The effects of artificial gender imbalance. Science & Society Series on Sex and Science

The use of reproductive technology to service a preference for male offspring has created an artificial gender imbalance, notably in Asian countries. The social effects of this large surplus of young men are not yet clear, but concerted action might be necessary to address the problemOne of the problems of sexual reproduction, especially in predominantly monogamous species that pair ‘for life'', is to ensure a balance between the birth rate of males and females. In humans, this balance has been remarkably even, but the past few decades have seen a substantial shift towards men, notably in some Asian countries. The reason, however, is not biological; there has simply been a cultural preference for sons in the affected societies, which together with recent availability of prenatal sex-selection technologies has led to widespread female feticide. The result has been a huge excess of males in several countries. Whilst it is not yet fully clear how a surplus of millions of men will affect these societies—perhaps even leading to civil unrest—some countries have already taken steps to alleviate the problem by addressing the underlying cultural factors. However, the problem is about to come to a crisis point, as a large surplus of men reach reproductive age. It will take many decades to reach a balanced representation of both sexes again.The sex ratio at birth (SRB) is defined as the number of boys born to every 100 girls. It is remarkably consistent in human populations, with around 103–107 male babies for every 100 female ones. John Graunt first documented this slight excess of male births in 1710 for the population of London, and many studies have since confirmed his finding [1]. Higher mortality from disease, compounded by the male tendency towards risky behaviours and violence, means that the initial surplus of boys decreases to roughly equal number of males and females during the all-important reproductive years in most populations.Researchers have studied a large number of demographic and environmental factors that could affect the SRB, including family size, parental age and occupation, birth order, race, coital rate, hormonal treatments, environmental toxins, several diseases and, perhaps most intriguingly, war [2,3,4]. It is well documented that wars are associated with a small increase in the sex ratio. This phenomenon occurs both during the war and for a short period afterwards. The best examples of this were reported for the First and Second World Wars in both the USA and Europe, and for the Korean and Vietnam Wars in the USA [5,6]. However, these findings were not reproduced in the more recent Balkan Wars and the Iran–Iraq war [7]. There have been several biological explanations for these increases. It has been proposed, for example, that the stress of war adversely affects the viability of XY-bearing sperm. Alternatively, a higher frequency of intercourse after prolonged separation during times of war is thought to lead to conception earlier in the menstrual cycle, which has been shown to result in more males [4,8]. There have been evolutionary explanations, such as the loss of large numbers of men in war leading to an adaptive correction of the sex ratio [4,9]. Nonetheless, the real causes of the altered SRB during war remain elusive: all of the discussed biological and social factors have been shown to cause only marginal deviations from the normal sex ratio.Whilst war has only slightly shifted SRB towards more male babies and only for a limited time period, cultural factors, namely a strong preference for sons, has been causing large distortions of gender balance during the past decades. Son preference is most prevalent in a band of countries from East Asia through to South Asia and the Middle East to North Africa [9]. For centuries, sons have been regarded as more valuable, because males can earn higher wages especially in agrarian economies, they generally continue the family line, are recipients of inheritance and are responsible for their parents in illness and old age. By contrast, daughters often become members of the husband''s family after marriage, no longer having responsibility for their biological parents [10]. There are also location-specific reasons for son preference: in India, the expense of the dowry, and in South Korea and China, deep-rooted Confucian values and patriarchal family systems [11].… cultural factors, namely a strong preference for sons, has been causing large distortions of gender balance during the past decadesUntil recently, son preference was manifest post-natally through female infanticide, abandonment of newborn girls, poorer nutrition and neglect of health care, all causing higher female mortality [12]. Studies have shown that unequal access to health care is the most important factor in differential gender mortality [13,14], especially in countries where health care costs are borne by the family [15]. As early as 1990, the Indian economist Amaryta Sen estimated that differential female mortality had resulted in around 100 million missing females across the developing world with the overwhelming majority of these in China, India, Pakistan and Bangladesh [16].

Science & Society Series on Sex and Science

Sex is the greatest invention of all time: not only has sexual reproduction facilitated the evolution of higher life forms, it has had a profound influence on human history, culture and society. This series explores our attempts to understand the influence of sex in the natural world, and the biological, medical and cultural aspects of sexual reproduction, gender and sexual pleasure.To make matters worse, during the 1980s, diagnostic ultrasound technology became available in many Asian countries, and the opportunity to use the new technology for prenatal sex selection was soon exploited. Indeed, the highest SRBs are seen in countries with a combination of son preference, easy access to sex-selection technologies and abortion, and a small family culture. The latter is important because where larger families are the norm, couples will continue to have children until they have a boy. If the couple plan, or are legally restricted, as in China, to only one or two children, they will use sex selection to ensure the birth of a son [17]. This combination has resulted in serious and unprecedented sex ratio imbalances that are now affecting the reproductive age groups in several countries, most notably China, South Korea and parts of India.South Korea was the first country to report a very high SRB, because the widespread uptake of sex-selection technology preceded other Asian countries. The sex ratios started to rise in the mid-1980s in cities; ultrasound was already widely available even in rural areas by 1990 [17]. By 1992, the SRB was reported to be as high as 125 in some cities.South Korea was the first country to report a very high SRB, because the widespread uptake of sex-selection technology preceded other Asian countriesChina soon followed. Here, the situation was further complicated by the one-child policy introduced in 1979. This has undoubtedly contributed to the steady increase in the reported SRB from 106 in 1979 to 111 in 1990, 117 in 2001, 121 in 2005 and as high as 130 in some rural counties [18]. The latest figures for 2010 report an SRB of 118 [19] (National Bureau of Statistics of China 2011), the first drop in three decades, suggesting an incipient downturn. However, the number of excess males in the reproductive age group will continue to increase for at least another two decades. Because of China''s huge population, these ratios translate into massive numbers: in 2005, an estimated 1.1 million excess males were born across the country and the number of males under the age of 20 might exceed females by around 30 million [18].These overall figures conceal wide variations across the country (Fig 1): the SRB is higher than 130 in a strip of heavily populated provinces from Henan in the north to Hainan in the south, but close to normal in the large sparsely populated provinces of Xinjiang, Inner Mongolia and Tibet. Some are sceptical about these high SRB figures or have suggested that, under the constraints of the one-child policy, parents might fail to register a newborn girl, so that they might go on to have a boy [20]. However, recent evidence shows that such under-registration explains only a small proportion of missing females and that sex-selective abortion undoubtedly accounts for the overwhelming majority [18].Open in a separate windowFigure 1Sex ratio at birth for China''s provinces in 2005.There are marked regional differences in SRB in India. Because incomplete birth registrations make the SRB difficult to calculate accurately, the closely related ratio of boys to girls under the age of six is used, showing distinct regional differences across the country with much higher levels in the north and west. According to the most recent census in 2010, the SRB for the whole country was 109, a marginal increase on the previous census in 2001, which showed an SRB of 108. These national figures, however, hide wide differences from a low SRB of 98 in the state of Kerala to 119 in Haryana State. The highest SRBs at district level for the whole of India are in two districts of Haryana state, where the SRBs are both 129 [21]. The Indian figures contrast with the Chinese in two ways: nowhere in China is the sex ratio low, and in India the sex ratio is higher in rural than urban areas, whereas the reverse is true for China [22].A consistent pattern in all three countries is a clear trend across birth order, that is first, second and subsequent children, and the sex of the preceding child. This is driven by the persistence of the ‘at least one boy'' imperative in these cultures. Where high fertility is the norm, couples will continue to reproduce until they have a boy. Where couples aim to restrict their family size, they might be content if the first child is a girl, but will often use sex selection to ensure a boy in the second pregnancy. This was shown in a large Indian study: the SRB was 132 for second births with a preceding girl, and 139 for third births with two previous girls. By contrast, the sex ratios were normal when the first born was a boy [23].The sex ratio by birth order is particularly interesting in China (18].

Table 1

Sex ratio at birth for China''s provinces in 2005.

Effect of nasal balloon autoinflation in children with otitis media with effusion in primary care: an open randomized controlled trial

Background:Otitis media with effusion is a common problem that lacks an evidence-based nonsurgical treatment option. We assessed the clinical effectiveness of treatment with a nasal balloon device in a primary care setting.Methods:We conducted an open, pragmatic randomized controlled trial set in 43 family practices in the United Kingdom. Children aged 4–11 years with a recent history of ear symptoms and otitis media with effusion in 1 or both ears, confirmed by tympanometry, were allocated to receive either autoinflation 3 times daily for 1–3 months plus usual care or usual care alone. Clearance of middle-ear fluid at 1 and 3 months was assessed by experts masked to allocation.Results:Of 320 children enrolled, those receiving autoinflation were more likely than controls to have normal tympanograms at 1 month (47.3% [62/131] v. 35.6% [47/132]; adjusted relative risk [RR] 1.36, 95% confidence interval [CI] 0.99 to 1.88) and at 3 months (49.6% [62/125] v. 38.3% [46/120]; adjusted RR 1.37, 95% CI 1.03 to 1.83; number needed to treat = 9). Autoinflation produced greater improvements in ear-related quality of life (adjusted between-group difference in change from baseline in OMQ-14 [an ear-related measure of quality of life] score −0.42, 95% CI −0.63 to −0.22). Compliance was 89% at 1 month and 80% at 3 months. Adverse events were mild, infrequent and comparable between groups.Interpretation:Autoinflation in children aged 4–11 years with otitis media with effusion is feasible in primary care and effective both in clearing effusions and improving symptoms and ear-related child and parent quality of life. Trial registration: ISRCTN, No. 55208702.Otitis media with effusion, also known as glue ear, is an accumulation of fluid in the middle ear, without symptoms or signs of an acute ear infection. It is often associated with viral infection.^1–3 The prevalence rises to 46% in children aged 4–5 years,⁴ when hearing difficulty, other ear-related symptoms and broader developmental concerns often bring the condition to medical attention.^3,5,6 Middle-ear fluid is associated with conductive hearing losses of about 15–45 dB HL.⁷ Resolution is clinically unpredictable,^8–10 with about a third of cases showing recurrence.¹¹ In the United Kingdom, about 200 000 children with the condition are seen annually in primary care.^12,13 Research suggests some children seen in primary care are as badly affected as those seen in hospital.^7,9,14,15 In the United States, there were 2.2 million diagnosed episodes in 2004, costing an estimated $4.0 billion.¹⁶ Rates of ventilation tube surgery show variability between countries,^17–19 with a declining trend in the UK.²⁰Initial clinical management consists of reasonable temporizing or delay before considering surgery.¹³ Unfortunately, all available medical treatments for otitis media with effusion such as antibiotics, antihistamines, decongestants and intranasal steroids are ineffective and have unwanted effects, and therefore cannot be recommended.^21–23 Not only are antibiotics ineffective, but resistance to them poses a major threat to public health.^24,25 Although surgery is effective for a carefully selected minority,^13,26,27 a simple low-cost, nonsurgical treatment option could benefit a much larger group of symptomatic children, with the purpose of addressing legitimate clinical concerns without incurring excessive delays.Autoinflation using a nasal balloon device is a low-cost intervention with the potential to be used more widely in primary care, but current evidence of its effectiveness is limited to several small hospital-based trials²⁸ that found a higher rate of tympanometric resolution of ear fluid at 1 month.^29–31 Evidence of feasibility and effectiveness of autoinflation to inform wider clinical use is lacking.^13,28 Thus we report here the findings of a large pragmatic trial of the clinical effectiveness of nasal balloon autoinflation in a spectrum of children with clinically confirmed otitis media with effusion identified from primary care.     

