The use of semen parameters to identify the subfertile male in the general population.

AIMS: To present a structured review of the literature published on semen parameters and in vivo fertility potential and to establish fertility/subfertility thresholds for sperm morphology using Tygerberg strict criteria, sperm concentration, and sperm motility. METHOD: The published literature comparing fertile and subfertile populations between 1983 and 2002 was reviewed. RESULTS: A total of 265 articles were identified by the sourcing methodology, but only four articles provided data that could be tabulated and analyzed. Using receiver-operating characteristics curves, morphology proved to be the best predictor of subfertility in 2 of the 4 articles, with concentration and motility also showing good predictive power. The thresholds calculated ranged between 4 and 10% for morphology, between 13.5 x 10(6)/ml and 34 x 10(6)/ml for concentration, and between 32 and 52% for motility. A second set of much lower thresholds was calculated in three of the articles using either a 15 or 50% prevalence of subfertility in the population or the tenth percentile of the fertile population. The adjusted thresholds were between 3 and 5% for morphology, between 9 x 10(6)/ml and 20 x 10(6)/ml for concentration, and between 20 and 30% for motility. CONCLUSIONS: Because these lower thresholds have a much higher positive predictive value, we suggest that thresholds of <5% normal sperm morphology, a concentration <15 x 10(6)/ml, and a motility <30% should be used to identify the subfertile male. The lower threshold for morphology also fits in vitro fertilization and intrauterine insemination data calculated previously. Using the parameters in combination increases the clinical value of semen analysis.

Physiological induction of the acrosome reaction in human sperm: validation of a microassay using minimal volumes of solubilized, homologous zona pellucida.

PURPOSE: To develop a method that could accommodate microvolumes of solubilized human zona pellucida (ZP) and sperm for assessing the induction of the acrosome reaction. METHODS: A microassay using 1 microliter of 2.5, 1.25, 0.6, 0.3, and 0.125 ZP/microliter incubated with 1 microliter of a highly motile sperm suspension for 60 min. As a control and parallel to the microassay a standard acrosome reaction technique was performed. RESULTS: No significant differences were observed between the percentage acrosome reacted sperm reported by the two assays under basal conditions (spontaneous) or after induction with a Ca2+ ionophore or solubilized ZP. At a ZP concentration of 0.6 ZP/microliter, the percentages of acrosome-reacted spermatozoa in both techniques were significantly higher compared to the spontaneous acrosome reaction results, namely, 18% and 17%, compared to 10% and 10%, respectively. An approximately 30% level of acrosomal exocytosis was induced with 2.5 ZP/microliter in both methods. CONCLUSIONS: This newly devised microtechnique is easy and rapid to perform, is repeatable and facilitates the use of minimal volumes of solubilized human ZP (even a single ZP) for assessment of the inducibility of the acrosome reaction of a homologous sperm population.

Physiological induction of the acrosome reaction in human sperm: validation of a microassay using minimal volumes of solubilized, homologous zona pellucida.

PURPOSE: The objective was to develop a method that could accommodate microvolumes of solubilized human (ZP) and sperm for assessing the induction of the acrosome reaction. METHODS: A microassay using 1 microliter of 2.5, 1.25, 0.6, 0.3, and 0.125 ZP/microliter incubated with 1 microliter of a highly motile sperm suspension for 60 min. As a control and parallel to the microassay a standard acrosome reaction technique was performed. RESULTS: No significant differences were observed between the percentage acrosome-reacted sperm reported by the two assays under basal conditions (spontaneous) or after induction with a Ca2+ ionophore or solubilized ZP. At a ZP concentration of 0.6 ZP/microliter, the percentages of acrosome-reacted spermatozoa in both techniques were significantly higher compared to the spontaneous acrosome reaction results, namely 18% and 17%, compared to 10% and 10%, respectively. Approximately a 30% level of acrosomal exocytosis was induced with 2.5 ZP/microliter both methods. CONCLUSIONS: This newly devised microtechnique is easy and rapid to perform, is repeatable, and facilitates the use of minimal volumes of solubilized human ZP (even a single ZP) for assessment of the inducibility of the acrosome reaction of a homologous sperm population.

In vitro fertilization failure: identification of gamete defects by investigation of sperm-zona pellucida binding capacity of unfertilized oocytes.

