Infertility 10...Unexplained vs. Idiopathic Male Infertility Unexplained male infertility is the inability to reproduce despite having a normal sexual history, physical exam, and semen

10Oxidative Stress in Unexplained Male Infertility

Sejal B. Doshi, Rakesh K. Sharma and Ashok Agarwal

A. Agarwal () · S. B. Doshi · R. K. SharmaCenter for Reproductive Medicine, Cleveland Clinic, 10681 Carnegie Avenue, Desk X11, Cleveland, OH 44195, USAe-mail: [email protected]

Introduction

Despite advances in modern reproductive technologies, in-fertility remains a common problem for couples worldwide. It is defined as the inability to conceive after one of year of unprotected sexual intercourse [1, 2]. In as many as 50 % of infertile couples, a male factor has been implicated as the sole or partial cause [3, 4]. Although there are specific male-related etiologies that can be addressed to correct the issue, in many cases, the cause of infertility cannot be identified, leading to a diagnosis of unexplained infertility (UMI). In fact, the diagnosis of unexplained infertility accounts for 10–30 % of infertility cases [5]. This chapter aims to shed light on the concept of UMI, particularly focusing on the di-agnosis, treatment, and implicating factors involved in the pathogenesis of this condition, including oxidative stress.

Unexplained vs. Idiopathic Male Infertility

Unexplained male infertility is the inability to reproduce despite having a normal sexual history, physical exam, and semen analysis on two or more occasions as well as no harmful toxin exposure. Its reported prevalence ranges from 6–27 % [2, 6]. Unexplained infertility is often loosely inter-changed with its counterpart, idiopathic infertility. Idiopathic infertility is defined as having an abnormal semen analysis in the absence of any identifiable cause [7]—this second category accounts for 40–50 % of male infertility cases [3]. For unknown reasons, the semen analysis of those with this type of infertility shows decreased sperm motility and sperm number and/or an increased number of spermatozoa with ab-normal morphology [3].

Due to the similarities in their definitions, studies often categorize unexplained and idiopathic infertility as one and the same [8]. However, further research on this topic has shown that there are significant distinctions between the two types of infertility (Table 10.1). Newer tests involving cytogenetic analysis and molecular genetics highlight these differences, which lie primarily in their specific etiologies [3]. Specifically, UMI has been associated with the follow-ing contributory factors: sperm dysfunction, auto-antibodies directed against sperm antigens, certain coital factors inter-fering with successful fertilization, and oxidative stress [9]. On the other hand, idiopathic infertility is suggested to be caused by age, environmental pollutants, mitochondrial al-terations, and infective agents such as Chlamydia trachoma-tis, herpes virus, and adenovirus [10, 11]. Additionally, post-testicular organs such as the epididymis have been shown to cause DNA methylation of cytosine–guanine nucleotides. This can lead to transcription repression and eventual idio-pathic infertility [10, 12]. Interestingly, recent reports state that sperm DNA damage and oxidative stress in the seminal plasma do not exist in certain males with idiopathic infer-tility due to differential expression of gene polymorphisms, such as the GSTM1 genotype. Specifically, males with this gene alteration have been associated with increased levels of oxidative-induced DNA damage [13, 14].

Although a singular cause of unexplained infertility can-not be identified in many cases, it is of great importance that a systematic infertility workup be performed (Table 10.1). This prevents the oversight of an underlying etiology of UMI [2]. Amidst the modern reproductive technologies of today, this chapter aims to shed light on the concept of UMI, partic-ularly focusing on the diagnosis, treatment, and implicating factors involved in the pathogenesis of this condition.

