"The use of automated systems may result in higher quality oocytes".
Author: Roberto de la Fuente
CRYOPRESERVATION AS A MEANS TO PRESERVE FERTILITY
Human reproduction has benefited from the possibilities offered by cryopreservation as much as any other discipline, in particular with the latest use of vitrification as opposed to slow freezing techniques (1). The increased efficiency of different techniques has progressively led to a “freeze-all” strategy within last years, which supports an improvement in clinical outcomes.
Current social trends imply a significant delay in the age of motherhood. There are a variety of factors to explain such shifts in demographic charts. However, not only social freezing has popularised the use of vitrification for female gametes; the reality is that research on oocytes, donor banks, or fertility preservation treatments in cases of cancer are possible thanks to cryopreservation.
Considering the efficiency of vitrification over slow-rate freezing methods when applied to embryos and oocytes (1), it is understandable how technology and research combined succeed in providing new and improved techniques. A good example of this is the development of devices for automated vitrification like GaviTM (2).
AUTOMATED OOCYTE VITRIFICATION
The basics of vitrification are the combination of an ultra-rapid drop of temperature and the use of high concentrations of cryoprotectants, with the aim of achieving the total elimination of ice crystal formation, both in the inside and the outside of the cell. The volume of the medium in which the oocyte is immersed also needs to be minimized in order to favour the fastest temperature drop. Closed systems (in which the oocyte is not in direct contact with liquid nitrogen) need to make up for the recipient and its volume to avoid further interference in this vein.
Prior to the ultra-fast cooling, the oocyte is exposed to different solutions that need to be adequately incorporated (and removed) to safely “equilibrate” the cell while avoiding toxic effects and osmotic shock (1, 3). To date, multiple open and closed systems to cryopreserve oocytes (and embryos) have been designed (4, 5), but all of them require a strict control of incubation times, steady hands and high expertise from the embryologist. However, automated systems allow treating every single cell in the exact same way, thus removing any external variable that may introduce artefactual variation between oocytes, and guaranteeing a consistent vitrification method. Potentially, procedures like this may represent a more efficient approach.
EVIDENCES FOR A MORE EFFICIENT VITRIFICATION: AUTOMATED SYSTEMS
Recently, the group of Dr. Montserrat Boada from Dexeus Mujer (Barcelona, Spain) has presented original results from an on-going investigation on the fertility potential of automated vitrified oocytes. According to the authors, there is strong evidence to support that the use of automated systems may result in higher quality oocytes than by performing the manual procedures currently standardised in clinics (6).
Improved data may be explained by the elimination of inter-oocyte variation, achieved by treating every single cell in exactly the same way; oocytes are first loaded in batches onto specific pods in the device, which automatically adds and removes the cryoprotectant and equilibration solutions. Additionally, the specific design guarantees minimization of the volume, any possible interference with the oocytes is eliminated, and pods are sealed to protect from direct contact with liquid nitrogen (7, 8). These and other features of the system represent a great advance as a whole, given the results achieved by the investigators (2, 6).
Oocytes that were automatically vitrified showed a slightly higher survival rate. This correlates with higher quality, even though the authors state that “(…) results were not significant due to the small number of oocytes utilized up to date (…)” (6). Nevertheless, fertilization rates were in fact notably higher than for manually vitrified oocytes when the experimental group was subsequently subjected to ICSI (6). Moreover, specific defects on oocytes were less frequent in those from automated vitrification (Table 1). Altogether, results shown are actually promising, and may point to the right path in the laboratory for future applications.
The study has been presented at the recent Congress of the American Society for Reproductive Medicine, held in San Antonio, Texas (US), and at the IX Congress of the ASEBIR in Barcelona (Spain), in which Dr. Miquel Solé, first author of the published study, was awarded the Best Cryobiology Communication Prize. Nonetheless, news got even better; the work of this group has resulted in the first baby in the world born from a (semi-)automatically vitrified oocyte. The achievement took place last September in Italy, thanks to the work done at Dexeus Mujer in Barcelona [click here to read the press release from Dexeus Mujer (in Spanish)]. The baby was born through natural labour and was perfectly healthy.
