Lise Frézal

Raphael Leblois
email: leblois@mnhn.fr

4 years of DNA barcoding : current advances and prospects

Keywords: DNA barcode, cytochrome c oxidase, COI, DNA taxonomy, species identification, international species databank, BOLD

L. Frézal and R

1-Introduction

Species identification and classification have traditionally been the specialist domain of taxonomists, providing a nomenclatural backbone and a key prerequisite for numerous biological studies. Indeed, today's society has to resolve many crucial biological issues, among which are the need to maintain biodiversity, to ensure bio-security, to protect species and to avoid pandemics. The achievement of such goals and the success of subsequent action programs require efficient global networks and rely on our capacity to identify any described species. As [START_REF] Dayrat | Towards integrative taxonomy[END_REF] clearly expressed, 'delineating species boundaries correctly -and also identifying species -are crucial to the discovery of life's diversity because it determines whether different individual organisms are members of the same entity or not'. The identification of species depends on the knowledge held by taxonomists whose work cannot cover all taxon identification requested by non specialists. To deal with these difficulties, the 'DNA Barcode of Life' project aims to develop a standardized, rapid and inexpensive species identification method accessible to non-specialists (i.e. non-taxonomists).

The idea of a standardized molecular identification system emerged progressively during the 1990s with the development of PCR-based approaches for species identification. Molecular identification has largely been applied to bacterial studies, microbial biodiversity surveys (e.g. [START_REF] Woese | Phylogenetic trees: whither microbiology?[END_REF][START_REF] Zhou | Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA[END_REF] and routine pathogenic strains diagnoses (e.g. Maiden et al., 1998[START_REF] Sugita | Identification of trichosporon asahii by PCR based on sequences of the internal transcribed spacer regions[END_REF][START_REF] Wirth | Sex and virulence in Escherichia coli: an evolutionary perspective[END_REF] due to a need for culture-independent identification systems.

PCR-based methods have also been frequently used in fields related to taxonomy, food and forensic molecular identification (Teletchea et al., 2005) and for identification of eukaryotic pathogens and vectors (e.g. Walton et al., 1999). Several universal systems for molecular-based identification have been used for lower taxa (e.g. nematodes, [START_REF] Floyd | Molecular barcodes for soil nematode identification[END_REF] but were not successfully implemented for broader scopes. The Barcode of Life project soon after became that attempt, aiming to create a universal system for a eukaryotic species inventory based on a standard molecular approach. It was initiated in 2003 by researchers at the University of Guelph in Ontario, Canada (http://www.barcoding.si.edu) and promoted in 2004 by the international initiative 'Consortium for the Barcode of Life' (CBOL). By then, it had more than 150 member organizations from 45 countries including natural history museums, zoos, herbaria, botanical gardens, university departments as well as private companies and governmental organizations. The DNA barcode project does not have the ambition to build the tree of life nor to perform molecular taxonomy (Erbach and Holdrege, 2005;[START_REF] Gregory | DNA barcoding does not compete with taxonomy[END_REF], but rather to produce a simple diagnostic tool based on strong taxonomic knowledge that is collated in the DNA barcode reference library [START_REF] Schindel | DNA barcoding a useful tool for taxonomists[END_REF]. The DNA Barcode of Life Data System (BOLD, http://www.boldsystems.org) has progressively been developed since 2004 and was officially established in 2007 [START_REF] Ratnasingham | BOLD: The Barcode of Life Data System (www.barcodinglife.org)[END_REF]. This data system enables the acquisition, storage, analysis and publication of DNA barcode records.

In the present paper we briefly review the current state of DNA barcode advances, trends and pitfalls. The main methods of the DNA barcoding approach are given. The feasibility of a universal barcoding approach and interest in the DNA barcoding approach for microbial studies are discussed.

2-The DNA Barcoding approach: definitions and objectives

2-1 DNA barcode definition and primary objectives

The DNA barcode project was initially conceived as a standard system for fast and accurate identification of animal species. Its scope is now that of all eukaryotic species [START_REF] Hebert | Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species[END_REF][START_REF] Miller | DNA barcoding and the renaissance of taxonomy[END_REF]. The DNA barcode itself consists of a 648 bp region 58-705 from the 5'-end of the cytochrome c oxidase 1 (COI) gene using the mouse mitochondrial genome as a reference. It is based on the postulate that every species will most likely have a unique DNA barcode (indeed there are 4 650 possible ATGC-combinations compared to an estimated 10 million species remaining to be discovered, [START_REF] Wilson | The encyclopedia of life[END_REF] and that genetic variation between species exceeds variation within species [START_REF] Hebert | Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species[END_REF]Hebert, 2004a). The two main ambitions of DNA barcoding are to (i) assign unknown specimens to species and (ii) enhance the discovery of new species and facilitate identification, particularly in cryptic, microscopic and other organisms with complex or inaccessible morphology [START_REF] Hebert | Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species[END_REF].

2-2 When is the DNA barcode useful?

Access to a public reference database of taxa allowing identification of a wide range of species will be beneficial whenever accurate taxonomic identifications are required. The DNA barcode can in this way be of great support to numerous scientific domains (e.g. ecology, biomedicine, epidemiology, evolutionary biology, biogeography and conservation biology) and in bio-industry.

The cost and time-effectiveness of DNA barcoding enables automated species identification, which is particularly useful in large sampling campaigns (e.g. Craig Venter's Global Ocean sampling team, [START_REF] Rusch | The Sorcerer II Global Ocean Sampling expedition: Northwest Atlantic through eastern tropical Pacific[END_REF]. In this way, DNA barcoding could also improve large surveys aiming at unknown species detection and identification of pathogenic species with medical, ecological and agronomical significance [START_REF] Armstrong | DNA barcodes for biosecurity: invasive species identification[END_REF][START_REF] Ball | DNA barcodes for insect pest identification: a test case with tussock moths (Lepidoptera: Lymantriidae)[END_REF]. Besides, it is important to be able to recognize, detect and trace dispersal of patented organisms in agrobiotechnology, either to certify the source organism (e.g. truffles, [START_REF] Rastogi | Species identification and authentication of tissues of animal origin using mitochondrial and nuclear markers[END_REF] or secure intellectual property rights for bioresources [START_REF] Gressel | Universal inheritable barcodes for identifying organisms[END_REF][START_REF] Kress | A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnHpsbA spacer region[END_REF][START_REF] Taberlet | Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding[END_REF].

One obvious advantage of DNA barcoding comes from the rapid acquisition of molecular data.

As a contrast, morphological data gathering can be time consuming, in some cases totally confusing and in others, almost impossible (e.g. Dinoflagellate taxonomy, [START_REF] Litaker | Recognizing dinoflagellate species using ITS rDNA sequences[END_REF]diatomea, Evans et al., 2007;earthworms, Huang et al., 2007). Furthermore, in three important situations, relevant species identification must necessarily be molecular-based. First, in determining the taxonomic identity of damaged organisms or fragments of (e.g. goods, food and stomach extracts). The DNA barcoding tool is thus potentially useful in the food industry, diet analyses, forensic sciences and in preventing illegal trade and poaching of endangered species (e.g. fisheries, trees, bushmeat). Second, molecular-based identification is necessary when there are no obvious means to match adults with immature specimens (e.g. fish larvae, [START_REF] Pegg | MtDNA barcode identification of fish larvae in the southern Great Barrier Reef, Australia[END_REF]amphibians, Randrianiaina et al., 2007;coleoptera, Caterino and Tishechkin, 2006;[START_REF] Ahrens | DNA-based taxonomy for associating adults and larvae in multi-species assemblages of chafers (Coleoptera : Scarabaeidae)[END_REF]; fungal sexual stage, [START_REF] Shenoy | Impact of DNA sequence-data on the taxonomy of anamorphic fungi[END_REF]. The third case is when morphological traits do not clearly discriminate species (e.g. red algal species, [START_REF] Saunders | Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications[END_REF]fungal species, Jaklitsch et al., 2006;and field-collected mosquito specimens, Kumar et al., 2007), especially when size precludes visual identification (i.e. 'unseeable animals', [START_REF] Blaxter | Defining operational taxonomic units using DNA barcode data[END_REF][START_REF] Webb | DNA barcoding: A molecular tool to identify Antarctic marine larvae[END_REF] or if species have polymorphic life cycles and/or exhibit pronounced phenotypic plasticity (e.g. Lamilariales, [START_REF] Lane | A molecular assessment of northeast Pacific Alaria species (Laminariales, Phaeophyceae) with reference to the utility of DNA barcoding[END_REF].

