Madalina Croitoru
email: madalina.croitoru@lirmm.fr

Stéphane Bazan
email: stefan.bazan@usj.edu.lb

Stefano Cerri
email: cerri@lirmm.fr

Hugh Davis

Raffaella Folgieri
email: raffaella.folgieri@unimi.it

Clement Jonquet
email: jonquet@lirmm.fr

François Scharffe
email: francois.scharffe@lirmm.fr

Steffan Staab
email: staab@uni-koblenz.de

Michalis Vafopoulos
email: vafopoulos@gmail.com

Thanassis Tiropanis

Su White

Negotiating the Web Science Curriculum through Shared Educational Artefacts

Keywords: K.3.2 [Computing Milieux] Computer and Information Science Education Design, Human Factors Web Science Education, Web Science Curriculum, Educational Repository, Negotiated Curriculum, Co-Creation

The far-reaching impact of the Web on society is widely recognised. The interdisciplinary study of this impact has crystallised in the field of study known as Web Science. However, defining an agreed, shared understanding of what constitutes web science requires complex negotiation and translations of understandings across component disciplines, national cultures and educational traditions. Some individual institutions have already established particular curricula, and discussions in the Web Science Curriculum Workshop series have marked the territory to some extent. This paper reports on a process being adopted across a consortium of partners to systematically create a shared understanding of what constitutes web science. It records and critiques the processes instantiated to agree a common curriculum, and presents a framework for future discussion and development.

INTRODUCTION

The need to study the Web in its complexity, development and impact led to the creation of web science. As a field of study web science is inherently interdisciplinary [START_REF] Berners-Lee | A Framework for Web Science[END_REF][START_REF] Halford | WebSci10: Extending the Frontiers of Society On-Line[END_REF][START_REF] Hendler | Web Science: An Interdisciplinary Approach to Understanding the Web[END_REF][START_REF] Shadbolt | Web Science Emerges[END_REF]. Its goals include:

•

Understanding the Web growth mechanisms; • Creating approaches that allow new powerful and more beneficial mechanisms to occur

The research community has organised itself, establishing an annual conference which for the first time in 2011 has acquired ACM status. Many eminent members of that research community are also active in the web science teaching community which has developed in parallel through the series of Web Science Curriculum Workshops. We consider teaching web science as an essential part of the project. One of the primary objectives is to help spread the knowledge of Web Science among students, thus encouraging new ideas, business research, cross-sectional expertise fostering connections with the business world and excellence in both academic achievements and technical knowledge acquisition. The curriculum itself is the natural manifestation of Web Science, serving to systemize knowledge and share approaches, current trends and future vision, to become the visible and practical part (i.e. the test bench) of the deep work in understanding and enhance Web Science

Teaching web science is a unique experience since the emerging discipline is a combination of two essential features. On one hand, the analysis of microscopic laws extrapolated to the macroscopic realm generates observed behaviour. On the other hand languages and algorithms on the Web are built in order to produce novel desired computer behaviour that should be put in context. Finding a suitable curriculum that is different to the decoupled study of language, algorithms, interaction patterns and business processes of established disciplines is thus an important and challenging task. The future of socio-technical systems will be in their innovative power (inventing new ways to solve problems), rather than their capacity to optimize current practices, Web Scientists need to be intellectually equipped to work with these phenomena. Some international collaboration is in place for PhD students through summer schools and funding of short-term visiting studentship. The Web Science Curriculum Development (WSCD) Project focuses European expertise in this interdisciplinary endeavour with the ultimate aim of designing a joint masters program for web science between the partner universities. The web science and engineering approach described by Berners-Lee [START_REF] Berners-Lee | A Framework for Web Science[END_REF] and then subsequently developed [START_REF] Berners-Lee | The Process of Designing Things in a Very Large Space: Kenote Presentation WWW[END_REF] to describe the combined functions of complexity and collaboration (see Figure1 below) is being consciously mirrored within the WSCD project.

Figure 1. Berners-Lee's science and engineering approach with magic [4] modified to show complexity and collaboration

There has been a longstanding agreement within the web science teaching community that a shared repository would be useful. We intend to use to the WSCD project to create a Web Science Curriculum Community Repository to be known as WSCCRpronounced whisker. We will extend the benefits of this initiative beyond the original collaboration to make the repository open to contributors who are outwith our initial European partnership.

The repository building magics'

Describing the way in which the project will mirror the 'two magics' paradigm, the process starts on the engineering side (right). From the technical design point of view the consortium is creating a semantically rich and open repository of shared educational artefacts. The system is based around EdShare 1 . which was developed at the University of Southampton [START_REF] Davis | Bootstrapping a Culture of Sharing to Facilitate Open Educational Resources[END_REF]. EdShare is designed for education and built on the EPrints repository engine. As WSCCR it will be used to collect, reference, annotate (and thus curate) the whole range of educational resources being used in our various programmes. As well as including resources created by the participating partners, there is a 1 http://edshare.soton.ac.uk facility to include links to extant resources 'in the wild', which is essential given the fast moving nature of the field of study.

Socially, these resources will be annotated collaboratively against the curriculum categorization published by Web Science Trust [START_REF] Vafopoulos | Web Science Subject Categorization (WSSC) Web Science Trust[END_REF]. At the same time, the curriculum is an evolving work which can be revised dynamically via a wiki hosted by the Web Science Trust [START_REF] Vafopoulos | Web Science Subject Categorization (WSSC) a Proposal for Discussion (Wiki)[END_REF] 2 . Work on this endeavour has been undertaken on behalf of the 'Web Science Curriculum Development' workshops and revisions to the proposed curriculum are discussed at workshop meetings. This additional process represents aspects of the complexity and collaboration located at the bottom of the diagram.

The resources items in the repository necessarily extend beyond artefacts used in the lecture and seminar room, such as slides, videos and hand-outs. They encompass artefacts associated with the range of administrative and organisational processes needed to assure the comparability of educational resources and underwrite the quality standards of the associated awards. These latter should prove to be particularly valuable in the task of individual curriculum design within and beyond the project. From the social point of view the contributions will be discussed and peer reviewed by members of the project consortium. Our intention is that by sharing the individual components of the teaching and educational process and quality assuring them by peer review we will provide concrete examples of our understanding of the discipline, so that the curriculum is effectively defined by a processes of co-creation, collaboration and co-evolution.

However, as Berners-Lee observes, it is in the move from the micro to the macro that the magic (complexity) is involved. The challenge for our consortium, once our community repository is adequately populated, is to involve the wider community in the contribution, discussion and annotation that will lead to the development of a negotiated and agreed, but evolving, curriculum for web science. At a macro level WSCCR can provide an Exploratorium which can be used by technologists and engineers as well as by social and human scientists. We cannot predict how it will evolve in the future, but we are hopeful that our approach of engineering a semantically rich, open and accessible repository for the community will provide succour for the creative ambitions of many different facets of the web science community.

To ensure that the repository is fit for purpose in its design, significant preparatory work has been necessary. Sections 2 and 3 provide an account of this activity. Section 4 describes WSCCR in more detail and explains the processes of the WSCD project in relationship to defining a curriculum for a particular institution and set of institutions. Subsequent sections discuss implications of this activity and point to conclusions and future work.

SHAPING THE REPOSITORY

Considerations in shaping the repository ranging from prior experience of repositories to aspirational motivations for the structure of the final system have to be addressed.

Curriculum development

Experience of community approaches to curriculum development already exists. For example, the Computer Science community established a repository of existing syllabi3 (CITIDEL) that enables designers of new courses to understand how others have approached the problem [START_REF] Tungare | Towards a Syllabus Repository for Computer Science Courses[END_REF].The Information Science community is using a wiki 4 [START_REF] Berners-Lee | The Process of Designing Things in a Very Large Space: Kenote Presentation WWW[END_REF] to enable the whole community to contribute to the dynamic development of the curriculum. What makes the WSCD project unique is that rather than taking a top down structured approach to curriculum definition it takes a bottom up approach, using the actual teaching materials as the basis on which to iteratively negotiate and refine the definition of the curriculum.

WSCD Aspirations

In our project, partner institutions are drawn from one Middle Eastern and five European institutions. Two of the partner institutions already teach a Masters in web science. All of the others in some way cover topics which are considered a part of web science through individual lectures or lecture series or special seminars. The topics are taught to students on existing academic programmes in academic areas in some way related to web science. Generally it is observed that different institutions and different academics have different understandings of what constitutes web science, accordingly there will be variations in the way they research, explain, define and contribute to web science. This is also true across our consortium. Furthermore, when offering a taught programme, local factors including specific faculty expertise; links with industrial partners; expectations associated with local educational culture; and prior experience of students, will all shape the nature of the particular interpretation of curriculum an institution puts on offer.

Existing experience of accreditation schemes with professional bodies or the matching of taught programmes against declared curricula such as the ACM Computer Science indicates that institutions will map their offered syllabus and curriculum against the published benchmark but an exact match is not expected. The process we describe working within the partnership however, as was noted in Section 1, is not typical. The project is working in such a way that the defined curriculum is emerging from the collected data supported by processes of collaborative definition; particularly appropriate since it is well aligned with processes observed at the heart of web science. Not only does the task of populating the shared repository serve as a means of providing a concrete representation of an agreed web science curriculum, it can also be analysed to identify the sets of trans-disciplinary competences which underpin web science.

CURRICULUM AND TEACHING

Inevitable when designing a repository, discussions arise as to how to construct the vocabulary which will be used to describe items and collections. Expertise and understanding of the shape of the web curriculum exists among the individual currently teaching web science -be it as a whole programme (Masters or Undergraduate) or as the whole or a part of a smaller part of a course or lecture series. The Web Science Trust has already established a wiki directory of web science courses 5 which lists around 20 courses (the authors acknowledge that there will be many more that are not known to the Web Science Trust).

