Infertility services are increasingly being utilized due to the later age of first pregnancy and associated reduction in female fertility. The considerable decline in fertility associated with advancing maternal age prompt the question whether advanced paternal age is also associated with compromised fertility. Changes in human reproductive behavior including prolonged life expectancy and improvements in assisted reproductive techniques have led to an increase in average paternal age [25]. Moreover, delayed childbearing is a common phenomenon in industrialized countries and age-related changes in the male reproductive system are becoming commonly recognized. Although men at any age can establish pregnancy in a woman, we still do not know how safe and wise it is to attain pregnancy through assisted reproductive techniques at an advanced age. Although the effect of male age is less prominent than of the female, this becomes especially significant when the female partner is also of advanced age [26].

Probably, men start contributing to a decline in the couple’s fertility in their late thirties and to a decrease in fecundity in early forties [27]. Changes in male reproductive and sexual physiology clearly occur with aging, but the real impact of these changes on male fertility is not completely established. Currently, there is evidence that birth defects, especially those arising from new autosomal mutations, increase with paternal age [28]. Also, advanced paternal age is associated with new mutations in paternal genome and increased risk of aneuploidy in the fetus. These findings suggest that genetic risks associated to increased paternal age should be of high interest to andrologists counseling older men who wish to father a child or those for whom ageing could be an associated cause of infertility.

Spermatozoa are susceptible to the damage induced by excessive reactive oxygen species (ROS) because their cytoplasm contains low concentrations of antioxidant scavenging enzymes [29]. It is already known that with advancing age an organism is under a greater oxidative stress (OS) as the result of impairment of the function of the mitochondrial respiratory chain [30].

Recent data showed that seminal ROS levels are significantly higher in healthy fertile men older than 40 years [31]. Also, these high levels of ROS are significantly correlated with age among fertile men. As high ROS levels have been associated with the pathogenesis of male infertility, these findings suggested that delayed fatherhood may reduce the chances of pregnancy.

Seminal OS causes impairment of semen quality by multiple mechanisms including damage of sperm DNA integrity. Recently, a significant age related increase of DNA fragmentation has been reported [32]. The risk of miscarriage in couples with paternal age over 40 years may be attributed to the greater DNA fragmentation found in older men, possibly as a result of a less efficient apoptotic mechanism [26, 32].

There are many other factors that could be involved in sperm dysfunction, independent of age. These factors include environmental pollution, lifestyle (smoking, caffeine intake, or alcohol), occupational exposure to industrial agents and heavy metals [33]. However, the andrologists should be aware that our population is aging and the age of prospective patients has increased. Therefore, there is an increasing concern about the notion that aging may affect spermatogenesis and fertility potential.

A detailed history should include a comprehensive assessment of environmental and occupational exposures that can impact fertility. Unfortunately, the impacts of environmental and occupational exposures on spermatogenesis are extremely difficult to prove and quantify.

Evidence indicative of harmful effects of occupational exposure on the reproductive system and related outcomes has gradually accumulated in recent decades, and is further compounded by persistent environmental endocrine disrupting chemicals [33]. Effects of chemicals on reproduction may be induced directly by a chemical itself on reproductive organs or indirectly through the influence in altering hormonal regulation, which is responsible for growth, sexual development, and many other essential physiological functions. A number of occupations are being reported to be associated with reproductive dysfunction in males as well as in females.

The overall functioning of the reproductive system is controlled by the nervous system and the hormones produced by the endocrine glands. The reproductive neuroendocrine axis of males involves principally the anterior pituitary gland and the testes. Toxicants that damage the Leydig cells can lead to reduced secretion of testosterone, which in turn affects the Sertoli cell function and spermatogenesis. Most reproductive toxicants are thought to act directly on the testes. There are some indications that substances interacting with the pituitary secretion of gonadotropin (FSH, LH) and hypothalamic neuroendocrine releasing factors may also play an important role in sperm quality [34].