2-3 DNA Barcoding as a driving force in biological sciences

More than being a species identification tool for non specialists, DNA barcoding is also of interest to specialists. To achieve the CBOL objectives, species have to be taxonomically described before their deposit in BOLD, which leads researchers to resolve analytical, technical and fundamental issues beforehand. It also brings together (and complements) taxonomy, molecular phylogenetics and population genetics (Hajibabaei et al., 2007b). According to [START_REF] Rubinoff | Between two extremes: mitochondrial DNA is neither the panacea nor the nemesis of phylogenetic and taxonomic inference[END_REF], DNA barcoding can be regarded as a 'tremendous tool' to accelerate species discovery and initiate new species descriptions [START_REF] Desalle | The unholy trinity: taxonomy, species delimitation and DNA barcoding[END_REF][START_REF] Desalle | Species discovery versus species identification in DNA barcoding efforts: Response to Rubinoff[END_REF]. Moreover, it re-opens the debate on species concepts [START_REF] Fitzhugh | DNA Barcoding: An Instance of Technology-driven Science?[END_REF]Rubinoff et al., 2006b;[START_REF] Balakrishnan | Species concepts, species boundaries and species identification: a view from the tropics[END_REF][START_REF] Miller | DNA barcoding and the renaissance of taxonomy[END_REF][START_REF] Vogler | Recent advances in DNA taxonomy[END_REF]. Unlike other well-known sequence libraries (e.g. NCBI) , B O L D i s a n interactive interface where deposited sequences can be revised and taxonomically reassigned. The compiling of sequences, from one or few common loci improves synergic studies at large geographic scales and across numerous genera (Hajibabaei et al., 2007b).

Such information on the global distribution of species, their genetic diversity and structure will enhance the speed and effectiveness of local population studies.

3-Advances in Barcoding

3-1 State of the art

By March 2008, the total available DNA Barcode records were at 363,584 sequences (50,039 species), of which 136,338 sequences (13,761 species) satisfied DNA barcoding criteria (i.e. minimum sequence length of 500 bp and more than 3 individuals per species). At this date, more than 65% of all barcoded specimens had been collected in the last five years. The majority of the specimens (over 98%) are from the animal kingdom with more than 65% representing Insecta. The International Barcode of Life project (iBOL) is now under development by the new Canadian International Consortium Initiative (ICI). Researchers from 25 countries will be involved in this large-scale and collaborative program, which aims at building a comprehensive DNA barcode registry for eukaryotic life. The program's starting date is tentatively set at January 2009 and within the first 5-year period there are plans to acquire DNA barcode records for 5 million specimens representing 500,000 species (out of more than an estimated 10 million species to be discovered).

So far, the COI gene has proved to be suitable for the identification of a large range of animal taxa, including gastropods [START_REF] Remigio | Testing the utility of partial COI sequences for phylogenetic estimates of Gastropod relationships[END_REF], springtails [START_REF] Hogg | Biological identification of springtails (Hexapoda : Collembola) from the Canadian Arctic, using mitochondrial DNA barcodes[END_REF], butterflies (Hebert et al., 2004a;Hajibabaei et al., 2006a), birds (Hebert et al., 2004b;[START_REF] Kerr | Comprehensive DNA barcode coverage of North American birds[END_REF], mayflies [START_REF] Ball | Biological identifications of mayflies (Ephemeroptera) using DNA barcodes[END_REF], spiders [START_REF] Greenstone | Barcoding generalist predators by polymerase chain reaction: carabids and spiders[END_REF], fish [START_REF] Ward | DNA barcoding Australia's fish species[END_REF], ants [START_REF] Smith | DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar[END_REF], Crustacea [START_REF] Costa | Biological identifications through DNA barcodes: the case of the Crustacea[END_REF] and recently, diatomea and Protista [START_REF] Evans | An assessment of potential diatom "barcode" genes (cox1, rbcL, 18S and ITS rDNA) and their effectiveness in determining relationships in Sellaphora (Bacillariophyta)[END_REF]. Hajibabaei et al. (2006a) showed that 97.9% of 521 described species of Lepidoptera possess distinct DNA barcodes and furthermore that the few instances of sequence overlap of different species involve very similar ones.

The efficiency of DNA barcoding has been reported in the detection and description of new cryptic species (Hansfield and Hansfield, 2006;Smith et al., 2006b;[START_REF] Anker | Revision of the Alpheus nuttingi (Schmitt) species complex (Crustacea: Decapoda: Alpheidae), with description of a new species from the tropical eastern Pacific[END_REF][START_REF] Bucklin | DNA barcodes for species identification of euphausiids (Euphausiacea, Crustacea)[END_REF][START_REF] Gomez | Mating trials validate the use of DNA barcoding to reveal cryptic speciation of a marine bryozoan taxon[END_REF][START_REF] Pfenninger | Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species[END_REF][START_REF] Tavares | Single mitochondrial gene barcodes reliably identify sisterspecies in diverse clades of birds[END_REF] and of sibling species [START_REF] Hogg | Biological identification of springtails (Hexapoda : Collembola) from the Canadian Arctic, using mitochondrial DNA barcodes[END_REF][START_REF] Amaral | A first approach to the usefulness of cytochrome c oxidase I barcodes in the identification of closely related delphinid cetacean species[END_REF][START_REF] Van Velzen | DNA barcoding reveals hidden species diversity in Cymothoe (Nymphalidae)[END_REF]. This identification tool can clearly give support to improve classifications and to critically examine the precision of morphological traits commonly used in taxonomy. Indeed, several studies have already illustrated the advances provided by the iterative processes between morphological-and DNA barcode-based studies in taxonomy (Hebert et al., 2004a;Hebert and Gregory, 2005b;[START_REF] Page | The taxonomic feedback loop: symbiosis of morphology and molecules[END_REF][START_REF] Carlini | A molecular systematic evaluation of the squid genus Illex (Cephalopoda: Ommastrephidae) in the North Atlantic Ocean and Mediterranean Sea[END_REF]Smith et al., 2006a;[START_REF] Smith | DNA barcodes affirm that 16 species of apparently generalist tropical parasitoid flies (Diptera, Tachinidae) are not all generalists[END_REF][START_REF] Van Velzen | DNA barcoding reveals hidden species diversity in Cymothoe (Nymphalidae)[END_REF].

3-2 New insights into ecology and species biology

New insights into ecology and species biology have already emerged from the DNA barcoding project. For example, the identification of organisms contained in stomach extracts allows the elucidation of wild animal diets, especially when behavioural studies are not feasible (e.g. Krill diets, [START_REF] Passmore | DNA as a Dietary Biomarker in Antarctic Krill, Euphausia superba[END_REF]; affirmation of polyphagy of the moth Homona mermerodes, [START_REF] Hulcr | DNA barcoding confirms polyphagy in a generalist moth, Homona mermerodes (Lepidoptera : Tortricidae)[END_REF]Xenoturbella bocki diet, Bourlat et al., 2008). DNA barcoding could also become an efficient tool to clarify host-parasite and symbiotic relationships [START_REF] Frézal | DNA barcoding of parasites and invertebrate disease vectors: what you don't know can hurt you[END_REF] and in turn give new insights on host spectra, as well as on the geographical distributions of species (host, parasites and/or endangered species). Moreover, the tool is suitable to elucidate the symbiont and parasite transmission pathways from one host generation to the next as illustrated in the interaction of beetles (Lecythidaceae) with their endosymbiotic yeasts (Candida spp. clades and other undescribed yeast species) [START_REF] Berkov | Yeasts isolated from neotropical woodboring beetles in SE Peru[END_REF]. Molecular dating of symbiotic relationships can also be deduced using barcoding tools [START_REF] Anker | Revision of the Alpheus nuttingi (Schmitt) species complex (Crustacea: Decapoda: Alpheidae), with description of a new species from the tropical eastern Pacific[END_REF].

3-3 Technical advances in barcoding

The purpose of the DNA barcoding project is to rapidly assemble a precise and representative reference library. Thus it is based on conventional and inexpensive protocols for DNA extraction, amplification and sequencing. With time, the reference library will become increasingly useful, enabling the rapid identification of low taxonomic level taxa with specific short-DNA sequences (i.e. mini-barcode 100bp, Hajibabaei et al., 2006c;300 bp, Min and Hickey, 2007.). It has been shown that species identity can be validated or inferred from a small number of polymorphic positions within the COI-barcode ('microcoding' of 25bp, [START_REF] Summerbell | Microcoding: the second step in DNA barcoding[END_REF]DNA arrays-based identification, Hajibabaei et al., 2007;SNP-based discrimination, Xiao et al., 2007). Other new molecular technologies used in bioengineering (e.g. silicon-based microarrays, nylon membranebased macroarrays, etc.) are becoming cheaper and may be integrated into the 'second step of DNA barcoding' [START_REF] Summerbell | Microcoding: the second step in DNA barcoding[END_REF]. Furthermore, new sequencing techniques such as pyrosequencing (454, Solexa, SOLID) enable rapid and representative analyses of mixed samples (e.g. stomach contents, food, blood or water columns. Largely used in the emerging field of metagenomics, this advance could be promising for future DNA barcoding initiatives.

DNA barcoding could also be used as a technical enhancer. Indeed, one condition for data submission to BOLD is the conservation of entire morphological reference for species (voucher).

Indeed, new techniques of non-destructive DNA extraction from recently collected specimens have already been developed [START_REF] Pook | Mitochondrial DNA sequences from dried snake venom: a DNA barcoding approach to the identification of venom samples[END_REF][START_REF] Frézal | Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens for DNA barcoding projects[END_REF][START_REF] Rowley | Vouchering specimens for documenting arthropod barcodes: a non-destructive method for DNA extraction[END_REF] and additional improvements in specimen conservation may arise. One major drawback of molecularbased studies as for example DNA barcoding is our incapacity to extract DNA from specimens conserved in formalin. Indeed, museum collections of animals represent the major part of voucher specimens from which species have been described and most of these are conserved in formalin. The ultimate challenge is to find the appropriate ways to extract DNA from formalin-conserved specimens and harvest DNA barcodes from them.

