Poster CC2 - In vivo dynamic 3D imaging of preimplantation embryo transport in the mouse oviduct for understanding tubal ectopic pregnancy

Introduction: The mammalian oviduct (fallopian tube) connects the ovary and the uterus and is essential for nature pregnancy. It transfers oocytes and sperm, hosts fertilization, and transports preimplantation embryos to the uterus for implantation. This entire process takes about four days in both human and mice, and it is believed to be driven by the beat of motile cilia lining the luminal epithelium and the contraction of smooth muscle surrounding the oviduct wall. Despite a long history of research, the specific roles of ciliary and muscular activities in driving the transport of embryos toward pregnancy remains unclear. In fact, little is known about this dynamic process inside its native environment. Studies have largely focused on evaluating the ending point, such as whether the embryos reach the uterus, but mechanistic assessment of the process is absent, primarily due to the difficulty in performing such analyses. As a result, there is a significant lack of knowledge on what the detailed functions of cilia and muscle are and how they act independently and/or cooperatively to orchestrate the transport of embryos in a timely fashion. This has led to the currently unknown etiology of tubal ectopic pregnancy, which affects 1-2% of all pregnancies, is life-threatening, and lacks clinical strategies in prevention, early diagnosis, and treatment for recurrence. Here, we addressed this lack of understanding by establishing a novel in vivo imaging approach in the mouse model to reveal the dynamics underlying the transport process, providing foundational and mechanistic insights into normal and ectopic pregnancies.

Materials and

Methods: We combined optical coherence tomography (OCT) and an intravital window for in vivo imaging of the mouse oviduct. The window is 3D printed, low-cost, and autoclavable. It bypasses the mouse body wall for optical access to the oviduct with a 10-mm-diameter aperture (Figure 1). The uterus on the window side presented successful embryo implantation and pregnancy, indicating that the window implantation does not affect oviduct physiology. We have developed a series of OCT-based imaging methods to quantitatively assess the oviduct structure and functions. In particular, our lab-built OCT system had a ~6 µm axial resolution, a ~9 µm lateral resolution, an imaging depth covering the entire oviduct lumen, and a 3D imaging speed at the Hz level, providing a unique capability to simultaneously capture and measure the movement of preimplantation embryos and the dynamics of the oviduct. This enables quantitative pumping analysis of the oviduct, which is otherwise extremely difficult, if not impossible. In this study, we focused on two regions of the oviduct: the isthmus that is the longest portion of the oviduct hosting the development and transport of preimplantation embryos, and the utero-tubal junction which connects the isthmus with the uterus. In vivo time-lapse volumetric OCT imaging was acquired, followed by 3D image reconstructions and quantifications. Padrin (prifinium bromide) was administered topically to inhibit smooth muscle contractions for determining the role of muscular activity in the transport process. All animal procedures have been approved by the Institutional Animal Care and Use Committee at Stevens Institute of Technology.

Results, Conclusions, and Discussions: We have made novel observations, producing high-quality visualizations that revealed the in vivo 3D transport process, for the first time to the best of our knowledge. Our quantitative analysis showed interesting pumping dynamics that were previously unknown. The in vivo oviduct contraction can be quickly and effectively inhibited by padrin, while motile cilia remained functional under padrin administration. Without muscular activities, embryos were found static, losing their typical bi-directional movements. This demonstrates that smooth muscle produces the driving forces for the embryo movements in both the isthmus and the utero-tubal junction. In the isthmus, the forward embryo movement (in the ovary-to-uterus direction) correlated with the contraction wave that initiated from the ampulla, which is the oviduct portion closer to the ovary. However, the backward embryo movement took place upon the relaxation of the ampulla. A snapshot of this dynamic process with the embryos in the upper isthmus tracked and labeled is shown in Figure 1. These suggest different pumping mechanisms, including peristalsis and suction, in generating the forward and backward movements of the embryos, respectively, in the isthmus. In contrast, bi-directional contraction waves were found in the utero-tubal junction, which drove both the forward and backward movements of the embryos. These results indicate a significant role of smooth muscle in the preimplantation embryo transport process, which was largely neglected before. Challenging the classical view where ciliary beating is believed to be the primary driver, this work opens a new avenue to build a more comprehensive understanding of the oviduct transport function in a pregnancy. With the emerging mouse models of embryo retention inside the oviduct, a prerequisite of tubal ectopic pregnancy, both the method and the insights presented here are useful to potentially elucidate the functional cause of tubal ectopic pregnancy.

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