With these data and the proven clinical success, it seems reasonable to think that automation of vitrification arises as a significant progress in assisted reproduction. Nevertheless, further clinical and technological research is needed to keep improving this type of approaches, and only time will tell how much automated procedures can serve assisted reproduction.
This article has been selected for publication in the Scientists in Reproductive Technologies (SIRT) Newsletter of The Fertility Society of Australia: DE LA FUENTE, R. (2017) Automated vitrification systems: the future of embryo/oocyte vitrification? Fertility Society of Australia - SIRT Newsletter 4(20): 23-24.
1. Edgar DH, Gook DA. A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Hum Reprod Update. 2012;18(5):536-54.
2. Roy TK, Brandi S, Tappe NM, Bradley CK, Vom E, Henderson C, et al. Embryo vitrification using a novel semi-automated closed system yields in vitro outcomes equivalent to the manual Cryotop method. Hum Reprod. 2014;29(11):2431-8.
3. De Munck N, Vajta G. Safety and efficiency of oocyte vitrification. Cryobiology. 2017. 10.1016/j.cryobiol.2017.07.009.
4. Vajta G, Rienzi L, Ubaldi FM. Open versus closed systems for vitrification of human oocytes and embryos. Reprod Biomed Online. 2015;30:325-33.
5. Momozawa K, Matsuzawa A, Tokunaga Y, Abe S, Koyanagi Y, Kurita M, et al. Efficient vitrification of mouse embryos using the Kitasato Vitrification System as a novel vitrification device. Reprod Biol Endocrinol. 2017;15(1):29.
6. Sole M, Polyzos N, Gonzalez Llagostera C, Carrasco B, Coroleu B, Veiga A, et al. Automatic vs manual vitrification of human oocytes. Preliminary results of the first randomised controlled trial using sibling oocytes. Fertil Steril. 2017;108(3 Supplement):e57.
Fibroblasts derived from trisomic mice can be reprogrammed into diploid iPSCs, differentiate into functional haploid sperm and generate normal fertile mice.
Author: Roberto de la Fuente
The common sex chromosome dosage in mammals is XX for females and XY for males. This implies that under normal circumstances each one of the X chromosomes in XX females comes from one of the parents, whereas in XY males the only X chromosome is always maternal, because the Y chromosome always comes from the father. Even though both X chromosomes in an XX individual behave as homologs, they may contain different variations of the same genes (alleles). Also, and because they come each from a different parent, they carry epigenetic information known as genetic imprinting, which allows to discriminate one from the other.
During spermatogenesis (sperm formation in the testis), an essential step called meiosis takes place. It consists of two rounds of cell divisions without DNA replication in between, separating first homologs and then chromatids, each into a single spermatid (the precursor of the spermatozoon). As a result, gametes with half the number of chromosomes are formed (haploid, as opposed to diploid somatic cells), hence the separation of X and Y chromosomes into different cells.
However, errors do occur, and so it happens that sex chromosomes do not always follow their normal behaviour. Instead, there are occasions in which both chromosomes may end up in the same spermatid (Fig. 1). If an XY-bearing spermatozoon fertilizes a normal X-bearing egg, the result is an XXY embryo. This sex chromosome trisomy (SCT) is one of the most common causes of infertility (in addition to other phenotypic traits), and it is known as Klinefelter syndrome. Similarly, other errors leading to nondisjunction of sex chromosomes can result in X0 females (Turner syndrome), XYY males (Jacobs syndrome) or even more complex combinations with multiple sex chromosomes (XXX, XXXX,...). Even though in most cases there are characteristic phenotypes associated to these chromosome abnormalities, mosaicism is also frequent (different cells containing different chromosome dosage), and so affected individuals may too have chromosomally normal children if their contributing gametes happen to be also genetically normal.
In a recent study led by sex chromosome expert Dr. James Turner (Francis Crick Institute in London, UK) (2), SCT-infertility has been overcome in mice. Researchers bred XXY and XYY infertile mice and used them to generate a line of cultured fibroblasts (with the original SCT complement), and then reprogrammed these into induced pluripotent stem cells (iPSCs). iPSCs were then differentiated into primordial germ cell-like cells (PGCLC) and later on into functional sperm (Fig. 2).
