Human Reproduction, Vol. 16, No. 1, 138-144,
January 2001
© 2001 European Society of Human Reproduction and Embryology
Clinical importance of zona pellucida-induced acrosome reaction and its predictive value for IVF
1 Andrology Laboratory, Drs du Buisson and partners, Pretoria and 2 Department of Obstetrics and Gynaecology, University of Stellenbosch, Tygerberg, South Africa
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The study aimed to establish zona pellucida induced acrosome reaction response (ZIAR) among 35 couples with normal and G-pattern sperm morphology and repeated poor fertilization results during assisted reproduction treatment. ZIAR tests were performed using 0.25 zona pellucida/µl co-incubated with spermatozoa for 60 min. Acrosome reactions were measured with FITC–PSA staining, and expressed as the difference between stimulated and unstimulated (spontaneous) sperm populations. Results were compared with IVF rates of metaphase II oocytes. Interactive dot diagrams divided the patients into two groups, i.e. ZIAR <15% and ZIAR > 15%, with mean fertilization rates of 49 and 79% respectively. The sensitivity and specificity for ZIAR results versus fertilization were 93 and 100% respectively. The area under the curve was 99% and the 95% confidence interval did not include 0.5 which implies that the ZIAR test is able to predict fertilization failure among IVF patients. In conclusion, the ZIAR test has diagnostic potential since it can assist the clinician to identify couples that will benefit from intracytoplasmic sperm injection therapy.
Key words: acrosome reaction/failed fertilization/zona pellucida
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The astounding success rates achieved by intracytoplasmic sperm injection (ICSI) (Van Steirteghem et al., 1993
), emphasized the need to refine sperm function evaluation. This is particularly true in cases of profound male factor infertility and therefore contemporary andrology laboratories should be able to select the most appropriate form of treatment for each couple (Oehninger et al., 1991, 1997; Kruger and Coetzee, 1999). Moreover, the past 10–15 years have brought not only an explosion in the number of laboratory tests for human sperm functions, but also the belief among many clinicians that sperm function testing is now irrelevant due to the advances in IVF technology. Ideally, an accurate and inexpensive test would be used to determine which men require ICSI and which do not. The current success with ICSI has stifled rather than stimulated the search for such a test. Also the history of abuse of sperm function tests has not helped; far too many have been heralded by their advocates as the best diagnostic approach and clinicians have been too quick to pronounce men fertile or infertile on the basis of a single favourite test.
At a consensus workshop in advanced andrology (Consensus Workshop, 1996) it was suggested that because of their validation and unquestioned clinical value, the homologous sperm–zona pellucida binding tests should be incorporated in the advanced stages of the work-up. Sperm–zona binding reflects multiple sperm function events depicting spermatozoa–zona pellucida interaction, i.e. completion of capacitation as manifested by the ability to bind to the zona pellucida and to undergo ligand-induced acrosome reaction. A diagnostic test underlining the ability of the spermatozoa to undergo the acrosome reaction in response to homologous zona pellucida would be a valuable additional tool in the male fertility work-up schedule (Franken et al., 1990; Oehninger et al., 1992; Liu and Baker, 1992).
During the consensus workshop it was agreed that better standardization of the currently-used acrosome reaction techniques should be implemented prior to their introduction as a routine clinical tool. The present report is an effort towards development and standardization of the acrosomal response as a clinical tool in the assisted reproductive programme. The most widely utilized method is the acrosome reaction (AR) ionophore challenge test (ARIC), during which the acrosome reaction is induced by calcium ionophore and then identified and defined by lectins in combination with indirect immunofluorescence (Consensus Workshop, 1996). The rationale for the development of this test was manifested in the precise timing of acrosomal response (Tesarik, 1989, 1996; Cummins et al., 1991). Guidelines for the interpretation of the acrosome response during the ARIC test defined AR prematurity in cases where >20% of spermatozoa show spontaneous AR after 3 h incubation under capacitating conditions. AR was reported to be normal if >15% difference existed among spontaneous and induced AR; <10% difference is abnormal and indicates a possible impairment of fertilization (WHO, 1999).
The ARIC test as well as the concept of acrosomal inducibility (Henkel et al., 1993, 1998) was used as a predictor of sperm fertilizing ability as compared with tests that simply measure the frequency of spontaneous AR.
The zona pellucida (ZP), both intact and solubilized, has been demonstrated to be a powerful and physiological inducer of the AR (Cross et al., 1988; Florman et al., 1989; Bielfeld et al., 1994; Liu and Baker, 1994; Franken et al., 1996, 1997). During fertilization, AR failure can be caused by multiple factors, such as: (i) inadequate sperm capacitation; (ii) an inability of the sperm membrane to undergo specific structural–functional changes after binding to the ZP; or (iii) an impaired capacity of the ZP of a specific oocyte to induce the acrosomal cascade.