How do we teach Web Science?

As part of this work we have analysed the extent and coverage of existing web science Curricula. We have done this by:

•

Analysing the information on the 20 courses registered with the Web Science Trust;

4 http://blogsandwikis.bentley.edu/iscurriculum/ 5 http://wiki.websciencetrust.org/w/Curriculum

• Surveying those who have attended Web Science curriculum workshops and other universities of whom we were aware or who advertised web science courses.

The survey 6 was carried out using an on-line form in March 2011 which invited participants to describe the nature of their web science course. The categories on which responses were gathered are shown in Table 1 at the end of this paper. There were 10 responses.

There are many different ways in which a curriculum can be explained and described. Individual institutions typically have an over-arching title for the programme, and then individual titles and codes for the component parts. Additionally students may be provided with the sense of a route or pathway through their degree programme. Figure 2 shows one such explanation.

Figure 2 Explaining a course's content and approach

However each degree programme will inevitably incorporate a 'perspective' on knowledge skills and understandings are of value, and what methods and methodologies need to be mastered and understood in order to engage in web science. As an interdisciplinary field of study, web science straddles the knowledge traditions and varying practices which Biglan identifies differentiating between Hard/Soft and Pure/Applied disciplines [START_REF] Biglan | The Characteristics of Subject Matter in Different Academic Areas[END_REF][START_REF] Biglan | Relationships between Subject Matter Characteristics and the Structure and Output of University Departments[END_REF]. These differences have been explored in general by Beecher and Trowler in their work on academic tribes and territories [START_REF] Becher | Academic Tribes and Territories : Intellectual Enquiry and the Cultures of Disciplines[END_REF], and were raised in relationship to web science in particular by Halford et al in 2010 [START_REF] Halford | WebSci10: Extending the Frontiers of Society On-Line[END_REF].

The web science subject categorisation [START_REF] Vafopoulos | Web Science Subject Categorization (WSSC) Web Science Trust[END_REF] identifies topic areas which describe the curriculum. For the survey we asked participants to identify the depth of coverage of each of the second level headings in the current curriculum categorisation.

For the purposes of the survey some of the details of the description were removed, and participants were not asked to comment on two categories which are deemed to be unrelated to instructional programmes.

Clearly the level of response is too small to make any firm statements about the development of web science curricula in practice. Nevertheless it is worth presenting and considering the findings so that they can form a basis for future discussions. Perhaps the most interesting insight drawn from of this study is the understanding that most courses that are describing themselves web science are modules (or sets of modules sufficient to constitute a 'minor') within a degree programme. The number of full degree courses, that we were able to discover, is still very small. The majority of the courses (modules or full degrees) were at master's level, but we identified a couple of emerging Bachelor's degrees.

It was also interesting to note that a couple of the universities who had played an significant role in defining and agreeing the web science curriculum categorisation had not yet been able to start their full degree courses due to economic constraints and anxiety within their institutions about the introduction of radical change.

In analysing the content in the courses in the study, we started with the hypothesis that since the majority of the courses were taught by computer science/informatics departments or were offered as part of such degrees, we might expect to see that, while the web technologies would be well covered, the Web Society Section (Section E in Table 1) might not be so well studied.

The analysis did not appear to support this assumption. Although most of the students appeared to come from scientific or business studies backgrounds, the people teaching the courses were by no means only from computing disciplines; teachers came from a range of appropriate disciplines. The courses themselves, as we had expected, covered the web technologies very thoroughly, but the 'Web Society' section was also well represented. Perhaps the least well-represented topics were Law (E5) and Philosophy (E4).

When asked what was missing from the current syllabus more than one respondent mentioned the need for interface design/ web design/ HCI to be represented more explicitly in the syllabus.

Another way in which the curriculum can be evaluated is by comparing its focus and extent to the research activities which have been discussed in the Web Science Conference to date. Data exists for 2009 and 2010, some data was generated top down (conference topic and tracks) other data is filtered by the opinions of the peer review college, but has 'emerged' as accepted papers, posters and workshops.

The tracks of 2009 can be seen as thematic -teaching and learning; trust and distrust; openness and control; tagging and search; social networking; web of data; government, citizens and law on the web. While the tracks of 2010 are perhaps more analytic -life on-line; web and society; web and communities; web and data; web and intelligence; web and methodology and can be seen to relate directly to the main top-level headings of the curriculum categorisation. In terms of paper abstracts and keywords, authors vary in their use of terms but emerging themes included economics, definition, analysis and technologies. Definition and analysis would perhaps point to the importance of methods and methodology while economics and technologies could remind us to strive for a social/technical balance in the curriculum

DESIGN AND BUILD

The way the curriculum categorisation has been defined, and the interface which is provided to that data has influenced our design.

The most recent development of this curriculum categorisation arranges the subject under the following four headings:

• Web History and Methodology • Web Technologies • Web Analysis

• Web Society:

Business, Economics, Social Engagement, Personal Engagement, Philosophy, Law, Politics and Governance:

Technically these heading and their subheadings have been realised in a SKOS binding and have been presented as a wiki 7 which is still open for discussion. The categorisation enables existing courses to be mapped against the categorisation. The 7 http://webscience.org/2010/wssc.html categorisation is formally published [START_REF] Vafopoulos | Web Science Subject Categorization (WSSC) Web Science Trust[END_REF]. There is an abridged version, Table 1 at the end of this paper.

As explained previously, the WSCCR repository will be built on EdShare. The EdShare system [START_REF] Davis | Bootstrapping a Culture of Sharing to Facilitate Open Educational Resources[END_REF] allows registered users to deposit their every day teaching materials, and then exposes these materials to search engines so that they can be located, downloaded and re-used by others. When the items are deposited the provider is encouraged to supply a description of the purpose of the materials, the language of instruction and annotation using the Curriculum Tags (free form folksonomy annotation is also encouraged).

Figure 3 WSCCR augments an existing courses page

Once the materials have been deposited in EdShare they will not only be found by Google search, or by searching EdShare specifically, but also dynamic links to the materials can be placed, for example, in third party websites or Virtual Learning Environments (VLEs). By way of example, Figure 3 shows the Web Science Trust's existing courses page supplemented by links to any material that has been deposited by teachers on that course.

Figure 4 shows the existing web science categorisation page dynamically supplemented by some course material items that have been deposited by teachers. Item annotated enables them to be automatically associated with this part of the curriculum. As the number of items in the web science repository grows it will be possible to judge the extent to which particular areas of the curriculum are most well represented across the range of known courses which are offered and the extent to which there are gaps. However, such analyses need to be treated with care -the EdShare team have already observed the extent to which teachers in some disciplines may be much less receptive to the idea of openly sharing their teaching resources than teachers in computer science, for example. We return to this consideration when discussing community in the section below.

Through WSCCR it should be possible to identify materials which do not seem to easily fit into the existing web science curriculum schema, as we can expect these to have only high-level annotation against the schema, but then use mostly folksonomic annotation.

Web Science Curriculum at a micro level

The WSDC project will lay the foundation stones for WSCCR by creating a coherent core of resources relating to a real and orchestrated set of overlapping courses in web science. In contributing to the repository, annotating and tagging the contents the partners will be formalising their understanding of the individual components of the 'modules', 'units' or 'lectures'/'seminar series' which collected together contribute to an entire programme of study/course. The open nature of the repository lends itself to sharing and peer-review of resources. The project will formalise peer review amongst its partners. Further opportunity for co-construction is afforded by linking the repository to a semantic wiki which provides an customisable platform which supports annotation and discussion beyond the constraints of the basic repository platform discussed above.

Engaging with these activities will assist each partner in identifying a complete instantiation or interpretation of the curriculum which covers the sets of topics and educational objectives appropriate to their individual educational context. Partners are keen to be able to share use and re-use educational material in ways that complement their available in house skillsets. Equally when faced with the task of developing assessment/evaluation activities such as individual or group courseworks there are considerable advantages (in terms of workload and enhanced quality) which can be derived from sharing templates and associated reference resources. Equally items can help in discovery and scoping tasks.

The partners all come from universities which recognise agreed standards for comparability of educational activities. So there is an additional advantage in this collaborative content creation and sharing which comes from the opportunity to calibrate the emerging units of instruction in terms of the European Credit Transfer and Accumulation System (known as ECTS-credits). This can therefore support mobility for masters' students enabling them to take different parts of their master's degree at different universities (although this is also dependent upon formal agreements which must be put in place). A motivation for the collaboration is the sharing of teaching and teaching resources.

Students can benefit if they are able to experience instruction from a wide range of academic perspectives whether face to face, virtually or in a blended form of interaction. Creating the repository is helping the partners identify their own expertise, and the repository as an artefact will serve such a purpose for the wider community.

The socio-technical process

Detail of the planning socio-technical aspects of the project may be of particular interest to those who wish to undertake their own curriculum design activity or who wish to make contributions to WSCCR themselves. The functional work of the project has been organised into seven areas or work packages as shown in Figure 5.

The diagram shows the task of project management and coordination outside of the network of interlinked activities which are more specifically curriculum related.

The task of course design generates activities which relate to teaching quality (how to measure, evaluate and thus assure the quality of any given educational process -wp4 quality assurance) and to educational objectives (labelled in the diagram as a map of competencies: wp3). Sets of educational objectives may be gathered together to become all or part of a course or lecture series. Decisions and insights which arise when designing such activities and processes at a micro level, will feed back into the overall course design (shown by the arrows in the diagram).

