1 Service dHistologie-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 dInformatique Médicale, CHU Saint-Louis, Assistance Publique Hôpitaux de Paris, 75475 Paris cedex 10, France
4 To whom correspondence should be addressed at: Service dHistologie-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
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Abstract |
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Key words: IVF/fertilization rate/pregnancy rate/spermzona pellucida binding assay/sperm motility
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Introduction |
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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.
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Materials and methods |
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Sperm preparation
Semen samples of patients were collected by masturbation after 25 days of sexual abstinence: (i) before IVF therapy for the spermZP binding test; and (ii) at the time of oocyte retrieval. For the spermZP 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, 1999
). Purified sperm from fertile donors were stained with fluorescein isothiocyanate (FITC) (104 g/ml) in a glucose (102 g/ml), KOH (1.12x104 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., 1988
). 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 1820 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.
SpermZP 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). 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% glycerolPBS 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 spermZP 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 Fishers 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 spermZP 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 spermZP binding index was <0.7. This threshold was selected using an ROC curve analysis, as maximizing the index of Youden (1950)
, 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., 1991
; Hayden and Brown, 1999
).
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).
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Results |
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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 spermZP 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 spermZP 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: 0.640.88) 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 spermZP binding index was found to maximize the Youdens index, with a sensitivity of 79% (exact 95% CI: 6589%) and a specificity of 63% (exact 95% CI: 4479%). This threshold was used in our study and the test was considered as negative when spermZP 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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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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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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Discussion |
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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, 1982). 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 spermZP 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 spermZP 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 spermZP 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., 1994
; Oehninger et al., 1997
).
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 spermZP 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., 1997). This suggest that, for patients with mild male factor infertility, the link between spermZP 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 spermZP binding and poor fertilization rates during IVF had normal semen analysis, according to WHO criteria (Liu and Baker, 2000
). Nevertheless, the spermZP 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 HZAs ability to predict oocyte fertilization outcome when associated with semen parameter abnomalies (Burkman et al., 1988; Liu et al., 1989
; Oehninger et al., 1989
, 1991
, 1997
, 2000
; Liu and Baker, 1992
; Franken et al., 1993
; Gamzu et al., 1994
).
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., 1988; Oehninger et al., 1989
, 1991
, 1997, 2000
; Franken et al., 1993
; Gamzu et al., 1994
) 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% (samples prevalence) to 24% (1 PPV). This finding clearly showed that, within the limits of our methodology, spermZP 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., 1991
; Hayden et al., 1999
).
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), 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 spermZP binding, which seemed to be predictive of a fertilization rate >20% in patients with normal semen parameters. Similarly, Liu et al. (2004)
showed an increased capacity to predict fertilizing ability of normal sperm but their additional tests, such as spermZP-induced acrosome reaction, seemed to us more difficult to perform than motility.
We conclude that our test, using spermZP 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 spermZP 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 1.072.59), 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.
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Submitted on January 6, 2005; resubmitted on April 27, 2005; accepted on April 29, 2005.
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