Research studies by the Cleveland Clinic have looked into the relationship between ROS levels and the time interval (1, 3, 5 and 24 h) between semen collection and analysis, as well as how levels of ROS generation fluctuate at different sperm concentrations (7.5, 15, 30 and 60 × 10⁶ ml) [22]. This study was conducted on semen samples from patients suspected of infertility and showed that ROS levels decreased significantly over time. This suggests that the time between sperm collection and analysis negatively affects sperm motility and viability, which could contribute to the decreasing ROS levels. In addition, ROS levels from the same specimen were found to vary when measured at different sperm concentrations. Thus, to obtain an accurate measurement of the actual amount of ROS generated, ROS levels in whole semen samples must be evaluated within an hour of sample collection. Following this study, sperm preparation techniques including centrifugation (time and g-force), resuspension, L4 filtration, swim-up, and washing were investigated to see if these techniques influenced ROS levels and/or damaged sperm [23–25]. Results showed that continuous washing, centrifugation, and vortexing of samples generated high levels of ROS; with washing causing the most harm [2, 23, 24]. These findings suggest that the overuse of these sperm preparation techniques should be avoided during ART procedures. However, sperm samples processed using techniques such as L4 filtration and swim-up yielded significantly lower ROS levels and with little to no sperm damage compared to that of the unprocessed sample. Both these methods were able to separate the highly motile sperm that produced less ROS from the original sample. In fact, ROS levels from samples processed using the L4 filtration and swim-up methods showed no statistically significant differences in ROS levels compared to each other [25].

Another study by Cleveland Clinic researchers compared post-wash and post-thaw (24 h) parameters of sperm to test the efficacy of the double density gradient media, PureSperm and swim-up (sperm migration), which are density gradient and motility checking techniques that help isolate sperm that are morphologically normal and motile [26]. The use of PureSperm provided more mature and motile sperm in fresh and cryopreserved semen compared to swim up. In another similar study, the differences between processing media (PureCeption, ISolate and SpermGrad-125) used during sperm preparation by density gradient were studied using samples from normal, healthy donors. All three gradients were found to produce sperm samples of good quality as they improved the percent motility, total motile sperm, percent recovery and DNA damage. However, the semen quality and percent recovery of morphologically normal sperm differed between these different commercially-available density gradient media. The DNA fragmentation that resulted from sperm preparation was negligible and did not differ significantly when different media was used in this controlled trial [27].

Cryopreservation of sperm cells is a common step involved in assisted reproduction procedures. However as a result of the freeze-thaw process, cryopreserved sperm are found to have damaged membranes due to membrane stress or lipid peroxidation. In one study, Cleveland Clinic researchers investigated the extent of lipid peroxidation (LPO) during cryopreservation, and the relationship between sperm concentration, motility and morphology with LPO in semen from healthy donors [28]. Results indicated that (1) cryopreservation-induced membrane damage was not related to LPO; (2) malonaldehyde (an indicator of lipid peroxidation) levels increased as sperm concentration increased; and (3) there was no correlation between motility, morphology, and malonaldehyde levels. As such, sperm handling, sperm freezing and the freezing medium seemed to not increase lipid peroxidation in the semen of normal men.

Assisted reproductive procedures (ART) is a common treatment for couples with infertility issues, however, the induction of OS can compromise the efficacy of these procedures [10]. Increasing levels of ROS without a simultaneous rise in antioxidant levels result in OS and consequently lipid, protein, and DNA damage. This oxidative state is thought to result in poor ART success rates [29]. In fact, ROS can have a significant impact on assisted reproduction outcomes and cause IVF failure if not kept in check (Fig. 8.4) [30]. In a prospective study, follicular fluid from patients undergoing IVF was studied to examine the association between LPO, TAC, oocyte maturity, embryo quality, fertilization, cleavage, and pregnancy rates [31]. LPO and TAC were positively correlated with pregnancy rates, and it was surmised that some amounts of LPO might be needed for pregnancy to be established. Furthermore, levels of LPO and TAC in patients who achieved a pregnancy through IVF could perhaps act as an indicator of the minimal metabolic activity needed within the pre-ovulatory follicle in order to be able to establish a pregnancy. In other studies by Cleveland Clinic researchers, the presence of ROS in follicular fluid and its role in pregnancy outcome, the relationship between levels of ROS in seminal fluid and fertilization rates, and role of cytokines in peri-ovulatory follicular fluid were all determined during and after IVF cycles [18, 32, 33]. Results showed that (1) ROS levels in spermatozoa had a significant correlation with fertilization rates after IVF [18]; (2) follicular fluid ROS at low concentrations might be predictive of a successful pregnancy in these IVF patients [32]; (3) high levels of IL-12 and low levels of IL-6 were associated with negative IVF outcomes; (4) IL-13 and tumor necrosis factor alpha (TNF-alpha) were absent in all patients; and (5) IL-1β was not significantly different for pregnant and non-pregnant cycles [33].

In cases of male infertility when IVF does not work, the ICSI method may be employed in which a sperm cell is injected into the oocyte’s cytoplasm [34]. Back in 2009, in-vitro maturation (IVM) emerged as a popular replacement for IVF because it reduces the need for ovarian priming and stimulation, which is a routine protocol in the conventional IVF process, thus reducing the risk of hyperstimulation syndrome [35].

In two studies by Cleveland Clinic researchers, the relationship between early human embryonic development parameters to day 1 ROS levels and day 3 ROS levels in culture media were examined in patients undergoing IVF and ICSI. ROS levels were measured by chemiluminescence using luminol as the probe. High day 1 ROS levels in culture media resulted in low blastocyst rate, fertilization rate, and cleavage rate respectively, as well as high embryonic fragmentation with ICSI but not with conventional IVF. Thus, lower pregnancy rates were observed in both IVF and ICSI cycles that was associated to high day 1 ROS levels. Similarly, high day 3 ROS levels in culture media correlated negatively with blastocyst development rate and pregnancy rate respectively. Researchers found day 3 ROS levels to be low in the pregnant cycles of IVF and ICSI patients. In addition, each 10 unit increase in day 3 ROS levels could decrease pregnancy rates by 41 % in these patients [20, 36]. Oxidative stress levels in the follicular fluid of ICSI patients was further assessed in another study which evaluated the association between the follicular fluid ROS (FFROS), TAC, ROS-TAC score and pregnancy after ICSI [37]. Higher follicular fluid TAC (FF TAC), higher FF ROS-TAC scores and lower FF ROS levels were associated with pregnancy after ICSI. These studies demonstrate that oxidative stress parameters may act as markers of metabolic activity within the follicle. However, supplementation with antioxidants can aid in reducing OS during sperm preparation techniques and thus increase the success of ART [38] (see Sect. 8.13).

A study conducted by Cleveland Clinic using the Endtz test demonstrated a strong positive correlation between ROS generation and polymorphonuclear granulocyte concentration, thus adding to the discourse about the controversial role of leukocytospermia in semen [48]. The results signified that the Endtz test is a simple and cost-effective test that indicates the excessive generation of ROS in semen.

In studies conducted by Cleveland Clinic, the efficacy of measuring total antioxidant capacity (TAC) to discriminate between fertile and infertile patients was tested. In these studies, proven fertile donors have higher TAC scores than infertile patients [49, 50], thus suggesting that measuring TAC in semen is an effective and simple test for diagnosing and managing male infertility. The colorimetric assay was also established as a cheaper, quicker and reliable method of measuring TAC when compared to the conventional chemiluminescence assay [51]. Further studies by Cleveland Clinic researchers found that a ROS-TAC score was superior to simply measuring ROS and TAC alone when distinguishing fertile and infertile men [52]. In the female reproductive system, increased blastocyst development rate, decreased embryonic fragmentation and enhanced cleavage rate were to some extent related to day 1 TAC in the culture media. As such, TAC in day 1 culture media can be a significant biochemical marker for early embryonic growth [21].