The objective of the study was to determine whether fertilization failure was due to spermatozoal or oocyte factors. Twenty-five unfertilized oocytes from 12 IVF/GIFT couples showing total or partial fertilization failure were evaluated for sperm zona binding potential under hemizonaeassay (HZA) conditions. Hemizonae were separately incubated with a sperm sample from the husband and that of a fertile control. Tight sperm binding to hemizonae was assessed. First, among the 12 patients, results showed a possible zona defect thought to be the cause of fertilization failure in five cases. Second, in two cases, fertilization failure was possibly caused by poor sperm binding potential of spermatozoa. Third, in two cases, fertilization failure was possibly caused by an oocyte defect, and fourth, three cases showed a mixture of possible causes. The results stress the need to develop a sequential analytic programme for those couples with repeated total or partial fertilization failure.

Sperm binding capacity of human zona pellucida derived from oocytes obtained from different sources.

The important contributions of sperm-oocyte interaction to infertility diagnostics is well established. Scientists are urged to search for methods to improve the assessment of gamete interaction. Sperm binding and penetration assays have frequented the literature, reporting on various aspects of sperm-oocyte interaction using either microbisected or whole human oocytes during the assay procedure. The objective of the study was to evaluate additional zona pellucida sources which can be used during zona binding studies. Hemizonae were obtained from the following oocytes: 1) experiment 1, prophase I oocytes from post-mortem ovarian tissue from different age groups namely, 7 months, 5 years, 7 years, 12 years and 30 years; 2) experiment 2 used donated immature Prophase I oocytes from the IVF treatment program and 3) experiment 3 evaluated zona binding for hemizonae which were previously used in hemizona assays. Results indicated that, in experiment 1, ovarian age does not have any influence on the zona pellucida's capacity to bind spermatozoa. The mean number of bound sperm among the different age groups did not differ significantly, namely 38.9 +/- 17 (7 months), 31.0 +/- 27 (5 years), 49.3 +/- 21 (7 years), 32.8 +/- 18 (12 years) and 39.5 +/- 17 (30 years). The pooled mean +/- SD binding for all the age groups in experiment 1 was 37.7 +/- 7. Likewise, the mean number of sperm bound (experiment 2) to zonae collected from oocytes using different ovulation induction regimes were 31.1 +/- 20 (unstimulated), 54.4 +/- 12 (HMG/HCG) and 15.3 +/- 9 (HMG alone).(ABSTRACT TRUNCATED AT 250 WORDS)

Hemizona index (HZI) demonstrates excellent predictability when evaluating sperm fertilizing capacity in in vitro fertilization patients.

The objective of this work was to assess the power of the hemizona assay to predict in vitro fertilization (IVF) results using receiving operating characteristics (ROC) analysis. In a tertiary medical center setting, 94 couples undergoing IVF were included in the study comparing their fertilization results to sperm function during the hemizona assay. Using ROC analysis, fertilization in IVF (mature oocytes only) was compared to performance of the sperm in the sperm-zona pellucida binding test as expressed by hemizona index (HZI; [number of test sperm bound/number of control sperm bound] x 100). The IVF results were as follows: 32 patients had no fertilization (0%), 55 patients fertilized all of the oocytes (100%), and 7 patients fertilized between 14% and 85% of the oocytes. Modified ROC analysis was used to determine the best threshold for fertilization success or failure, depending upon how the data are viewed. A HZI of 40-43 separated the 100% fertilization group from the other groups, with a true positive rate of 87% and a true negative of 83%. Conversely, a HZI of 4-5 separated the 0% fertilization versus partial or total fertilization, with a true positive rate of 100% and true negative rate of 71%. Sperm performance in the hemizona assay as measured by the HZI reflects performance in IVF and benefits couple evaluation and counseling.

Wheat germ agglutinin receptors on human sperm membranes and sperm morphology.

Sperm membrane components play an important role in the determination of sperm fertilizing ability. During spermatogenesis, epididymal transit, and capacitation, the sperm membrane undergoes various subtle changes which are important maturational events for the production of viable spermatozoa and hence partly determine fertility. Localization and intensity of wheat germ agglutinin (WGA) receptors are reported to be directly associated with male fertility. We examined the correlations and differences between the distribution patterns and intensities of WGA receptors of 75 semen samples, classified according to the strict criteria described for sperm morphology, namely P-pattern (0-4% normal forms, n = 19), G-pattern (5-14% normal forms, n = 41) and normal samples (> 15% normal forms, n = 15). Results indicate distinct differences in WGA receptor intensity in all of the three groups as well as significantly (P < 0.05) lower per-cent staining of the equatorial segment amongst the P-pattern group when compared to the other two groups. It would appear that there is a close relationship between WGA receptors on human sperm membranes and sperm morphology.

