Hemizona Index Research Articles

Suitability of the hemi-zona assay for the evaluation of the effect of the length of the equilibration period before cryopreservation

The aim of this study was to test the suitability of the interspecific hemizona assay (HZA) to predict the fertilizing capacity of bovine sperm after modifying the length of the equilibration period before freezing and thawing. Ejaculates from 10 proven fertile bulls were split after dilution, equilibrated at 4 °C for either 24 h (control sperm = CS) or 6, 48, 72 or 96 h (test sperm = TS) and cryopreserved. Hemizona (HZ) pairs from in vitro matured pig oocytes were used for the heterologous HZA: After thawing and swim-up (1 h) CS and TS were co-incubated with matching HZ (125,000 S/HZ in 25 μL Fert-TALP) for 4 h. Spermatological analyses (progressive motile sperm (PMS), plasma membrane- and acrosome-intact sperm (PMAI), sperm showing a high degree of DNA fragmentation (%DFI)) were performed after 0 and 3 h of incubation after thawing. After an equilibration time of 48 h and 72 h values for PMAI0h were higher (P < 0.05) compared to PMAI0h values of sperm equilibrated for 6 h, and %DFI3h values were higher after 96 h (P < 0.05) compared to 6 h equilibration. Between 12 and 90 TS and 13–97 CS were tightly bound to each HZ, respectively. The mean Hemizona Index (HZI) after a sperm equilibration for 48 h (HZI = 92.3 ± 12.7) or 72 h (HZI = 98.9 ± 16.23) was higher (P < 0.01) than after an equilibration for 6 h (HZI = 73.3 ± 13.93) or 96 h (HZI = 81.3 ± 11.41). The HZI for 96 h equilibration was moderately negatively related to PMS0h and PMS3h (r < −0.35, P < 0.05). Furthermore the HZI for 6 h equilibration was highly negatively correlated with DFI0h (r = −0,46, P < 0.01). On the basis of these results it can be concluded that the hemi-zona assay is a suitable test to detect alterations in the fertilizing capacity of bovine sperm after modifying the equilibration period.

Involvement of the prostate and testis expression (PATE)-like proteins in sperm-oocyte interaction

The prostate and testis expression (PATE)-like family of proteins are expressed mainly in the male genital tract. They are localized in the sperm head and are homologous to SP-10, the acrosomal vesicle protein also named ACRV1. Our aim was to characterize the expression and functional role of three PATE-like proteins in the testis and ejaculated sperm. The expression and localization of PATE-like proteins in human testis biopsies (n= 95) and sperm cells were assessed by RT-PCR, immunohistochemistry and immunofluorescence staining (at least 600 sperm cells per specimen). The function of the PATE protein was tested by the hemizona assay and hamster egg penetration test (HEPT). PATE and PATE-M genes and proteins were present almost exclusively in germ cells in the testis: immunoflourescence showed that the percentage of germ cells positive for PATE, PATE-M and PATE-B was 85, 50 and 2%, respectively. PATE and PATE-M proteins were localized in the equatorial segment of the sperm head, while PATE-B protein was localized in the post-acrosomal region. A polyclonal antibody (Ab, at 1:50 and 1:200 dilutions) against the PATE protein did not inhibit sperm-zona binding in the hemizona assay (hemizona index of 89.6 ± 10 and 87 ± 36%, respectively). However, there was inhibition of sperm-oolemma fusion and penetration in the HEPT (penetration index: without Ab 7 ± 3.9; Ab dilution of 1:100, 4 ± 3.5; Ab dilution of 1:20, 0.6 ± 1.2, P < 0.001). Our data suggest that PATE protein is involved in sperm-oolemma fusion and penetration but not sperm-zona binding.

Effect of sperm preparation with TEST yolk buffer on sperm-binding capacity under hemizona assay conditions.