4-What can be learnt from the limitations of DNA barcoding?

Despite the promises of the global barcoding initiative, some crucial pitfalls must be mentioned. We believe that these limitations should be clearly identified and resolved in the library construction phase, otherwise the BOLD database will not ever become universally relevant.

4-1 The under-described part of biodiversity

The sampling shortage across taxa can sometimes lead to 'barcoding gaps' [START_REF] Meyer | DNA barcoding: Error rates based on comprehensive sampling[END_REF], which highlights the care that must be accorded to sampling quality during the database construction phase [START_REF] Wiemer | Does the DNA barcoding gap exist? -a case study in blue butterflies (Lepidoptera: Lycaenidae)[END_REF]. The individuals chosen to represent each taxon in the reference database should cover the major part of the existing diversity. Indeed, in the interrogation of BOLD, identification difficulties arise when the unknown specimens come from a currently under-described part of biodiversity (Rubinoff, 2006;[START_REF] Rubinoff | Are plant DNA barcodes a search for the Holy Grail?[END_REF]. [START_REF] Meyer | DNA barcoding: Error rates based on comprehensive sampling[END_REF] estimated the error rates for specimen assignment in well-characterized phylogenies and in partially known groups. They showed that the DNA barcode exclusively promises robust specimen assignment in clades for which the taxonomy is well understood and the representative specimens are thoroughly sampled. Their conclusions are totally concordant with the example of the Muntjac described in [START_REF] Desalle | The unholy trinity: taxonomy, species delimitation and DNA barcoding[END_REF].

4-2 Inherent risks due to mitochondrial inheritance

The diversity of mitochondrial DNA (mtDNA) is strongly linked to the female genetic structure due to maternal inheritance. The use of mitochondrial loci can thus lead to overestimate sample divergence and render conclusions on species status unclear. For instance, in Homona mermerodes (Lepidoptera) mtDNA polymorphism is structured according to the host plants on which females feed, and the two clades produced by phylogenetic analyses are artefacts of female nutritional choice [START_REF] Hulcr | DNA barcoding confirms polyphagy in a generalist moth, Homona mermerodes (Lepidoptera : Tortricidae)[END_REF]. Heteroplasmy and dual uniparental mitochondrial inheritance (e.g. Mussels, [START_REF] Terranova | A mitochondrial phylogeography of Brachidontes variabilis (Bivalvia: Mytilidae) reveals three cryptic species[END_REF] are further misleading processes for mitochondrion-based phylogenetic studies.

The mitochondrial inheritance within species can also be confounded by symbiont infection.

Firstly, indirect selection on mitochondrial DNA arises from linkage disequilibria with endosymbionts, either obligate beneficial micro-organisms, parasitically-or maternally-inherited symbionts [START_REF] Funk | Intraspecific phylogenetic congruence among multiple symbiont genomes[END_REF][START_REF] Whitworth | DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera : Calliphoridae)[END_REF]. Such symbionts are very common in arthropods (e.g. Wolbachia infects at least 20% of Insecta and 50% of spiders, [START_REF] Hurst | Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts[END_REF]; Cardinium infects around 7% of arthopods, [START_REF] Weeks | From parasite to mutualist: Rapid evolution of Wolbachia in natural populations of Drosophila[END_REF] and are probably widespread in many other Metazoa. Secondly, interspecific hybridization and endosymbiont infections can generate transfer of mitochondrial genes outside an individual's evolutionary group [START_REF] Dasmahapatra | DNA barcodes: recent successes and future prospects[END_REF]. Examples are the cross-generic mitochondrial DNA introgression observed between Acreae (Lepidoptera) and Drosophila (Diptera) coming from the vertically transmitted Wolbachia [START_REF] Hurst | Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts[END_REF], or the cross-kingdom horizontal mtgene transfer detected between sponges and their putative fungal symbionts [START_REF] Rot | Putative cross-kingdom horizontal gene transfer in sponge (Porifera) mitochondria[END_REF]. Finally, one host species can bear different symbionts (e.g. european populations of Adalia bearing three symbionts, Spiroplasma, Rickettsia and Wolbachia, [START_REF] Hurst | Male-killing Wolbachia in two species of insect[END_REF], leading to intraspecific (i.e. inter-population) variation in mtDNA sequences.

In all these cases, nuclear loci are required to resolve phylogenetic relationships and may serve as a validating tool during the database construction stage. Besides, special care must be accorded to the compilation of reference sequences (i.e. DNA barcode), especially for species with already known disturbed mitochondrial inheritance. The presence of such potentially misleading effects should be explicitly indicated in the BOLD. However, unknown endosymbionts or exclusive causes of mtDNA inheritance disturbance could also be revealed during the DNA barcoding database filing.

4-3 Nuclear copies of COI (NUMTs)

Nuclear mitochondrial DNAs (NUMTs) are nuclear copies of mitochondrial DNA sequences that have been translocated into the nuclear genome (Williams and Knowlton, 2001). In eukaryotes, the number and the size of NUMTs are variable, ranging from none or few in Anopheles, Caenorhabditis and Plasmodium, to more than 500 in humans, rice and Arabidopsis [START_REF] Richly | NUMTs in sequenced eukaryotic genomes[END_REF]. As reported by Ann Bucklin (Oral comm., the 3 rd international Cons. Gen. symposium, New York, 2007) using DNA barcoding in investigations on marine zooplankton, and by [START_REF] Lorenz | The problems and promise of DNA barcodes for species diagnosis of primate biomaterials[END_REF] performing primate DNA barcoding, nuclear COI copies can sometimes greatly complicate the straightforward collection of mitochondrial COI sequences. Disturbance due to NUMTs must be seriously considered, in both DNA barcode library construction and further specimen identification. Owing to their particular codon structure, non-synonymous mutations, premature stop codons and insertion-deletions [START_REF] Strugnell | A barcode of life database for the Cephalopoda? Considerations and concerns[END_REF], NUMTs can be recognized in the sequence and in the amino acid alignments. In the sequence acquisition stage, NUMTs can be detected by the sequence checking process proposed in BOLD (i.e. rejection of inconsistent amino acid alignment), and in such cases, their occurrence should be referenced in BOLD. Only recently integrated NUMTs that are difficult to detect [START_REF] Thalmann | Unreliable mtDNA data due to nuclear insertions: a cautionary tale from analysis of humans and other great apes[END_REF], could be ignored. Although it is more difficult, it is nevertheless possible to get the true mtCOI sequence of voucher specimens with the reverse transcription [START_REF] Collura | A quick, direct method that can differentiate expressed mitochondrial genes from their nuclear pseudogenes[END_REF]. In the diagnostic stage, there may be cases where NUMT occurrence is unknown, which highlights the care that should be taken in DNA barcode alignments.

4-4 Rate of evolution in COI

The rate of genome evolution (mitochondrial or nuclear) is not equal for all living species. Notably, molluscs have a higher evolutionary rate than other bilateral metazoans [START_REF] Strugnell | A barcode of life database for the Cephalopoda? Considerations and concerns[END_REF]. In contrast, diploblast sponges and cnidarians have an evolutionary rate 10-20 times slower than in their bilaterian counterparts, a consequence of which is the lack of COIsequence variation that prevents distinction below the family level [START_REF] Erpenbeck | CO1 phylogenies in diploblasts and the 'Barcoding of Life' -are we sequencing a suboptimal partition?[END_REF]. The rate of evolution can even differ at the ordinal level, as shown between six dermapteran (Insect) species [START_REF] Wirth | Directional Substitution and Evolution of Nucleotide Content in the Cytochrome Oxidase II Gene in Earwigs (Dermapteran Insects)[END_REF]. In the same way, the level of variation in mitochondrial sequences in the plant kingdom excludes species identification based on COI sequence polymorphism [START_REF] Kress | Use of DNA barcodes to identify flowering plants[END_REF].

More generally, the lack of resolving power of COI-sequence reported for some taxa has led the CBOL to envisage the transition from the primary single-gene method (i.e. BARCODE) to a multiregion barcoding system, when it is justified (i.e. in cases where COI is not species specific, or for taxa with low mitochondrial evolutionary rates) taxon-specific reference regions (i.e. nuclear plus/or organelle genes), also called non-COI barcode (Bakker, Second International Barcode of Life Conference, TAIPEI, September 2007).

4-5 The intra-specific geographical structure should be taken into account

Geographical structure, if ignored, can blur and distort species delineation. Actually, high rates of intraspecific divergence can derive from geographically isolated populations [START_REF] Hebert | Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species[END_REF], and thus, must be considered in the setting up of the DNA barcode reference database. This point stresses a key challenge for the DNA barcoding initiative, from both the fundamental and analytical points of view. What is the boundary between a population and a species? Does it exist? To solve this issue, wide-ranging intra-specific sampling should be integrated in the reference database, and one must consider species boundaries not as a definitive but as a revisable concept. The relevance of the reference DNA barcode database depends on the exhaustiveness of intra-taxon sampling.

