreversible cognitive and behavioral disability characterized the fetal alcohol spectrum disorder.Animal studies have shown that chronic paternal alcohol consumption has damaging effect on mental and physical offspring development without alcohol exposure during their uterine life.Primarily, FASD is attributed to maternal alcohol use during pregnancy but this study was to establish a link between alcohol use in men with methylation in sperm DNA.It has been documented that alcohol reduced DNA methyltransferases.This enzyme is a key enzyme that establish and maintain methylation pattern.The reduction of the enzyme activity leads to DNA hypomethylation and activation of normally silenced genes.The findings have shown that alcohol affected an important gene that regulate fetal development.It also has identified a useful method for studying paternal effect in inherited epigenetic modifications (Ouko et al., 2009).
Both human study has slightly different age of the subject.Age is one of the cofounding factors that contribute an important role in determining the status of DNA damage in human as aging has been proven scientifically as one of the factor that could contribute to higher sperm DNA damage in men (Das et al., 2013;Pharm et al., 2014).
In prospective study involving human subject, age factor cannot be manipulated.Due to this age variation, it would be better if classification of age is done for further evaluation of alcohol doses on status of DNA damage in the future.
Based on this review, availability of study on the sperm DNA damage in association with alcohol consumption is very sparse.This situation claims a vast investigation in the future.Even though there were reports stating that conventional semen parameters could be reversible upon discontinuation of alcohol consumption (Vicari et al., 2002;Sermondade et al., 2010;Guthauser et al., 2014), conclusion of this same effect on the sperm DNA need to be further investigated in the future.

Table 1 .
Characteristic of studies on sperm DNA damage in association with alcohol consumption in preclinical and clinical sample Journal of A gure 1. Flow ch e reported on t perm DNA.A d that the DNA ectrophoresis ( s have evalua blue (TB) and l., 2016).Thes TB and AO s