81G. L. Schattman et al. (eds.), Unexplained Infertility, DOI 10.1007/978-1-4939-2140-9_10, © Springer Science+Business Media, LLC 2015

82 S. B. Doshi et al.

Assessment of Males with Unexplained Infertility

When a couple seeks assistance for infertility issues, a thor-ough clinical evaluation of both partners should be performed to rule out identifiable causes and focus the investigation [15, 16]. The initial infertility workup includes a detailed history with an emphasis on the couple’s previous fertility record. This may include a history of recurrent miscarriages and ectopic pregnancies, the period of time in which the pa-tient has been infertile, successful previous pregnancies, and any complications that may have occurred before, during, or after delivery [2, 17]. Furthermore, a thorough coital history from both partners is necessary to reveal any difficulties in regard to intercourse, such as improper sexual technique or inappropriate timing of intercourse [18]. In addition to male infertility evaluation, the female partner should undergo a separate workup, in order to assess the patency of both fal-lopian tubes, consistency of the cervical mucus, and recep-tivity of the endometrium to blastocyst implantation [2, 6].

Once a thorough sexual and fertility history has been completed, a comprehensive physical exam should be per-formed, which can help to exclude anatomical causes of in-fertility [2). The physician should carefully examine the male patient for any physical aberrations within the structures of the male reproductive system. The penis, in addition to the epididymis, testis, and spermatic cord, should be palpated in order to rule out conditions such as epididymitis, orchitis, and varicocele [19–21]. In particular, all of these conditions promote the aggregation of free radicals or reactive oxygen species (ROS), eventually leading to sperm dysfunction and infertility [19].

Routine Semen Analysis

After the completion of an extensive medical history and physical examination, a routine semen analysis should be the first laboratory test conducted in the infertility workup. This is a cost-effective, non-invasive test that has been integral in the evaluation of male factor infertility for decades [22]. The efficacy of this test lies in the parameters for which it tests, which include pH, volume, color, total count, motility, concentration, and morphology.

Sperm count and motility are the first and most impor-tant predictors of fertility potential [23]. Normal values for all semen parameters have been highly associated with im-proved fertility outcomes and therefore are used for the as-sessment of infertility [24]. Although semen analysis is the first diagnostic step routinely employed in the evaluation of UMI, it does not identify the exact cause behind the infertil-ity [23, 25].

Therefore, in addition to a comprehensive history, clinical examination, and initial lab assessment, sperm function tests should be performed [26, 27]. Moreover, etiologies related to endocrine or genetic abnormalities should also be explored in the infertile individual [28]. Overall, it is clear that a rou-tine semen analysis cannot be used alone to diagnose infer-tility. Additional tests are required for the investigation and evaluation of the subfertile male [4].

Upon obtaining a normal semen analysis concurrent with an unremarkable history and physical examination, the phy-sician can make the diagnosis of UMI. At this point, the phy-sician should then explore potential contributing factors im-plicated in the pathogenesis of the UMI [2, 4]. This includes immunologic factors, genetic integrity defects, fertilization defects, and oxidative stress. The former three are explained in greater detail in subsequent chapters whereas oxidative-induced UMI is the prominent focus of this chapter.

Table 10.1 Unexplained male infertility versus idiopathic male infertilityParameter Unexplained male infertility Idiopathic male infertilitySemen analysis [1, 2, 6] Normal AbnormalHistory, physical exam, and endocrine assess-ment [2, 3, 14]

Normal Normal

Female factor infertility [2, 6] Ruled out (no tubal patency, no cervical hos-tility, or good endometrial receptivity)

Ruled out (no tubal patency, no cervical hos-tility, or good endometrial receptivity)

Percentage of total cases of male infertility [1, 3, 5]

10–30 % 40–50 %

Contributory causes [9–12] 1. Sperm dysfunction2. Auto-antibodies directed against sperm

antigens3. Coital factors interfering with successful

fertilization4. Oxidative stress

1. Age2. Environmental pollutants3. Mitochondrial alterations4. Infective agents (i.e., Chlamydia trachoma-

tis, herpes virus, and adenovirus)

History of harmful toxin exposure [6, 7] None NoneInfertility Status [3–5] Unknown KnownCategorized under [1, 2] Male infertility of unknown origin Male infertility of unknown origin

8310 Oxidative Stress in Unexplained Male Infertility

Immunologic Factors

The immune system of the human body naturally protects its cells from the harmful effects of an autoimmune reaction and various pathogens [29]. One such defense mechanism is the blood-testis-barrier, which protects sperm cells undergoing the process of spermatogenesis from an autoimmune attack [30]. However, if this barrier is broken, sperm antigens will come into contact with the immune system and lead to the formation of antisperm antibodies and eventually autoim-mune infertility [31].

