Abstract
Purpose
There is a need to improve our understanding of the cut‐off value of the direct immunobead test (D‐IBT).
Methods
The subjects were 26 D‐IBT‐positive and 140 D‐IBT‐negative males. The results of post coital tests (PCTs) for each subject were examined.
Results
A significant difference was observed in abnormal PCTs between values <20 % and those ≥20 % (P = 0.02). However, there was no significant difference in abnormal PCTs between values <50 % and those ≥50 % (P = 0.084).
Conclusions
A cut‐off value of 20 % was correlated with the possibility of conception on treatment with IUI. The D‐IBT is a screening test, and the value of 20 % initially suggested by Bronson et al. seems to be more appropriate than that of 50 % in the criteria defined by the World Health Organization.
Keywords: Antisperm antibody, Cut‐off value, Direct immunobead test, Male infertility, Postcoital test
Introduction
Sperm have long been known to be antigenic. Antisperm antibodies (ASA) are found in both males and females. We reported the incidences of ASA in 3.54 % of the former [1] and 2.3 % of the latter [2]. ASA are produced as autoantibodies in males and as isoantibodies in females, and they can be a cause of immunological infertility. Candidate antigens for ASA are mainly glycoproteins on the sperm plasma membrane or seminal fluid [3], but the reason why only some individuals produce ASA still remains unclear. Some of the antibodies play important roles in infertility by inhibiting sperm passage in the female genital tract [4, 5], inhibiting the fertilization process [6, 7], and impairing embryo development [8, 9].
The direct immunobead test (D‐IBT) [10] is a screening method for detecting ASA bound to the sperm surface in infertile males. If the D‐IBT result is positive, a postcoital test (PCT) and the hemizona assay (HZA) [11] are performed for couples, and the results are used for therapeutic planning according to our strategy [1].
Originally, this test was used to screen for ASA bound to sperm, with immunobead (IB) binding of ≥20 % being considered positive. However, the World Health Organization amended this cut‐off value to ≥50 % without providing any reason or evidence in 1999 [12] and also 2010 [13]. Therefore, this study was performed to investigate which value is more appropriate for decision‐making in male immunological infertility by analyzing the results of the PCT.
Materials and methods
Subjects
In order to investigate male immunological infertility, D‐IBT was performed using sperm from 1,144 infertile males who visited the Center for Reproductive Medicine, Jichi Medical University Hospital, between May 1999 and August 2010. Twenty‐six (2.3 %) infertile males had ASA. One hundred and forty control males with a negative D‐IBT result visited our center between January 2009 and December 2010, and males whose wives had ASA were excluded from this study. Therefore, a total of 166 couples were enrolled in this study.
Their characteristics are summarized in Table 1. Twenty‐six males with IB binding of ≥20 % were considered positive, and 140 males with IB binding of <20 % were considered negative controls.
Table 1.
