08 August 2014
In an important paper published August 8th 2014 in Human Reproduction, Prof Bruce Campbell and his team at the School of Medicine, Nottingham set out some exciting results on the restoration of fertility - the cryopreservation and transplantation of whole adult sheep ovaries. This is the first time that a whole organ from an adult large mammal has been successfully frozen, successfully transplanted and successfully produced immediate results.
Premature ovarian failure affects 1–2% of all women and is due to a number of causes such as genetic predisposition and cancer treatment - and this loss has serious clinical and psychological effects. Using a new method, a Planer controlled rate freezer and different post operative regimes, Prof Campbell restarted 100% of the ovarian function and produced high rates of natural fertility in his sheep: pregnancy rate 64%; live birth rate 29%.
Freezing and subsequent transplantation require successful cryo-preservation of both the ovary and its vascular supply. Work to date has not been so successful; five years ago a delivery was made, but 545 days after transplantation. Additionally the follicular survival rate in the grafted ovaries was just 1.7%–7.6%. The Nottingham work involved adult sheep, rather than lambs, where ovaries are more typical of human ovaries but much harder to freeze and was immediately successful.
What the Nottingham team discovered was that the duration of cryo-protectant perfusion was highly significant and that the degree of this penetration - and the maintenance of post- operative vascular viability - were the critical determinants of success.
Transplanting fragments of ovarian cortex for human fertility is a new procedure and has its problems - both practically and because these fragments contain only a fraction of a woman's ovarian reserve, and so can only provide the recipient with a relatively brief fertile window before the supply of oocytes contained within their graft is used up. The problem of longevity of the actual graft may be exacerbated by the freeze-thaw process and high rates of follicle loss occur. So, grafting pieces of ovarian cortex may have drawbacks as a fertility treatment with, for example, older patients in whom follicle density is already low. The cryogenic preservation of the whole ovary, complete with vascular pedicle, for later transplantation provides a very promising alternative strategy.
All methods of tissue cryopreservation require the replacements of intra-cellular water with penetrating cryo-protective agents. During organ preservation this has usually been tried by vascular perfusion. The new Nottingham technique, suing Planer special slow rate freezing equipment, has the advantage of providing very short diffusion distances for the cryo- protectant to penetrate, but has the potential to cause microvascular damage. The cellular injury and loss caused by the formation of intra-cellular ice during cooling and warming can usually be avoided using controlled rate slow freezing methods, whereas the formation of ice in extra-cellular locations, and particularly in blood vessels, can produce serious injury and prevent organ function.
Before this technology can be used in human preservation the perfusion rates and the levels of anti-coagulant will need to be optimised for different sized ovaries and of course trials on the normality of offspring will be required.
This method holds great promise for the preservation of fertility in women. Additionally it could also perhaps be applied to the cryopreservation of other reproductive organs or even major ones such as kidneys where there are considerable difficulties in storing donated tissue.
Whilst small, the ovary is an organ, with multiple cell types and a separate blood supply. So far attempts to freeze and store whole organs such as the heart, liver and kidney have proved uniformly unsuccessful. The primary challenge with whole organ cryopreservation is that of preserving an intact vascular system.
Fertility drugs used to stimulate ovulation did not increase the chances of breast cancer for most women in a long-term study of around 10,000 women in the USA.
However, an elevated risk was noted in women who were treated with high doses that are no longer recommended and a subset of women who did not become pregnant after treatment.
As previous studies have produced mixed results on the effect of fertility treatment on breast cancer risk, lead study author Dr Louise Brinton of the National Cancer Institute, USA, said her team set out to look at any long-term effect 'after controlling for other factors that have been shown to be correlated with both breast cancer risk and use of those drugs'.
She added: 'Overall, our data show that use of fertility drugs does not increase breast cancer risk in this population of women, which is reassuring'.
The study looked at women who were evaluated or treated at five clinics in the USA between 1965 and 1988. Just under half received fertility treatment with clomifene citrate and/or gonadotrophins. All patients were followed up over the course of 30 years and breast cancer rates were compared between groups. The researchers took into account confounding factors like a family history of breast cancer.
Most women who received clomifene citrate did not show an increased risk of breast cancer compared with women not given fertility drugs. However, those who received 12 or more cycles of this drug were one-and-a-half times more likely to develop invasive breast cancer.
Breast cancer risk was also doubled in women who failed to become pregnant following treatment with both clomifene citrate and gonadotrophins. This finding counters that from a separate, smaller study published two years ago, which found a reduced breast cancer risk for women given fertility drugs who did not become pregnant (see BioNews 666).