Various behaviors and lifestyles have been associated with increased ROS production. An association between cigarette smoking and reduced seminal quality has been identified [35]. Harmful substances including alkaloids, nitrosamines, nicotine, cotinine, and hydroxycotinine are present in cigarettes and produce free radicals [36]. In a prospective study, Saleh et al. compared infertile men who smoked cigarettes with nonsmoker infertile men [37]. Smoking was associated with a significant increase (approximately 50 %) in seminal leukocyte concentrations, a 107 % ROS level increase, and a 10 point decrease in reactive oxygen species and total antioxidant capacity (ROS-TAC score). Also, infertile men who smoke cigarettes present higher seminal ROS levels than infertile nonsmokers, possibly due to the significant increase in leukocyte concentration in their semen. An earlier study also reported an association between cigarette smoking in infertile men and increased leukocyte infiltration in the semen [38]. Significantly higher levels of DNA strand breaks have also been identified in men who smoke. DNA strand breaks may be resulting from the presence of carcinogens and mutagens in cigarette smoke [39].

In recent decades, evidence suggestive of the harmful effects of occupational exposure to endocrine disruptive chemicals on the reproductive system has gradually accumulated [34]. Environmental pollution is a major source of ROS production and has been implicated in the pathogenesis of poor sperm quality [40]. OS is hypothesized to play an important role in the development and progression of adverse health effects due to such environmental exposure [41].

Sperm quality can be influenced for all potential causative factors mentioned above and another that has not received much attention to date. It is well known that weight gain in men, particularly the deposition of adipose tissue around the waist, can depress serum total testosterone levels and increase serum estradiol levels [42]. The interaction between obesity and fertility has received increased attention owing to the rapid increase in the prevalence of obesity in the developed world [43]. Increase in mean male weight coupled with type 2 diabetes and metabolic syndrome could explain some of the observed declines of sperm quality in specific populations of men studied. The relationship between high levels of body mass index and changes in altered standard semen analysis parameters are already described in the literature [44, 45]. Furthermore, more recent studies revealed that increased body mass index values are associated with decreased mitochondrial activity and progressive motility and increased DNA fragmentation [46].

Usually, behavior and lifestyle modification should be the first steps in reducing ROS. Unfortunately, only some data link changes in these exposures to decrease in OS and subsequent increases in human fertility. Although it is likely good medical practice to recommend modifications of unhealthy lifestyles or exposures, definitive evidence awaits additional studies.

The testis is an interesting organ in which it is an immunologically privileged site, probably owing to the blood–testis barrier. The tight Sertoli–cell junctions provide the testis with a barrier that prevents the immune system from coming in contact with the post-meiotic germ cells. Autoimmune infertility may be a result of certain conditions such as previous genital tract infection, testicular biopsy, testicular trauma, testicular torsion, and vasectomy [47−49]. After the blood–testis barrier is broken, the body is exposed to sperm antigens resulting in an immune response presented as antisperm antibodies (ASA). An immunologic basis for some cases of infertility has been identified in a significant number of infertile men, suggesting that ASA may have a harmful effect on fertilization [50, 51].

Immunologic infertility is characterized by the presence of antibodies against spermatozoa in three locations; serum, seminal plasma, and sperm surface. Among these, sperm surface antibodies are the most clinically relevant and the antibody classes that appear to be clinically relevant include immunoglobulin G (IgG) and IgA, as IgM has high molecular weight and cannot penetrate the blood–testis barrier. The IgG antibody is derived from local production and from transudation from the bloodstream, whereas IgA is thought to be purely locally derived. It is thought that antibodies bound to the sperm head might interfere with sperm–egg interaction and fertilization capacity, whereas tail bound antibodies may be more likely to influence sperm transport through the female reproductive tract [52].

Testing for antisperm antibodies is classically indicated when: (1) the semen analysis reveals aggregates of sperm; (2) there are isolated asthenospermia; or (3) there is a risk of autoimmune infertility (i.e. prior testicular trauma or torsion). Indications of ASA tests are listed in Table 21.2. Moreover, in men with unexplained infertility, it has been suggested that ASA should be routinely tested due to the high frequency of normal routine seminal parameters in men with elevated ASA [53]. Although, approximately 10 % of infertile men will present with ASA as compared with 2 % of fertile men [54], ASA formation has been reported in up to 42 % of men with unexplained infertility [55, 56].

