Anti-sperm antibodies detection by a modified MAR test: Towards a better definition of its indications

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Anti-sperm antibodies detection by a modified MAR test: Towards a better definition of its indications BIOGRAPHY

Nicolas Gatimel has been working as an embryologist at CHU Toulouse’s assisted reproduction unit since 2011. He obtained a specialist degree in medical biology after a 4-year residency in medical biology, specializing in assisted reproductive technologies. His PhD thesis was on the interest of human sperm morphology assessment. Nicolas Gatimel1,2,*, Jessika Moreau1,2, François Isus1,2, Nathalie Moinard1,2, Jean Parinaud1,2, Roger D. Leandri1,2

KEY MESSAGE

Anti-sperm antibodies detection should not be systematically recommended in investigations of fertility status and before assisted reproductive technology treatment, but reserved for when sperm agglutination is found during conventional sperm examination or if the patient has a history of scrotal trauma or of inguinal surgery. ABSTRACT

Research question: Anti-sperm antibodies (ASA) have been shown to reduce male fertility but consensus about the precise situations in which tests should be carried out are lacking. In infertility investigations, should the mixed antiglobulin reaction (MAR) test be a first-line test? Should it be carried out systematically before assisted reproductive technology (ART)? What are the risk factors for ASA? Design: All infertile patients (n = 1364) were tested with SpermMar (modified MAR test) between July 2013 and June 2017. Intra-patient variability of the MAR test was also assesed by comparing two tests within the same year in selected patients (n = 101). Results: The main factor that influenced the percentage of ASA was the presence or absence of sperm agglutination. In the presence of agglutinations, 27 out of 72 (37.5%) patients were positive for ASA compared with 33 out of 1292 (2.6%) in the absence of agglutinations (P < 0.0001). When one risk factor was present (spontaneous sperm agglutination, history of scrotal trauma or inguinal surgery), 33 out of 179 (18.44%) tests were positive for ASA (≥50% coated spermatozoa), whereas only 27 out of 1242 (2.2%) were positive when no risk factor was present (P < 0.0001). Conclusions: ASA detection should not be systematically recommended in investigations of fertility status and before ART but reserved for when sperm agglutination is found during conventional sperm examination, or if the patient has a history of scrotal trauma or has undergone inguinal surgery. 1  Service

de Médecine de la Reproduction, Hôpital Paule de Viguier, CHU Toulouse, 330 avenue de Grande Bretagne, Toulouse 31059, France 2  Université Paul Sabatier Toulouse-III, Groupe de Recherche en Fertilité Humaine (EA 31694, Human Fertility Research Group), 330 avenue de Grande Bretagne, Toulouse 31059, France © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. *Corresponding author. E-mail address: [email protected] (N Gatimel). https://doi.org/10.1016/j.rbmo.2018.09.011 1472-6483/© 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. Declaration: The authors report no financial or commerical conflicts of interest.

KEY WORDS

ART Anti-sperm antibodies Infertility Prevalence

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INTRODUCTION

T

he presence of anti-sperm antibodies (ASA) in human semen can reduce fecundity (Ayvaliotis et al., 1985). Diverse ASA bind to the sperm surface, and their effect on fertilization may differ. Some sperm-bound antibodies are related to inhibitory effects on fertilization (Shibahara and Koriyama, 2013), but not all ASA cause infertility. The main immunoglobulin classes for ASA are immunoglobulin G (IgG) and immunoglobulin A (IgA) (Mazumdar and Levine, 1998). It has been assumed that immunoglobulin M (IgM) would only be found in rare situations as it is a large molecule. Several investigators have found a decreased fertilization rate when more than 80% of the sperm head was coated with IgA or IgG (Clarke et al., 1985b; Pattinson and Mortimer, 1987). Also, no consensus has been reached on the importance of the location of IgG or IgA and sperm function (Mandelbaum et al., 1987b; Sukcharoen and Keith, 1995; Chiu and Chamley, 2004). The value of data on the frequency of ASA in infertile men may depend on the testing method used (Clarke et al., 1985a; Cerasaro et al., 1985; Kamieniczna et al., 2003). In a population of infertile men (without exclusion criteria and without a clear description of semen parameters), ASA was detected with the MAR test on more than 40% of the motile spermatozoa in about 13% of men (Sinisi et al., 1993). Various pathogeneses explain why ASA may lead to infertility. The presence of ASA decreases sperm penetration in cervical mucus (De Almeida et al., 1986; Menge and Beitner, 1989). Some ASA can reduce the ability of spermatozoa to undergo capacitation or acrosome reaction through inhibition of changes in the sperm membrane (Fusi and Bronson, 1990). Moreover, the proportion of spermatozoa bound with ASA has been shown to be related to the fertilization ability of sperm. Among IVF trials assessing the effect of ASA on fertilization (detected by the MAR test), six out of seven trials showed a decrease in the fertilization rate (Chiu and Chamley, 2004). The effect of ASA on sperm– oocyte fusion is still under debate, as shown in studies on binding and penetration of hamster zona-free eggs (Primakoff and Hyatt, 1986; Aitken et al.,

