Effect of in vitro embryo culture and embryo transfer on the expression of toll-like receptor (TLR)-9 in adult mouse lung tissue

Various assisted reproductive technologies (ART), mainly in vitro fertilization and embryo transfer, are widely used in infertility treatment all over the world. However, recent studies have indicated that children born following ART are more sensitive to infections and allergic respiratory diseases like asthma compared with children born through normal pregnancy. Innate immune system is the first line of defense against pathogens and foreign antigens. It is known that toll-like receptors are an important component of the innate immune system. Among these, TLR-9 plays an important role in the detection of bacterial/viral CpG-DNA and in the establishment of an immune response. The aim of the present study was to examine if there is any effect of in vitro embryo culture and embryo transfer on the expression of TLR-9 in adult lung tissue. Two groups were included in the study: The Control group consisted of adult mice obtained through mating of female mice that did not receive PMSG + hCG administration with male mice of the same age. The Experimental group consisted of adults derived from the transfer of in vitro produced blastocysts. To this end, fertilized egg cells (zygotes) were cultured under atmospheric oxygen concentration until the blastocysts stage of development, blastocysts were transferred to pseudo-pregnant mice and pups born were reared to adulthood to be included in the Experimental group. Crossman's triple staining method was used to demonstrate the general histological structure of the adult lung tissue. Immunohistochemistry and immunofluorescence methods were used to determine cell types that express TLR-9 in both Control and Experimental groups. In addition to those methods, real-time PCR and western blot techniques were used to determine expression level of TLR-9 at the mRNA and protein levels, respectively. The results of immunohistochemistry/immunofluorescence analyses demonstrated that cells within and around the air ways, epithelium lining the alveoli and blood vessels express TLR-9 and that the pattern of staining was similar between the two groups. Results obtained from western blot analysis indicated that the expression level of the TLR-9 protein in the Experimental group was 1.25 fold higher compared to the Control group (p=0,015). On the other hand, real-time PCR analyses demonstrated that the expression of TLR-9 transcripts decreased 1.38 fold in the Experimental group when compared to the Control group (p>0,05). Taken together, results obtained from the present study demonstrate that TLR-9 is expressed in certain cell types including Type II pneumocytes and macrophages in the adult lung tissue and suggest that in vitro embryo culture and embryo transfer may affect the immune response of the lung by altering expression of TLR-9.