Zona-free hamster ovum penetration assay as a screening procedure for in vitro fertilization

Twenty couples with primary sterility due to mechanical factors entered our program of IVF. In addition to routine sperm evaluation, an SPA was performed in all cases. Whenever the SPA was pathologic, none of the human ova were fertilized in vitro, while 77% of the patients with a normal SPA had IVF of at least one human ovum. The good correlation between pathologic SPA and failure of IVF stresses the clinical value of this assay. The use of the SPA for patient selection for IVF is suggested.     

Human sperm penetration assay as an indicator of sperm function in human in vitro fertilization

Performance of spermatozoa in a hamster oocyte/human sperm penetration assay (SPA) was correlated with the results of in vitro fertilization (IVF). Forty-two patients underwent 50 IVF cycles. SPA scores were obtained before IVF cycles (screening SPA, n = 30) and, where practical, on the semen sample used for IVF (IVF SPA, n = 26). Screening SPA score did not correlate to IVF result, fertilization and cleavage rates were similar between normal (n = 17) and low (n = 13) SPA groups, pregnancy rates were 35 and 46%, respectively. In addition, SPA score at the time of IVF did not correlate with IVF result. Pregnancy rates were 33% for the normal group (n = 16) and 30% for the low SPA group (n = 10). Overall, the low SPA group (n = 16) exhibited a 78% fertilization rate and a 38% pregnancy rate, which was not different from the normal SPA group: 76 and 29%, respectively. The results of this study indicate that SPA score is a poor indicator of sperm function in IVF.     

Prediction of in vitro fertilization outcome by sperm penetration assay with TEST-yolk buffer preincubation.

OBJECTIVE: To evaluate sperm penetration assay (SPA) value as a screening tool before in vitro fertilization (IVF). DESIGN: Follow-up study comparing sperm variables and IVF outcome. SETTING: Infertile couples in an academic research environment. PATIENTS, PARTICIPANTS: Two hundred forty-one infertile couples scheduled for IVF. INTERVENTIONS: Sperm penetrating assay with cold Tes-TRIS (TEST) Yolk buffer semen preincubation and IVF. MAIN OUTCOME MEASURES: Percent of egg penetration recoded into poor and good category (0% to 20%, 21% to 100%) and compared with fertilization, embryo transfer, and pregnancy rate (PR) in IVF, as well as sperm count motility and morphology. RESULTS: Sperm penetrating assay predicted fertilization with a high negative (74%) and positive (82%) predictive rate and good specificity value (0.96). One of 31 patients in the poor SPA category (3%) fertilized less than 50% of eggs; no pregnancy occurred. In the good SPA category, 87 of 210 patients (41%) fertilized greater than or equal to 50% of eggs with 34.3% PR. Sperm penetrating assay correlated better with fertilization rate than did sperm count and motility but not morphology. CONCLUSIONS: Sperm penetrating assay is a useful screening assay before IVF together with sperm morphology.     

Correlation between the zona-free hamster egg sperm penetration assay and human in vitro fertilization

The association between in vitro fertilization (IVF) and the zona-free hamster egg sperm penetration assay (SPA) was studied in 134 couples. The indications for IVF were tubal disease in 82 couples, unexplained infertility in 23, male infertility in 10, and combined mechanical and male factors in 19. In general, a positive SPA was a good predictor of subsequent IVF (91 of 107 couples). Specificity (number of couples with fertilization in vitro divided by the number of couples with a positive SPA times 100) was 94% for tubal infertility, 76% for unexplained infertility, and 46% for male infertility. A negative SPA predicted an overall fertilization failure of 78% (21 of 27 couples). Sensitivity of the test (number of couples with IVF failure divided by the number of couples with a negative SPA times 100) was 100% for tubal and unexplained infertility but only 63% for male infertility. The high predictive value of the SPA for subsequent IVF outcome in tubal infertility and unexplained infertility warrants its routine use for prescreening in IVF programs. Because of the lack of association between SPA and IVF in oligoasthenospermia, the bioassay should not be relied upon for predicting IVF outcome in male subfertility. The overall high association between the animal model and human IVF reinforces the use of the SPA for both basic research and clinical decision-making.     