The study was conducted to evaluate the diverse effect and clinical significance of TEST yolk buffer treatment on sperm samples of 128 infertile men. Sperm samples were incubated with TEST yolk buffer and control medium (Ham's F-10) at room temperature for 2 h. The hemizona indices (mean +/- SE) of the TEST yolk buffer and medium-treated sperm samples were 29 +/- 2.3% and 22 +/- 1.6%, respectively. Inspection of the individual response of each sperm sample to TEST yolk buffer revealed that 63 samples (49%) improved (double the interassay variation = 28%) their binding to zona pellucida, 36 (28%) remained unchanged, whereas the binding capacity of 29 samples (23%) decreased. Furthermore, TEST yolk buffer treatment of 24 samples (19%) resulted in an increased binding beyond the hemizona index threshold set up at 23%. This level was previously shown to be the cut-off point between fertile and infertile sperm samples. It was concluded that when applied to an unselected group of infertile men, TEST yolk buffer significantly increased sperm binding capacity to the zona pellucida. However, only 19% of the sperm samples showed improvement with clinical significance. The other sperm samples may have improved, remained unchanged or even deteriorated independently on basic sperm variables. Thus, the effect of TEST yolk buffer treatment on sperm binding should be tested prior to its clinical use to avoid possible damage to certain sperm samples.

Sperm binding and ultrasound changes after operative repair of varicocele: correlation with fecundity.

The study was conducted to evaluate the changes in sperm binding capacity and ultrasound measurements of the internal spermatic vein, after operative repair of a varicocele. In order to clarify the effect of a varicocele on fertility, these changes were correlated to pregnancy achievement. Twelve infertile males with subnormal semen parameters and varicocele, underwent operative repair. Pre- and post-operatively, all had semen analysis, hemizona assay and ultrasound of the internal spermatic veins. The patients were divided into three subgroups according to pregnancy outcome, and the changes in the different evaluation tests after the operation were compared. Sperm concentration and motility improved post-operatively in all three subgroups, whereas the hemizona index and ultrasonographic measurements improved significantly only in the subgroup that achieved early pregnancies (the mean post-operative percentage of normal morphology was significantly higher), compared to the subgroup without pregnancies. Unlike sperm parameters which improve after operative repair of the varicocele, but have no correlation to conception, sperm binding and ultrasound measurements of the internal spermatic veins improve significantly in cases that achieve early pregnancies. The use of these tests, as well as measuring the percentage of normal morphology, are recommended in all cases of infertility-related varicocele.

Rapid freezing versus slow programmable freezing of human spermatozoa

To compare the efficacy of rapid freezing (RF) and slow programmable freezing (SPF) of human spermatozoa. Experimental study. University-based assisted conception laboratory. Semen from 30 normospermic men. Semen was processed through density gradients and divided into three groups: nonfrozen control, RF, and SPF. Sperm motility, kinematics, and morphology were assessed by a computer-assisted semen analyzer; viability by eosin-Y staining and the hypo-osmotic swelling test; DNA integrity by comet assay; and sperm binding by hemi-zona assay. Post-thaw sperm motility (53.9%, 37.0%, and 75.5% for RF, SPF, and controls, respectively) and sperm vitality (hypo-osmotic swelling test 60.1%, 44.1%, and 77.9%; eosin-Y staining 64.8%, 50.4%, and 81.8% for RF, SPF, and controls, respectively) were higher in the RF than in the SPF group, but lower than in the nonfrozen control group. There was no significant difference in post-thawed normal sperm morphology (14.9%, 14.4%, and 16%) and sperm DNA integrity by comet assay (93.6%, 94.5%, and 94.2%) in the RF, SPF, and controls, respectively. The hemi-zona index was no different between the two cryopreserved groups. The RF gave superior post-thaw motility and cryosurvival than SPF.