Fig. 2. Illustration of the process detailed in Hirota et al (2017). Fibroblasts from infertile, trisomic XXY and XYY mice are cultured and reprogrammed into iPSCs. During the process, the extra sex chromosome is lost. iPSCs are induced to differentiate into PGCLCs and then transplanted into germ cell-deficient testes from sterile W/Wv mice. Spermatogenesis is fully restored and functional sperm is formed. Normal sperm differentiated from XXY-/XYY-derived iPSCs is then injected into a normal X-bearing egg through ICSI, generating euploid, fertile offspring.
Interestingly, both in XXY and XYY iPSCs lines, spontaneous loss of a sex chromosome was observed. In both cases, a high percentage of cells had lost the extra duplicated sex chromosome, leaving the remaining complement as a normal XY. Such loss was also observed in normal XX and XY control lines, yet in a significantly much lower rate.
Following chromosome loss back to “normal”, XXY- and XYY-derived iPSC lines were differentiated into male germ cells (PGCLC), which were subsequently transplanted into testes from infertile mice lacking the germline. The result was the restoration of spermatogenesis for all cell lines used. Furthermore, functional sperm originated from these lines was successfully employed to fertilize eggs through ICSI, giving rise to genetically normal embryos and healthy, normal and fertile offspring.
The authors also showed promising results in preliminary experiments with human cells, demonstrating that returning cells to their original normal chromosome complement is possible. However, they warn about all risks associated with induced pluripotent stem cell manipulation and transplantation, as well as legal and ethical issues on these matters.
This said, it is undeniable that current technologies can help treat specific genetic conditions resulting in infertility, thus giving hope to patients who had found no alternative, so far. The future is becoming present, faster and faster every day.
1. Maiburg M, Repping S, Giltay J. The genetic origin of Klinefelter syndrome and its effect on spermatogenesis. Fertil Steril. 2012;98:253-60.
2. Hirota T, Ohta H, Powell BE, Mahadevaiah SK, Ojarikre OA, Saitou M, et al. Fertile offspring from sterile sex chromosome trisomic mice. Science. 2017;pii: eaam9046.
Author: Roberto de la Fuente
Genetic modifications are still seen with both excitement and reticence by general population. This poses a challenge, being scientists and media responsible for the information available to the public. Besides GMOs (genetically modified organisms) for industry or food, the last decade has witnessed a growing interest in genetic modifications and genome-editing techniques applicable to humans. So, it is important to note that people who are not specialists in the field have every right to be aware of the progress on these technologies and to have access to breaking-news on the subject.
The latest groundbreaking achievement has been just published online this week in Nature by an international collaboration of different groups (1). The paper reports the “correction” of a mutated gene in human embryos that leads to a severe heart disease in adults. This mutation consists in a four-nucleotide deletion in the MYBPC3 gene, which encodes a protein essential for the proper development and structure of the heart (cardiac myosin-binding protein C or cMyBP-C) that affects contraction and relaxation of the cardiac fibres. Individuals carrying this mutation suffer from hypertrophic cardiomyopathy or HCM, and it is the cause of unusual cardiac growth, arrhythmias and a high percentage of sudden death due to heart failure in otherwise healthy people. Because it is an autosomal dominant mutation, the disease is manifested with just one of the two alleles mutated. Additionally, it is normal that the individual has already fathered offspring when the disease manifests, which implies the mutation has been passed on to the next generation.
This is an example of those diseases in which prevention is not yet possible, and the only viable treatments are symptomatic attenuation. Therefore, a current possible approach for preventing transmission of the mutation to the offspring is preimplantation genetic diagnosis (PGD), which allows for discarding affected embryos (theoretically 50% of them because of the dominant inheritance).
Scientists found the goldmine for genetic modifications at the beginning of the 21st century with the development of the ZFN (zinc finger nucleases) that led to TALENs (transcription activator-like effector nucleases). These systems gave enormous expectations on the possibilities for editing the genome. A multitude of studies were carried out on these elements in different species, but one of the methodological obstacles was the need to design specific proteins that would recognize specific DNA sequences in each case, which resulted in promising but eventually relatively low efficient approaches in many cases. Moreover, the difficulty for the exonucleases to enter the cell was a problem to deal with and not always appropriately solved.