Typically performed with antibody coated, polyacrylamide spheres, an ASA test with at least 50 % of sperm bound with antibodies is considered clinically significant. The presence of multiple ASA can lead to impaired sperm transport through the reproductive tract, immobilization, and/or agglutination of spermatozoa, which blocks sperm–egg interaction. They can also prevent implantation, and/or arrest embryo development [49, 57].

The real significance of ASA in infertile men is controversial and currently, there are no standardized treatment regimens [58]. Oral corticoids are commonly used to suppress antibody production, but to date; no double-blind, randomized trial has confirmed their efficacy. Intracytoplasmic sperm injection (ICSI) is considered to be the treatment of choice for patients with severe sperm autoimmunity [59].

The prevalence of leukocytospermia (> 10⁶ WBC/mL semen) among male infertility patients is approximately 10–20 % [60]. Under a wet mount microscopy, both leukocytes and immature germ cells have a similar appearance and are properly termed “round cells.” Although many laboratories improperly report all round cells as white blood cells, the clinician must make sure that the two types of cells are differentiated. Leukocytes are difficult to differentiate from immature germ cells without the use of traditional cytology staining and immunohisthochemical techniques [61]. The World Health Organization (WHO) considers leukocytospermia to be a condition in which leukocyte levels are equal to or exceed 1 × 10⁶/mL [62]. As a consequence, all seminal analysis containing leukocyte levels below this limit are considered “normal”. In spite of that, recent studies reported that leukocyte counts below 1 × 10⁶/mL were significantly correlated with the production of seminal ROS as well as decreased sperm DNA integrity, despite the seminal parameters between the reference ranges [63−66]. All men with elevated seminal white blood cell levels (> 1 × 10⁶/mL) should be evaluated for a genital tract infection or inflammation, and a semen culture should be performed. Unexpectedly, approximately 80 % of leukocytospermic samples are microbiologically negative [60, 67].

The significance of white blood cells in semen is controversial. Most studies found that leukocytospermia is associated with decreased sperm motility and fertilization capacity [68−72]. However, El-Demiry et al. reported no association between standard seminal parameters and the leukocyte concentration in human semen [73]. This discrepancy may be due to the fact that different techniques were used to determine the leukocyte concentration in semen. In addition, the studies differed in regard to the lower leukocyte concentration responsible for sperm damage [63, 65, 74]. Infections located in the testis and epididymis produce ROS that are particularly harmful to sperm due to their lack of a pro-oxidant defense system.

The most commonly found Gram-positive and Gram-negative bacteria are Streptococcus fecalis and Escherichia coli, respectively [75]. Also, Chlamydia trachomatis and Ureaplasma urealyticum are often involved. Once the responsible microorganism has been identified, antibiotic therapy is initiated. However, culture-negative patients should be treated with anti-inflammatory therapy and frequent ejaculation because empiric antibiotic therapy generally provides no benefit and may be harmful [76, 77]. In cases of refractory leukocytospermia, sperm washing can be performed before intrauterine insemination to remove the white cells. Although antibacterial therapy can reduce inflammatory influences when administered in patients with genital tract infection, there are no available studies on this subject that show improved pregnancy rates [78].

A carefully performed semen analysis is the primary source of information on sperm production and reproductive tract patency. However, it is not a measure of fertility. An abnormal semen analysis simply suggests the likelihood of decreased fertility and normal seminal parameters are not assuredness of fertility.

Routine semen parameters such as sperm count, percentage motility, and morphology have a limited value mainly because there is not any consistent data that could distinguish between fertile and infertile samples in both in vitro and in vivo [79]. There are just reference values determined by the WHO over the last decades [62, 80, 81].

Approximately half of men presenting for an infertility evaluation will have seminal parameters between the “normal” reference values , representing a particularly difficult task to assign an etiology for subfertility and reinforcing the inherent inability of standard seminal parameters to assess sperm function.

We must keep in mind that the interpretation of the new reference ranges for seminal parameters proposed by the WHO, in 2010, requires an understanding that seminal parameters within the 95 % reference interval do not guarantee fertility nor do the values outside those limits necessarily indicate male infertility [80]. However, as the new lower reference limits are even lower than the previous “reference” values, clinicians will more frequently face men with semen parameters within the “normal” reference limits. Due to these seminal parameters markedly lower, a higher percentage of men will not be even referenced for an andrologic evaluation [82]. They will be inaccurately diagnosed as potential unexplained male infertility and sent to an in vitro fertilization (IVF) clinic for treatment. This may illustrate an urgent need for new diagnostic tools in the evaluation of these men.