This study aimed to evaluate and establish, among non-male factor couples with repeated `poor' or no fertilization, their AR to solubilized human ZP during IVF treatment. The results will be used to define: insufficient AR, i.e. where the difference between spontaneous and zona induced AR (ZIAR) is <15%; and normal AR response where the difference between spontaneous AR and induced AR is 
15%.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Patients
All patients in the study signed an informed consent form after Institutional Review Board approval was obtained. The inclusive criteria for patients accepted into the study were: (i) females diagnosed as tubal factors; (ii) having a normal FSH/LH ratio on day 3 of the menstrual cycle (<10 U/l); (iii) producing three or more pre-ovulatory metaphase II oocytes at retrieval; (iv) having husbands with normal sperm parameters as well as either normal or G-pattern (4–14% normal forms) morphology; and (v) a history of repeated poor (<30% metaphase II oocytes fertilized) fertilization or complete fertilization failure, during two previous cycles. The mean (±SD) age and number of oocytes retrieved per patient was 30 ± 3 years and 8.3 ± 2 oocytes respectively. During the 12 month study period, a total of 35 from 382 (9%) couples adhered to the above inclusive criteria.
Ovulation induction
Ovulation induction protocols were adapted to individual needs. Thirty-three patients in the study were treated with a `short' protocol, while two who did not respond well on the short protocol were treated with the `long' protocol. The long protocol started with 0.5 mg/day Buserelin® [gonadotropin releasing hormone (GnRH) analogue; Suprefact, Hoechst IHD, Johannesburg, Republic of South Africa] or Lucrin® (GnRH analogue; Abbott IHD, Johannesburg, Republic of South Africa) on day 21 of the menstrual cycle and continued until the day of human chorionic gonadotrophin (HCG) administration. Oestradiol was checked 14 days after the first GnRHa injection. If oestradiol concentrations were <100 pg/ml, the GnRHa dose was reduced thereafter to 0.2 mg/day until HCG injection. Patients are given 225 IU (3 ampoules) Gonal F® (recombinant FSH; Serono Pty Ltd, Centurion, Republic of South Africa) daily for 6 days. In the short protocol patients received 0.3 mg GnRHa and Gonal F® (3 ampoules) at the same time on day 2 of the cycle. These hormones were administered daily until two dominant follicles of 18 mm diameter were ready on ultrasound, after which ovulation was triggered with 10 000 IU HCG (Profasi®; Serono) administration.
Oocyte evaluation
Metaphase I and II oocytes were identified according to their nuclear maturity status. In metaphase II oocytes a first polar body was present while in metaphase I oocytes no polar body was observed. Following oocyte retrieval, great care was taken to ensure that the evaluation and classification of the oocytes were meticulously carried out by the embryologist. Under microscopic vision, metaphase II oocytes were characterized by their round, even shape and presence of light colour and homogeneous granularity. Metaphase II oocytes were always associated with expanded, luteinized cumuli and `sun-burst' corona radiata. The membranes of the granulosa cells harvested along with the metaphase II oocytes were also luteinized, loosely aggregated and had mature features (Veeck, 1988).
In-vitro fertilization
Metaphase II oocytes were cultured as suggested in the IVF Science Product Manual (Granberg, 1999). Briefly, culture dishes (Falcon 1006®; Becton Dickinson SA Scientific, Randburg, Republic of South Africa) were prepared using 24 h pre-equilibrated culture media (IVF-50; Scandinavian IVF Science Products, Gothenburg, Sweden and universal IVF medium; Medi-Cult, Mollehaven, Jyllinge, Sweden) and Sigma mineral oil (Sigma-M-8410, embryo tested; Sigma-Aldrich, Johannesburg, Republic of South Africa). A total of 3 ml IVF-50 medium was incubated in tissue culture grade plastic tubes (Falcon 2058®; SA Scientific, Randburg, Republic of South Africa) for the final swim-up procedure. Spermatozoa were washed in ASP-100 (Scandinavian IVF Science Products) and layered with 1.5 ml IVF-50 medium. Oocyte insemination was performed with standard sperm concentration of 100 000 motile spermatozoa/ml/oocyte in 3 ml IVF-50 culture medium. On day 1, oocytes were denuded and evaluated by the embryologist for fertilization status.
Rescued intracytoplasmic sperm injection
The oocytes of patients where no fertilization was reported 1 day after aspiration were subjected to rescued ICSI. Prior to injection the unfertilized oocytes were placed in 10 µl HEPES buffered droplets under oil in 1006 Falcon Petri dishes. Rescued ICSI was performed as a therapeutic procedure and before the zona pellucida induced acrosome reaction response (ZIAR) results were available. Briefly, a small volume (2–3 µl) of prepared spermatozoa was deposited in a droplet of polyvinylpyrrolidone (PVP, Medicult®; Harrilabs, Randburg, Republic of South Africa). Narishige micromanipulators mounted on a Nikon inverted microscope were used to perform the sperm injection. The oocyte was stabilized by applying negative suction to a holding pipette (Cook, Brisbane, Queensland, Australia). An individual morphologically normal spermatozoon was immobilized and sucked into a Cook injection pipette. The spermatozoa was injected into the ooplasma and the injected oocyte was then incubated according to the IVF protocol.