A new computerized method of reading sperm morphology (strict criteria) is as efficient as technician reading.

OBJECTIVES: To compare the ability of a computerized method of sperm morphology with the manually recorded method in predicting in vitro fertilization (IVF) results, to compare results obtained by both methods, and to determine the intraobservation variability. DESIGN, SETTING, PATIENTS: Forty-three stained semen slide preparations from two large level-three academic institutions' reproductive endocrinology units (IVF programs) were blindly evaluated, and the sperm were classified into normal and amorphous forms. RESULTS: Experiment 1: Twenty-one slide preparations from the Tygerberg gamete intrafallopian transfer program were manually evaluated; the fertilization rates for the groups with < 14% and > 14% normal sperm forms were 33.3% (15/45 oocytes) and 76.6% (46/60 oocytes), respectively. Corresponding fertilization rates with FERTECH were 46.8% (30/64) and 75.6% (31/41). Experiment 2: Twenty-two slide preparations from the Norfolk IVF program were evaluated. The manual method reported a fertilization rate in the group with < 14% normal forms of 27.4% (14/51 oocytes) compared with 90.0% (127/141 oocytes) in the group with > 14% normal forms. Corresponding figures for the FERTECH method were 33.9% (18/53) and 88.4% (123/139), respectively. Experiment 3: When the 43 slide preparations were blindly evaluated using both methods, 84% of the FERTECH evaluations correlated well with the manual method and FERTECH ability to diagnose the subfertile male (< 14% normal forms) was 95% (sensitivity). Experiment 4: A total of 16 different slides (8 per group) were randomly selected and analyzed five times (100 cells per reading) by the computerized method. The slides were obtained from men with normal sperm morphology of < 14% and > 14% as classified by the manual method. In the first group (< 14%) 97.5% (39/40) of the readings classified the sperm in the proper category, whereas in the second group (> 14%) 95% (38/40) of the cases were correctly identified. CONCLUSION: Using strict criteria for morphology evaluation, there is a positive and significant correlation between FERTECH evaluation and manual assessment. The reproducibility of the computerized method and the ability to distinguish between fertile and subfertile groups using those criteria are good.

Defining the valid hemizona assay: accounting for binding variability within zonae pellucidae and within semen samples from fertile males.

OBJECTIVE: To achieve a better understanding of the variability in sperm and oocyte binding capacities will optimize use of the hemizona assay (HZA) as a predictor of sperm function. DESIGN: Limitations of the HZA were more clearly delineated by current studies: (1) variability of sperm binding capacity of men over a 90-day interval; (2) variability of sperm binding using different oocytes; and (3) lower limits of the number of sperm bound from the fertile control in two laboratories. PATIENTS: Semen was obtained from proven fertile men and one subfertile individual. MAIN OUTCOME MEASURE: The number of sperm tightly bound to the hemizona were measured and compared. RESULTS: In the initial study, 6 fertile control men exhibited a similar degree of variability in zona binding when studied over a 90-day interval. Average sperm binding for individuals ranged from 68 to 127. Second, 3 of the 15 simultaneous assays showed very low numbers of sperm bound, indicating that 20% of the zonae had poor binding. Third, from 18 men who had 0% fertilization in an in vitro fertilization system using mature oocytes, evaluation of their sperm by HZA was performed. The sperm bound poorly and the 95% confidence interval was 20 sperm bound. Thus, the fertile controls should bind greater than 20 sperm to distinguish them from the infertile group in the HZA system resulting in a valid assay. CONCLUSIONS: With these guidelines, applications of the HZA may be made with greater reassurance of a valid bioassay of sperm fertilizing potential.

The gonadotropin-releasing hormone agonist stimulation test--a sensitive predictor of performance in the flare-up in vitro fertilization cycle.

