Ovotoxicity Screening Using Banked Encapsulated In Vitro Follicle Growth

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The basic functional unit of female reproduction, the ovarian follicle, is derived from a pool established before birth and is non-regenerable. Environmental contaminants containing endocrine disrupting chemicals such as pesticides, metals, some additives or contaminants in food, and personal care products can be toxic to follicles. Some pharmaceutical compounds such as chemotherapeutics are also toxic. These chemicals can lead to ovotoxicity increasing women’s risks of premature ovarian failure, hormonal imbalance, and infertility. New chemical entities are therefore required to be tested for endocrine disruption.

The gold standard of ovotoxicity testing uses laboratory animal models which are time consuming, costly, and by their nature potentially harmful to the animals. The primary argument for in vivo models is that they more faithfully represent human biology than in vitro models. Whereas in vitro models have clear advantages in terms of cost, throughput, and animal welfare. Against this backdrop there were somewhere between 25,000 and 84,000 chemicals in commerce in the United States in 2014 and a backlog in testing.

Efficient and faithful in vitro models have therefore been sought, including 3D alginate hydrogel encapsulation to grow both mouse and human immature follicles, termed encapsulated in vitro follicle growth (eIVFG). The process of preparing fresh eIVFG takes several weeks so it is not suitable for rapid high-throughput studies. The authors of a new study with corresponding author Shuo Xiao of University of South Carolina, Columbia, published in the journal Reproductive Toxicology, sought to determine whether eIVFG could be rendered suitable for high-throughput studies by vitrification and banking prior to high-throughput analysis.

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A closed vitrification system with cooling in liquid nitrogen vapor and a two-phase warming procedure was selected based on successful prior studies using the technique. Using the optimal protocol nearly all vitrified mouse follicles showed normal morphology.

Freshly harvested follicles and vitrified banked follicles were comparable. The size and viability of eIVFG generated for eight days using banked follicles was comparable to freshly harvested follicles. Vitrified follicles also had normal ovarian steroidogenesis during eIVFG. They showed normal in vitro ovulation and oocyte meiotic maturation. The expression of oocyte-specific genes were comparable.

Vitrified follicles with eIVFG can determine ovotoxicity. Doxorubicin (DOX), a commonly used chemotherapeutic, consistently induced follicular cell DNA damage and apoptosis in vitrified follicles with eIVFG as expected. Microcystins (MCs) are an emerging category of environmental contaminants associated with harmful algal blooms. MC-LF was found to be the most ovotoxic MC congener tested using vitrified follicles and exhibited dose-dependent ovotoxicity. MC congeners exhibited comparable ovotoxicities between vitrified and fresh follicles.

“In summary, our optimized vitrification protocols can be used to establish a long-term-storage and ready-to-use follicle bank, enabling for a high-throughput in vitro follicle culture, chemical exposure, and ovotoxicity screening. This platform is not meant to replace in vivo animal models, but it can efficiently and significantly help prioritize chemicals with high ovotoxicity concern for more targeted and mechanistic in vitro and in vivo toxicity assessments,” concluded the authors.


  1. https://www.sciencedirect.com/science/article/pii/S0890623820300137
  2. https://www.ncbi.nlm.nih.gov/books/NBK268889/