World Health Organization grade ‘a’ motility and zona-binding test accurately predict IVF outcome for mild male factor and unexplained infertilities

C. Sifer1, T. Sasportes1, V. Barraud1, C. Poncelet2, J. Rudant3, R. Porcher3, I. Cedrin-Durnerin2, B. Martin-Pont1, J.N. Hugues2 and J.P. Wolf1,4

1 Service d’Histologie-Embryologie-Cytogénétique, Laboratoire de Biologie de la Reproduction,2 Service de Médecine de la Reproduction, Hôpital Jean Verdier, Assistance Publique – Hôpitaux de Paris, 93140 Bondy and 3 Service de Biostatistique et d’Informatique Médicale, CHU Saint-Louis, Assistance Publique – Hôpitaux de Paris, 75475 Paris cedex 10, France

4 To whom correspondence should be addressed at: Service d’Histologie-Embryologie-Cytogénétique-Laboratoire de Biologie de la Reproduction, Hôpital Jean Verdier, 93140 Bondy, France. E-mail: jean-philippe.wolf{at}jvr.ap-hop-paris.fr


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The aim of this study was to determine the pronostic value of a sperm–zona pellucida (ZP) binding assay, combined with World Health Organization (WHO) grade ‘a’ sperm motility on the day of the IVF attempt, to predict sperm fertilizing ability in unexplained and moderate male factor infertilities. METHODS: In total, 84 couples (64 unexplained infertility; 20 male factor) underwent both a sperm–ZP binding assay and an IVF attempt, irrespective of the test’s result. The test was negative when grade ‘a’ motility was #5% and/or the ZP binding index was <0.7. Fertilization and pregnancy rates were related to the test’s results. RESULTS: Thirty-one patients had a negative test (group N) and 53 a positive test (group P). A difference was observed concerning the fertilization rate [median (range): 0 (0–75%) versus 50 (0–100%); P = 0.0001] and the number of cycles with fertilization rate <20% (65 versus 23%; P = 0.0002) between groups N and P respectively. In the group of unexplained and male factor infertilities, the test showed a sensitivity of 83 and 60%, specificity of 50 and 90%, positive predictive value of 76 and 86%, and negative predictive value of 61 and 69% respectively. CONCLUSION: Sperm–ZP binding test, combined with WHO grade ‘a’ motility assessment, is relevant to prevent IVF fertilization failures in unexplained infertility and, most particularly, in moderate male factor infertility.

Key words: IVF/fertilization rate/pregnancy rate/sperm–zona pellucida binding assay/sperm motility