Accurate assessment of ROS levels in semen are essential to determine because they can aid in the diagnosis of OS-induced infertility [53]. The chemiluminescence assay is a commonly used method for the direct measurement of intracellular and extracelluluar ROS generation [42]. Studies conducted by the Cleveland Clinic researchers have validated the chemiluminescence assay as a reliable and accurate diagnostic test that minimizes intra- or inter-assay variation and prevents interference from extraneous factors [54]. Moreover, the Clinic researchers have specified that the assay is both accurate and reliable only when the sperm concentration is greater than 1 × 10⁶ ml⁻¹ and the samples are analyzed within the first hour after specimen collection [55].

Further studies conducted by Cleveland Clinic researchers have found that in certain instances, flow cytometry has a higher specificity for intracellular ROS production by spermatozoa and thus some samples which tested negative by chemiluminescence might still have high levels of intracellular hydrogen peroxide [56]. In cases such as these, flow cytometry would be a more accurate method to measure ROS generation.

Further insight into ROS generation in semen can be provided by the nitroblue tetrazolium reduction test, which is an effective method of assessing the contributions of defective spermatozoa and seminal leucocytes to the overall ROS generation in semen. Moreover, the NBT reduction test is easily available and performed, has high sensitivity and is cost-effective [57].

The sperm deformity index (SDI) score indicates the quality of sperm morphology, which can serve as a more powerful predictor of male factor fertility and of IVF outcome compared to the assessment of the percentage of sperm with normal morphology [58]. High SDI scores are associated with OS [59, 60] and apoptosis [61] in spermatozoa.

Thus, the SDI is also a useful method to identify infertile men with abnormal levels of OS-induced DNA damage in spermatozoa [59, 62]. Researchers at Cleveland Clinic have found that SDI scores were positively correlated with the percentage increase in sperm DNA damage when sperm samples treated with NADPH (which is known to cause an increase in ROS production) were incubated for 24 h; thus validating the SDI [59]. In an in vitro study, exposure of sperm to peritoneal fluid from patients with endometriosis was associated with significantly increased DNA damage after 24 h of incubation. The SDI scores assessed showed significant correlation with sperm DNA damage at 24 h post-incubation [63].

The oxidative insult to spermatozoa due to the over production of ROS can result in LPO [5]. During LPO, over 60 % of the fatty acid chains in the plasma membrane can deteriorate thus reducing sperm membrane integrity [13, 82]. One of the byproducts of LPO is MDA, which has been used in numerous studies by the Cleveland Clinic to monitor the degree of peroxidative damage to sperm membranes. One such study observed that as sperm concentration increased, so did MDA levels, thus indicating that sperm concentration is positively correlated with LPO [28]. Antioxidants that break free radical chain reactions, such as Vitamin E, inhibit LPO [73].

A controlled prospective study conducted by Cleveland Clinic researchers compared the mitochondrial membrane potential (MMP) with ROS levels in nineteen infertile men and seven healthy donors. MMP is an important indicator of the functional integrity of the spermatozoa, and is positively correlated with sperm motility and viability. Results of the aforementioned study showed that abnormal semen parameters and ROS levels were negatively correlated with MMP, thus indicating that the measurement of MMP in spermatozoa provides useful insight into a man’s fertility potential [66].

Sperm DNA damage is observed more frequently in infertile men than in healthy fertile men thus suggesting that it could be a major cause of male infertility [83]. In a study at the Cleveland Clinic, the sperm chromatin structure assay was used to assess DNA damage in human spermatozoa at different stages of maturation from males undergoing infertility evaluation [84]. The study showed that chromatin alterations were interconnected with leukocyte concentration in immature and mature sperm as well as with immature germ cell concentration and abnormal forms of semen. In other studies, Cleveland Clinic researchers reviewed the relationship between sperm chromatin integrity, hormone levels, seminal plasma TAC, and sperm parameters in men with male factor and non-male factor infertility. These studies suggested that male factor infertility associated with sperm chromatin damage may be related to sperm protamination and to serum DHEA [85, 86].

Sperm DNA damage has been implicated as a leading cause of male infertility. In two studies by Cleveland Clinic researchers, the effect of ROS generation on DNA damage and apoptosis (by stimulating DNA damage) in men classified as idiopathic infertile was examined [87, 88]. As apoptosis showed little correlation with sperm DNA damage, it seemed unlikely that apoptosis was the cause of ROS-induced DNA damage in these studies. Instead, it was ROS generation that showed a significant contribution towards DNA damage in spermatozoa.