An evaluation of various treatments to increase sperm penetration capacity for potential use in an in vitro fertilization program.

OBJECTIVE: To select in vitro fertilization (IVF) patients with a low sperm penetration assay (SPA) value and to determine if the penetration rate, fertilization rate, and the pregnancy rate (PR) can be improved in these patients by treating sperm with refrigeration, calcium ionophore A23187, prostaglandin E1, prostaglandin E2, or heparin. DESIGN: The study consists of three parts: identification of patients with poor SPA values, analysis of treatments to improve the SPA value, and evaluation of the treatments to improve fertilization and PRs. RESULTS: The data indicate that treatment of sperm with refrigeration can improve fertilization and PRs during IVF in selected patients previously shown to have an improved SPA value with the treatment. CONCLUSIONS: Three points are emphasized: (1) the treatments analyzed in this study can improve SPA values in some of the patients with low sperm penetration capacity; (2) of the treatments studied, sperm refrigeration resulted in the largest improvement in sperm penetration capacity; and (3) sperm refrigeration can increase fertilization and PRs during IVF in this select group of patients.     

Penetration of zona-free hamster oocytes using human sperm aspirated from the epididymis of men with congenital absence of the vas deferens: comparison with human in vitro fertilization.

OBJECTIVES: To assess the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes. To directly compare the performance of human epididymal sperm in the zona-free hamster oocyte sperm penetration assay (SPA) with the results of human in vitro fertilization (IVF). DESIGN: Sperm penetration assay was carried out with epididymal sperm retrieved microsurgically, and with ejaculated sperm obtained from fertile donors (internal controls). For direct comparison, SPA was performed with the same epididymal sperm sample used for IVF. PATIENTS, PARTICIPANTS: Men with congenital absence of the vas deferens undergoing sperm aspiration as part of their infertility treatment and control donors who provided ejaculated sperm. RESULTS: Epididymal sperm penetrated SPA with a score of 0% to 30%. The SPA scores for internal controls using ejaculated sperm was 30% to 71%. Linear regression analysis of the association between penetration scores in SPA and fertilization rate in IVF indicated a positive correlation that was highly significative. CONCLUSIONS: These findings using SPA confirm previous reports on the fertilizing potential of human epididymal sperm and its ability to produce normal pregnancies. The good correlation between SPA and human IVF using epididymal sperm suggest that SPA is an excellent bioassay to test laboratory experimental conditions for improving fertilizing capacity of human epididymal sperm.     

The predictive value of zona-free hamster egg sperm penetration assay for failure of human in vitro fertilization and subsequent successful zona drilling

The value of various sperm parameters and the zona-free hamster egg sperm penetration assay (SPA) in predicting human in vitro fertilization (IVF) failure and subsequent successful fertilization with zona drilling was assessed. In 19 couples, throughout 31 IVF cycles, a total of 153 oocytes failed to be fertilized. In subsequent 12 cycles with zona drilling, 33 of 131 (25%) were fertilized. The incidence of teratospermia and asthenospermia was significantly higher in the study group than in the control, 74% versus 32% and 42% versus 5%, respectively. Although the mean values for the performance of sperm in SPA and fertilization of human eggs after zona drilling were remarkably similar (28 +/- 6 versus 28 +/- 4), there was no correlation between individual parameters (r = 0.15). Thus, whereas male factor infertility is more likely to be associated with teratoasthenospermia, neither the SPA nor other sperm parameters have any predictive value for failure in IVF. In addition, no criterion of sperm function has yet been identified that would eliminate oligoteratoasthenozoospermic males from consideration of IVF with zona drilling.     

Importance of sperm motility after capacitation in interpreting the hamster ovum sperm penetration assay

Routine semen analysis and a zona-free hamster ovum sperm penetration assay (SPA) were carried out for 220 men from a group of infertile couples. The grade of sperm motility was determined after a preincubation period. For both the normal and abnormal semen samples, the number of positive SPAs was significantly higher when the postcapacitation motility was normal. This difference was observed also when the routine semen characteristics of density, motility, and morphologic features were considered separately regarding their relationship with the SPA. Motility after preincubation showed the most significant positive correlation with the SPA result. Therefore, the SPA score should be limited to semen samples showing adequate postincubation motility.     