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Binding of mammalian sperm to the zona pellucida (ZP) and the induction of the acrosome reaction are prerequisites for successful oocyte fertilization. In standard IVF, sperm function is essential for normal fertilization. Sperm must be able to bind to the ZP, undergo the acrosome reaction, penetrate the ZP and fuse with the oolemma before fertilization takes place (Yanagimachi, 1994Go; Wassarman, 1999Go). In contrast, most sperm functions are not required for fertilization in ICSI since this technique bypasses the ZP and oolemma by injecting a single spermatozoon directly into cytoplasm of the oocyte. Therefore, the decision to treat one patient with either IVF or ICSI is mostly dependent on evaluation and assessment of sperm function. However, conventional semen analyses do not provide accurate information about sperm fertilizing ability. This has been shown especially in unexplained or male factor infertilities, since many patients with subtle sperm defects cannot be detected. Failed fertilization occurs in 5–10% of IVF cycles and 2–3% of ICSI cycles (Mahutte and Arici, 2003Go). In our laboratory, a 0% fertilization rate was observed in 8.9% of IVF cycles and 3.7% of ICSI cycles (unpublished data). Furthermore, a fertilization rate <20% is considered by most French IVF centres, and also those in other countries (Miller et al, 1998Go; Benadiva et al., 1999Go; van der Westerlaken et al., 2005Go), as an indication to perform an ICSI at the subsequent IVF attempt. Many studies reported a statistically significant decrease of oocyte fertilization rates during IVF attempts occuring in unexplained (Mahadevan et al.Go, Mackenna et al., 1992Go; Ruiz et al., 1997Go; Hull et al., 1998Go; Omland et al., 2001Go) and moderate male factor infertilities (Plachot et al., 2002Go; Tournaye et al., 2002Go) indicating an increased risk of complete fertilization failure in these cases. Furthermore, we analysed in our laboratory the IVF outcome related to grade ‘a’ sprem motility (rapid progressive motility), determined according to World Health Organization guidelines (WHO, 1999Go). We observed that grade ‘a’≤5% was clearly linked with a poor fertilization rate (<20%) (unpublished data). Hence, failed fertilization could result from impaired sperm, oocyte deficiencies, or defects in the in vitro sperm/oocyte culture medium. In the IVF setting, most cases are caused by male factor deficiencies, whereas failure of oocyte activation is the most common cause of failed fertilization after ICSI. Thus, more advanced sperm function tests are required to detect sperm defects that may lead to fertilization failure in standard IVF, so that patients could be directed to an ICSI programme. However, they need to be the most relevant since conventionnal IVF will be preferentially done rather than ICSI because of uncertainties concerning its safety. Indeed, the risks of disturbing the spindle during introduction of the pipette (Asada et al., 1995Go; Blake et al., 2000Go; Dumoulin et al., 2001Go), the possible asynchronized decondensation of sperm chromosomes (Terada et al., 2000Go), the reduced capacity for blastocyst formation in vitro, particularly in cases of poor sperm motility and morpholology (Griffiths et al., 2000Go; Miller and Smith, 2001Go), the lower survival (Schnorr et al., 2001Go) and implantation rates of frozen–thawed embryos originating from ICSI compared to those of embryos obtained by IVF (Macas et al., 1998Go), the malformations and chromosomal abnormalities observed in the fetus (Bonduelle et al., 1999Go; Wennerholm et al., 2000Go) and the increased risk of transmission of infertility and other genetic defects to the offspring are still open to debate (Chang et al., 1999Go). A previous study showed that both the mean number of sperm bound to ZP at day 1 of IVF attempts and the percentage of motile sperm were significantly higher when fertilization rates were >0% (Liu and Baker, 1992Go). Furthermore, although the standard semen analysis has limited ability to predict fertilization failure, rapid and linear progressive motility (Bollendorf et al., 1996Go; Donnelly et al., 1998Go; Verheyen et al., 1999Go) classified as grade ‘a’ according to WHO (1999) criteria and sperm–ZP binding ratios, determined either by the hemizona assay (HZA) (Burkman et al., 1988Go; Oehninger et al., 1989Go, 1991Go, 1997Go, 2000Go; Franken et al., 1993Go; Gamzu et al., 1994Go) or tests using entire ZP (Liu et al., 1988Go, 1989, 1990Go, 2004Go; Liu and Baker, 1992Go, 2000Go), provide increased capacity to avoid this outcome.However, all these previous studies have focused on the relationship between HZA and standard semen parameters. They compared the fertilization rate during an IVF attempt between patients with oligoasthenoteratozoospermia (OAT) and those with normal sperm, used as controls. These reports were not specifically interested in the ability of the HZA to predict fertilization outcome, especially according the subgroups of IVF indications such as moderate OAT or unexplained infertilities.

