Noninvasive Evaluation of Mouse Embryo Development Using Time-lapse Optical Coherence Microscopy

Introduction: In vitro fertilization (IVF) has been conducted over 190,000 cases in 2017 in the United States to overcome infertility, but the birth rate is still less than 40% [1]. To enhance the success, embryo screening has been performed based on mostly bright field (BF) microscopy of static developmental time points by trained embryologists, which introduces the problems such as human subjectivity and suboptimal culture conditions induced by imaging [2]. Time-lapse imaging has then been proposed to monitor the dynamic behavior, but the current two-dimensional (2D) BF platforms have not converged to an effective scoring system [3]. Optical Coherence Microscopy (OCM) as a high-resolution, non-invasive three-dimensional (3D) modality has been used for time-lapse mammalian embryo imaging as a proof of principle, but the full development due to the culture condition was not completely covered [4]. Here, we acquired time-lapse OCM images with co-registered BF imaging inside the incubator on thawed cryopreserved mouse embryos starting from one-cell stage. Our preliminary results demonstrate the capability of OCM to detect the early structural features for the stage classification and blastocyst scoring. In brief, OCM-based time-lapse technology is promising to provide complementary information about early embryo development and facilitate embryo selection in the IVF clinics.

Materials and

Methods: Our dual-modal imaging system, shown in Fig. 1. (A), consists of a standard bright-field microscopy and a Spectral-Domain OCM (SD-OCM) in a concurrent manner inside the incubator. The broadband optical light source has a center wavelength at 850 nm with a spectral bandwidth of 168 nm (cBLMD-T-850-HP, Superlum). The axial resolution and lateral resolution of OCM were ~2 and ~1 μm in the air with a 20× objective lens. The axial scan (A-scan) rate of the OCT system is set at 80 kHz, with a frame rate as ~115 Hz. 10-12 embryos were imaged per experiment every ten minutes with OCM 3D images and BF images. As shown in Fig. 1. (B), the embryos were then transferred into the imaging dish (V005001, IVF store) in 100 μL Multipurpose Handling Medium-Complete (MHM-C) with Gentamicin (90166, FUJIFILM) covered with 3 mL mineral oil (9305, FUJIFILM). For each embryo, it took about one minute to complete the stage movement, auto-tracking, BF/OCM imaging and data transfer, demonstrated in Fig. 1. (C). The field of view was ~300×300 μm2. OCM images were reconstructed by lab-customed software, and visualization and segmentation were achieved by Amira (Thermo Fisher Scientific). One-cell stage cryopreserved mouse embryos were purchased from IVF store (B1-10) and thawed in the lab for monitoring. Four 4-minute washes were performed after a 2-minute thawing to the room temperature. The embryos were kept in the same media in the incubator at 5% O2, 6% CO2 for over 6 days.

Results, Conclusions, and Discussions:

Results: Our preliminary results have shown that OCM can reveal 3D microstructures throughout all developmental stages, providing a comprehensive view of the embryo’s development, as shown in Fig. 2. (A-D). From the classification of the stages, we were able to group the difference between timing and spot out the abnormalities during the dynamic development (Fig. 2. (E)). OCM can not only provide insights for the intracellular features at their few-cell stages, but also the 3D view of inner cell mass (ICM) and trophectoderm (TE) which are important for blastocyst grading. The hatching site specificity is also well-highlighted by OCM images (See Fig 2. (F)), which was proved to significantly influences pregnancy outcomes in mice [5].

Conclusions: We have constructed a dual-modality (BF and OCM) imaging system that can automatically perform time-lapse imaging every 10 minutes. From there, we analyzed the time-lapse information and morphological features to evaluate the quality of the mouse embryos that developed into the blastocyst state. We will accumulate more datasets with proper labeling and the feature analysis as a library for future time-lapse 2D and 3D information analysis. Overall, this research will facilitate in vitro fertilization, which has profoundly impacted reproductive medicine, by the dual-modality, 3D, time-lapse imaging platform to monitor the embryo development process to predict successful blastocyst formation.

Acknowledgements (Optional): We acknowledge the funding source: a start-up fund from Washington University in St. Louis and NIH grants R01EB025209 and 1R21EB03268401A1.
Reference
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