A Systematic Evaluation of Methods to Separate X- and Y- Bearing Sperm

This project was initiated to determine if was possible to enrich either X- or Y- bearing sperm, and hence to preselect the sex of a child. Two of the possible reasons why couple might want to select the sex of a child are firstly because of a family history of an X-linked recessive genetic disorder, which usually only affect sons, and secondly families who have had several children of one sex. For this study, men with three or more children of the same sex were recruited following the publication of an article in The West Australian newspaper. The percentage of X- and Y- bearing sperm within the semen samples of men with three or more children of the same sex was determined using dual colour fluorescence in situ hybridisation (FISH). The aim of the investigation was to determine if these men had an altered ratio of X- to Y- bearing sperm, which would explain why these men had a predominance of children of one sex. Comprehensive analyses were also carried out on the semen samples. The reliability of the dual colour FISH technique was established using a number of standard metaphase spreads; from male and female subjects and an individual with Klinefelters syndrome. It was determined that dual colour FISH was a suitable technique for determining the percentage of X- or Y- bearing sperm within a sample. The semen samples were processed using one of two protocols. Samples from men with three or more daughters were treated using Human serum albumin columns, with the intention of increasing the percentage of Y-bearing sperm within the final fraction. It has been suggested that this method enriches the Y- bearing sperm from a sample due to the differential motility exhibited by the X- and Y- bearing sperm, although this characteristic has not been proven. Samples from men with three or more sons were processed using 8-layer ISolate® discontinuous gradients, with, the aim of enhancing the amount of X- bearing sperm within the final fraction. This method is based on the formation of the discontinuous gradients because Percoll has not been approved for the production of sperm fractions for human insemination. It has been suggested that the X- and Y- bearing sperm can be enriched using such gradients either as a result of differences in their velocity of sedimentation or due to a greater nett negative charge on the surface of X- bearing sperm. However, neither of these theories have been validated. As it has also been proposed that the survival rate of X- bearing sperm is slightly longer than that for Y- bearing sperm, this was also investigated. In summary no statistically significant enrichment of X- or Y- bearing sperm was observed following the treatment of the semen samples with either the ISolate® discontinuous gradient or the Human serum albumin column protocols. Nor was there any enrichment in X- bearing sperm due to their suggested greater survival time

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

ORIGINAL RESEARCH

The density of human semen and the validation of weight as an indicator of volume: a multicentre study