We decided to set up a zona binding test to predict IVF outcome in couples known to be at risk of fertilization failure. This easy-to-perform test was done using unfertilized oocytes that had been donated by patients after informed consent. As the sperm parameters can vary from day to day, we added the grade ‘a’ motility value of the sperm the day of the IVF attempt. Thus, the aim of this study was to determine how rapid-linear progressive sperm motility the day of IVF attempt and ZP binding test results, taken together, could be used as an additional discriminant diagnostic test to predict in vitro sperm fertilizing ability specifically in mild male factor and unexplained infertilities.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Eighty-four women (mean age = 33.7 ± 4.6 years) scheduled for IVF were included in this study. The causes of infertility in these patients were unexplained in 64 and male factor in 20 cases. Controlled ovarian stimulation (COS) was done using a classical long protocol. Pituitary down-regulation was achieved after i.m. administration of GnRH agonist (triptorelin; Ipsen-Biotech, Paris, France), started at the mid-luteal phase of the previous cycle, and was assured by serum estradiol levels at ≤30 pg/ml. COS was performed with s.c. administration of recombinant human FSH (75 IU per ampoule) (Gonal F®; Serono, Boulogne, France) started with 2 ampoules per day and adapted from day 6 on an individual basis according to the ovarian response as assessed by sequential transvaginal ultrasonography and serum estradiol measurements. hCG (10 000 IU; Organon, Saint Denis, France) was administered i.m. when a consistent rise in serum estradiol concentration was associated with the presence of ≥4 follicles ≥16 mm in diameter. Transvaginal oocyte retrieval was scheduled 36 h after hCG administration under ultrasound guidance and standard IVF techniques were performed. Luteal support was initiated on day 1 after oocyte retrieval with 400 mg/day of progesterone. In the absence of ovarian hyperstimulation syndrome (OHSS) risk, 2500 and 1500 IU of hCG were administered 1 and 3–6 days after oocyte retrieval respectively. This study was approved by the Regional Ethics Committee and each couple included in this study was asked to sign an approval consent form.

Sperm preparation
Semen samples of patients were collected by masturbation after 2–5 days of sexual abstinence: (i) before IVF therapy for the sperm–ZP binding test; and (ii) at the time of oocyte retrieval. For the sperm–ZP binding test, semen samples of 84 proven fertile donors, known to have fathered a pregnancy within 2 years or to provide a fertilization rate ≥50% in a recent conventional IVF attempt, were also collected and used as controls. All had signed an informed consent form. Discontinuous PureSperm® (Nidacon International, Gothenburg, Sweden) separation (90 and 45% layers) was performed on all semen samples and the 90% layer was washed in B1 medium (CCD, Paris, France). Following the procedure, sperm concentration and motility were assessed before and after migration, according to WHO (1999) criteria. Sperm morphology and vitality were performed only before migration. Patients were classified in the male factor infertility group when: (i) grade ‘a’ motility was <25%; (ii) sperm concentration was <20x106/ml; and/or (iii) normal morphological forms of sperm were <30% at the time of routine sperm examination. For all couples, a conventionnal IVF attempt was indicated as the concentration of selected motile sperm was >0.5x106 per treated ejaculate (Arrêté du 12 Janvier, 1999Go). Purified sperm from fertile donors were stained with fluorescein isothiocyanate (FITC) (10–4 g/ml) in a glucose (10–2 g/ml), KOH (1.12x10–4 g/ml) phosphate-buffered saline (PBS) buffer, before sperm concentration and motility were determined. FITC has previously been shown not to impair sperm motility or fertilizing ability in the absence of UV light (Liu et al., 1988Go). Briefly, 200 µl of purified sperm were gently mixed with 100 µl of FITC solution, then incubated at 37°C for 15 min. Sperm were washed with 5 ml of PBS and centrifuged at 600 g for 5 min. Supernatants were discarded and 300 µl of embryo culture media were added to pellets.

IVF procedures
The IVF attempts were performed irrespective of the test value. Gamete insemination and embryo culture were performed in 20 µl equilibrated drops of commercially available culture media overlaid with paraffin oil (Medicult, Lyon, France) at 37°C in a 5% CO2 atmosphere as described below. Oocytes were inseminated with a concentration of 150 000 progressive motile sperm cells/ml and checked 18–20 h later. Fertilization was assessed by the presence of two pronuclei and two polar bodies. Embryos were cultured for 2 more days, with development monitored daily. We recorded: the mean number of retrieved oocytes; the percentage of metaphase II stage oocytes determined at day 1, including oocytes with one polar body or exhibiting one, two or three pronuclei; the fertilization and pregnancy rates. The number of cycles with a fertilization rate <20% was also recorded.