In a further study conducted by the Clinic, the correlation between abnormal sperm morphology ROS levels and sperm DNA damage was evaluated using the SDI [59]. NADPH (a primary source of ROS) was found to play a key role in ROS-mediated DNA damage in infertile patients’ semen samples containing high levels of abnormal spermatozoa. Other studies by Cleveland Clinic researchers have also found that immature spermatozoa have higher incidences of NADPH-induced DNA damage [77]. In fact, the relative proportion of immature sperm producing ROS was directly correlated with increased DNA damage in mature sperm. However, the researchers found no correlation between DNA damage and sperm morphology in mature sperm. They concluded that the increased DNA damage in mature sperm could be due to oxidative damage resulting from ROS producing (immature) sperm that occurs as sperm migrates from the seminiferous tubules to the epididymis [89].

Finally, levels of sperm DNA damage and OS in infertile men with varicocele were studied. Results showed significantly high levels of DNA damage, which seemed to be related to the high levels of OS found in the patients’ semen [90].

Research conducted at the Cleveland Clinic has linked oxidative stress and the production of ROS with the expression of cytokines. In such studies, infertile patients with varicocele and patients having undergone vasectomy reversal had elevated levels of IL-6, which was positively correlated with ROS production [91, 92]. Another study found that alteration to microtubule spindle structure and chromosomal alignment in mouse metaphase II oocytes due to tumor necrosis factor is augmented by the induction of oxidative stress [93].

Numerous studies have been conducted by Cleveland Clinic researchers to examine the relationship between apoptosis and ROS (Fig. 8.6). In one study, a positive correlation between ROS levels and the percentage of cells undergoing apoptosis was observed [87]. Another study examined the mechanism by which ROS might increase apoptotic cell death. Researchers reported that an increase in ROS levels was associated with an increase in caspase 3, caspase 9, and cytochrome c, which are proteins that mediate apoptosis [94]. Moreover, the significant positive correlation between ROS and these apoptosis-mediating proteins also suggested the possibility of DNA damage through increased ROS production. Anti-heat shock protein antibodies (anti-HSP) also have an adverse effect on embryonic cell development by increasing the incidence of apoptosis in these cells [95]. However, ROS levels were unaffected by variation in anti-HSP antibodies. Thus, the increased incidence of apoptosis occurred independent of ROS level fluctuation [95].

The presence of l-carnitine (an amino acid derivative) in the peritoneal fluid of endometriosis patients lowered the incidence of mouse embryo apoptosis, therefore suggesting that carnitines could play a protective role against apoptotic cell death. The improvement seen in the level of embryo apoptosis after the addition of l-carnitine may be due to the potent antioxidant properties of l-carnitine and its role in downregulating cytokines present in the peritoneal fluid of endometriosis patients [96]. Besides protection from apoptotic cell death, l-carnitine has free radical-scavenging activity and inhibits LPO, thereby protecting the cell membrane and DNA against damage induced by free oxygen radicals. Another study using mouse pre-implantation embryos showed that the incidence of apoptosis was reduced by supplementation of culture media with protein (serum substitute). In addition, protein supplementation reduced ROS levels and helped improve the hatching rate in these embryos [97].

Research by the Cleveland Clinic indicates an active role for poly (ADP-Ribose) polymerase (PARP) in maintaining normal sperm cell physiology by ensuring normal sperm maturation and preventing OS as well as apoptosis [98, 99]. PARP is an abundant nuclear enzyme that plays a role in DNA repair and transcriptional regulation. It also helps detect DNA strand breaks caused by ROS. Cleaved poly (ADP-ribose) polymerase (cPARP) plays a significant role in interrupting the DNA repair process and activating caspase-3, thus inducing apoptotic cell death. Cleaved PARP is present in ejaculated human spermatozoa and plays a role in OS-induced damage of spermatozoa [99].