A structural road map to unveil basal body composition and assembly

EMBO J 31 3, 552–562 (2012); published online December132011The Basal Body (BB) acts as the template for the axoneme, the microtubule-based structure of cilia and flagella. Although several proteins were recently implicated in both centriole and BB assembly and function, their molecular mechanisms are still poorly characterized. In this issue of The EMBO journal, Li and coworkers describe for the first time the near-native structure of the BB at 33 Å resolution obtained by Cryo-Electron Microscopy analysis of wild-type (WT) isolated Chlamydomonas BBs. They identified several uncharacterized non-tubulin structures and variations along the length of the BB, which likely reflect the binding and function of numerous macromolecular complexes. These complexes are expected to define BB intrinsic properties, such as its characteristic structure and stability. Similarly to the high-resolution structures of ribosome and nuclear pore complexes, this study will undoubtedly contribute towards the future analysis of centriole and BB biogenesis, maintenance and function.The microtubule (MT)-based structure of the cilium/flagellum grows from the distal part of the Basal Body (BB), which in many animal cells develops from the mature centriole in the centrosome. Electron microscopic (EM) images of chemically fixed resin-embedded centrioles and basal bodies (CBBs) suggest that their ultrastructure is similar, and that their key components are MTs. The mechanisms underlying the organization of CBB MTs, comprising highly stable closed and open MTs, are likely to hold many surprises as they are remarkably different from other microtubular structures in the cell. Additionally, non-MT-based structures are also part of the CBB, including a cartwheel in the proximal lumen region that reinforces CBB symmetry (reviewed in Azimzadeh and Marshall, 2010 and Carvalho-Santos et al, 2011).Several centriole components and BB proteins were identified by comparative and/or functional genomics and proteomics studies of purified CBBs (reviewed in Azimzadeh and Marshall, 2010 and Carvalho-Santos et al, 2011). Advances in our understanding of the molecular mechanisms of CBB assembly depend on high-resolution comparative studies of wild-type (WT) and mutant structures, as well as characterization of the localization of molecular complexes within the small CBB structure. Despite the existence of beautiful ultrastructure data acquired from chemically fixed specimens (Geimer and Melkonian, 2004; Ibrahim et al, 2009), high-resolution structures of native CBBs were missing. Using electron cryo-tomography and 3D subtomogram averaging, Li et al (2012) solved the structure of the near-native BB triplet at 33 Å resolution. A pseudo-atomic model of the tubulin protofilaments at the core of the triplets was built by fitting the atomic structure of α/β-tubulin monomers into the BB tomograms.The 3D density map reveals several additional densities that represent non-tubulin proteins attached, both internally and externally, to all triplet MTs, some linking MTs inside the triplets and/or MTs in consecutive triplets (Li et al, 2012; for a summary, see Li et al, 2012; Geimer and Melkonian, 2004; Ibrahim et al, 2009), but with less detail and complexity. The authors speculate that some of the additional densities present at the A- and B-tubule inner wall might correspond to proteins of the tektin family, probably conferring rigidity to the BB triplet (Amos, 2008).

Table 1

Characteristics of the non-α/β-tubulin structures reported in Li et al (2012) in this issue of The EMBO journal