Patients’ characteristics
| Characteristics | IBT <20 % | IBT ≥20 % | P | IBT <50 % | IBT ≥50 % | P |
|---|---|---|---|---|---|---|
| No. of patients | 140 | 11 | 143 | 8 | ||
| Age (y/o) of patients | 39.0 (35.0–43.6) | 38.0 (35.2–49.4) | NS | 39.0 (35.5–44.5) | 37.5 (30.6–45.5) | NS |
| Age (y/o) of wives | 36.0 ± 4.5 | 34.0 ± 6.9 | NS | 35.8 ± 4.7 | 36.2 ± 6.6 | NS |
| Primary infertility (%) | 111 (79.3) | 9 (81.8) | NS | 114 (79.7) | 6 (75.0) | NS |
| Infertility period (months) | 24.0 (19.6–42.9) | 36.0 (19.6–76.3) | NS | 24.0 (19.6–42.9) | 27.0 (15.6–39.8) | NS |
| Causes of infertility | ||||||
| Endocrine (%) | 20 (14.3) | 3 (27.3) | 22 (15.4) | 1 (12.5) | ||
| Male (%) | 27 (19.3) | 9 (81.8) | 28 (19.6) | 8 (100) | ||
| Uterine (%) | 13 (9.3) | 0 (0) | 13 (9.1) | 0 (0) | ||
| Cervical (%) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | ||
| Tubal (%) | 10 (7.1) | 2 (18.2) | 11 (7.7) | 1 (12.5) | ||
| Unexplained (%) | 30 (21.4) | 0 (0) | 30 (21.0) | 0 (0) | ||
| Semen characteristics | ||||||
| Volume (ml) | 3.9 (3.0–5.0) | 3.0 (2.7–4.2) | NS | 3.9 (3.1–5.0) | 2.6 (2.4–3.6) | NS |
| Concentration (106/ml) | 84.2 (69.1–175.1) | 121.7 (48.15–171.0) | NS | 83.9 (70.5–176.6) | 131.7 (42.7–181.9) | NS |
| Motility (%) | 53.0 (36.7–66.7) | 27.0 (17.2–45.0) | 0.01 | 53.0 (37.2–66.7) | 16.5 (14.0–32.9) | 0.001 |
Causes of infertility included overlapping factors
Data are shown as mean ± SD and as median (interquartile range)
Assay for detecting antisperm antibodies in the subjects’ semen
D‐IBT was performed using the semen samples following the method of Bronson et al [10]. Briefly, 5 μl of washed sperm suspension was placed on a glass slide and 5 μl of each type of IB including rat antihuman IgG, IgA, and IgM (Irvine Scientific, Santa Ana, CA, USA) were added to the sperm suspension and mixed well using either a pipette tip or the edge of a cover slip. A cover slip (22–22 mm2) was placed on each of the mixtures. After keeping the slide in a moist chamber for 10 min, the sperm were observed at 400× magnification under a phase‐contrast microscope. At least 200 motile sperm were counted in duplicate per preparation. The percentage of motile sperm that had one or more attached IBs was recorded. A cut‐off value of 20 % was adopted in this study.
Postcoital test
PCTs were performed in 151 couples. Since the PCT cannot be performed in anovulatory females, the data included only females with an ovulatory cycle. Furthermore, the females who had cervical factors were excluded. PCT results were available for 11 of 26 D‐IBT‐positive men.
The PCT was used to assess the penetration of sperm through the cervical mucus. Using PCT evaluation according to the World Health Organization [14], the subjects were instructed to have intercourse on a day that was estimated to be near the day of ovulation based on various clinical criteria, including the usual cycle length, basal body temperature, cervical mucus change, and follicular diameter examined by transvaginal ultrasound. As a general rule, the test was conducted 3–10 h after intercourse. Cervical mucus was analyzed under 400× magnification. An abnormal PCT result was defined as that in which less than 10 motile sperm were detected in the cervical mucus.
Statistical methods
Prior to statistical testing, the Shapiro–Wilk test was performed to determine whether each variable was normally distributed. We used the t test or Mann–Whitney U test. Statistical analyses of the data were performed using the Chi‐square test or Fisher's exact test. All statistical analyses were carried out using the Statistical Package for Social Science (SPSS) for personal computers, version 17.0 for Windows (SPSS Institute Inc., Chicago, IL, USA). A value of P < 0.05 was defined as significant.
Results
Patients’ characteristics
The mean ages of the male patients and their wives were 35.0 ± 6.1 years (range 26–58 years) and 32.5 ± 5.3 years (range 23–47 years), respectively. The mean duration of infertility was 29.6 ± 28.1 months (range 2–192 months), and 147 patients (88.6 %) had primary infertility. Causes of infertility included overlapping factors (Table 1).
Immunobead binding in the direct immunobead test
According to Bronson's criteria [10], 140 of 166 patients showed no IB binding, 4 patients showed 20–29 % IB binding, 1 patient showed 30–39 % IB binding, 3 patients showed 40–49 % IB binding, 2 patients showed 60–69 % IB binding, 3 patients showed 70–79 % IB binding, 6 patients showed 80–89 % IB binding, 4 patients showed 90–99 % IB binding, and 3 patients showed 100 % IB binding (Fig. 1).