The mechanism of action of the CRISPR-Cas9 system is based on the recognition of specific DNA sequences by a guide RNA, and their cleavage by the Cas9 nuclease. CRISPR stands for clustered regularly interspaced short palindromic repeats. These DNA sequences were originally discovered in the 90's by Dr. Francis Mojica (2), and later developed by other groups into the genetic tool that is nowadays giving so many surprises. The use of this technology allows for modification, repair or substitution of virtually any sequence within the genome. Cleavage sites of DNA by the nuclease are marked as DNA double-strand breaks (DSBs), which are repaired by specific cell mechanisms. One of them is usually the non-homologous end-joining (NHEJ), which essentially restores the DNA double strand. However, this method commonly induces mutations because there is no actual fidelity when it comes to DNA repair, and the result is the introduction of insertions and/or deletions (“indels”), so the DNA sequence is largely altered. From the point of view of therapeutic applications, this mechanism is clearly not an option if the aim is to “fix” the mutation and restore the gene to the original non-mutated form (“wildtype allele”).
On the contrary, there is another DNA repair mechanism that the cell can also use, depending on the activation molecular pathway, and the result is the so-called homology-directed repair (HDR), which literally performs a “copy’n’paste” from a template. Thus, using a homologous DNA sequence from an exogenous sample, the mutated gene can be modified; the CRISPR-Cas9 system recognizes the target sequence, cuts it off and replaces it with a new “healthy” sequence using the provided sample as template. Unfortunately, the efficiency of HDR is not high yet, so therapeutic applications do not seem to be currently viable.
WHAT DID THE AUTHORS ACTUALLY DO?
In the paper, the authors used heterozygous zygotes in which the mutant allele had been contributed by the sperm, while the copy from the oocyte came from healthy donors. Interestingly, DNA breaks introduced in the MYBPC3 paternal gene were mostly repaired by using the oocyte DNA as a template (HDR), which suggested a preferential or specific mechanism of DNA repair in the germline. This may represent a milestone for future approaches in the gene-editing strategy for curing genetic diseases in embryos. Treated embryos later developed into blastocysts with a similar success rate to that from control non-treated embryos, and the overall efficiency rate of gene-editing from the mutated form to the original wildtype form was about 70%.
These results seem to be a promising start, but they also mean real therapeutic applications are still far down the road. Also, other problems such as off-target hits (mutations induced by the CRISPR-Cas9 system in different DNA sites) or embryo mosaicism due to gene-editing are an obstacle for the purpose of clinical applications. Thereby, even if these effects are minimized, they still need to be completely eliminated before any similar approach can be brought into the clinic to cure a genetic disease.
This is where we are now as for the scientific and physical reality. It is clear that extreme caution needs to be a must in the laboratory when performing these experiments. However, what about ethical concerns? Similarly to other common practices, it is necessary to step back for a minute and think twice whether gene-editing is a good idea, and if so, when and why. We talk here about genetic problem solving, improved gene therapy and definite solutions to currently lethal or sublethal conditions. Again, the idea is that, potentially, any gene could be eventually modified in order to turn a genetically affected individual into a perfectly healthy person. Diabetes, Huntington’s disease, cystic fibrosis, hypertrophic cardiomyopathy... any epidemiologically significant condition could be subjected to removal from a population. That is the current main goal of genome-editing.
THE ETHICAL IMPLICATIONS
Generally speaking, this sort of treatments and techniques must be performed at some embryo stage, and research on human embryos is forbidden in most countries (meaning forbidden in those countries that have an actual regulation on this issue; there are too many countries that lack the proper regulation to act on these matters, and so they could potentially perform certain kind of operations that might be doubtful from the ethical point of view). The current situation has been catching attention from the media since a group in China published their experiments on human embryos in 2015 (3). Based on the author's claims, research was perfectly legal and it had been approved by the corresponding Chinese authorities. Plus, the embryos used for the experiments were actually not viable (3PN embryos), so there was no issue about any potential future human being. The end point of the Chinese group was to demonstrate that this research was already possible, doable anywhere and by anyone, and so it must be regulated worldwide. Besides other experiments on human somatic line material, genetic modification of human embryos hit the media again in February 2016, when Dr. Kathy Niakan’s group from the Francis Crick Institute in London specifically asked the HFEA (Human Fertility and Embryology Association) for permission to conduct experiments on human embryos. Permission was granted, and so things are since running.
