T.A.E. Stout

The Potential Health and Environmental Risks of 3D-engineered Polymers

Polymer engineering, such as in three-dimensional (3D) printing, is rapidly gaining popularity, not only in the scientific and medical fields but also in the community in general. However, little is known about the toxicity of engineered materials. Therefore, we assessed the toxicity of 3D-printed and molded parts from five different polymers commonly used for prototyping, fabrication of organ-on-a-chip platforms, and medical devices. Toxic effects of PIC100, E-Shell200, E-Shell300, polydimethylsiloxane, and polystyrene (PS) on early bovine embryo development, on the transactivation of estrogen receptors were assessed, and possible polymer-leached components were identified by mass spectrometry. Embryo development beyond the two-cell stage was inhibited by PIC100, E-Shell200, and E-Shell300 and correlated to the released amount of diethyl phthalate and polyethylene glycol. Furthermore, all polymers (except PS) induced estrogen receptor transactivation. The released materials from PIC100 inhibited embryo cleavage across a confluent monolayer culture of oviduct epithelial cells and also inhibited oocyte maturation. These findings highlight the need for cautious use of engineered polymers for household 3D printing and bioengineering of culture and medical devices and the need for the safe disposal of used devices and associated waste.

Proteomic Analysis of Pregnant Mare Uterine Fluid

The maternal recognition of pregnancy (MRP) is the physiological process whereby the conceptus signals its presence to its maternal host. From approximately day 6 to day 16 after fertilization the equine conceptus continually migrates throughout the uterus. It contacts the endometrium during this migration but relies exclusively on the uterine fluid for nourishment and survival. It is anticipated that throughout this 10-day passage the yet unknown message triggering MRP in the mare may pass through the fluid. Gene expression and transcriptomic studies have been instrumental in characterizing early embryo-endometrium interactions. Now, the sequencing of the equine genome and the advent of mass spectrometry (MS) have opened new doors for proteomics investigation, with recent studies having looked at the equine oviductal proteome [1]. None to our knowledge have investigated the intrauterine fluid. This study sets out to profile the proteomic composition of the early gestational equine uterine fluid, the content and dynamics of which are critical to the success of the pregnancy, but have not yet been comprehensively examined. The findings generate rationale for further investigation and indicate that the pregnant mare uterine luminal fluid can provide a rich source of information about the mechanisms at play in the recognition and support of early pregnancy.

Developing a dynamic test to identify stallions with reduced sperm DNA stability

Sperm DNA integrity is usually evaluated using a ‘static’ test. However, recent studies suggest that a ‘dynamic’ test to examine how rapidly spermDNA degenerates at 37 C is a more sensitive index of (sub)-fertility. This study validated a ‘DNA stress’ test by examining whether sperm DNA from different stallions degraded repeatedly at different rates during incubation at 37 C. After daily semen collection for 1 week to reach daily sperm output, semen was collected on three consecutive days from each of eight 3-year old Warmblood stallions, i.e. three ejaculates per stallion. All ejaculates had >60% morphologically normal and >60% motile sperm. The semen was diluted to 50x106 sperm/mL in an egg yolk-skimmed milk extender (Spervital EVD ; Spervital) and divided into two portions; one portion was centrifuged (800xg, 20min) and re-suspended. Centrifuged and non-centrifuged samples were split again and incubated at 37 C and 5 C. Semen was assessed during 37 C incubation after 4, 8, 24 and 48h; during 5 C incubation after 48, 96, 144 and 192h. At each time-point, sperm motility was evaluated using a computerized system (SpermVision ; Minitub), viability by SYBR14/PI staining assessed by fluorescence microscopy, and DNA integrity by SCSA . Differences in percentage of motile, viable or DNAintact sperm were compared between incubation temperatures, times and stallions using analysis of variance. Percentages of motile and viable sperm at 5 C were higher (p<0.05) for centrifuged than non-centrifuged semen, and there were no between-stallion differences in the rate of loss of motility or viability. Centrifugation did not reduce the rate at which the percentage of motile or viable sperm decreased during 37 C incubation. The rate of change of sperm DNA degradation, as reflected by the alpha t value, did not differ between these 8 stallions when semen was