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12 February 2018

By Professor Robin Lovell-Badge
Appeared in BioNews 937


The problem of fertility preservation for girls and women undergoing cancer treatments has been a subject of research for many decades. The recent study by McLaughlin and colleagues from Professor Evelyn Telfer's lab at the University of Edinburgh, UK, is aimed at finding a solution to this problem, with the claim that they have produced mature human egg cells in the lab for the first time (see this week's BioNews story).

Egg cells or oocytes begin their development in the ovary as a small cell contained within a single layer of granulosa cells, together making up a 'primordial follicle'. For these to develop to a stage when they can be fertilised requires a period of growth of the oocyte (from 15 to about 100 micrometres) and development of a multi-layered and complex follicle, which after puberty usually takes a minimum of about four weeks. This is then followed by a phase referred to as maturation when the fully-grown oocyte completes the first meiotic cell division (MI) and divides asymmetrically to give a large MII oocyte and a very small first polar body, which usually dies within a day and does not contribute to any subsequent development of an embryo.

In vitro maturation (IVM) to go from a fully-grown to an MII oocyte is already a fairly common procedure (although still formally an experimental one), sometimes for cancer patients, but also for other cases where standard IVF procedures are difficult, such as polycystic ovary syndrome (PCOS). The use of this technique has led to many hundreds of apparently healthy babies. Therefore, for women who have fully grown and/or mature oocytes (eggs), it is now possible to freeze these, thaw them and carry out IVF. It is also possible to freeze pieces of ovary and then to thaw and graft these back to the ovary (or a nearby site) after cancer treatment. Care has to be taken with the latter approach, however, in case the ovary tissue contains some cancer cells, which is likely if the cancer is, for example, a leukaemia or there has been metastasis.

For prepubertal girls, however, they will not have any fully-grown oocytes; indeed they will almost all be at the primordial follicle stage. This McLaughlin et al. paper claims to have developed a method for growing and maturing these all the way through to oocytes that might be capable of being fertilised (metaphase II, or MII). Despite considerable effort form several labs, this had been done previously only in the mouse, with the first demonstration some 20 years ago, and with substantial improvements made in following years.

Nevertheless, several of the steps had already been achieved with human material, notably going from primordial follicles to secondary (multi-laminar) follicles and from secondary follicles to MII oocytes, so the novel aspect of this current work is to have joined these into one continuous process.

However, there are several problems. It was really quite inefficient, with perhaps 385 early follicles (of which about 80 percent were primordial) giving 87 secondary follicles suitable for the second stage culture method. Of these, 54 developed a typical fluid-filled cavity suggesting that they were progressing normally, but of these, only 32 gave fully grown oocytes. These were then transferred to typical IVM conditions. However, only nine gave rise eventually to mature fully-grown MII oocytes.

Critically, the pieces of ovarian tissue that the authors began with were not from prepubertal girls, but were biopsies from adult women, which clearly contained some follicles that had already begun to develop. The authors can't be certain that the ones that did make it to fully-grown MII oocytes actually arose from primordial follicles; they could have developed from later stage 'primary follicles' or even some of the already growing secondary oocytes present in the original tissue. The timing from beginning to end was also relatively fast compared with what is thought to happen in vivo, which could suggest that the mature oocytes were indeed not coming from primordial follicles. If they did originate from primordial follicles, their development may have been too fast, then this might lead to abnormal oocytes.

The characterisation of the oocytes obtained was rather minimal, but it was very obvious that they were not quite right. Usually, when the fully-grown oocyte divides to give rise to an MII oocyte, this is a very asymmetric cell division producing the large oocyte and a very small 'first polar body'. The latter contains a nucleus, but has too little cytoplasm to support its survival for long. However, the 'polar bodies' associated with the MII oocytes in this paper were very large. (The first meiotic cell division had not been sufficiently asymmetric.) In addition to suggesting that the oocytes had not developed normally, these large polar bodies could probably survive for longer and may even be capable of being fertilised.

If both the oocyte and the polar body are fertilised (by separate sperm) this can lead to the development of a mosaic individual, composed of cells with two distinct genotypes. ICSI (intracytoplasmic sperm injection) could be used to fertilise just the larger cell, but this will be smaller than normal, and it would be necessary to know that it wasn't deficient in some way. The large 'polar body' might also interfere with development of any embryo.

Moreover, although the authors showed evidence for a spindle (the cellular structure on which the MII chromosomes line up, moving to opposite ends of the cell when it divides in two), they did not check to see if there were any problems with the chromosome and their subsequent segregation. They did not see if the oocytes could be fertilised, which would have required an HFEA (Human Fertilisation and Embryology Authority) licence, or ask what would happen if they were activated. All the final IVM steps have been done before to yield normal fertilisable eggs that have given children, so the fact that the oocytes in this study were abnormal suggests that there were problems with the steps that were used to promote the early in vitro growth.

Finally, for clarification, this work is not describing the development of mature oocytes all the way from pluripotent stem cells in vitro. It is beginning with small oocytes that are already enclosed in follicles that have been obtained from adult ovaries. In this respect it is also not clear whether the methods will be of use for the purpose of allowing fertility preservation from stored ovarian tissue obtained from prepubertal girls prior to cancer treatments, which is one of the main justifications for the research. The follicles in prepubertal ovaries will be at an early stage, not yet primed for growth, and perhaps quite different from those in an adult.
The authors were aware of the limitations of their work and gave balanced comments. But the media interest seems to have been egged-on by an over-enthusiastic press release from the University of Edinburgh. This was not very responsible given the topic and the desperate need for workable solutions. A more balanced judgement might be that the paper indicates that it should be achievable, but don't count your chickens before they have hatched.