To prevent misleading results, the current data format for submission to BOLD should be complemented with new fields related to the limitations mentioned above (i.e. NUMT occurrence, known endosymbiont, available insight on molecular clock, genetic structure and geographical distribution). Besides the biological limitations, DNA barcoding raises analytical and statistical issues.

5-DNA-Sequence Analysis, a double trend: pure assignment vs delimitation of species

5-1 Query sequence assignment

Frézal, L., Leblois, R. Infection, Genetics and Evolution

The main and unambiguous objective of DNA barcoding analysis is to assign one query sequence to a set of referenced tagged-specimen sequences extracted from BOLD. The method currently used in BOLD combines similarity methods with distance tree reconstruction in the following way: (i) First, the query sequence is aligned to the global alignment through a Hidden Markov Model (HMM) profile of the COI protein [START_REF] Eddy | Profile hidden Markov models[END_REF], followed by a linear search of the reference library. The 100 best hits are selected as a pre-set of "closely related tagged-specimens";

(ii) Second, a Neighbor-Joining tree is reconstructed on this preset plus the query sequence to assess the relationship between the query sequence and its neighboring referenced sequences [START_REF] Kelly | DNA barcoding using chitons (genus Mopalia)[END_REF]. The query sequence is then assigned to the species name of its nearest-neighboring referenced sequence, whatever the distance between the two sequences. This method is direct and rapid, but its main shortcomings are high prevalence of sampling-dependent accuracy, high rates of false-positive assignments [START_REF] Koski | The closest blast hit is often not the nearest neighbor[END_REF] and the fact that there is no other way to infer the reliability of the query assignment than computing percentages of similarity or genetic distances, two measures that are known to be irrelevant for taxonomic relationship [START_REF] Ferguson | On the use of genetic divergence for identifying species[END_REF]. The loss of character information is also inherent in distance methods, as computing distances erase all character-based information [START_REF] Desalle | Species discovery versus species identification in DNA barcoding efforts: Response to Rubinoff[END_REF]. Moreover, as both similarity and distance methods strongly depend on the disparity between intra-and inter-specific variations, incomplete taxonomic sampling (i.e. barcoding gaps) will artificially increase the accuracy of such methods. [START_REF] Matz | A likelihood ratio test for species membership based on DNA sequence data[END_REF][START_REF] Nielsen | Statistical approaches for DNA barcoding[END_REF][START_REF] Abdo | A step toward barcoding life: A model-based, decision-theoretic method to assign genes to preexisting species groups[END_REF].

The question here is whether it is worthwhile to adopt a biological, populational and/or phylogenetic rationale for DNA barcode sequence analyses or, whether pure statistical approaches are more efficient to assign a query sequence to a species name. Note that character-based methods (either character-based phylogenetics, i.e. not distance-based, or statistical classification) are consistent with the phylogenetic species concept [START_REF] Goldstein | Phylogenetic species, nested hierarchies, and character fixation[END_REF], whereas distance-based methods are not [START_REF] Lipscomb | The intellectual content of taxonomy: a comment on DNA taxonomy[END_REF]. CAOS of [START_REF] Sarkar | Characteristic attributes in cancer microarrays[END_REF] is an example of character-based analysis, in which the nucleotide sequence is considered as a chain of characters. In the same way, [START_REF] Desalle | The unholy trinity: taxonomy, species delimitation and DNA barcoding[END_REF] proposed the combination morphological and molecular characters, which has the advantage of bridging the gap between the classical taxonomy and 'molecular-taxonomy' and the DNA barcoding approach.

At present, global comparisons between all these approaches are clearly missing. However, few studies have already compared some of these algorithms [START_REF] Elias | Limited performance of DNA barcoding in a diverse community of tropical butterflies[END_REF][START_REF] Ross | Testing the reliability of genetic methods of species identification via simulation[END_REF].

For example, Austerlitz et al., (Second International Barcode of Life Conference TAIPEI, September 2007) compared phylogenetic tree reconstruction with various supervised classification methods (CART and Random Forest, Support Vector Machines and Kernel methods, [START_REF] Breiman | Classification and Regression Trees[END_REF] on both simulated and real data sets. Their main conclusions are: (i) maximum likelihood phylogenetic (PhyML, [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF] approaches always seem to be more accurate than distancebased (Neighbor-Joining) phylogenetic inferences; (ii) computation times are much higher for maximum likelihood phylogenetic reconstruction than for statistical classification; and (iii) the accuracy of all the methods strongly depends on sample size and global variability of the taxa. Supervised classification methods outperform phylogenetic analyses only when the reference sample per species is large (n > 10).

Rigorous assignment relevancy depends on our capacity to estimate the probability of a falseassignment event. False species assignments can be due to three types of errors [START_REF] Nielsen | Statistical approaches for DNA barcoding[END_REF]): (i) the true species may not be represented in the database; (ii) the random coalescence of lineages in populations and species may not necessarily lead the query sequence to be the most closely related to the true species sequence; (iii) the random process at which mutations arise on lineages may cause the sequence representing another species to be more similar to the query.

Population genetics theory, and more specifically coalescent theory, can help to assess the probability of the occurrence of the last two events. Recently, model-based decision theory framework based on the coalescence theory [START_REF] Matz | A likelihood ratio test for species membership based on DNA sequence data[END_REF][START_REF] Nielsen | Statistical approaches for DNA barcoding[END_REF][START_REF] Abdo | A step toward barcoding life: A model-based, decision-theoretic method to assign genes to preexisting species groups[END_REF] has been established, and should lead to greater accuracy in query sequence assignment with an estimation of the degree of confidence with which this assignment can be made.

However, the major drawbacks of such model-based decision tools are high computation times and the requirement of large data sets (e.g. more than 10 sequences per species) for enough genetic information to perform accurate analyses. Moreover, the mitochondrial neutrality has recently being put into question [START_REF] Bazin | Population size does mitochondrial genetic in animals[END_REF], which may invalidate inferences using neutral coalescent processes.

To conclude on the query assignment method, it would be advisable to adopt a sequential investigation. Firstly, to search the complete database with similarity methods thus reducing the total data set to the genus or family of the query sequence. Then, to use statistical classification and/or phylogenetic tools to more precisely assign the query sequence to a given species. If still no obvious assignment emerges, it should then be made using population genetic methods based on coalescence.

However, even if the assignment with classification or phylogenetic methods seems unambiguous, coalescent-based methods running on the closest neighbours of the query sequence should give an idea of the degree of uncertainty associated with an identification.

5-2 Delimitation of species

The second and more controversial objective of DNA barcode analyses is to define clusters of individuals and consider them as species, in other words, to do molecular taxonomy on unidentified taxa. Unlike the approaches mentioned above, clustering is an unsupervised learning problem that involves identifying homogeneous groups in a data set. Beside all the well-justified discussions between taxonomists about the molecular delimitation of species, such a clustering approach is much more complicated than pure assignment to a pre-identified taxonomic group. Three main approaches have been put forward so far. Hebert et al. (2004b) first proposed the use of a divergence-threshold to delimit species. The underlying idea was that intra-species divergence is lower than inter-species divergence. The standard divergence threshold value advised was of ten times the mean intraspecific variation ('10fold rule') with the reciprocal monophyly. Despite the efficiency of the threshold approach reported for fishes [START_REF] Ward | DNA barcoding Australia's fish species[END_REF], crustaceans [START_REF] Lefebure | Relationship between morphological taxonomy and molecular divergence within Crustacea: Proposal of a molecular threshold to help species delimitation[END_REF], North American birds (Hebert et al., 2004b), tropical lepidopterans (Hajibabaei et al., 2006a) and cave-dwelling spiders [START_REF] Paquin | The power and perils of "molecular taxonomy": a case study of eyeless and endangered Cicurina (Araneae: Dictynidae) from Texas caves[END_REF], the use of thresholds in species delineation has been strongly discouraged. Indeed, the divergence-threshold methods lack strong biological support and undoubtedly could not become a universal criterion suited to animal species delineation [START_REF] Meyer | DNA barcoding: Error rates based on comprehensive sampling[END_REF][START_REF] Hickerson | DNA barcoding will often fail to discover new animal species over broad parameter space[END_REF][START_REF] Wiemer | Does the DNA barcoding gap exist? -a case study in blue butterflies (Lepidoptera: Lycaenidae)[END_REF]. By their literature survey of mitochondrial DNA studies on low taxonomic-level animal phylogeny, [START_REF] Funk | Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA[END_REF] detected species-level paraphyly or polyphyly in 23% of 2,319 assayed species, demonstrating that NJ-tree analysis will fail to assign query sequences in a significant proportion of cases [START_REF] Ross | Testing the reliability of genetic methods of species identification via simulation[END_REF]. The question is thus to clearly characterize the proportion of non-monophyletic species and the relationship between intra-and inter-specific variability in various taxa to globally assess the relevance of such threshold approaches.

The second approach to delimitate species has been developed by [START_REF] Pons | FlgM anti-sigma factors: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence-structure-function relationships[END_REF] using a mixed model combining a coalescent population model with a Yule model of speciation. Their approach is based on the differences in branching rates at the level of species and populations. Such a model allows them to infer a time of branching regime change, from the coalescent rate to the speciation rate, and to define species as being the clusters for which all individuals are branched inside the coalescent time frame. Even if their approach is currently oversimplified because they consider a unique rate of coalescence (i.e. all population sizes are the same) and a unique shift from population to species processes, it is a promising step that combines the principles of population genetics and those of speciation processes.

