Antioxidant(s)
Study
Treatment dosage (per treatment or day)
Treatment details
Parameters improved
SOD
Rossi et al. [19]
SOD (100 U/ml) and catalase (100 U/ml)
Added to fresh semen before freezing
• Semen parameter, especially progressive motility) (SOD and catalase)—due to their combined and simultaneous action on superoxide anion and hydrogen peroxide
Catalase
Li et al. [20]
Ascorbate (300 or 600 μM) and catalase (200 or 400 IU/ml)
Added to semen before freezing
• Reduced ROS levels and ROS-induced damages in post-thaw spermatozoa [ascorbate (300 μM) and catalase (200 and 400 IU/l)]
Catalase
Chi et al. [21]
Catalase (1, 10, 100 U/ml) or EDTA
(1, 10, 100 μM/ml)
Added to medium used during sperm wash
• Sperm motility (10 μM/ml EDTA)
• Acrosome reaction rate of the spermatozoa (catalase)
• Decreased DNA fragmentation rate of the spermatozoa (EDTA and catalase)
Vitamin E
Kalthur et al. [22]
Vitamin E 5 mM
Added to cryomedia prior to freeze-thaw
• Post-thaw motility
• DNA integrity
Vitamin E
Taylor et al. [23]
Vitamin E 100 or 200 μmol
Added to cryomedia
• Post-thaw motility
Vitamin E
Cicek et al. [24]
Vitamin E 400 IU orally to women with unexplained infertility undergoing ovarian stimulation and IUI
Between day 3 and 5 of menstrual cycle until hCG injection
• Endometrial thickness on hCG day
Vitamin E + selenium
Moslemi and Tavanbakhsh [25]
Vitamin E 400 IU + Selenium 200 μg orally in men with idiopathic asthenozoospermia
100 days
• Sperm motility
• Sperm morphology
• Spontaneous pregnancy rates
Vitamin C
Akmal et al. [26]
2000 mg vitamin C in oligozoospermic men
2 months
• Mean sperm count
• Sperm motility
• Sperm morphology
Vitamin C + vitamin E
Greco et al. [27]
Oral 1 g vitamin C and 1 g vitamin E in men with elevated sperm DNA fragmentation (15 %) and prior failed ICSI attempt
2 months
• Reduced DNA damaged sperm
• Implantation rates
• Implantation rates
• Clinical pregnancy rates
Vitamin C
Henmi et al. [28]
Oral ascorbic acid 750 mg in women with luteal phase defects
1st day of the third cycle until a positive urinary pregnancy test
• Clinical pregnancy rates
Vitamin C
Crha et al. [29]
Vitamin C 500 mg in women undergoing IVF-ET
Gradual release over 8–12 h
• Improved pregnancy rates
Vitamin C
Branco et al. [30]
Ascorbic acid 10 mM
Semen before freezing
• Reduced DNA damage
Melatonin
Eryilmaz et al. [31]
Melatonin 3 mg in IVF-ET patients
3rd to the 5th day of the menstrual cycle until hCG
• Mean number of retrieved oocytes
• Mean number of MII oocyte counts
• G1 embryo ratio
Melatonin
Tamura et al. [32]
Melatonin 3 mg in women with prior IVF-ET failure
Between the 3rd and the 5th day of the menstrual cycle until hCG injection
• Oocyte quality
• Fertilization rates
Melatonin
Unfer et al. [33]
Myo-inositol 4 g and melatonin 3 mg and folic acid 400 mcg in women with failed IVF cycle
3 months
• Number of MII oocytes retrieved
• Total and top-quality embryos transferred
• Fertilization rate
Coenzyme Q10
Nadjarzadeh et al. [34]
CoQ10 200 mg in iOAT patients
12 weeks
• Increased TAC
• Reduced LPO
Coenzyme Q10
Safarinejad [35]
CoQ10 600 mg in iOAT patients
12 months
• Sperm quality (concentration, progressive motility, morphology)
• Pregnancy rates
l-Carnitine
Lenzi et al. [36]
l-Carnitine 2 g
2 months
• Semen quality
l-Carnitine and acetyl l-Carnitine
Cavallini et al. [37]
l-Carnitine 2 g and acetyl-l-Carnitine 1 g
6 months
• Semen quality
• Pregnancy rates
l-Carnitine with Vitamin E
Wang et al. [38]
l-Carnitine 2 g and vitamin E
3 months
• Percentage of forward motile sperm after the treatment
• Pregnancy rate
l-Carnitine and acetyl l-Carnitine
Vicari and Calogero [39]
l-Carnitine 2 g and acetyl-l-Carnitine 1 g
3 months
• Sperm forward motility
• Sperm viability
• Reduced ROS production
l-Carnitine
Khademi et al. [40]
l-Carnitine 3 g in men with idiopathic sperm abnormalities
3 months
• Percentile of motile and grade A sperm
• Percentile of normal-shaped sperms decreased significantly
l-Carnitine
Abdelrazik et al. [41]
l-Carnitine 0.3 and 0.6 mg/ml
