16 July 2021

Mom from a test tube

How artificial follicles and eggs will help rhinos and us

Polina Loseva, N+1

It is forbidden to grow artificial human embryos, except as part of an experiment. But what about the germ cells? Japanese scientists started with mice: they collected a piece of mouse ovary from stem cells, grew eggs in it, fertilized them – and eventually got healthy mice. Article by Yoshino et al. Generation of ovarian follicles from mouse pluripotent stem cells published in the journal Science

The editor of N+1 reflects on which stories from the world of reproductive technologies can get a new impetus if the success of the Japanese with mice continues with rhinos and humans.


Four-week-old mice born as a result of the experiment. Drawings from an article in Science.

Cloning people is not only illegal, but also technically difficult. Embryos do not grow well without a mother – or rather, without a uterus and extra-embryonic tissues with which the embryo is attached to it. Alone, embryonic tissues cannot agree on where to grow the head and where to grow the tail. In order to orient themselves in space, they need to "feel" the mother.

The ban on creating clones, however, does not apply to embryos in a test tube – if they are intended for experiments. This requires not only a biological mother (more precisely, her uterus), but also a genetic mother – an egg donor. And it, as it turned out, is also not easy to grow in vitro. Like the embryo, it does not develop alone – it needs a follicle: a support group of non-ovarian germ cells. The follicle not only supplies the oocyte (the future egg) with food and protects against external dangers, but also signals when to start maturation and prepare for fertilization.


Follicles derived from stem cells.

Growing oocytes from stem cells began back in the 2000s. But with follicular cells, without which an egg will not grow from an oocyte, it did not work out: it was always easier to get them from real ovaries, mouse or human. Then artificial follicles were molded from donor cells, which supported the transformation of the oocyte into an egg. But in clinical practice, such technology will not work: every time doctors decided to help some infertile woman conceive a child, they would have to take part of the ovary from another woman (and at the same time make sure that they do not take eggs with her). This problem, in fact, has just been solved by scientists from Japan. They took mouse embryonic stem cells, grew oocytes and follicular cells separately from them, and then collected full-fledged follicles from the resulting cells, in which real eggs ripened.


The scheme of the experiment.

This means that a donor is no longer needed to create an artificial ovary: you can simply take any cells from a woman, reprogram them into stem cells, and then make an incubator for eggs from them. And now let's see where it can be useful to us.

Let's save the northern white rhino

There are only two northern white rhinos left on Earth – the female Najin and her daughter Fatu. The last male who could provide them with offspring died in 2014, leaving scientists with frozen sperm of not the best quality. It is impossible to fertilize Najin and Fatu with this sperm – both females are already too old and therefore infertile. All hope is for the remaining oocytes in their ovaries and females from a neighboring species – also white rhinos, but southern.

The plan to save the rhinos looks like this: stimulate the ovaries of Najin and Fatu so that they grow and expel the last eggs from themselves. Fertilize them in vitro with thawed spermatozoa. The resulting embryos should be planted to surrogate mothers, southern rhinoceros – they are already being trained to bear cubs conceived "in vitro".

Zoologists have very few attempts left. So far, only three viable-looking embryos have been constructed from the remains of sperm and extracted eggs. Whether new rhinos will be born from them is unknown. It is unclear whether it will be possible to "squeeze" an additional supply of oocytes from northern white rhinoceroses without harming their health. So it's impossible to predict how much time we have left. It would be much easier if rhino eggs could be produced in a laboratory. It would be possible to take samples of any other tissue from Najin and Fatu – for example, to draw from blood or fat – turn them into stem cells and assemble an incubator for the eggs of the northern southern rhinoceros. Then we will have at least an order of magnitude more attempts – and scientists who are trying to urgently give birth to descendants of Najin and Fatu will stop sweating their palms.

Let's replenish the stock of eggs

In humans, as in northern rhinos, oocytes are in short supply – their reserve in the ovary is limited from birth. And then it only gets smaller: some oocytes are lost during ovulation, some acquire chromosomal abnormalities, and some simply die under the burden of mutations. Depletion of the reserve leads to the fact that a woman becomes infertile – not because she is physiologically unable to bear and give birth, but because she has no healthy eggs left.

Embryologists are trying different ways to replenish this stock. Some are trying to stop mutagenesis in developing oocytes so that they die less often and live to maturity more often. And others at this time are looking for oocyte progenitor stem cells in the ovaries. If they find it, it turns out that our ideas about the finiteness of eggs are incorrect – and also that these progenitor cells can be extracted (or somehow propagated inside the ovary).

Moreover, in some works they seemed to be able to detect them. But when several groups of scientists went through the human ovary cells at once, nothing stem was found in it. And the question of whether it is possible to increase the supply of eggs in the body remained open.

If an artificial follicle can be grown for humans, as well as for mice, all these searches for the "grail of oocytes" will become completely unnecessary. And those who are currently engaged in them will be forced to switch to something more practical - because even if the progenitor cells of oocytes are hidden somewhere in the depths of the ovaries, it is easier to multiply eggs in vitro than to "wake" them inside the female body.

