Oviduct-on-Chip improves fertilization and reproduction research

Tuesday, 26 November 2019

How can we better understand the process of fertilization in mammals? Thanks to a new member of the Organs-on-Chip family, Dr Bart Gadella and his colleagues at the faculty of Veterinary Medicine at Utrecht University are able to study fertilization and reproduction. They developed an Oviduct-on-Chip that enables them to use cow gametes obtained from slaughterhouses to achieve fertilization and early embryonic development for their research. This replaces in vivo fertilization. In addition to contributing to further knowledge of reproduction, they have also reported important findings that are essential for anyone who uses 3D printing technology in combination with living cells. The latest Newsletter of the 3Rs Centre ULS reports their findings.

Although it was long thought that the oviduct, or fallopian tube, played a passive role in fertilization, it has become increasingly clear in recent years that it actually has an active function in this process. Before fertilization can take place in the female mammalian reproductive organs, essential morphological changes take place in the oviduct epithelial cells. These changes allow a sperm cell to bind to the epithelium, which activates it to allow the sperm cell to fertilize an egg. To better study these processes, Dr Gadella and his colleagues have developed an oviduct-on-a-chip.

"The need to develop an oviduct-on-a-chip model had multiple causes," says Dr Gadella. "From a fundamental science viewpoint, it was necessary to improve the current cell culture methods to study the function and morphology of oviduct epithelial cells. These cells are difficult to grow in a Petri dish; they lose their cilia, secretory activities and three-dimensional structure, and with that their ability to guide fertilization and the further development to the embryonic stage."

Secondly, the model was also needed when improving in vitro fertilization (IVF). This is currently the most common method to realize fertilization in humans that have fertilization problems, but it fails in some other species like the horse. Dr. Gadella: "IVF is not always effective, partly because the embryo skips the oviduct in this process. In most animal models studied, the omission of the oviduct frequently results in no or low embryo development. In order to address these issues, my colleagues, Dr. Marcia Ferraz and Dr. Heiko Henning developed an oviduct-on-a-chip model."

IVF problems tackled

The prototype of the oviduct-on-chip was developed in collaboration with prof. Dr. Jos Malda and Dr. Pedro Ferreira da Costa within the Utrecht Advanced In Vitro Models (U-AIM) hub. The model is an air-liquid interface (ALI) in a 3D-printed system, which comprises a filter (called an "insert"), on which the oviduct epithelial cells are grown. On one side of this filter the blood circulation is simulated, which can resemble hormonal changes that take place during ovulation, for example.

The other side of the filter provides the ideal conditions for the epithelial cells to preserve their properties to promote fertilization and early embryonic development. On this side, sperm cells can be inserted into the system, and behave like they would in an in vivo oviduct: they bind to the epithelium, are activated by this binding, then are released and can consecutively fertilize the egg that was already matured in vitro and added into the same (fertilization) compartment. The process resembles in vivo fertilization much better than classical IVF does, since three stimulating factors must be present to the sperm and egg cells to achieve fertilization in IVF. These factors often cause adverse effects, such as polyspermia (fertilization by multiple sperm cells) and parthenogenesis (cleavage without fertilization), which are both lethal to the embryo. In the oviduct-on-a-chip system of Dr Gadella and his colleagues, there is no need to add any of these factors. "The epithelial cells are able to condition the chip-system on their own, in such a way that fertilization can take place. This reduces the risk of lethality, which is great progress, and a proof of principle that the system works."

Warning for 3D-bioprinting colleagues

Despite this first success, Dr Gadella experienced a major problem. Although fertilization was successful, the embryos in the system were consistently destructed during the four-cell stage. Because this doesn't take place in IVF, it was suspected that the cause could be found in the 3D-printed chip system. By using mass spectrometry analysis, they found a toxic dose of diethyl phthalate and poly-ethylene-glycols in the polymers used for 3D-printing, both compounds known as being endocrine disrupters. They published their findings in an environmental magazine, in which they emphasize the need for caution when selecting polymers for 3D bioprinting and bioengineering of culture and medical devices. After this discovery, Dr. Gadella in collaboration with Dr. Severine Le Gac (University of Twente) switched to other types of polymers, which do not contain these toxic compounds.