P-402: Fertility loss in the sperm of tobacco smokers may be reversed after washing with a cannabinoid agonist

Human sperm carry a nicotinic acetylcholine receptor and respond to nicotine from tobacco. They also have cannabinoid (CB) receptors and respond to marijuana and endocannabinoids or synthetic CB chemicals. We previously reported that sperm from 2/3 of tobacco smokers showed a significant drop in fertilizing capacity, using the Hemizona Assay (HZA). Their sperm showed reduced binding to the zona pellucida (cover) of the human egg. In Part II, we hypothesized that treatment of the smoker’s sperm with a CB agonist would improve sperm binding capacity. We have studied the effects of a CB agonist (AM-1346; analog of the endocannabinoid anandamide). After obtaining a positive response when donor sperm were treated with nicotine plus a CB agonist, we studied the sperm from 8 chronic tobacco smokers in 24 in vitro HZA tests. Each smoker was previously tested in the HZA, where his sperm fertilizing function was compared to a non-smoking donor. Four of 8 smokers (Group I) had shown normal sperm fertilizing function (HZI > 65). The other 4 smokers (Group II) had failed the zona assay. In Part II, each new test used the sperm from one tobacco smoker. His sperm were washed with and without a CB ligand (Control had no ligand present). We tested the effects of a potent CB1 agonist, AM-1346 (n = 17; 100 pM to 100 nM). After a “swim-up,” the motile sperm fraction was used for the HZA. Here, a nonviable human oocyte was cut in half. One half was incubated with smoker’s sperm that had been treated with a CB ligand. The matching Hemizona (HZ) was placed with that smoker’s Control sperm. Post-incubation, each HZ was rinsed and counted for the number of sperm tightly bound to the outside. The ratio of [# bound for the treated sperm / # bound for the Control sperm] was calculated as the Hemizona Index (HZI). HZI = 100 indicates that the treated sperm bound to the zona as well as the Control sperm. We hypothesized that the HZI would improve with in vitro CB treatment. After exposing the sperm to AM-1346, at any concentration, we found that Group II (low fertility) responded with markedly improved zona binding, compared to the Control sperm (4 out of 8 experiments). Specifically, when semen quality was poor, Group II showed a mean HZI of 177% (77% improvement compared to the untreated sperm). However, the sperm from smokers with normal fertility (Group I) behaved differently. Here, only 1 of 9 tests showed a zona binding enhancement (p < 0.05). In Groups I & II, there were 7 experiments where the smoker was tested with a low concentration of AM-1346 (one assay) and also tested at a higher 1346 level (second assay). In 6 of 7 cases, the higher 1346 dose produced a better HZI score (improved binding). We conjecture that most smoking men would show a stimulation of zona binding if the dose of AM-1346 is high enough. Based on our previous data, and published literature, it is clear that a majority of tobacco smokers will exhibit a small or significant decline in fertility potential. Infertility clinics worldwide are trying to meet the challenge of male and female tobacco smokers who have only borderline fertility. Smoking cessation is the best solution. However, the in vitro use of synthetic cannabinoid analogs may significantly improve the fertilizing capacity of tobacco smokers who cannot quit.

Predictive value of the hemizona assay for pregnancy outcome in patients undergoing controlled ovarian hyperstimulation with intrauterine insemination

The hemizona assay (HZA) is an established functional test that examines in vitro sperm-zona pellucida binding capacity with high predictive power for fertilization outcome in IVF. The objective of this study was to evaluate the value of the HZA as a predictor of pregnancy in patients undergoing controlled ovarian hyperstimulation (COH) and intrauterine insemination (IUI). Prospective clinical study. Academic center. Eighty-two couples with unexplained or male factor infertility that underwent 313 IUI cycles. Basic semen analysis and HZA were performed within three months of starting COH/IUI therapy. Hemizona index (HZI) and clinical pregnancy. Overall, patients with an HZI of <30 had a significantly lower pregnancy rate compared to patients with an HZI of > or =30 (11.1% vs. 40.6%, respectively; P<.05; relative risk for failure to conceive: 1.5 (confidence interval 1.2-1.9)). In all patients combined, and in the range of HZI 0-60, the duration of infertility (P=.000) and the HZI (P=.004) were significant determinants of conception (receiver operating characteristics (ROC) analysis). In couples with male infertility, the average path velocity and HZI were significant predictors of conception (P=.001 and P=.005, respectively, ROC analysis). The negative and positive predictive values of the HZA for pregnancy were 93% and 69%, respectively. Logistic regression analysis provided models of HZI (P=.021) and duration of infertility (P=.037) with highest predictability of conception in male factor and unexplained infertility groups, respectively. The HZA predicted pregnancy in the IUI setting with high sensitivity and negative predictive value in couples with male infertility. Results of this sperm function test are useful in counseling couples before allocating them into COH/IUI therapy.

