Review of Clinical Trials on Effects of Oral Antioxidants on Basic Semen and Other Parameters in Idiopathic Oligoasthenoteratozoospermia

Infertility affects 50 to 80 million people worldwide. Male factor is a cause of infertility in almost half of cases, mainly due to oligoasthenoteratozoospermia (OAT). With common diagnostic methods no cause can be found in approximately 30% of cases of male infertility due to OAT and these are considered idiopathic. Reactive oxygen species (ROS) play an important role in male infertility and are proved to be higher in infertile men; antioxidants could oppose their effect. The aim of this paper was to review the literature on clinical trials in the period from year 2000 to year 2013 studying the effects of various types of antioxidant supplements on basic and other sperm parameters and pregnancy rates in subfertile males with idiopathic OAT. The majority of studies were randomized and placebo controlled and confirmed beneficial effect of antioxidants on at least one of the semen parameters; the biggest effect was determined on sperm motility. In many of these trials combinations of more antioxidants were assessed. The optimal dosages of one or more antioxidants were not defined. We concluded that antioxidants play an important role in protecting semen from ROS and can improve basic sperm parameters in case of idiopathic OAT.


Introduction
Almost 15% of all couples trying to conceive are affected by infertility, and in almost half of these cases male infertility is the sole or a contributing factor [1]. While conditions such as varicocele, cryptorchidism, and hypogonadism are definable causes for infertility, no cause may be determined for an abnormal semen analysis in over 25% of cases [2]. Such idiopathic infertility and oligoasthenoteratospermia (iOATs) is a condition in which sperm concentration, the proportion of motile sperms, and the proportion of morphologically normal sperms are below the World Health Organization (WHO) reference values [3].
Elevated reactive oxygen species (ROS) levels in the semen may be an etiologic factor for male infertility [4]. It is estimated that 25% of infertile men possess high levels of semen ROS, whereas fertile men do not have high levels of semen ROS [5,6]. ROS are needed for capacitation, the acrosome reaction, and ultimately fertilization [7]. However, their uncontrolled production is detrimental to cell function as they damage a variety of biomolecules such as lipids, amino acids, carbohydrates, protein, and DNA and adversely affect sperm function [8] due to DNA damage [9,10], reduced motility [11], and defective membrane integrity [12,13]. Spermatozoa are particularly susceptible to oxidative injury due to the abundance of plasma membrane polyunsaturated fatty acids. These unsaturated fatty acids provide fluidity that is necessary for membrane fusion events (e.g., the acrosome reaction and sperm-egg interaction) and for sperm motility [14]. The human ejaculate contains a number of potential sources of ROS. These include leukocytes, germ cells, or abnormal sperms [15]. At the same time, a number of cellular molecules called antioxidants, which protect the cell from excessive ROS-induced lipid peroxidation, are also present within the ejaculate [16]. Studies have shown that seminal antioxidant capacity is suppressed in infertile men with high ROS levels compared to men with normal levels of ROS [17,18].

Materials and Methods
We searched PubMed with keywords, including combinations of search terms such as "male infertility" and "antioxidants. " We searched for reviews, controlled and randomized controlled clinical studies. From the numerous search results for the period between 1st January 2000 and 31st December 2013, 32 primary studies on idiopathic oligoasthenoteratozoospermia (OAT) were chosen and their data were gathered in order to provide a complete overview of the literature. Given the different antioxidants used (both alone and in combination), the different dosages, different duration of treatment, and various number of participants (from very small groups to large researches), we looked up for statistical significance of changes in basic sperm parameters and pregnancy rates.

Results and Discussion
The review of the studies on antioxidants in clinical studies is illustrated in Table 1.

Sperm DNA Fragmentation and Chromatin Integrity.
ROS can cause sperm DNA damage and integrity of sperm DNA can be measured with DNA fragmentation. The levels of sperm-derived ROS (measured in sperm preparations having minimal leukocyte contamination) have been associated with sperm DNA damage [53]. High level of denatured DNA in spermatozoa with large nuclear vacuole could arise from precocious decondensation and disaggregation of sperm chromatin fibers [54]. Dietary antioxidants may be beneficial in reducing sperm DNA damage, particularly, in men with high levels of DNA fragmentation [5]. Five out of 32 studies confirmed that the usage of different antioxidants had important influence on DNA fragmentation and chromatin integrity [20,[42][43][44][45][46]. Song et al. showed that combination of Chinese medicine Compound Xuanju Capsule with vitamin E taken for 3 months decreased degree of DNA fragmentation index (DFI) after therapy to 29.57 ± 12.19 compared just to BioMed Research International

Negative or No Effect on Sperm Parameters.
In this review we find out also rare negative effects of antioxidants on sperm parameters or no effect. Pycnogenol caused nonsignificant fall in baseline sperm count by 10% [41]. Similarly, treatment with vitamins C and E, ß-carotene, zinc, and selenium significantly increased sperm decondensation [43]. Large research on saffron showed no statistically significant improvements in any of the studied semen parameters [48].

Other Parameters.
We looked at the basic sperm parameters but there were also many other positive influences; for example, CoQ10 and pentoxifylline caused improvements in total antioxidant capacity and acrosome reaction [22,25,49,50]; FSH value [22,23] decreased after CoQ10 treatment, semen leucocyte concentration decreased [36], and level of ROS [50] decreased after antioxidant mixtures. Antioxidants protect unsaturated fatty acids and so provide fluidity that is necessary for membrane fusion events like the acrosome reaction. Although hormonal abnormalities are not always evident, iOAT is sometimes associated with lower serum testosterone and inhibin levels and higher serum estradiol, LH, and FSH levels [55,56]. The increased serum FSH level in men with azoospermia or severe oligozoospermia indicates damaged seminiferous tubule [57] and is inversely associated with sperm concentration, motility, and morphology [58]. ROS has been found in the seminiferous tubules and seminal plasma of most patients with iOAT [59]. Decreased levels of ROS due to antioxidant consumption can cause fall in serum FSH level. Leukocytes are potential source of ROS and due to protective influence of antioxidants their concentration may decrease [15]. In addition, studies have found an increase in inhibin B value [23] and in superoxide-dismutase-(SOD-) like and catalase activity [21,25], which among others represent the total antioxidant capacity of seminal plasma [60]. Inhibin B in positively correlated with sperm concentration and is, like FSH, thought to be a marker of spermatogenesis and Sertoli cell function [61,62].

Conclusions
Most of the published studies were randomized and placebo controlled. The majority of studies confirmed beneficial effect of different antioxidants on at least one of the semen parameters and the biggest effect was determined on sperm motility. In many of these trials combinations of more antioxidants were assessed. The optimal dosages of one or more antioxidants were not defined. Most commonly antioxidants studied were vitamin E, vitamin C, selenium, CoQ10, N-acetyl-cysteine, L-carnitine, and zinc and their favorable effect was confirmed. According to this review favorable effects on iOAT have been determined with CoQ10, vitamin E, selenium, and also vitamin C and N-acetyl-cysteine treatments. In case of oligozoospermia vitamin E and CoQ10 were most often proved to be effective. Favorable effects on asthenozoospermia have most often been determined with vitamin E, CoQ10, and selenium treatments. In teratozoospermia selenium and CoQ10 treatments were most often proved to be effective. In addition, combination of vitamin C and E showed the biggest favorable effect on DNA fragmentation; similar effects were determined with zinc and selenium treatments.
In conclusion, antioxidants play an important role in protecting semen from ROS and can improve basic sperm parameters in case of idiopathic oligoasthenoteratozoospermia.