Male immune infertility: a million dollar question in medically assisted reproduction

2.1 Infections and inflammation

Infections of the reproductive tract can be the cause of disruptions in the blood-testis barrier (BTB) and for local inflammation, both of which may contribute to ASA formation. However, the exact role of inflammatory processes in the male genital tract and the formation of ASA remains a matter of debate. Inflammation of the prostate could be related to the formation of ASA [36]. However, Hoover and Naz [37] reported no correlation between prostatic hyperplasia and prostate cancer with the formation of ASA. Nevertheless, considering that these types of prostate disorders usually lead to an increase of circulating prostate-specific antigen (PSA), which is responsible for dissolving the seminal coagulum and allowing sperm to swim freely, we could expect that men with increased PSA levels would also exhibit ASA in seminal plasma, potentially affecting fertilization. Naz and Butler [38] supported this in their study, although they could not determine whether it is a direct cause of infertility or merely an associated finding. Another factor worth considering are the cases of epididymal abnormalities, such as epididymitis, since ASA-positive patients exhibit some chronic epididymal inflammation [39].

We can divide infections into two main groups: viral and bacterial infections. On one hand, viral infections include cases of human papillomavirus (HPV), adeno-associated virus (AVV), and hepatitis C virus (HCV). These infections can affect the semen and the reproductive tract tissue, they may trigger different autoimmune reactions and an increase in ASA production, which then results in higher levels of ASA on the sperm surface and causes a significant decrease in sperm motility [40]. Several studies [41, 42, 43] have confirmed this in HCV and HPV infertile men, suggesting that viral infections are a significant contributing factor to male infertility. Hussein et al. [41] conducted a study on 30 HCV-infected individuals and 30 healthy control subjects. They measured liver enzymes and reproductive hormones, performed computer-assisted semen analysis (CASA), determined IgG and IgA in semen, and measured HCV-RNA in serum. IgG and IgA levels were higher in HCV patients, and sperm concentration, total motility, and progressive motility were lower in HCV patients. Correlations between examined semen parameters and viral load were nonsignificant, but still, they claim HCV may be responsible for increased IgG and IgA levels. Garolla and co-workers [42] included 151 infertile couples with the detection of HPV in semen, counselled to receive adjuvant HPV vaccination (79 accepted vaccination, 72 did not and became the control group). They evaluated the effect on reproductive outcome of HPV vaccination by recording progressive sperm motility, ASA levels, spontaneous pregnancies, miscarriages, and live births. Their results show that progressive sperm motility and ASA levels were improved in the vaccine group, and that adjuvant vaccination was associated with enhanced HPV healing in semen cells and an increased rate of natural pregnancies and live births. The question of whether the presence of HPV in semen is associated with impairment of semen quality, was analyzed in a large cohort study by Luttmer et al. [43]. They tested male partners for HPV-DNA, counted sperm concentration and motility, and determined ASA levels with MAR test. Their study concluded that the presence of HPV in semen was not associated with the age of the participants, seminal pH, semen volume, total sperm count, sperm concentration, progressive motility, or the presence of antisperm antibodies. Among the viral infections mentioned above, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also needs to be mentioned. We found no specific literature claiming that SARS coronavirus is correlated with higher ASA levels, but recent evidence report that SARS coronavirus could also lead to fertility damage by utilizing the angiotensin-converting enzyme 2 (ACE-2) receptor expressed on testicular tissue [44]. Taken together, these studies underscore the need for further investigation to elucidate the underlying pathophysiological mechanisms and clarify the clinical significance of respiratory virus infections in male fertility.

