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Objective To review the clinical value of sperm-oocyte interaction tests for the diagnosis and management of infertility by standard IVF or intracytoplasmic sperm injection (ICSI). Design Review of recent publications on relationships among sperm-oocyte interaction tests, sperm characteristics, and results of IVF and determination of frequency of defective sperm-oocyte interaction in infertile men. Main outcome measure(s) Fertilization rates with IVF, sperm characteristics, sperm–zona pellucida (ZP) binding, ZP-induced acrosome reaction (AR), and sperm-ZP penetration. Result(s) Sperm defects associated with low sperm-ZP binding or impaired ZP-induced AR and sperm-ZP penetration are the major causes of failure of fertilization when all or most oocytes from a couple do not fertilize in standard IVF. There is a high frequency of defective sperm-ZP interaction in men with oligozoospermia ( 6 /mL) and severe teratozoospermia (strict normal sperm morphology ≤5%). Sperm morphology correlates with sperm-ZP binding, and sperm concentration correlates with ZP-induced AR in infertile men with sperm concentrations >20 × 10 6 /mL. Defective ZP-induced AR may cause infertility in up to 25% men with idiopathic infertility. These patients require ICSI despite the normal standard semen analyses. Conclusion(s) Sperm-oocyte interaction tests are useful for diagnosis of subtle sperm defects that cause infertility in men without severe abnormalities of semen analysis. Pre-IVF diagnosis of these sperm defects will assist in the clinical assignment of patients to treatment with either standard IVF or ICSI.

BACKGROUND: Standard semen analysis has low objectivity and reproducibility and is not closely related to fertility. We assess the prognostic value of automated measurements of sperm motility and morphology. METHODS: During 1997‐1999, 1191 infertile couples with no known absolute barrier to conception were assessed by conventional semen analysis, and automated measurements of average straight-line velocity (VSL) and the percentage of sperm with characteristics that conform to those of sperm which bind to the zona pellucida of the human oocyte (%Z). During follow-up to 2001, there were 336 natural pregnancies. RESULTS: Only %Z, VSL and female age were independently significantly related to pregnancy rate by Cox regression analysis. Pregnancy rate was higher with above average %Z and VSL, indicating a continuous rather than a threshold relationship. The likelihood of pregnancy within 12 cycles can be evaluated for specific values of %Z, VSL and female age using the Cox regression model. CONCLUSIONS: The automated semen measures of sperm morphometry (%Z) and velocity (VSL) are related to pregnancy rates in subfertile couples and should assist clinicians in counselling subfertile patients about their prognosis for a natural pregnancy. Objective automated methods should replace the traditional manual assessments of semen quality.

Results of zona pellucida (ZP)-induced acrosome reaction (AR) are reported for 186 normospermic men with unexplained infertility and compared with 34 normal fertile men and 54 patients with disordered ZP-induced AR (DZPIAR) diagnosed after failure of standard IVF. For each ZP-induced AR test, four oocytes that failed to fertilize in IVF were incubated for 2 h with 2x10(6)/ml motile spermatozoa. Spermatozoa tightly bound to the ZP were recovered by aspirating the oocytes with a pipette and the AR assessed using pisum sativum agglutinin labelled with fluorescein. The standard deviation of the difference was 5.2% for repeated tests for ZP-induced AR on different ejaculates from 54 men. The ranges for the ZP-induced AR were 3-98% for normospermic infertile men, 24-95% for fertile men and 0-16% for DZPIAR patients. In the normospermic group, there was a significant correlation between ZP-induced AR and sperm concentration (Spearman r = 0.238, P < 0.001). Using ZP-induced AR < or =16% as the threshold for diagnosis of DZPIAR, the frequency of this condition in normospermic infertile men would be 25%. Thus DZPIAR is common with normospermic idiopathic infertility and this condition should be diagnosed before assisted reproductive technology since it requires intracytoplasmic sperm injection.

The cervical mucus penetration tests aid research and determine the clinical importance of positive sperm antibody tests. Limited availability and variability of human cervical mucus have instigated the search for mucus substitutes for these tests. This study compares sperm migration in cervical mucus with that in artificial media including hyaluronate solution, egg white and albumin Tyrode solution. Results were quantified by measuring the migration distance (the maximum distance of capillary migration from a semen reservoir by spermatozoa after 1 h) and the sperm concentration at half the migration distance. The mean of both measures for cervical mucus and hyaluronate solution were equivalent [4.4 +/- 1.1 (SD) versus 4.3 +/- 1.0 cm and 118 +/- 51 versus 111 +/- 44x10(3)/ml], and higher than in egg white and albumin Tyrode solution. Antisperm antibodies impaired sperm penetration in cervical mucus and hyaluronate solution in a similar manner (r = 0.92). These results suggest that hyaluronate solution sufficiently resembles human cervical mucus in terms of penetrability that it may be used as a substitute for mucus in capillary tube tests of sperm function. The higher penetrability of cervical mucus and hyaluronate solution is probably related to a channelling effect due to their polymeric structure.