The Cleveland Clinic researchers have looked at the effect of OS on the developing embryo. In one study, ROS was positively correlated with blastocyst development rate [100], thus suggesting that ROS has a physiological role in embryonic development. However, another study found that when embryonic cells were cultured, high day 1 ROS levels were associated with low blastocyst development rate, embryonic fragmentation, and low fertilization rate in intra-cytoplasmic injection (ICSI) cycles [20]. In essence, while ROS play a physiological role in oocyte development and maturation, excessive ROS production can be harmful to embryonic cells [2, 35, 101]. This is because OS can alter steroidogenesis in the ovaries, which increases androgen production and disturbs follicular development [82]. Moreover, excess ROS levels in the oocyte, follicular fluid and embryo result in poor oocyte quality, alter the activity of cumulus mass cells and result in embryonic development block and retardation [10].

Patients undergoing assisted reproduction using conventional IVF or ICSI cycles were included in a study by Cleveland Clinic researchers. The results showed that high day 1 ROS levels were associated with high embryonic fragmentation only during ICSI cycles and not IVF, due to the early exposure of embryos to ROS in ICSI cycles [60].

For the first time, Cleveland Clinic researchers characterized the presence of ROS, TAC and LPO in hydrosalpingeal fluid (HSF) aspirated from infertile women undergoing laparoscopic salpingectomy. The blastocyst development rate of mouse embryos incubated in human HSF correlated positively to concentrations of ROS but did not correlate significantly with LPO. It was found that increasing concentrations of HSF decreased the blastocyst development rate, thus suggesting a possible role for OS in the embryotoxicity of HSF [100]. In a follow up study, the researchers demonstrated for the first time the presence of cytokines IL-1beta, IL-8, IL-6 and TNF-alpha, and the absence of IL-13 in human HSF. Of these cytokines, IL-6 correlated positively to the blastocyst development rate of mouse embryos [102].

Spermatogenesis is a temperature-dependent process, which occurs ideally at temperatures slightly lower than that of the body. As such, thermoregulation is essential to maintain an adequate testicular temperature. A higher testicular temperature negatively effects spermatogenesis and the resulting spermatozoa. Therefore, thermoregulatory failure leading to testicular heat stress can result in poor sperm quality and thus increase the risk of infertility [103]. Moreover, elevated temperature in the testis can result in germinal atrophy, spermatogenic arrest as well as germ cell apoptosis [103]. One study by Cleveland Clinic researchers that confirms these heat-induced changes observed that sperm motility decreased dramatically after sperm samples were incubated at low temperatures (4 °C) [67] However, this decrease in percentage motile sperm was less dramatic when the sperm sample was incubated at 27 and 37 °C. The maintenance of sperm motility at higher temperatures was partially explained by the observation that seminal ROS levels were lowest in the sperm sample incubated at 37 °C. Thus, it can be surmised that when the temperature of a sperm sample deviates from the physiological temperature of the body, spermatozoa are adversely affected largely due to an increase in ROS production [67].

Cleveland Clinic researchers have investigated the effect of hydrogen peroxide (H₂O₂) exposure on spermatozoa and found that exogenous H₂O₂ increases intracellular ROS and NO production, and subsequently, decreases sperm motility [104]. A study was conducted to test the effect of H₂O₂ on mouse metaphase II oocyte microtubular spindle structure [93]. It was recorded that even at low concentration, H₂O₂ exposure increased the odds of abnormal microtubule and chromosomal alignment by 93 %. Furthermore, the intensity and probability of the damage to the microtubules and chromosomal alignment increased as the incubation period increased. It was also noted that chromosomal alignment in metaphase II oocytes was more resistant to the effects of H₂O₂-induced OS compared with microtubules [93]. In another study, Mahfouz et al. further examined the effects of H₂O₂ exposure on immature and mature sperm fractions from samples by healthy volunteers of unproven fertility [16]. Exposure to H₂O₂ decreased the viability in both sperm fractions and increased the number of apoptotic sperm. Thus, this study indicated that intracellular H₂O₂ might induce apoptosis [16]. The pathology of H₂O₂ is related to the ability of it to diffuse through the membrane of sperm cells and reduce the activity of certain enzymes [73]. However, under high ROS conditions, intracellular H₂O₂ was positively correlated with the number of viable sperm. This is because high ROS conditions may lead to an increase in defense mechanisms in spermatozoa, which help to counter and adapt to the increased presence of H₂O₂ [16].