Bacterial infections, on the other hand, have been correlated with Streptococcus agalactiae, Staphylococcus aureus, and Enterococcus faecalis. In most cases, ASA production increases due to molecular mimicry between sperm and bacteria, leading to cross-reactivity issues between bacterial antibodies and ASA [45]. Based on epidemiological and experimental evidence, there has been a growing awareness of the role of infectious diseases in autoimmunity via molecular mimicry and cross-reactivity. Molecular mimicry is one of the leading mechanisms by which infectious agents may induce autoimmunity, and this occurs when similarities between self and foreign-peptides favour an activation of autoreactive B or T cells by foreign-derived peptides. Currently, four major criteria are used to define molecular mimicry: “(1) similarity between a host epitope and an epitope of a microorganism or environmental agent, (2) detection of antibodies or T cells that cross-react with both epitopes in patients, (3) epidemiological link between exposure to the environmental agent or microbe, and (4) reproducibility of autoimmunity in an animal model following sensitization with the appropriate epitopes either following infection with the microbe or exposure to the environmental agent.” [46]. Although these criteria have been in place for many years, they remain difficult to demonstrate conclusively in humans. Challenges include insufficient epidemiological power, study limitations, and issues such as long latency between exposure and disease onset. Additionally, humans encounter numerous infections throughout life, yet only a small subset lead to autoimmune responses [47]. In increased ASA production related to bacterial infections, heat shock proteins (HSP), also known as chaperones, are potential causative agents, because these proteins are released in response to stressful conditions. The bacterial heat shock protein 60 (HSP60) protein, the major antigenic determinant during an infection, has approximately 50% homology with human HSP60, which can lead to cross-reactivity and may result in reproductive issues through ASA production [48]. The existence of molecular mimicry leading to cross-reactivity between sperm and bacteria was mentioned in the study by Prabha et al. [49], where they claimed that sperm immobilization factor isolated from Staphylococcus aureus immobilizes human spermatozoa, as well as motile bacteria, showing that sperm immobilization factor receptor might be shared by bacteria and human spermatozoa. Their results show that ASA in humans cross-reacted with bacterial antibodies, sperm immobilization factor, isolated from Staphylococcus aureus, immobilized spermatozoa, and motile bacteria, higher concentrations of sperm immobilization factor even caused sperm death. With all gathered facts, they concluded that molecular similarity between bacteria and spermatozoa exists. Interestingly, the most well-known sexually transmitted bacteria, Chlamydia trachomatis, is not reported in correlation with ASA production [50].

Although Mycoplasma spp. and Ureaplasma spp. are natural inhabitants of the male urethra, they can become pathogenic and contribute to genital infections related to male infertility. In andrology, the most relevant species are Mycoplasma genitalium and Mycoplasma hominis. These two pathogens may trigger inflammatory processes that could theoretically lead to ASA formation, but no correlation with ASA levels has been demonstrated. The only identified consequence of the pathogen binding to spermatozoa was the occurrence of sperm agglutination [51].

Mycoplasma hominis is usually associated with changing semen parameters, particularly density and motility, as shown by Rose and Scott [52], who incubated spermatozoa with mycoplasma overnight and observed pathogen binding to sperm head, tail, and midpiece. On the contrary, Diaz-Garcia and coworkers [53], reported that a short-term semen exposure to Mycoplasma hominis does not reduce sperm viability.

Mycoplasma genitalium is linked to male urethritis, prostatitis, infertility, and may induce sperm DNA damage. Some in vitro studies show that its attachment to spermatozoa can cause sperm agglutination and reduced motility [54]. Svenstrup et al. [55], noted that Mycoplasma genitalium can bind to the midpiece region of spermatozoa, and then be transported by motile sperm.

2.2 Varicocele

Varicocele is a well-known factor contributing to infertility and abnormal semen parameters. It is often detected during routine examinations, where an abnormal enlargement of the spermatic cord veins can be observed. Several possible mechanisms have been proposed to explain the impact of varicocele on fertility, including blood stasis leading to toxin accumulation, hormonal imbalances, temperature dysregulation, oxidative stress, and testicular hypoperfusion [56]. Even though varicocele has been linked with levels of ASA, findings still remain inconsistent. Veräjänkorva et al. [57] claim there is no correlation between levels of ASA and varicocele, a conclusion supported by Bozhedomov et al. [58]. However, they noted that while varicocele itself is not an immediate cause of autoimmune reactions against spermatozoa, it represents a very important cofactor of immune infertility due to testicular trauma in varicocele patients. They also demonstrated a significant decrease in semen quality correlated with levels of ROS in ASA-positive varicocele patients, which were notably higher than in ASA-negative varicocele patients. A recent study [59] investigated ASA positivity in the semen of men with and without varicocele (control group) and compared different detection tests—MAR test, immunobead (IB) direct and indirect test, and enzyme-linked immunosorbent assay (ELISA) testing. After reviewing 151 abstracts, 6 articles met the inclusion criteria and were included in the meta-analysis. With MAR test 39.8% of patients with varicocele tested positive for ASA, compared to 17% in control group (p = 0.04); with IB test 50% of patients with varicocele tested positive for ASA, compared to 15.4% in control group (p = 0.09); with ELISA test 37.1% patients with varicocele tested positive for ASA, compared to 15.8% in control group (p < 0.01). They confirmed that ASA positivity is significantly higher among men with varicocele than those without varicocele when tested by MAR test or ELISA test. This suggests the presence of an immunological pathology in infertile men with varicocele. Despite the limited number of studies available, current evidence supports the view that varicocele plays an important role in ASA levels.