Preparation of zonae pellucidae
Solubilization of ZP was performed using 20 oocytes at a time depending on the number of ZIAR tests to be performed. This method was developed during previous reports and results revealed it to be optimal in our laboratory, since (i) the small volumes of solubilized zonae can be stored (7–10 days) at 4°C (Franken et al., 1996, 2000), thus avoiding loss of precious zona material and (ii) since the ZIAR tests were performed blindly on the day of the oocyte retrieval, the use of small volumes of zonae was more practical. Unpublished data from our laboratory showed that the acrosome inducing potential of the different ZP batches did not differ between batches. Unfertilized oocytes (no pronuclei or second polar body) donated by diagnosed male factor couples from the in-vitro fertilization programme were used to induce the AR in the study. Oocytes with <10 spermatozoa attached to the zona pellucida were rinsed in Dulbecco's phosphate buffered saline (DPBS; Sigma-Aldrich). Twenty oocytes were placed in 0.1 ml DPBS in an Eppendorf 0.5 ml micro tube (Micro Tubes, Safe-Lock®; Eppendorf-Netheler-Hinz-GmbH, Hamburg, Germany) at 4°C. The oocytes were washed by centrifugation for 10 min at 600 g. Using a stereoscope, DPBS was then gently aspirated with a glass drawn Pasteur micropipette. DPBS was added and oocytes were centrifuged again. A total of 20 µl acid Tyrode's (T-1788, Tyrode's Solution Acidic, embryo tested; Sigma Aldrich) solution was gently added to the oocyte pellet after removal of the DPBS. The zonae were subsequently dissolved at room temperature (45–60 min) in acid Tyrode's by agitating the zonae solution every 5 min with a glass drawn Pasteur Pipette. The reaction was neutralized by adding 20 µl 1 mmol/l NaOH. Zonae solutions with a concentration of 0.5 ZP/µl were stored for up to 2 weeks at 4°C (Franken et al., 2000). Previous reports indicated that their adequate AR stimulation occurred using micro-volumes of spermatozoa and solubilized zona pellucida. During these studies, serial dilutions of disaggregated human ZP (obtained from post mortem derived prophase I oocytes), i.e. 2.5, 1.25, 0.6 and 0.3 ZP/µl induced 32, 26, 18 and 14% of spermatozoa to acrosome react. Spontaneous AR values during these studies were 10% (Franken et al., 1996, 2000).
Preparation of spermatozoa
The semen from 10 fertile sperm donors (14 samples) were used, during a pilot study, as controls to establish the baseline AR response among a fertile population, not attending an assisted reproductive programme. Spermatozoa from 35 patients taking part in our IVF programme with normal and G-pattern morphology were used. Liquefied semen was washed in ASP-100 (Scandinavian IVF Science Products) for 10 min at 400 g. ASP-100 medium is a modified human tubal fluid culture medium containing standard concentrations of 25 mmol/l sodium bicarbonate supplemented with 10 mg/ml human serum albumin. The sperm pellet was layered with 1.5 ml IVF-50 medium (Scandinavian IVF Science Products) and incubated for 30 min at 37°C and 5% CO2 to achieve a swim-up separation of motile cells. After the incubation period, 1.0 ml of the top sperm suspension was aspirated, a 0.2 ml portion of highly motile spermatozoa was then removed and placed in a plastic tube (Falcon 2058; Becton Dickinson) for the ZIAR. This fraction was then washed once at 400 g for 10 min with DPBS and the pellet was resuspended in 100 µl DPBS, whilst the remaining volume was used for insemination purposes in the IVF laboratory.
ZIAR test
Prior to the study, technician and sperm specimen variation was recorded, by establishing intra- and interassay/technician coefficients of variations (CV) for PSA–FITC (Pisum satiruma agglutinin–fluorescein isothiocyanate) staining during ZIAR testing. Intra-assay and technician variation was determined by evaluating 100 cells on five different microscopic fields (total 500 cells) from the same semen specimen by each of three technicians. Interassay and technician variation was accomplished by evaluating staining by counting 100 cells on slides prepared from five different sperm specimens by the same sperm donor. CVs for both intra- and interassay and intra- and inter-technician values were calculated by dividing the mean with SDx100% for each observation. The inter- and intra-assay as well as inter- and intra-technician CV was <15 and <10% among the slides and three technicians respectively. Results were discarded in cases where the CV for assay and technician exceeded 15 or 10%.