The media and food-risk perceptions

Food scares are prime examples of how the media can sway public perceptions of risk. Scientists and regulators need to understand the complex relationship between the media and their audience if they seek to counter scare stories and put risks and benefits into context.In 1996, at the height of the scandal about mad cow disease in the UK, a guest on Oprah Winfrey''s talk show claimed that meat produced in the USA could cause bovine spongiform encephalopathy (BSE). “That just stopped me cold from eating another burger,” Winfrey responded. Later, beef farmers from Texas sued Winfrey''s show, claiming that it was partly responsible for the steep decline in beef prices in the USA during the following months, even though the country did not have a single case of BSE. This episode demonstrates not only the power of the media and its influence on the public, but also how easily the public is swayed, particularly by fear, even in the absence of information.Nevertheless, more information is not necessarily a panacea for disinformation. Households in developed countries have greater access to information than ever before—through television, newspapers, journals, radio and the internet—yet the public remains, ironically, poorly informed. This is most evident when consumption of a food dramatically declines after media reports about contamination or harm, or when European consumers vehemently oppose genetically modified food, despite accumulating scientific evidence that these products do not harm the environment and are safe for human consumption.There are various understandable causes of public reactions to food scares or food-health stories in the media, but the media itself sets the stage for the public''s response by choosing which information to present and, perhaps more importantly, how to present it. Extensive media coverage affects consumer perceptions of products and risks and, consequently, can influence demand for services and products.There are various understandable causes of public reactions to food scares or food-health stories in the media, but the media itself sets the stage for the public''s response…The function of the media is not to foster the public good or to reassure the public that they are safe. Most television stations and newspapers are now privately owned—many of them by one of a few huge companies. The media therefore has its own financial and other interests, and needs to please both shareholders and audiences by providing the kind of information and analysis that mass audiences expect. Similarly, other sources of information—such as agriculture and biotechnology companies, universities and farmers—have equally powerful incentives that could bias the information they are willing to share and the conclusions they seek to draw. In the USA, news coverage has always been largely commercial in this way, whereas in Europe, private companies have only become the dominant source of information during the past two decades. Moreover, the structure of the media market itself has changed with the growth of 24-hour news and the internet—notably in terms of blogs, social media and the ability to distribute videos online.The function of the media is not to foster the public good or to reassure the public that they are safeOne criticism that is often levelled at the media is that it sensationalizes news and is biased against positive news stories. Instead, the media seems to focus on negative news stories and shun careful and balanced analysis of an issue, favouring ‘sound bites'' and simplistic conclusions. Commercial news reporting tends to focus on events, such as a sudden food-safety problem or an organized event accompanying the launch of a new product or policy.The overall concern is that the increasing commercialization of the media has led to a ‘dumbing down'' of the news; that is, lower-quality journalism and less coverage of complex issues, driven by competitive pressures that have forced media companies to cut back on reporting and editorial staff in areas that do not attract many readers or viewers (Alterman, 2008; Zaller, 1999). The emergence of the 24-hour news cycle might even have further weakened journalistic standards; modern news reports have been found to contain an increasing number of factual errors (Pew, 2004).These concerns have caused many European governments to continue their subsidized public broadcasting, in order to maintain the overall quality and reliability of news and information. However, if subsidized public media cover the high-quality news market, it might further decrease the quality of coverage offered by commercial companies (Canoy & Nahuis, 2005). This argument is supported by studies of the US media market, which show that the regional expansion of so-called ‘quality'' newspapers such as The New York Times and The Washington Post has led to a reduction in the quality of local and regional newspapers (George & Waldfogel, 2006).All of this is particularly relevant in the context of food, as most consumers primarily receive information about food and biotechnology through the popular press and television (Hoban & Kendall, 1993; Marks et al, 2003). Extensive media coverage of an event can contribute to a heightened perception of risk and amplify its consequences. Food scares are prime examples of this effect: they are typically accompanied by a flood of media coverage and lead to a decline in demand for the product in question, often concomitant with a level of panic that scientists would argue is not appropriate, given the real risks.Accordingly, social scientists and psychologists have conducted research into how information shapes and determines perceived risks of food. Generally, most consumers are “rationally ignorant” (McCluskey & Swinnen, 2004); they rationally choose not to fully inform themselves about an issue. In other words, although consumers have access to huge amounts of information, they choose to be less than fully informed. There are three explanations for this attitude. First, if it costs money to access the news and doing so only provides limited benefits, it is rational not to purchase the information. Second, although reducing the price of news will make information more accessible, acquiring and processing it takes time, energy and attention. Consequently, consumers reach a threshold at which the cost of processing the information is larger than the benefit. The third reason has to do with the information source: ideological bias or distrust of a news source might cause consumers not to inform themselves fully.…although consumers have access to huge amounts of information, they choose to be less than fully informedThe decision about how much information is enough also depends on consumers'' ex ante (previous) risk perceptions. In one of the first surveys of consumer perceptions of health risks in food, van Ravenswaay (1990) concluded that most consumers acknowledge the existence of risks, but perceive them to be small. Although the public adjust their risk perceptions in the light of new information, they are only willing to pay modest amounts for information that would reduce perceived food risks. One explanation is that the cost of risk avoidance is low because consumers can stop purchasing a specific food if they learn that it poses a higher risk than they thought.In fact, ex ante beliefs tend to have a stronger influence on risk perceptions than news or other types of information. For example, many consumers think that organically produced products—which carry a higher risk of mycotoxins—are safer than more-intensively farmed crops, irrespective of information about management activities (Loureiro et al, 2001). Generally, consumers perceive natural risks as being easier to manage because they seem to be less threatening than technological risks.In general, risk perception varies between consumers, owing to many factors. Gender and education are consistent demographic predictors of food-risk perceptions. Non-demographic predictors include the nature of the perceived threat, trust in regulatory authorities, the source of the information and the way in which it is distributed, and health and environmental concerns (Ellis & Tucker, 2009). For example, consumers of organic foods perceive greater risks from pesticide residues than other consumers.Both social and individual factors can amplify or dampen perceptions of risk (Flynn et al, 1998; Koné & Mullet, 1994), and the media is an important mechanism in this process. Slovic (1987) suggests that risk perception is influenced by two factors: dread and unknown risks. Dreaded risks are those deemed to be uncontrollable, involuntary and affect many people with potentially catastrophic consequences. Unknown risks are new, uncertain and unobservable, or might have delayed effects. Food scares are often rated highly as dreaded risks, but because they are understood they receive lower ratings as unknown risks. By contrast, new food technologies, such as genetically modified foods, are rated highly as unknown risks. Thus, differences in consumer knowledge might influence risk perceptions; most scientists tend not to think that genetically modified foods are risky.Previous beliefs also have an important role in the selection and processing of information provided by the media. Poortinga & Pidgeon (2004) studied the perception of genetically modified food in the UK and found a strong confirmatory bias—selecting information that agrees with your previous beliefs; those with positive or negative beliefs interpret the same events as being in line with their attitude. Frewer et al (1997) also found that the initial attitude to genetic engineering is the most important determinant of how people assess new information about it. These attitudes remain stable, even if persuasive arguments against them are provided. In fact, initial attitudes also affect perception of the quality of information; respondents with a negative view are likely to perceive positive information about the technology as less accurate and more biased than people with positive views.The nature of the information also matters. In general, consumers give more weight to negative than positive information. This is ironic because one often-heard complaint about the media is that news coverage is too negative. This tendency is actually driven by demand (McCluskey & Swinnen, 2004), as the value of information is higher for consumers if it concerns an issue with a negative effect on welfare. The rationale is that consumers can use negative information to make decisions in order to avoid losses. As media companies care about profits, they will inevitably offer more negative stories.…consumers give more weight to negative than positive informationSiegrist & Cvetkovich (2001) conducted psychological experiments to assess this bias towards negative information in regard to health risks in food. They found that people place greater trust in results that indicate a health risk, and that confidence in the results increases with a higher indication of risk. The authors suggest three possible explanations: diagnosticity—negative information is more diagnostic than positive information, and might therefore be given greater weight; loss aversion—for most people it is important to avoid losses; and credibility—negative information might be more credible than positive information because positive information can be regarded as self-serving, whereas negative information often seems to lack this quality. However, critics of these studies warn against confusing negativity bias and confirmatory bias in explaining how information shapes citizens'' perceptions. Yet, after controlling for confirmatory bias, negativity bias still has a role: negative items have more impact than positive ones.The source of information is also important for shaping risk perception, as distrust of the institution providing the information increases the perception of risk (Renn, 2005). There is some debate about the importance of source credibility. Some studies find that source credibility has a key role in determining the impact of a message on public opinion, while others find that source credibility seems to have a limited effect and is less important than initial attitudes. Kumkale et al (2010) show in a meta-analysis that the credibility of the source matters mostly for attitude-formation conditions, whereas its impact in attitude-change conditions is lower. Conversely, recent studies show that internet users pay little or no attention to source credibility when they seek health information.Many people, in fact, anticipate that information from the media might be biased and take this into account when evaluating it. However, several behavioural studies conclude that even when viewers know that media sources are biased, they do not sufficiently discount the information to account for this bias. Exposure to media can thus systematically alter or reinforce beliefs and consumer behaviour. In conclusion, the impact of bias in media reporting on consumer attitudes is bidirectional and complex. Consumer bias in personal preferences and beliefs affect the media''s reporting strategies to convince these consumers to buy their media products. Similar complex interactions occur between the media and politicians and between the media and business.Although the media''s effects on public perception are complex, their impact can be significant. Curtis et al (2008) argue that differences in the structure of the media between countries might have important implications for food-risk perceptions. The negative attitude towards genetically modified foods that is typical of consumers in rich countries is in contrast to attitudes in poorer countries, where studies have found that consumer attitudes towards genetically modified foods are not as negative, and in many cases even positive. The authors claim that this might be partly explained by differences in the organization of the media. In poorer countries, information is more expensive and scarce and people often have less time to read and acquire information, which leads to an overall lower level of information. Moreover, government control of the media in poorer countries tends to be more extensive and might lead to more-positive coverage of biotechnology, if the government has a positive attitude.An important issue is the dynamics of the media market—that is, not only whether, but when to publish news. The structure of the mass media encourages fast, concentrated coverage. As collecting information requires time, effort and other costs, publishing a story on the basis of incomplete information risks biasing reports, which might hurt the reputation of the media outlet, and thereby future profits. However, covering a story early on might yield market share and profits if an outlet can be the first to provide information on a new issue. Consumers also face a trade-off. They might be willing to take the risk of getting biased information, as long as they get whatever information is available. In other words, any news is better than no news.These issues are particularly important in food scares. A case in point is the 1989 Alar controversy in the USA. Alar was the trade name for daminozide, a plant growth-regulator used to stimulate the growth, appearance and ripening of fruits, primarily apples. In February 1989, the US news programme 60 Minutes covered the Natural Resources Defence Council''s report, which said that Alar poses a cancer risk to children. Most US media organizations followed suit. As a result, supermarkets took apples off their shelves and schools removed apples from their cafeterias. US apple growers lost millions of dollars in revenues and announced a voluntary ban on Alar, which became effective in the autumn of 1989. In hindsight, analysts argue that the media confused a long-term cumulative effect with an imminent danger, resulting in unnecessary panic and financial losses (Negin, 1996).BSE, commonly known as mad cow disease, is another example. In March 1996, the UK government announced that mad cow disease was the likely cause of death for ten people. In April 1996, coverage of BSE on the Oprah Winfrey show in the USA was followed by a steep decline in beef prices in the following month, even though there were no BSE-infected cattle in the USA.Tabloid newspapers and the popular press typically worry less about their reputation in terms of quality, and more about being the first to publish or broadcast a story. The elite press worries more about quality. However, there is an interesting dynamic component: once one media company reports a story—no matter how biased their coverage is—it can initiate a chain reaction. If the issue is important enough, competitive forces will cause elite press organizations to follow suit, even before they are able to verify the story. The first story becomes the basis of their reporting.There are two reasons for this dynamic. First, competition and consumer choice force the media to pay attention to an issue, otherwise consumers ask why their preferred media source is not covering the story and will go elsewhere. The second reason is that by commenting on a story that was launched by another media company, more-reputable media outlets are covered if things go wrong—that is, when the primary information turns out to be biased. They can hide behind the fact that they were not the first to cover it, and only reflected on a story launched by someone else. The first factor minimizes the immediate losses from waiting too long, and the second limits future negative effects on reputation. These dynamics are summarized by the following quote, “Even apparently responsible papers […] contribute to building up [food] scares. When the scare has run its course, they will argue against it. But when the scare dynamic is up and running, [the quality press] will join with the throng and become more tabloid than the tabloids” (North, 2000).Although competition for audiences leads to an intensification of media attention in the early reporting of a story, it also induces a rapid decline in attention afterwards. The popular press is often first to report on a crisis and more intense in its initial coverage, but quickly loses interest. Thus, competition in the commercial media intensifies the scale of the scare, as well as bringing it to a fast—and often premature—conclusion.…competition in the commercial media intensifies the scale of the scare, as well as bringing it to a fast—and often premature—conclusionThere is also evidence that early claims, even when they are false, are reported more extensively than later corrections. Swinnen et al (2005) examined the media response to two food-safety crises: the 1999 dioxin crisis, and the 2001 foot and mouth disease outbreak. Comparing tabloids and the elite press, they found that overall coverage was almost the same, but that tabloids initially responded more quickly and intensely and also lost interest more quickly. They also found that initial errors in the news were not properly corrected when new facts emerged and initial interest had waned.The short-term impacts of food-safety information on consumer demand can be significant. One example is BSE, which had a negative effect on the consumer demand for beef, the severity of which was increased by the media. Verbeke & Ward (2001) found considerable misperception of the problem by consumers, a lack of knowledge about the relevant science and biased perception of the scientific criteria relevant to the safety of meat. Television coverage of meat safety had a negative effect on the demand for red meat after the BSE outbreak (Verbeke et al, 2000), and younger people were most susceptible to negative media coverage.However, in the long run, consumption and sales typically recover if the problems are addressed (Henneberry et al, 1999; Piggott & Marsh, 2004), although the effects on policy can be lasting. In 1993, after an Escherichia coli outbreak at the Jack in the Box restaurant chain, 144 people were hospitalized and three died. The restaurant chain almost went out of business in the wake of the event, but after two years, sales had recovered to pre-scare levels (Entine, 1999). By contrast, the legislative repercussions on burger restaurant chains have persisted.The most-significant long-term effect of mass-media reporting is its impact on public policy. By invoking strong responses in their audiences through concentrated, emotionally charged coverage, media outlets put pressure on governments to react to situations, effectively setting the agenda on a certain issue; this is sometimes called the ‘CNN factor'' (Hawkins, 2002). Similarly, an absence of media coverage of even important events or problems lowers their priority in legislative agendas. Robinson (2001) suggests that the media has great power to lead policy-makers, especially when there is uncertainty or limited information. For example, in the wake of the media frenzy surrounding the Jack in the Box E. coli outbreak, US President Bill Clinton called congressional hearings about the safety of the food supply. The US Food and Drug Administration raised the recommended internal temperature of cooked burgers to 155 ° fahrenheit (68 °C). It is now almost impossible to order a burger cooked less than ‘medium'' in US restaurants.…the media has great power to lead policy-makers, especially when there is uncertainty or limited informationAnother interesting example is the use of the precautionary principle in regulation in the EU and the USA. The precautionary principle is now used as a major regulatory tool in food safety issues in the EU, in particular to regulate genetically modified foods. However, it was used more in the USA from the 1960s to the mid-1980s (Vogel, 2003). Several European food scares in the 1990s, heavily publicized in the mass media, changed this. It pushed politicians to introduce a series of new regulations and it caused consumers to be more concerned about food safety. Although ex post studies showed that several of these food-safety problems were exaggerated, the massive press coverage induced strong political reactions, leading to regulations and shifts in consumer preferences that are having long-lasting effects on perceptions of food risk and the regulation of the food system in Europe (Swinnen & Vandemoortele, 2010).The examples considered above and the power of the media to influence an ignorant public—willfully or otherwise—have important implications for risk communication, education and management. First, because initial beliefs are important—affecting not only overall risk perceptions, but also the way in which consumers process new information—it is important to enhance consumer understanding of risk through education and by providing early information. This should create a realistic framework within which people can assess risks once an event occurs. Pre-emptive risk communication and the establishment of institutions that are responsive to problems can mitigate negative, long-term consequences on public policy or consumer preferences.Second, businesses, scientists and governments should be prepared to provide accurate, open and understandable information when crises occur. The media will report on the issues regardless and will draw on whichever ‘expert'' they can find if companies, scientists and governments are not ready to put events and facts into perspective.Third, the growth of the internet as a source of information and a communication tool not only imposes challenges, but also provides important opportunities. It enables direct communication with the public to provide information without depending on the mass media as brokers. Hence, even if the media do not report—or do so with a lack of nuance—companies, scientists and governments can communicate correct and nuanced information through the internet.Even if the commercial media provide simple and clear messages, consumers might realize that reality is more complexFourth, it is generally considered that successful risk management in regard to food safety critically depends on communication. Yet communication about food risk is difficult because the science is complex, uncertain and ambiguous. Even if the commercial media provide simple and clear messages, consumers might realize that reality is more complex. For example, Frewer et al (1997) have found that an admission of scientific uncertainty, which seems to reflect honesty, has a positive effect on the efficiency of communication. Risk communication should aim to enable citizens to make their own judgements, without trying to convince them that a certain risk is (in)tolerable. In order to be successful, communication should integrate documentation, information, dialogue and participation, and these four elements should be tailored towards meeting the three challenges of complexity, uncertainty and ambiguity (Renn, 2005).Finally, there seem to be cultural variations in the impact of the media and risk-communication strategies and in how food risks are perceived. Van Dijk et al (2007) found variation in the impact of communication strategies, even among western European countries: the communication of uncertainty has a positive impact in Germany, whereas the same information has a negative impact in the UK and Norway. Hence, effective risk-communication strategies depend on the culture in which the scientist, company or government is operating.Scientists, businesses, interest groups and politicians can also influence public perception, in particular by using the internet to circumvent the mass mediaFood scares are serious issues that have a significant impact in terms of consumer behaviour, economics and politics. Nevertheless, it would be wrong to blame the media for disproportionate public responses to such stories, although their influence is important and sometimes detrimental to public understanding. Scientists, businesses, interest groups and politicians can also influence public perception, in particular by using the internet to circumvent the mass media as the main source of information. As such, it is important for all parties to work together to become better at communicating with the public and providing education. In this way, the public should enjoy a heightened baseline of knowledge that will allow them to assess critically the sensationalist reports that appear in the media, and perhaps reduce the demand for such reporting in the first place.? Open in a separate windowJohan SwinnenOpen in a separate windowJill McCluskey     