Objective To develop a fully automated image analysis system to provide objective, quantitative, and reproducible assessment of the head morphology of human spermatozoa. Design Shorr-stained smears of washed sperm were imaged with a microscope (×100) and black and white video camera and digitized for computer analysis. Automatically selected sperm head images are analyzed in terms of 32 parameters that reflect size, shape, and staining heterogeneity. Unique features of the analysis include extraction of a 40% density contour in addition to the usual boundary contour and parameterization of optical density profiles. The morphometry of a semen sample is summarized by evaluation of the sample mean and standard deviation for each morphometric parameter. In addition, each analyzed sperm in a sample is assessed for conformity to a reference morphometric data set. Results The system filters seminal debris, superimposed cells, and lysed sperm, with an average sperm recognition error of 2.3% and contour interpretation error of 1.7%. The average proportion of variance attributable to slide preparation and analysis methods (2.6%) was low relative to that between infertility patients (12.4%). The relatively slow assessment times would be improved substantially with upgraded hardware. Conclusion The precision and sensitivity offered by the detailed image analysis of this system provides a powerful tool for morphometric semen analysis.

To determine whether adverse perinatal outcomes are increased in subfertile women.Cohort study.Two tertiary assisted reproductive technologies (ART) centers; Victorian births register.Records of women who registered with the clinics (1991-2000), but did not have an infant using ART, were linked to the birth register (1991-2004) to identify singleton non-ART births within 5 years of registration (N = 2171). Controls, matched by maternal age and year of infant's birth, were selected randomly from birth records (N = 4363).None.Adverse obstetric and perinatal outcomes.After adjusting for confounders, compared with controls, subfertile women had increased odds of hypertension or preeclampsia (adjusted odds ratio [OR] 1.29, 1.02-1.61), antepartum hemorrhage (adjusted OR 1.41, 1.05-1.89), perinatal death (adjusted OR 2.19, 1.10-4.36), low birth weight (adjusted OR 1.44, 1.11-1.85), preterm birth37 weeks (adjusted OR 1.32, 1.05-1.67) or31 weeks (adjusted OR 2.37, 1.35-4.13), and cesarean delivery (adjusted OR 1.56, 1.37-1.77). There was weak evidence for increased birth defects (adjusted OR 1.30, 0.98-1.72) and gestational diabetes (adjusted OR 1.25, 0.96-1.63). No increased risk was found for prelabor rupture of membranes, small for gestational age, or postpartum hemorrhage.Subfertile women with singleton births are at increased risk of several adverse outcomes. These risks should be considered during their antenatal care and when analyzing adverse effects of ART.

background: The reasons for increased birth defect prevalence following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are largely unknown. Classification of birth defects by pathology rather than organ system, and examination of the role of embryo freezing and thawing may provide clues to the mechanisms involved. This study aimed to investigate these two factors. method: Data on 6946 IVF or ICSI singleton pregnancies were linked to perinatal outcomes obtained from population-based data sets on births and birth defects occurring between 1991 and 2004 in Victoria, Australia. These were compared with 20 838 outcomes for singleton births in the same population, conceived without IVF or ICSI. Birth defects were classified according to pathogenesis. results: Overall, birth defects were increased after IVF or ICSI [adjusted odds ratio (OR) 1.36; 95% CI: 1.19– 1.55] relative to controls. There was no strong evidence of risk differences between IVF and ICSI or between fresh and thawed embryo transfer. However, a specific group, blastogenesis birth defects, were markedly increased [adjusted OR 2.80, 95% CI: 1.63 –4.81], with the increase relative to the controls being significant for fresh embryo transfer (adjusted OR 3.65; 95% CI: 2.02 –6.59) but not for thawed embryo transfer (adjusted OR 1.60; 95% CI: 0.69 –3.69). conclusion: Our findings suggest that there is a specific risk of blastogenesis birth defects arising very early in pregnancy after IVF or ICSI and that this risk may be lower with use of frozen-thawed embryo transfer.

In the literature there is still confusion whether acrosome-reacted sperm in medium can initiate primary binding to human zona pellucida (ZP). The ability of acrosome-reacted sperm to bind to ZP in vitro can be deduced by measuring the acrosome reaction (AR) of ZP-bound sperm compared with sperm in medium after incubation under different conditions inhibiting the ZP-induced AR. Motile sperm from fertile men, normospermic men and infertile men diagnosed with disordered ZP-induced AR (DZPIAR) were selected by swim-up (2 x 10(6) in 1 mL medium) and incubated for 1-2 h with four oocytes from failed in vitro fertilization (IVF). The acrosome status of sperm was assessed using pisum sativum agglutinin labelled with fluorescein. The ZP-induced AR was inhibited in experiments using sperm from DZPIAR patients, hyper-osmotic medium (400 mOsm/kg) and medium containing soybean trypsin inhibitor (SBTI; 4 mg/mL). Pre-treatment with calcium ionophore was used to create a sperm population with elevated AR. In all experiments with factors inhibiting the ZP-induced AR, the AR was significantly lower for ZP-bound sperm compared with sperm in medium: DZPIAR patients 4% vs. 15%, hyper-osmotic medium 3% vs. 12%, SBTI 2% vs. 12% and SBTI 3% vs. 23% after treatment with calcium ionophore. In conclusion, acrosome-reacted sperm in vitro have significantly reduced, in fact probably zero ability to bind to the ZP.