In regard to the role of autoimmunity in infertility, studies have shown that antisperm antibodies penetrate the blood-testis-barrier, bind to spermatozoa, and reduce their fertil-ization capacity. These antibodies may also inhibit the ac-rosome reaction, activate the complement cascade system to lyse sperm cells, and interfere with the sperm’s ability to recognize particular binding sites on the zona pellucida [32]. Furthermore, they enter the cervical mucus and inhibit the ability of spermatozoa to penetrate it. Overall, the improper cellular or humoral immune response against sperm antigens causes dysfunction of spermatozoa and is a possible contrib-uting factor to the unexplained infertility seen in males.

Defects in Genetic Integrity

Compromises in genetic integrity have been significantly correlated with UMI. Such compromises in DNA can take the form of insertion or deletion of bases, cross-linkage of strands, chromosomal anomalies, as well as single or double-stranded breaks [33]. Current research suggests that there is a causal relationship between defective spermatozoa DNA and male infertility [34]. This was demonstrated in a study that performed microarray analyses on spermatozoa mRNA, which illustrated differential expression of many genes be-tween normozoospermic infertile men and fertile men. This indicates that males with unexplained infertility have distinct genome expression profiles specific only to UMI [35–37].

Overall, it is evident that large amounts of DNA damage in spermatozoa can interfere with a man’s ability to achieve a natural, viable pregnancy. Further research needs to be conducted on the effects of DNA damage on pregnancy out-comes as well as the threshold of damage that allows for normal functioning of spermatozoa [33].

Fertilization Defects

Fertilization of the oocyte is a complex and arduous process carried out by spermatozoa. It is a multi-step event beginning with (1) capacitation, (2) hyperactivation, (3) sperm-zona pellucida binding, (4) acrosome reaction, (5) penetration of

the zona pellucida, (6) sperm-oocyte fusion, (7) cortical and zona reaction, and finally (8) post-fertilization events. Each component of this process must be carried out in a precise manner because interference with any of these steps can po-tentially lead to infertility [38]. Specifically, those with UMI have been shown to have reduced levels of protein phosphor-ylation, which is necessary for the process of capacitation [39]. Moreover, normozoospermic infertile men have dem-onstrated decreased hyperactivation of spermatozoa as well as defects in the proteins necessary for the zona pellucida induced-acrosome reaction [40–42]. Overall, a variety of anomalies can take place during the process of fertilization and contribute to unexplained infertility in males.

Oxidative Stress

Oxidative stress is defined as an imbalance between reactive oxygen species (ROS) and antioxidant defense mechanisms in the body [43]. ROS comprise a class of radical and non-radical oxygen derivatives [44]. Not only do ROS include oxygen radicals such as the hydroxyl radical, superoxide radical, and hydrogen peroxide but also a subclass of nitro-gen-containing compounds collectively known as reactive nitrogen species (RNS). Examples of RNS include peroxyni-trite anion, nitroxyl ion, nitrosyl-containing compounds, and nitric oxide [44, 45]. The most common ROS that is pro-duced by spermatozoa is the superoxide anion radical; this is turn forms hydrogen peroxide (strong oxidizer) on its own or by the action of superoxide dismutase (SOD).

Physiologic Role of Free Radical Species Studies have shown that physiological levels of ROS are required for many baseline bodily functions [44]. Moreover, appropriate concentrations of ROS allow for proper signal transduction, mediation of cytotoxic events, and facilitation of inflamma-tion via prevention of platelet aggregation and neutrophil adherence to endothelial cells [44]. These free radical spe-cies also serve as signaling molecules or second messengers, as well as aid in the production of hormones, regulation of tight junctions, and mediation of apoptosis. In regard to the male reproductive system, low ROS levels are necessary for capacitation, hyperactivation, acrosome reaction, zona pel-lucida binding and fertilization capacity of spermatozoa and promote normal semen parameters, such as sperm motility, morphology, and viability [19, 28, 46].