Figure 5. WSCD Methodology

Each individual partner, academic or institution, has their own set of expertise (competencies) for which they are able to design and instruct particular topic areas. By representing this expertise as a map of competencies the project partners will be able to negotiate how they might share activities, or how they might work to overcome some perceived gap or shortcoming. Of course, in terms of educational resources the partners can also make use of resources deposited by colleagues outwith the consortium, as well as resources which are 'in the wild'. Similarly because the partners aspire to support their students in studying at different institutions across the consortium, the partner implementation is being formally defined. This is a process which will be facilitated by the repository rather than actually being represented within the repository (wp5).

The process of publishing the repository is an essential aspect of enabling dissemination of the contents. As has been previously stated, the repository is designed to be semantically rich and open, active dissemination of the artefacts and the project activity is shown as wp6. Ways in which the repository might be further enriched is discussed later in the paper.

Through the socio-technical processes, each partner is therefore able to design their own curriculum, but it will be possible to represent the total curriculum as a whole incorporating each of the individual instantiations.

SUSTAINING COMMUNITY

A major challenge for the WSCCR repository is to accrete significant and useful content and to grow in a sustained manner.

Incorporating prior experience

The Southampton partners in the WSCD consortium have particularly relevant experience of building and using repositories [START_REF] Davis | Bootstrapping a Culture of Sharing to Facilitate Open Educational Resources[END_REF][START_REF] Hitchcock | Digital Preservation Service Provider Models for Institutional Repositories: Towards Distributed Services[END_REF][START_REF] Mcsweeney | The Mechanisms and Impact of Encouraging Community Engagement in Teaching Repositories Open Repositories[END_REF][START_REF] Millard | The Language Box: Re-Imagining Teaching and Learning Repositories International Conference on Advanced Learning Technologies[END_REF][START_REF] Millard | Phantom Tasks and Invisible Rubric: The Challenges of Remixing Learning Objects in the Wild[END_REF][START_REF] White | Making It Rich and Personal: Crafting an Institutional Personal Learning Environment[END_REF]. Experience gained from these activities is discussed below. This work was initially focussed on establishing research archives, and subsequently to support and enhance the educational process. Because of the nature of teaching at Masters level, and the fast moving development of the web science literature, both aspects of this experience are equally useful and relevant. The implementation of the academic and research repositories at Southampton together with the development of associated environments have progressed through a number of different contexts following a series of clear stages:

•

Starting from a research perspective faculty in Electronics and Computer Science were among the first few communities to systematically archive their academic publications;

•

The practice for archiving research outputs was then implemented across the entire university creating experience of working with a variety of academic disciplines; • Subsequently researchers in Electronics and Computer Science implemented an in-house teaching repository for university-wide use; • A number of subject specific repositories were built and have been used across subject communities throughout the UK.

•

A data driven learning environment has been developed in Electronics and Computer Science which integrates local administrative information with tagged data taken directly from the educational repository;

•
This approach is now being developed as a university-wide data driven learning environment is being developed which will integrate administrative data, tagged data from the educational repository and a variety of other items, some of which are found 'in the wild'.

Accounts and analyses of the activities outlined above have been published documenting the emerging expertise and understandings. Early accounts looked at institutional aspects [START_REF] Hitchcock | Digital Preservation Service Provider Models for Institutional Repositories: Towards Distributed Services[END_REF], those which followed examined EdShare use in Southampton [START_REF] Davis | Bootstrapping a Culture of Sharing to Facilitate Open Educational Resources[END_REF][START_REF] White | Making It Rich and Personal: Crafting an Institutional Personal Learning Environment[END_REF]. Using EdShare as a platform for subject specific implementations providing additional experiences for comparison [START_REF] Mcsweeney | The Mechanisms and Impact of Encouraging Community Engagement in Teaching Repositories Open Repositories[END_REF][START_REF] Millard | The Language Box: Re-Imagining Teaching and Learning Repositories International Conference on Advanced Learning Technologies[END_REF][START_REF] Millard | Phantom Tasks and Invisible Rubric: The Challenges of Remixing Learning Objects in the Wild[END_REF]. What emerges is a clear underlying perspective which identifies the importance of recognising and using the strong socio-technical affordances associated with repositories. It is noticed that such affordances enhance the value of the repositories to their user communities. It is clear from the accounts that this understanding has been influential in shaping successive development approaches. The lessons learnt from these experiences are being consciously incorporated into the activity of establishing the community repository for web science. It has become clear that the core tasks of repository building, use, and development are necessarily socio-technical endeavours which exhibit strong inter-relationships between the functionality of the repositories and the cultural processes of the communities in which they are used. Some early studies of educational repositories in the area of computer and information science point to problems which may be encountered in establishing educational repositories. They suggest for example a number of barriers which need to be overcome such as issues associated with trust, authority and findability [START_REF] Mitchell | Assessing the Value of Computer Science Course Material Repositories[END_REF]. Since that time, social software use has grown and a common understanding of online communities has become very clearly established. We see that routine use of metadata has increased, systems are purposefully engineered to expose data. The more recent experience of the Greenroom repository, like the experiences in Southampton with Language Box and HumBox [START_REF] Mcsweeney | The Mechanisms and Impact of Encouraging Community Engagement in Teaching Repositories Open Repositories[END_REF][START_REF] Millard | The Language Box: Re-Imagining Teaching and Learning Repositories International Conference on Advanced Learning Technologies[END_REF] demonstrate how carefully engineered environments which incorporate highly focussed discipline based endeavours produce active repositories which cultivate a strong sense of loyalty and community identity. It has been observed at Southampton that there is some variability in participation with the teaching repository according to home academic discipline. More generally, some discipline communities (for example chemistry and physics) have strong reputations for community cohesion and use of shared repositories. As Xia evidences, it is not necessarily true that repository deposit practice will vary across disciplines, if there are strong drivers to make use of the repository [START_REF] Xia | Assessment of Self-Archiving in Institutional Repositories: Across Disciplines[END_REF]. We suggest that the web science community, although diverse, is still coherent. The continued existence and activities of the web science curriculum workshops provide evidence that there are strong drivers and commitment to create a community repository. For this reason we believe the community is well placed to grow and sustain an active repository with high levels of functionality. We suggest that the creativity of the web science community, and the alignment between the structure of the repository and the key paradigm of 'the two magics' will play a significant part in this future.

Interdisciplinary community

In the case of this repository, the will to share resources is strong, and the fundamental inter-disciplinarity and trans-disciplinarity of web science means that the support process is well aligned with the philosophical underpinnings of the emerging discipline.

In the case of research it is argued that "the repository also represents a new kind of scholarly activity, the curation and management of an institution's intellectual output on behalf of its own scholars for the benefit of the whole scholarly community". [START_REF] Carr | Could the Web Be a Temporary Glitch?[END_REF]. In the case of web science, the way in which institutions educate and develop the researchers and thought leaders of tomorrow has a similar role representing intellectual output to the whole scholarly community. The web science curriculum repository has additional importance because of the fact that web science is emerging, the fact that there are many different flavours of web science, and the fact, as is argued by Halford et al [START_REF] Halford | WebSci10: Extending the Frontiers of Society On-Line[END_REF] , that web science could be enriched by making use of pivotal concepts from social sciences issues associated with heterogeneity in epistemologies and their associated research methods.

Ensuring Web science is world wide too

We have initially taken a two-part approach to the structure of our repository. A semantic wiki functions as an external component which we are using in conjunction with a supporting repository built using the ePrints engine. Thus WSCCR provides the space to deposit and tag the resources, while the semantic wiki provides the interface which automatically serves the items and which provides an intellectually and linguistically rich context from which the repository items can be viewed.

One of the strengths of the dual approach of using a repository and semantic wiki is that, from the very start we are accommodating the need for plurilingualism: "The ability to use languages for the purposes of communication and to take part in intercultural interaction...." [START_REF]Council of Europe Common European Framework of Reference for Languages[END_REF] p168. We do well to remember, that the web is world wide. There are many more languages besides English in which web science is being manifested. Not only are the effects of the web international but also the phenomena which we call web science, the technical, engineering and social components of the web, are being analysed in many different linguistic and intellectual traditions.

Our Masters level degrees are intended to educate the thought leaders and decision makers of tomorrow. In the same way that the conception of web science is inherently interdisciplinary, so the evidence will necessarily be pluriingual. Since the learners and the resources will be drawn from many different linguistic traditions, it is naturally that infrastructure around learning resource is plurilingual. The value of plurilingualism is not only an issue for the European Union, it is also recognised by the United Nations and UNESCO. These bodies have all, in various ways, pointed to the particular strengths of plurilingualism in terms of its role of ensuring that participants in debates from different linguistic and intellectual traditions are enabled to take and equal part in the on-going discourse. These values and objectives are well aligned with the already observed manifestation and needs of web science as a fundamentally interdisciplinary field of study [START_REF] Berners-Lee | A Framework for Web Science[END_REF][START_REF] Halford | WebSci10: Extending the Frontiers of Society On-Line[END_REF][START_REF] Hendler | Web Science: An Interdisciplinary Approach to Understanding the Web[END_REF][START_REF] Shadbolt | Web Science Emerges[END_REF]

CONCLUSIONS AND DISCUSSION

Teaching web science should ideally equip the researchers and thought leaders or tomorrow in every part of the globe. For example recent experiences in the Arab world have attracted much debate, and there is already a web science initiative underway to develop research from a perspective close to the ground [START_REF] Bazan | Web Science in the Context of the Arab near East WebSci10: Extending the Frontiers of Society On-Line[END_REF]. We must increasingly turn to effective analysis from local cultural and societal perspectives. If Web Science is about understanding the impact of the Web on society, then teaching Web Science should take in consideration contextual learning. The Web is by definition a global phenomenon, its technical dimension is unique, unified, but its reality is the large sum of micro-experiments, which confirm each other in an infinity of places and produce an infinity of forms and connections.