Let's give cancer patients a chance

Some women risk becoming infertile very early, despite their oocyte reserves – when they have to undergo chemotherapy. And if this therapy is associated with a tumor in the reproductive system, then doctors face a difficult task: they need to somehow save the patient's oocytes in order to give her the opportunity to get pregnant after she is cured. At the same time, getting them in the usual way – "pumping" a woman with hormones, forcing several oocytes to mature at once and causing superovulation – is too risky, because hormones can spur the growth of tumor cells.

Not so long ago, embryologists came up with a clever way to cope with this: they cut out a section of the ovary with oocytes from the patient and grew them in vitro, watering the hormones and serum of the woman herself. It would be possible to return them to the ovary after that – as soon as the chemotherapy is over and remission occurs – but the patient did not agree to this. Therefore, the mature eggs were frozen, and five years later, when the woman despaired of getting pregnant naturally, they were thawed and used for IVF.

But this method is not without risks. At each stage of the procedure, part of the eggs may die, and the more steps on the way from an immature oocyte to IVF, the higher the chance that there will be no healthy eggs left. All this could have been avoided if we had been able to grow follicles in a test tube. We take a sample of another tissue from the patient, which does not suffer so much from therapy – and collect follicles on request.

Let's edit the person (and not offend anyone)

There are several ways to cure a child of a hereditary disease before it develops. This is, for example, genetic editing during fertilization – but the world scientific community does not approve of this idea yet. Or mitochondrial transfer – if the disease is caused by a mutation in the maternal mitochondria, they can be replaced with donor ones. This method is also controversial, but in some countries, "children from three parents" have been giving birth for a long time.

The problem with these technologies is that they involve direct intervention in the genome of the future person. When we treat a disease that has not yet arisen in an unborn child, we "correct" his nature without his permission. With "children from three parents" everything is relatively simple: we do not climb into the nuclear genome with our hands, but only change the faulty mitochonodria to a donor one. But when it comes to reshaping DNA, the consequences can be much more serious. And in order to evaluate them, we need experiments – and for this sooner or later we will have to grow "test" embryos and check how they develop. Until recently, all such "tests" were limited to the 14th day of development, and this period was not enough to assess the viability of the embryo.

Perhaps some ethical disputes could be avoided if genes and mitochondria were edited not in the embryo, but in the egg. At the same time, it could be argued that we are not treating an embryo, but a separate cell – and the disease is already present in it in the form of a genetic mutation.

Recently updated international guidelines do not prohibit genetically modifying human germ cells. True, the scientific community does not yet recommend getting embryos from them, which will then be transferred to a woman's uterus, but it admits that one day we will do it.

Let's pick up a child

Even before there were reports of the first genetically edited children, there was talk in the scientific community about how it would be possible to "improve" a person in principle. But no one knows how to do it reliably and accurately enough to seriously take a swing at the "refinement" of the genome (or does not speak about it publicly). But we already know how to choose which embryo to become a child – with the help of preimplantation genetic diagnostics.

Not all the traits that someone might want to see in their children are easy to assess by the sequence of genes - it is impossible to predict even simple signs, like growth, with accuracy. In addition, in some countries, it is prohibited to select embryos for signs unrelated to hereditary diseases. But somewhere there is no such prohibition – and from time to time there are companies that promise to give a detailed description of your unborn child by the embryo genome so that you can choose who to give birth to.

However, even the absence of this prohibition does not mean that all parents will rush to choose their children "better". Since such a diagnosis works only in conjunction with IVF, for its sake mothers will have to go through the entire prescribed cycle: hormone therapy, maturation, superovulation, egg extraction surgery. These procedures can be quite painful and carry certain risks for a woman's body – for example, high doses of hormones can be fraught with the development of tumors.

This inconvenience, however, will cease to be an obstacle if we learn how to grow eggs from stem cells. Moreover, the number of options from which parents will be able to choose will become potentially infinite (although it will remain, of course, within the "capabilities" of the parent genome) – the only question will be how long you are willing to pay for new attempts. But technology, as you know, tends to get cheaper over time.

 But not right now

Skeptics, of course, are in no hurry to project the results obtained on mice, on humans, and even more so rhinos. After all, a person (and his oocytes at the same time) develops much longer than a mouse, and his follicular cells may be more capricious in culture.

In addition, before applying this method to humans, its developers will have to prove for a long time and carefully that eggs are completely "identical to natural", especially with regard to genes. For example, it will be necessary to check that the cells that are taken from an adult woman are completely reprogrammed – and the DNA of the resulting egg does not carry epigenetic markers characteristic of fat or blood.

And you will also need to make sure that the resulting eggs carefully overcome the division stage and approach fertilization with a full set of chromosomes. Japanese scientists have already coped with this in their experiment: the mice that turned out in their laboratory are quite healthy and fertile, they did not get any genetic anomalies. Maybe we should hurry up, at least for the sake of rhinos?

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