Scientific dissent and public policy

The temptation to silence dissenters whose non-mainstream views negatively affect public policies is powerful. However, silencing dissent, no matter how scientifically unsound it might be, can cause the public to mistrust science in general.Dissent is crucial for the advancement of science. Disagreement is at the heart of peer review and is important for uncovering unjustified assumptions, flawed methodologies and problematic reasoning. Enabling and encouraging dissent also helps to generate alternative hypotheses, models and explanations. Yet, despite the importance of dissent in science, there is growing concern that dissenting voices have a negative effect on the public perception of science, on policy-making and public health. In some cases, dissenting views are deliberately used to derail certain policies. For example, dissenting positions on climate change, environmental toxins or the hazards of tobacco smoke [1,2] seem to laypeople as equally valid conflicting opinions and thereby create or increase uncertainty. Critics often use legitimate scientific disagreements about narrow claims to reinforce the impression of uncertainty about general and widely accepted truths; for instance, that a given substance is harmful [3,4]. This impression of uncertainty about the evidence is then used to question particular policies [1,2,5,6].The negative effects of dissent on establishing public polices are present in cases in which the disagreements are scientifically well-grounded, but the significance of the dissent is misunderstood or blown out of proportion. A study showing that many factors affect the size of reef islands, to the effect that they will not necessarily be reduced in size as sea levels rise [7], was simplistically interpreted by the media as evidence that climate change will not have a negative impact on reef islands [8].In other instances, dissenting voices affect the public perception of and motivation to follow public-health policies or recommendations. For example, the publication of a now debunked link between the measles, mumps and rubella vaccine and autism [9], as well as the claim that the mercury preservative thimerosal, which was used in childhood vaccines, was a possible risk factor for autism [10,11], created public doubts about the safety of vaccinating children. Although later studies showed no evidence for these claims, doubts led many parents to reject vaccinations for their children, risking the herd immunity for diseases that had been largely eradicated from the industrialized world [12,13,14,15]. Many scientists have therefore come to regard dissent as problematic if it has the potential to affect public behaviour and policy-making. However, we argue that such concerns about dissent as an obstacle to public policy are both dangerous and misguided.Whether dissent is based on genuine scientific evidence or is unfounded, interested parties can use it to sow doubt, thwart public policies, promote problematic alternatives and lead the public to ignore sound advice. In response, scientists have adopted several strategies to limit these negative effects of dissent—masking dissent, silencing dissent and discrediting dissenters. The first strategy aims to present a united front to the public. Scientists mask existing disagreements among themselves by presenting only those claims or pieces of evidence about which they agree [16]. Although there is nearly universal agreement among scientists that average global temperatures are increasing, there are also legitimate disagreements about how much warming will occur, how quickly it will occur and the impact it might have [7,17,18,19]. As presenting these disagreements to the public probably creates more doubt and uncertainty than is warranted, scientists react by presenting only general claims [20].A second strategy is to silence dissenting views that might have negative consequences. This can take the form of self-censorship when scientists are reluctant to publish or publicly discuss research that might—incorrectly—be used to question existing scientific knowledge. For example, there are genuine disagreements about how best to model cloud formation, water vapour feedback and aerosols in general circulation paradigms, all of which have significant effects on the magnitude of global climate change predictions [17,19]. Yet, some scientists are hesitant to make these disagreements public, for fear that they will be accused of being denialists, faulted for confusing the public and policy-makers, censured for abating climate-change deniers, or criticized for undermining public policy [21,22,23,24].…there is growing concern that dissenting voices can have a negative effect on the public perception of science, on policy-making and public healthAnother strategy is to discredit dissenters, especially in cases in which the dissent seems to be ideologically motivated. This could involve publicizing the financial or political ties of the dissenters [2,6,25], which would call attention to their probable bias. In other cases, scientists might discredit the expertise of the dissenter. One such example concerns a 2007 study published in the Proceedings of the National Academy of Sciences USA, which claimed that cadis fly larvae consuming Bt maize pollen die at twice the rate of flies feeding on non-Bt maize pollen [26]. Immediately after publication, both the authors and the study itself became the target of relentless and sometimes scathing attacks from a group of scientists who were concerned that anti-GMO (genetically modified organism) interest groups would seize on the study to advance their agenda [27]. The article was criticized for its methodology and its conclusions, the Proceedings of the National Academy of Sciences USA was criticized for publishing the article and the US National Science Foundation was criticized for funding the study in the first place.Public policies, health advice and regulatory decisions should be based on the best available evidence and knowledge. As the public often lack the expertise to assess the quality of dissenting views, disagreements have the potential to cast doubt over the reliability of scientific knowledge and lead the public to question relevant policies. Strategies to block dissent therefore seem reasonable as a means to protect much needed or effective health policies, advice and regulations. However, even if the public were unable to evaluate the science appropriately, targeting dissent is not the most appropriate strategy to prevent negative side effects for several reasons. Chiefly, it contributes to the problems that the critics of dissent seek to address, namely increasing the cacophony of dissenting voices that only aim to create doubt. Focusing on dissent as a problematic activity sends the message to policy-makers and the public that any dissent undermines scientific knowledge. Reinforcing this false assumption further incentivizes those who seek merely to create doubt to thwart particular policies. Not surprisingly, think-tanks, industry and other organizations are willing to manufacture dissent simply to derail policies that they find economically or ideologically undesirable.Another danger of targeting dissent is that it probably stifles legitimate crucial voices that are needed for both advancing science and informing sound policy decisions. Attacking dissent makes scientists reluctant to voice genuine doubts, especially if they believe that doing so might harm their reputations, damage their careers and undermine prevailing theories or policies needed. For instance, a panel of scientists for the US National Academy of Sciences, when presenting a risk assessment of radiation in 1956, omitted wildly different predictions about the potential genetic harm of radiation [16]. They did not include this wide range of predictions in their final report precisely because they thought the differences would undermine confidence in their recommendations. Yet, this information could have been relevant to policy-makers. As such, targeting dissent as an obstacle to public policy might simply reinforce self-censorship and stifle legitimate and scientifically informed debate. If this happens, scientific progress is hindered.Second, even if the public has mistaken beliefs about science or the state of the knowledge of the science in question, focusing on dissent is not an effective way to protect public policy from false claims. It fails to address the presumed cause of the problem—the apparent lack of understanding of the science by the public. A better alternative would be to promote the public''s scientific literacy. If the public were educated to better assess the quality of the dissent and thus disregard instances of ideological, unsupported or unsound dissent, dissenting voices would not have such a negative effect. Of course, one might argue that educating the public would be costly and difficult, and that therefore, the public should simply listen to scientists about which dissent to ignore and which to consider. This is, however, a paternalistic attitude that requires the public to remain ignorant ‘for their own good''; a position that seems unjustified on many levels as there are better alternatives for addressing the problem.Moreover, silencing dissent, rather than promoting scientific literacy, risks undermining public trust in science even if the dissent is invalid. This was exemplified by the 2009 case of hacked e-mails from a computer server at the University of East Anglia''s Climate Research Unit (CRU). After the selective leaking of the e-mails, climate scientists at the CRU came under fire because some of the quotes, which were taken out of context, seemed to suggest that they were fudging data or suppressing dissenting views [28,29,30,31]. The stolen e-mails gave further ammunition to those opposing policies to reduce greenhouse emissions as they could use accusations of data ‘cover up'' as proof that climate scientists were not being honest with the public [29,30,31]. It also allowed critics to present climate scientists as conspirators who were trying to push a political agenda [32]. As a result, although there was nothing scientifically inappropriate revealed in the ‘climategate'' e-mails, it had the consequence of undermining the public''s trust in climate science [33,34,35,36].A significant amount of evidence shows that the ‘deficit model'' of public understanding of science, as described above, is too simplistic to account correctly for the public''s reluctance to accept particular policy decisions [37,38,39,40]. It ignores other important factors such as people''s attitudes towards science and technology, their social, political and ethical values, their past experiences and the public''s trust in governmental institutions [41,42,43,44]. The development of sound public policy depends not only on good science, but also on value judgements. One can agree with the scientific evidence for the safety of GMOs, for instance, but still disagree with the widespread use of GMOs because of social justice concerns about the developing world''s dependence on the interests of the global market. Similarly, one need not reject the scientific evidence about the harmful health effects of sugar to reject regulations on sugary drinks. One could rationally challenge such regulations on the grounds that informed citizens ought to be able to make free decisions about what they consume. Whether or not these value judgements are justified is an open question, but the focus on dissent hinders our ability to have that debate.Focusing on dissent as a problematic activity sends the message to policy-makers and the public that any dissent undermines scientific knowledgeAs such, targeting dissent completely fails to address the real issues. The focus on dissent, and the threat that it seems to pose to public policy, misdiagnoses the problem as one of the public misunderstanding science, its quality and its authority. It assumes that scientific or technological knowledge is the only relevant factor in the development of policy and it ignores the role of other factors, such as value judgements about social benefits and harms, and institutional trust and reliability [45,46]. The emphasis on dissent, and thus on scientific knowledge, as the only or main factor in public policy decisions does not give due attention to these legitimate considerations.Furthermore, by misdiagnosing the problem, targeting dissent also impedes more effective solutions and prevents an informed debate about the values that should guide public policy. By framing policy debates solely as debates over scientific facts, the normative aspects of public policy are hidden and neglected. Relevant ethical, social and political values fail to be publicly acknowledged and openly discussed.Controversies over GMOs and climate policies have called attention to the negative effects of dissent in the scientific community. Based on the assumption that the public''s reluctance to support particular policies is the result of their inability to properly understand scientific evidence, scientists have tried to limit dissenting views that create doubt. However, as outlined above, targeting dissent as an obstacle to public policy probably does more harm than good. It fails to focus on the real problem at stake—that science is not the only relevant factor in sound policy-making. Of course, we do not deny that scientific evidence is important to the develop.ment of public policy and behavioural decisions. Rather, our claim is that this role is misunderstood and often oversimplified in ways that actually contribute to problems in developing sound science-based policies.? Open in a separate windowInmaculada de Melo-MartínOpen in a separate windowKristen Intemann     