Detrimental Role of Reactive Oxygen Species Although ROS are necessary for normal physiological functions, excess levels overwhelm the body’s natural antioxidant capacity. Due to the unpaired electron in their outer orbit, ROS are highly reactive and interact with a variety of lipids, proteins, and nucleic acids in the body. Such reactions are


extremely harmful for reproductive potential and possibly contribute to testicular dysfunction, decreased gonadotro-pin secretion, and abnormal semen parameters [47]. While ROS affect a variety of reproductive functions, their reactive nature leads to the generation of more free radicals. This, in turn, perpetuates a chain of reactions creating tissue damage in the form of oxidative stress [45].

In the male reproductive system, ROS are mainly pro-duced by immature spermatozoa, macrophages, and poly-morphonuclear leukocytes. Specifically, the latter two rep-resent the majority of seminal leukocytes that generate ROS [48]. Conditions such as varicocele and leukocytospermia stimulate these leukocytes, among other inflammatory cells, to produce large amounts of ROS. Moreover, lifestyle habits such as smoking are strongly correlated with increased ROS production [49]. Additionally, these free radical species have been associated with cardiovascular disease due to the oxi-dation of low density lipoprotein (LDL) within the vascular endothelium. Oxidative stress also contributes to reperfusion injury following ischemia well as tissue injury after radia-tion therapy. Furthermore, infections such as Helicobacter pylori and neurodegenerative diseases such as Alzheimer’s and Huntington’s disease have been also been linked to the accumulation of oxidative stress [48].

Role of Oxidative Stress in Unexplained Infertility In addi-tion to contributing to a variety of conditions, there is grow-ing evidence that oxidative stress is involved in many aspects of UMI (Fig. 10.1). Studies have shown that when com-pared to fertile men, normozoospermic infertile males have elevated ROS levels measured by the malonaldehyde lev-els and protein carbonyl groups. [1, 50]. This is also evident by lower reactive oxygen species-total antioxidant capacity (ROS-TAC) scores in patients with UMI, which indicates elevated levels of seminal oxidative stress [51, 52]. More-over, studies report high ROS dysfunction in UMI patients. This dysfunction can be in the form of reduced fertilization capacity, impairment of sperm metabolism, and lipid per-oxidation of polyunsaturated fatty acids within the sperm plasma membrane [53]. Detrimental effects of high ROS on semen parameters such as motility, viability, morphology have been demonstrated by numerous studies [54, 55]. Not only does oxidative stress have negative consequences on sperm parameters but also on the DNA of sperm cells [50]. Studies have shown that patients with UMI have a signifi-cantly higher DNA fragmentation index ( > 30 %) induced by toxic levels of ROS when compared to those who are fertile [56]. Specifically, a fragmentation index > 30 % is associated with lower chances of achieving pregnancy by natural con-ception or by insemination [57]. This finding of increased ROS levels may indicate that seminal oxidative stress may be involved in the pathogenesis of sperm DNA damage in these patients [56]. It has also been suggested that specific

genetic polymorphisms can promote the accumulation of oxidative stress within the seminal plasma, such as the glu-tathione S-transferase Mu-1 (GSTM1) gene polymorphism. Studies have reported that men with idiopathic and unex-plained infertility who have the GSTM1 null genotype had significantly higher levels of seminal oxidative stress than those who possessed the GSTM1 gene. Therefore, in patients suffering from infertility, the GSTM1 polymorphism might be integral to determining the susceptibility of spermatozoa to oxidative damage [58]. Additionally, studies report that UMI patients with excess levels of ROS have numerous mutations on genetic analysis of sperm mitochondrial DNA. Examples of such alterations include nucleotide changes in the ATPase and nicotinamide adenine dinucleotide dehydro-genase genes. These DNA mutations may be the underlying etiology of a male’s unexplained infertility [59]. Further-more, reactive oxygen species-induced DNA damage may accelerate the process of germ cell apoptosis, leading to the decline in sperm counts associated with male infertility [60]. Overall, it is clear that the oxidative stress may play a role in UMI via the mechanism of spermatozoa DNA damage.