A total of 20 µl of the zonae solution and 20 µl prepared spermatozoa (5x106 spermatozoa/ml) were mixed in a microplate (Greiner Lab and Scientific Equipment, North Riding, Republic of South Africa). The mixture was aspirated into a Hamilton Pipette Tip® (Hamilton, Separations, Republic of South Africa) using a 1.0 ml sterile, non-pyrogenic latex free syringe (Becton Dickinson). The tip containing the spermatozoa–zonae solution was incubated in a Falcon dish (Falcon 3003®; Becton Dickinson) for 60 min at 37°C and 5% CO2. After the incubation period the spermatozoa were expelled onto the glass slide and coded `test' spermatozoa, while `control' spermatozoa were aspirated from the DPBS resuspended sample described above. Pilot studies have indicated that NaOH neutralized acid Tyrode's solution had similar effect on the AR compared with that recorded with DPBS, namely 13.7% ± 2 for neutralized acid Tyrode's compared with 14.6% ± 3 for DPBS (P > 0.5). We therefore used DPBS to resuspend the control spermatozoa in all forthcoming experiments. Slides were air-dried and fixed in 100% ethanol for 24 h. Previous experiments in our laboratory indicated that prolonged exposure of spermatozoa to ethanol during the fixation period increased the sensitivity of the PSA–FITC staining. This allowed us to record AR mediated by lower ZP concentrations (0.3 ZP/µl) generally reported in the literature, i.e. 2 ZP/µl (Liu and Baker, 1996b). Thereafter the spermatozoa were stained for 2 h at room temperature with 30 µg/ml PSA labelled with FITC (L-0770; Sigma- Aldrich). Finally, slides were washed in DPBS and mounted with DPBS. A minimum of 100 spermatozoa were counted under a Nikon fluorescent microscope (Labophot 2, Nikon; IMP, Johannesburg, Republic of South Africa), Filter Ex 465–495 0 with x400 magnification. The following staining patterns were evaluated as acrosome reacted spermatozoa: (i) patchy staining on acrosomal region, (ii) distinct staining in the equatorial region occurring as an equatorial bar and (iii) and no staining observed over entire sperm surface. Since we used only swim-up sperm samples, the motility was in all cases >80%. AR data present the findings recorded for live spermatozoa. Spermatozoa with patchy FITC–PSA staining were classified as a population of spermatozoa where the AR was initiated and all were classified as acrosome reacted. The slides were evaluated on the day of oocyte aspiration prior to the IVF outcome in order to ensure that the AR results were recorded blindly by the technicians. The zona induced AR was calculated as the difference between zona induced AR minus the spontaneous (unstimulated) AR results (A minus B, Table I).
|
Statistical analyses
The diagnostic accuracy of the ZIAR results was illustrated with the interactive dot diagram, while the positive and negative predictive values were recorded with the receiver operating characteristics (ROC) curve (Altman and Bland, 1994
; Schoonjans et al., 1995). |
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The sperm parameters and AR (mean ± SD) obtained from fertile sperm donors are depicted in Table I
. All the AR data were obtained by ZP from donated IVF metaphase II oocytes, which, compared with zonae from prophase I oocytes, had not only an enhanced acrosome inducing capacity, but also exhibited increased sperm–zona binding potential (Oehninger et al., 1992; Franken et al., 1994). The range for ZP induced AR, namely 18–25% in Table I, was within the range for metaphase II oocytes. However, the dose–response curve for the induction of the AR that we have established (Franken et al., 2000) was recorded using post-mortem retrieved prophase I oocytes. We routinely use the prophase I dose–response curve as reference (0.3 ZP/µl: positive control) in all experiments related to ZP mediated AR. The semen parameters, ZIAR as well as the IVF results of the 35 couples are depicted in Table II.
|
The correlation coefficient between the ZIAR and fertilization rates was r = 0.95 (P = 0.0001). The interactive dot diagram (Figure 1
) illustrates the diagnostic power of ZIAR. In order to evaluate the clinical importance of the ZIAR data that was recorded for the 35 couples, a ZIAR cut-off value of 15% was chosen for the group. This value was chosen based on the following: (i) shifting the cut-off points for ZIAR (from 7–19%) during the ROC analyses the 15% level revealed optimum sensitivity and specificity for morphology (i.e. 60 and 68% respectively) and 100 and 90% respectively for fertilization; (ii) furthermore, the mean ZIAR value recorded for the group was 12.8 ± 2%, which was rounded off to 15% when using 1 xSD above the mean; and (iii) there was a clear cut in the data at 15%.
|
The IVF rates were calculated as the total fertilization rate (total number of pre-ovulatory oocytes fertilized/total number of pre-ovulatory oocytes inseminated) at 88.0 ± 19% (mean ± SD). The minimum total fertilization rate (mean minus 2 xSD) that could be considered normal in our IVF programme was therefore 50%. Total fertilization values <50% were considered to be abnormal for the IVF laboratory (Franken et al., 1993
). From the ROC analyses, the optimum sensitivity and specificity was calculated at 50% for the best separation between the two groups, ZIAR >15% and ZIAR <15%.