The health risks of ART

Assisted reproductive technologies enable subfertile couples to have children. But there are health risks attached for both mothers and children that need to be properly understood and managed.Assisted reproductive technology (ART) has become a standard intervention for couples with infertility problems, especially as ART is highly successful and overall carries low risks [1,2]. The number of infants born following ART has increased steadily worldwide, with more than 5,000,000 so far [3]. In industrialized countries, 1–4% of newborns have been conceived by using ART [4,5], probably owing to the fact that couples frequently delay childbearing until their late 30s, when fertility decreases in both men and women [2]. Considering the possibility that male fertility might be declining, as Richard Sharpe has discussed in this series [6], it is likely that ART will be even more widely used in the future. Yet, as the rate of ART and the total number of pregnancies has increased, it has become apparent that ART is associated with potential risks to the mother and fetus. The most commonly cited health problems pertain to multiple gestation pregnancies and multiple births. More recently, however, concerns about the risks of birth defects and genetic disorders have been raised. There are questions about whether the required manipulations and the artificial environments of gametes and embryos are potentially creating short- and long-term health risks in mothers and children by interfering with epigenetic reprogramming.Notwithstanding, ART represents a tremendous achievement in human reproductive medicine. The birth of Louise Brown, the first ‘test tube baby'' in 1978, was the result of the collaborative work of embryologist Robert Edwards and gynaecologist Patrick Steptoe [7]. This success was a culmination of many years of work at universities and clinics worldwide. An initial lack of support, as well as criticism from ethicists and the church, delayed the opening of the first in vitro fertilization (IVF) clinic in Bourn Hall near Cambridge until 1980. By 1986, 1,000 children conceived by IVF at Bourn Hall had been born [8]. In 2010, Edwards received the Nobel Prize in Medicine for the development of IVF. Regrettably, Steptoe had passed away in 1988 and could not share the honour.…as the rate of ART and the total number of pregnancies has increased, it has become apparent that ART is associated with potential risks to mother and fetusOver the next decades, many improvements in IVF procedures were made to reduce the risks of adverse effects and increase success rates, including controlled ovarian stimulation, timed ovulation induction, ultrasound-guided egg retrieval, cryopreservation of embryos and intracytoplasmic sperm injection (ICSI)—a technique in which a single sperm cell is injected into an oocyte using a microneedle. In addition, there were further improvements such as assisted hatching and in media composition, such as sequential media, which allow the in vitro culture of the embryo to reach the blastocyst stage [8].Current IVF procedures involve multiple steps including ovarian stimulation and monitoring, oocyte retrieval from the ovary, fertilization in vitro and embryo transfer to the womb. Whereas the first IVF cycles, including the conception of Louise Brown, used natural ovulatory cycles, which result in the retrieval of one or two oocytes, most IVF cycles performed today rely on controlled ovarian stimulation using injectable gonadotropins—follicle stimulating hormone and luteinizing hormone—in supraphysiological concentrations for 10–14 days, followed by injection of human chorionic gonadotropin (hCG) 38–40 h before egg retrieval to trigger ovulation. This updated protocol makes it possible to grow multiple follicles and to retrieve 10–20 oocytes in one IVF cycle, thereby increasing the number of eggs available for fertilization.Post-retrieval, the embryologist places an egg and sperm together in a test tube for fertilization. Alternatively, a single sperm cell can be injected into an egg by using ICSI. This procedure was initially developed for couples with poor sperm quality [9], but has become the predominant fertilization technique used in many IVF clinics worldwide [8]. The developing embryos are monitored by microscopy, and viable embryos are transferred into the woman''s womb for implantation. Louise Brown, as with many embryos today, was transferred three days after egg retrieval, at approximately the eight-cell stage. However, using sequential media, many clinics advocate culturing embryos until day five when they reach the blastocyst stage. The prolonged culture period allows self-selection of the most viable embryos for transfer and increases the chance of a viable pregnancy. Excess embryos can be cryopreserved and transferred at a later date by using a procedure known as frozen embryo transfer (FET). In this article we use the term ART to refer to IVF procedures with or without ICSI and FET.