Studies on sperm donors with known fertility status reveal a significant overlap in the sperm characteristics between fertile and subfertile men [54, 83]. The current normal values fail to satisfy clinical and statistical standards and pose the risk of misclassifying a subject’s true fertility status [83]. Moreover, introduction in clinical practice of new values likely result in a reclassification of many infertile couples [82]. Specifically, those couples previously classified as having male factor infertility with sperm parameters greater than the new reference limits but less than the previous values will now be diagnosed as having unexplained or female factor infertility. In fact, using the WHO actual cutoff values most likely some patients previously categorized as having an abnormal semen analysis will now be considered “normal,” with referral for evaluation postponed or not undertaken [82, 84].

To reach the site of fertilization, the spermatozoa must be able to successfully cross the cervix and the cervical mucus . The cervical mucus demonstrates cyclical changes in consistency being highly receptive around the time of ovulation. Increase in penetrability is often observed one day before the LH surge. Also, cervical mucus has been shown to protect the spermatozoa from the hostile environment of the vagina. The postcoital test (PCT) is a conventional test to evaluate the cervical environment as a cause of infertility. The PCT is the microscopic examination of the cervical mucus, performed shortly before expected ovulation and within hours after intercourse, to identify the presence of motile sperm in the mucus. So, accurate timing is crucial because it must be conducted when the cervical mucus is thin and clear just before ovulation.

In this test, cervical mucus is examined 2–8 h after normal intercourse. Progressively motile sperm superior than 10–20 per HPF is classified as normal. Practical guidelines of the American Society of Reproductive Medicine recommend PCT in a few situations including hyperviscous semen, unexplained infertility, or low-volume semen with normal sperm count [85]. The medical history and semen analysis can predict the result of the PCT in approximately 50 % of the subfertile couples with a regular cycle, without compromising its potential to predict pregnancy [85]. Impaired seminal parameters most likely will result in poor PCT. Despite that, couples with an abnormal PCT may benefit from intrauterine insemination which bypasses the hostile cervical factors [54]. There are other causes of irregular PCT including anatomic abnormalities, improperly performed intercourse, inappropriate timing of the test, abnormal seminal parameters, and cervical or seminal mucus antisperm antibodies. Of note, persistently abnormal PCT in the presence of adequate seminal parameters should indicate poor cervical mucus quality. The finding of good quality mucus with non-motile spermatozoa demonstrating shaking motion should call attention to evaluate both partners for the presence of antisperm antibodies. Occasionally, antisperm antibodies in the cervical mucus may inhibit sperm motility in vivo and prevent fertilization . This situation can be quantified with indirect antisperm antibody testing of the cervical mucus , although an in vivo assessment of the compatibility of sperm with the cervical mucus can be provided with the PCT.

Although the PCT utility and predictive value have been seriously questioned, some practitioners still consider it a useful diagnostic test since it may help to identify ineffective coital technique or a cervical factor not otherwise suspected on the basis of history and physical examination [86, 87]. Also, a more recent study showed that the PCT has prognostic value but does not add substantially as a prognostic tool for spontaneous pregnancy [88].

Contemporary treatments for otherwise unexplained infertility, such as intrauterine insemination or in vitro fertilization , successfully reverse any unrecognized cervical factors. Currently, PCT is not recommended routinely, especially for men who have abnormal semen analyses. In addition, the test may be reserved for patients in whom results will influence treatment strategy.

The removal of the zona pellucida from hamster oocytes allows human spermatozoa to fuse with hamster ova. This procedure is termed as sperm penetration assay (SPA). The SPA determines the functional capacity of the spermatozoa necessary to fertilize an oocyte, which determines the ability of sperm to successfully undergo capacitation, acrosome reaction, membrane fusion with oocytes, and chromatin decondensation. The zona pellucida is stripped, allowing cross-species fertilization. Normally, 10–30 % of ova are penetrated [62].