The technical and social reality of the Web varies according to the viewpoint of the observer. The reality of the Web is complex; the local actors of the Web create new original practices, the fruits of multiculturalism, multilingualism and the search for an identity to be built in a globalized social space in full transformation. This reality has too often been observed from far away, with for effect the construction of a prescriptive discourse: But teaching Web Science adds a new means to succeed in defining not only an interdisciplinary methodological frame of observation, but also a new epistemological contextual positioning, which might finally fulfil Anderson's "deficit of differentiation" [START_REF] Anderson | Des CommunautéS Virtuelles ? Vers Une Théorie « Techno-Pratique » D'internet Dans Le[END_REF].

The Web Science Curriculum Development Project aims to make real many long term ambitions harboured by the web science teaching community. Our partners are all intent on establishing their own particular flavours of web science in a rich and enlarging landscape. What they will actually be like is difficult to predict. The number of existing courses specifically tagging themselves as web science is still too small to deduce any significant conclusions or a trend. However, we hope that this paper sets a base line for later analysis and examining emerging change.

We hope this paper will stimulate discussion and prompt the community to elicit further information on the nature and extent of web science teaching internationally. We urge readers to contribute to our survey and actively engage in the community. An improvement we would suggest in conducting any further surveys is to be much more explicit in the difference between surveying individual modules and whole degree courses.

We hope too, that the infrastructure we will provide through WSCCR will enable some of that data to emerge and thereby feed a discourse which is complementary to that taking place within the web science research community. We are not predicting how WSCCR will develop in the future but we can hope the Exploratorium will cultivate innovations such as automated semantic enrichment, spider driven candidacy alongside (particularly international) user generated content Considering that discourse may take people into unfamiliar and perhaps uncomfortable territory we might do well in reminding ourselves of Korzybski's often quoted observation "the map is not the territory" [START_REF] Korzybski | Science and Sanity: An Introduction to Non-Aristotelian Systems and General Semantics[END_REF], at the same time we may help deepen our various understandings of what we mean by Web Science and how we should go about teaching it if we were too redouble our efforts map the territory in as many different ways as possible.

Figure 4

 4 Figure 4 Dynamically supplementing the categorisation.

http://wiki.websciencetrust.org/

http://www.citidel.org/

https://www.isurvey.soton.ac.uk/2290

ACKNOWLEDGMENTS

Our thanks are due to all those past participants in the web science curriculum workshops who have built and sustained the initiative to define the web science curriculum. In this case we want to give special acknowledgement to one of our authors Michalis Vafopoulos who with assistance from his fellow contributors (Ioannis Antoniou and Anastasia Dimou, Aristotle University of Thessaloniki, George Metakides, University of Patras, Leslie Carr, University of Southampton, Jacek Kopecky, The Open University and Kieron O'Hara, Web Science Trust, UK) developed the SKOS curriculum currently published by the Web Science Trust as The Web Science Subject Categorisation


Siolian Liewang

Rita Ball

David M Winder

Katie Vaughan

Nashat Hanna

Jonathan Andre Michel

Salomon Levy

Jane Sterling

Margaret A Stanley

Peter K C Goon

Siolian L R Ball

Jonathan Levy

Analyses of human papillomavirus genotypes

Keywords: HPV, Genital warts, E6 DNA viral loads, E6 mRNA HPV. Genital warts. E6 DNA viral loads. E6 mRNA. 17

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction 1

Infection with Human papillomaviruses (HPVs) results in a major global disease burden. In 2 particular, HPVs infecting mucosal tissue cause very significant morbidity and mortality. Those 3 HPVs fall into 2 groups; the so-called 'low risk' strains that cause warts and the 'high risk' 4 (oncogenic) strains (particularly HPV 16 and 18) that are the cause of ano-genital and oro-5 pharangeal cancers [START_REF] Winder | Sensitive HPV detection in oropharyngeal cancers[END_REF]zur Hausen, 2002]. 6 In the uterine cervix, multiple HPV infections are frequent [START_REF] Schmitt | Abundance of Multiple 32 High-Risk Human Papillomavirus (HPV) Infections found in cervical cells analyzed by use of 33 an ultrasensitive HPV genotyping assay[END_REF]. Studies using 7 highly sensitive PCR based methods for HPV strain detection in ano-genital lesions indicate that 8 multiple types (including both high and low risk strains) can also be detected in Condylomata. In 9 such circumstances it is difficult to assign causality to a specific HPV strain, although over 90% 10 of these lesions are positive for HPVs 6 and 11 DNA [START_REF] Brown | Detection of multiple human 5 papillomavirus types in condylomata acuminata lesions from otherwise healthy and 6 immunosuppressed patients[END_REF]; [START_REF] Aubin | Human papillomavirus genotype distribution in external 3 acuminata condylomata: A large French national study (EDiTH IV)[END_REF]; 11 [START_REF] Chan | Distribution of human 11 papillomavirus types in anogenital warts of men[END_REF] and there has been a significant decline in incident wart disease since the 12 introduction of a quadrivalent L1 VLP vaccine (Gardasil™) raised against HPVs 6 and 11 together [START_REF] Potocnik | Distribution of human papillomavirus (HPV) 14 genotypes in gentital warts from males in Slovenia[END_REF] with the oncogenic HPVs 16 and 18 in 2007 ( [START_REF] Fairley | Rapid decline in 24 presentations for genital warts after the implementation of a national quadrivalent human 25 papillomavirus vaccination program for young women[END_REF]; [START_REF] Munoz | Impact of human papillomavirus 5 (HPV)-6/11/16/18 vaccine on all HPV-associated genital diseases in young women[END_REF]). 14 Clarification of the clinical relevance of the multiple HPV strains found in a wart sample from an [START_REF] Coleman | Histone modifications 16 associated with herpes simplex virus type 1 genomes during quiescence and following ICP0-17 mediated de-repression[END_REF] individual patient is important for understanding which HPV strain is causal and these data will [START_REF] Qian | Expression profiling of CD34+ 17 hematopoietic stem/ progenitor cells reveals distinct subtypes of therapy-related acute 18 myeloid leukemia[END_REF] be informative in any cases of vaccine failure. 17 In this study, the HPV strain prevalence, the frequency of more than 1 type in condylomata and 18 the relevance of viral DNA load to early gene expression have been determined in order to in HPV16 could be multiplexed with the additional GAPDH internal control but all other HPV 2 amplification reactions were performed separately in parallel with control reactions. Standard 3 curves were constructed using purified placental human DNA (Sigma-Aldrich, Gillingham, UK) 4 and purified plasmids containing the HPV of interest. Serial dilutions of 1 in 5 were constructed 5 (containing 100ng to 6.4pg human DNA and 10 5 to 6.4 copies of HPV per reaction). The 6 specificity of primers was determined under identical conditions with genomic HPV DNA (HPVs 7 1a, 2a, 3, 5, 6, 7, 8, 10,11,14,[START_REF] Qian | Expression profiling of CD34+ 17 hematopoietic stem/ progenitor cells reveals distinct subtypes of therapy-related acute 18 myeloid leukemia[END_REF]17,[START_REF] De Boer | High 19 Human papillomavirus oncogene mRNA expression and not viral DNA load is associated with 20 poor prognosis in cervical cancer patients[END_REF]20,[START_REF] Schmitt | Abundance of Multiple 32 High-Risk Human Papillomavirus (HPV) Infections found in cervical cells analyzed by use of 33 an ultrasensitive HPV genotyping assay[END_REF][START_REF] Munoz | Impact of human papillomavirus 5 (HPV)-6/11/16/18 vaccine on all HPV-associated genital diseases in young women[END_REF]50 or 57) at a concentration of 10 6 8 copies/µL. No significant non-specific reactions were observed. Viral load was expressed as 9 number of virus particles/cell assuming 1ng DNA ~150 cells [START_REF] Leyva | Measurement of DNA in cultured human cells[END_REF]. Viral loads The second half of the bisected tissue sample was shredded at the time of excision and stored

as per qPCR with supplementary internal control primers (Supplementary table S II). 2

To enable multiplexing of mRNA internal controls, primer-pairs and probes were examined for 3 any potential primer-dimer pairs using 4 Autodimer™(http://www.cstl.nist.gov/div831/strbase/AutoDimerHomepage/AutoDimerProgra 5 mHomepage.htm). RT-qPCR was performed for all HPVs as a single-plex assay using primers and 6 probe as listed in Table I.