Diagnosis and treatment of immunologically infertile males with antisperm antibodies.

The presence of antisperm antibodies (ASA) can reduce fecundity in both males and females. The present review describes a strategy, established by investigations of the diverse inhibitory effects of ASA on fertility, for the appropriate diagnosis and treatment of infertile males with ASA. For infertile males with ASA, diagnosis using the direct-immunobead test (D-IBT), the postcoital test (PCT) and the hemizona assay (HZA) should be carried out as the basis for decision-making. If the patient with ASA has an abnormal hemizona index, it seems reasonable to advise selecting intracytoplasmic sperm injection-embryo transfer (ICSI-ET) as a primary treatment. However, it has been shown that some immunologically infertile males with normal fertilizing ability established pregnancy by timed intercourse (TI) or intrauterine insemination (IUI). In such patients with ASA having normal hemizona index, TI or IUI can be selected based on the PCT result. Therefore, the treatment strategy for males with ASA is similar to that for infertile males with oligozoospermia or asthenozoospermia. In conclusion, it should be emphasized that a diversity of ASA exists and their effects on fertility in infertile males. Although there is an argument that routine testing for ASA in males is not always necessary, one should be aware that in some cases of failed IUI or IVF, ICSI is selected afterward because of the diagnosis of ASA. (Reprod Med Biol 2005; 4: 133-141).

Effect of Antisperm Antibodies Present in Human Follicular Fluid upon the Acrosome Reaction and Sperm–Zona pellucida Interaction

To determine the ability of IgGs isolated from follicular fluids (hFFIgGs) to induce the acrosome reaction (AR) in human spermatozoa and to inhibit sperm-zona pellucida (ZP) interaction. Incubation of capacitated spermatozoa with hFFIgGs (n = 40) and assessment of their effect on the AR or hemizona (HZ) assay in a condition that allows sperm-ZP interaction, avoiding acrosomal exocytosis. hFFIgGs from different women varied in their ability of inducing the AR. Those hFFIgGs with the highest AR-inducing capacity evoked the exocytotic response in most of the different sperm donors tested [high Induction Frequency (IF)]. Some of these antibodies were also able of inhibiting sperm binding to ZP [low HZ Index (HZI)]. A significant correlation was found between the IF and the HZI for each hFFIgG. Human follicular fluid contains antibodies capable of inducing the AR and inhibiting sperm-ZP binding, suggesting that they could be directed towards ZP receptors. hFFIgGs would constitute a tool for the identification of sperm entities involved in fertilization.

Diversity of the inhibitory effects on fertilization by anti-sperm antibodies bound to the surface of ejaculated human sperm.

The presence of anti-sperm antibodies (ASA) in males can reduce fecundity. However, it has been shown that there is a diversity of ASA bound to the sperm surface. This study was performed to investigate the inhibitory effects on fertilization by ASA in males. ASA were detected using the direct-immunobead test (D-IBT) in 509 semen samples. In some cases, the direct-sperm immobilization test (D-SIT) was carried out. The fertilizing ability of infertile males with ASA was determined as follows; (i) an IVF fertilization rate of >/=50%, (ii) a hemizona index (HZI) of >/=50%, and (iii) pregnancy established without the use of ART. In total, 18 (3.54%) infertile males had ASA on the sperm surface. Except for one male with an absolute indication for ICSI because of severe asthenozoospermia and two males who dropped out of this study, fertilizing ability in 15 males could be determined. Four (26.7%) men did not satisfy the criteria. The existence of sperm immobilizing antibodies on the surface of ejaculated sperm had no impact on fertilization. In four (57.1%) of seven patients who had IB-bound sperm of >/=80%, fertilizing ability was inhibited, while none of the eight patients who had <80% IB-bound sperm had an inhibitory effect on fertilization. There was a significant difference between the two groups (P = 0.01). Some sperm-bound antibodies are related to the inhibitory effects on fertilization, indicating that a diversity of sperm-bound antibodies exists in males. This result might be one of the reasons for the controversy of the relationship between ASA and male immunological infertility. Based on the present study, a sperm-zona pellucida binding assay should be performed for appropriate decision making in infertile males with ASA.