What does this mean? For a large part of the scientific community, the Chinese opened the Pandora box. But many others think that paper proofed the inevitable, and the British group took the lead to demonstrate that genetic modification must be studied in humans in order to know exactly what the consequences and limitations are. On these grounds, in September 2015, the Hinxton Group (a compound of specialists in different fields and topics in experimental and social sciences, as well as philosophers and humanists) had previously met in Manchester and had released a statement in which they supported research on human embryos for genetic modification, and of course they also explained why. Essentially, in the text they referred to the needs of understanding the right tools and biological models for research, the consequences and the responsibilities, and they made emphasis on the need and urgency of letting the general population know about genetic modification, genome-editing technologies, reasons and consequences.
In 2015, Dr. Jennifer Doudna, world expert on CRISPR systems from University of California, Berkeley, exposed her ideas on human embryo genome-editing (4). A summary of her main points would be as follows:
1) Safety. Need for ensuring efficiency with no off-target hits to reproduce experiments.
2) Communication. Essential to make scientific progress accessible to scientists, public and society to make them aware of the importance of the new and reach properly based opinion.
3) Guidelines. Collaboratively elaborate policies along with scientists and experts for standards and to establish what is ethically acceptable in research.
4) Regulation. Apply those guidelines to appropriately conduct research and look out for the compliance of the agreement.
5) Caution. Technology cannot be applied to routine practice yet (such as for assisted reproduction).
WHAT TO EXPECT IN THE FUTURE
The research published this week is not the only one on human embryo genetic modification. Different groups keep publishing evidences that show these techniques are working well enough to invest properly and dedicate substantial efforts to improve it and eliminate every single problem mentioned earlier (5). It is certain that many more publications are yet to come, and the latest paper in Nature is only part of the beginning. This publication has once again shaken the scientific community opinion, but favourable reactions have also been immediate. In fact, two important institutions such as the US National Academy of Science and National Academy of Medicine have released their own report as well, backing research on gene-editing of human embryos.
Genetic engineering is since many years ago an exciting and still developing field with unimaginable possibilities. We need to step back out of the box, have a look at the big picture and think. Then, work on it, get better on it and become successful in (1) identifying reasons/circumstances to apply genetic modifications, (2) perform genome-editing without error or off-target hits, and (3) explaining why this is important to the general public. Given differences between countries, legislation, lifestyle, economic resources and technological progress, it is likely that the debate will remain open for a long time. But society must be aware of the progress of science, and people need to know the hows and whys of scientific research and how it can affect society.
If you want to know more about CRISPR-based technology, do not miss the videos from our section.
1. Ma H, Marti-Gutierrez N, Park SW, Wu J, Lee Y, et al. Correction of a pathogeneic gene mutation in human embryos. Nature. 2017; doi:10.1038/nature23305
2. Mojica, FJM, Díez-Villaseñor C, Soria E, Juez G. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol. 2000; 36:244-246
3. Liang P, Xu Y, Zhang X, Ding C, Huang R, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015; 6(5):363-372
4. Doudna J. Editing needs scrutiny. Nature. 2015; 528(7580):S6
5. Tang L, Zeng Y, Du H, Gong M, Peng J, Zhang B, et al. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein. Mol Genet Genomics. 2017; 292(3):525-33.
Author: Javier Del Río Riego
One of the scientific advances made in recent years is 3D printing of human organs. The creation of an organ from a suitable material often allows medical doctors to decide on the best way to perform a surgical procedure. In addition, it opens up new possibilities for the use of 3D printing technologies in organ transplant programs. Now, researchers from Northwestern University Feinberg School of Medicine and McCormick School of Engineering have developed prosthetic ovaries made of gelatin, which allowed mice to conceive and give birth to healthy offspring.
The bioprosthetic ovaries are constructed of 3D-printed scaffolds that house immature eggs, and have been successful in boosting hormone production and restoring fertility in mice. The material used for building up these ovaries is a biological hydrogel made from decomposed collagen naturally found in animal ovaries. This material is degradable to allow cellular remodelling, contains cell adhesion sites and shows soft durable mechanical properties. Scientists built the ovaries by printing various patterns of overlapping gelatin filaments on glass slides. Modulating the distance between these filaments, as well as the advanced angle between layers, allowed for the generation of different pore sizes and geometries.
