Virginia Research Day 2021

Two Cell to Hatching Blastocyst Development During E-cig Fluid Condensate Media Exposure King K. Ampofo (OMS-II), Aubrey A. Jackson (OMS-II), VicLouis C. Arreola (OMS-II), Bo Liu, PhD, R. James Swanson, PhD

Methods Vaped condensate, figure 1 was diluted at either 2% or 4% in M16 media. 2-cell zygotes were collected 32hrs post- mating from mice and placed into 96-well plate containing control M16 media and either 2% or 4% condensate fluid. Using the Keyence BZ- X810 microscope, figures 2 and 3 and Zeiss Inverted phase microscope, figure 4, development was recorded, and an image was captured every 10 mins for 120 hrs. Images were analyzed for developmental milestone based on morphological characteristics. Time points were recorded for developmental milestones, and each stage was given a Morph score(M-S) based on morphological characteristics such as embryo diameter, zona pellucida thickness, perivitelline space; individual blastomere shape, and overall blastomere organization. The M-S semi quantitative values are 1-4 with 1 being normal and 4 being fragmented or degenerated.

Results

Abstract The emergence of electronic cigarettes (e-cigs) as devices touted to be helpful in the cessation or reduction of smoking have prompted concerns about the prevalence and consequences of their use during pregnancy. 1 Studies have shown that e-cigs, vaping, and hookah use are increasing among adolescents and young adults, but the long-term health impacts on pregnancy remain unknown. Women who smoke tobacco products may therefore be motivated to switch to vaping during pregnancy in order to alleviate the commonly known hazards of smoking. 2 Use of e-cigs is rapidly increasing in popularity within the U.S. and around the world. 3 Since vaping is marketed as a better alternative to smoking cigarettes, smoking women who are planning to get pregnant or have become pregnant may be more likely to consider using e-cigs in assisting in smoking cessation. 4 Due to the paucity of literature on vaping effects on the embryo and pregnancy, both health practitioners and the public, especially pregnant women, are misinformed about the health risks associated with vaping. 5 A successful pregnancy begins with healthy embryos. This study investigated the impact of e-cig fluid condensate on the developing embryo. Twenty mice were divided into 4 groups: 1) control, 2) mango flavored fluid with 0% nicotine (NC), 3) mango flavored fluid with 2.5% nicotine, 4) spearmint flavored fluid with 2.5% nicotine, and 5)absolute zero flavored fluid with 2.5% nicotine. The mice were injected to super ovulate with PMSG and hCG 6 and individually placed with one male mouse on day 4. Two-cell embryos were collected from vaginal plug-positive mice on day 6 and plated on four 96-well plates with M16 media alone for control and M16 with filtered condensate from E- cig fluid for experimental groups. The stage of development for each embryo was recorded twice a day for four days. The results showed that there was a greater percentage of embryos in the control group (p≤0.0048) reaching blastocyst than any of the experimental groups. On both plates, none of the embryos in the 2.5 % mango with nicotine or 2.5 % spearmint with nicotine groups reached the 4- cell stage (p≤0.0001). This suggests there was a harmful effect of e-cig fluid condensate on embryo development from post- fertilization until the hatching blastocyst stage. Footnotes 1.Nan Jiang, Lily Lee, Judith T. Zelikoff and Michael Weitzman, E-Cigarettes: Effects on the Fetus Pediatrics in Review March 2018, 39 (3) 156 - 158; DOI: https://doi.org/10.1542/pir.2017-0112 2.US Dept of Health & Human Services. E-cigarette Use Among Youth and Young Adults: A Report of the Surgeon General. (2016) (Atlanta, GA) 3.National Academics of Sciences Engineering and Medicine. Public Health Consequences of E-cigarettes. The National Academics Press, Washington, DC (2018) 4.Andrea McCubbin, Amanda Fallin-Bennett, Janine Barnett, Kristin Ashford, Perception and use of e-cigs in pregnancy, Health Education Research , Vol. 32, Issue 1, 1 Feb 2017, pp. 22 -32, https://doi.org/10.1093/her/cyw059 5.Xi Wang, Nora L. Lee, Igor Burstyn, Smoking and use of e -cigs (vaping) in relation to preterm birth and small-for-gestational-age in a 2016 US national sample, Received 27 Nov 2019, Revised 3 Feb 2020, Accepted 22 Feb 2020, Available online 24 Feb 2020. 6) Mitchell MH, Swanson RJ, Hodgen GD, Oehninger S, J Soc Gyn Inv , 1994 1 215-9. Aim Determine electronic cigarette carrier fluid influence (with and without high concentrations of nicotine) on embryonic development using 10-minute time-lapse micrographs at 400x over 120hrs

Figure 1. Vaping Condensate Apparatus

Figure 2. Keyence BZ-x810

Conclusion Control embryos (unexposed) had a higher rate of blastocyst development, suggesting that vaping condensate exposure beginning with 2-cell mouse embryos has an arresting effect on normal development to blastocyst stage. The difference between 4% and 2% spearmint with NC suggests these effects are dose dependent. The lack of significant difference between NC and non-NC groups of the same flavor suggests more factors outside of NC are involved. A difference between spearmint and other flavors at the same NC level and concentration suggests that flavor may also impact the extent of harm to the embryo. Histochemical fluorescent tagging research is ongoing to parse out these additional potential causes.

Figure 3. Keyenze BZ-x810 Universal Stage Figure 4. Zeiss Inverted Phase Microscope

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