Measurement of Oxidative Stress

A number of methods have been utilized in the laboratory setting to measure ROS [45]. Direct methods include cy-tochrome c reduction, electron spin resonance, and nitro-blue tetrazolium technique (NBT, and xyenol orange-based assay). Indirect methods of measurement include the Endtz

Fig. 10.1 Schematics of unexplained male infertility resulting in pro-duction of reactive oxygen species, genetic defects as well as fertiliza-tion defects


test, redox potential (GSH/GSSG), measurement of lipid peroxidation levels, chemokines and measurement of DNA damage, and measurement of reactive nitrogen species by Greiss reaction and fluorescence spectroscopy [61].

Assessment of ROS by Chemiluminescence The chemilumi-nescence assay is one of the most commonly used techniques to measure seminal ROS levels in clinical andrology labora-tories. The two commonly used probes are luminol (5-amino-2,3-dihydro-1,4-phthalazinedione and 3-aminophthalic hydrazide) and lucigenin ( N, N’-dimethyl-9,9’-biacridinium dinitrate). Both H2O2 and O2

●− are involved in luminol-depen-dent chemiluminescence because both catalase and SOD can disrupt the luminol signal very efficiently. A luminescent sig-nal is produced with luminol through a one-electron oxida-tive event mediated by hydrogen peroxide (H2O2) and either endogenous peroxidase or by addition of horse radish peroxi-dase. Superoxide anion (O2

●−)is an essential intermediate for the luminol-dependent chemiluminescence. Also, the redox cycling activity associated with this probe allows the signifi-cant amplification of the signal and allows easy measurement of H2O2. There are a variety of luminometers available and these may be single tube or multiple tube luminometers [61].

Luminol measures both intracellular and extracellular ROS such as hydrogen peroxide, and superoxide anion, on the other hand, lucigenin measures only extracellular ROS, and in particular superoxide anion.

For the actual assay, luminol (5 millimolar) is used to measure ROS in a clinical andrology lab setting. Chemilumi-nescence is measured using an instrument called luminom-eter (Fig. 10.2). Luminol is sensitive to light and the assay is performed in indirect light or in the dark. The samples are run in duplicate or in triplicate with appropriate negative and positive controls. Test samples are comprised of 400 µL of completely liquefied seminal ejaculate + 10 µL luminol; positive control: 400 µL of phosphate buffered saline (PBS)

and 50 μL hydrogen peroxide (30 %) + 10 μL luminol; and negative control: 400 μL PBS and 10 μL luminol. (Fig. 10.3) The measurement is for 15 min. The results are expressed as relative light units (RLU)/s/106 sperm. ROS levels > 20(RLU)/s/106 sperm are considered as positive.

Flow Cytometry Flow cytometry is a laboratory method used for analyzing the expression of cell surface markers and intracellular molecules. Oxidation of 2, 7 dichlorofluo-rescein diacetate (DCFH-DA) by ROS, which is generated within the cell, makes them highly fluorescent and can be used to measure formation of intracellular levels of hydrogen peroxide. Hydroethidine (HE) is another fluorescent probe that can be used for the measurement of intracellular levels of superoxide [61, 62].

Griess Test and Spectrophotometry The Griess test is one of the most sensitive methods for detecting ROS in the form of nitrite or nitrous acid. Specifically, the presence of these NO-containing compounds can be assessed by reacting them with sulfanilic acid. The resulting product is a diazonium compound, which combines with alpha-naphthylamine to produce pink azo dye that is highly absorbent. In particular, the formation of this dye can be used to measure amounts of any substance that will yield nitrite in known proportions. The amount of nitrogenous compounds absorbed by the azo dye is measured colorimetrically [63, 64].