Establishment of TEST-yolk buffer enhanced sperm penetration assay limits for fertile males

TEST-yolk buffer has been shown to enhance sperm penetration of zona-free hamster eggs. Review of sperm penetration assay (SPA) data from a fertile population was undertaken to determine a normal range for SPA with TEST-yolk buffer enhancement. Thirty-eight intrauterine insemination patients and 4 artificial insemination donors who had successfully initiated a pregnancy within 18 months of SPA analysis were examined. All 42 enhanced SPAs demonstrated penetration of greater than 10%, and 37 of these (88%) yielded SPA values of greater than or equal to 20%. Thirty-three percent (14/42) of these individuals achieved 0% penetration in the SPA without TEST-yolk buffer. The SPA performed with the TEST-yolk modification has fewer false negatives than the assay done with the original methodology.     

Male partner screening before in vitro fertilization: preselecting patients who require intracytoplasmic sperm injection with the sperm penetration assay

Objectives: To determine the diagnostic accuracy of the sperm penetration assay (SPA) and standard semen parameters for subsequent fertilization in in vitro fertilization-embryo transfer (IVF-ET).

Design: Prospective study.

Setting: Andrology Laboratory, and university research laboratory.

Patients: Two hundred sixteen couples undergoing male-partner screening before IVF-ET (265 cycles).

Intervention(s): Male-partner screening (semen analyses [SA] and SPA), standard IVF-ET procedures, follow-up of fertilization in IVF-ET.

Main Outcome Measure(s): Diagnostic accuracy of SA and SPA for prediction of fertilization in IVF-ET.

Result(s): The SPA predicted IVF fertilization with high negative (84%) and positive (98%) predictive rates, and correct prediction in 88% of cycles. In contrast, sperm concentration, motility, morphology, and complete SA showed poor diagnostic accuracy, with correct prediction of IVF fertilization in 64%, 65%, 45%, and 68% of cycles, respectively.

Conclusion(s): Very low sperm concentration and/or motility were good predictors of poor IVF fertilization, however, low to normal semen parameters were not predictive of successful IVF fertilization. The SPA is a useful screening tool that predicts IVF fertilization with high diagnostic accuracy. The SPA may be useful to discriminate between those couples with a high probability of normal fertilization in IVF and those with a low probability of normal fertilization that may benefit from assisted fertilization by intracytoplasmic sperm injection (ICSI).     

Testing the fertilizing ability of motile spermatozoa separated by Percoll in patients with abnormal sperm analysis or sperm penetration

The Percoll density-gradient technique was used to select motile sperm from men who exhibited abnormal semen analysis or unexplained infertility. The fertilizing ability of the spermatozoa was evaluated by the hamster ova sperm penetration assay (SPA). The current work is a novel approach in the usage of Percoll in an attempt to improve the quality of the sperm of nonfertile patients and to examine its results by SPA. The method succeeded in improving the penetration score of the fertile controls by 15% to 30%. However, no improvement of the low penetration score of the nonfertile patients was demonstrable. The difficulties inherent in using the Percoll density gradient technique are discussed.     

The relationship between the human sperm hypoosmotic swelling test, routine semen analysis, and the human sperm zona-free hamster ovum penetration assay

The functional integrity of sperm membranes of 270 semen samples collected from fertile men and the male partners in couples with infertile marriages was assessed by the hypoosmotic swelling test and the results correlated with routine semen analysis and the human sperm zona-free hamster ovum penetration assay. Semen samples with abnormal semen parameters had lower values of percentage of swollen sperm after hypoosmotic treatment in comparison with those with normal semen parameters. A weak positive correlation was observed between sperm swelling and sperm morphologic features (r = 0.32, P less than 0.05) and between sperm swelling and sperm motility (r = 0.22, P less than 0.05). Insignificant correlation was observed between sperm swelling and in vitro sperm fertilizing capacity, as assessed by the zona-free hamster ovum penetration assay. The results indicate that the sperm swelling test and the zona-free hamster ovum penetration assay are evaluating different functional qualities of sperm that are apparently not associated with each other.     