Cumulus cells reduce the spermatozoa–zona binding inhibitory activity of human follicular fluid

Objective To investigate the effects of human follicular fluid cultured with cumulus cells to inhibit the binding of spermatozoa to the zona pellucida of oocytes. Design Controlled experimental laboratory study. Setting University gynecology unit. Patient(s) Women undergoing assisted reproduction program and men visiting the subfertility clinics. Intervention(s) Culture medium and human follicular fluid were used to culture cumulus cells in vitro for specified time periods. Main outcome measure(s) Zona binding capacity and motility of spermatozoa after incubation with cumulus cells treated culture medium or human follicular fluid. Result(s) Compared with the control medium, spent culture media after culturing cumulus cells for 3, 5, and 7 hours did not affect the motility and zona binding capacity of the treated spermatozoa. Significantly more spermatozoa treated with human follicular fluid that had been preincubated with cumulus cells for 5 and 7 hours bound onto hemizona in hemizona binding assay when compared with those preincubated in human follicular fluid without cumulus treatment. The hemizona index increased with the increase in the duration of cumulus cell treatment. Human follicular fluid with or without cumulus cells maintained sperm motility to similar extent for 3 hours. Conclusion(s) Cumulus cells reduced the inhibitory effect of human follicular fluid on spermatozoa–zona binding in vitro in a time-dependent manner.

Application of a 1.48-microm diode laser for bisecting oocytes into two identical hemizonae for the hemizona assay.

Laser systems are very promising new technical tools in assisted reproduction. It was investigated if laser radiation can replace the mechanical cutting procedure via micromanipulator in the hemizona assay (HZA), a commonly used bioassay to determine the sperm-zona pellucida binding capacity. An oocyte was bisected precisely into two identical hemizonae with approximately 20 laser pulses (pulse length 30 msec) using a 1.48-microm diode laser. Compared with the conventional method using microscalpels for zona bisection, laser treated hemizonae showed equivalent sperm-binding and within the two groups there was no detectable difference between matching hemizonae in their capacity for tight sperm-binding. To evaluate whether laser radiation affects the outcome of the HZA when effects of certain substances are investigated, the spermatozoa were preincubated with human follicular fluid (hFF), which inhibits the binding of spermatozoa to zona pellucida in vitro. Supplementation with follicular fluid exerted an inhibitory effect in both groups. The hemizona index (HZI) showed no statistical differences between the two methods. Therefore, the 1.48-microm diode laser is a suitable new instrument for generating equally sized hemizonae. There is no use for holding pipettes and microscalpels, on the contrary, for performing the HZA the laser is a precise, very quick and easy to use new working tool.

Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound.

Spermatozoa-zona pellucida binding selects for human spermatozoa with progressive motility, normal morphology and functional competency. We postulated that this gamete interaction would also act to select against spermatozoa with chromosomal numerical aberrations. Spermatozoa from 41 men participating in the intracytoplasmic sperm injection (ICSI) programme were evaluated for the incidence of aneuploidy of chromosomes 18, X and Y. The hemizona assay was utilized to determine whether zona-bound spermatozoa from these patients have a reduced incidence of aneuploidy compared with those selected by motility only in a standard swim-up procedure. Using multicolour fluorescence in-situ hybridization (FISH) with DNA probes specific for chromosomes 18, X and Y, the disomy rates for chromosomes 18, X, Y and XY were found to be 0.31, 0.27, 0.29 and 0. 14% respectively in the swim-up motile fraction, and 0.31, 0.33, 0. 32 and 0.19% respectively in the pellet fraction. Analysing the zona-bound spermatozoa, the disomy rates for chromosome 18, X, Y and XY were found to be 0.02, 0.15, 0.12 and 0.07% respectively. The zona-bound spermatozoa had a significantly lower frequency of aneuploidy than the swim-up motile fraction or the pellet fraction (P < 0.0001). The incidence of chromosome 18 aneuploidy, including both chromosome 18 disomy and nullisomy, in the swim-up motile fractions was significantly increased in patients with an abnormal or borderline hemizona index compared with those with a normal hemizona index (P < 0.05). We also found that a high incidence of sperm aneuploidy was associated to a certain extent with low fertilization rate, and with failure to achieve pregnancy through ICSI. This study suggests that the human zona pellucida has the capacity to select against aneuploid spermatozoa by an as yet undetermined mechanism.

