While washing of human sperm cells by centrifugation and resuspension is a procedure in widespread use, there have been indications that this procedure per se may be harmful to the cells. The objective of this study was to investigate this question. To this end, a method for the clean separation of motile human spermatozoa from seminal plasma in the absence of centrifugation was developed, using a modified swim-up procedure, in which liquefied semen was mixed with an equal volume of 30 mg/ml dextran in medium, and the mixture overlaid with medium containing 5 mg/ml bovine serum albumin, forming two discreet layers with stable interface. The percentage of motile cells in a given sample was consistently > 80% immediately after recovery. Damage to the cells was assessed by loss of motile cells during incubation up to 96 h post-recovery. Comparison of aliquots of spermatozoa obtained by the dextran swim-up procedure showed that the aliquot subjected to centrifugation had 4 +/- 3% motile cells after 48 h, while the untreated aliquot had 52 +/- 12%. The aliquots showed no difference 1 h post-recovery. Similar results were obtained with spermatozoa that had been centrifuged in seminal plasma and resuspended in fresh plasma, then recovered by dextran swim-up. The delayed onset of motility loss in the centrifuged samples implies that this treatment induces sublethal damage in the cells. Comparison of the standard swim-up and Percoll gradient methods for sperm recovery, both of which involve centrifugation steps, showed decline in motility of the samples similar to that seen with dextran swim-up of centrifuged cells. We conclude that centrifugation per se induces sublethal damage in human spermatozoa, independently of treatment method, and suggest that recovery methods for human spermatozoa which avoid centrifugation might partially alleviate the damage incurred by these cells during cryopreservation.

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Zona pellucida thickness was measured daily in zygotes and cleavage stage embryos. Measurements were performed on a Nikon inverted microscope equipped with Hoffman modulation optics, using an ocular micrometer. Zona thickness of each zygote/embryo was measured four times, the zygote/embryo was then “rolled over,” and four more measurements were repeated for a total of eight. The zygotes/embryos were photographed daily and the measurements repeated on the prints. Subsequently, the mean zona thickness for each stage was calculated. A total of 81 patients (mean age 33.8 ± 4.2) participated in the study. A total of 427 embryos were evaluated. Categorical data differences between groups were evaluated by ANOVA and multiple linear regression. For nominal data, the Kruskal‐Wallis test was applied; when P < 0.05 the differences were considered to be significant. We found that the average zona thickness on day 1 of in vitro culture was 17.7 ± 0.14 μm; 16.3 ± 0.14 μm on day 2 and 14.9 ± 0.14 μm on day 3 (P < .0001). When the zona thickness was analyzed in relation to the number of blastomeres on day 3 of culture, there was a highly significant correlation with blastomere number (P < .0001). Similarly, there was a highly significant correlation with embryo grade (P < .005) and fragmentation (P < .001). The data were also analyzed for embryos transferred that resulted in a successful pregnancy, revealing that embryos in a pregnancy cycle had significantly thinner zonae pellucidae (P < .0001), when compared to embryos that were not transferred or from nonconceptual cycles. The average zona thickness also decreased with age, and was most apparent after 35 years. Changes in zona thickness correlated with the number of blastomeres, grade, fragmentation, age and were more evident in embryos transferred from cycles resulting in successful pregnancies. Therefore, zona pellucida measurements should be included in the overall assessment of embryo quality, since this information may be useful in the selection of optimal embryos for transfer. Mol. Reprod. Dev. 47:99–104, 1997. © 1997 Wiley‐Liss, Inc.

mRNA phenotyping by the reverse transcription-polymerase chain reaction (RT-PCR) method was used to compare the patterns of expression of insulin and insulin-like growth factor (IGF) ligand and receptor genes in preimplantation bovine embryos with those established previously for preimplantation murine embryos. In the early bovine embryo, transcripts for IGF-I, IGF-II and mRNAs encoding receptors for insulin, IGF-I and IGF-II were all detectable at all embryo stages from the 1-cell zygote to the blastocyst. In the mouse, IGF-II ligand and receptor mRNAs were not expressed until the 2-cell stage, and the insulin and IGF-I receptor mRNAs were not detectable until the 8-cell stage. Since transcriptional activation of the embryonic genome occurs at the 8- to 16-cell stage in the bovine embryo and at the 2-cell stage in the murine embryo, it is suggested that these transcripts are products of both the maternal and embryonic genomes in the bovine embryo whereas in the mouse they are present only after activation of the embryonic genome. Transcripts for insulin were not detected in preimplantation embryos of either species. Colloidal-gold immunocytochemistry with antibodies directed against the insulin receptor, IGF-I receptor and IGF-I ligand has confirmed the presence of these molecules in bovine blastocysts. RT-PCR and indirect immunofluorescence procedures demonstrated that the glucose transporter (GLUT) isoform 1 is present in murine embryos from the oocyte to blastocyst stage whereas GLUT 2 expression begins at the 8-cell stage.

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