1988; Shibahara et al., 1996). Some (but not all) of the ASA have been shown to decrease fertilization ability. Other studies suggest ASA could act directly on antigen sperm surface antigens, or have an indirect action mediating the release of cytokines that affect sperm function (Mazumdar and Levine, 1998; Lombardo et al., 2001). The deleterious effect of ASA on embryo implantation has been shown in a few studies (Witkin and Chaudhry, 1989; Check et al., 2000), but the mechanism by which ASA could interfere with embryo implantation is not clear. Commonly used tests to detect seminal ASA are the mixed antiglobulin reaction test (MAR) and immunobead test. Current tests, however, cannot differentiate ASA that cause infertility from those that do not. A commercially available modified MAR test (SpermMar, FertiPro, Beernem, Belgium) is widely used during routine semen analysis in andrologic laboratories because of its technical advantages, such as the application of unwashed fresh semen and its rapidity. Even if indirect tests to detect ASA in serum have good sensitivity and specificity (Mazumdar and Levine, 1998), they are increasingly neglected in routine practice in favour of quicker and cheaper direct tests. The MAR test has been routinely used for several decades; however, its indications are not well defined. The World Health Organization (WHO) laboratory manual for the examination and processing of human semen includes the testing for antibody coating of spermatozoa in its ‘Standard Procedures’ chapter not in its ‘Optional Procedures’ (WHO, 2010). Introducing this testing, the manual stipulates that ‘If spermatozoa demonstrate agglutination … the presence of sperm antibodies may be the cause.’ Immediately after, it states that ‘Sperm antibodies can be present without sperm agglutination’. Therefore, we examined the application of routine testing for ASA as a first-line test in an infertility investigation, and for risk factors for ASA, by systematically testing all men in an infertile couple with SpermMar who were seen in our centre between July 2013 and June 2017. The aim of this study was to explore whether SpermMar should be carried out systematically in infertility investigations and before assisted reproductive technology and to detect any risk factors for ASA.

Furthermore, because it has been poorly assessed previously, we tested for the intra-patient variability in the MAR test results over time.

MATERIALS AND METHODS Study population Infertile men were defined as men in a relationship who failed to achieve a pregnancy after 12 months or more of regular unprotected sexual intercourse. All infertile men (n = 1364) who were seen in our centre between July 2013 and June 2017 were tested for the presence of ASA, except in cases of severe oligoasthenozoospermia, defined as a sperm concentration below 1.106/ml or progressive motility less than 10%. All patients were asked about their medical, surgical and andrologic history by an andrologist physician in our fertility centre. Andrologic history includes history of scrotal trauma, scrotal or inguinal surgery, genital infectious disease such as epididymo-orchitis and history of prostatitis. No men who had undergone a vasectomy reversal were included in this study, as vasectomy in France is not current practice compared with other countries. To assess intra-patient variability, we conducted a second analysis comparing the results of two MAR tests conducted in the same year in 101 patients who planned to return to the centre for other consultations or examinations. The patients had no medical, andrologic or surgical treatment history between the two assessments. Anti-sperm antibody detection Determination of ASA was carried out directly after complete semen liquefaction for 15–60 min at 37°C using the SpermMar IgG kit ® (FertiPro, Beernem, Belgium). This method is based on the binding reaction of latex particles sensitized with human IgG to live spermatozoa in the presence of an antiserum against the Fc fragment of human IgG. Ten microlitres of fresh untreated semen, 10 µL of SpermMar Test IgG Latex Particles and 10 µL of SpermMar Test IgG Antiserum were placed separate to each other on a microscope slide. The sample and the latex reagent were mixed five times with the edge of a cover glass and then the antiserum was mixed with the previous mixture. The cover glass was placed on the final mixture and the mixture