A cut-off value for the fertilization rates of 50% was therefore chosen for the optimum separation between the two groups ZIAR >15% and ZIAR <15%. The corresponding sensitivity and specificity was 93 and 100% respectively. Using the interactive dot diagram as guide we then divided the couples according to the ZIAR test outcome. The mean (±SD) sperm parameters, acrosome response of the ZIAR groups >15% and <15% are depicted in Table III.
|
Due to the design of the study no correlation could be established between normal morphology and fertilization rates. The interactive dot diagram (Figure 2
) illustrates a sensitivity of 60% and specificity of 68%. These results were expected since normal and G-pattern morphology was an inclusive criterion for the study.
|
ROC curve analyses
The discriminating power of the ZIAR to identify fertilization failure among the 35 couples was calculated with ROC analyses. The power of the ZIAR (>15% and <15% acrosome reacted) of a given sperm sample to predict the fertilization is represented in Figure 3
. The area under the ROC curve, for the ZIAR data of 99%, illustrated the predictive power of the test. An area under the curve of 0.99 implied that a randomly selected individual with ZIAR result of <15% would have fertilization failure in 99% of cases. The confidence interval (0.89–1.00) does not include 0.5, implying that ZIAR results have the ability to distinguish between fertilization rates of >50% and <50%.
|
Rescued ICSI
The oocytes (n = 146) of 16 couples were used during a rescued ICSI procedure after fertilization failure was reported in the IVF laboratory. The results of the ZIAR test, as well as the fertilization data during IVF and rescued ICSI, are presented in Table IV
. The fertilization rate achieved during rescued ICSI was 39.8 ± 28%. Retrospectively, the ZIAR test results, i.e. 3.3 ± 2%, were indicative of fertilization failure among these couples. During the following cycle, two couples from this group were prospectively referred to the ICSI programme on account of their ZIAR data as well as the IVF fertilization history. During this cycle, the retrieved oocytes were randomly allocated to undergo either IVF or ICSI treatment. A total of 21 and 12 oocytes were retrieved from these patients, of which 11 and six respectively were allocated to the IVF programme. The rest of the oocytes, namely 10 and six respectively received ICSI treatment. Complete fertilization failure was again reported for both patients during IVF, while 7/10 (70%) and 4/6 (66%) fertilization rates were reported during the ICSI treatment.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Substantial information concerning the mammalian sperm acrosome reaction (AR) and its relevance during fertilization processes has accumulated during the past few years (Bielfeld et al., 1994
; Liu and Baker 1994, 1996a; Brucker and Lipford, 1995; Franken et al., 1996, 1997). The data include information on the mechanism of the AR the role in the fertilization process, the characterization of AR abnormalities compromising fertility, and methods whereby these abnormalities can be diagnosed and treated (Liu and Baker, 1996a,b). Recent studies have evaluated the nature of the receptors involved in the response and the way AR-inducing signals are transduced from the receptors to the membrane fusion effectors responsible for the ensuing exocytotic reaction (Luconi et al., 1998). Progesterone and zona pellucida glycoprotein (ZP3) have been identified as natural oocyte-associated AR-inducing ligands, and their sequential action has been shown to underlie the physiological AR (Melendrez et al., 1994; Roldan et al., 1994; Franken et al., 1997).
The ARIC test identifies two types of AR pathology, namely, `AR insufficiency' (Tesarik and Mendoza, 1993) and `AR prematurity' (Tesarik and Mendoza, 1995). AR insufficiency describes cases in which the difference in frequency of AR between ionophore-treated and untreated aliquots of a capacitated sperm population is <15%, while AR prematurity defines cases in which the frequency of spontaneous AR is >20% (Tesarik and Mendoza, 1995). Both of the mentioned pathologies can occur in the same patient. AR insufficiency as revealed by the ARIC test can only reflect downstream anomalies of the calcium influx in the signal transduction cascade responsible for AR induction (Tesarik, 1996).