Added to cryomedia
• Blastocyst development rate
• Reduced DNA damage
Folic acid and zinc sulphate
Wong et al. [42]
Folic acid 5 mg and zinc sulphate 66 mg in subfertile men
26 weeks
• Folate concentrations in seminal plasma
• Total normal sperm count
Myo-inositol and folic acid
Papaleo et al. [43]
Myo-inositol 4 g and 400 μg folic acid twice a day
Continuous from the day of GnRH administration
• Reduced mean number of immature oocytes at pick up
• Reduced mean number of degenerated oocytes at pick up
• Number of retrieved oocytes maintained
Myo-inositol and folic acid
Ciotta et al. [44]
Myo-inositol 4 g and 400 μg folic acid twice a day
3 months
• Number of oocytes recovered at pick-up
• Reduced number of immature oocytes
Combination of antioxidants
Wirleitner et al. [45]
FertilovitRMplus twice daily
2 months
• Sperm motility
• Reduced percentage of immotile sperm cells
• Total sperm count
• Percentage of class I sperm
Combination of antioxidants
Tunc et al. [46]
1 Menevit capsule
3 months
• Pregnancy outcome
Combination of antioxidants
Tremellen et al. [47]
Menevit
3 months
• Viable pregnancy rate
Combination of antioxidants
Omu et al. [48]
Zinc 5 mg, Vitamin E + zinc 10 mg and Zinc + Vitamin E + C 200 mg
3 months
• Sperm parameters
Combination of antioxidants
Rizzo et al. [49]
Myo-inositol 4 g and folic acid 200 mg and melatonin 3 mg
Continuous from the day of GnRH administration
• Oocyte quality
• Number of morphologically mature oocytes at ovum pick up
In general, AOX help to maintain a balance between ROS and oxidative damage by acting as a defense mechanism. Any deficiency in AOX may lead to imbalance in this process [7]. In addition to the known diseases leading to infertility, lifestyle choices, including diet, alcohol consumption and smoking may contribute to an increased risk of AOX deficiency. Overall, infertility patients have either an increase in ROS levels, which lead to OS, lipid peroxidation and cellular damage, or a decrease in the antioxidant capacity, or both [1, 8–10].
Based on the AOX properties of defending against ROS, their use has gained increasing interest among practitioners as adjuvant agents to counteract the detrimental effects of OS [9, 11–14]. In this chapter, we will discuss the role of AOX as potential agents to alleviate OS-related male and female infertility.
7.2 Therapeutic Role of Antioxidants (AOX) in the Treatment of Infertility in the Male
7.2.1 Evidence from In Vitro Studies
7.2.1.1 Superoxide Dismutase (SOD)
The testes contain enzymatic antioxidants that protect spermatozoa from excessive ROS. An example is SOD, which is an endogenous enzyme that converts superoxide radicals (∙O2−) to hydrogen peroxide (H2O2) [15]. Hence, SOD neutralizes intra and extracellular superoxide anions.
SOD was shown to preserve sperm motility and reduce lipid peroxidation, as evidenced by the decrease in malondialdehyde (MDA) concentrations. In one study, sperm incubation with high concentrations of SOD (2000 μ/ml) preserved motility in mouse spermatozoa and enhanced mice embryo development. On the contrary, lower concentrations of SOD (<2000 μ/ml) had no remarkable effect [16]. In another study evaluating the effects of SOD on sperm viability, motility and morphology, the authors found that SOD was associated with improved semen quality [17]. Interestingly, a study conducted by de Lamirande et al., which evaluated sperm capacitation, hyperactivation and acrosome reaction, demonstrated that a higher proportion of sperm exposed to superoxide anion exhibited capacitation and hyperactivation as opposed to those unexposed to SOD [18].
7.2.1.2 Vitamin C
Vitamin C (ascorbic acid) is a water-soluble vitamin present in dietary sources. It is considered a synthetic antioxidant because it cannot be produced by the human body enzymatically. Vitamin C is found at ten times higher concentrations in the seminal fluid of fertile men than in infertile men and is therefore assumed to play an important role as a seminal AOX [51].