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was observed under a light microscope (x400 magnification at phase contrast). The results were read after 3 min. One hundred spermatozoa were counted to determine the percentage of reactive spermatozoa (two or more latex particles attached to motile spermatozoa). If no bead attachment to spermatozoa was observed, the slide was read again after 10 min. According to WHO guidelines (WHO, 2010), the test is considered positive if at least 50% of spermatozoa are coated with antibodies. As the proportion of patients with a positive reaction in the SpermMar Test IgA in the absence of any reaction in the SpermMar Test IgG is rare (Mazumdar and Levine, 1998), we decided to routinely use only the IgG-class kit in our centre. Semen analysis Semen analyses were conducted by four technicians from the same laboratory. Briefly, analyses were carried out after 15– 60-min liquefaction. An initial microscopic examination of the sample at phase contrast with a x20 objective was carried out to detect agglutination. The difference between agglutination (=spontaneous agglutination) and aggregation was noted. As stated in the WHO 2010 guidelines, agglutination specifically refers to motile spermatozoa sticking to each other, head-to-head, tail-to-tail or in a mixed way with a shaking motion in most cases. The adherence either of immotile spermatozoa to each other or of motile spermatozoa to mucus strands, non-

sperm cells or debris is considered to be aggregation (WHO, 2010). Semen volume was measured by a graduated pipette (in laboratory validated technique after comparison to the reference method by weighing; data not published). Spermatozoa were counted on two replicates (at least 200 spermatozoa per replicate) in a Malassez chamber. Sperm motility was measured using phase-contrast optics at x400 magnification and expressed as percentage progressive motility. Sperm viability was assessed by nigrosin and eosin staining (Sigma Aldrich) and expressed as percentage viability. Statistical analysis Data were extracted from the Gynelog clinical database used in our department. StatView software (Abacus Concepts Inc., Berkeley, CA) was used for statistical analyses. Data were presented as means ± SD. Percentages were compared using chi-squared test. Means were compared using Student’s t-test or the Mann– Whitney test according to the normality of data distribution. P < 0.05 was considered statistically significant. Ethical approval Data were extracted from the ART centre patient database. This database was approved by the French National Commission for Information Technology and Civil Liberties to be used for clinical research. Patients are aware that their

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data can be used for anonymous clinical studies unless they specifically state otherwise. According to new French law (2016-1537), non-interventional studies, such as from clinical databases, do not need to be submitted to an ethical committee.

RESULTS The total prevalence of positive ASA (≥ 50%) in our population was 60 out of 1364 (4.4%); 35 out of 1364 (2.6%) patients had a high percentage of coated spermatozoa (≥75%); 637 out of 1364 (46.7%) had 0% of ASA; 626 out of 1364 (45.9%) had 1–24% of ASA-coated spermatozoa; and 41 out of 1364 (3.0%) had 25–49% ASA-coated spermatozoa (FIGURE 1). The conventional semen parameters were not significantly different according to the percentage of spermatozoa coated with antibodies (TABLE 1). The clinical factors and the presence or absence of spontaneous sperm agglutination according to the proportion of ASA-coated spermatozoa are presented in TABLE 2. The main factor that influences the percentage of ASA is the presence or absence of sperm agglutination. Applying the threshold (>50%) for a positive detection in the presence of agglutination, as defined by WHO (2010), 27 out of 72 (37.5%) patients were positive for ASA compared with 33 out of 1292 (2.5%) patients

FIGURE 1  Distribution of percentage of anti-sperm antibodies coated spermatozoa in a population of 1364 infertile men.

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TABLE 1  COMPARISON OF CONVENTIONAL SEMEN PARAMETERS ACCORDING TO THE PERCENTAGE OF ANTIBODIES COATED SPERMATOZOA a Percentage of antibodies coated spermatozoa ± SD <50%; n = 1304

≥50%; n = 60

Age (years)

35.2 ± 6.2

35.4 ± 6.1

Volume (ml)

3.4 ± 1.7

3.2 ± 1.6

Sperm concentration (106/ml)

51.5 ± 56.0

40.5 ± 38.1

Total sperm count

(106

156.9 ± 172.8

127.9 ± 133.6

Progressive motility (%)

per ejaculate)

34 ± 12

33 ± 12

Vitality (%)

70 ± 13

69 ± 14

a

  No statistically significant differences were observed between the two groups for all parameters.

when no agglutination was present (P < 0.0001). When at least one of the three risk factors were present (spontaneous sperm agglutination or history of scrotal trauma or of inguinal surgery), 33 out of 179 (18.4%) tests were positive, whereas only 27 out of 1185 (2.3%) were positive when no risk factor was present (P < 0.0001).

were found to be negative in the first test, three (3.8%) were found to be positive in the second test (TABLE 3), whereas, of the 22 patients who were found to be positive in the first test, 10 (45%) were found to be negative in the second test.

