An in vitro model for purging of tumour cells from ovarian tissue.

BACKGROUND: Cryopreservation and autografting of ovarian tissue may preserve fertility after cancer treatment, but could be hampered by tumour cell contamination. Epithelial tumour cell lysis can be obtained with cytotoxic T cell retargeting through the bispecific antibody BIS-1, with combined affinity for the T-cell receptor and epithelial glycoprotein-2 (EGP-2). Our aim was to study the concept of tumour cell purging in the setting of a suspension of ovarian tissue. METHODS: Human ovarian tissue was brought into suspension by mechanical and enzymatic treatment. Cells of the MCF-7 breast cancer cell line and activated human lymphocytes were co- incubated for 4 h with or without BIS-1 and with or without ovarian suspension. After incubation, MCF-7 cell death and cell growth were evaluated by fluorescent cell detection and MTT assay, respectively. Ovarian tissue morphology was evaluated immunohistochemically. RESULTS: MCF-7 cell death and cell growth inhibition increased with increasing ratios of lymphocytes to MCF-7 cells. BIS-1 addition gave further augmentation, with a maximum depletion of growing MCF-7 cells of 89% (SD 11%) versus without BIS-1, 23% (SD 15%; P < 0.001). Follicles remained morphologically intact. CONCLUSIONS: Purging of added epithelial tumour cells from ovarian tissue with BIS-1 is possible in vitro. Morphologically, follicles remain intact after this procedure. This method may contribute to safer replacement of ovarian tissue in female cancer survivors.

Variation in semen parameters derived from computer-aided semen analysis, within donors and between donors.

The development of computer-aided semen analysis (CASA) has made it possible to study sperm motility characteristics objectively and longitudinally. In this 2-year study of 8 sperm donors, we used CASA to measure 7 semen parameters (concentration, percentage of motile spermatozoa, curvilinear velocity, average path velocity, straight-line velocity, amplitude of lateral head displacement, and beat/cross frequency). The frequency distributions of the 7 parameters in the semen samples of each donor were investigated. All parameters but one were normally distributed; concentration was distributed log-normally. Variation within individual donors and between donors was studied. Analysis of variance demonstrated that variation between donors was not explained by the longitudinal variation within individual donors. Variations in motility characteristics between donors were substantial, which may make motility characteristics of limited value as a tool for establishing fertility. Strong correlations were found between the 7 parameters, partly because by definition, motility characteristics are interdependent. Fisher's discriminant analysis demonstrated that each donor appeared to have his own set of semen characteristics and, more specifically, his own motility signature. From this data set it can be predicted that in order to find population means among sperm, it may be more efficient to measure more subjects than to increase the number of samples per subject.

Protein involvement in the fusion between the equatorial segment of acrosome-reacted human spermatozoa and liposomes.

Artificial membranes (liposomes) can interact with the equatorial segment (ES) of human spermatozoa, provided that the acrosome reaction (AR) has occurred [Arts, Kuiken, Jager and Hoekstra (1993) Eur. J. Biochem. 217, 1001-1009]. Using fluorescently labelled liposomes, this interaction can be seen as either punctate fluorescence in the ES (lip-ESp), reflecting only bound liposomes, or as diffuse fluorescence in this region (lip-ESd), indicating that the liposomes have fused with the ES membrane. Only equatorial segments that still contain constituents of the acrosomal matrix have the capacity to bind liposomes and eventually to fuse with them. Since the exposure of such intact equatorial segments is the exclusive result of induction of the AR under physiological conditions, these results imply that liposomes can be used for the rapid detection of acrosome-reacted spermatozoa. The lip-ESp and lip-ESd patterns were shown to be reflections of two distinct properties of the ES. Proteolytic treatment after AR completely inhibited the formation of a lip-ESd pattern, whereas formation of the lip-ESp pattern was only marginally inhibited by the proteolytic treatment. The same results were obtained using anti-sperm antibodies, which did not react with acrosome-intact spermatozoa. Proteolytic treatment of spermatozoa before AR induction had no effect on the fusion capacity of the ES after subsequent AR, which implies that the putative fusion protein is not accessible before AR. Thus fusion of liposomes with the ES of human spermatozoa is mediated by a sperm protein(s), whereas the lip-ESp pattern is not likely to represent the liposome-binding stage that precedes the fusion step.

Evaluation of a computer-aided semen analysis system with sperm tail detection.

The aim of this study was to evaluate the Stroemberg-Mika cell motion analyser (SM-CMA) which uses tail detection in order to discriminate between immotile spermatozoa and other particles. Analysis of the spermatozoa by the SM-CMA can easily be checked on a video monitor. The semen samples were from donors and patients visiting the fertility unit of the University Hospital, Hanzeplein, The Netherlands. Both fresh semen samples and purified sperm suspensions were used to estimate sperm counts and motility characteristics. We tested the use of the x10 objective instead of the x20 and we assessed the ways in which motility characteristics were influenced by temperature. We found a considerable discrepancy between sperm concentrations measured manually and by computerized analysis, both in semen samples and in purified sperm suspensions. The SM-CMA correctly recognizes motile spermatozoa, but underestimates immotile ones. Although temperature affects motility characteristics, in routine measurements the influence of short cooling periods, which are unavoidable, was nil. We found that using the x10 objective can be useful, especially at low sperm concentrations. In our opinion, the SM-CMA system is, despite some shortcomings in its user-interface, a useful and versatile instrument for examination of human semen samples, with desirable features.