The third methodology, which also uses principles of population genetics, is the extension of the coalescent-based models of [START_REF] Matz | A likelihood ratio test for species membership based on DNA sequence data[END_REF], [START_REF] Nielsen | Statistical approaches for DNA barcoding[END_REF] and, [START_REF] Abdo | A step toward barcoding life: A model-based, decision-theoretic method to assign genes to preexisting species groups[END_REF]. This approach has not yet been fully developed but is suggested in the three abovementioned papers. The underlying idea is that maximum likelihood or Bayesian inference, using coalescent models, should help to assess divergence times and/or presence or absence of gene flow between the clusters considered. Estimates of divergence times and gene flow can then be used to infer species status of clusters, based on the biological definition of species. As for the assignment methods, the main drawback of such coalescent-based methods are computation times and large intra-specific sampling requirements.

We emphasize that, whatever approach is used, every taxonomic decision using DNA Barcode data should be validated by other independent lines of evidence.

6-What level of universality can the DNA barcode reach?

6-1 The choice of the genome region(s)

The main difficulty of DNA barcoding is to find the ideal gene that discriminates any species in the animal kingdom. [START_REF] Hebert | Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species[END_REF]Hebert et al. ( , 2004a,b) ,b) argue in favor of the mitochondrial 5' COI region [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF], a choice justified by its great resolving-power for birds, lepidopteran and dipteran species discrimination. Ideally, a single pair of universal primers (e.g. Folmer primers, Folmer et al., 1994) would amplify the DNA barcode locus in any animal species. The development of taxon-specific primers and their combinations are however sometimes necessary to obtain greater intra-generic accuracy (e.g. coral reef, [START_REF] Neigel | DNA barcoding as a tool for coral reef conservation[END_REF], as illustrated by the primer combinations and cocktails required to obtain DNA barcodes from fish species [START_REF] Ward | DNA barcoding Australia's fish species[END_REF][START_REF] Ivanova | Universal primer cocktails for fish DNA barcoding[END_REF], or the primer sets needed to distinguish between primate genera [START_REF] Lorenz | The problems and promise of DNA barcodes for species diagnosis of primate biomaterials[END_REF]. The COI amplification does not always ensure the success of the specimen identification. Indeed, the COIbased identification sometimes fails to distinguish closely related animal species, underlining the requirement of nuclear regions (e.g. Cytb and Rhod to identify all teleost fish species, [START_REF] Sevilla | Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes[END_REF]: the idea of a multi-locus DNA barcoding approach is progressively emerging.

The extension of DNA barcoding to other kingdoms is also progressing. The efficiency of COI-based barcoding has been documented for few groups of fungi (e.g. Penicillium sp., [START_REF] Seifert | Prospects for fungus identification using C01 DNA barcodes, with Penicillium as a test case[END_REF], macroalgae (Rhodophyta, Saunders, 2005) and two ciliophoran protists genera (Paramecium and Tetrahymenas, [START_REF] Barth | Intraspecific genetic variation in Paramecium revealed by mitochondrial cytochrome c oxidase 1 sequences[END_REF]Lynn and Strüder-Kypke, 2006;[START_REF] Chantangsi | Barcoding ciliates: a comprehensive study of 75 isolates of the genus Tetrahymena[END_REF], suggesting that the DNA barcode standardization may be harder to reach than expected. It is now commonly accepted that the universality of the initial COI-based CBOL project is unlikely. Indeed, considering mitochondria solely would not solve problems of differential evolutionary rates among close genera, of inheritance discrepancy, of mtDNA introgression processes and of the intron-size variations that prevent COI-sequence alignment (e.g. fungi, plants). Besides, methods of sequence assignment based on a single-locus will often lack accuracy [START_REF] Elias | Limited performance of DNA barcoding in a diverse community of tropical butterflies[END_REF]. The ineluctable future trend for species identification through DNA barcoding is to develop a multi-locus system, including COI-region or/and independent markers [START_REF] Rubinoff | Between two extremes: mitochondrial DNA is neither the panacea nor the nemesis of phylogenetic and taxonomic inference[END_REF][START_REF] Dasmahapatra | DNA barcodes: recent successes and future prospects[END_REF][START_REF] Kress | A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnHpsbA spacer region[END_REF][START_REF] Smith | DNA barcodes affirm that 16 species of apparently generalist tropical parasitoid flies (Diptera, Tachinidae) are not all generalists[END_REF][START_REF] Sevilla | Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes[END_REF].

Additional molecular markers have already been proposed, among which the nuclear subunit ribosomal RNA genes are promising candidates because of their great abundance in the genome and their relatively conserved flanking regions. Moreover, the use of rRNA allows efficient species distinction (e.g. for amphibians, [START_REF] Vences | Performance of 16S rRNA in DNA barcoding of amphibians[END_REF][START_REF] Vences | Deciphering amphibian diversity through DNA barcoding: chances and challenges[END_REF]for truffles, El Karkouri et al., 2007), and can sometimes provide classifications into molecular taxonomic units, MOTU (e.g. for nematodes, [START_REF] Floyd | Molecular barcodes for soil nematode identification[END_REF][START_REF] Blaxter | Defining operational taxonomic units using DNA barcode data[END_REF].

In higher plants, the mitochondrial genome evolves much more slowly than in animals. The COI-region is thus inappropriate for plant species distinction [START_REF] Rubinoff | Are plant DNA barcodes a search for the Holy Grail?[END_REF]. The CBOL plant working group (PWG) agrees that plant barcoding will be multi-locus, with one 'anchor' (i.e. universal across the plant kingdom) and 'identifiers' to distinguish closely related species (Bakker, Second International Barcode of Life Conference TAIPEI, September 2007). Several combinations of DNA regions have been recently proposed [START_REF] Kress | Use of DNA barcodes to identify flowering plants[END_REF]Chase et al., 2006;[START_REF] Kress | A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnHpsbA spacer region[END_REF][START_REF] Pennisi | Wanted: A Barocde for Plants[END_REF]Lee et al., Second International Barcode of Life Conference TAIPEI, September 2007). At present, there is still no consensus on which candidate markers are the best Frézal, L., Leblois, R. Infection, Genetics and Evolution plant DNA barcoding region [START_REF] Pennisi | Wanted: A Barocde for Plants[END_REF]. The future combination will certainly contain noncoding intergenic spacers (e.g. trnH-psbA, [START_REF] Kress | Use of DNA barcodes to identify flowering plants[END_REF]Chase et al., 2006;[START_REF] Kress | A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnHpsbA spacer region[END_REF] and plastidial coding sequences (e.g. matK, [START_REF] Chase | A proposal for a standardised protocol to barcode all land plants[END_REF]. Recently, [START_REF] Lahaye | DNA barcoding the floras of biodiversity hotspots[END_REF] working on a large representative sample (>1600 plants specimens) strongly converged with [START_REF] Chase | A proposal for a standardised protocol to barcode all land plants[END_REF] conclusion, and advocates the matK locus as the best universal 'anchor' for DNA barcoding of plant taxa. However, they also agree with the need for extra loci (i.e. 'identifiers') to resolve lower taxon identification. In addition, [START_REF] Taberlet | Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding[END_REF] focused on the feasibility of barcoding plants from highly degraded DNA that is of interest for ancient DNA studies (e.g. permafrost samples) and other applied fields (e.g. processed food, customs, medicinal plants). They promoted the chloroplast trnL (UAA) intron or a shorter fragment of this intron (the P6 loop, 10-143 bp), which, despite the relatively low resolution, can be amplified with highly conserved primers.

If the prior universality of a single locus and a single primer set remains utopian, the use of a few common loci is still a great advance for future diversity assessments within higher taxa. Steady common features of the DNA barcoding approach will remain, but will certainly evolve in kingdomor even lower taxon-specific technical approaches.

6-2 The challenge: barcoding microscopic biodiversity

One of the greatest challenges for the Barcode of Life project is to account for the diversity of unicellular life (i.e. archea, bacteria, protists, and unicellular fungi). As a matter of fact, with an evolutionary history dating back to 3.5 billion years, microscopic life (<1mm) represents the largest part of biodiversity. Besides, microscopic species are the causal agents of numerous diseases and are keys to the functioning of trophic networks [START_REF] Chantangsi | Barcoding ciliates: a comprehensive study of 75 isolates of the genus Tetrahymena[END_REF]. In oceans, microbial life is responsible for 98% of the primary production and the mediation all the biogeochemical cycles [START_REF] Sogin | Microbial diversity in the deep sea and the underexplored "rare biosphere[END_REF]. One of the striking characteristics of the microbiosphere is the unstable population size over short periods of time, one population can be dominant at a specific time or location, but rare, and thus difficult to survey at another time or location. The low-abundance populations (e.g. rare biosphere) that account for most of the phylogenetic diversity are masked by the dominant populations, leading to an underestimation of the diversity of microbial life [START_REF] Sogin | Microbial diversity in the deep sea and the underexplored "rare biosphere[END_REF].

