A research group led by Specially Appointed Assistant Professor Takehiro Hiraoka (at the time of the research, currently a visiting researcher at the University of Tokyo) and Professor Masahito Ikawa at the Research Institute for Microbial Diseases at the University of Osaka announced that they have developed a mouse ex vivo uterine system and successfully reproduced implantation. They maintained the in vivo implanted uterine environment ex vivo and reproduced implantation processes at a high level, including embryonic attachment, invasion, and embryogenesis. The system enables the possibility of studying implantation ex vivo and is expected to contribute to technological development for improving implantation rates and other applications. The results were published in the July 1 issue of Nature Communications.
Adhesion of the embryo and subsequent development (top). Formation of epiblast-like structures and ectoderm-like structures outside the embryo was observed whilst maintaining implantation (bottom).
Provided by the University of Osaka
In Japan, demand for human assisted reproductive technology (ART) is increasing annually, with approximately 1 in 10 people born through the technology. While fertilization can expect success rates of over 80% through in vitro fertilization and intra cytoplasmic sperm injection, implantation has success rates below 50% and decreases with age, creating barriers to success. There are no effective treatments for recurrent implantation failure, where implantation repeatedly fails even when good quality embryos are transferred. Meanwhile, implantation is a complex biological phenomenon that occurs through interactions between blastocysts consisting of multiple differentiated cell masses and the endometrium consisting of multiple cell types in the deep parts of the uterus, making research difficult even in experimental animals.
Blastocysts (mature fertilized eggs) first attach to the endometrium, then continue embryogenesis through a process of invasion into the endometrium. Previous research using pseudo-blastocysts and uterine organoids has been conducted, but actual implantation could not be reproduced.
In this study, the group aimed to create a new experimental model for reproducing implantation ex vivo that more closely resembles conditions in vivo. Previous research by Professor Yasushi Hirota and his colleagues at the University of Tokyo had shown that multiple endometrial factors are essential for implantation in mice. Due to this, they considered constructing a system that combined the ex vivo culture of actual uterine tissue with embryo culture.
Specifically, they established a technique to obtain pre-implantation stage blastocysts from mice and isolate only the endometrium from another mouse onto a sheet. They sectioned the endometrium into fragments and cultured them together with blastocysts. For the culture, they adopted an air-liquid interface culture method that enabled culture medium supply. Additionally, they devised a culture system capable of oxygen supply by using PDMS (polydimethylsiloxane), a new gas-permeable material.
They placed the endometrial fragments on an agar medium set in culture dishes with culture medium, placed the blastocysts on top, and covered them with thick sheet-like PDMS from above. This created a system where blastocysts are retained on endometrial fragments, culturing endometrial fragments on agar while supplying oxygen through the PDMS. Furthermore, the group examined and optimized various conditions including the culture medium composition and device size.
As a result, they induced attachment between endometrium and blastocysts with over 90% probability. Blastocysts attached within 24 hours after mating, and embryonic development was observed while maintaining implantation at 96 hours.
Post-implantation blastocysts formed epiblasts (fetal components) and extraembryonic ectoderm that will become the placenta, with the appearance of visceral endoderm, parietal endoderm, and yolk sac cavity. They could also confirm the appearance of embryonic cells invading endometrial cells through differentiation.
To further confirm whether these phenomena were reproduced on the uterine side, they performed single-cell RNA sequencing on the endometrium. As a result, they confirmed gene expression in the endometrium that is also expressed in vivo. Subsequently, through immunostaining, they confirmed the existence of uterine glandular structures, which are three-dimensional structures observed during in vivo implantation within endometrial fragments and also reproduced factors such as blood cells related to immune tolerance important for implantation. The temporary avascular state for angiogenesis that occurs when blastocysts implant was also reproduced.
Using this system, they examined whether it is possible to analyze the previously unknown mechanism by which the implantation factor COX-2 (an enzyme that produces prostaglandins) in the uterus controls implantation.
As a result, they confirmed that, as in living organisms, implantation was inhibited by functional inhibition of the uterine implantation factor COX-2 and recovered with supplementation. They also revealed that COX-2 activates AKT molecules in embryonic trophoblast cells through downstream signaling. Currently, development beyond 96 hours cannot be reproduced, and it is believed that other factors not included in the system are involved beyond this point.
Hiraoka commented: "In the future, by reproducing and verifying various pathological conditions of implantation failure ex vivo, we believe we will be able to discover treatments for implantation failure. We also expect improvements in implantation success rates in ART."
Journal Information
Publication: Nature Communications
Title: An ex vivo uterine system captures implantation, embryogenesis, and trophoblast invasion via maternal-embryonic signaling
DOI: 10.1038/s41467-025-60610-x
This article has been translated by JST with permission from The Science News Ltd. (https://sci-news.co.jp/). Unauthorized reproduction of the article and photographs is prohibited.