Improving IVF and fighting extinction of species

Now that the prototype of the oviduct-on-a-chip is successful in fertilization and early embryonic development, it has the potential to be used for several purposes. In addition to being an improved model for studying fertilization and reproduction, it could reduce the number of animal procedures necessary for embryo transfers in cattle, for example. Embryo transfer is a common procedure in the cattle industry, which has the goal to get certain genetic characteristics from one population (for example, Argentina) into another (for example, the USA), without having to transport the cow.

The most successful method to obtain embryos for transfer is through in vivo fertilization, after which oviducts are washed out in order to obtain the embryos. These will be frozen, transported and transferred into the acceptor cow to obtain offspring with a specific genetic profile. Another method is to obtain mature oocytes from the donor cow by ovum pick up (OPU), these can be fertilized in vitro and developed in vitro into embryos as well.

Both the flushing of an embryo from the cow's oviduct as well as the OPU method are considered to be animal experiments as they might compromise animal welfare. It is also possible to obtain pre-mature oocytes out of ovaries obtained from an abattoir (this procedure is not regarded as an animal experiment) but currently, such embryos are of lower quality. In 2013 the total number of embryo transfers worldwide was 1,275,000 (from approx. 200,000 donor cows with an annual expected increase of approx. 5 %). Of these embryos 725,000 were fertilized in vivo and flushed from donor cows, 550,000 were obtained by OPU from the donor cows and fertilized, while only 30,000 embryos were prepared after IVF on oocytes obtained from abattoir derived ovaries. Dr Gadella: "In our approach, we make use of ovaries obtained from abattoir to collect eggs and oviducts from the same culled animals to culture oviducts epithelia. The oviduct-on-a-chip allows successful fertilization of these oocytes. When commercially applicable, one can imagine how this can reduce the number of animal experiments on a yearly basis".

The oviduct-on-chip can also be used as a new method for fertilization in horses. "For unclear reasons, regular IVF does not deliver viable embryos in horses. We expect the absence of the oviduct in this process may be the cause. If this is the case, the oviduct-on-chip might be a successful alternative. We are currently investigating this, in collaboration with Dr Heiko Henning and Dr Bart Leemans (from the department of Equine Sciences of Utrecht University and from the University of Ghent, respectively)," says Dr. Gadella.

We are also investigating possible application in dogs: a canine gamete enters the oviduct after ovulation and will stay there for approximately seven days before it can be fertilized. This process cannot be modelled with IVF, but the oviduct-on-a-chip might provide a solution.

Dr Gadella and his colleagues have ambitious plans if they succeed: "We would like to develop a 'biobank of embryos', that can be used to protect endangered dog species from extinction, like the maned wolf, for example. Dr Marcia Ferraz is currently working as a postdoc in Washington to further investigate this possibility."

Furthermore, Dr Gadella expects that their discovery of toxic compounds in currently used 3D printing polymers not only validates the use of his new chips for novel screening methods of components on reprotoxicity (collaboration with Prof. Dr Majorie van Duursen, VU Amsterdam), but also to have a positive impact on the quality of 3D bioprinting itself. "Within the field of bioengineering, an incredible amount of pioneering is happening. I honestly think we have just scratched the surface of the countless possibilities in this area, so these are certainly interesting times to practice science. Take the organoid revolution, for example. The latest developments there are the possibility to create a channel-like structure consisting of multiple organoids. With our oviduct-on-a-chip, we now have a system that can generate a flow on both sides of the cells. Therefore, I think the chip and organoid fields will be much more intertwined in the future. However, evolving in this field involves a lot of trial and error, pioneering and taking leaps of faith. Therefore, we need to keep collaborating, also internationally, in order to achieve the progress we are all aiming for."

Source: 3Rs-Centre ULS Newsletter nr. 5-2019

PhD thesis about this subject:
Oviduct-on-Chip, Marcia Ferraz (UU) concerns 3D printing and nanofluidic perfusion technology to develop a 3D Oviduct-on-Chip culture system to study bovine gamete interaction and early embryo development.

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