OBJECTIVE: To evaluate the initial versus early pattern of estradiol (E2) change after administration of a gonadotropin-releasing hormone agonist (GnRH-a), i.e., the GnRH-a stimulation test versus E2 pattern, respectively, as predictors of ovarian response and pregnancy in in vitro fertilization (IVF) patients stimulated with a flare-up protocol. DESIGN: Prospective study in a consecutive group of patients. SETTING: Tertiary care, institutional setting. PATIENTS: Two hundred twenty-eight patients entered and completed the study. The only patients excluded from study were those anticipated to have polycystic ovarian disease, those with a single ovary, or those with an ovarian cyst(s). INTERVENTIONS: Patients were stimulated with a GnRH-a flare-up protocol beginning on menstrual day 2. MAIN OUTCOME: Evaluation of the GnRH-a stimulation test and the E2 pattern as predictors of the number of mature oocytes retrieved and pregnancy. RESULTS: The GnRH-a stimulation test but not the E2 pattern was predictive of the number of mature oocytes retrieved (r = 0.53, P less than 1 X 10(-5) and pregnancy (chi 2 = 8.5, P = 0.04). The E2 pattern was predictive of the duration and number of ampules of gonadotropin required for stimulation. CONCLUSION: The GnRH-a stimulation test is a sensitive predictor of performance in the flare-up IVF cycle.

Critical assessment of sperm morphology before and after double wash swim-up preparation for in vitro fertilization.

Critical assessment of sperm morphology using specific and stringent criteria is predictive of the subsequent ability of those sperm to fertilize oocytes in in vitro fertilization (IVF). Previous studies have evaluated sperm morphology prior to sperm preparation and, thus, have not assessed the actual sperm used for insemination. We studied the impact of a double wash swim-up technique used for IVF on sperm morphology using the strict criteria of Kruger et al. [5, 6] in 73 consecutive patients undergoing IVF. Pre- and postswim-up morphological assessments were done in a prospective, randomized, and blinded fashion. The mean percentage of normal forms pre- and postswim-up was 19.8% and 23.4%, respectively, an improvement of 18% (p less than 0.05) with 62 of 73 patients showing improvement. Significantly, analysis of the 27 patients with abnormal morphology on initial assessment (normal forms less than or equal to 14%) revealed an improvement in percentage of normal forms from 9.0% to 21.5%, a mean increase of 239% (p less than 0.005). Eighteen of these 27 patients showed improvement in their percentage of normal forms, eight were unchanged, and only one patient had a decline in their percentage of normal forms. We conclude that the double wash swim-up preparation used for IVF substantially improves the percentage of sperm with normal morphology, and that the benefit is most substantial in those cases where the percentage of normal morphology is abnormal.

Hemizona assay using salt-stored human oocytes: evaluation of zona pellucida capacity for binding human spermatozoa.

Human oocytes were stored (25 degrees C) in 1.5 M MgCl2 for 6-30 days, then utilized in the new hemizona assay (HZA) for tight binding of human spermatozoa [Burkman et al.: Fertil Steril 49:688-697, 1988]. We have compared 1) the ability of matching salt-treated hemizonae or dimethylsulfoxide (DMSO)-treated hemizonae to distinguish between sperm from semen having normal versus subnormal characteristics and, 2) the kinetics of fertile sperm binding to salt-treated or DMSO-treated hemizonae. After sperm preparation one salt-treated hemizona was incubated with normal spermatozoa and the matching hemizona was placed with sperm from the subnormal group. As a control, DMSO-treated hemizonae were incubated in additional sperm droplets. After 4 hours, the number of sperm tightly bound to each hemizona was counted. Within the normal semen group, there was equivalent binding to salt- or DMSO-treated hemizonae (54.0 +/- 12 and 49 +/- 14, respectively, mean +/- SEM). Similarly, tight binding of sperm from the subnormal group was not affected by the zona storage method (21 +/- 8 and 17 +/- 5, respectively). For either storage approach, binding of subnormal sperm was significantly less (P less than 0.01) compared with the number of normal sperm attached to the matching hemizona. For the kinetics study, the hemizona binding of proven fertile spermatozoa was followed throughout 8.5 hours. The shape of the binding curve was the same for zonae stored by either method and was consistent with our published kinetics data. Salt storage offers a simple and inexpensive means for accumulating and transporting human zonae pellucida; the resulting hemizonae function effectively in the HZA for estimating sperm binding potential.