Science & Society Series on Sex and Science

Sex is the greatest invention of all time: not only has sexual reproduction facilitated the evolution of higher life forms, it has had a profound influence on human history, culture and society. This series explores our attempts to understand the influence of sex in the natural world, and the biological, medical and cultural aspects of sexual reproduction, gender and sexual pleasure.Embryos can also be screened for chromosomal aneuploidies—missing or extra chromosomes—by preimplantation genetic diagnosis (PGD) when indicated and when available. PGD can also be used to test fertile couples at increased risk of genetic disorders. To perform PGD, a single cell is obtained from three-day-old embryos for molecular testing, for example sequencing for inherited monogenic disorders or fluorescent in situ hybridization for chromosomal abnormalities [8]. Only embryos with a normal chromosomal constitution, and without the genetic disorder in question, would then be transferred into the woman''s womb.Despite tremendous progress during the past three decades, people undertaking ART still face a considerable risk of failure to achieve parenthood. The rate of clinical pregnancies in Bourn Hall between 1980 and 1985 was 24% and 14% in women younger and older than 40 years, respectively [10]. The reported rates for clinical pregnancies and live births vary by country; the average delivery rate is 22.4%, 23.3% and 17.1% for IVF, ICSI and FET cycles, respectively [11]. According to the last Centers for Disease Control and Prevention report in 2009, the average live-birth rate was 35% per fresh ART cycle, although it sharply declines with age, from 45% among women younger than 35 years to 7% among women older than 42 years [5]. The reasons include poor response to ovarian stimulation, ovarian hyperstimulation syndrome and failure of eggs to fertilize. However, these failures occur in only a minority of patients and the success rate of egg retrieval and fertilization leading to embryo transfer is a remarkable 90% [12].Implantation remains the least understood process and is a key rate-limiting step in ART. Poor embryo quality is considered to be the main cause of implantation failure and it reflects a high incidence of chromosomal aneuploidies, which increases with maternal age [13]. One obvious solution to improve implantation rates is to transfer more embryos. However, this also increases the risk of multiple births, and related morbidity and mortality in newborns. An alternative approach is to select for good-quality embryos by culturing them to the blastocyst stage, because it seems that aneuploid embryos arrest by this stage and that blactocysts are more likely to have a normal chromosomal complement. There is ongoing research aimed at identifying viable embryos through PGD and metabolic profiling [13].Despite tremendous progress during the past three decades, people undertaking ART still face a considerable risk of failure to achieve parenthoodIt has also been suggested that failure to implant could be caused by the inability of the embryo to hatch out of a glycoprotein layer surrounding the embryo, known as the ‘zona pellucida''; this layer hardens if the embryo is cultured or frozen. Assisted hatching by rupturing the zona pellucida before embryo transfer does increase clinical pregnancy rates, especially for thawed embryos [13]. Another factor linked to the failure of implantation is endometrial receptivity. The endometrium consists of multi-layered mucosa cells in the inner wall of the uterus, which undergoes coordinated remodelling during the menstrual cycle and there is a specific time window when it is receptive to embryo implantation. Several research studies have identified molecular biomarkers of poor endometrial receptivity, showing that prostaglandins, cell adhesion molecules, mucins and cytokines are important [13].When it comes to health risks for mothers and infants, the use of ART increases the risk of multiple births, including higher rates of caesarian sections, prematurity, low birth weight, infant death and disability. More recently, concerns regarding elevated risks of birth defects, genetic abnormalities, neurodevelopmental disorders and imprinting disorders have been reported; however, not all are substantiated. There are still many unanswered questions regarding the potential short- and long-term health risks of ART for women and children, and there are tremendous challenges in studying the safety of ART procedures. Apart from the subset of individuals undergoing ART for social reasons—single parents or same sex couples—most patients are subfertile couples. Subfertility, defined as a failure to conceive naturally after 12 months of unprotected intercourse, affects 8–20% of couples [2], and it can occur for a variety of unknown or known reasons including maternal factors—endocrine, hormonal, endometriosis and blocked fallopian tubes—and paternal factors such as spermatogenesis abnormalities.Most studies have assessed the risks of ART by comparing the outcomes of ART-conceived pregnancies to naturally conceived pregnancies. There is emerging evidence that underlying maternal or paternal subfertility might be an important factor in obstetric, neonatal and childhood outcomes in the ART population. Therefore, to determine the specific health risks associated with the ART process itself, the outcomes of ART-conceived pregnancies should be assessed in comparison with naturally conceived pregnancies in subfertile parents, which is methodologically difficult. Alternatively, studying the health risks of ART in fertile couples—for instance, same-sex couples and couples at risk of genetic disorders—would be informative, but the number of such couples is relatively small.Women who undergo ART are at risk of ovarian hyperstimulation syndrome (OHSS). OHSS is a complication of ovulation induction resulting in enlargement of ovaries and retention of fluids leading to various secondary complications, which normally resolve within two weeks, but can persist if pregnancy occurs. Patients with OHSS can be offered embryo cryopreservation and frozen embryo transfer when symptoms resolve. Moderate forms of OHSS occur in 5% of patients undergoing ART; 2% of patients require hospitalization. Death occurs with an incidence of approximately 3 per 100,000 ART cycles [14]. OHSS is predominantly caused by human chorionic gonadotropin injection used for inducing final oocyte maturation and ovulation. Research is focused on optimizing alternative stimulation protocols [14].The use of supraphysiological concentrations of hormones during ovarian stimulation has also raised concerns that ART can increase cancer risks linked to hormonal fluctuations. These include breast, ovarian, endometrial, cervical and colon cancers, as well as melanoma. Studies evaluating the risks of cervical cancers, colon cancers and melanoma have not demonstrated increased risks for women undergoing ART [1]. The data for breast, ovarian and endometrial cancer is more complex, however, and more research is required to conclusively determine whether there is an increased risk.The perinatal and obstetric risks of ART are most significantly influenced by multiple pregnancies. These are at a more than 60% risk of low birth weight or premature delivery [2], and related risks of pregnancy complications such as gestational diabetes, abnormal placentation and hypertensive disorders [1]. Multiple pregnancies occur in 1% of naturally conceived pregnancies and 25–50% of ART pregnancies, owing to multiple embryo transfer. In the Western world, about 30–50% of all twin pregnancies result from ART [2]. Whilst double or triple embryo transfer is still common, the development of cryopreservation techniques and extended blastocyst culture has increased the use of single embryo transfer (SET), especially for younger women. Many European countries and the province of Quebec, in Canada, where ART is publicly funded, have adopted a policy of SET, which has dramatically decreased the incidence of multiple pregnancies. In Belgium and Quebec, SET policies have reduced multiple pregnancies from 19% to 3% and from 27% to 6%, respectively. It has been argued that SET results in a lower live-birth rate than a double-embryo transfer, but this is almost completely overcome by an additional single frozen embryo cycle [2].…there are tremendous challenges in studying the safety of ART proceduresThe question of whether ART increases the risks of pregnancy complications, including prematurity and low birth weight in singletons, remains unresolved; several studies have found an increased risk, but others have not replicated these findings [1,2]. It has been suggested that the fertility history of patients undergoing ART is an important factor, as there is an association between the length of time to conception and prematurity and birth weight [15]. Prematurity and low birth weight are also known to be associated with long-term health effects, including adult onset coronary artery disease, hypertension, obesity and type 2 diabetes [16,17].Various studies have also reported a higher incidence of congenital anomalies in ART-conceived children, with a suggested 30% increase of malformations [2]. However, this is another risk that might be attributable to parental subfertility, as a study comparing children conceived by ART to subfertile parents and children conceived naturally to subfertile parents did not find any significant difference in the congenital anomaly rate [2]. Findings from another study of the risks of birth defects in children conceived naturally to women with and without a history of subfertility compared with children conceived with the assistance of ART also suggest that it is subfertility, rather than ART, that is associated with an increased risk of birth defects [18].Several studies reported an increased risk of cerebral palsy and other neurological abnormalities in children conceived by ART [2]. But again, these findings are mainly attributed to complications resulting from multiple pregnancies including prematurity and low birth weight. The increased utilization of SET is therefore expected to result in fewer multiple pregnancies, which should result in a concomitant decrease in neurological complications. Further evidence that neurological complications in ART children are not exclusively related to ART came from studies that have assessed neurodevelopmental outcomes, such as locomotion, cognition, language and behavioural development of ART children in comparison with naturally conceived children. These analyses did not reveal any differences when adjusted for confounding factors of low birth weight and prematurity. In a similar vein, numerous studies have investigated whether there is an increased incidence of autism in ART-conceived children, but these have been inconclusive [19].There are potential concerns regarding the fertility of ART children. However, this requires future studies as most of this population is younger than 30 years of age. There is some evidence that boys conceived through ICSI have an increased rate of genital anomalies [2] and that males with severe infertility, such as low sperm counts, are more likely to carry chromosomal abnormalities, which could be passed on to their children conceived through ICSI [15].It has also been suggested that there might be an increased risk of cancers in ART-conceived offspring. Although multiple studies have identified no such risk, a large Swedish study reported a marginally increased risk of cancer, including haematologic, eye, nervous system, solid tumours and histiocytosis [2]. Similarly to other ART-related adverse health outcomes, it has been suggested that the increased risk of cancer could be attributed to prematurity, a recognized risk factor for cancer, rather than to the ART procedure itself. Further long-term studies are required to determine if there is truly an increased risk of adult cancers in ART offspring.…there remain unanswered questions about both the health risks associated with ART and the potential mechanisms that could account for these findingsOne thing is clear from the available evidence to date: there remain unanswered questions about both the health risks associated with ART and the potential mechanisms that could account for these findings. One possible explanation is that the exposure of gametes and preimplantation embryos to the various steps of ART might affect growth and development of offspring through dysregulation of epigenetic pathways [20]. In addition, there is evidence that genetic and epigenetic alterations might be inherited from the gametes of subfertile parents, which would reinforce assertions that subfertility itself might play a role in ART-related health outcomes [1,20].Epigenetics refers to heritable changes in gene expression without alterations to the underlying DNA sequence. DNA methylation and modifications of histones are epigenetic modifications that determine active against repressive conformation of chromatin structure, thereby regulating gene expression and driving essential processes such as embryonic development, fetal organ development, cell differentiation and tissue-specific gene expression [21]. Genomic imprinting is a type of epigenetic gene regulation that uses epigenetic marks to silence specifically one of the parental alleles. There are approximately 100 known imprinted genes in humans [22]. Most imprinted genes are found in clusters across the genome and are regulated by parent-specific DNA methylation and histone modification marks at cis-acting imprinting centres, as well as non-coding RNAs. Most of the known imprinted genes have functions related to growth and behaviour; disruption of the normally programmed parental expression of imprinted genes can therefore result in disorders related to growth and neurodevelopment.Gametogenesis and embryogenesis are important stages of mammalian development that require genome-wide epigenetic reprogramming. During spermatogenesis, protamines replace most histone proteins to create a highly compacted DNA. Establishment of DNA methylation imprints at paternally methylated imprinting centres is complete in males at the time of birth. In females, the establishment of maternally methylated imprinting centres begins during puberty and is almost complete in ovulated oocytes. After fertilization, the paternal genome undergoes rapid active DNA demethylation in which protamines are replaced by histones, whilst the maternal genome is passively demethylated, so that DNA methylation patterns are lost through cell divisions. Although, the whole genome undergoes demethylation, parent-specific DNA