Morphology and the sperm penetration assay

OBJECTIVE: To assess the ability of Kruger morphology to predict the outcome of the sperm penetration assay (SPA). DESIGN: A retrospective review using univariate and multivariate analysis, t tests, and logistic regression was performed to examine the correlation between Kruger morphology and the optimized zona-free hamster egg sperm penetration assay (O-HESPA). SETTING: University hospital. PATIENT(S): One hundred fifteen private-practice patients who underwent semen analysis, including Kruger morphology and O-HESPA, as part of an infertility evaluation between 1997-2000 were retrospectively reviewed. INTERVENTION(S): Retrospective analysis of the sperm penetration assay. MAIN OUTCOME MEASURE(S): Univariate and multivariate analysis, Student's t test, and logistic regression were performed to analyze Kruger morphology, count, and viability and their relationship to SCI result. RESULT(S): Univariate analysis demonstrated a statistically significant correlation between SCI and sperm count, viability, and Kruger morphology. Multivariate analysis demonstrated statistically significant correlations between SCI and count and viability. There was no correlation between Kruger morphology and SCI. Logistic regression was performed on the SCI results, using count, Kruger morphology, and viability. Sperm count was found to be the only variable that was statistically significant with respect to SCI results. CONCLUSION: This study demonstrated that Kruger morphology assessment cannot be used to predict the results of SCI or replace it in the management of the infertile couple.     

Effect of limited proteolysis with α-chymotrypsin on semen with an abnormal sperm penetration assay and possible application for in vitro fertilization or intrauterine insemination

Purpose This study was designed to assess the impact of limited proteolysis with -chymotrypsin on the sperm penetration assay (SPA) of infertile patients and to identify a group whose results would normalize with this pretreatment. Further, the application of this treatment to semen of these patients during in vitro fertilization (IVF) and intrauterine insemination (IUI) was reported.Results Three groups were identified. In one, SPA was abnormal and improved to normal with pretreatment; in the second, SPA was abnormal and did not normalize; and in the third, SPA was normal and improved significantly (included in this group are three known fertile controls).Conclusion Chymotrypsin pretreatment and repeat SPA are advocated for patients with abnormal SPA. If normalization occurs, pretreatment of semen for IVF or IUI is a therapy to be considered.     

In vitro fertilization in couples with male factor infertility

The data from 83 consecutive in vitro fertilization-embryo transfer cycles were examined with emphasis on the presence of subfertile male parameters, including abnormal sperm density, motility, morphologic features, or an abnormal result in the sperm penetration assay (zona-free hamster ova penetration). There were 25 cycles (21 couples) in which there was a solitary or coexistent abnormal male factor. The presence of a male factor was associated with a lower fertilization rate and fewer embryos transferred. Statistically important reductions in the fertilization rate occurred with reduced sperm density and abnormal motility, but not with abnormal morphologic features. The parameter that was most significantly associated with human ova fertilizability was the ability to exceed two penetrations per egg in the sperm penetration assay.     

Enhanced gamete interaction in the sperm penetration assay after coincubation with pentoxifylline and human follicular fluid.

OBJECTIVE: To evaluate the effect of pentoxifylline and heat-inactivated human follicular fluid (FF) on performance in the sperm penetration assay (SPA) as a paradigm for the effect of these agents on human sperm-egg interaction in vivo and in vitro fertilization. DESIGN: Semen specimens from men undergoing SPA testing for evaluation of suspected male factor infertility were coincubated with neat medium or media supplemented with pentoxifylline or human FF in a nonblinded manner. PARTICIPANTS: Twenty male factor infertility patients. INTERVENTIONS: Semen specimens were preincubated with: [1] pentoxifylline 0.25 mg/mL; [2] 10% human FF; [3] pentoxifylline+human FF; and [4] neat Biggers, Whitten, and Whittingham medium. MAIN OUTCOME MEASURES: Differences in the rate of penetration of zona-free hamster oocytes. RESULTS: Preincubation with either human FF or pentoxifylline produced a significant improvement in hamster egg penetration rates. Coincubation with a combination of human FF and pentoxifylline resulted in a significant enhancement of penetration as compared with single agent treatment. CONCLUSIONS: Coincubation of sperm with human FF and pentoxifylline may provide a means of enhancing sperm activity for insemination and assisted reproduction.     