Notwithstanding, the effects of vitamin C on semen parameters have yielded conflicting results. A study by Askari et al. demonstrated that 10 mmol/l of vitamin C added to TEST yolk buffer failed to preserve sperm motility during the cryopreservation process [52]. On the other hand, Verma et al., reported a dose-dependent effect of vitamin C on sperm motility. In their study, sperm motility reached its peak after six hours incubation with vitamin C at a concentration of 800 μmol/l [53]. Not only did vitamin C enhance sperm motility, but it also played a role in protecting sperm DNA from oxidative damage. Donnelly et al. demonstrated that in vitro sperm incubation with vitamin C at concentrations ranging from 300 to 600 μmol/l provided full protection to sperm against damage inflicted by H 2 O 2 [54].
Fraga et al. reported an inverse association between vitamin C and sperm DNA damage. By testing normal semen samples, it was noted that increased 8-OHdG levels in the seminal fluid was associated with decreased levels of vitamin C. When administration of vitamin C reduced from 250 mg to 5 mg/day, a marked increase in 8-OHdG accompanied by a decrease in vitamin C levels in seminal fluid was noted. Similarly, saturation of seminal fluid by vitamin C led to a decrease of 8-OHdG levels and increase in vitamin C levels [55]. This result suggested that vitamin C may protect sperm from OS-induced DNA damage.
7.2.1.3 Vitamin E
Vitamin E actually pertains to a family of vitamins in the tocopherol group. The major type within this group is α-tocopherol. Vitamin E is a fat-soluble vitamin essential for reproduction. Like other AOX, vitamin E plays a role in protection of the cell membrane, prevention of protein modification and protection against DNA damage. With regards to male infertility, the function of vitamin E depends on selenium levels. During the scavenging process vitamin E utilizes glutathione peroxidase, a selenium-dependent enzyme, to reduce hydrogen peroxide molecules [56].
In an in vitro study performed by de Lamirande and Gagnon, vitamin E was shown to protect spermatozoa from damage by OS [57]. Moreover, addition of vitamin E to semen during cryopreservation also protected spermatozoa from OS [23]. In a series of studies by Aitken and colleagues, the role of vitamin E as a sperm motility enhancer and AOX was investigated. Using a 10 mmol dose, vitamin E decreased lipid peroxidation and preserved sperm motility [58, 59]. Furthermore, Verma and Kanwar reported that that 1 and 2 mmol/l dosages were related to improvements in sperm motility in vitro in a dose-dependent manner [60].
Addition of vitamin E to the cryomedia was also shown to preserve sperm motility during the freezing-thawing process [61, 62]. In contrast, vitamin E at a dose of 1 mmol had no effect on lipid peroxidation [63]. When combined with vitamin C, vitamin E may have pro-oxidative effects as shown by Hughes and colleagues [64]. Others have found contrary results in which the combination of vitamin E and C resulted in lower sperm DNA damage in normal and asthenozoospermic men [54, 65, 66].
7.2.1.4 Carnitine
l-Carnitine (LC) is a non-enzymatic AOX. It is produced by the liver and transported to the epididymis through blood circulation [67], where it is found in high concentrations. It has been suggested that LC is a key factor in promoting sperm motility during epididymis transit [12, 68]. Both LC and acetyl-l-carnitine (ALC) play a major role in providing energy for sperm, thereby affecting sperm function.
Several animal and human studies were conducted to explore the role of carnitine on sperm motility in vitro. l-Carnitine, alone or combined with ALC, was shown to increase sperm motility when added to sperm medium [69]. It seems though that l-carnitine has to be converted to ALC to exert its effects [70–72].
7.2.1.5 Carotenoids
Carotenoids are mainly xanthophyll and carotene. Lycopene, found in vegetables, fruits, as well as in the testes and seminal plasma, is the most powerful AOX within this group [73]. Lycopene exerts its effects by donating electrons to free radicals, thus balancing and neutralizing the latter [74]. Another mechanism of action is regulation of the cell cycle [74, 75].
7.2.1.6 Glutathione (GSH)
Glutathione, supplied by N-acetyl cysteine (NAC) during removal of free radicals, is found in large quantities in the epididymis [7, 67, 77]. In an early study, sperm incubation with NAC for 20 min enhanced motility and decrease ROS levels [78]. Subsequently, the combination of GSH and hypotaurine was tested in both asthenozoospermic and normal sperm at different concentrations and incubation periods, but no effect on sperm motility was noted [79].