Sperm–ZP binding test
In order to evaluate its prognostic value, this test was performed within 3 months prior to the IVF attempt. Salt-stored or fresh human oocytes were used: (i) at the metaphase I stage; (ii) reaching metaphase II stage after in vitro maturation; or (iii) unfertilized after ICSI. This test was performed according to an adaptation of that previously described by Liu et al. (1988)Go. Briefly, four intact ZP were rinsed and incubated separately in 20 µl culture media droplets. Individual ZP were inseminated with a mixture of 4000 FITC-stained control and 4000 unstained test motile sperm under classical IVF culture conditions. After an 18 h incubation, each ZP was washed to remove loosely bound sperm, and mounted on a glass slide in a 5 µl 5% glycerol–PBS droplet. Each ZP was observed at x400 magnification: (i) first, under a fluorescent light microscope (Axiophot; Zeiss, Le Peck, France) measuring the number of FITC-stained bound sperm resulting from each control; and (ii) subsequently under a phase-contrast microscope (Optiphot-2; Nikon, Champigny sur Marne, France) determining the number of total bound sperm resulting both from controls and patients. A sperm–ZP binding index (number of sperm bound for patients/controls) was calculated.

Statistical analysis
Data are presented as counts and percentages for categorical variables and median (range) for continuous variables.

A successful fertilization was defined by a fertilization rate ≥20%. The association of indication and semen parameters with a successful fertilization was evaluated by comparing the distribution of each parameter according to the success of fertilization, using either Fisher’s exact tests or Wilcoxon rank-sum tests. Logistic regression models were used to estimate odds ratios and areas under the receiver operating characteristics (ROC) curve (AUC), as a measure of discrimination between unsuccessful and successful fertilization. To combine the sperm–ZP binding index with WHO grade ‘a’ motility on the day of IVF attempt, we considered a test as negative when grade ‘a’ motility value was ≤5% and/or the sperm–ZP binding index was <0.7. This threshold was selected using an ROC curve analysis, as maximizing the index of Youden (1950)Go, defined as the difference between the true-positive and the false-positive rates, i.e. sensitivity + specificity – 1. Additionally, positive predictive values (PPV; i.e. ability of a positive test to predict successful fertilization) and negative predictive values (NPV; ability of a negative test to predict a poor fertilization <20%) were computed, together with positive likelihood ratios [LR+ = sensitivity divided by (1 – specificity)], which indicate the impact of the proposed test on the post-test probability of successful IVF (Simel et al., 1991Go; Hayden and Brown, 1999Go).

All tests were two-sided with P < 0.05 considered significant. Analyses were carried out using R 2.0.1 software (The R Development Core Team).


    Results
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 Introduction
 Materials and methods
 Results
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 References
 
The semen parameters the day of the IVF attempt, sperm–ZP binding index, and fertilization outcome of the 84 patients and their assisted reproductive treatment indication are summarized in Table I. A statistically significant difference was shown between patients with male factor and unexplained infertility with a decrease in all semen parameters and the fertilization rate. However, sperm–ZP binding index was not significantly different between these two groups, although a decrease was shown in the male factor group. A fertilization rate <20% (38%) occurred in 32 of the 84 IVF attempts, 22 from the 64 unexplained infertilities (34%) and 10 from the 20 moderate male factors (50%) (non-significant).