methylation is maintained at imprinting centres. Subsequently, the genome is remethylated and cell-type-specific epigenetic patterns are established as embryonic development proceeds. The parent-specific DNA methylation at imprinting centres is maintained in somatic cells, but it is erased and re-established in the gametes starting a new cycle of imprinting (Fig 1; [23]). As the establishment and maintenance of imprinting marks coincides in timing with important stages of ART, such as oocyte maturation under supraphysiological hormone concentrations and embryo culture, it has been proposed that ART can lead to imprinting errors [24].Open in a separate windowFigure 1Life cycle of genomic imprinting and assisted reproductive technology. Erasure, re-establishment and maintenance of genomic imprinting occur during gametogenesis and preimplantation embryo development. Blue and red solid lines show paternal and maternal methylation at imprinting centres through gametogenesis and early stages of preimplantation development. Imprinting marks are erased at early stages of gametogenesis. Re-establishment of imprinting occurs throughout gametogenesis, but finishes much later in oocytes compared with sperm. During preimplantation development, both maternal and paternal imprinting marks are maintained whilst the rest of the genome is demethylated. The paternal genome is demethylated rapidly and actively (dashed blue line) whilst the maternal genome is demethylated at a slower rate passively through cell division (dashed red line). Various steps of assisted reproductive technology such as ovarian stimulation, ovulation induction, gamete and embryo manipulation and culturing create unusual environments for gametes and embryos and thus, can interfere with proper establishment of imprinting marks in oocytes or maintenance of imprinting marks in embryos. Subfertility can be associated with epigenetic errors in imprinting erasure and/or establishment in both oocytes and sperm. Adapted from [23].In 2001, the first evidence that genomic imprinting can be perturbed during ART procedures came from studying sheep fetuses derived from in vitro cultured embryos that presented with large offspring syndrome (LOS; [25]). LOS occurs sporadically in cattle and sheep conceived by IVF and is characterized by a 20–30% increase in birth weight frequently accompanied by congenital anomalies and placental dysfunction [24]. Owing to phenotypic similarities of LOS to the human overgrowth disorder Beckwith–Wiedemann syndrome (BWS), which is caused by the dysregulation of gene expression within an imprinted cluster on chromosome 11p15.5, the authors hypothesized that genes from the orthologous cluster in sheep or a closely related pathway could be dysregulated in LOS. They tested expression of the insulin-like growth factor 2 (IGF2) gene known to be overexpressed in BWS, and the IGF2R receptor gene, which is involved in clearance of IGF2 from the circulation. IGF2R is imprinted in sheep but not in humans. In sheep with LOS, no differences for IGF2 were found, but reduced expression of IGF2R was observed after loss of DNA methylation at the imprinting centre for this gene [25].In the following decade, several studies provided further evidence that children conceived by ART might be at increased risk of imprinting disorders. The strongest case has been made for BWS and Angelman syndrome. BWS is the most common human overgrowth syndrome characterized by prenatal and postnatal overgrowth, congenital anomalies and tumour predisposition [26]. Angelman syndrome is a neurodevelopmental disorder characterized by microcephaly, severe intellectual disability and a unique behavioural profile including frequent laughter, smiling and excitability [27]. Multiple case reports from various countries indicate an increased frequency of BWS and Angelman syndrome in ART children (3–10-fold) compared with the general population. However, two cohort studies failed to replicate this association [28]. The low incidence of both BWS (1 in 13,700) and Angelman syndrome (1 in 15,000) in the general population [28] makes epidemiological studies difficult—the two cohort studies reported 2,492 and 6,052 ART children, respectively, and are probably underpowered to detect an increased risk of BWS and Angelman syndrome. However, even if there might be increased relative risks for these syndromes in ART children, the absolute risks in this population remain low.The molecular causes of BWS and Angelman syndrome are heterogeneous. They include genomic (deletion, uniparental disomy and gene mutation) and epigenetic (loss of imprinting due to aberrant DNA methylation) alterations at imprinted gene clusters on chromosomes 11p5.5 and 15q11–q13, respectively. These alterations occur with specific frequencies for each of the two disorders [26,27]. Results of molecular testing in children with these syndromes and conceived using ART, reveal an excess of epigenetic compared with genetic molecular alterations. For example, loss of DNA methylation at imprinting centre 2 occurs in about 50% of BWS cases in the general population, whereas several studies found loss of DNA methylation at imprinting centre 2 in 96% (27/28) of BWS ART-conceived children. In Angelman syndrome, approximately 3% of cases in the general population have loss of methylation at 15q11–13, whereas 5 out of 19 (26%) Angelman syndrome children conceived by ART or naturally by parents with a history of subfertility had loss of DNA methylation at 15q11–13 (Fig 2).Open in a separate windowFigure 2Enrichment of epigenetic alterations in Beckwith–Wiedemann syndrome and Angelman syndrome after assisted reproductive technology. Loss of methylation (LOM) at imprinting centre 2 (IC2) on chromosome 11p15.5 contributes to 50% of Beckwith–Wiedemann syndrome (BWS) cases in the general population, whereas LOM at IC2 is found in 27 out of 28 cases (96%) in the BWS assisted reproductive technology (ART) population, which represents a 1.9-fold enrichment of this epigenetic defect. For Angelman syndrome (AS), methylation disruption at the 15q11–q13 imprinting centre contributes to 3% of AS cases, and in the AS ART and subfertility population it was found in 5 out of 19 cases (26%; eight fold enrichment). Data from the following publications were used for these calculations, BWS [31,32,33,34,35] AS [35,36].The data for loss of DNA methylation in Angelman syndrome cases conceived naturally by subfertile parents highlights the fact that epigenetic alterations could, at least in part, result from underlying parental subfertility. Indeed, several studies have shown that abnormalities of spermatogenesis, such as oligospermia (low sperm concentration), low sperm motility or abnormal sperm morphology are associated with altered DNA methylation at imprinted loci. These occur in both maternal and paternal alleles of imprinting centres in sperm and could be transmitted to offspring conceived by ART [26]. One study of chromosomally normal fetuses spontaneously aborted at six to nine weeks of gestation found that DNA methylation alterations at imprinted loci were sometimes inherited from sperm. Thus, it is possible that this dysregulation of imprinting in male gametes might be one cause of the association between imprinting disorders and ART.Studies of other known imprinted syndromes, such as Prader–Willi syndrome, Russell–Silver syndrome, maternal and paternal uniparental disomy of chromosome 14, pseudohypoparathyroidism type 1b and transient neonatal diabetes mellitus, have either not demonstrated an association with ART or have been inconclusive owing to their small size [29]. A link has also been suggested between ART and the newly defined ‘multiple maternal hypomethylation syndrome'', which clinically presents either as BWS or transient neonatal diabetes mellitus, and is associated with loss of DNA methylation at multiple maternally methylated imprinting centres; loss of methylation at paternal imprinting centres has not been reported so far. Thus, human imprinting disorders that have been observed with increased relative frequency in ART offspring are confined to loss of DNA methylation at maternally methylated imprinting centres, similar to epimutations of IGF2R in LOS. One could propose that ART has a greater impact on female than male gametes, as the eggs are subjected to more environmental exposures—supraphysiological doses of hormones—and more manipulation than the sperm. However, studies of mouse in vitro cultured embryos and ART-exposed human and mouse gametes suggest that ART can also be associated with either loss or gain of DNA methylation on both maternal and paternal alleles [23].Mouse models are a valuable method to investigate which stages of ART procedures can disrupt normal imprinting patterns. The advantage of using mouse models is the ability to investigate each of the parameters of ART—ovulation stimulation and embryo culturing—separately and at different stages of development. Furthermore, mouse models allow investigators to alter ART parameters, such as concentration of hormones or media for embryo culturing. Most importantly, studies in animal models have shown that ART procedures without the confounding factor of subfertility do have a negative impact on imprint regulation [23].The exposure of maturing oocytes from mice to abnormally high doses of gonadotropins has been suggested to alter imprint establishment. Yet, studies performed directly on superovulated oocytes are inconclusive, as not all of them have demonstrated increased rates of DNA methylation errors at imprint centres compared with spontaneously ovulated oocytes. Interestingly, studies of DNA methylation in mouse blastocysts harvested from superovulated mothers identified an increased rate of DNA methylation errors at imprint centres. This included loss of DNA methylation at the paternally methylated H19—the imprinting centre on human chromosome 11 and mouse chromosome 7 implicated in BWS and the related undergrowth Russell–Silver syndrome. It suggests that superovulation also impairs imprinting maintenance; probably by affecting the ability of the oocyte to synthesize and store sufficient maternal factors (RNA and proteins; [23]). In support of this hypothesis, four maternal effect proteins have been previously identified that are involved in imprinting maintenance in preimplantation embryos. It was also found that imprint errors arise in blastocysts in a dose-dependent manner—higher doses of hormones resulted in DNA methylation errors in a larger number of embryos [23].As the establishment and maintenance of imprinting marks coincides in timing with important stages of ART […] it has been proposed that ART can lead to imprinting errorsAnother factor that might contribute to imprinting errors is the micromanipulation of gametes during IVF and ICSI procedures. Evidence supporting this hypothesis includes the observation in mouse models that a higher number of IVF embryos—resulting from superovulation alone or superovulation and embryo culturing—have aberrant H19 DNA methylation compared with in vivo conceived embryos [23]. Media with varying compositions are used in ART clinics, and whilst all of the media are suboptimal for normal maintenance of all DNA imprints in mouse embryos, the number of embryos with aberrant DNA methylation at imprinting centres varies depending on the media [23]. Interestingly, it was also found that embryos with faster rates of development are more prone to loss of DNA methylation at imprinting centres [23].Though it is not yet clear how these findings relate to ART in humans, the mouse research is crucial for informing human studies about which variables should be addressed to optimize the safety and efficacy of ART procedures. Apart from ART itself, it has been shown that compromised fertility in mice results in loss or delay of DNA methylation acquisition in one of three tested imprinted genes. The compromised fertility is induced by genetic manipulation of a gene involved in communication between oocytes and surrounding follicular cells, which is crucial for proper oocyte maturation. The results suggest that the observed loss of DNA methylation could be caused by impaired transport of metabolites from follicular cells to oocytes, which is important for imprint establishment [23].Data linking dysregulation of imprinted loci and ART is limited to several imprinted gene clusters associated with clinically recognizable syndromes. However, there are more genes in the human genome that have been discovered to be, or are predicted to be, imprinted [22] but are not yet known to be associated with clinical phenotypes. Potentially, ART can lead to dysregulation of these imprinted genes, which might be another, as yet unrecognized factor contributing to neonatal and long-term health problems of ART-conceived children. At this point, it is also not clear whether epigenetic disruption during ART is limited to imprinted genes or has more global effects on the genome. The data for genome-wide DNA methylation analysis are limited in both human and mouse to individuals with no apparent disease phenotype. So far, these data have been inconclusive [23,28].One could propose that ART has a greater impact on female than male gametes, as the eggs are subjected to more environmental exposures […] and more manipulation than the spermDespite significant advances in the efficacy and success of ART procedures during the past few decades, the health risks, especially related to long-term outcomes in ART-conceived children, remain poorly understood. Moreover, the phenomena known as ‘fetal programming''—when maternal and in utero exposures can lead to various adult onset disease susceptibilities—have been suggested to be transmissible to the next generations, probably through epigenetic mechanisms [30]. In the case of ART procedures, the effect of ‘unusual'' environments during gametogenesis and early embryonic development on adult-onset disease and trans-generational inheritance is still not clear. Additional research is needed to elucidate the effects of ART on genome-wide epigenetic patterns and their link to human disease. As ART will continue to be an important medical intervention and the number of children born with the help of ART procedures will probably continue to rise in the future, it is crucial to understand the associated health risks and underlying molecular mechanisms of these technologies. This will increase the safety of this intervention and enable couples using ART to be fully informed regarding both present and future health-related risks.? Open in a separate windowDaria GrafodatskayaOpen in a separate windowCheryl CytrynbaumOpen in a separate windowRosanna Weksberg     

