Acceptable microorganisms concentration in a semen sample for in vitro embryo production

Abstract

The aim of this study was to report that the acceptable concentration of microorganisms in a semen sample for insemination may not be safe for an in vitro fertilization procedure. It seems that the semen sample should be completely germ-free, because of the excellent microorganism proliferation condition promoted by the in vitro environment. Key words: semen, microorganisms, in vitro fertilizationFundamental procedures currently used in in vitro embryo production (IVP) include transvaginal aspiration and oocytes in vitro maturation; spermatozoa in vitro capacitation and in vitro fertilization (IVF) (4). Infectious agents in IVP systems might reduce the number and the quality of embryos generated and result in transmission of disease to recipients and offspring, or even confound findings for research (3).Semen is a potential source of non-pathogenic, as well as pathogenic microorganisms. Semen collection can be relatively free of contaminants, but it is not necessarily germ-free (3). Units of semen containing Stenotrophomonas maltophilia, Pseudomonas putida, Pseudomonas aeroginosa, Enterobacter cloacae, Staphylococcus sciuri, Acinectobacter cacloaceticus, Pantoeau agglomerans and Flavobacterium spp have been reported as contaminants in IVF embryo cultures (2). The aim of this work was to report that the acceptable concentration of microorganisms in a semen sample for insemination may not be safe for an IVF.Routine IVF procedures were performed with oocytes collected from ovaries derived from slaughtered cows, and maturated for 24h in 5% CO 2 atmosphere at 39ºC. The semen samples were thawed at 37ºC/30' and processed in Percoll gradient (Cultilab, Campinas, Brazil) for spermatozoa capacitation. After maturation time, the oocytes were washed in Human Tubal Fluid (HTF -Cultilab, Campinas, Brazil), a fertilization medium, supplemented with 10% bovine fetal serum (BFS) and kept with the processed spermatozoa for fertilization (18-21h). However, after fertilization, in two consecutive IVF procedures, performed with two different semen samples that had presented normal evaluation parameters, the presence of contamination was detected. To locate the main focus of contamination, a new IVF procedure was performed, with the same semen samples, following the pattern protocol (1).One fertilization dish was used for each semen sample test. They were prepared with four HTF medium drops (50 µL) each. In the first drop a conventional IVF procedure was done (in vitro matured oocytes with processed spermatozoa), in the drop 2 just oocytes were kept and in the drop 3 just processed semen. In the fourth drop HTF medium was left to be used as negative control. The dishes were maintained in 5% CO 2 environment for 18h. The presence of contamination was confirmed in drops 1 and 3 of each dish, showing that probably the semen was the contamination focus. The drops containing the oocytes and