7.2.2 Evidence from In Vivo Studies
7.2.2.1 Vitamin C
In a randomized control trial conducted on 30 men suffering from infertility but otherwise healthy, patients were divided into three groups according to the following prescription given for one month: (1) Vitamin C 1000 mg/day; (2) Vitamin C 200 mg/day; and (3) placebo. At both high and low dosages vitamin C was shown to improve sperm motility, viability and morphology compared to placebo [80]. In another study involving smokers, 75 healthy men were divided into three groups and treated as aforementioned. Semen analysis revealed that only morphology was improved on those individuals who received high dosages [81]. In two subsequent studies exploring the dosage of 200 mg/day, vitamin C was given for 6 months but no improvements on semen parameters were noted [82, 83].
7.2.2.2 Vitamin E
7.2.2.3 Carnitine
Carnitine was also shown to improve sperm parameters in the mice [86, 87]. In humans, one study involving 100 infertile men treated by either placebo or LC (2 g/day) for 6 months revealed that sperm concentration and motility were improved [36]. In another study involving 47 infertile men treated for 3 months with LC (3 g/day), similar effects were observed with regard to sperm count and motility [88]. On the contrary, no detectable differences were noted in semen parameters of 20 men with idiopathic infertility treated with ALC (4 g/day) for sixty days [89].
7.2.2.4 Carotenoids
In an experimental study involving rats, lycopene (7 mg/kg) was shown to protect against cisplatin toxicity [90]. In humans, lycopene at a dosage of 4 mg/day given to 30 infertile men for 3 months resulted in a positive effect on sperm motility, morphology, and concentration [91]. Furthermore, in a randomized clinical trial involving 30 men with unexplained infertility treated with either 16 mg/day of astaxanthin or placebo for three months, sperm motility was increased whereas ROS levels were decreased. However, there was no impact on sperm morphology or concentration [92].
7.2.2.5 Glutathione (GSH)
In a placebo-controlled, double blind, cross-over trial of glutathione therapy involving 20 infertile men, Lenzi et al. observed that patients receiving 600 μg of GSH daily for two months had a positive impact on sperm morphology and motility [93]. These authors’ results were corroborated by another study involving 11 infertile men who have taken the same GSH scheme [94]. In general, GSH seems to have a positive impact on semen parameters, but further studies are needed to evaluate its effects on male fertility.
7.2.2.6 Combined Antioxidant Treatment
The synergistic effect of combined AOX therapy has been explored in a few studies. In one study involving 54 infertile men, selenium (225 μg/day) and vitamin E (400 mg/day) were given to 25 patients whereas vitamin B group (4.5 g/day) was administered to the remaining 26 men. The authors observed that the combination of selenium and vitamin E resulted in increased sperm motility and viability and decreased lipid peroxidation [95]. In another study, Piomboni et al. examined the effects of AOX combination (vitamin C, lactoferrin and vitamin E) given to a group of 51 asthenoteratozoospermic patients. After a 3-month treatment period, sperm motility and morphology was increased whereas sperm DNA damage was reduced [96]. Comhaire et al. also examined the effect of AOX combination in a group of 27 infertile men. Different formulation involving N-acetyl-cysteine, vitamins A with E and a combination of fatty acids were given for 6 months. The authors noted an increase in sperm concentration and decrease in ROS levels with no change on sperm morphology or motility [97].
Several studies investigated the combination of vitamin C and vitamin E and their role in DNA protection and enhancement of male fertility. In a study conducted by Greco and colleagues, 64 infertile men with high levels of sperm DNA damage were randomized to receive 1000 mg/day of both vitamin C and E for two months or placebo. Although no differences were observed in conventional semen parameters, the group receiving AOX combination was shown to have lower levels of sperm DNA damage [27].
7.3 Assisted Reproductive Technology (ART) and AOX Therapy
Assisted reproductive technology (ART) has been widely used to treat infertility due to severe male factor [98]. In these settings, the use of AOX has been suggested to reduce the levels of ROS generated from many sources such as immature spermatozoa and leucocytes [99, 100]. Reduction in DNA fragmentation [30], improvement in sperm motility [101] and viability [102] after adding vitamin C to sperm culture media during ART have been reported. Catalase added to sperm culture media was also shown to be associated with reduction in ROS levels, DNA fragmentation and increase in acrosome reaction rate [21].
In addition, antioxidants have been used as cryomedia supplements aimed at protecting sperm from the freeze-thawing process as AOX can antagonize the ROS by preventing its formation or removing the already formed ROS [13, 99, 103]. The addition of vitamin E to sperm cryomedia was shown to have positive effects on sperm motility [25, 95] and morphology (Fig. 7.1) [23].