1

DNA from all 31 genital wart samples was positive for β-globin and HPV using the Linear Array 2 assay. All the warts harboured at least one low-risk HPV (Table I), and, as expected, HPV 6 and 3 11 dominated and were present in 97%, with HPV 6 in 28 (90%) and HPV 11 in 10 lesions (32%). In 9 lesions (29%), only HPV 6 was found. The frequency distribution of HPV strains in this 5 cohort of tissue is featured in figure 1. 6 7 22 of the warts (71%) were multiply infected (Table II). In 7 (22%), more than one low-risk HPV 8 was detected and in 15 (48%) a mixture of low-and high-risk types were found. 11 (35%) of the 9 samples contained HPV16 or 18 or both. Only one sample (sample 25) was not infected by HPV 10 6 or 11, but was infected by other low risk types 40, 84 and CP6108 in addition to the oncogenic 11 strains 16, 18, 33 and possibly 52. Other low and high risk strains detected are shown in Table II. 12 13 Viral load was assayed using 10ng of sample DNA as standardization experiments had shown 14 that the Taqman™ qPCR was unbalanced with 100ng or more DNA. A threshold level for copy 15 numbers of specific HPV E6/E7 DNA was assumed to be 10 copies/cell indicating the HPV in 16 question to have been replicated and so likely to be involved in the pathogenesis of the clinical 17 lesion [START_REF] Doorbar | Molecular biology of human papillomavirus infection and cervical cancer[END_REF]. In addition, the sample DNA was obtained from tissue containing both 18 epithelium and stroma, so the concentration of HPV in infected keratinocytes is likely to be 19 higher than the values obtained for the full biopsy. Of the 28 warts positive for HPV 6 in Linear Array, 18 had a copy number of HPV 6 above 1 threshold value. 5 warts, in which there was co-detection of HPV 11, had a copy number of HPV 2 11 exceeding 10 copies/cell (Table II). 8/31 samples (26%) did not have viral loads above 3 threshold value of either HPV 6 or 11. In 3 of these, however, HPV 16 was detected at a level 4 above 10 copies/cell and was also positive in the Linear Array assay. In 4 of these 8 samples, 5 other low risk HPV strains, which were not tested by qPCR, had been detected in the Linear 6 Array assay and so potentially could have been causal in these lesions. CP6108 mRNAs meant that their contribution to the pathogenesis of this lesion could not be 14 discounted and it would be incautious to conclude that HPV 16 caused this lesion. 15 16 Inter-assay concordance values were derived using Cohen's κ algorithm (Table III) [Viera and 17 [START_REF] Viera | Understanding interobserver agreement:The Kappa statistic[END_REF]. For all comparisons agreement was considered better than 'moderate' (κ>0.41), 18 except between Linear Array results and relative mRNA levels for HPV 6 which was determined 19 to be 'fair'. These data suggest that the presence of HPV 6 L1 DNA as determined by the Linear 20 Array assay does not predict E6 genomic expression as measured by the RT-qPCR. It was no surprise to find HPV 6 was the most frequently detected in these lesions with HPV 11 14 as the second most prevalent type. This is in accordance with previous data for other studies 15 using the Linear Array assay in genital wart swabs [START_REF] Aubin | Human papillomavirus genotype distribution in external 3 acuminata condylomata: A large French national study (EDiTH IV)[END_REF][START_REF] Chan | Distribution of human 11 papillomavirus types in anogenital warts of men[END_REF]] and 16 tissue [START_REF] Potocnik | Distribution of human papillomavirus (HPV) 14 genotypes in gentital warts from males in Slovenia[END_REF]. The finding of high risk HPVs in 35% of samples in this 17 immunocompetent group is in keeping with other reports of high risk types detected in 14-44% 18 of non-immunosuppressed individuals [START_REF] Brown | Detection of multiple human 5 papillomavirus types in condylomata acuminata lesions from otherwise healthy and 6 immunosuppressed patients[END_REF][START_REF] Potocnik | Distribution of human papillomavirus (HPV) 14 genotypes in gentital warts from males in Slovenia[END_REF] but below that 19 found in patient groups with a higher HIV positive prevalence where high risk HPVs may be 20 present in 47-100% of genital warts [START_REF] Brown | Detection of multiple human 5 papillomavirus types in condylomata acuminata lesions from otherwise healthy and 6 immunosuppressed patients[END_REF] The inter-assay agreement levels between the Linear Array assay and qPCR was good and 8 validated the use of either assay for screening. reported case [START_REF] Chrisofos | HPV 16/18-associated condyloma 13 acuminatum of the urinary bladder: first international report and review of literature[END_REF] based on DNA analysis alone, suggests that it may. The 14 DNA analysis shows 3 samples in which HPV 16 DNA was detected by the Linear Array assay and 15 also at a significant level by qPCR, suggesting that this oncogenic type is amplified within these 16 lesions, but this can only be confirmed by quantitation of the mRNA which was not performed 17 in this study. analysis is required to discount the contribution of HPV 6 and/or 11 when DNA loads are low. In 1 order to confirm these findings, a prospective study including larger numbers of patients, rather 2 than an unselected collection of warts should be performed. It is likely that analysis of HPV 3 mRNA would provide data informative for HPV vaccines and will be especially useful in the 4 event of vaccine failure. Supplementary table I.

1

  Clinical specimens and sample preparation. 2 The study was approved by the local Research Ethics Committee and all patients provided 3 written informed consent for their tissues to be used for research. A total of 31 4 immunocompetent male and female patients attending the Department of GU/HIV Medicine, 5 St Mary's Hospital, London, UK, were recruited to the study. Genital wart samples were 6 obtained under local anaesthesia and the tissue bisected and snap frozen at the time of biopsy 7 for DNA and RNA extraction. 8 DNA extraction and HPV detection.

9 12 Measurement of viral load 13 For

 1213 DNA was extracted with DNeasy™ blood and tissue kit (Qiagen, Crawley, UK), following the 10 manufacturer's instructions. HPV types in 62.5ng of sample DNA were identified using the 11 Roche Linear Array (Roche Diagnostics Ltd, Burgess Hill, UK)[START_REF] Woo | The use of HPV Linear Array Assay for 49 multiple HPV typing on archival frozen tissue and DNA specimens[END_REF]. Taqman TM qPCR, primers and probes were synthesised by Sigma Genosys, Gillingham, UK. 14 The primers and probes for human GAPDH [Coleman et al., 2008], HPV6, 11 and 18 E6/7 DNA 15 and β-globin (A) [Tucker et al., 2001] and for HPV16 E6/7 DNA, and human β-Globin (B) [de 16 Boer et al., 2007] have been previously described (Supplementary table S I). Amplification was 17 performed in 20µL using 10ng sample DNA and Hotstart Taq (Qiagen, Crawley, UK) with cycling 18 conditions as follows: initial Taq activation and template denaturation at 95°C for 15 min, 19 followed by 45 cycles of 15s at 95°C and 60s at 60°C, with acquisition of fluorescent signal at 60 20 seconds. Amplification was performed on a Rotor-Gene 3000 (Corbett Life Science, Qiagen, 21 Crawley, UK) and data analysed using the Rotor-Gene software, v6.1. All optimisation 22 in triplicate at least 3 times. It was determined that HPV18 and 1

10 < 0

 100 .01 copies/cell were deemed as insignificant. 11 mRNA extraction, purification and cDNA synthesis.

12

 12

13 at-

 13 80°C in RNAlater™ (Qiagen, Crawley, UK). The tissue was later resuspended in 200µL Trizol™ 14 (Invitrogen, Paisley, UK) in a lysing matrix D tube (MP Biomedicals, Solon OH, USA) and 15 pulverised using a Bulletblender™ machine (Next Advance, Averill Park, NY,USA). RNA was 16 precipitated with isopropranol and resuspended in 50µL H 2 O. After DNAse I digestion, RNA was 17 recovered by column purification using PureLink™ RNA extraction kit (Invitrogen, Paisley, UK) 18 following manufacturer's instructions and a maximum of 5µg reverse transcribed using 19Bioscript™ (Bioline, London, UK) after pre-incubation with random hexamer primers for 5min at 20 65°C. Reactions were performed as follows: 25°C for 10min, 42°C for 60min and 70°C for 15min.[START_REF] Doorbar | Molecular biology of human papillomavirus infection and cervical cancer[END_REF] No-RT controls were performed without the addition of enzyme. The resulting cDNAs were 22 10 with pure water before qRT-PCR analysis. RT-qPCR for HPV 6 and 11 E6/E7 was 1 performed

7

  Measurement of viral RNA load8 Values of HPV mRNA copy number were determined by standard curves of genomic HPVs (as 9 described for DNA viral load). Relative cellular mRNA was determined by normalising CT values 10 of TBP (TATA-Box binding protein), YWHAZ (tyrosine 3-mono-oxygenase/tryptophan 5-mono-11 oxygenase activation protein, zeta polypeptide) and HMBS (hydroxymethylbilane synthase) 12 triplex RT-qPCR to calibrator sample (sample 9). Primers and probes sequences were first 13 reported for TBP, YHWAZ and HMBS by [Radonic et al., 2005], [Ohl et al., 2006] and [Qian et al., 14 2002] respectively. 15 Data analysis and statistics 16 The results from the Linear Array assay were compared to those obtained by qPCR. Unweighted 17 Kappa statistics were calculated by online software 18 (http://www.graphpad.com/quickcalcs/kappa1.cfm). Kappa values determined were ranked as 19 moderate, substantial or near perfect as described by Viera et al. [Viera and Garrett, 2005].

4

 4

13 14 21 Page

 1321 11 E6/E7 transcripts were detected and quantified in 23 samples. No tissue was 9 available for RNA extraction in 3 cases. In all samples where HPV mRNA was quantified at a 10 measurable level, the same HPV type had been found by the Linear Array assay. Only sample 8 11 had very low HPV6/11 DNA and mRNA levels, suggesting that one or more of the other low risk 12 HPVs present (55, 62, or 84) could have been causal in these lesions. These data show that relatively small DNA viral loads are capable of producing high mRNA 15 levels as, for example, sample 1 had only 2 copies of HPV 6 per cell, but generated a relative 16 amount of 1163 copies of HPV 6 mRNA per reaction. The converse is also true, as the sample 17 with the largest HPV 6 DNA viral load (namely sample 15 with 870 copies/cell), did not yield the 18 largest E6/7 mRNA load. Five multiply infected samples (namely 1, 4, 8, 17 and 22) had low HPV 19 6 DNA viral loads (<10 copies/cell) suggesting that another HPV was responsible for the lesion.20However, analysis of relative mRNA levels revealed measurable HPV 6 E6 transcription in 4 4, 17 and 22) thus HPV 6 could still be causing these lesions. As HPV 6 E6 mRNA was 1 not measurable in sample 8, other low risk HPVs present (55, 62 or 84) may have been causal.