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Table I. Semen parameters on the day of IVF attempt, sperm–zona pellucida (ZP) binding index and fertilization rate in IVF

 

The ability of the different parameters considered to predict a fertilization rate ≥20% is presented in Table II. Among the different semen characteristics, only grade ‘a’ motility and the sperm–ZP binding index were found to be significantly associated with a successful fertilization. Sperm concentration was found to be lower in cases with a fertilization rate <20%, but with moderate discriminative properties, as the AUC was limited to 0.62. Indication and sperm morphology did not seem to be able to predict a successful fertilization in our study. The ROC curve, computed for varying thresholds over the range of the sperm–ZP binding index, according to a value of grade ‘a’ motility ≤5%, is presented Figure 1. The area under the curve (AUC) was 0.76 (95% CI: 0.64–0.88) indicating fair performance of the test to predict sperm fertilizing ability. Results also showed an improved predictive performance as compared to grade ‘a’ motility or ZP-binding index alone (Table II). A threshold of 0.7 for the sperm–ZP binding index was found to maximize the Youden’s index, with a sensitivity of 79% (exact 95% CI: 65–89%) and a specificity of 63% (exact 95% CI: 44–79%). This threshold was used in our study and the test was considered as negative when sperm–ZP binding index was <0.7 and/or grade ‘a’ motility was ≤5%, thus defining two groups: group N (negative test) or P (positive test).


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Table II. Ability of semen characteristics to predict fertilization rate

 


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Figure 1. Receiver operating characteristic (ROC) curve for prediction of fertilization outcome using combined zona pellucida (ZP) binding index with World Health Organization grade ‘a’ motility value in the sperm of patients. The test was considered as negative when (i) grade ‘a’ motility was <5% in the sperm of tested patients or (ii) the sperm–ZP binding index was inferior to a given threshold. The point with sensitivity 100% and specificity 0% was obtained by relaxing the condition grade ‘a’ motility value <5%.

 

As summarized in Table III, we showed a statistically significant difference between groups N and P concerning: (i) the distribution of infertility indications; (ii) fertilization rates and number of cases with fertilization rate <20%; and, as a consequence, (iii) number of embryos transferred per ovarian puncture; and (iv) clinical pregnancy rate.


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Table III. Characteristics of patients from groups N (negative test) and P (positive test) according to a 0.7 threshold

 

Sensitivity, specificity, PPV and NPV of the test according to IVF indications are given on Table IV. Interestingly, the test showed a specificity and a PPV of 50 and 76% for unexplained infertility and 90 and 86% for moderate male factor infertility. According to our results, the overall false-positive rate was 23%, 14% in moderate male factor and 24% in unexplained infertilties. The overall false-negative rate was 35%, 31% in moderate male factor and 39% in unexplained infertilities. The overall correct predictive ability of our combined test for fertilization outcome was 72.6% (75% in male factor and 71.9% in unexplained infertility). The LR+ were 1.67 for unexplained infertilities and 6.0 for moderate male factor.


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Table IV. Sensitivity, specificity and positive and negative (PPV/NPV) predictive values of the proposed test according to the indication of infertility

 


    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Previous studies have shown that unexplained and mild male factor infertilities were associated with an increased risk of poor fertilization outcome during a conventionnal IVF attempt (Mahadevan et al., 1983Go; Mackenna et al., 1992Go; Ruiz et al., 1997Go; Hull et al., 1998Go; Omland et al., 2001Go; Plachot et al., 2002Go; Tournaye et al., 2002Go). Furthermore, rapid and linear progressive motility (Bollendorf et al., 1996Go; Donnelly et al., 1998Go; Verheyen et al., 1999Go) and sperm–ZP binding ratios (Burkman et al., 1988Go; Liu et al., 1988Go; Liu et al., 1989Go, 1990, 2004; Oehninger et al., 1989Go, 1991Go, 1997, 2000; Liu and Baker, 1992,Go 2000Go; Franken et al., 1993Go; Gamzu et al., 1994Go) have already separately shown their interest in predicting fertilizing ability of semen samples. However, a poor fertilization rate was defined as <20% in our laboratory, as is the case for most French IVF centres, representing an indication to perform ICSI. This represents a different approach of our study compared to all previous studies relating HZA index to fertilization rate during IVF, since a fertilization rate was considered as poor when it was <60–66% (Burkman et al., 1988Go; Oehninger et al., 1989Go, 1991Go, 1997, 2000; Franken et al., 1993Go; Gamzu et al., 1994Go). Furthermore, Liu et al. (2001)Go used additional tests to sperm–ZP binding, such as sperm–ZP-induced acrosome reaction, to predict a poor fertilization rate, defined as ≤30%, in infertile men with normal semen analysis.