During the present study we defined normal AR in cases where the ZIAR was significantly correlated with IVF and ZIAR outcome was >15%. Insufficient AR was identified in cases where ZIAR was <15% and typically associated with fertilization failure. The percentage of spermatozoa among the group of men showing an insufficient AR also had an elevated spontaneous AR of 17.9%. These findings, i.e. patients with normal conventional semen analyses, failure to fertilize with standard IVF and low ZIAR test results, may have a similar condition to that described by Liu and Baker (1994). They reported on 10 couples with long-standing infertility presenting reduced frequency of the AR of spermatozoa bound to the ZP, concluding that the possibility of the existence of a zona bound sperm population with a disordered AR causing impaired fertility (Liu and Baker, 1994). In close agreement with our findings, Liu and Baker (1997) concluded that patients with disordered ZIAR can be treated with ICSI. Patients with a long history of idiopathic infertility or complete fertilization failure should be tested for this condition using spermatozoa–zona pellucida interaction tests. Reduced ZIAR patients should be directed to ICSI rather than to standard IVF.
Tesarik and Mendosa (1995) described a `premature' AR which might be an indication of dysfunctional capacitation processes leading to a `premature' AR manifesting in an increased number of spontaneous acrosome reacted cells. We were unable to identify a `premature AR' group in our study since the ZIAR <15% group did not differ statistically between stimulated and spontaneous AR, i.e. 21.6 ± 11% and 17.9 ± 13% (P > 0.05, unpaired t-test). There was also no difference between normal controls (14.8 ± 2.7, Table I) and those patients with low ZIAR (17.9 ± 13, Table III).
It is important to note that among the ZIAR >15% group, the percentage of normal spermatozoa was reported to be 12.9 ± 4% (95% confidence interval, 11.2–14.6%). The reported fertilization rates among men with 5–14% normal spermatozoa and in those cases where no corrective measurements were taken (increase insemination concentration) were 64% (Kruger et al., 1986). The ZIAR test therefore identified a sperm population that has normal sperm parameters, with dysfunctional AR to solubilized human ZP. ZIAR test results can be clinically used to distinguish between couples that will benefit from IVF treatment, but moreover seem to be an indicator for ICSI therapy. The 16 couples with complete fertilization failure during IVF of 146 metaphase II oocytes, and who were treated with a rescued ICSI procedure (Table IV), had a decreased ZIAR of 3.3 ± 2% (mean ± SD). ZIAR results in these cases could possibly be seen as a powerful indicator of fertilization failure. On account of the ZIAR results, the metaphase II oocytes of two couples from rescued ICSI group, who did not achieve success during the first treatment period, were randomly divided and used for IVF or ICSI treatment during the following cycle. Both couples had failed fertilization during IVF, whereas 70 and 66% fertilization rates respectively for patient 1 and 2 were recorded during ICSI.
The fundamental utility of the ZIAR test stems from its being a functional bioassay of sperm performance in relation to the human ZP. Together with tight binding to the ZP, the ZIAR potential of a sperm population can be seen as a requisite for IVF. The ZIAR results provide useful discrimination between men capable of achieving fertilization in vitro versus those who are unlikely to be successful. When a larger number of patients have been tested in comparable prospective studies, the ZIAR test may become a valuable tool in the diagnostic scheme of the consulting clinician, since the outcome of the test underlines the most effective clinical option, namely IVF or ICSI.
|
Acknowledgments |
|---|
The authors wish to extend their gratitude to the andrology laboratory at du Buisson and partners for the opportunity to complete the study and to Dr P.J.Becker, CERSA, Division Biostatistics (Pretoria), for his keen interest and assistance in the study.
|
Notes |
|---|
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, 3rd Floor, Tygerberg Hospital, Tygerberg, 7505, South Africa. E-mail: dfranken{at}gerga.sun.ac.za
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Altman, D.G. and Bland, J.M. (1994) Diagnostic test 3: receiver operating characteristic plots. Br. Med. J., 309, 188.
Bielfeld, P.P., Faridi, A., Zaneveld, L.J.D. and de Jonge, C.J. (1994) The zona pellucida induced acrosome reaction of human spermatozoa is mediated by protein kinases. Fertil. Steril., 61, 536–541.[Web of Science][Medline]
Brucker, C. and Lipford, G.B. (1995) The human sperm acrosome reaction: physiology and regulatory mechanisms. An update. Hum. Reprod. Update, 1, 51–62.
Consensus Workshop on Advanced Diagnostic Andrology Techniques (1996) ESHRE Andrology Special Interest Group. Hum Reprod, 11, 1463–1479.
Cross, N.L., Morales, P, Overstreet, J.P. and Hanson, F.W. (1988) Induction of the acrosome reaction by the human zona pellucida. Biol. Reprod., 38, 235–244.[Abstract]
Cummins, J.M., Pember, S.M., Jequier, A.M. et al. (1991) A test of the human sperm acrosome reaction following ionophore challenge. Relationship to fertility and other seminal parameters. J. Androl., 12, 98–103.