Measurement of Other Parameters of Oxidative Stress

Total antioxidant capacity In addition to measuring levels of ROS, total antioxidant capacity and DNA damage can also be evaluated to study the impact of oxidative stress on UMI. Total antioxidant capacity can be measured in the seminal plasma samples using an antioxidant assay kit (Cayman Chemical Company, Ann Arbor, Michigan). Its principle is based on the ability of aqueous and lipid-based antioxidants in seminal plasma to inhibit oxidation of the ABTS (2,2’-Azino-di-[3-ethylbenzthiazoline sulphonate]) to ABTS●+. Under the reaction conditions used, the antioxi-dants in the seminal plasma suppress absorbance at 750 nm to a degree that is proportional to their concentration. The capacity of the antioxidants in the sample to prevent ABT-Soxidation can be compared with that of Trolox, a water-soluble tocopherol analog, and the results are reported as micromolar trolox equivalents [65].

Measurement of DNA damage DNA damage can be mea-sured by the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) assay. In

Fig. 10.2 A multitube Autolumat 953 plus luminometer used in the measurement of ROS by chemiluminescence assay. Multiple tubes can be loaded simultaneously for measuring ROS. (Reprinted with permis-sion, Cleveland Clinic Center for Medical Art & Photography © 2011–2013. All rights reserved)


this assay, sperm DNA fragmentation is evaluated using a TUNEL assay with an Apo-DirectTM kit (Pharmingen, San Diego, CA) as described earlier [66, 67]. An aliquot of well liquefied seminal ejaculate is used to assess DNA damage using the TUNEL assay. Briefly, 1–2 million spermatozoa are washed in (PBS) and resuspended in 3.7 % paraformalde-hyde; the sperm concentration is adjusted to 1–2 × 106 sperm/mL. Spermatozoa are washed to remove the paraformalde-hyde and then resuspended in 70 % ice-cold ethanol. Positive and negative kit controls provided by the manufacturer are run in addition to the lab internal control specimen (speci-mens from donors and patients with known DNA damage) with each run. Following a second wash with “Wash buf-fer” to remove ethanol, the sperm pellets are resuspended in 50 µL of freshly prepared staining solution for 60 min at 37 °C [66, 67]. The staining solution contains terminal deoxytransferase (TdT) enzyme, TdT reaction buffer, fluo-rescein isothiocynate tagged deoxyuridine triphosphate nucleotides (FITC-dUTP) and distilled water. After washing in “Rinse buffer” they are re-suspended in 0.5 mL of prop-idium iodide/RNase solution, and incubated for 30 min in the dark at room temperature followed by flow cytometric analysis. The samples are next analyzed within an hour after PI/RNase staining.

All fluorescence signals of labeled spermatozoa are ana-lyzed by the flow cytometer FACScan (Becton Dickinson, San Jose, CA). About 10,000 spermatozoa are examined for each assay at a flow rate of < 100 cells/s. The excitation

wavelength is set at 488 nm supplied by an argon laser at 15 mW. Green fluorescence (480–530 nm) is measured in the FL-1 channel and red fluorescence (580–630 nm) in the FL-2 channel. The percentage of positive cells (TUNEL-positive) is calculated on a 1023-channel scale using the flow cytometer software FlowJo Mac version 8.2.4 (Flow-Jo, LLC, Ashland, OR) [66, 67] (Fig. 10.4). Samples with > 19 % DNA damage are considered as positive or abnormal.

Therapeutic Options for UMI

Upon receiving a diagnosis of UMI, a couple must decide if they would like to identify the precise cause of the infertil-ity. This decision is quite important, for it allows the most appropriate treatment option to be considered [7]. However, in the face of no functional abnormalities, watchful waiting is suggested as a valid treatment option for UMI if the fe-male partner is younger than 35 years and the couple has had infertility problems for less than three years. This is due to the high spontaneous conception rate seen in couples who fall into these criteria [68]. If chances of achieving a sponta-neous pregnancy are minimal, the clinician should continue trying to find the cause of the infertility via testing.