The microbiodiversity DNA barcoding has yet been poorly studied. The emerging community genomics [START_REF] Frézal | Community genomics among stratified microbial assemblages in the ocean's interior[END_REF] and the metagenomics approaches promise great insights on prokaryote biodiversity and molecular evolution [START_REF] Delong | Microbial population genomics and ecology: the road ahead[END_REF][START_REF] Tyson | Community structure and metabolism through reconstruction of microbial genomes from the environment[END_REF]Venter et al., Frézal, L., Leblois, R. Infection, Genetics and Evolution 2004;[START_REF] Tringe | Comparative metagenomics of microbial communities[END_REF][START_REF] Leclerc | Intestinal metagenomics and nutrition[END_REF]. But, for microscopic eukaryotes, the routine use of the metagenomics approach is not technically conceivable and too expensive today, highlighting the interest of DNA barcoding on pooled samples (i.e. where multiple species are present or communities) to assess eukaryotic microbiodiversity (Johnson and Slatkin, 2008).

Exploring the microscopic eukaryotic life diversity can be achieved by the COI-based barcode [START_REF] Blaxter | Defining operational taxonomic units using DNA barcode data[END_REF][START_REF] Chantangsi | Barcoding ciliates: a comprehensive study of 75 isolates of the genus Tetrahymena[END_REF][START_REF] Evans | An assessment of potential diatom "barcode" genes (cox1, rbcL, 18S and ITS rDNA) and their effectiveness in determining relationships in Sellaphora (Bacillariophyta)[END_REF]. But as for the macroscopic species, a specific and multi-locus barcoding approach may be required. The 5'-end region (600 bp) of the small ribosomal subunit has already been reported to assign isolates to specific subtypes of the human parasite Blastocyctis hominis [START_REF] Scicluna | DNA barcoding of Blastopystis[END_REF] and successfully reached types or species in the deep seas [START_REF] Sogin | Microbial diversity in the deep sea and the underexplored "rare biosphere[END_REF].

6-3 Universality of the Barcode Of Life Data system (BOLD)

The universality of the Barcode of Life mainly resides in the synergic and standard approach for data acquisition and their compilation into BOLD, which is the central connection of the CBOL initiative. The current format for data submission to BOLD is composed of five fields for voucher specimen characterization: (i) the specimen identifier (the catalogue and collection codes, the institution responsible for providing the specimen samples); (ii) the taxonomic status; (iii) the specimen characteristics (sex, life stage, reproduction); (iv) the collection data (collector, collection date and location with GPS coordinates); (v) the DNA barcode sequence (gene name and location, trace file, alignment details, primers used to generate the amplicon). Next to this, pictures of vouchers and the trace of their DNA Barcode must be submitted. All the guidelines are specified on the BOLD website (http://www.barcodinglife.com/docs/boldtutorial.html).

Recently the international sequences depository (NCBI) have accepted to put in place a BARCODE keyword into their search facilities for entries that conform to the minimal CBOL requirements (i.e. traces file, collection location). The BOLD barcode Submission tool associates the 'Barcode submissions' with a further submission to NCBI using the 'My NCBI' user name. Note that for data that satisfies the BARCODE-keyword conditions, the NCBI taxonomy browser gives the direct link to the BOLD taxonomy browser. A keyword related to the concept of 'non-COI barcode' may be soon envisaged.

BOLD will provide an increasing amount of DNA barcode records (either COI-or non-COI barcodes) to clearly identify unknown specimens, which will enable accurate query assignments and will facilitate comparison between data obtained in geographically dispersed institutions. Recently, an increasing amount of initiatives for global data recording have been proposed to manage clinical and molecular information about infectious diseases (epiPATH, [START_REF] Amadoz | epiPATH: an information system for the storage and management of molecular epidemiology data from infectious pathogens[END_REF], but also to focus on either human pathogenic bacteria (e.g. pathoMIPer, [START_REF] Thiyagarajan | PathogenMIPer: a tool for the design of molecular inversion probes to detect multiple pathogens[END_REF]pyloriBASE, Ahmed et al. 2007;VectorBase, Lawson et al., 2007;DengueInfo, Schreiber et al., 2007) or on plant pathogens (e.g. PhiBASE, Winnenburg et al., 2006). BOLD could serve as the universal starting point for species identification, which would convey users to refer to specialized databases (e.g. pathogenic strains, disease vector species and endangered species). The CBOL has already initiated the new International Network for the Barcoding of Invasive and Pest Species (INBIPS; www.barcoding.si.edu/INBIPS.htm) that will help to coordinate the collection of barcode data on pest species around the world [START_REF] Ball | DNA barcodes for insect pest identification: a test case with tussock moths (Lepidoptera: Lymantriidae)[END_REF].

7-Conclusion

After four years, the animal DNA barcoding approach has become less controversial, its relevancy is now supported by numerous successes and by the increasing amount of DNA barcoding projects, among which the barcoding of 500,000 species planned by the iBOL. However, clear limitations arise from the incomplete coverage of the existing diversity, the inherent characteristics of the mitochondrial DNA (evolutionary rate, inheritance, introns, neutrality) and the single-locus initial strategy. With its enlargement to all eukaryote taxa, the Barcode of life project has also evolved to a more flexible framework. The approach reveals to be more complex than the system projected by the CBOL initially. The multi-locus barcoding approach is now commonly accepted, particularly to discriminate between low level taxa and to increase the power of the sequence assignments.

The BOLD data system is central to the DNA barcoding approach. The specificities of BOLD are (i) to assemble standard information on voucher specimens using common description fields (DNA tag, specimen taxonomy, specimens details, collection information, voucher pictures), and, (ii) its dynamic status that allows taxonomic revisions and reassignment of the deposited sequences.

The final point concerning the Barcode of Life project is that, beyond the construction of a standard approach based on the existing taxonomic knowledge, it has enhanced communication between different scientific communities, including taxonomists, phylogeneticists and population geneticists.

  Various alternative methods have been proposed to analyse DNA barcode data amongst which we can distinguish four main categories of approaches: (i) similarity approaches, based solely on the similarity between the total DNA barcode sequences or small parts of them (e.g. oligonucleotide motifs,Dasgupta et al., 2005;[START_REF] Little | A comparison of algorithms for the identification of specimens using DNA barcodes: examples from gymnosperms[END_REF]; (ii) classical phylogenetic approaches, using either genetic distances or maximum likelihood / Bayesian algorithms and assuming different mutational models (e.g. Neighbor-Joining, phyML, MrBayes,[START_REF] Elias | Limited performance of DNA barcoding in a diverse community of tropical butterflies[END_REF]; (iii) multiple-character based analysis (DeSalle et al., 2005) (vi) pure statistical approaches based on classification algorithms without any biological models or assumptions (CAOS, Sarkar et al., 2002 a, b); and (v) genealogical methods based on the coalescent theory using demo-genetic models and maximum likelihood / Bayesian algorithms

Acknowledgments

We thank Kevin Bleakley, Pascale Chesselet, Olivier David, Mark Stevens, Michel Veuille and Thierry Wirth for constructive comments and usefull discussions on the manuscript. L.F. was financially supported by a ATER grant from the Ecole Pratique des Hautes Etudes.


Edouard Ribot
email: ribotted2@gmail.com

Julie Berbis

Ilyes Hamouda

Daniel Cohen

Aubert Agostini

Blandine Courbiere
email: blandine.courbiere@ap-hm.fr

Pregnancy outcomes after in vitro fertilization for moderate and severe endometriosis. A case-control study

Keywords:

Introduction: Previous international studies showed that endometriosis could have a link with obstetrical complications, as an increased risk of preterm birth, gestational diabetes mellitus, and cesarean section. However, the results are difficult to interpret because biases are common, such as heterogeneity in the severity of the endometriosis cases included. That's why some complications as risk of miscarriage and post-partum hemorrhage are still debated. Our objective was to study pregnancy outcome after In Vitro Fertilization (IVF) in women suffering from rAFS stage III and IV endometriosis. Methods: We conducted a case-control study between 2009 and 2019. We compared pregnancy outcomes after IVF in two groups of women matched by age, body mass index and smoking in two hospital centers. Group A was constituted by singleton pregnancies following ART for moderate and severe endometriosis (rAFS stage III and IV endometriosis). Group B was composed of singleton pregnancies in women with no endometriosis following ART for another reproductive disease. All women achieved pregnancy after 22 weeks. Results: A total of 240 pregnant women were included: 80 singleton IVF pregnancies (group A) were compared with 160 singleton IVF pregnancies (group B). We observed an increased risk of placenta previa (12.5% Vs 1.9%; p = 0,001), and cesarean section (49.4% (n = 39) Vs 29.6% (n = 47) p = 0,004). Rate of postpartum hemorrhage was not significantly different in endometriosis group (11.2% Vs 7.5% p = 0.47). Conclusion: Despite conflicting results in literature due to a lot of confounding variables, the impact of endometriosis on pregnancy is still debated in women suffering from rAFS III and IV endometriosis. In our study, we observed statistically higher rates of placenta previa and cesarean section but not an increased risk of postpartum hemorrhage. Further larger series are needed to confirm our findings and a possible link with other obstetrical complications. However, we think that an ART pregnancy in a context of severe endometriosis should be considered at risk of adverse obstetrical outcomes.