We performed ROC curve analysis to assess the best threshold of our test to use. The AUC obtained from our study indicated fair performance of the test in predicting sperm fertilizing ability. Actually, values of the AUC between 0.7 and 0.8 or values >0.8, indicate fair and high discriminative properties, respectively. (Hanley and McNeil, 1982Go). Although the specificity of the test is not particularly high per se, the selected 0.7 threshold corresponded to the highest possible specificity over the range of sperm–ZP binding index. To avoid attempting IVF in patients with a poor oocyte fertilization rate, the number of false positives had to be reduced and the most specific test was preferred. This confirmed the selection in our study of the 0.7 threshold to conclude the test as negative or positive with a valid statistical methodology.

The aim of our study was to evaluate the ability of a sperm–ZP binding assay combined with WHO grade ‘a’ motility on the day of the IVF attempt, to avoid poor oocyte fertilization rate for IVF indications known to be at risk of fertilization failure. We used a linear logistic model to compare all classical semen parameters and infertility indications. As a result, only grade ‘a’ motility and the sperm–ZP binding index were found to be significantly associated with a successful fertilization. Furthermore, results also showed an improved predictive performance of our combined test as compared to ‘a’ motility or ZP binding index alone. Furthermore, our study showed that the number of ‘a’ sperm (motility ≤5%) was statistically significantly increased when a fertilization rate <20% was observed, confirming: (i) previous unpublished data from our laboratory; and (ii) previous studies which clearly reported that motility plus HZA index is a better predictive model of fertilization in vitro than HZA index alone (Gamzu et al., 1994Go; Oehninger et al., 1997Go).

In spite of a significant decrease of classical semen characteristics and fertilization rates in the group of male factor infertilities, compared with those of unexplained infertilities, we did not show a significant difference in the sperm–ZP binding index between these two groups of infertility indications. This result is in contradiction with a previous study which showed a decrease of the HZA index in abnormal semen parameters (Oehninger et al., 1997Go). This suggest that, for patients with mild male factor infertility, the link between sperm–ZP binding index and conventional semen analyses is not well defined. Indeed, we hypothesized that all semen samples, with similar standard semen parameter values, resulting from men with a mild male factor infertility as well as an unexplained infertility, have not the same ability to bind to ZP and so, as a consequence, to fertilize an oocyte during an IVF attempt. This is supported by a previous study which showed that ~50% of the patients with poor sperm–ZP binding and poor fertilization rates during IVF had normal semen analysis, according to WHO criteria (Liu and Baker, 2000Go). Nevertheless, the sperm–ZP binding ratio, in our study, was statistically significantly decreased when a fertilization rate <20% was observed; this, independently of the IVF indication. This leads to the ability of this index to predict poor fertilization, and fully justifies to perform it as in our study, whatever the IVF indication was, in order to detect a fertilization failure risk.

To fulfil our main objective, we classified patients according to our combined test results. We report a statistically significant decrease in the fertilization rates and, as a consequence, the mean number of embryos transferred/puncture and clinical pregnancy rates in the group of patients with a negative test. Interestingly, we showed that the distribution of infertility indications in this group was represented by male factor significantly more than in the group of patients with a positive test, with an increase in the fertilization rate <20%. This suggests that our test better predicts successful fertilization during an IVF attempt for the subgroup with male factor infertility than for those with unexplained infertility. This point corroborates all previously reported data analysing the HZA’s ability to predict oocyte fertilization outcome when associated with semen parameter abnomalies (Burkman et al., 1988Go; Liu et al., 1989Go; Oehninger et al., 1989Go, 1991Go, 1997Go, 2000Go; Liu and Baker, 1992Go; Franken et al., 1993Go; Gamzu et al., 1994Go).

