Chemo- and radiotherapy destroy cancer cells but they also destroy eggs. This gonadal toxicity results in early menopause and ovarian failure. Cryogenically freezing the patient’s own ovarian tissue prior to treatment and reimplanting it afterward has been successful—80 children have been born, including the first at UCL’s Saint-Luc University Hospital in 2004—but with some cancers malignant cells in the reimplanted ovarian tissue can induce a recurrence. Since 2008, Christiani Amorim and her ECRI team have focused on an alternative: inventing an artificial ovary.
Fickle follicles
It all begins with the follicle. The ovarian follicle is a tight cluster of somatic cellssomatic cells around an immature egg cell called an oocyte. The somatic cells’ job is to support the oocyte as it grows. In this early stage of egg development, the follicles are referred to as primordial.
‘The mammalian ovary produces millions of primordial follicles,’ Prof. Amorim explains, ‘but most die and few ovulate’—that is, leave the ovary for the purpose of fertilisation. ‘The question is how to make human follicles survive in an artificial ovary, and get them to grow sufficiently to be able to liberate a mature egg. We start by isolating primordial follicles and ovarian cells from the ovary, graft them together, and, basically, try to build a matrix for them.’
A matrix is the material in which cells are embedded to keep them together. Finding the right material to build a matrix for follicles has proved difficult, because follicles are fickle things. For one, a follicle needs a rigid matrix. ‘The human follicle needs to maintain a three-dimensional structure. If it loses it, it dies.’ And yet the matrix can’t be too rigid. ‘The primordial follicle grows rapidly: from only 30 microns across in the primordial stage to 8 millimetres when it’s ready to ovulate. So while the matrix needs to be rigid enough to help maintain the follicle’s three-dimensional structure, it also needs to be “soft” enough and have a proper rate of degradation to accommodate growth.’ Degradation is chemical breakdown that supports cell proliferation. ‘An ovary is made of soft, but not too soft, tissue. Synthetic materials could be used, but my feeling is that something natural will be more familiar to the follicle.’
Bonding with blood clotter
Research teams elsewhere tried a few materials, including collagen, alginate and bioengineering materials. The UCL team also tried alginate but found its degradation rate insufficient. So they opted for fibrin. ‘It’s a natural polymer, our bodies produce it.’ Thus the idea is to encapsulate primordial follicles and ovarian cells in a matrix made of the same fibrous protein that rushes in to stop a cut from bleeding by forming a clot. ‘We use a commercially available fibrin kit and we can play with its fibrinogen and thrombin concentrations to make the matrix softer or harder, which can influence follicle survival and growth, and also to play with matrix degradation. In the end, the fibrin matrix containing follicles and ovarian cells looks like a tiny white clot. This is what we implant. This is the artificial ovary prototype.’
It’s not built to last. By design, the fibrin matrix is like a skin soon to be shed. This is why ‘playing with degradation’ is so crucial. The fibrin matrix, once transplanted, must endure long enough to allow what’s within it to grow and eventually replace it from the inside out: a whole new matrix called the extracellular matrix.
‘So after implantation, the tiny white clot degrades and is replaced by this new tissue formed by the ovarian cells, the new extracellular matrix and follicles. We don’t yet know the optimal degradation rate for the fibrin matrix, but perhaps two weeks minimum, because by this time the ovarian cells grafted with the follicles proliferate and synthesise the extracellular matrix—which is actually the original matrix present in the ovarian tissue.’ In other words, given that what is in the fibrin matrix is genuine ovarian cells producing new tissue, the extracellular matrix is the original ovarian matrix: the artificial ovary—which houses transplanted, proliferating ovarian cells—has spawned a real one.
Just a few more years
‘In the last year we started implanting our fibrin matrix containing human follicles in mice, to get a feel for how the follicles survive and grow.’ In an article recently published in Reproductive Biomedicine Online, she and her team presented the results: 22% of the follicles survived for one week and some started growing. ‘That may not sound like much, but back when we started transplanting ovarian tissue into mice, we had a survival rate of 27%, so we’re not so far behind with artificial tissue.’
The follicles that didn’t make it, she and her team hypothesise, may have lost their three-dimensional structure—the matrix was too soft. They will now experiment further with fibrin. ‘We’ll make it stiffer and repeat the experiment in mice. If everything goes well and we achieve 21 days of survival, then we’ll repeat the experiment again and aim for six months’ survival, which is the time necessary to make an egg ready to ovulate. If everything’s alright after six months, then we’ll attempt it on another animal, a sheep or primate, and mimic a patient’s situation, that is, remove the animal’s ovary, isolate its ovarian follicles and cells, encapsulate everything in the matrix, and then implant our matrix to see if fertility is restored and if the animal can become pregnant. This would be the pre-clinical phase, before conducting human trials. We’re slowly building up a list of patients interested in participating, some have tissue frozen in our bank, but it’s not really our focus right now, because we’re still a few years away.’
Is there hope for infertile women who aren’t cancer survivors?
‘This is something we can consider if our results with the artificial ovary are superior to those of ovarian tissue transplantation. In this case, the artificial ovary could replace the ovarian tissue transplantation for women with benign or autoimmune diseases.’
Lee Gillette
A glance at Christiani Amorim'bio
Prof. Christiani Amorim received her PhD from Brazil’s Federal University of Santa Maria. She then worked at Florence University (Italy) and subsequently served as Associate Professor at Brasília University (Brazil). Currently, she is professor at the Université Catholique de Louvain. In recent years, Prof. Amorim has focused her attention on the development of a transplantable artificial ovary to restore fertility in cancer patients. Her pioneering studies helped launch the field of ovarian tissue engineering and she has been actively leading the first reproductive tissue engineering team. Prof. Amorim is also an associate editor of the journal Human Reproduction and Annals of Biomedical Engineering.