2.3 Autoimmune diseases

Autoimmune diseases are health conditions that occur when the immune system mistakenly produces antibodies that attack the body’s own cells [60]. Considering that “immune infertility” is also an autoimmune disorder, we could predict that ASA levels are elevated in men with other autoimmune diseases [18]. One such condition is lupus erythematosus, in which increased levels of ASA have been reported. Shiraishi et al. [61] compared men with and without systemic autoimmune diseases and found higher levels of ASA in the group of men with autoimmune diseases than in men without, suggesting that systemic autoimmune diseases may represent a risk factor for ASA development in men. Conversely, ankylosing spondylitis, juvenile dermatomyositis, and antiphospholipid syndrome have not been associated with higher ASA [62, 63]. One of the world’s most well-known autoimmune diseases, multiple sclerosis (MS), has likewise not been linked with higher ASA levels to date. However, MS is a chronic disease that can cause a wide range of symptoms and disabilities, raising important questions regarding its impact on fertility and reproduction. Massarotti et al. [64] addressed three important questions in their study; (a) Is multiple sclerosis correlated with conditions that increase the risk of infertility? (b) Does multiple sclerosis cause infertility per se? (c) Do disease-modifying therapies (DMTs) or other therapies for multiple sclerosis impact gonadal function in men? They concluded that the issue of fertility in men with MS has not been thoroughly investigated, and that only indirect data from population registries are available. Nonetheless, some data indicate that up to 70% of men with MS experience erectile dysfunction, and up to 50% of men exhibit alterations in ejaculation [65]. Despite these findings, no links were observed between MS and ASA levels. Similarly, Li and coworkers [66] conducted a Mendelian randomization study to assess the causal relationship between MS and abnormal spermatozoa. Their results indicated that patients with MS have a higher risk of sperm abnormalities, and that reproductive and fertility issues in men with MS warrant special attention from clinicians. The most relevant factor identified was the higher levels of ROS, which is linked to chronic inflammation; however, this study also found no correlation with ASA levels. In summary, men with MS could be subfertile due to disease-related ejaculation and/or erection dysfunction and/or inflammatory effects, but a clear correlation with higher ASA levels has not yet been determined.

2.4 Vasectomy

Vasectomy is a surgical procedure performed to prevent fertility in males. It can lead to an immunogenic response, which may result in the production of ASA. While researchers hold differing opinions regarding the above mentioned risk factors for elevated ASA levels; they unanimously agree that vasectomy is one of the most firmly established factors associated with increased ASA levels [67]. In a study by Lee et al. [68] that included 484 men with male infertility who had undergone ASA testing, serum ASA levels were found to be higher in men after vasectomy. Similarly, Azizi et al. [69] examined 110 vasectomized men and reported the presence of ASA in 95% of participants. These findings demonstrate that the association between vasectomy and ASA levels is well-documented and firmly supported as one of the major risk factors. However, regardless of the reason for performing this procedure in the first place, we can still say it has a negative effect on fertility, but it’s not the reason for primary infertility associated with ASA.