Florman, H.M., Tombes, R.M., First, M.L. and Babcock, D.F. (1989) An adhesion-associated agonist form the zona pellucida activates G protein-promoted elevation of internal calcium and pH that mediate mammalian sperm acrosomal exocytosis. Dev. Biol., 135, 133–136.[Web of Science][Medline]
Franken, D.R., Kruger, T.F., Menkveld, R. et al. (1990) Hemizona assay and teratozoospermia: increasing sperm insemination concentrations to enhance zona pellucida binding. Fertil. Steril., 54, 497.[Web of Science][Medline]
Franken, D.R., Acosta, A.A., Kruger, T.F. et al. (1993) The hemizona assay: its diagnostic role in male factor infertility. Fertil. Steril., 59, 1075–1080.[Web of Science][Medline]
Franken, D.R., Kruger, T.F., Oehninger, S.C. et al. (1994) Sperm binding capacity of human zona pellucida derived from oocytes obtained from different sources. Andrologia, 26, 277–281.[Web of Science][Medline]
Franken, D.R., Morales, P. and Habenicht, U-F. (1996) Inhibition of G protein in human sperm and its influence on acrosome reaction and zona pellucida binding. Fertil. Steril., 66, 1009–1011.[Web of Science][Medline]
Franken, D.R., Bastiaan, H.S., Kidson, A. et al. (1997) Zona pellucida mediated acrosome reaction and sperm morphology. Andrologia, 29, 311–317.[Web of Science][Medline]
Franken, D.R., Bastiaan, H.S. and Oehninger, S.C. (2000) A micro-assay for sperm acrosome reaction assessment. J. Assist. Reprod. Genet., 17, 156–161.[Web of Science][Medline]
Granberg, M. (1999) Recommended Use of Scandinavian IVF Science Products, 2nd edn. Gothenburg.
Henkel, R., Müller, C., Miska, W. et al. (1993) Determination of the acrosome reaction in human spermatozoa is predictive of fertilization in vitro. Hum. Reprod., 12, 2128–2132.
Henkel, R., Franken, D.R. and Habenicht, U.F. (1998) Zona pellucida as physiological trigger for the induction of the acrosome reaction. Andrologia, 30, 275–280.[Web of Science][Medline]
Kruger, T.F. and Coetzee, K. (1999) The role of sperm morphology in assisted reproduction. Hum. Reprod. Update, 5, 172–178.
Kruger, T.F., Menkveld, R., Stander, F.S.H. et al. (1986) Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil. Steril., 46, 1118–1123.[Web of Science][Medline]
Liu,D.Y. and Baker, H.N.G. (1992) Morphology of spermatozoa bound to the zona pellucida of human oocytes that failed to fertilize in vitro. J. Reprod. Fertil., 94, 71–84.
Liu, D.Y. and Baker, H.W.G. (1994) Disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona penetration and reduced fertilization in vitro. Hum. Reprod., 9, 1694–1700.
Liu, D.Y. and Baker, H.W.G. (1996a) A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona pellucida: lack of relationship with ionophore A23187-induced acrosome reaction. Hum. Reprod., 11, 551–557.
Liu, D.Y. and Baker, H.W.G. (1996b) Relationship between the zona pellucida (ZP) induced acrosome reaction and the ability of sperm to penetrate the ZP in men with normal sperm-binding. Fertil. Steril., 66, 312–315.[Web of Science][Medline]
Liu, D.Y. and Baker, H.W.G. (1997) High fertilization and pregnancy rates after intracytoplasmic sperm injection in patients with disordered zona pellucida induced acrosome reaction. Fertil. Steril., 67, 955–958.[Web of Science][Medline]
Luconi, M., Barni, T., Vannelli, G.B. et al. (1998) Extra-cellular signal-regulated kinases modulate capacitation of human spermatozoa. Biol. Reprod., 58, 1476–1489.
Melendrez, C.S., Meizel, S. and Berger, T. (1994) Comparison of the ability of progesterone and heat solubilized porcine zona pellucida to initiate the porcine acrosome in vitro. Mol. Reprod. Dev., 39, –438.
Oehninger, S., Acosta, A., Veeck, L. et al. (1991) Recurrent failure of in vitro fertilization: role of the hemizona assay in the sequential diagnosis of specific sperm–oocyte defects. Am. J. Obstet. Gynecol., 164, 1210–1215.[Web of Science][Medline]
Oehninger, S.C., Franken, D.R., Alexander, N.J. and Hodgen, G.D. (1992) The hemizona assay and its impact on the identification and treatment of human sperm dysfunction. Andrologia, 24, 307–312.[Web of Science][Medline]
Oehninger, S.C., Franken, D.R. and Kruger, T.F. (1997) Approaching the next millenium: how should we manage andrology diagnosis in the intracytoplasmic sperm injection era? Fertil. Steril., 67, 434–436.[Web of Science][Medline]
Roldan, E.R.S., Murase, T. and Shi, Q-X. (1994) Exocytosis in spermatozoa in response to progesterone and zona pellucida. Science, 266, 1578–1581.