Introduction

Endometriosis is a pelvic disease that affects 6 to 15% of women worldwide [START_REF] Burney | Pathogenesis and pathophysiology of endometriosis[END_REF]. Endometriosis is defined by ectopic endometrial tissue and is often responsible of pelvic pain and infertility. The eutopic endometrium of women with endometriosis has shown molecular and cellular alterations that are believed to alter pregnancy outcome in these women [START_REF] Benaglia | Pregnancy outcome in women with endometriosis achieving pregnancy with IVF[END_REF]. All ART pregnancies could be considered at higher risk of adverse outcome [START_REF] Stern | Adverse pregnancy and birth outcomes associated with underlying diagnosis with and without assisted reproductive technology treatment[END_REF].

Moreover, some studies have reported an increasing risk of unfavorable pregnancy outcome in women with endometriosis such as preterm birth, small for gestational age (SGA), preeclampsia and cesarean section rate [START_REF] Fernando | Preterm birth, ovarian endometriomata, and assisted reproduction technologies[END_REF][START_REF] Jacques | Adverse pregnancy and neo-natal outcomes after assisted reproductive treatment in patients with pelvic endometriosis: a case-control study[END_REF]. In contrast, some other studies did not show higher risk of preterm birth even with a significant sample size [START_REF] Benaglia | Pregnancy outcome in women with endometriosis achieving pregnancy with IVF[END_REF]. These observations confirm difficulty to obtain homogeneous population and reproducible results. If endometriosis seems to be associated with more obstetrical complications, it is unclear if obstetrical complications depend of endometriosis stage. In most studies, all endometriosis stages are included, leading to heterogeneity of the studied population. Some studies compared pregnancy outcomes of women with endometriosis conceived by ART with those who conceived naturally.

In endometriosis, chronic inflammation leads to a rise of inflammatory cytokines like interleukins (IL) and tumor necrosis factor alpha (TNFa) in peritoneal tissue. IL1 and TNFa have an effect on myometrium receptors and could induce uterine contractions [START_REF] Petraglia | Inflammation: a link between endometriosis and preterm birth[END_REF][START_REF] Carassou-Maillan | Adverse pregnancy outcomes after Assisted Reproduction Technology in women with endometriosis[END_REF]. This situation may explain more threatened preterm labor in pregnant women with endometriosis [START_REF] Bonuccelli | Premature Birth in Women with Endometriosis: a Systematic Review and Meta-analysis[END_REF]. Likewise, more women suffering from several endometriosis may have increased internal bleeding. Endometriosis located at the level of the uterine isthmus may weaken the posterior uterine wall, predisposing it to uterine rupture during pregnancy or postpartum hemorrhage [START_REF] Vigano | Beyond infertility: obstetrical and postpartum complications associated with endometriosis and adenomyosis[END_REF]. Most studies support the idea that dysfunctional critical uterine changes during implantation process can trigger a defective process of decidualization and placentation, with a cascade of events resulting in defective remodeling of the spiral arteries [START_REF] Brosens | Risks of adverse pregnancy outcome in endometriosis[END_REF].

The main objective of our study was to assess obstetrical complications, in women with stage III and IV endometriosis who conceived following IVF.

Materials and methods

Studied population

In our study, only stages III and IV endometriosis were included, according to the revised American Society for Reproductive Medicine (rASRM) classification of 1997 [START_REF]Revised American Society for Reproductive Medicine classification of endometriosis: 1996[END_REF]. Women's data concerning IVF pregnancy and neonatal outcome were collected retrospectively from medical database between 2009 and 2019 in two Departments of Gynecology-Obstetric and Reproductive Medicine of two Public Hospitals. Two groups of pregnant women between 18 and 42 years with a history of infertility > 12 months and pregnant after IVF or ICSI were constituted: group A was constituted by women pregnant following IVF for rASRM stage III and IV endometriosis Vs Group B of IVF pregnant women with no endometriosis. We also excluded ART pregnant women after intra-uterine inseminations, gametes donation, and embryo transfer from vitrified oocytes. Twin pregnancies were excluded. A personal phone call was performed to every woman in order to collect their oral and written consent to participate to the study and to complete obstetrical or neonatal information when needed. We excluded all women who didn't consent to participate to the study and women who didn't understand French well enough to give their consent.

For group A, diagnosis of endometriosis was established during a laparoscopic exploration and/or Magnetic resonance imaging (MRI), as previously described [START_REF] Millischer | Magnetic resonance imaging presentation of deep infiltrating endometriosis nodules before and after pregnancy: a case series. Bergsland N, editeur[END_REF]. Data from MRI were available for most women in order to determine deep infiltrating endometriosis combined (DIE)). Group B was the control group composed by women without endometriosis and who underwent IVF during the same period as women from group A. For each group, we included both pregnancies obtained after fresh embryo transfer (ET) and those obtained after frozen-thawed embryo transfer (FET). Transferred embryos were 2, 3 or 5 days embryos. All supernumerary good quality embryos were frozen with a slow-freezing program at Day 2, 3 or 5. Among pregnancies of more than 22 weeks of gestation, groups A and B were paired 1:2 for exploration of pregnancy outcomes.

Studied parameters

Concerning women's clinical characteristics, we collected age, body mass index (BMI) and smoking status. Smoking status was considered as an active smoking status for one or more cigarettes per day. IVF or ICSI was decided according to sperm parameters. Abnormal sperm parameters included oligospermia (<39 million spermatozoa per ml of semen), asthenozoospermia (<50% of mobile sperm), and teratozoospermia (<4% with normal morphology). We defined a positive pregnant test as a positive level of hCG 14 days after ET. A clinical ongoing pregnancy was defined by an embryo with a cardiac activity at 8 weeks of gestation (WG). Secondly, we collected obstetrical diseases during pregnancy after 22 weeks of gestation: placenta previa, placental abruption.

Placenta previa was defined by a placenta implant in the lower segment of the uterus unless 20 mm of cervix. Concerning obstetrical and neonatal outcomes, we collected gestational age at delivery (gestational weeks), rate of postpartum hemorrhage (PPH) (defined as bleeding < 500 mL after delivery), rate of cesarean section, birth weight and neonatal hospital admission for each baby.

Outcome measures

Primary outcome measure was all obstetrical complications at delivery considered together (PPH, placenta previa, placental abruption and cesarian section). Secondary outcomes were rate of postpartum hemorrhage, rate of placenta praevia, rate of cesarian section, gestational age at delivery and neonatal hospitalization rate.

Statistical methods

Descriptive data were expressed as counts and percentages for qualitative variables, and as means and standard deviations (SD) for continuous variables. The search for association between two independent variables was done using the X 2 test for homogeneity, the FISHER's exact test for qualitative variables, the Student's t-test for two independent groups for the comparison of continuous variables, with the determination of significance thresholds at 0.05.

We performed univariate and multivariable analyses. Variables in the univariate analysis with p < 0.20 were introduced in the multivariable regression model. A backward selection method was performed with an alpha significance level of 0.05 (both for entry and retention). The results are shown as odds ratios (ORs) with 95% confidence intervals (CIs). The matching of case and control cases was performed using greedy matching (1:2 nearest neighbor) [START_REF] Kosanke | gmatch: match 1 or more controls to cases using the GREEDY algorithm[END_REF] from three criteria: age, BMI and smoking status. The statistical analysis was performed using the software IBM SPSS Statistics V20.0.0. Bergstralh, EJ and Kosanke JL (1995); Section of Biostatistics Technical Report 56. Mayo Foundation.

Ethics statement

This study was approved by the Ethical Committee of Aix-Marseille University (2017-05-04-005).

Results

Study population

We included 240 women who achieved pregnancy after IVF or ICSI: 80 singleton pregnant women after IVF for severe endometriosis (group A) were compared with 160 IVF singleton pregnancies in women with no endometriosis (group B). The two groups were comparable in terms of age, BMI and smoking status and rates of fresh or frozen embryo transfers were comparable among the two groups, and so were gestational age at delivery and birth weight (Table 1). All women achieved pregnancy after 22 WG. Concerning type of ART, 66 women received IVF (82.5%) in group A Vs 88 women (55%) in group B (p<0.001). Conversely, more group B women had undergone ICSI (n = 72, 45%) than patients suffering from endometriosis (n = 14, 17.5%). Moreover, we observed no difference between fresh and frozen ET in the two groups (p = 0.42) (Table 1).

Endometriosis features

In population with several stages of endometriosis, 65 (81.2%) women underwent surgery in order to diagnose or treat peritoneal and ovarian lesions of endometriosis. Surgical managements included resection, ablation or drainage of ovarian endometrioma, deep infiltrating endometriosis ablation and bowel surgery. Although rASRM classification is defined by a laparoscopic assay, we considered that the MRI evaluation is effective for diagnosing severe endometriosis when performed by experienced radiologists [START_REF] Guerriero | Transvaginal ultrasound vs magnetic resonance imaging for diagnosing deep infiltrating endometriosis: systematic review and meta-analysis[END_REF].