The ovaries are referred to as "bioprosthetic" organs because they contain both living material (ovarian cells) and nonliving material (gelatin). They release hormones in the same way a normal ovary would, allowing the animal to go through its natural cycle, including ovulation. Also, this scaffolding needed to be made of organic materials, stiff enough to be handled during surgery and sufficiently porous to interact naturally with the tissues of the mouse body. In the study, after having placed the biosynthetic ovaries in the mouse, 40-50 follicles were carefully implanted into each scaffold.
Vascularization in the ovary is critical because it provides oxygen and nutrients to the follicles, and because it allows follicle-produced hormones to circulate in the blood stream. Since gelatin is a natural material, the body can recognize the implant as a regular body part and allows blood vessels to grow into it.
Although mice had been sterilized by the removal of both ovaries, the reproductive tract (including the oviduct), had been left intact to assess fertility through natural mating after the surgical introduction of the 3D-printed organs. The study culminated in the pairing of a dozen female mice, two mice with sham controls (bioprosthetic ovary without follicles) and seven mice with bioprosthetic ovaries were mated with males who had previously sired pups. Three bioprosthetic ovary recipients had litters of one or two pups each, while none of the sham controls had pups. In all cases, the mothers produced milk to feed them.
FUTURE APPLICATIONS IN HUMANS
Researchers hope in particular to help cancer survivors whose treatments had damaged their ovaries. The 3-D printed bioprosthetic scaffold can be repopulated using patient's previously extracted own follicles or donated samples from ovarian tissues. Another possible use would be helping women with impaired ovaries, so that symptoms of menopause could be alleviated. Rather than using synthetic hormones, which often entail unpleasant side effects, women could have an all-natural source of hormones implanted directly into her body.
The team is now preparing a similar trial on pigs, which are closer to humans in size and biology. They believe that it might be a challenge to scale up the 3D-printed structure to the size needed for human use.
Original publication: Laronda MM, Rutz AL, Xiao S, Whelan KA, Duncan FE, Roth EW et al. A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice. Nat Commun. 2017; 8:15261.
(1) Available from: http://www.npr.org/sections/health-shots/2017/05/20/528646323/scientists-one-step-closer-to-3-d-printed-ovaries-to-treat-infertility. [Cited 22 May 2017].
Authors: Shuyana Deba Rementeria, Javier Del Río Riego and Sara Sanz Juste
Since 2015, the fertility-related company OvaScience from Canada has recently launched a new technique called AUGMENT, which has resulted in high controversy around the world.
WHAT IS AUGMENT?
It is a new IVF technique designed to improve women’s oocyte quality.
How does it work? Through this technique, doctors have to biopsy tissue from the lining ovaries of a woman normally under general anaesthesia, the entire procedure being performed via laparoscopic surgery. Then, samples are transported to the OvaScience laboratory, where the mitochondria from egg precursor cells are isolated and subsequently transferred through ICSI into a mature egg, as well as the sperm.
Serving patients in a total of six countries: Canada, Spain, Japan, Panama, Turkey and United Arab Emirates.
What do you think about it? Do you think it is a useful technique? Would you offer this approach at your fertility clinic?
You can read more about OvaScience and their treatments in the following links. If you cannot find any paper, let us know and we will send it to you.
In the scientific area:
Fakih M. The AUGMENTSM Treatment: Physician Reported Outcomes of the Initial Global Patient Experience. Journal of Fertilization: In Vitro - IVF-Worldwide, Reproductive Medicine, Genetics & Stem Cell Biology. 2015;03(03).
Couzin-Frankel J. Eggs unlimited. Science. 2015;350(6261):620-624.
Imudia A, Wang N, Tanaka Y, White Y, Woods D, Tilly J. Comparative gene expression profiling of adult mouse ovary-derived oogonial stem cells supports a distinct cellular identity. Fertility and Sterility. 2013;100(5):1451-1458.e2.
Woods D, Tilly J. Isolation, characterization and propagation of mitotically active germ cells from adult mouse and human ovaries. Nature Protocols. 2013;8(5):966-988.
In the media:
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