  22 and 25) had copies of HPV 16 DNA viral loads above 10 copies/cell, raising the 4 possibility that these lesions could be caused by HPV 16. For sample 3, HPV 11 and 16 were 5 detected by the Linear Array assay, subsequent qPCR and RT-qPCR revealed HPV 11 E6 DNA and 6 E6 mRNA levels to be negligible whilst HPV 16 DNA was present at 177 copies/cell. These data 7 suggest HPV 16 is causing the lesion, although in the absence of HPV 16 mRNA measurement it 8 can only be speculated. In sample 22 (positive for HPV 6 and 16 DNA) HPV 6 mRNA was 9 abundant even though the HPV 6 DNA load was <10 copies/cell.

10 11

 10 Sample 25, however was negative for HPV 6 and 11 on the Linear Array assay, but positive for 12 HPV 40, 84, CP, 16, 18, 33 and 52. The absence of measurement of the low risk HPVs 40, 84 and 13

21

 21

  and RNA analysis of HPVs in genital warts attempted to identify not just the 2 HPV strains present in or on the lesion, but also to gather evidence of the HPV strains that could 3 be causing the lesions and producing infective virions. To our knowledge, there are no previous 4 published data for this type of analysis in condylomata acuminata. Most studies of HPV types in 5 genital warts have used surface swabs and PCR amplification with sequencing or hybridization 6 methods to identify multiple infections [Chan et al., 2009; Greer et al., 1995; Giuliano et al., 7 2008; Sanclemente et al., 2007]. These sensitive methods can also detect HPV DNAs in the 8 anogenital region in asymptomatic individuals [Nielson et al., 2009]. The potential risk from 9 analysis of surface samples is that the HPVs detected may reflect carriage but not necessarily 10 permissive viral growth. The analysis of lesion tissue should minimize false positive results due 11 to other non-lesion associated HPVs within this body area.

  ; Müller et al., 2010; Schlecht et al., immunocompetent patients show that 71% excised wart samples contained dual 2 or multiple HPV types, agreeing with previous estimates [Brown et al., 1999; Aubin et al., 2008, 3 Chan et al., 2009]. The Linear Array assay has the advantage of simultaneously detecting the 4 presence of 37 genital HPV types [Woo et al., 2007] and is considered the most sensitive test of 5 its kind [van Ham et al., 2005; van Hamont et al., 2006]. The advantage of qPCR is the ability to 6 estimate viral load, allowing comparison of specific HPV subtypes in multiply infected samples.

7

 7

9 10 The

 10 identification of HPV strains other than 6 or 11 causing genital warts will be important for 11 future vaccination strategies, although the reduction of anogenital warts since the introduction 12 of the quadrivalent vaccine, Gardasil™ [Fairley et al., 2009; Munoz et al., 2010], supports 13 confirmation presented here of the role of HPV 6 or 11 in causing lesions, even in multiply 14 infected samples or when their DNA loads are low. One concern is that if lesions caused by HPV 15 6 and 11 are eradicated, other HPVs may become more prevalent and therefore more 16 important for identification, and subsequent confirmation of which HPV is causal. Confirmation 17 and quantitation of other HPV mRNAs would be desirable, but as yet there is no equivalent of 18 the Linear Array assay for HPV mRNA analysis and this study was limited to the primer and 19 probe sets in the laboratory. The PreTect™ HPV Proofer and Aptima™ systems are designed for 20 the detection of high-risk HPV mRNA only [Halfon et al., 2010; Molden et al., 2007; Keegan et 21 al., 2009] and so would be unsuitable for Condylomata.

  22

  E6/E7 transcript in this study was based on their known roles in the viral 2 infectious cycle[START_REF] Doorbar | Molecular biology of human papillomavirus infection and cervical cancer[END_REF]. Studies investigating the DNA and mRNA loads of oncogenic 3 HPVs in cervical intraepithelial neoplasia (CIN) have concluded that E6 RNA load is a better 4 predictor of lesion severity [de Boer et al., 2007; Cattani et al., 2009; Ho et al., 2010] and 5 progression levels [de Boer et al., 2007], and although malignant progression was not likely in 6 these lesions, E6 RNA abundance probably reflects viral contribution to lesion pathogenesis. As 7 demonstrated in this report, DNA loads of low-risk subtypes, HPV 6 and 11, do not correlate 8 with their respective mRNA E6 transcript levels and is similar to that observed in HPV 16-9 associated CIN [de Boer et al., 2007; Cattani et al., 2009]. Therefore, viral genome copy number 10 does not directly reflect viral activity. 11 12 HPV16 is not thought to produce Condyloma-like lesions [zur Hausen, 2002] although one 13

18 19ThesePage

 18 data provide evidence for multiple subtype infection as a common event in the natural 20 history of Condylomata acuminata, but with significant replicative activity of usually HPV 6 or 21 11. DNA viral loads alone are insufficient to truly determine which HPV is causal, and mRNA 22

7

  Primers and probe sets used in qPCR assay of viral load. 8 Primer/probe sets used for the detection of HPV and Human DNA in DNA samples 9 generated from wart tissue. Sequences are represented 5'-3' and the 5' fluorescent labels 10 indicated (6FAM -6-carboxyfluorescin, TET -tetrachloro-6-carboxyfluorescein or Cy5 -11 cyanine). Tetramethylrhodamine (TAMRA), Black Hole Quencher 1 (BHQ1) or Black Hole 12 Quencher 3 (BHQ3) was incorporated at the 3' end of each probe.

76

  Figure 1

7

  All occurrences of HPV were scored, regardless of being single or multiple infections.

  This work was supported by grants from the British Skin Foundation and Cancer Research UK to 8 PKCG. PKCG and MAS act as consultants to Sanofi Pasteur-MSD, Lyon, France and are in receipt 9 of an unrestricted educational grant. MAS also acts as consultant to Merck Research 10 Laboratories, Westpoint, USA, and GSK Biologicals, Rixensart, Belgium.

	Page 13 of 22			Journal of Medical Virology
	1	Figures
	2
	3	Supplementary table S I
	4
							Optimised
		Target	Accession Number	Forward Primer	Reverse Primer	Probe	primer concentration
							(nM)
						[6FAM]-
	5	HPV6	AF092932	GTTCATAAAGCTAAATTG TACGTGGAA	TGTGAATCTTGTCCGTC CACTT	[TAMRA] ACAATATCCTTTAGGGTAAC ATGTCTTCCATGCATG -	75
						[6FAM]-
	6	HPV11	M14119	GCTTCATAAAACTAAATA ACCAGTGGAA	TGCGTCTTGTTTGTCCA CCTT	CTATATCCTTTAGGGTAACA AGTCTTCCATGCATGTTG-	75
						[TAMRA]
	7	Disclosure of Conflicts of Interest F o r F o r F o r HPV16 K02718 CCGGACAGAGCCCATTAC AAT F HPV18 NC_001357 CAACCGAGCACGACAGG AA o r β-globin (A) NG_000007 CAGGTACGGCTGTCATCA CTTAGA	ACGTGTGCTTTGTACG CAC CTCGTCGGGCTGGTAA ATGTT CATGGTGTCTGTTTGA GGTTGCTA	[6FAM]-TGTTGCAAGTGTGACTCTAC GCTTCGGT-[BHQ1] [6FAM]-AATATTAAGTATGCATGGA CCTAAGGCAACATTGCAA-[BHQ1] [TET]-GCCCTGACTTTTATGCCCAG CCCTG-[BHQ1]	100 100
	11			P P P P e e e e
				e r e r e r e r
					R R R R
					e e e e
					v i e v i e v i e v i e
						w w w w
				John Wiley & Sons

John Wiley & SonsJournal of Medical Virology

Supplementary table S II

Primer/probe sets used for the detection of internal control housekeeping transcripts in cDNA 7 samples. Sequences are represented 5'-3' and the 5' fluorescent labels indicated (6FAM -6-8 carboxyfluorescin, TET -tetrachloro-6-carboxyfluorescein or Cy5 -cyanine). Tetramethylrhodamine 9 (TAMRA), Black Hole Quencher 1 (BHQ1) or Black Hole Quencher 2 (BHQ2) was incorporated at the 10 3' end of each probe. Amplicon size is in brackets (number of base pairs). 11 12 13 Table I Samples were assayed for the appearance and DNA and RNA viral load of HPV as described in the methods 6 section. (-) denotes not significantly positive during assay (less than 1 copy/cell or 5 copies/reaction total RNA). 7 CP represents CP6108. 52? denotes positivity at the 52/33/35/58 band on the Linear Array, but also for 8 individual 33 and 58 markers, but where the presence of 52 cannot be discounted as described in the 9 manufacturer's instructions. NA denotes no tissue available for RNA analysis. 10


PharmD, PhD Sidonie Lambert-Niclot

Catherine Poirot

Roland Tubiana

Allal Houssaini

Cathia Soulié

Stephanie Dominguez

Benoit Schubert

Marie Prades

Manuela Bonmarchand

Vincent Calvez

Tubiana

Effect of antiretrovirals on semen quality

Keywords: Antiretrovirals drugs, semen quality, penetration score Antiretrovirals drugs, semen quality, penetration score 46