Figure 1.
Distribution of number of patients related to IB binding. The D‐IBT (direct immunobead test) consists of polyacrylamide beads that are coated with a specific anti‐immunoglobulin. According to Bronson's criteria [10], 140 of 166 patients showed no IB binding, 4 patients showed 20–29 % IB binding, 1 patient showed 30–39 % IB binding, 3 patients showed 40–49 % IB binding, 2 patients showed 60–69 % IB binding, 3 patients showed 70–79 % IB binding, 6 patients showed 80–89 % IB binding, 4 patients showed 90–99 % IB binding, and 3 patients showed 100 % IB binding
Comparison of the PCT results between cut‐off values of 20 and 50 %
Among the 166 couples, PCTs were performed for 151 couples except for those with endocrine and tubal factors. The patients’ characteristics are summarized in Table 1 (categorized in two groups: cut‐off value of 20 or 50 %).
When the cut‐off value of 20 % was selected for the D‐IBT, 11 males were positive and 140 males were negative in PCTs. No significant differences were observed in the age of males, age of females, duration of infertility, and semen volume and concentration. Sperm motility was significantly lower for cut‐off values of ≥20 % (Table 1).
Abnormal PCTs were noted in 29 (20.7 %) of 140 males with IB binding of <20 %, and in 6 (54.5 %) of 11 males with IB binding of ≥20 %. There was a significant difference in the frequency of abnormal PCTs between the two groups (P = 0.02) (Fig. 2).
Figure 2.
Comparison of the PCT results between cut‐off value of 20 and 50 %. Among the 166 couples, PCTs were performed in 151 couples. Abnormal PCTs were noted in 29 (20.7 %) of 140 males with IB binding of <20 %, and in 6 (54.5 %) of 11 males with IB binding of ≥20 %. There was a significant difference in the frequency of abnormal PCTs between the two groups (*P = 0.02). Abnormal PCTs were noted in 31 (21.7 %) of 143 males with IB binding of <50 %, and in 4 (50 %) of 8 males with IB binding of ≥50 %. There was no significant difference in the frequency of abnormal PCTs between the two groups (P = 0.084)
When the cut‐off value of 50 % was selected for the D‐IBT, 8 males were positive and 143 were negative in PCTs. No significant differences were observed in the age of males, age of females, duration of infertility, and semen volume and concentration. Sperm motility was significantly lower for cut‐off values of ≥50 % (Table 1). Abnormal PCTs were noted in 31 (21.7 %) of 143 males with IB binding of <50 %, and in 4 (50 %) of 8 males with IB binding of ≥50 %. There was no significant difference in the frequency of abnormal PCTs between the two groups (P = 0.084) (Fig. 2).
To determine the cut‐off value of D‐IBT, the sensitivity and specificity of the data were evaluated in both 20 and 50 %. When the cut‐off value of 20 % was selected, the sensitivity and specificity were 17.1 and 95.7 %, respectively. When the cut‐off value of 50 % was selected, the sensitivity and specificity were 11.4 and 96.6 %, respectively.
Discussion
The presence of ASA can reduce fecundity in both males and females. In males, methods for detecting ASA on the surface of sperm include the D‐IBT and mixed antiglobulin reaction test (MAR test) [12]. As the D‐IBT is the most practical of these methods, we have been using it in clinical practice in conjunction with the cut‐off value of 20 % proposed by Bronson et al [10]. We have used the D‐IBT in initial screening and proposed therapeutic strategies. Although the WHO raised the cut‐off value for the D‐IBT to 50 % [12], this change was not well supported. We therefore investigated whether a cut‐off value of 20 % is more appropriate than 50 %.
D‐IBT cut‐off values vary from 20 % [3, 15, 16] to 50 % [12, 13, 17, 18]. However, there have been no reports describing the relationship between cut‐off values for the D‐IBT and PCT results and they have yet to be fully standardized.
