ASA, the hallmark of autoimmune male infertility, are implicated in sperm dysfunction by their direct effect on various sperm antigens. ASA may induce sperm apoptosis and untimely AR [3]. ASA may also hinder fertilization by interfering with sperm penetration into the cervical mucus, zona pellucida (ZP) binding, and sperm–oocyte fusion. Furthermore, ASA may alter chaperone function, protein folding, and disulphide bonds [4]. These alterations ultimately decrease the chances of achieving a natural conception [5]. As a matter of fact, Ayvaliotis et al. reported a significantly lower spontaneous conception rates (15.3 %) in couples where autoimmunity to sperm was detected compared with those without clinically relevant levels of ASA (66.7 %, p < 0.05) [5].

Sperm agglutination and/or immobilization is usually observed in association with ASA, and may limit sperm progression through the female genital tract [6]. The findings of sperm agglutination, “shaking motility,” or both, during conventional semen analysis are highly suggestive of elevated ASA titers [7]. The term “agglutination” describes spermatozoa that are stuck to each other by antibodies. Sperm agglutination usually occurs in specific manners such as head-to-head, midpiece-to-midpiece, tail-to-tail, or tail-tip-to-tail-tip [8]. On the other hand, “shaking motility” is a pattern of sperm movement characterized by a very strong tail movement associated with a nonprogressive motility that results in immobilization [8].

Although immunoglobulin subclasses IgA, IgG, and IgM can be found in human ejaculates, IgA seems to be the most important one from a biological standpoint [9, 10]. IgA bound to the sperm surface significantly impair sperm progression through the cervical mucus [10]. Nevertheless, immunoglobulins can adhere to various sperm sites regardless of their subclasses [5].

It is out of our scope to provide a comprehensive review on immunologic male infertility since it is discussed in Chap. 9. Our focus is otherwise to present the tests to determine ASA, as well as their advantages and limitations.

Concerning the testing for ASA in UMI, the specimen of interest is the semen . ASA can also be assessed in the seminal plasma when low sperm counts and/or motility limits the applicability of the assay. However, such conditions are associated with abnormal semen analysis results, and therefore do not meet the criteria of UMI. The immunobeads binding test (IBT) and the mixed agglutination reaction (MAR) test are the most commonly used among andrology laboratories performing ASA determination (Table 11.2) [11].

The sperm fertilizing potential is related to its ability of undergoing capacitation, ZP binding, and AR, which ultimately leads to sperm penetration into the oocyte and fusion with the oolema .

In vivo, ejaculated spermatozoa from all Eutherian mammals are unable to fertilize until they have undergone capacitation, which allows the AR to take place, when they approach or contact the oocyte [17]. Capacitation has been recognized as a time-dependent phenomenon, with the absolute time course being species-specific [18]. Capacitation prepares the sperm to undergo AR with the accompanying release of lytic enzymes and exposure of membrane receptors, which are required for sperm penetration through the ZP and for fusion with the oolema [18]. Sperm transport through the female genital tract can occur quite rapidly (times as short as 15 to 30 min have been reported in humans), whereas the capacitation process can take from 3 to 24 h [18]. It is speculated, therefore, that capacitation is not completed until after spermatozoa have entered the cumulus oophorus. This delay is physiologically beneficial because spermatozoa do not respond to AR-inducing signals until they have approached the ZP, thus preventing premature AR that ultimately leads to sperm inability to timely penetrate the egg vestments [18, 19]. Sperm capacitation is a postejaculatory modification of the sperm surface, which involves the mobilization and/or removal of certain surface components of the sperm plasma membrane, such as glycoproteins, decapacitation factor, acrosome-stabilizing factor, and acrosin inhibitor. Sperm capacitation involves major biochemical and biophysical changes in the membrane complex and energy metabolism . The presence of high concentrations of cholesterol in the seminal plasma, which maintains high cholesterol concentration in the sperm membranes, seems to be the most important factor for inhibition of capacitation [20]. Capacitation is associated with increased membrane fluidity caused by removal of cholesterol from sperm plasma membrane via sterol acceptors from the female tract secretions [21, 22]. A marked change in sperm motility, named hyperactivation (HA), is the first step in this complex process. Hyperactivated spermatozoa exhibit an extremely vigorous but nonprogressive motility pattern, as a result of a Ca²+ influx, which causes increased flagellar curvature [23] and extreme lateral movement of the sperm head [8]. Proteasome participates in activating calcium channels that also leads to increased membrane fluidity and permeability [24–27]. These events are followed by or occur simultaneously with (i) a decrease in net surface charge, (ii) the absence of intramembrane protein and sterols area, and (iii) increased concentrations of anionic phospholipids [27, 28]. Hyperactivated motility is essential for sperm penetration into the intact oocyte-cumulus complexes in vitro and in vivo [29, 30]. Various components of the female reproductive tract act as physiological stimuli for HA, such as progesterone, ions, and secretions in the oviduct luminal fluid [31]. The extent of which sperm is hyperactivated is positively correlated with the sperm’s ability of zona binding, AR, zona-free oocyte penetration, and thus fertilizing capacity in vitro [31] .

