Washing mineral oil reduces contaminants and embryotoxicity Dean E. Morbeck, Ph.D., Zaraq Khan, M.B.B.S., David R. Barnidge, Ph.D., David L. Walker, M.S.  Fertility and Sterility  Volume 94, Issue 7, Pages 2747-2752 (December 2010) DOI: 10.1016/j.fertnstert.2010.03.067 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Response of two-cell mouse embryo assay (MEA) and Human Sperm Motility Assay (HSMA) to oils SMO71 and SMO44. (A) Sperm motility was not affected by SMO71-conditioned HTF when compared to control oil. Sperm did not survive to 24 hours when exposed to media conditioned with SMO44 (P<.001). (B) Development of two-cell mouse embryos to blastocyst was reduced for test oils at all times observed compared to control oil (P<.01). (C) Two-cell MEA results after contaminated oil lots (SMO71 and SMO44) were washed with human tubular fluid (HTF). Embryos passed at >70% blastocyst rate when they were cultured in media that was exposed to washed oil lot SMO71. No embryo growth was seen in the media that was exposed to washed oil lot SMO44 (P<.01). Fertility and Sterility 2010 94, 2747-2752DOI: (10.1016/j.fertnstert.2010.03.067) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 The LC-MS chromatograms from aqueous extracts of three different types of oil and a culture media control. (A)The peak at 14.68 minutes in Lot SMO71 chromatogram is clearly visible and is not present in any of the other extracts or the media control. (B) The ions present in the unique peak of Lot SMO71 shows a series of peaks representing ions that are 44 Da apart, which is indicative of a polymer with substituted polyethylene (–H2C–O–CH2–)n subunits. Fertility and Sterility 2010 94, 2747-2752DOI: (10.1016/j.fertnstert.2010.03.067) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) One-cell MEA after washing dSMO44. Embryos passed at >70% blastocyst rate for each washing agent and were different from the unwashed SMO44 (P<.01). (B) The TX-100 area under the curve after washing SMO71. Washing oil was equally effective in reducing the amount of TX-100 irrespective of the washing agent (P<.001). (HTF = human tubular fluid; HSA = HTF + 5 mg/dL of 5% human albumin; Water = distilled water). Fertility and Sterility 2010 94, 2747-2752DOI: (10.1016/j.fertnstert.2010.03.067) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions