Oct 2025 | Russack, J., Huynh, H., Urrutia, A., and Worrilow, K.C.
To be presented as a poster presentation at ASRM, Oct. 2025.
OBJECTIVE:
Current In Vitro Fertilization (IVF) guidelines recommend that clinics maintain volatile organic compounds (VOCs) between 400-800 ppb. Yet, there is emerging evidence that VOCs below that threshold can adversely affect specific metrics of embryogenesis. The current study assesses the impact of low-level VOCs on embryo morphokinetics and development.
MATERIALS AND METHODS:
Frozen-thawed, two-cell mouse embryos were placed in microwells containing antioxidant-free (GTL) or -containing (GXTL) media and overlaid with oil. Embryos were cultured for 72 hours in a time-lapse incubator pre-gassed with 480-485 ppb acetaldehyde (nGTL = 16; nGXTL = 14), styrene (nGTL = 13; nGXTL = 15) or control (no VOCs; nGTL = 12; nGXTL = 15). Time-lapse images were analyzed for time to 3-cell, 4-cell, 8-cell, morula
(compaction and loss of cellular junctions), blastocyst (formation of the blastocoel), and hatching blastocyst (initial penetration of the zona pellucida). The percent of embryos progressing to each subsequent developmental stage was recorded. Five representative embryos were stained for nuclei, senescence, and reactive oxygen species (ROS) with DAPI, GFP, and CY5, respectively, and 3D-imaged using an EVOS M5000 (Fig 2). Fluorescent images were analyzed using Celleste™ and Aivia™. Data were analyzed by Kruskal Wallis and Wilcoxon pairwise tests.
RESULTS:
Morphokinetic differences were observed at morula and blastocyst stages
(Prob>Chi Square = 0.0045; Prob>ChiSquare = 0.0119). Pairwise differences are summarized in Figure 1A. No significant differences were observed in the number of embryos progressing to 3-, 4-and 8-cell, morula, and blastocyst stages (p > 0.05). However, only 33% of styrene-GTL treated embryos reached the hatching stage by the end of the incubation period, as opposed to 77% of styrene GXTL embryos (p = 0.025), 81% of control GTL (p = 0.017) and 71% of control GXTL embryos (p = 0.0084) (Fig. 1B).
No differences were observed in nuclei counts between treatment groups (p > 0.05) (Fig. 3A). Expression of senescence marker GFP was greater in the acetaldehyde GTL group over the control GTL group (p = 0.0081) (Fig. 3B). Acetaldehyde GTL embryos had significantly higher CY5 signaling than both control GTL (p = 0.037, Z = 2.088) and GXTL (p = 0.036, Z = 2.10) groups. Acetaldehyde GXTL embryos expressed higher CY5 than control GTL embryos (p = 0.036, Z = 2.10) (Fig. 3C), indicative of increased oxidative stress.
CONCLUSIONS:
The present study demonstrates that low-level VOCs can adversely affect timing, progression, and cellular stress during embryo development. Exposure to acetaldehyde was correlated with altered morphokinetic timing and increased ROS. Styrene exposed embryos exhibited delayed compaction and blastocoel formation, as well as significantly reduced hatching rates, which were all recoverable with the addition of antioxidants. Differing responses to styrene and acetaldehyde indicate distinct mechanisms of action.
IMPACT STATEMENT:
The present study counters current guidelines and suggests that lower levels of VOCs play a role in preimplantation embryo toxicology, and further supports the use of antioxidants in IVF culture media. Future studies will continue to investigate the effect and mechanism of various VOCs in IVF and aim to develop methods to mitigate the impact of environmental VOCs.