Varicocele is the dilation of veins around the spermatic cord [13]. This condition has been closely linked with OS, because the testis responds to varicocele-associated stressors at the expense of ROS production [105, 106]. Contemporary evidence points towards a dominant role of ROS and OS in the pathogenesis of varicocele-associated male infertility despite the actual mechanisms not being elucidated yet. Excessive ROS present in varicocele patients is associated with sperm DNA fragmentation, both of which could result in poor sperm function and fertilization outcome [107]. The latest systematic review and meta-analysis by Cleveland Clinic researchers showed that varicocele is a significant risk factor that exerts a negative impact on semen quality [108].

Studies by the Cleveland Clinic researchers have found that semen samples from fertile donors have higher sperm concentration and motility than those from varicocele patients [88, 92, 109]. One clinical trial found that infertile patients with varicocele had a greater degree of impaired sperm parameters when compared to infertile patients without varicocele [90]. This study clarifies that although infertile men produce excessive amounts of ROS regardless of the presence of varicocele, varicocele exacerbates the condition of OS in infertile patients [110]. When compared to infertile patients with vasectomy reversal and idiopathic infertility, infertile patients with varicocele had the highest levels of ROS [71]. Another study compared infertile patients with varicocele to fertile patients with varicocele using healthy fertile donors as controls [111]. It was found that the infertile and fertile patients with varicocele had significantly higher ROS levels, lower TAC levels, lower ROS-TAC scores and lower semen quality scores compared to the controls; however, the ROS-TAC score between the fertile and infertile varicocele patients respectively did not differ [111]. These findings indicate that the presence of varicocele exclusively impairs sperm function regardless of the whether the patient was originally fertile or not.

Furthermore, a specific study found that the grading of varicocele affects the levels of seminal OS present in patients with varicocele [109, 112]. Patients with grade 3 varicocele had higher seminal ROS levels than those with grade 1 varicocele [109, 112]. Literature on Cleveland Clinic studies also suggests that the contribution of varicocele to increase OS levels may be limited to only infertile patients. One study analyzed the semen from fertile patients with and without varicocele and found that there was no significant difference in seminal ROS levels between the two groups. Thus, this study indicates that the adverse effect of increased ROS levels on sperm parameters in varicocele patients may be predictable depending on whether the patient is initially fertile. Moreover, this study demonstrates that the presence of clinical varicocele does not associate with higher seminal ROS levels or abnormal semen parameters in fertile men. In fact, in these fertile men, ROS levels were not correlated with varicocele grade or testicular volume [113].

The mechanism by which varicocele leads to the extensive generation of ROS is partly explained by studies which found that infertile varicocele patients (compared to fertile donors) have elevated ROS levels and inflammatory cytokines, such as IL-6, as well as decreased TAC [71, 90–92, 109, 114, 115]. An increase in pro-inflammatory cytokines leads to higher seminal ROS levels due to a significant decrease in TAC [13]. On the other hand, functional proteomic analysis conducted by the Cleveland Clinic has provided insight into the mechanisms by which varicocele can impair sperm function. Infertile men with unilateral and bilateral varicocele have various overexpressed and underexpressed proteins which impair proteins involved in fundamental reproductive processes such as capacitation, hyperactivation, sperm-zona binding and acrosome reaction [116, 117]. These proteins can serve as potential biomarkers for unilateral and bilateral varicocele.

Varicocele treatment reduces ROS levels as well as improves semen parameters and fertilization rates [118]. The most effective treatment option for infertile men with varicocele is microsurgical spermatic vein ligation [47].