In assessing intra-patient variability (TABLE 3), when the tests were repeated a second time in the same year in the same patient, of the 79 patients who

Some investigators have argued in favour of an association between ASA and reduced male fertility potential (Mazumdar and Levine, 1998; Lombardo

DISCUSSION

et al., 2001; Chiu and Chamley, 2004). Good evidence has also shown that prevalence of ASA is higher in infertile men than in fertile men (Sinisi et al., 1993). Although these in-vitro studies have shown that ASA can impair various mechanisms, such as motility, spermoocyte fusion or acrosome reaction, the relationship between the presence of ASA and spontaneous fertility is not clear. In the study by Ayvaliotis et al. (1985), the spontaneous pregnancy rate for couples whose male partners had more than 50% sperm-bound antibodies

TABLE 2  FACTORS INFLUENCING THE PERCENTAGE OF SPERMATOZOA COATED IN ANTIBODIES Statistical comparisona

Spermatozoa coated in antibodies (%) 0

1–24%

25–49%

50–74%

≥75%

Yes (n = 72)

12 (16.7)

22 (30.6)

11(15.3)

9 (12.5)

18 (25.0)

No (n = 1292)

625 (48.4)

604 (46.7)

30 (2.3)

16 (1.2)

17 (1.3)

Yes (n = 54)

23 (42.6)

22 (40.7)

3 (5.6)

3 (5.6)

3 (5.6)

No (n = 1310)

614 (46.9)

604 (46.2)

38 (2.9)

22 (1.7)

32 (2.4)

Yes (n = 8)

3 (37.5)

2 (25.0)

2 (25.0)

1 (12.5)

0 (0)

No (n = 1356)

634 (46.7)

624 (46.0)

39 (2.9)

24 (1.7)

35 (2.5)

Yes (n = 52)

21 (40.4)

26 (50.0)

1 (1.9)

2 (3.8)

2 (3.8)

No (n = 1312)

616 (47.0)

600 (45.7)

40 (3.1)

23 (1.8)

33 (2.5)

Yes (n = 62)

17 (27.4)

35 (56.5)

3 (4.8)

2 (3.2)

5 (8.1)

No (n = 1302)

620 (47.6)

591 (45.4)

38 (2.9)

23 (1.8)

30 (2.3)

Yes (n = 17)

8 (47.1)

9 (52.9)

0 (0)

0 (0)

0 (0)

No (n = 1347)

629 (46.7)

617 (45.8)

41 (3.0)

25 (1.9)

35 (2.6)

Yes (n = 179)

52 (29.1)

78 (43.6)

16 (8.9)

13 (7.3)

20 (11.2)

No (n = 1185)

585 (49.4)

548 (46.2)

25 (2.1)

12 (1.0)

15 (1.3)

Spontaneous sperm agglutination, n (%) P < 0.0001

History of scrotal trauma, n (%) NS

History of epididymo-orchitis, n (%) NS

History of scrotal surgery, n (%) NS

History of inguinal surgery, n (%) P = 0.008

History of prostatitis, n (%) NS

Spermatozoa agglutination or history of scrotal trauma or of inguinal surgery, n (%)

a

  Comparing all groups using the chi-squared test.

P < 0.0001



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TABLE 3  RESULTS OF A SECOND ANTI-SPERM ANTIBODIES TEST AS A FUNCTION OF THE FIRST TESTa Second test, n (%) First test

0%

1–24%

25–49%

50–74%

≥75%

0% (n = 41)

23 (56.1)

16 (39.0)

0

1 (2.4)

1 (2.4)

1–24% (n = 33)

8 (24.2)

23 (69.7)

1 (3.0)

0

1 (3.0)

25–49% (n = 5)

0

3 (60.0)

2 (40.0)

0

0

50–74% (n = 12)

0

4 (33.3)

4 (33.3)

2 (16.7)

2 (16.)