Quantification of changes in semen may give insight into the testosterone (T)-induced disruption of spermatogenesis in man. A model analogous to flushing of sperm from the genital tract after vasectomy was used to quantify the time course of semen changes in subjects participating in male contraceptive trials using 800 mg T-implant (n = 25) or 200 mg weekly intramuscular injection (IM-T; n = 33). A modified exponential decay model allowed for delayed onset and incomplete disruption to spermatogenesis. Semen variables measured weekly during a 91-day period after initial treatment were fitted to the model. Sperm concentration total count motility and morphometry exhibited similar average decay rates (5 day half-life). The mean delay to onset of decline in concentration was 15 (IM-T) and 18 (T-implant) days. The significantly longer (P < 0.005) delays deduced for the commencement of fall in normal morphology (41 days) normal morphometry (40 days) and sperm viability (43 and 55 days) and the change of morphometry to smaller more compact sperm heads are consistent with sperm being progressively cleared from the genital tract rather than continued shedding of immature or abnormal sperm by the seminiferous epithelium. A significant negative relationship was found between lag time and baseline sperm concentration consistent with longer sperm-epididymal transit times associated with lower daily production rates. (authors)

To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests.The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed.RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZP1 was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZP1 was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed.RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZP1 from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.

BACKGROUND Sperm binding to the zona pellucida (ZP) is required for human fertilization. Under experimental conditions not limited by ZP binding sites, the cumulative numbers of sperm binding tightly to the ZP will asymptote with time to the total number of sperm in the insemination medium capable of binding. METHODS Numbers of ZP-bound sperm were counted after groups of 10 oocytes were incubated with 2x10(4) motile sperm in 20 micro l droplets. The time-course of sperm binding was measured in three consecutive 2 h incubation periods using fresh oocytes for each period (n = 12). Using the kinetic theory of gases to model sperm-oocyte collision rates, the time-course results were extrapolated to give the total proportion of motile sperm capable of binding to the ZP. ZP binding of sperm after 4 h incubation was studied in 20 fertile and 20 normozoospermic subfertile men. RESULTS The percentage of motile sperm capable of binding was for fertile men: mean 14% (range 8-25) and for the subfertile: 4.3% (range 0.1-13, P < 0.001). Sperm morphology correlated with the proportion of ZP-bound sperm. CONCLUSIONS More than 75% of motile sperm from fertile men have no ability to bind to the ZP. This finding has important implications for improvement of semen analysis.

Objective To determine whether adverse perinatal outcomes are increased in subfertile women. Design Cohort study. Setting Two tertiary assisted reproductive technologies (ART) centers; Victorian births register. Patient(s) Records of women who registered with the clinics (1991–2000), but did not have an infant using ART, were linked to the birth register (1991–2004) to identify singleton non-ART births within 5 years of registration (N = 2171). Controls, matched by maternal age and year of infant's birth, were selected randomly from birth records (N = 4363). Interventions None. Main Outcome Measure(s) Adverse obstetric and perinatal outcomes. Result(s) After adjusting for confounders, compared with controls, subfertile women had increased odds of hypertension or preeclampsia (adjusted odds ratio [OR] 1.29, 1.02–1.61), antepartum hemorrhage (adjusted OR 1.41, 1.05–1.89), perinatal death (adjusted OR 2.19, 1.10–4.36), low birth weight (adjusted OR 1.44, 1.11–1.85), preterm birth Conclusion(s) Subfertile women with singleton births are at increased risk of several adverse outcomes. These risks should be considered during their antenatal care and when analyzing adverse effects of ART.

Objective To obtain quantitative measures of morphometric selectivity of the human spermzona pellucida binding process as determined by light microscopy. Design Fully automated sperm head morphometry based on a 32-dimensional parameterization of images of Shorr-stained sperm. Zona pellucida selected sperm removed from reinseminated oocytes that previously failed IVF. Setting Academic research group associated with a tertiary infertility service. Patient(s) Semen samples from 51 infertile patients. Intervention(s) None. Main Outcome Measure(s) Differences in morphometric parameter means observed before and after swim-up and binding to zonae pellucidae. Result(s) Significant differences between insemination and bound sperm were observed in 17 parameter means and 21 standard deviations. The sperm-zona pellucida binding process preferentially selects sperm heads with a large anterior region with relatively low optical density, as well as high axial symmetry and minimal neck anomalies. Bias against sperm with pyriform morphology was not observed. Conclusion(s) A causal link has been established between sperm head morphometry, particularly within the acrosomal region, and the ability of sperm to bind to the human zona pellucida. As sperm-zona binding is necessary for fertility, it is possible to derive a physiologically based assessment for clinical diagnosis of male infertility using the "zona-preferred" morphometry results. Fertil Steril ® 1997;67:362-71 Key Words Sperm-zona binding, sperm morphometry, automated image analysis, zona selectivity, acrosomal region