Compact Parkin only: insights into the structure of an autoinhibited ubiquitin ligase

EMBO J 32 15, 2099–2112 doi:10.1038/emboj.2013.125; published online May312013Mutations in Parkin represent ∼50% of disease-causing defects in autosomal recessive-juvenile onset Parkinson''s disease (AR-JP). Recently, there have been four structural reports of autoinhibited forms of this RING-IBR-RING (RBR) ubiquitin ligase (E3) by the Gehring, Komander, Johnston and Shaw groups. The important advances from these studies set the stage for the next steps in understanding the molecular basis for Parkinson''s disease (PD).Regulated protein degradation requires that E3s and their access to substrates be exquisitely controlled. RBR family E3s provide striking examples of this regulation. The complex and compact structures of Parkin (Riley et al, 2013; Spratt et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013) as well as another RBR E3, human homologue of Ariadne (HHARI) (Duda et al, 2013), demonstrate extraordinarily intricate inter-domain arrangements. These autoinhibited structures ensure that their functions are restricted until activated.Until recently, RBR E3s were believed to be a subclass of RING E3s, which allosterically activate E2 conjugated with ubiquitin (E2∼Ub). However, Wenzel et al (2011) determined that they are actually hybrid E3s, containing an E2 binding site in RING1 and a catalytic cysteine residue in the domain designated as RING2. The catalytic cysteine is an acceptor for an ubiquitin from RING1-bound E2∼Ub forming an intermediate (E3∼Ub) that leads to substrate or autoubiquitination. In this way, RBRs resemble HECT E3s, which also form catalytic intermediates in ubiquitination. There are 13 human RBR family E3s. Besides Parkin, two notable RBRs are HOIL-1 and HOIP, which form part of a complex integral to NF-κB activation (Wenzel and Klevit, 2012).In addition to causal roles in AR-JP, single allele mutations of Parkin are found in some sporadic cases of PD (references in Wauer and Komander, 2013). Mutations in the Parkin-associated kinase PINK1, which is upstream of Parkin, also account for a significant number of AR-JP cases (Hardy et al, 2009; Narendra et al, 2012; Lazarou et al, 2013). A number of diverse Parkin substrates have been postulated to be associated with PD. There is substantial evidence that one role for Parkin is at mitochondria. Once activated and recruited to damaged/depolarized mitochondria by PINK1, it ubiquitinates exposed mitochondrial proteins leading to both proteasomal degradation and mitophagy (Narendra et al, 2012; Sarraf et al, 2013). Parkin has also been implicated in cell surface signalling and as a tumour suppressor (see references in Wauer and Komander, 2013).Parkin encodes five structured domains, beginning with an N-terminal ubiquitin-like domain (UbLD) and followed by four domains that each bind two zinc (Zn) atoms (Figure 1A). The most N-terminal of the Zn-binding domains is RING0. C-terminal to this is the RBR, consisting of RING1, the IBR and RING2. The crystal structures of inactive Parkin from Riley et al (2013), Trempe et al (2013) and Wauer and Komander (2013) show remarkable congruity. Spatially, the IBR is at the complete opposite end of the molecule from RING2, to which it is connected by a partially unstructured ∼37 residue linker. This linker includes a two-turn helix, referred to as the repressor element of Parkin (REP) or tether, which binds and occludes the E2 binding face of RING1. RING1 occupies the central position in these structures, and RING0 separates RING1 from RING2 (Figure 1B and C). The latter contains the residue identified by Wenzel et al (2011), and confirmed by all three groups, to be the catalytic cysteine, C431. A lower resolution structure also includes the UbLD and places this domain adjacent to RING1 (Trempe et al, 2013). A second unstructured linker connects the UbLD and RING0. UbLDs are involved in a number of protein–protein interactions and small angle X-ray scattering confirms that this domain is integral to the core structure of Parkin (Spratt et al, 2013; Trempe et al, 2013). Biophysical characterization of Parkin and HHARI suggests that each is a monomer in solution.Open in a separate windowFigure 1Schematic and spatial representation of Parkin. (A) Primary structure and domain designations of Parkin, including the REP sequence within the otherwise unstructured IBR-RING2 linker. (B) Structural representation of full-length Parkin (PDB 4K95) highlighting the complex domain interactions in the three-dimensional structure, the catalytic C431 residue, and residue W403 within the REP, which plays a role in stabilizing the autoinhibited form of Parkin. (C) A model of Parkin with the E2 UbcH5B/Ube2D2 bound (devised using PDB 4K95 and PDB 4AP4 to mimic the position of an E2 bound to RING1) to illustrate the required displacement of UbLD and REP and the large distance between the E2∼Ub attachment site of the E2 and the catalytic active site of Parkin. Note that in this conformation the catalytic Cys within RING2 (C431) remains buried by RING0.RING1 is the only bona fide RING domain. All NMR and crystal structures of IBR domains from Parkin, HHARI and HOIP (PDB ID: 2CT7) are in good agreement. The Parkin and HHARI RING2s are structurally highly homologous and share a common Zn-coordinating arrangement with IBR domains. In contrast to the IBR and RING2, RING0 has a distinct arrangement of Zn-coordinating residues (Beasley et al, 2007; Duda et al, 2013; Riley et al, 2013; Spratt et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013) (see Figure 1F of Trempe et al (2013) for the various Zn coordination arrangements).All of the Parkin crystal structures represent inactive forms of the E3. This is imposed by the quaternary positioning of the domains, which precludes activity in multiple ways. RING0 plays two obvious roles to maintain Parkin in an inactive state. RING0 shares an interface with RING2 and buries C431, making it unavailable as an ubiquitin acceptor. Moreover, RING0 intervenes between RING1 and RING2, creating an insurmountable separation of >50 Å between the active site Cys of an E2 bound to RING1 and C431 (Figure 1B and C). Thus, RING0 must be displaced for ubiquitin transfer to occur. Accordingly, deletion of RING0 results in a marked increase in Parkin autoubiquitination and in C431 reactivity (Riley et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013). In HHARI, these two inhibitory functions are fulfilled by the C-terminal Ariadne domain, which similarly interposes between RING1 and RING2 (Duda et al, 2013).Additional inhibition is provided by the REP, which binds to RING1 at the canonical RING-E2 binding site and prevents E2 binding. This provides at least a partial explanation for the impaired ability of Parkin to bind E2 when compared to HHARI, which lacks this element (Duda et al, 2013). A disease-associated REP mutant (A398T) at the RING1 interface increases autoubiquitination (Wauer and Komander, 2013). The significance of inhibition by REP-RING1 binding was verified by mutating a critical RING1-interacting REP residue (W403A). This increased autoubiquitination and E2 binding (Trempe et al, 2013). Consistent with the requirement for charging C431 with ubiquitin in mitochondrial translocation (Lazarou et al, 2013), Parkin association with depolarized mitochondria is accelerated with this mutation (Trempe et al, 2013). Interestingly, W403 also interacts with the C-terminal Val of Parkin within RING2, and could therefore potentially further stabilize the autoinhibited form of the protein (Riley et al, 2013), consistent with previous observations (Henn et al, 2005).The quaternary structure of full-length Parkin also suggests that displacement of its N-terminal UbLD must occur for full activation (Trempe et al, 2013). The positioning of the UbLD adjacent to RING1 indicates that it would provide a steric impediment to E2∼Ub binding (Figure 1B and C). Additionally, displacement of the UbLD could be important to relieve interactions with the IBR-RING2 linker, which, as suggested in a previous study (Chaugule et al, 2011), might help to maintain Parkin in an inactive state. Finally, the crystal structure of the full-length Parkin indicates that the UbLD is not available for interactions with other proteins. This would limit Parkin''s range of intermolecular interactions.RBR E3s have at least two domains critical for sequential ubiquitin transfer and full activity, RING1 and RING2. The RING1 of Parkin, as well as all other RBR E3s, is notable in lacking the basic residue in the second Zn coordinating loop (or its equivalent in U-box proteins), which has recently been implicated in RING-mediated transfer of Ub from E2∼Ub (Metzger et al, 2013). This suggests that other factors play compensatory roles in positioning ubiquitin for transfer from E2∼Ub to C431. A non-mutually exclusive possibility is that the lack of this basic residue in RING1 limits unwanted attack on the E2∼Ub linkage, thereby minimizing the unregulated ubiquitination. Turning to RING2, the area surrounding the active site C431 of Parkin is notable in that it includes a sequence recognizable as a catalytic triad, similar to that in deubiquitinating enzymes. The Cys-His-Glu grouping, found in Parkin and other RBR E3s, contributes to in vitro activity (Riley et al, 2013; Wauer and Komander, 2013). Interestingly, however, the Glu was dispensable in a cellular assay (Riley et al, 2013). This triad is conserved in HHARI, where an Asn between the Cys and His residues (found in a number of RBRs but not conserved in Parkin), was found to be important for catalysis (Duda et al, 2013).The advances made in these studies impart significant information about an important and clinically relevant E3. However, Parkin, as well as HHARI, has been captured in their inactive, unmodified forms. One obvious question is how does Parkin transition between inactive and active states. PINK1 is implicated in phosphorylating Parkin on its UbLD and potentially other sites, with evidence that phosphorylation contributes to Parkin activation (Narendra et al, 2012). How phosphorylation could contribute to protein interactions that might facilitate Parkin activation, potentially including Parkin oligomerization (Lazarou et al, 2013), is unknown. Regardless, it is evident that considerable unwinding of its quaternary structure must take place.While there is much work ahead to understand these processes, one important interface that must be disrupted for activation is that between the REP and RING1. It is intriguing to consider that such interruption might be associated with other alterations in the IBR-RING2 linker, potentially facilitating the movement of the UbLD from RING1 and contributing to activation. Related to activation is the all-important question of how Parkin recognizes and targets specific substrates. While the UbLD represents a potential site of interaction, most purported substrates are not known to have UbLD-interaction domains. Although interactions involving the UbLD could occur indirectly, through bridging molecules, there is also evidence that other regions of Parkin, including the RBR region, might recognize substrates either directly or indirectly (Tsai et al, 2003) and that some substrates may be phosphorylated by PINK1 (Narendra et al, 2012). Conformational changes induced by substrate interactions, particularly in the IBR RING2 linker, could, as above, represent an important aspect of activation.There are over 75 missense mutations of Parkin associated with AR-JP, most of these inactivate the protein, but there are also some that are activating (Wauer and Komander, 2013). Activating mutations presumably result in pathology at least partially as a consequence of increased autoubiquitination and degradation (e.g., A398T). The current studies help to provide a classification of missense mutations into those that affect (i) folding or stability, (ii) catalytic mechanism, and (iii) interactions between domains. Interdomain mutations might inactivate or contribute to constitutive activation leading to autoubiquitination and degradation.Finally, we know little about how the autosomal recessive and the much more prevalent sporadic forms of PD overlap in their molecular pathology. However, mitochondrial dysfunction is increasingly a common theme. Thus, with the structure of the inactive protein in hand, there is hope that we can begin to consider ways in which domain interactions might be altered in a controlled manner to activate, but not hyperactivate, this critical E3 and lessen the progression of PD.