Use of failed-fertilized oocytes for diagnostic zona binding purposes after sperm binding improvement with a modified medium.

Because the availability of prophase oocytes for zona binding testing is limited, we compared sperm binding to the zona of failed-fertilized intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) oocytes after incubation in a standard IVF medium and a specially composed binding improvement medium. Semen samples from nine patients and nine fertile donors were separated in parallel by the standard swim-up method in both media. Subsequently, hemizona assays were performed with prophase, failed-fertilized ICSI and IVF oocytes. Sperm separation resulted in a significantly higher sperm count (P < 0.01) and progressive motility (P = 0.018) in binding improvement medium. Moreover, spermatozoa coincubated with hemizonae (prophase, failed-fertilized ICSI and IVF oocytes) in binding improvement medium bound significantly more to hemizonae than in the controls (P < 0.01). However, the hemizona index did not differ. Thus, the limited number of human zonae can be increased by the use of oocytes that failed to fertilize during ICSI or IVF. This will lead to a qualitative improvement of the diagnostic spectrum in male-factor infertility.

Pre-freezing sperm preparation does not impair thawed spermatozoa binding to the zona pellucida.

The present study was conducted to assess the fertilizing potential of frozen-thawed spermatozoa, which were cryopreserved after separation on a Percoll gradient, or washed out of seminal plasma. For this purpose, binding to the zona pellucida and other characteristics of the treated sperm cells were compared with those of cryopreserved spermatozoa from the same original sample which were not manipulated before freezing. Semen specimens were obtained from 80 candidates for sperm donation. Percoll-treated sperm samples compared with the sibling, unprocessed controls had significantly higher values of sperm motility characteristics and per cent of cells with normal morphology after freezing and thawing. Sperm binding ability to the zona pellucida was not statistically different (109 +/- 8.1% and 94 +/- 6.7% in unprocessed and Percoll-treated samples respectively). Sperm specimens processed by washing had significantly higher values for motility characteristics than untreated sibling samples, but no differences were found between the treated and untreated samples for morphology and binding to the zona pellucida (hemizona index of 75 +/- 7.0% and 76 +/- 6.7% in unprocessed and washed samples respectively). These findings suggest that, judged by the binding assay, the aforementioned pre-freezing separation processes have no adverse effect upon the fertilizing potential of the thawed sperm cells. These procedures make it possible to optimize the progressive motile sperm cell concentration of the frozen specimen, which facilitates the storage of samples with good quality, even when the features of the original semen are sub-optimal.

Preliminary results of hemizona assay (HZA) as a fertility test for canine spermatozoa