The tests for determining infertility are quite specific and manifold. Thus, in order to save the patient time and money, it is essential to narrow down the underlying cause of UMI so that unnecessary tests are not performed [69]. Subsequent

Fig. 10.3 Schematics of sample preparation for the ROS measure-ment. A total of 11 tubes are labeled from S1–S12: blank, negative control, patient sample, and positive control. Luminol is added only to all tubes except blank. Hydrogen peroxide is added only to the positive control


chapters will go into further details regarding the battery of tests available for detecting the functionality of spermatozoa, defects in genetic integrity, as well as the fertilization poten-tial of sperm [70, 71].

Once the contributing cause has been determined, a va-riety of treatment options can be used. Specifically, when considering infertile males with antisperm antibodies, DNA damage, fertilization defects, or oxidative stress, studies have shown that intracytoplasmic sperm injection (ICSI) is the most successful treatment of choice [1]. In this assisted reproductive technique (ART), a spermatozoon is directly injected into an oocyte’s cytoplasm. This allows for direct fertilization of the oocyte and bypasses any barriers that spermatozoa may encounter during the natural fertilization process [72]. Nevertheless, this technique does not come without its risks. If a defective sperm happens to be selected for ICSI, the resulting pregnancy has a risk of giving rise to mutations after fertilization. This is because damage to the spermatozoa, often in the form of compromised genetic in-tegrity, cannot be fixed during the early stages of embryonic development as ICSI directly injects the spermatozoon into the egg. As a result, there are no natural protections avail-able to the spermatozoa on its journey to the oocyte [73]. Although ICSI is a promising treatment for UMI due to a myriad of causes, further research is still needed on the com-plications of this technique in regard to embryo development and pregnancy outcomes [72].

When considering oxidative stress as the sole cause of man’s unexplained infertility, a multitude of treatment op-tions exist. One such treatment includes dietary antioxidant supplementation. Specifically, antioxidants neutralize ROS and prevent subsequent tissue damage as a result of oxida-tive stress [74]. Helpful antioxidants include carnitine, se-lenium, zinc, lycopene, vitamin E, and vitamin C. Adding these antioxidants to the diet of males with unexplained

infertility has shown to substantially improve sperm DNA damage, pregnancy rates, semen parameters, and live birth outcomes [75]. However, more research needs to be done to determine the dose and duration of antioxidant therapy that will eliminate oxidative stress without causing any harmful side effects.

Other helpful methods for ROS-induced UMI include a variety of lifestyle modifications: consuming more fruits and vegetables, exercising, and avoiding tobacco products [2, 75].

Conclusion

Upon reviewing the literature, it can be concluded that nor-mal sperm parameters do not guarantee full fertilization ca-pacity of spermatozoa. Only after performing a thorough his-tory and physical exam of both partners in the face of a nor-mal semen analysis should UMI be considered. Specifically, doing this can help rule out female infertility factors, coital and genetic problems, as well as any physical aberrations. It is also important to fully comprehend the process of fertil-ization and the various components required for maximum fertilization potential of sperm. Upon confirming a diagnosis of UMI, a variety of factors should be explored as sugges-tive causes of infertility. These include autoimmune antibod-ies, DNA damage, fertilization defects, and oxidative stress. Modern andrology has developed innovative treatment op-tions specific to the case of UMI. Overall, further molecular and genetic studies are needed to better understand the phys-iology of spermatozoa and the overall fertilization process. Finally, long-term clinical trials need to be performed in order to assess the effects of various reproductive techniques on pregnancy rates and embryo outcomes.

Fig. 10.4 A typical curve show-ing sample with a: Negative DNA damage and b: positive DNA damage


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Infertility 10...Unexplained vs. Idiopathic Male Infertility Unexplained male infertility is the inability to reproduce despite having a normal sexual history, physical exam, and semen