Comparative flow cytometric analysis of the human sperm acrosome reaction using CD46 antibody and lectins.

PURPOSE: Our purpose was to determine the most suitable marker for the human sperm acrosome reaction, based on detection of CD46 antibody binding compared with lectin binding. METHODS: Flow cytometric analysis of CD46 antibody versus lectins (PNA, PSA, and Con A) was used to quantify the acrosome reaction of human sperm. RESULTS: Neither PSA nor Con A was able to detect significant changes in the spontaneous and ionophore-induced acrosome reactions compared to CD46 antibody. However, PNA was found to exhibit a binding pattern similar to that observed with CD46 and could be used to quantify measurable changes in acrosomal response to ionophore, albeit of a lower magnitude than the responses detected by CD46. CONCLUSIONS: We conclude that PNA binds to the inner acrosomal membrane of acrosome-reacted sperm and is suitable for use as a marker of the acrosome reaction by flow cytometry. Data are presented which clarify the assessment of the acrosome reaction when CD46 and lectins are used.

Twin pregnancy following transmyometrial-subendometrial embryo transfer for repeated implantation failure.

Most in-vitro fertilization (IVF) failures are due to failure of implantation. We present a case of a successful attempt at transvaginal/transmyometrial/subendometrial embryo transfer in a patient with seven previous failures in assisted conception cycles. We demonstrate achievement of a twin pregnancy, which resulted in the delivery of healthy twin girls in January 1997, following a novel technique of embryo transfer.

Progesterone does not potentiate the acrosome reaction in human spermatozoa: flow cytometric analysis using CD46 antibody.

The study was designed in order to investigate the action of progesterone on the spontaneous and ionophore-induced human spermatozoa acrosome reaction in vitro. The principle of the assay system is flow cytometric analysis of CD46 antibody binding to the inner acrosomal membrane. The technique is a simple and objective method of analysis, allowing fluorescent analysis of a large segment (5000 spermatozoa) of the spermatozoa population under investigation, with concomitant isolation of the live fraction of the spermatozoa population. Four concentrations of progesterone (1, 25, 50, and 100 microg/ml) were examined for their effects on spermatozoa capacitated for 4 and 24 h. In addition, motility parameters were examined by the CellSoft 2000 automated semen analyser system. Analysis of variance revealed that progesterone had no effect on either the spontaneous acrosome reaction or the ionophore-induced acrosome reaction at both 4 h and 24 h of spermatozoa capacitation times. Further, no effects on sperm motility parameters or on spermatozoa viability could be attributed to progesterone. We therefore conclude that progesterone has no objectively measurable effects on either the sperm acrosome reaction or sperm motility parameters, as measured in normal sperm populations.

Is Percoll safe for in vivo use?

Fourteen virgin female rabbits were injected with 60% Percoll solution in the right ovary and uterine horn and sperm prepared with Percoll in the left ovary and uterine horn. Histologic examination after 4 weeks showed no inflammatory cell infiltration in either uterine horns or ovaries.

Pentoxifylline potentiates ionophore (A23187) mediated acrosome reaction in human sperm: flow cytometric analysis using CD46 antibody.

The study was designed to investigate the effects of pentoxifylline on the acrosome reaction of human spermatozoa in vitro, and to determine whether the reaction is differently modulated after sperm selection by multiple tube swim-up and Percoll buoyant density centrifugation. The acrosome reaction was induced in vitro by using calcium ionophore (A23187) and was detected by measuring the fluorescence of FITC-conjugated goat anti-mouse immunoglobulin bound to CD46 antibody (which binds to the CD46 receptor site on the inner acrosomal membrane) by flow cytometry. Spermatozoa separated on Percoll displayed significantly lower spontaneous acrosome reactions (P = 0.002) than did those separated by the swim-up technique. Pentoxifylline did not, by itself, induce acrosome reaction, but after induction with ionophore, it significantly increased the reaction (P = 0.003) and this increase was seen to be greater when Percoll separation was used as compared to the swim-up technique (P = 0.0002). We therefore conclude that Percoll selection of motile spermatozoa together with pentoxifylline treatment may be of value in assisted reproductive techniques, as an increased ARIC score arose after both treatments, and that flow cytometry allows a precise and rapid quantification of the acrosome reaction.

Percoll semen preparation enhances human oocyte fertilization in male-factor infertility as shown by a randomized cross-over study.

The aim of this study was to compare two methods of semen preparation: multiple tube swim-up and Percoll separation, using a randomized cross-over clinical study, in which sperm parameters, oocyte fertilization rates, embryo quality and cell stage were analysed. Overall, there was no difference between the two preparation methods in the normozoospermic cycles. In the male-factor cycles, Percoll extracted a higher total number of spermatozoa (P = 0.02), increased the concentration of motile spermatozoa (P = 0.02), increased the total number of motile spermatozoa per sample (P = 0.02), and enhanced the recovery rate of motile spermatozoa (P = 0.04) compared to swim-up. There was a significant improvement in fertilization rates (P = 0.0006), in the percentage of embryos over 2-cell stage on day of transfer (P = 0.004), and in the number of replaced embryos per transfer (P = 0.01) in the Percoll as compared to swim-up cycles. There was no significant difference in embryo quality. We conclude, therefore, that in advanced reproductive procedures where sperm dysfunction exists, semen preparation with Percoll should replace the swim-up technique.