Infertile sperm would be expected to penetrate as well as fertilize a lower fraction of eggs than normal sperm. The indications for the diagnostic SPA are limited but could be used to further evaluate couples with unexplained infertility and to help couples decide whether to undergo with intrauterine insemination, when presenting good SPA result, or to proceed to IVF. Although SPA has low predictive power, but is positively correlated with spontaneous pregnancy outcomes [89]. The monthly fecundity rate at any time during a 30-month interval of follow-up is twice as great for men with normal SPA values as for those with abnormal values [89]. However, this test should be reserved for patients in whom results may influence treatment strategy. Many versions of the SPA have been used clinically, and the value of the test results depends, in part, on the experience of the laboratory performing the assay [90].

Compared with SPA, the zona binding test uses oocytes that failed to fertilize in IVF clinics. A meta-analysis of sperm function assays by Oehninger and colleagues showed a high predictive power of sperm zona pellucida binding assays over SPA for fertilization and IVF outcome [91]. Also, the findings indicated a poor clinical value of the SPA as predictor of fertilization. On the other hand, the need for human oocyte supply remains an important limitation to the use of zona binding test in the clinical settings.

The acrosome is a membrane-bound organelle that covers the anterior two thirds of the sperm head. After capacitation, the sperm fuses with the ovum plasma membrane and releases acrosomal enzymes that will allow sperm penetration and fertilization. Acrosome reaction is an essential precondition for successful fertilization. Although transmission electron microscopy is the procedure of choice to detect acrosome reaction defects is a labor-intensive and also an expensive test.

The acrosome reaction test may be recommended in cases of profound abnormalities of head morphology or in the setting of unexplained fertility in patients with poor IVF cycles results [92]. Samples presenting normal seminal parameters demonstrate spontaneous acrosome reaction rates of less than 5 % and induced acrosome reaction rates of 15–40 %. On the other hand, infertile populations have shown high spontaneous rates of acrosome-reacted sperm and low rates of induced-acrosome reactions. Despite of that, acrosome reaction testing is not widely practiced in laboratories and only remains a research interest.

Free radicals are a group of highly reactive chemical molecules that have one or more unpaired electrons and can oxidatively modify biomolecules that they encounter. This causes them to react almost instantly with any substance in their vicinity [93]. Generally, free radicals attack the nearest stable molecule, “stealing” its electron, beginning a chain reaction. Once the process is started, it can cascade and ultimately lead to the disrupting of living cells.

Human semen consists of different types of cells such as mature and immature spermatozoa, round cells from different stages of the spermatogenic process, leukocytes, and epithelial cells. Of these, leukocytes (neutrophils and macrophages) and immature spermatozoa are the two main sources of ROS [94, 95]. Leukocytes can be activated by infection and/or inflammation, in which situation they are capable of producing 100 times superior amounts of ROS than inactivated leukocytes [74].

Spermatozoa produce small amounts of ROS that are essential to many of the physiological processes such as capacitation, hyperactivation, and sperm–oocyte fusion [29, 96]. In the context of human reproduction, an equilibrium usually exists between ROS production and antioxidant scavenging activities in the male reproductive system. Minimal amounts of ROS remain in the system since they are needed for the regulation of normal sperm functions such as sperm capacitation, the acrosome reaction, and sperm–oocyte fusion [97].

In most cases, free radical-induced damage can be repaired. Unfortunately, spermatozoa are unable to repair the damage induced by ROS because they lack the cytoplasmic enzyme systems required to accomplish this [98, 99]. The pathological levels of ROS detected in the semen of infertile men are more likely caused by increased ROS production than by the reduced antioxidant capacity of the seminal plasma [29] .

The production of excessive amounts of ROS in semen can overwhelm the antioxidant defense mechanisms of spermatozoa and seminal plasma causing oxidative stress. OS is a common condition caused by biological systems in aerobic conditions that results in an extreme generation of ROS, which damages cells, tissues, and organs [100, 101]. Numerous assays for ROS measurement have been introduced in the last decade [97]. The chemiluminescence method is the most commonly used technique for measuring ROS produced by spermatozoa [102]. This assay quantifies both intracellular and extracellular ROS. Depending on the probe used, this method can differentiate between the production of superoxide and hydrogen peroxide by spermatozoa .