In the present study, when a cut‐off value of 20 % was used for the IB binding rate in the PCT, abnormal PCTs were observed in 20.7 % of patients with IB binding <20 %, and observed in 54.5 % of patients with IB binding ≥20 %. When a cut‐off value of 50 % was used for the IB binding rate, abnormal PCTs were observed in 21.7 % of patients with IB binding <50 %, and observed in 50.0 % of patients with IB binding ≥50 %. A significant difference between the two groups was noted only when a cut‐off value of 20 % was used (Fig. 2). The sensitivity and specificity of the cut‐off value between 20 and 50 % were similar. As D‐IBT is carried out as the first screening test for detecting ASA bound to the sperm surface in infertile males, the cut‐off value of 20 % seems to be useful as a clinical test.
The PCT is a common method of testing for infertility. While the PCT is an important test, many of the patients referred to our university hospital from other facilities have undergone the PCT and IUI numerous times, and some of them consider it meaningless to perform PCTs for cases of repeated IUI failure. However, for patients who tested positive on screening using the D‐IBT, evidence‐based therapeutic strategies can be proposed by performing the PCT and HZA [11]. In addition, even if repeated IUI failures have occurred in previous clinics, further IUI attempts may be worthwhile in patients with a negative D‐IBT and normal PCT. In other words, we believe that the role of the D‐IBT for decision‐making regarding infertility treatment and its cut‐off value are of considerable importance.
Our therapeutic strategy is as follows: When performing semen analysis, the D‐IBT is performed to screen for ASA binding to the surface of ejaculated sperm. Bronson et al. [10] introduced the D‐IBT, which can detect ASA regardless of the biological activity. Routine D‐IBTs showed that some of the difficulties associated with elucidating the clinical significance of ASA in infertile men are associated with ASA diversity. The immunoglobulin (Ig) antibody classes (IgG, IgA, and IgM), sperm binding sites (head, mid‐piece, and tail), ratio of antibody‐bound motile sperm among total motile sperm, and biological activities (such as sperm immobilization) are all involved [16].
Of course, the therapeutic strategy should not be based solely on a positive D‐IBT result, namely ASA binding to ejaculated sperm in infertile men, as not all ASAs detected in this manner are involved in infertility.
Therefore, we always assess sperm motility, particularly the ability of sperm to penetrate the cervical mucus using the PCT and fertilization ability using the HZA in D‐IBT‐positive men. If the hemizona index (HZI) is abnormal or fertilization ability is low, then intracytoplasmic sperm injection (ICSI) is theoretically essential. On the other hand, if HZI is normal or the fertilization ability is high, PCT results are reviewed. For a normal PCT, timed intercourse is recommended, while IUI is recommend for an abnormal PCT. However, if IUI fails after a few attempts, then assisted reproductive technologies (ART) are considered, but since the HZA rules out fertilization failure, ICSI is not needed [16].
The HZA is an efficient assay for assessment of the zona binding ability of sperm. Hence, we examined whether or not D‐IBT findings can predict fertilization failure. The results showed that, regardless of the Ig class, fertilization failed only in patients with a D‐IBT value ≥80 % [16].
As for immunologically infertile females with ASA, Kobayashi et al. [19] reported that those with a quantitative SIT (SI50: 50 % sperm immobilization unit titer) higher than 10 did not conceive by IUI except when treated with in vitro fertilization‐embryo transfer (IVF‐ET), while those with SI50 titers lower than 10 could conceive by IUI. Since then, a cut‐off value of 10 for the SI50 titer has been used when clinical decisions about treatment are made.
In conclusion, with respect to standardization of the D‐IBT cut‐off value, the value of 20 % proposed by Bronson et al. [10] seems to be more appropriate than that of 50 % recommended by the WHO [12]. Evaluation of the D‐IBT cut‐off value should be useful for decision‐making in infertile couples regarding whether they have a chance to conceive through timed intercourse. Further studies are required to clarify whether there is a D‐IBT cut‐off value for IVF.
Acknowledgments
We express special thanks to Mr. Hiromichi Tsunoda and Mrs. Chieko Yamaguchi (Center for Reproductive Medicine, Jichi Medical University Hospital) for their technical support and useful suggestions.
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