2.5 Cryptorchidism and testicular trauma

Cryptorchidism, the most prevalent congenital abnormality involving male genitalia, is a well-known factor of infertility. Also referred to as an undescended testis, it results from the failure of one or both testes to descend into the scrotum. Cryptorchidism is also found in patients diagnosed with Prader-Willi Syndrome (PWS). Genital abnormalities are the rule in PWS patients and cryptorchidism in particular is present in 85–100% of cases [70, 71]. Due to the elevated intra-abdominal temperature, we could presume that some immune reactions might occur; however, cryptorchidism is primarily associated with impaired germ cell maturation [72]. No clear association between cryptorchidism and ASA levels has been shown, and infertility associated with cryptorchidism is not immunological [73]. As mentioned above, elevated temperature could induce some immune reactions, which can be influenced by an individual’s genetic constitution. Niepiekło-Miniewska et al. [74] studied gene system encoding immunoglobulin receptors (KIRs) and found that there were no differences among cryptorchidic patients and healthy controls in terms of ASA levels.

Another potential cause of ASA production described in the literature is testicular trauma, which may disrupt the blood-testis barrier and expose sperm antigens to the immune system. However, the association between these conditions and ASA remains controversial [35].

2.6 Inguinal hernia or hernia repair

Inguinal hernia is a relatively common medical condition that can be quickly resolved with a minor surgical procedure. An inguinal hernia is a hernia (protrusion) of abdominal cavity contents through the inguinal canal. Some risk factors for developing a hernia are smoking, obstructive pulmonary disease, collagen vascular disease, obesity, strenuous physical activity, chronic constipation, connective tissue disorder, etc. Some predispositions to hernias can also be genetic [75]. Because surgical intervention is required, there is a potential risk of BTB disruption, local inflammatory response, and alterations in testicular blood flow, which could, in turn, have an impact on ASA development and infertility. Currently, two standardized surgical techniques are widely used: open hernia repair with mesh placement to close the hernia and laparoscopic repair. Generally, no significant differences in postoperative quality of life between these two methods are reported [76]. However, there are some links between immunological reactions triggered by these surgical procedures and some inflammatory processes. Štula and coworkers [77], on one hand, investigated testicular disorders related to infertility following inguinal hernia mesh repair. They included 43 male patients and analyzed ASA levels, but they found no significant differences and concluded that inguinal hernia mesh repair does not have a clinically significant influence on ASA levels. On the other hand, Negri et al. [78] conducted a study with 2258 infertile male patients who underwent ASA testing and urologic examination; among them, 191 had a history of inguinal hernia repair, and found increased ASA levels in patients who underwent andrological surgery or hernia repair. In summary, we can conclude that because of the limitations of the existing evidence and some conflicting results in studies, we cannot determine for sure if inguinal hernia repair does or does not lead to clinically significant immunological reactions. Nevertheless, assessing ASA levels could be useful in patients with a history of hernia repair in order to provide better insight into unexplained infertility.

3.1 The mixed antiglobulin reaction (MAR) test

The mixed antiglobulin reaction (MAR) test provides less information than other tests, but it is quick, inexpensive, and sufficiently sensitive to yield valid results [82]. For the MAR test, an unwashed sample of semen is mixed with latex beads coated with anti-human antibodies, and in the presence of ASA, the anti-human antibodies on the beads will bind to the antibodies on the sperm surface. As a result, motile spermatozoa become coated with beads, which can be observed under the microscope. Usually, spermatozoa continue to move around with the beads attached, but if agglutinates become massive, the movement of spermatozoa can be blocked. The aim is to determine the percentage of motile spermatozoa with attached beads. Spermatozoa that lack surface antibodies can be seen swimming freely among the particles. The recommendation is that at least 200 motile spermatozoa should be evaluated. The criteria for a positive ASA result vary among sources. According to the 5th edition of the WHO Laboratory Manual, the reference value is 50% for both IgG and IgA [81]. The 6th edition of the manual, however, does not specify an exact reference value for a positive ASA result; instead, they recommend that each laboratory establish its own reference value based on testing a large population of fertile and infertile men to distinguish between normal and pathological semen samples [17]. However, as per the manufacturer of SpermMar test from FertiPro, a positive ASA result is when at least 40% or more of spermatozoa are bound to latex beads.