The reliability of a given diagnostic test is governed by its sensitivity and specificity. In our study, we wanted to determine the most specific test to predict the fertilzation outcome since false-positives needed to be minimized to prevent a fertilization failure occurring after a positive test. However, false-negatives need to be kept to a minimum to avoid performing unnecessary ICSI. Indeed, uncertainties concerning the safety of ICSI suggest that it should be used cautiously and judiciously. Here, we reported that our test could be of great benefit in cases of mild male infertilities. In this subgroup, the specificity was increased to 90% with a PPV of 86% that led us to perform conventional IVF rather than ICSI when the test is positive. This finding confirmed all previous studies using HZA to predict fertilization outcome, with similar specificity (Burkman et al., 1988Go; Oehninger et al., 1989Go, 1991Go, 1997, 2000Go; Franken et al., 1993Go; Gamzu et al., 1994Go) but with a more relevant definition of a poor fertilization outcome during IVF in our study (<20% rather than <60%). However, we observed that our test reached a specificity of only 50% in unexplained infertilities. Despite its specificity of 50%, the test proposed yielded a PPV of 76%. Although this predictive value is less than that obtained for mild male infertilities, using the test in this subgroup of patients would enable a decrease in the rate of IVF failure by approximately one-third, from 34% (sample’s prevalence) to 24% (1 – PPV). This finding clearly showed that, within the limits of our methodology, sperm–ZP binding index combined with grade ‘a’ motility on the day of the IVF attempt could be used to predict fertilization outcome in this kind of IVF indication. However, for unexplained infertilities, the LR+ was estimated at 1.67, which indicates a small but statistically significant impact on the post-test probability of successful IVF, whereas the LR+ was 6.0 in mild male infertilities, which represents a better, though moderate, impact on this probability (Simel et al., 1991Go; Hayden et al., 1999Go).

HZA used by most of the workers interested by this topic is known to be difficult to perform. Indeed, each ZP needs to be cut into equal hemispheres by micromanipulation. Subsequently, one droplet exposes a hemizona to abnormal sperm, while the control droplet contains the matching hemizona and sperm from normal semen. This generates a potential risk of heterogeneous binding of tested sperm since exposed hemizona surfaces are not strictly identical. Here, we used a technique much easier to perform, developed previously by Liu et al. (1988)Go, using entire untreated ZP from unfertilized and/or in vitro matured oocytes, leading to more homogeneous results since sperm-binding from tested patients and controls occcurred on the same ZP surface. Furthermore, we combined grade ‘a’ sperm motility with sperm–ZP binding, which seemed to be predictive of a fertilization rate >20% in patients with normal semen parameters. Similarly, Liu et al. (2004)Go showed an increased capacity to predict fertilizing ability of normal sperm but their additional tests, such as sperm–ZP-induced acrosome reaction, seemed to us more difficult to perform than motility.

We conclude that our test, using sperm–ZP binding index combined with WHO grade ‘a’ linear-progressive motility, is an excellent predictor of sperm fertilizing potential in cases of mild male factor infertility and should be incorporated as a functional test to direct patients to IVF or ICSI at their first attempt. Even though we obtained statistical significance, the relevance of our test in mild male factor infertilities needs to be confirmed on a larger, external series of patients. Indeed, we thought that it would not be ethical still to perform IVF rather than ICSI, especially in cases of male factor presence, when a sperm–ZP binding test was determined as negative. Furthemore, PPV of 76% for unexplained infertility with normal standard sperm parameters, and the positive LR of 1.67 (95% CI 1.07–2.59), allowed us to use this test in these cases. This original last result leads us to recommend the performance of this test in unexplained infertilities also. Indeed, we now routinely use this test in unexplained or mild male factor infertilities to manage our patients. We have observed an overall decrease of ~50% concerning numbers of fertilization rates <20% when the test was positive.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on January 6, 2005; resubmitted on April 27, 2005; accepted on April 29, 2005.





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