≥79% (n = 10)

1 (10.0)

1 (10.0)

0

2 (20.0)

6 (60.0)

a  Distribution

of percentage of anti-sperm spermatozoa coated in antibodies in two tests carried out within the same year. Significance is P < 0.0001, comparing all groups

using the chi-squared test.

was significantly decreased (15.3% versus 66.7%; P < 0.005). The results of studies evaluating the effect of ASA on reproductive outcomes, however, are somewhat controversial (Chiu and Chamley, 2004). The MAR test is currently a rapid (3–10 min), easy to perform diagnostic test on unwashed fresh semen. One limitation is that it can only be carried out if motile spermatozoa are present in the semen, as samples with poor motility may have false negative results. In cases of severe impairment in sperm parameters, determination of ASA is not contributive, either for the diagnosis of infertility or for treatment (choice of ART technique). In the present study, we found that 4.4% of infertile patients had positive ASA (≥50% coated spermatozoa) using SpermMar test. Our results are similar to those of Sinisi et al. (1993), who detected IgG bound to spermatozoa (with >40% ASA binding threshold) in 6.2% of infertile men. In a study using serum assays, Collins et al. (1993) found ASA in 8.1% of infertile men. Studies using indirect assays, however, should be interpreted with caution because they may not reflect the presence of ASA on spermatozoa (Bronson et al., 1987). On the other hand, ASA detected by SpermMar are present in 0.9% of fertile men (Sinisi et al., 1993). Immunobead tests for IgG, IgA, and IgM were carried out by Pattinson and Mortimer (1987) on 300 sperm samples from infertile men. Thirty-two (10.7%) showed positive results (using a threshold >10% of coated spermatozoa). These latter results should be interpreted with caution because, as demonstrated by Barratt et al. (1989), low to moderate levels of ASA have a poor prognostic value. In the present study, the main factor that influenced the percentage of ASA

is the presence or absence of sperm agglutination. The association between the presence of ASA and agglutination was demonstrated over 40 years ago (1974) and, since then, by others (Kunathikom et al., 1995). In this latter study, the incidence of positive MAR results in samples with and without spontaneous sperm agglutination was 15 and 0%, respectively (P < 0.05); however, the prevalence of spontaneous agglutination was high in this study (80/160). Our results highlight the importance of agglutination evaluation on fresh semen during the assessment of conventional sperm parameters. Sperm antibodies, however, can be present without sperm agglutination, and agglutination can be caused by factors other than sperm antibodies, such as bacterial infections or some antibiotic therapies (Carranza-Lira et al., 1998; Kaur and Prabha, 2013). In relation to andrologic factors that could influence the presence of ASA, we did not find an association between a history of prostatitis and the presence of ASA. These results are not in agreement with the metaanalysis by Jiang et al. (2016). The investigators of this meta-analysis found a combined odds ratio of the ASApositive rate in patients with chronic prostatitis and normal controls of 3.26 (95% CI 1.86 to 5.71). An immune pathogenicity for chronic prostatitis has been suspected. It may be mediated by lymphocyte infiltration in the semen of patients with autoimmune prostatitis, or by a semen prostatic liquid reflux phenomenon (Batstone et al., 2002; Ausmees et al., 2013; Jiang et al., 2016). The absence of correlation between chronic prostatitis and ASA in our study could be due to the small number of patients with chronic prostatitis. In agreement with some studies (Marconi et al., 2009), we found no effect of

history of epididymo-orchitis on ASA formation: however, our results are not in accordance with others studies that showed a correlation between some inflammation, infection situations (as acute epididimytis), or both, and presence of ASA (Ingerslev et al., 1986; Heidenreich et al., 1994). We found that testis trauma is associated with a trend of more frequent ASA-positive semen. During testis trauma, the breaching of the blood–testis barrier may induce an exposure of immunogenic sperm antigens to the immune system, resulting in ASA formation (Mandelbaum et al., 1987a; Mazumdar and Levine, 1998; Bohring and Krause, 2003). The occurrence of these testis traumas must be systematically sought and, even in early childhood, because ASA may be produced in children during puberty despite ‘quiescent’ spermatogenesis (Domagala et al., 2006). Childhood inguinal herniorrhaphy with vas-deferens obstruction has also been reported to be associated with ASA (Matsuda et al., 1993). In the present study, we found a significantly higher prevalence of ASA in patients who had undergone inguinal surgery. Applying the threshold (>50%) for a positive detection, in the presence of a history of inguinal surgery, seven out of 62 (11.3%) patients were positive for ASA compared with 53 out of 1302 (4.1%) patients when no history of inguinal surgery was present (P = 0.008). Vasectomy has been reported to induce ASA (Awsare et al., 2005). As vasectomy is not current practice in France compared with other countries, we were not able to determine the prevalence of these antibodies in men after vasectomy reversal. Our results show that the presence of at least one of the three risk factors (spontaneous sperm agglutination or history of scrotal trauma or of inguinal