3.2 The direct and the indirect immunobead test

The direct immunobead (IB) test is less commonly used and is more time-consuming than the MAR test, but it provides information about antibodies on spermatozoa that have been masked before. In this test, a washed sperm sample is used and mixed with covalently-bound anti-human immunoglobulins against IgG and IgA. If the beads bind to motile spermatozoa, this indicates the presence of surface-bound antibodies on the spermatozoa. The indirect immunobead test is used to detect ASA in sperm-free fluids (serum, seminal plasma, testicular fluid), and is useful in semen samples where motility is not adequate for the MAR test. In this test, antibody-free donor spermatozoa bind to ASA that is present in the tested fluid; the sample is then evaluated in the same manner as the direct IB test. Both procedures are different from the MAR test. According to the 5th edition of the WHO manual, the consensus value of 50% motile spermatozoa with bead particles is considered a threshold value [81].

3.3 Detection of ASA in seminal plasma by ELISA technique

An enzyme-linked immunosorbent assay (ELISA) has been developed and evaluated for the detection of equine ASA. It could detect ASA by the antigen absorbed in the solid phase, and antihuman globulin conjugated to alkaline phosphatase as a developing reagent. The ELISA assay detects IgM, IgG, and IgA in serum, cervical mucus, and seminal plasma [30]. Typically, the ELISA plate is coated with a mixture of spermatozoa proteins, which are recognized by ASA. Samples and standards are pipetted into the wells and then incubated, allowing any ASA present to bind to the immobilized sperm proteins. After all initial steps of the procedure, the color of the solution changes, and the intensity of this color change is measured. The ELISA method is a more standardized test demonstrating the correlation between seminal and sperm-bound ASA values. It also has the advantage of long-term sample storage ability, unlike MAR and IBT, which require fresh samples. However, it is used less frequently in routine diagnostics because MAR and IBT tests are faster and more cost-effective [83, 84]. But this is not the only reason for not being used so frequently, ELISA test also requires fixation of whole sperm or use of membrane extracts. Fixation may lead to membrane damage or denaturation of sperm antigens, which can result in a false-positive or false-negative result [30].

3.4 Sperm agglutination tests and sperm immobilization test (SIT)

The sperm agglutination test requires fresh motile spermatozoa and includes the tray agglutination test, gelatin agglutination test, and glass agglutination test. Agglutinates can be formed by any amorphous material present in the semen sample, nonspecific immunoglobulins, as well as nonimmunoglobulin proteins, which can all lead to false-positive results. The class of immunoglobulin cannot be identified; that’s why this method is not recommended for routine use.

Sperm immobilization test (SIT) means counting the motile spermatozoa under the microscope, which makes this method highly subjective. Because sperm-immobilization assays are limited in their detection of IgA and can give false-negative results, this method is also not recommended for routine use [30].

3.5 Immunofluorescence assays (IFAs)

As a conclusion from the study by Bohring et al. [85] immunofluorescence assays in the diagnosis of immune infertility should be encouraged, particularly if MAR test is positive and the acrosome function is questioned. However, there are some shortcomings for these assays. The method is unable to detect the number of antibody molecules or antigens involved in the binding. It can only detect the immunoglobulin class of the antibody concerned [18]. Despite some positive facts about this method, IFAs are not recommended for routine use, because the results based on IFAs are unreliable [30].

3.6 Flow cytometry and radiolabeled antiglobulin assay (RIA)

One of the not recommended methods for routine use in ASA detection is flow cytometry. Flow cytometry was used as a promising method for the determination of the number of sperm binding ASA, because it may be able to determine the exact amount of IgG and IgA in individual sperm samples [86]. However, Nikolaeva et al. [87] concluded that it does not always give an objective result, and for that, the practical use of flow cytometry has been limited.

Radiolabeled antiglobulin assay (RIA) provides no information about the proportion of antibodies bound to sperm or the region where antibodies bind, similar as ELISA. Also, antigens are not biochemically identified and those relevant for the process of fertilization might not be contained in the antigen mixture.