Schoonjans, F., Zalata, A., Depuydt, C.E. and Comhaire, F.H. (1995) Medcalc: a new computer program for medical statistics. Comput. Meth. Programs Biomed., 48, 257–262.
Tesarik, J. (1989) Appropriate timing of the acrosome reaction is a major requirement for the fertilizing spermatozoon. Hum. Reprod., 4, 957–961.
Tesarik, J. (1996) Acrosome reaction testing. In Consensus Workshop on Advanced Diagnostic Andrology Techniques. ESHRE Andrology Special Interest Group. Hum. Reprod., 11, 5–8.
Tesarik, J. and Mendoza, C. (1993) Sperm treatment with pentoxifylline improves the fertilizing ability in patients with acrosome reaction insufficiency. Fertil. Steril., 60, 141–146.[Web of Science][Medline]
Tesarik, J. and Mendoza, C. (1995) Alleviation of the acrosome reaction prematurity by sperm treatment with egg yolk. Fertil. Steril., 63, 153–157.[Web of Science][Medline]
Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, 1061–1066.
Veeck, L.L. (1988) Atlas of the Human Oocyte and Early Conceptus. Williams and Wilkins, Baltimore, vol. 2, p. 3.
World Health Organization (1999) Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge, pp. 11–17.
Submitted on June 8, 2000; accepted on September 22, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  D. Y. Liu, M. L. Liu, C. Garrett, and H.W. G. Baker Comparison of the frequency of defective sperm-zona pellucida (ZP) binding and the ZP-induced acrosome reaction between subfertile men with normal and abnormal semen Hum. Reprod., July 1, 2007; 22(7): 1878 - 1884. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Canovas, P. Coy, and E. Gomez First Steps in the Development of a Functional Assay for Human Sperm Using Pig Oocytes J Androl, March 1, 2007; 28(2): 273 - 281. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Caballero-Campo, M. Chirinos, X. J. Fan, M. E. Gonzalez-Gonzalez, M. Galicia-Chavarria, F. Larrea, and G. L. Gerton Biological Effects of Recombinant Human Zona Pellucida Proteins on Sperm Function Biol Reprod, April 1, 2006; 74(4): 760 - 768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Pixton, E. D. Deeks, F. M. Flesch, F. L.C. Moseley, L. Bjorndahl, P. R. Ashton, C. L.R. Barratt, and I. A. Brewis Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: Case report Hum. Reprod., June 1, 2004; 19(6): 1438 - 1447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Y. Liu and H.W.G. Baker High frequency of defective sperm-zona pellucida interaction in oligozoospermic infertile men Hum. Reprod., February 1, 2004; 19(2): 228 - 233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Moldenhauer, G. C. Ostermeier, A. Johnson, M. P. Diamond, and S. A. Krawetz Diagnosing Male Factor Infertility Using Microarrays J Androl, November 1, 2003; 24(6): 783 - 789. [Full Text] [PDF]
|
|
|
|
|
|
D. Y. Liu and H.W.G. Baker Frequency of defective sperm-zona pellucida interaction in severely teratozoospermic infertile men Hum. Reprod., April 1, 2003; 18(4): 802 - 807. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Makkar, E. H. Y. Ng, W. S. B. Yeung, and P. C. Ho The significance of the ionophore-challenged acrosome reaction in the prediction of successful outcome of controlled ovarian stimulation and intrauterine insemination Hum. Reprod., March 1, 2003; 18(3): 534 - 539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Oehninger and R. G. Gosden Should ICSI be the treatment of choice for all cases of in-vitro conception?: No, not in light of the scientific data Hum. Reprod., September 1, 2002; 17(9): 2237 - 2242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Schuffner, H. S. Bastiaan, H. E. Duran, Z.-Y. Lin, M. Morshedi, D. R. Franken, and S. Oehninger Zona pellucida-induced acrosome reaction in human sperm: dependency on activation of pertussis toxin-sensitive Gi protein and extracellular calcium, and priming effect of progesterone and follicular fluid Mol. Hum. Reprod., August 1, 2002; 8(8): 722 - 727. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Jagannathan, E. L. Punt, Y. Gu, C. Arnoult, D. Sakkas, C. L. R. Barratt, and S. J. Publicover Identification and Localization of T-type Voltage-operated Calcium Channel Subunits in Human Male Germ Cells. EXPRESSION OF MULTIPLE ISOFORMS J. Biol. Chem., March 1, 2002; 277(10): 8449 - 8456. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Y. Liu, G. N. Clarke, M. Martic, C. Garrett, and H.W.G. Baker Frequency of disordered zona pellucida (ZP)-induced acrosome reaction in infertile men with normal semen analysis and normal spermatozoa-ZP binding Hum. Reprod., June 1, 2001; 16(6): 1185 - 1190. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||