The Hemizona assay (HZA) is considered to be an effective test for predicting the fertilizing potential of spermatozoa. It is a functional test that distinguishes the zona-binding capacity of spermatozoa from fertile and infertile males. The objective of this study was to validate the HZA for canine spermatozoa, as a test for diagnosing canine male fertility status. Various parameters that affect binding capacity were examined: the presence of an adequate number of capacitated and motile spermatozoa for an HZA, the influence of fertility status, sperm-binding variability within fertile dogs over 60 d, variability in sperm-binding capacity of different oocytes, the lower limit number of spermatozoa binding to a zona from the fertile control, and evaluation of HZI to determine the fertilizing capacity of spermatozoa. Hemizonae were obtained from frozen oocytes of spayed bitches. The oocytes were manually cut into nearly equal halves. Spermatozoa were capacitated by swim-up and 1 h incubation at 37°C in modified Ham's F10 medium. Spermatozoa and hemizonae were co-incubated in 100-μL drops at 37°C for 1 h. Spermatozoa from 7 fertile and 3 infertile dogs were used for this study. The optimal sperm concentration for hemizona insemination was 1x10 6/mL capacitated and motile spermatozoa. A significant difference (P<0.001) was found the number of tightly zonabound spermatozoa between fertile and infertile dogs. Although there was a small difference in zona binding capacity between ejaculates of the same fertile dog (44 ± 18.24), the main cause for the difference mentioned above was that of poor zona pellucida-binding capacity of spermatozoa from infertile dogs. We found a maximum of 14.28% bad oocytes when we compared sperm samples from 3 fertile and 3 infertile dogs in 56 HZA replicates. To avoid the effect of bad zona on sperm binding we calculated 37 (95% Cl) bound spermatozoa from infertile dogs in 56 replicates. Thus, an HZA experiment in which a control dog had <37 zona bound spermatozoa was repeated. Based on a minimum of 37 bound spermatozoa for fertile males (controls), a differential zona-binding capacity and hemizona index (HZI) between fertile and infertile dogs and between 2 fertile dogs was determined. The binding differential between fertile and infertile dogs was 64.92 ± 24.29, while between 2 fertile dogs it was 22.38 ± 10.02 (P<0.001). According to the HZI values, a value equal to or less than 41.11 indicated an infertile test dog, while an HZI value equal to or greater than 57.95 indicated a fertile test dog. Any value between these two could indicate either fertility or infertility. The evaluation of fertilizing potential of spermatozoa can be improved using the HZA protocols described above.

Direct evidence for the involvement of carbohydrate sequences in human sperm-zona pellucida binding.

Several lines of evidence indicate that mammalian fertilization is initiated via a binding process that is dependent upon the recognition of oligosaccharide sequences associated with zona pellucida (ZP) glycoproteins. Here, specific chemical and enzymatic methods were employed to modify human ZP and to test their effects on sperm binding in the hemizona assay system (HZA). Periodate oxidation of human ZP under very mild conditions (10 min, 0 degrees C, 1 mM sodium m-periodate) that attacks only terminal sialic acid resulted in a 30% loss of human sperm binding in the HZA [hemizona index (HZI) = 70.2 +/- 10.9, n = 22; P < 0.05]. Periodate oxidation under mild conditions (1 h, 23 degrees C, 10 mM sodium m-periodate) caused a 40% decrease in binding (HZI = 60.8 +/- 10.3; n = 24; P< 0.01). Treatment of human ZP with neuraminidase caused a substantial increase in sperm binding to human ZP (HZI = 297 +/- 45, n = 22; P < 0.01). These findings indicate that there are sialic acid dependent binding sites coexisting with binding sites that are obscured by sialic acid. To determine the periodate sensitivity of these obscured sites, hemizona were first digested with neuraminidase and subsequently subjected to mild periodate oxidation. The combined enzymatic and chemical treatments caused a 79% decrease in sperm binding compared to control hemizona (HZI = 20.7 +/- 4.4, n = 16; P < 0.001). Human sperm-ZP interaction was also increased by digestion of human ZP with endo-beta-galactosidase (HZI = 710 +/- 232, n = 14; P < 0.01), indicating that potential binding sites for spermatozoa are also obscured by lactosaminoglycan sequences. These studies support a definitive role for the involvement of ZP-associated glycans in the binding of human spermatozoa to oocytes.