4.1 IUI

A primary mechanism whereby ASA can interfere with fertility is the ability of the sperm to penetrate through the cervical mucus. With IUI, we can bypass the cervical mucus, which is the main reason IUI has been widely used as the first-line treatment [83]. However, uncertainties remain regarding the effectiveness of IUI. Although data vary among studies, evidence suggests that IUI can overcome some of the mechanisms of ASA interference and should be an approach for selected patients who have been diagnosed with so-called male subfertility [92]. In a retrospective cohort study, Barbonetti et al. [93] investigated the relationship between the percentage of ASA levels in natural and IUI-assisted live births. Men in this study had to be at least 50% positive at the IgG MAR test and were divided into two groups (100% and 50–99% MAR test). In the first group (100% MAR), the live birth rate per couple after IUI was significantly higher than the natural live birth rate (LBR) (p = 0.0004). In 38 out of 44 couples with 100% MAR, there were 14 live births after IUI (LBR = 36.8%), and the natural live birth per couple in the same group was only 4.5%. In contrast, among the men in the 50–99% MAR group, LBR after IUI was not higher. The findings emerging from this study indicate that in a group with couples where a male partner has a 100% positive IgG MAR test, the percentage of natural LBR is very low, compared with the percentage of natural LBR in a group with couples where male partner has a 50–99% positive IgG-MAR. This finding indicates that different ASA levels have different impacts on reproduction outcomes. They also showed that IUI can represent an effective treatment even in 100% positive IgG-MAR. Similar results were presented by Francavilla et al. [92]. Earlier, Ombelet et al. [94] compared the results between IUI and IVF treatment in couples where the male partner had to have at least a 50% positive MAR test (IgG or IgA). Both IUI and IVF achieved high pregnancy rates (64% and 47%), but the authors concluded that IUI should be the first-line therapy in male immunological infertility. Although all these studies had relatively small sample sizes and differences in the ASA positivity thresholds used, their results consistently support IUI as an effective first-line treatment for carefully selected couples.

4.2 IVF

Another technique used for semen samples with positive ASA is in vitro fertilization (IVF). After semen collection, the sample is washed and then subjected to swim-up procedure, which may help decrease the number of antibodies. Several studies have investigated whether positive ASA levels have an impact on fertilization rate and live birth rate after IVF. Lu et al. [95] conducted a study with 399 couples comparing the fertilization rate, pregnancy rate, and live birth rate between couples with ASA-positive male partners (n = 39) and ASA-negative male partners (n = 360) after IVF. ASA was detected in serum using the ELISA test. Their results show that in the ASA-positive group, the fertilization rate was lower than in the ASA-negative group (41.7% vs. 54.8%, p = 0.03), alongside the quality of the embryos, which was lower in the ASA-positive group (18.9% vs. 35.2%, p < 0.001). Pregnancy rate (38.5% vs. 61.1%, p = 0.01) and live birth rate (20.5% vs. 42.5%, p = 0.01) were also lower in the ASA-positive group. Similarly, Clark [96] confirmed that high levels of ASA reduce fertilization rate after IVF. He was focusing on IgA antibodies and compared a group of male partners with high levels of IgA with a group of male partners with negative ASA levels. A similar study was made by Vujisić et al. [97], where they compared fertilization rate and pregnancy rate between ASA-positive and negative groups. Their samples were analysed by direct MAR test, but their results did not give such a clear difference between those two groups; fertilization rate and pregnancy rate were not significantly different. Comparable findings were reported by Zini et al. [98], who examined reproductive outcomes after 106 cycles of IVF and found no significant relationship between ASA-positive levels and reproductive outcomes after IVF. Direct ASA levels were measured in fresh semen by MAR test and the test was considered positive if >50% of the motile sperm were covered with antibody (IgG or IgA). The fertilization rates and clinical pregnancy rates were also not significantly different in ASA-positive and ASA-negative groups (68% vs. 66%), and (42% vs. 52%). Overall, the data about the impact of ASA levels on reproductive outcomes is still controversial; some studies indicate that the presence of ASA negatively affects fertilization and live birth rates, whereas others found no difference. The different results in the studies mentioned in this paragraph may be a consequence of using different methods for ASA detection, usually MAR test or ELISA, different thresholds for positive sperm samples, and small groups of ASA-positive male partners, because the majority of the study population were ASA-negative male partners.