Over 90 % of men with spinal cord injury are infertile, indicating that spinal cord injury is related to infertility and possibly OS [13]. In one study, the Cleveland Clinic investigated the relationship between the generation of ROS and semen characteristics in men with spinal cord injury. To stimulate a burst of ROS production from leukocytes, N-formyl-methionyl-leucylphenylalanine (FMLP) was used, while ROS production from spermatozoa was stimulated by 12-myristate 13-acetate phorbol ester (PMA) in the semen of both male patients with spinal cord injury and normal men [119]. When the semen was compared, it was found that the levels of ROS and the white blood cell count were higher in spinal cord injury patients than in normal men. Sperm morphology and motility were also lower in spinal cord injury patients [119]. These results indicate that a link exists between spinal cord injury and OS.

ROS is present in both fertile and infertile females with endometriosis (Fig. 8.7) [80]. Studies by Cleveland Clinic researchers have revealed that a vicious cycle exists in the case of endometriosis whereby OS is induced which in turn causes the implantation of the ectopic endometrium (a primary characteristic of endometriosis). This action triggers the generation of more ROS which further triggers OS [120]. However, it is important to note that some studies found that the presence of ROS in the peritoneal fluid of females with endometriosis is not statistically different from the ROS production from healthy female donors [80]. Thus, the presence of ROS in the peritoneal fluid may not have a significant effect on females with endometriosis in the context of infertility. In contrast, other studies observed increased levels of OS markers (MDA and pro-inflammatory cytokines) in the peritoneal fluid of patients with endometriosis [121] thus suggesting that endometriosis can induce OS.

Antioxidants, such as vitamins C and E neutralize ROS and thus could be a form of treatment for patients with endometriosis [120].

Vasectomy reversal patients have elevated ROS and IL-6 levels when compared to normal controls [91, 122, 123], thus indicating that vasectomy reversal may induce seminal OS. ROS levels are also higher in infertile vasectomy reversal patients than fertile vasectomy reversal patients [123]. However, vasectomy reversal does not seem to affect TAC levels [122]. As expected due to the excessive generation of ROS, the patients who have undergone vasectomy reversal have decreased sperm concentration and motility [91]. Moreover, levels of creatine kinase (CK), a biochemical marker for cellular maturity were elevated in infertile patients with vasectomy reversal when compared to patients with idiopathic infertility, testicular cancer, and normal fertile donors. This indicates that spermatozoa from post-vasectomy reversal patients may be biochemically immature, and therefore have a lesser chance of fertilizing the ovum [124]. This study conducted by Cleveland Clinic researchers suggests that vasectomy reversal may be associated with OS.

Studies reported by the Cleveland Clinic researchers have linked poor semen quality, DNA mutations [125] and an increase in disorders such as autism and schizophrenia, with increased paternal age [126]. Elevated ROS levels related to aging and infertility can result from decreased antioxidant levels and impaired Leydig cell function [3]. A study on 98 fertile men who were candidates for vasectomy was conducted to evaluate the effect of age on ROS generation [127]. Participants in the study were divided into two groups: individuals under the age of 40 (n = 78) and individuals over the age of 40 (n = 20). Positive controls consisted of 46 infertile patients. The mean age of the men was 35.1 ± 5.6 years. The group of individuals above the age of 40 had higher ROS levels than the younger group of men and the controls. Thus, ROS levels in the whole ejaculate have a positive age-dependent correlation among fertile men [127].

Cigarettes have over 4000 chemicals, some of which can cause excessive generation of ROS, thus inducing a state of OS [39]. Levels of OS were evaluated in infertile men with a history of smoking, which demonstrated that infertile men who smoked had higher OS levels compared to non-smoking infertile men [128]. In this study, smoking was associated with increased ROS levels, seminal leukocyte concentrations, and decreased ROS-TAC score. Another study by the Cleveland Clinic analyzed the semen from infertile smokers, infertile non-smokers, and healthy fertile donors [128]. It was determined that smoking increased seminal leukocyte concentrations by 48 %, increased ROS levels by 107 % and lowered ROS-TAC scores by 10 points [39, 128]. In the female reproductive system, smoking can also adversely alter follicular and oocyte maturation [129]. A recent evidence based review by Cleveland Clinic researchers studied the data on smoking and male fertility and reiterated that couples who are trying to conceive in particular should adhere to the preventive approach of discouraging smoking and eliminating exposure to tobacco smoke [130].