Effects of Low Concentrations of Nitric Oxide on the Zona Pellucida Binding Ability of Human Spermatozoa

Objective: To investigate the direct effects of sodium nitroprusside, a nitric oxide donor, on the fertilizing ability of human spermatozoa in vitro. Design: Prospective, controlled in vitro study. Setting: IVF Unit, Medical College Hospital. Patient(s): Women undergoing conventional IVF. Intervention(s): Human spermatozoa samples were incubated with a nitric oxide donor and a nitric oxide quencher, carboxy-imidazolineoxyl N-oxides. Main Outcome Measure(s): The capacitation and the acrosome reaction rates were determined by chlortetracycline assay. Sperm zona pellucida binding and sperm penetration into oocytes were determined using the hemizona assay and the human aged zona-free oocyte sperm penetration assay. Result(s): High concentrations of sodium nitroprusside (10 −3 and 10 −5 M) inhibited sperm motility and viability. In contrast, low concentrations of sodium nitroprusside (10 −7 and 10 −8 M) did not affect motility and resulted in increased capacitation (47 ± 6% at 10 −7 M, 42 ± 6% at 10 −8 M, and 24 ± 4% in controls, respectively, P< 0.01). A twofold increase in the hemizona index occurred compared to the matched control. However, low levels of sodium nitroprusside treatment did not affect the acrosome reaction and human zona-free oocyte sperm penetration rates. Conclusion(s): Low concentrations of nitric oxide may have some physiologic role in fertilization through the enhancement of capacitation and zona pellucida binding but not by the induction of the acrosome reaction or the facilitation of penetration into oocytes.

Impact of fertilization history and semen parameters on ICSI outcome.

The objectives of this study were (1) to investigate intracytoplasmic sperm injection (ICSI) outcome according to its indications, i.e., a history of failed or poor fertilization and unsuitable sperm parameters for conventional IVF, and (2) to examine the impact of a female's age, sperm concentration, motility, morphology, presence of antisperm antibodies, and hemizona assay (HZA) results on overall outcome. Two hundred seventy-nine ICSI cycles performed in 207 couples were retrospectively evaluated. Clinical pregnancy and delivery rates were 36.8 and 29.8% for patients with prior failed fertilization, 23.2 and 17.8% for patients who had prior poor fertilization, and 28.6 and 21.3% for patients with unsuitable sperm parameters. The differences among all groups were found to be insignificant. There was a significant, negative correlation between a female's age and pregnancy results. No difference was found in the three basic sperm parameters between those patients who produced and those who did not produce a pregnancy, but the fertilization rate was significantly higher in patients with more adequate sperm parameters. Although there was a trend toward a better fertilization rate in patients with a hemizona index (HZI) greater than 30 (indicative of a superior sperm-zona pellucida binding capacity) than in those with a HZI less than 30, the difference was not significant. There were no differences in fertilization rate according to the presence or absence of antisperm antibodies. Fertilization history in a conventional IVF cycle has no effect on success rates following ICSI, and there is no correlation among the basic sperm parameters, the presence of antisperm antibodies, and pregnancy rates.

The effect of seminal plasma on human sperm-zona pellucida binding.

The aim of this study was to determine the effect of different concentrations of seminal plasma (SP) in insemination medium on human sperm-zona pellucida (ZP) binding. The effects of fresh SP [50% (n = 10) and 1% (n = 9)], frozen-thawed versus fresh SP [5% (n = 10)] and frozen-thawed SP [5% (n = 10) and 1% (n = 9)] on sperm-ZP binding were examined using hemizona assays. The validity of the hemizona assay as performed in this study was also examined. Relative sperm binding percentages were determined for respective hemizonae, and the hemizona index was calculated for each experimental group and compared statistically. The 50% native SP inhibited sperm-ZP binding by approximately 70% (P = 0.0051), while 1% native SP enhanced sperm-ZP binding by approximately 350% (P = 0.0051). Significantly more spermatozoa (mean +/- SD, 172.00 +/- 54.12) bound to zonae in the presence of 5% frozen-thawed SP than with 5% SP that had not been frozen (mean +/- SD, 127.00 +/- 69.18; P = 0.0431). The 1 and 5% frozen-thawed SP stimulated sperm-ZP binding by approximately 400 and approximately 250% respectively (P = 0.0077 and 0.0051 respectively). It is concluded that SP concentrations found in insemination media during assisted reproductive techniques do not inhibit but in fact enhance sperm-ZP binding.