4.3 ICSI

ICSI has become an alternative for managing couples affected by ASA, because microinjection of sperm into the oocyte cytoplasm can minimize the effects of ASA on spermatozoa-zona pellucida binding. Similar to those on IVF, there are studies that assessed ICSI and reproductive outcomes, typically by comparing groups of ASA-positive male partners to groups of ASA-negative male partners and examining if there are any differences in reproductive outcomes. Lu et al. [95] conducted the same study for ICSI as for IVF mentioned above, evaluating fertilization rate, pregnancy rate, and live birth rate between the ASA-positive group and the ASA-negative group. They included 155 couples for ICSI cycles, where 19 male partners were ASA-positive and 136 were considered ASA-negative. Their results show that all the three outcome factors were comparable, with no significant differences between the groups. Pregnancy rate was 52.6% in ASA-positive group and 61.8% in ASA-negative group, and live birth rate was 47.4% vs. 44.1%. Esteves et al. [99] performed a retrospective study to analyze the influence of ASA on the outcome after ICSI. They used the direct IB test for evaluation of ASA levels in 351 semen samples. Male participants were divided into four groups according to their percentage of ASA levels (I. (n = 194): 0–10% ASA; II. (n = 107): 11–20% ASA; III. (n = 33): 21–50% ASA and IV. (n = 17): 51–100% ASA) and compared fertilization rates (I. = 80%, II. = 75.0%, III. = 75.0%, IV. = 82.4%), clinical pregnancy rates (I. = 53.5%, II. = 52.8%, III. = 52.0%, IV. = 50%), and miscarriage rates (I. = 21.7%, II. = 10.8%, III. = 23.0%, IV. = 25.0%), between the four groups. The results showed no significant differences among groups, suggesting that varying levels of autoimmunity against spermatozoa do not affect ICSI outcomes. Results in favour of better outcomes after ICSI for ASA-positive male partners were presented in the study by Mercan et al. [100], where they found that fertilization rate was seemingly higher in ASA-positive group (70%) than in the ASA-negative group, 63% (p = 0.06). However, there was no difference regarding pregnancy rate (36% vs. 39%) and live birth rate (36% vs. 30%). Similar findings were observed by Nagy et al. [101], who also noted a higher fertilization rate in the ASA-positive group than in the ASA-negative group (p = 0.046) but found a greater proportion of poor-quality embryos. They concluded that fertilization, embryo development, and pregnancy rates after ICSI are not significantly affected by the proportion of antibody-bound spermatozoa; however, ICSI should be considered the treatment of choice for patients with high levels of ASA in their semen. They claim that ICSI is the better choice because antisperm antibodies do not interfere with the fertilization process when we deposit spermatozoon into the cytoplasm of the egg, so we get better fertilization rates as in standard IVF. It is difficult to find an explanation for this outcome, but one of the possible explanations might be that immunoglobulins against spermatozoa facilitate the acrosome reaction and thus increase the fertilization of the microinjected spermatozoon [102]. Additionally, like we know, ASA can cause sperm agglutination and interfere with sperm motility, which could make the affected spermatozoa incapable of successful fertilization through standard IVF procedures. In such cases, ICSI may provide better outcomes.

4.4 IVF or ICSI?

To achieve optimal reproductive outcomes, there is always the question of how to best overcome male immunological infertility. To find an answer to that question, several researchers decided to compare IVF and ICSI outcomes in couples with a known male immune infertility factor. Zini et al. [98] examined the relationship between ASA levels and reproductive outcomes, specifically fertilization and pregnancy rates, following IVF or ICSI. They analyzed IVF and ICSI cycles separately and found no relationship between ASA levels and either fertilization (67% vs. 59%) or pregnancy rate (55% vs. 47%). They concluded that ASA levels are not related with improved reproductive outcomes. This finding was further supported by their subsequent systematic review and meta-analysis, in which they suggested that both IVF and ICSI remain viable options for infertile couples with semen ASA [103]. A trend in the increase of the rates of live births in ICSI versus IVF within the ASA-positive group was observed by Lu et al. [95], however, it lacked a statistical significance. Live birth rate in IVF cycles was 20.5% and in ICSI cycles 47.4%, (p = 0.07). This may be due to the relatively small number of patients with ASA-positive results included in both the ICSI and IVF groups, so future studies with larger cohorts are therefore warranted to determine whether this trend achieves statistical significance.