Fig. 7.1
Sources of ROS in ART
AOX can also be given as oral supplements during or prior to ART to reduce the levels of endogenous and exogenous sources of ROS [104–108]. Improvement in sperm motility [85, 109] and decrease in lipid peroxidation after vitamin E administration have been reported in previous prospective studies and RCTs. Similarly, positive effects were found with folic acid and zinc sulphate on sperm concentration [42]. Furthermore, oral intake of coenzyme Q10 resulted in increased sperm concentration and motility [110].
7.4 Therapeutic Role of Antioxidants (AOX) in the Treatment of Female Infertility
In this section, the focus will be on the therapeutic role that antioxidants play in the female reproductive system and how these interventions can affect the fertility status of infertile women suffering from different reproductive diseases, either by increasing the chances of regular conception or increasing the chances of successful ART (Table 7.1).
ROS act as stimulants for oocyte maturation and are required for meiosis I completion in the dominant oocyte. Although ROS are needed for the completion of meiosis I, antioxidants are essential for the initiation and completion of meiosis II [111]. Follicle stimulating hormone acts on pathways leading to catalase generation, which has a protective effect against the pro-apoptotic effects of ROS. Glutathione has also a role in neutralizing ROS.
7.4.1 Endometriosis
Endometriosis is a female reproductive disease in which the endometrium grows outside of the uterine cavity. The most common site of endometriosis is presence in the ovaries or peritoneum [112]. Endometriosis is a very common cause of infertility and is prevalent in 30 % of infertile women who are diagnosed with this condition [113]. It remains unclear how endometriosis has an effect on infertility; however, Sampson’s well established theory suggests that the presence of endometrial tissue in the peritoneum can be explained by retrograde menstruation [114]. There are different treatment strategies to treat endometriosis related infertility; therapeutic laparoscopy and ART are the most widely used approaches [115].
Many studies suggest that women suffering from endometriosis have increased oxidative stress [116]. This can be explained by increased numbers of macrophages present in the peritoneum as a result of inflammatory response by endometrial tissue. Metabolic processes occurring in the macrophages are believed to be the cause of excessive ROS production leading to oxidative stress [116]. In addition to excessive ROS production, other inflammatory mediators such as cytokines, chemokines, metalloproteinases and prostaglandins are involved in the pathophysiology of endometriosis-related infertility [117]. Similar to a positive feedback mechanism, inflammatory processes allow for the development of endometrial tissue by means of increased ROS which leads to even further production of ROS thus making OS levels very high [118]. Nevertheless, the association between endometriosis patients and oxidative stress in the peritoneum is not unequivocal [119].
To further understand the relationship between endometriosis and OS, studies were conducted to test whether the levels of antioxidants in the peritoneal and follicular fluids of endometriosis patients were decreased. Overall, the levels of superoxide dismutase and glutathione peroxidase are usually decreased in endometriosis patients compared to controls [120–122]. Animal models have been used to test the effects of low molecular weight antioxidants such as vitamins C and E on induced endometriotic implants. In one study, vitamin C decreased the number of natural killer cells thus resulting in decreased levels of OS [123]. Another well-established method to measure the effect of antioxidants on OS is through the various OS markers such as malondialdehyde (MDA) (Table 7.1).
In a randomized controlled trial conducted by Mier-Cabrera et al. involving endometriosis patient, vitamins C and E were shown to decrease the levels of MDA and lipid hydroperoxides [124]. In another study conducted by Santanam et al. the authors tested the effect of various antioxidant vitamins administered for 8 weeks to women with history of endometriosis and infertility in a randomized placebo-controlled trial. Several inflammatory markers were measured in the peritoneal fluid, including interleukin 6 (IL6), monocyte chemotactic protein (MCP), normal T-cell expressed and secreted (RANTES) [125]. In addition to that, pelvic pain was measured using a pain scale. The results of this study indicate that antioxidant treatment significantly decreases inflammatory markers in the peritoneal cavity of women suffering from endometriosis. Regarding the effects of antioxidants on pelvic pain, 43 % of those in the antioxidant group reported decreased pain in contrast to 0 % in the placebo group. Moreover, 52 % of patients in the antioxidant group reported no change in pain compared to 100 % of the placebo group. While the study lacked any comments on the relieving of infertility in endometriosis patient, the reduction in the levels of inflammatory markers suggests a corresponding decrease in OS, which could result in alleviation of infertility symptoms.
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