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The aim of this cross-sectional study was to determine which antiretroviral drugs (ARVs) are 31 associated with changes in the characteristics of semen and the impact of these ARVs 32 according to their score penetration into the male genital compartment. Data from 144 men 33 infected with HIV-1 enrolled in an Assisted Reproductive Technology program were 34 analyzed retrospectively. A seminal penetration score of ARV was based on the available 35 literature. The nonparametric Kruskal-Wallis test and Khi-2 test were used. There was no 36 difference on sperm parameters between NRTI, NNRTI or PI regimen. In patients receiving 37 NRTIs or PIs no differences were observed between antiretrovirals of these classes. However 38 in patients receiving NNRTIs, nevirapine (n=22) was associated with a higher percentage of 39 progressively motile spermatozoa (p<0.0001) versus efavirenz (n=38) as well as vitality 40 (p=0.0004). No relationship was observed between semen quality and the penetration score. 41 NRTIs and PIs were not associated with any semen changes. Nevirapine was associated with 42 a better quality of semen versus éfavirenz. It would be of interest to validate, improve and test 43 our penetration score in a prospective study. 44

Introduction 55

Potent combination antiretroviral therapy has increased the life expectancy and quality of life 56 of patients infected with HIV-1. Currently, some couples request medical assistance to 57 achieve conception while reducing to a minimum the risk of HIV-1 transmission. This 58 technical requirement for Assisted Reproductive Technology program raises the question of 59 semen characteristics in men infected with HIV-1 under antiretroviral treatment. Several 60 studies have assessed semen quality in men infected with HIV-1 [START_REF] Bujan | 221 Decreased semen volume and spermatozoa motility in HIV-1-infected patients under 222 antiretroviral treatment[END_REF]Dulioust 61 et al., 2002;[START_REF] Nicopoullos | The effect of human 245 immunodeficiency virus on sperm parameters and the outcome of intrauterine 246 insemination following sperm washing[END_REF]. However the use of antiretroviral drugs (ARVs) did not 62 appear to be correlated with any sperm characteristics while most of the patients were 63 receiving antiretroviral therapy. A practical method was developed for quantifying the 64 penetration in genital tract of the many different ARV drug regimens currently prescribed, as 65 estimated by the penetration score. This method uses publicly available information about 66 ARV drug characteristics, measured semen concentrations, and effectiveness in the genital 67 tract to rank drugs relative to one another. Antiretroviral drugs showed different penetration 68 score in the male genital tract and may therefore influence differently spermatogenesis, or 69 semen quality which is a key factor for reproductive success. 70

The aim of this cross-sectional cohort study was undertaken to determine which ARVs were 71 associated with changes in semen characteristics and the impact of these ARVs according to 72 their penetration score into the male genital compartment. 73 September 2007 were studied retrospectively. HIV-1 RNA quantitation was determined by 78 using the Amplicor Monitor assay (Cobas 1.5; Roche Diagnostics, Basel, Switzerland), which 79 has a detection limit of 200 copies/ml in seminal plasma. 80

All semen samples were tested in the Unit of Reproductive Biology according to standardized 81 methods throughout the study period and according to the World Health Organization (WHO) 82 guidelines [WHO, 1992]. 83

Semen samples were collected in the laboratory by masturbation into a sterile graduated 84 container after 3-5 days of sexual abstinence. After 30 minutes at 35°C, each sample was 85 examined. The volume of the seminal fluid, semen pH and viscosity were determined. 86

Motility was graded (a) rapid progressive motility; (b) slow progressive motility, (c) non-87 progressive motility and (d) no motility according the WHO guidelines. The percentage of 88 motile spermatozoa was determined at T0 (30-45 min after semen collection) and at T1 (4-5 89 hours after semen collection). Sperm and round cell concentrations were determined using a 90 Kova Slide (HYCOR Biomedical, Garden Grove, California, USA). Sperm vitality was 91 assessed after staining with Eosin-Nigrosin. The multiple abnormalities index represents the 92 ratio of the number of sperm abnormalities, over the number of abnormal spermatozoa. 93

Teratospermia is the opposite of the percentage of morphologically normal spermatozoa. 94

The present study was carried out in accordance with the declaration of Helsinki and was 95 approved by ANRS AC11 Resistance Study Group scientific committee. All patients gave 96 written informed consent. 97 Penetration of antiretroviral drugs was characterized using a hierarchical approach based on 98 the best available evidence. Data on chemical and pharmacokinetic characteristics were 99 6 reviewed for antiretroviral drugs approved by national agencies using package inserts, drug 100 references, published papers, and international conference abstracts [START_REF] Pereira | The pharmacokinetics of amprenavir, zidovudine, 249 and lamivudine in the genital tracts of men infected with human immunodeficiency 250 virus type 1 (AIDS clinical trials group study 850)[END_REF]Solas 101 et al., 2003;[START_REF] Taylor | Antiretroviral drug concentrations in semen of HIV-infected men: differential 260 penetration of indinavir, ritonavir and saquinavir[END_REF]. Using this approach, antiretroviral drugs were classified into 102 three categories. Individual antiretroviral drugs were assigned a rank based on penetration 103 category (0=lowest, 0.5=intermediate and 1=highest penetration) (table2). The final rank was 104 then determined by adding the individual penetration scores for each ARV drug in a regimen. 105 Continuous variables were compared using the Kruskal-Wallis test while categorical variables 106 were compared using Khi-2 test. All analyses used a 2-sided α of 0.05. Statistical analyses 107 were performed with SAS release 9.1 for Windows XP Pro. 108 The main characteristics of the study population are described in Table 1. At the time of 110 semen collection, 124 men (86%) were receiving ARVs (dual therapy: 3 men; triple therapy: 111 109 men; ≥4 drugs: 12 men). The median number of drugs used was 2 nucleosides reverse 112 transcriptase inhibitors NRTIS (range, 0-4), 1 protease inhibitor PI (range, 0-1) and 0 non 113 nucleoside reverse transcriptase inhibitors NNRTI (range, 0-1). 114

Semen characteristics are shown in Table 1. The characteristics of seminal fluid ranged from 115 0.13-8.50 mL (mean 3.5) for volume and 7.4-9.0 (mean 8.0) for pH. Majority of ejaculates 116 83.9% (115/137) had a normal viscosity. The concentration of spermatozoa ranged from 2.61-117 676 million per millilitre (mean 116). The concentration of round cells ranged from 0-36 118 10 6 /mL (mean 2.79). The total sperm count was ranged from 1.3-2000 10 6 per ejaculate 119 (mean 385.3). The mean percentage of rapid progressively motile spermatozoa was 16.6 120 (range 0-60) and the mean percentage of slow progressively motile spermatozoa was 27.3 121 (range 5-75). The mean percentage of vitality was 78.6 (range 35-98). The mean of multiple 122 abnormality index was 1.70 (range 1.17-2.86). The percentage of abnormal sperm 123 morphology was ranged from 30 to 99 (mean 69). Twenty four % of patient (35 out of 144) 124 had all their sperm characteristics strictly normal according to WHO standards. 125 Studying the different variables of the quality of semen, no relationship between semen 126 quality and the penetration score was found. There was no difference in the characteristics of 127 sperm between patients receiving NRTI, NNRTI or PI regimen. In patients receiving NRTIs 128 or PIs no differences were observed between the antiretroviral drugs of these classes. No 129 association was shown between thymidine analogues (D4T or AZT) or other NRTIs and 130 semen characteristics. However, considering only the NNRTI containing regimen, it was 131 shown that nevirapine was associated with a higher percentage of both progressively motile 132 spermatozoa (at T0 (immediately after liquefaction) and at T1 (4 hours after liquefaction)) and 133 In the present study, the characteristics of sperm were the same as those found in other studies 144 [START_REF] Bujan | 221 Decreased semen volume and spermatozoa motility in HIV-1-infected patients under 222 antiretroviral treatment[END_REF][START_REF] Dulioust | Semen alterations in HIV-1 infected men[END_REF][START_REF] Nicopoullos | The effect of human 245 immunodeficiency virus on sperm parameters and the outcome of intrauterine 246 insemination following sperm washing[END_REF]van Leeuwen et al., 2008b] 145 except for sperm concentration which was higher. The same trend was observed for the total 146 sperm count. Several studies have assessed semen quality in men infected with HIV-1 in 147 comparison to HIV-1 seronegative men [START_REF] Bujan | 221 Decreased semen volume and spermatozoa motility in HIV-1-infected patients under 222 antiretroviral treatment[END_REF][START_REF] Dulioust | Semen alterations in HIV-1 infected men[END_REF]Nicopoullos 148 et al., 2004]. The study by [START_REF] Bujan | 221 Decreased semen volume and spermatozoa motility in HIV-1-infected patients under 222 antiretroviral treatment[END_REF] demonstrated decreases in the semen volume, 149 spermatozoa motility, and total motile sperm count and increases in the pH values and 150 multiple anomaly indices of patients infected with HIV. In the study by [START_REF] Dulioust | Semen alterations in HIV-1 infected men[END_REF] 151 the most significant semen alterations were a decrease of the rapidly progressive motile 152 spermatozoa (motility a) and an increase of less rapidly progressive spermatozoa (motility b) 153 the patients infected with HIV. In the study by [START_REF] Nicopoullos | The effect of human 245 immunodeficiency virus on sperm parameters and the outcome of intrauterine 246 insemination following sperm washing[END_REF], ejaculate volume, 154 sperm concentration, total count, progressive motility and normal morphology were 155 decreased. However, none of these studies related these semen alterations to HIV infection or 156 ARV therapy. Another study explored the characteristics of semen before and after the start of 157