Dr Brinton remarked: 'The observed increase in risk for these small subsets of women may be related to persistent infertility rather than an effect of the medications. Nevertheless, these findings stress the importance of continued monitoring of women who are exposed to fertility drugs'.
Although the increases in breast cancer risk in this population were relatively modest, those who went on to develop breast cancer did so at an average age of 53 years, younger than that seen in the general population.
As well as suggesting that this group of women should continue to be monitored, the authors say that further work is needed to investigate the effect of current fertility drug regimes on breast cancer risk.
The study was published in the journal Cancer Epidemiology, Biomarkers and Prevention.
Scientists at the University of Oxford, UK, believe they have identified the way in which embryos implant in the uterus, providing essential information which may be used in the future for infertility treatments and offering hope to thousands of infertile couples.
Implantation of an embryo to the lining of the mother's uterus is an essential process that takes place at an early stage of development. The embryo initially attaches and forms a contact with the uterus lining, which triggers a cascade of signals in both the embryo and the uterus. This allows cells from the embryo to start moving across into the uterus, finding blood vessels in the mother and eventually forming the placenta.
Problems in the implantation process can lead to loss of potential pregnancies, even in couples trying to conceive without infertility problems. Current estimates suggest that infertility affects one in seven couples in the UK, with around 32,000 couples seeking infertility treatment each year. It is thought that a significant number of these patients could be infertile as a result of implantation problems.
The team of scientists, led by Professor Helen Mardon from the Nuffield Department of Obstetrics and Gynaecology at Oxford, along with Professor Anne J Ridley at King's College, London, added embryos to a layer of cells from uterus lining in a culture dish to mimic events in the womb. They were then able to video embryos implanting themselves in the cell layer, allowing the scientists to dissect the molecular processes involved. Their findings were published in the journal Proceedings of the National Academy of Sciences.
Their investigation led them identify two proteins that are essential players in the implantation process. They are from the Rho GTPase family of proteins, and ensure that cells in a particular part of the uterus lining move out of the way of the 'invading' embryonic cells.
Professor Mardon said: 'We have shown that two proteins, called Rac1 and RhoA, control the invasion. The first stimulates cells in the womb lining to move and allow the embryo to invade and implant properly while the second inhibits this. We believe this controlled balance of the two proteins is critical for successful implantation of the embryo. If the balance of Rho GTPases is altered, the cells of the womb lining don't migrate and the embryo doesn't implant'.
The findings bring new hope to people with infertility issues. The new information will help the understanding of how this process works, and therefore aid 'the development of drugs to help embryos implant properly', said Prof Mardon.
This novel way of creating sperm cells could bring about new therapies for male infertility, which is thought to be the problem in roughly half of couples who have trouble conceiving.
'This research provides an exciting and important step for the promise of stem cell therapy in the treatment of azoospermia, the most severe form of male factor infertility', said Dr Michael Eisenberg, director of male reproductive medicine at Stanford University, where the study was conducted. 'Being able to efficiently convert skin cells into sperm would allow this group to become biologic fathers'.
The research looked at men with a condition known as azoospermia, who were unable to produce healthy mature sperm due to missing genes on their Y chromosomes.
The team took skin samples from five men, three of whom had azoospermia and two of whom were fertile. They transformed them into induced pluripotent stem cells, and when these were transplanted into mouse testes, they developed into early-stage sperm cells. These may have become fully mature sperm cells if the stem cells had been transplanted into the men's testes, the researchers believe.
'We saw better germ-cell differentiation in this transplantation model than we've ever seen', said Dr Renee Reijo Pera, who led the study. 'We were amazed by the efficiency. Our dream is to use this model to make a genetic map of human germ-cell differentiation, including some of the very earliest stages'.
Dr Allan Pacey from the University of Sheffield commented to the Telegraph: 'The received wisdom is that if you have a Y chromosome defect like this you don't make sperm. Until I read this paper I would have said if you take skin cells from an infertile guy it won't work. But what this seems to suggest is that it could'.
Warning that sons born using such a procedure would be at risk of inheriting infertility, Dr Pacey added: 'Obviously the outcome is poorer than if you don't have the defect, but the fact that they could do this at all is quite exciting. At the moment the door is closed to these men'.
Azoospermia is thought to affect one percent of men worldwide, but the team hopes that other men affected by infertility could one day be treated using this technique.
'[This study] provides very intriguing possibilities for men rendered sterile after cancer treatments', said Dr Eisenberg. 'Infertility is one of the most common and devastating complications of cancer treatments, especially for young boys and men'.