The spermatozoon binds to the ZP with its intact plasma membrane after capacitation and penetration into the cumulus oophorous. Sperm binding occurs via specific receptors to ZP glycoproteins located over the anterior sperm head [32]. Glycosylation of ZP glycoproteins is important in sperm-ZP interaction. It is believed that human ZP glycoprotein-3 (ZP3) has a central role in initiating the AR [33]. However, human ZP1 and ZP4 are also implicated in the process [21, 22, 34–37]. ZP3-induced AR involves the activation of T-type voltage operated calcium channels (VOCCs), whereas ZP1- and ZP4-induced ARs involve both T- and L-type VOCCs. Chiu et al. [38] reported that glycodelin-A, a glycoprotein present in the female reproductive tract , sensitizes spermatozoa to undergo ZP-induced AR. The process involves the activation of the adenylyl cyclase/PKA pathway along with up-regulation of ZP-induced calcium influx and suppression of extracellular signal-regulated kinase activation. It is, therefore, suggested that glycodelin-A may be important in vivo to ensure full responsiveness of human spermatozoa to the ZP. Defective ZP-bound sperms are present in approximately 15 % of subfertile men with normal semen analysis, and it may explain the low success of IVF and intrauterine insemination (IUI) when these methods are applied in certain cases of UI [39, 40]. The presence of defective sperm–ZP binding in infertile men with normal semen parameters may be due to either a defective signal transduction pathways upstream of protein kinases A and C, or downstream disorders, structural defects, or absence of sperm receptors for ZP binding [12]. Such defects are not identified unless specialized sperm function tests are used [41].

The AR is a stimulus-induced process that involves fusion of the sperm plasma membrane with the outer acrosomal membrane. This process leads to the release of exocytotic proteolytic enzymes in response to sperm–ZP binding [42, 43]. The mammalian sperm head is composed of two main structures, the acrosome and the nucleus. The acrosome is a membrane-bound organelle derived from the Golgi complex located in the anterior portion of the sperm head. In humans, the acrosome occupies 40–70 % of the sperm head and its contents include proteases such as proacrosin, hyaluronidase, and phospholipase A2. Spermatozoa bind to the ZP with intact acrosomes. Then, multiple fusions between the outer acrosomal membrane and the plasma membrane result in the release of hydrolytic enzymes (mostly acrosin) and exposure of new membrane domains, both of which essential for fertilization . The released hydrolytic enzymes digest the ZP, allowing the spermatozoa to penetrate the oocyte [32].

The AR seems to be physiologically induced by natural stimulants such as follicular fluid (FF), progestin, progesterone, and hydroxy progesterone, all of which bind to sperm receptors [28]. FF and cumulus cells have protein-bound progesterone, and this hormone is considered among the most important AR-inducing agents [43, 44]. However, other sperm-deriving factors may also play a role in this process. For instance, a link between estradiol locally produced by ejaculated spermatozoa, sperm capacitation, and AR have been demonstrated [45]. These stimulatory signals are intracellularly transduced via second messengers ultimately leading to exocytosis [46]. A number of second-messenger pathways have been identified in human spermatozoa, including those that result in the activation of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and phospholipid dependent protein kinases [27, 47–49]. These kinases are called, respectively, protein kinases A, G, and C. It is possible that these pathways interact to assure an optimal response at the correct place and time during the fertilization process.

The AR is a time-dependent phenomenon that cannot take place prematurely or too late [47]. As such, acrosome integrity is crucial for normal fertilization both in vivo and in vitro. In fact, a high proportion of sperm with intact acrosomes is seen in ejaculates of normal men. In such individuals, ~ 5–20 % sperm cells exhibit spontaneous AR devoid of clinical significance [48]. In contrast, abnormal conditions affecting the sperm may lead to decreased fertilization ability. One example is acrosomeless round-headed spermatozoa (globozoospermic spermatozoa) that are unable to fertilize. Increased percentages of morphologically abnormal acrosomes have also been related to fertilization failure in assisted conception using conventional IVF [49]. In addition, premature AR and sperm inability to release the acrosomal contents in response to proper stimuli (AR insufficiency) have been associated with unexplained male infertility [50]. Although the cause of premature AR is unknown, the premature (stimulus independent) initiation of acrosomal exocytosis seems to be related to a perturbation of the plasma membrane stability. In this context, the AR may not involve a premature activation of the receptor-mediated process, but rather reflect an inherent fragility of the sperm membrane, leading to a receptor-independent acrosomal loss [51].