Relationship between fertility, semen analysis and human sperm penetration of zona-free hamster eggs

Spermatozoa from 12 fertile donors, 37 infertile patients with normal spermiogram and 61 infertile men with abnormal semen analysis were assessed with zona-free hamster eggs penetration test, according to Barros. We obtained significant differences in positive penetrations in the 3 groups tested: 96 per cent of positive results for the first group, 59 per cent for the second and 21 per cent for the third group. A significant relationship between penetration results and sperm count, motility and morphology is evidenced.     

The human sperm-hamster egg penetration assay: prognostic value

Males from 227 infertile couples were evaluated using the human sperm-hamster egg penetration assay (SPA). Indications for the SPA were abnormal semen analyses, poor postcoital tests, documented autologous sperm antibodies, and long-term unexplained infertility. Normal results defined as greater than or equal to 11% penetration were seen in 58.6% of couples. Penetration rates of 1% to 10% were observed in 25.6%, and 15.9% failed to penetrate any of the oocytes. During the follow-up period, with a mean of 17.9 months, 26.9% conceived at least once with or without treatment. Monthly fecundity was 0.014 for normal SPA patients and 0.007 for the abnormal SPA group when calculated by the Kaplan-Meier survival curve with Cox correction for unequal follow-up. The monthly fecundity rate at any time during the 30-month interval of follow-up was twice as great for men with normal SPA values as for those with abnormal values, regardless of male or female diagnosis or therapy indicating the prognostic value of the SPA in an infertile population.     

Correlations between the human sperm-hamster egg penetration assay and in vitro fertilization results

The human sperm-hamster egg penetration assay (SPA) has been used to evaluate male fertility under in vivo conditions. The test is not only unstandardized, but agreement does not exist as to the lower limits of normal penetration rates. Therefore, it is not surprising that there is considerable doubt as to whether any correlation exists between SPA results and the behavior of sperm in an in vitro laboratory situation. We investigated this question by examining the SPA results and the subsequent fertilization and cleavage rates in the in vitro fertilization program at Pennsylvania Hospital. The sperm from men whose SPA results were 11% or more fertilized 86% of the morphologically normal and mature human oocytes to which they were exposed. Men whose SPA scores ranged from 1% through 10% fertilized 65% of human oocytes, and men whose sperm failed to penetrate hamster eggs still were able to fertilize 41% of the human oocytes. The differences between these groups are statistically significant. This information can be of practical prognostic value during in vitro fertilization cycles in which the number of mature oocytes seen ultrasonographically appears to be at a minimum.     

Effect of aging and cold temperature storage of hamster ova as assessed in the sperm penetration assay

The penetration of zona pellucida-free hamster ova by human spermatozoa has been used to quantitate sperm penetration potential. However, since mammalian eggs in vitro have limited viability, the effect of in vitro aging on the ability of hamster ova to be penetrated by human spermatozoa was examined. Zona-free ova maintained at room temperature (25 degrees C) lost their ability to be subsequently penetrated with a half-life of 50.1 +/- 8.8 minutes. This was partly the result of removing the zona pellucida by trypsin digestion, since zona-free oocytes in the presence of trypsin inhibitor or zona pellucida-intact oocytes had half-lives of 99.1 +/- 15.2 and 120.5 +/- 17.4 minutes, respectively. Reduction in penetration rates associated with ovum aging did not appear to be due to loss of viability and could be completely prevented by maintaining the ova on ice (4 degrees C). In the presence of TEST-yolk buffer at 4 degrees C, ova retained (100%) their ability to be penetrated for up to 24 hours and were morphologically indistinguishable from fresh ova. These observations show that ovum aging in vitro at 25 degrees C is much greater than previously anticipated. This may result in artifactually low and variable scores in the penetration bioassay.