ARV with different results and demonstrated a statistically significant reduction in the 158 percentage of progressively motile spermatozoa in patients with HIV-1 infection during 159 treatment with ARV therapy [van Leeuwen et al., 2008b]. They demonstrated previously that 160 there was no detectable change in semen quality in patients during a period of untreated 161 asymptomatic HIV-1 infection with similar proportion of progressively motile spermatozoa 162 [van Leeuwen et al., 2008a]. These observations suggest that the reduction in progressively 163 motile spermatozoa, which occurred in the current study during treatment, was related to the 164 use of ARV and not to HIV infection. In these studies, it is not clear if some semen alterations 165 could be the result of antiretroviral treatment or the result of HIV infection. Indeed various 166 studies have demonstrated a relationship between mitochondria and sperm motility [Donnelly 167 , 2000;[START_REF] Chretien | 242 Increased sperm mitochondrial DNA content in male infertility[END_REF]. Since several ARVs have mitochondrial toxicity, the 168 observed changes in motility could be due to the ARV itself. However no association was 169 found in the current study between quality of semen and the thymidine analogue containing 170 treatment. 171 However it was found that nevirapine was associated with a higher percentage of 172 progressively motile spermatozoa at T0 and at T1 and a better vitality in comparaison with 173 efavirenz. The proportion of progressively motile spermatozoa under nevirapine or PI were 174 normal according to WHO standards whereas in the efavirenz group this percentage was 175 decreased. Concerning vitality, nevirapine, PI or efavirenz remained according to WHO 176 standards although the efavirenz group presented vitality significantly inferior to nevirapine 177 or the PIs groups. The percentage of progressively motile spermatozoa and vitality are related 178 measures of semen quality. Nevirapine has a better penetration score than efavirenz and is a 179 priori more efficient in this compartment, a better control of the virus may explain a better 180 quality of semen. Another explanation could be a direct toxicity of efavirenz on cells 181 producing spermatozoa. Indeed there is no study on sperm characteristics before and after 182 infection. Despite no relationship with the penetration score it would be interesting to improve 183 this score and to try to validate it in a prospective study. 184

In conclusion, NRTIs and PIs were not associated with any semen changes. However, in 185 patients receiving NNRTIs, nevirapine was associated with a better quality of semen versus 186 efavirenz. This study confirms the fact that semen alterations are frequent in patients infected 187 with HIV and suggests that these alterations could be related to ARV treatment. 188 [START_REF] Dumond | First Dose and Steady-state Genital Tract Pharmacokinetics of 232 Ten Antiretroviral Drugs in HIV-infected Women: Implications for Pre-and Post-233 Exposure Prophylaxis[END_REF]Kashuba et 214 al., 1999;[START_REF] Pereira | The pharmacokinetics of amprenavir, zidovudine, 249 and lamivudine in the genital tracts of men infected with human immunodeficiency 250 virus type 1 (AIDS clinical trials group study 850)[END_REF][START_REF] Reddy | Roundtable report: importance of 252 antiretroviral drug concentrations in sanctuary sites and viral reservoirs[END_REF] ; 2: [START_REF] Dumond | First Dose and Steady-state Genital Tract Pharmacokinetics of 232 Ten Antiretroviral Drugs in HIV-infected Women: Implications for Pre-and Post-233 Exposure Prophylaxis[END_REF]Ghosn et al., 215 2004;[START_REF] Reddy | Roundtable report: importance of 252 antiretroviral drug concentrations in sanctuary sites and viral reservoirs[END_REF]; 3: [START_REF] Dumond | First Dose and Steady-state Genital Tract Pharmacokinetics of 232 Ten Antiretroviral Drugs in HIV-infected Women: Implications for Pre-and Post-233 Exposure Prophylaxis[END_REF][START_REF] Ghosn | Penetration of enfuvirtide, tenofovir, efavirenz, and 237 protease inhibitors in the genital tract of HIV-1-infected men[END_REF][START_REF] Kashuba | Antiretroviral-239 drug concentrations in semen: implications for sexual transmission of human 240 immunodeficiency virus type 1[END_REF]216 Pereira et al., 2002;[START_REF] Reddy | Roundtable report: importance of 252 antiretroviral drug concentrations in sanctuary sites and viral reservoirs[END_REF][START_REF] Solas | 255 Discrepancies between protease inhibitor concentrations and viral load in reservoirs 256 and sanctuary sites in human immunodeficiency virus-infected patients[END_REF][START_REF] Taylor | Antiretroviral drug concentrations in semen of HIV-infected men: differential 260 penetration of indinavir, ritonavir and saquinavir[END_REF]van Leeuwen et 217 al., 2007;[START_REF] Van Praag | Enhanced 272 penetration of indinavir in cerebrospinal fluid and semen after the addition of low-273 dose ritonavir[END_REF]; 4: [START_REF] Ghosn | Penetration of enfuvirtide, tenofovir, efavirenz, and 237 protease inhibitors in the genital tract of HIV-1-infected men[END_REF]

  et Marie Curie University, UMR943, INSERM U943, AP-HP Pitié 10 Salpêtrière Hospital, Virology Laboratory, Paris, France. 11 2 UPMC Pierre et Marie Curie University, ER9 UPMC, AP-HP Pitié Salpêtrière Hospital, 12 Biology of Reproduction Unit, Paris, France. 13 3 INSERM U943, AP-HP Pitié Salpêtrière Hospital, Infectious Diseases Department, Paris, 14 France. 15 4 INSERM U943, AP-HP Pitié Salpêtrière Hospital, Paris, France 16 5 AP-HP, Pitié Salpêtrière Hospital, Biology of Reproduction Unit, Paris, France. 17

  6 AP-HP, Pitié Salpêtrière Hospital, Internal Medecine department Paris, France. 18 7 EA 449, Denis Diderot University and AP-HP, Bichat Claude-Bernard Hospital, Clinical 19

  forty four men infected with HIV-1 attending the Pitié-Salpêtrière Hospital 76 in the multidisciplinary assisted reproductive technology program between January 2002 and 77

  . Regarding the mean percentage of progressively motile spermatozoa 134 both atT0 and T1, nevirapine (22.05 and 15.24) and PI (17.31 and 12.71) provided similar 135 values while efavirenz (7.92 and 3.81) led to smaller values. The same trend was observed in 136 the percentage of vitality with a mean of 85.3 for nevirapine, 72.2 for efavirenz and 80.1 for 137 PI. The difference observed between nevirapine and efavirenz does not seem to be related to 138 the NRTI associated treatment. Indeed thymidine analogues were distributed equally between 139 nevirapine and efavirenz groups. The known characteristics of patients were similar between

  to thank G. Le Mallier and P. Grange for their technical assistance. 191 192 Funding section 193 This work was supported by ANRS (Agence Nationale de Recherche sur le SIDA et les 194 Hépatites Virales) and ARVD (Association de Recherche en Virologie and Dermatologie). 195 The research leading to these results has received funding from the European Community's 196 Seventh Framework Programme (FP7/2007-2013) under the project 'Collaborative HIV and 197 Anti-HIV Drug Resistance Network (CHAIN)' -grant agreement n° 223131. 198 199 Ethics 200 The present study was carried out in accordance with the declaration of Helsinki and was 201 approved by the ANRS AC11 Resistance Study Group scientific committee. All patients gave

Table 1

 1 Characteristics of patients and semen. 208

	Variable of patients	n	Mean (SD)
	Age	144	39.00 (5.75)
	Viral load, mean log 10 copies/mL CD4 + cell count, means cells/mm 3	103 93	2.48 (0.89) 497.00 (211.71)
	Semen characteristics
	Ejaculate volume	143	3.50 (1.70)
	pH	144	8.00 (0.30)
	Viscosity	137
	Low (%)	7 (5.10)
	Normal (%)	115 (83.90)
	High (%)	15 (10.90)
	Concentration of spermatozoa (cell per 10 6 /mL)	144	116.00 (109.74)
	Round cells (10 6 /mL)	144	2.79 (4.67)
	Total sperm count (10 6 per ejaculate)	144	385.30 (371.10)
	Rapid progressively motile spermatozoa at T0 (%) 144	16.60 (12.80)
	Slow progressively motile spermatozoa at T0 (%)	144	27.30 (12.80)
	Progressively motile spermatozoa at T1 (%)	135	11.40 (9.70)
	Slowly motile spermatozoa at T1 (%)	135	22.30 (10.00)
	Vitality (%)	143	78.60 (14.11)
	Multiple abnormality index	143	1.70 (0.29)
	Teratospermia (%)	144	69.00 (15.00)
	209

Table 2

 2 Penetration score of antiretroviral drugs. 210

			Penetration score of antiretroviral drugs
			1 (>50%)	0.5 (10-50%)	1 (<10%)
		NRTI 1	Tenofovir
			Zidovudine
			Abacavir
			Emtricitabine
			Lamivudine
			Didanosine
		NNRTI 2	Nevirapine		Efavirenz
					Etravirine
		PI 3	Indinavir/r	Amprenavir/r	Nelfinavir
				Atazanavir/r	Ritonavir
				Darunavir/r	Lopinavir/r
					Saquinavir/r
					Tipranavir/r
		FI 4			Enfuvirtide
		CCR5 inhibitor	Maraviroc
		INSTI	Raltegravir
	211
	212	NRTI: nucleoside reverse transcriptase inhibitor; NNRTI: non nucleoside reverse
	213	transcriptase inhibitor; PI: protease inhibitor; FI: fusion inhibitor; CCR5: chemokine
		coreceptor 5 ; INSTI: integrase strand transfer inhibitor. 1:

Journal of Medical Virology