ASA may also adversely affect the ability of sperm to undergo capacitation and AR [15]. Chang et al. [52] reported a reduction in fertilization either by the action of IgG directly bound to sperm or IgM present in the female serum. Moreover, the combination of IgG and IgA has synergistic negative effects on fertilization [53–56]. Gonadotoxic substances can also influence AR. High concentrations of dietary phytochemicals, such as genistein isoflavone and β-lapachone, were shown to suppress the AR in a dose- and time-dependent manner in the rat model [57]. Inhibition of AR by genistein seems to involve the protein kinase C pathway while β-lapachone has a direct cytotoxic effect on the sperm cell membrane. It is suggested that genistein and β-lapachone may impact male fertility via AR suppression in high doses and AR induction in low doses [57]. Calcium channel blockers may also interfere with the AR exocytic event. Sperm incubation with different blockers, such as trifluoperazine (calmodulininhibitor), verapamil (Ca²⁺ channel inhibitor), and nifedipine (voltage-dependent Ca²+ channel inhibitor) significantly reduces the ability of hamster sperm to undergo AR [58]. The frequency of acrosome-reacted spermatozoa is also increased by the recreational use of marijuana [59]. Falzone et al. [60], studying the effect of pulsed 900-MHz GSM mobile phone radiation on the AR of human sperm , observed that despite not adversely affecting the AR rate, radiofrequency electromagnetic fields (RF-EMF) exposure significantly impaired sperm morphometry and the ability of sperm binding to the hemizona. Finally, Mukhopadhyay et al. evaluated the in vitro effect of benzo[a]pyrene, a substance present in cigarettes, on sperm HA and acrosome status of normozoospermic semen specimens. They observed a significant increase in sperm HA as well as in the premature AR rates in the presence of benzo[a]pyrene [61]. In conclusion, exogenous factors may affect the sperm’s ability to undergo timely AR, thus hindering such gametes infertile.

Several tests can be used to measure sperm fertilization defects at different levels, as summarized in Table 11.3.

ROS are byproducts of oxygen metabolism and energy production that act as regulators of vital physiological intracellular processes. In the male reproductive tract, the primary producers of ROS are the leukocytes and immature spermatozoa. Small quantities of ROS have important roles in sperm function including regulation of capacitation, AR, HA, and the fusion of spermatozoa with the oocyte [94]. In sperm, ROS are generated by two ways, (1) NADPH oxidase system at the level of the sperm plasma membrane [95], and (2) NADH-dependent oxido-reductase system at the mitochondrial level, which is the major source of ROS in sperm of infertile men [96, 97]. When ROS levels disproportionately increase, mainly by the presence of superoxide (O₂ ^–), hydroxyl radicals (OH), or nitric derivatives (NO), compared with the neutralizing capacity of intracellular and extracellular antioxidants, or when a reduction in the antioxidant capacity occurs, oxidative stress usually follows and target lipids, proteins, sugars, and nucleic acids through a variety of mechanisms .

In sperm, ROS primarily target membranes and DNA. By having an available valence electron, oxidative species interact with the cell membrane and cause lipid peroxidation of which the final product is malondialdehyde (MDA), a measurable compound used to determine the extent of ongoing oxidative modification of membrane-polyunsaturated fatty acids (PFA). The peroxidation of PFAs has detrimental effects on the structure of the sperm head and midpiece membranes, causing changes in the sperm membrane fluidity that ultimately lead to suboptimal sperm motility and fertilization [94]. Oxidative species also target DNA molecules and cause a variety of defects in the cell genetic material. ROS can inflict serious damage to DNA, including point mutations, polymorphisms, deletions, chromosomal rearrangements, frame shifts, and single-stranded or double-stranded breaks [98]. The OS-induced sperm damage has been suggested to be a significant contributing factor in 30–80 % of all cases of male infertility [99]. Likewise immunologic infertility and fertilization defects , oxidative stress cannot be detected in the routine semen analysis .

The methods used for ROS detection are broadly divided into two major categories based on their ability to directly or indirectly measure oxidative radicals (Table 11.4). Indirect measurements involve the assessment of lipid peroxidation products (MDA), protein oxidation products (such as protein carbonyl) and oxidized DNA (8-hydroxy-2′-deoxyguanosine [8-OHdG]) [105] whereas direct oxidative-stress measurements include total or specific ROS level in semen and total antioxidant capacity (TAC) .