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surgery) significantly increased the probability of positive test for ASA. Finally, in assessing intra-patient variability in ASA detection using the SperMar test, our results (TABLE 3) suggest that it is not necessary to repeat the test a second time if it is negative on the first determination. This is in accordance with a previous study on a smaller population showing that among 29 patients first tested as negative (ASA threshold ≤40%); all of them were still negative in two supplementary tests, carried out respectively with a mean interval of 4 and 10 months after the first test (Paschke et al., 1994). On the other hand, we show that if a first test is positive, 45% of those patients are detected negative within a year. This is also in agreement with the study y Paschke (1994) in which 25 patients that first tested as positive, nine (36%) became negative on the following tests. No data have been published on how to manage such patients. It would be of interest to assess the clinical effect of ASA in these patients. A routine diagnostic test for immunologic infertility does not assess the specificity of the antibodies, nor does it provide information on the proportion of ASA bound to clinically relevant antigens. As stated by WHO guidelines, fifth edition (WHO, 2010), ‘The mere presence of sperm antibodies is insufficient for a diagnosis of sperm immunologic infertility. It is necessary to demonstrate that the antibodies severely interfere with sperm function’. A sperm–mucus penetration test has typically been used for this. Unfortunately, these sperm– mucus penetration tests should no longer be carried out, given their lack of clinical relevance and analytical reliability, as recommended by learned societies (Crosignani and Rubin, 2000; Smith et al., 2003). In 2018, it is still difficult to know the clinical value of ASA determination before ART treatment. In relation to the effect of ASA on ART outcomes, the results have been heterogeneous. Some studies report that ASA are associated with a reduced probability of pregnancy after intrauterine insemination (Check and Bollendorf, 1992; van Weert et al., 2005). A more recent study reported pregnancy rates of 18.4% (seven out of 38) after intrauterine insemination, with or without ovarian stimulation in cases

of immunologic male subfertility (with >90% antibody-coated spermatozoa), suggesting that the ASA clinical interference with fertility could be overcome by bypassing the cervical mucus (Francavilla et al., 2009). Studies assessing the effect of ASA on fertilization and pregnancy rates after conventional IVF have reported contradictory results (Junk et al., 1986; de Almeida et al., 1989; Ford et al., 1996; Culligan et al., 1998; Rajah et al., 1993; Vujisic et al., 2005). In other studies that examined the effect of ASA on ICSI outcomes, ASA was shown to have no influence on intracytoplasmic sperm injection (ICSI) pregnancy rates (Lahteenmaki et al., 1995; Mazumdar and Levine, 1998; Check et al., 2000). As far as we know, however, no comparative study of different ART in patients with ASA has been published (probably due to the difficulty of recruiting patients with an ASA-positive test). On the basis of published research, ASA alone has a poor correlation with conventional IVF or ICSI outcomes. In previously cited studies on the effect of ASA on ART outcomes, however, the cut-off value for an ASA positive test varied from 1 to 80%. A systematic review and metaanalysis indicated that ASA are not related to pregnancy rates after IVF or ICSI (Zini et al., 2011). Our results do not assess the effect of ASA on clinical outcomes in ART; however, in view of the low prevalence of ASA in our study in the absence of a risk factor, and of literature data demonstrating controversy about the efect of ASA on clinical outcomes, we do not recommend systematic determination of ASA before ART. In conclusion, routine direct assays to detect ASA in spermatozoa have been used for several decades and are easy to use; however, their indications are still not well defined. We conclude that ASA detection should not be recommended as a systematic test for the investigation of fertility status and before ART, unless sperm agglutinations are found during conventional sperm examination, or if there is a history of scrotal trauma or of inguinal surgery. This study, however, has been conducted using the SperMar test and results obtained using a different technique, such as the immunobead test, may not be comparable.

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