Obstetrical outcomes after 22 WG

Analysis of primary outcomes showed that postpartum hemorrhage rate was not higher in endometriosis group (11.2% (n = 9) versus 7.5% (n = 12), p = 0.47) (Table 2). Group A had an increased risk of placenta praevia with 12.5% (n = 10) Vs 1.9% (n = 3), in group B (p<0.001) (Table 2). The cesarean section rate was increased in group A (49.4% (n = 39)) Vs group B 2 (9.6% (n = 47)) (p = 0.004). Neonatal hospitalization rate was similar in group A (22.1% (n = 17)) and in group B (14.7% (n = 21), (p = 0.19)) (Table 2). Multivariate analysis of risk factors associated to all obstetrical outcomes considered together (PPH, placental abruption (RPH), PP and cesarean section), were endometriosis ([adjusted OR] 2.47, CI95% [1.38-4.44], p-value 0.002), BMI ([OR] 1.15, CI95% [1.06-1.25], p-value < 0.001) and gestational age at delivery ([OR] 0.78, CI95% [0.68-0.90], p-value 0.001) (Table 3).

Discussion

In our study, 80 singleton IVF pregnancies (group A) were compared with 160 singleton IVF pregnancies (group B). We observed an increased risk of placenta previa (12.5% Vs 1.9%; p = 0,001), and cesarean section (49.4% (n = 39) Vs 29.6% (n = 47) p = 0,004). Rate of postpartum hemorrhage was not significantly different in endometriosis group (11.2% Vs 7.5% p = 0.47).

Decreased fertility observed in women suffering from endometriosis has been suggested to be due to several pathogenic consequences of this gynecological disease: e.g., mechanical defects, chronic inflammation and ovarian dysfunction [START_REF] Brosens | Risks of adverse pregnancy outcome in endometriosis[END_REF][START_REF] Gupta | Pathogenic mechanisms in endometriosis-associated infertility[END_REF]. Moreover, structural changes in the junctional zone including abnormal remodeling of spiral arteries may cause deficient deep placentation, therefore increasing risk of adverse pregnancy outcomes [START_REF] Brosens | Risks of adverse pregnancy outcome in endometriosis[END_REF]. In our retrospective cohort study, we decided to only include women with moderate and severe endometriosis (rASRM stage III and IV) who was matched by age, BMI and smoking statue to control group (Table 1). We excluded twin pregnancies that are associated with their own adverse pregnancy outcomes, as compared to single pregnancies [START_REF] Vercellini | The ominous association between severe endometriosis, in vitro fertilisation, and placenta previa: raising awareness, limiting risks, informing women[END_REF]. We observed a higher rate of adverse obstetrical outcomes in singleton IVF pregnancy in women with moderate and severe endometriosis, namely placenta previa and cesarean section (Table 2). In agreement, the meta-analysis of Breintoft et al. also reported evidence highlighting the association of endometriosis and placenta previa, cesarean section but also preeclampsia, gravidic hypertension, placental abruption, and stillbirth [START_REF] Breintoft | Endometriosis and Risk of Adverse Pregnancy Outcome: a Systematic Review and Meta-Analysis[END_REF]. Interestingly, in agreement with our findings, results of this meta-analysis didn't show a significant risk of PPH. However, our study presents two major limitations: with a retrospective study of a small sample size. Moreover, we acknowledge the fact that it cannot be excluded that patients in the control group may have had asymptomatic endometriosis; they did not undergo MRI. Nevertheless, the precise potential biological mechanisms underlying any differences between symptomatic and asymptomatic endometriosis, remain poorly characterized [START_REF] Prescott | A prospective cohort study of endometriosis and subsequent risk of infertility[END_REF].

Our study presents several strengths: we only included stages III and IV endometriosis and our two study groups were matched according to maternal characteristics in order to assure comparability. In an effort to study association of obstetrical outcomes with endometriosis as an independent factor not influenced by ART, we compared IVF-pregnancies with or without endometriosis. Importantly, our results agree with previously published data.

Several mother-child morbidities associated with endometriosis are still subject to debate, mainly due to heterogeneity in the design and populations of these studies. For instance, an increased rate of miscarriage in deep and ovarian endometriosis group has been reported, but authors didn't compare miscarriage rate to a control group without endometriosis [START_REF] Vercellini | Pregnancy outcome in women with peritoneal, ovarian and rectovaginal endometriosis: a retrospective cohort study[END_REF]. While, Santulli et al. also found an increased rate of spontaneous miscarriages in endometriosis women [START_REF] Santulli | Increased rate of spontaneous miscarriages in endometriosis-affected women[END_REF], a recent retrospective cohort study of 1006 women with endometriosis, showed that the risk of miscarriage did not statistically increase in women with endometriosis who achieved pregnancy through IVF fresh cycles. The miscarriage rate between women with and without endometriosis was similar [START_REF] Yang | Risk of miscarriage in women with endometriosis undergoing IVF fresh cycles: a retrospective cohort study[END_REF]. These observations showed that the difficulties to conclude on links between endometriosis and obstetrical morbidity.

Previous studies have reported impact of endometriosis on late pregnancy outcome, and have often described an increased risk of premature birth. However, this outcome also remains controversial. Values are presented as numbers (%). P-value less than or equal to 0.05 was considered as statistically significant.

During our study, in accordance with the retrospective study of Benaglia et al. [START_REF] Benaglia | Pregnancy outcome in women with endometriosis achieving pregnancy with IVF[END_REF] we didn't observe a higher risk of preterm delivery < 37 weeks of gestation (Table 1). Conversely, the national Swedish cohort study of Stephansson et al., including 1 442 675 singleton live-births with 13 090 singletons born from 8 922 women diagnosed with endometriosis found a higher risk of preterm birth, antenatal bleeding, preeclampsia and cesarian section [START_REF] Stephansson | Endometriosis, assisted reproduction technology, and risk of adverse pregnancy outcome[END_REF]. After having stratified their statistical analysis, they found that the risk of preterm birth was lower among ART-pregnancies Vs spontaneous pregnancies. Other studies have also reported an increased risk of preterm birth in singleton ART pregnancies obtained in women with endometriosis [START_REF] Breintoft | Endometriosis and Risk of Adverse Pregnancy Outcome: a Systematic Review and Meta-Analysis[END_REF][START_REF] Jacques | Adverse pregnancy and neo-natal outcomes after assisted reproductive treatment in patients with pelvic endometriosis: a case-control study[END_REF]. Two studies have addressed the relationship between adenomyosis and pregnancy outcome. Adenomyosis could be associated with an increased risk of preterm birth and premature rupture of membranes [START_REF] Juang | Adenomyosis and risk of preterm delivery[END_REF][START_REF] Mochimaru | Adverse pregnancy outcomes associated with adenomyosis with uterine enlargement[END_REF]. However, to our knowledge, no studies have yet considered association between endometriosis and adenomyosis as a possible confounding factor. Despite placental complications seem more frequently observed in pregnancies with endometriosis, several studies didn't find a relationship between endometriosis and preeclampsia [START_REF] Lin | Obstetric outcomes in Chinese women with endometriosis: a retrospective cohort study[END_REF][START_REF] Hadfield | Is there an association between endometriosis and the risk of pre-eclampsia? A population based study[END_REF].

In most studies, placenta previa would be more frequent in endometriosis pregnancies. Vercellini et al. reported an influence of deep infiltrative endometriosis on obstetrical outcome. They observed most placenta previa when endometriosis was located at the level of the uterine isthmus [START_REF] Vercellini | Pregnancy outcome in women with peritoneal, ovarian and rectovaginal endometriosis: a retrospective cohort study[END_REF]. Several retrospective cohort studies reported that endometriosis increased risks of placenta previa [START_REF] Benaglia | Pregnancy outcome in women with endometriosis achieving pregnancy with IVF[END_REF] and preterm labor [START_REF] Lin | Obstetric outcomes in Chinese women with endometriosis: a retrospective cohort study[END_REF]. The prospective cohort study of Harada et al. found an increased risk of placenta praevia and PROM [START_REF] Harada | Obstetrical complications in women with endometriosis: a cohort study in Japan[END_REF] among 330 women with history of endometriosis but only 29 women had received ART therapy with 16 IVF and 13 ICSI, making it difficult to conclude. The retrospective cohort study of Fujii et al. also suggested a correlation between endometriosis stages and adverse outcomes during the second and third trimester of ART-pregnancies. They found a high risk of preterm birth and placenta previa in women with endometriosis rASRM stage IV [START_REF] Fujii | Assisted reproductive technology pregnancy complications are significantly associated with endometriosis severity before conception: a retrospective cohort study[END_REF]. Also, the retrospective cohort study of Farella et al. reported that placenta previa was associated with history of stage III and IV endometriosis in ART-conceived pregnancies [START_REF] Farella | Pregnancy outcomes in women with history of surgery for endometriosis[END_REF]. Concerning neonatal morbidity associated to endometriosis, Fernando and al. reported that preterm birth and SGA rates were increased in case of endometriomas [START_REF] Fernando | Preterm birth, ovarian endometriomata, and assisted reproduction technologies[END_REF]. Stern and al. observed only a higher rate of prenatal hospitalizations and not a higher rate of preterm delivery or SGA in endometriosis with ART group [START_REF] Stern | Adverse pregnancy and birth outcomes associated with underlying diagnosis with and without assisted reproductive technology treatment[END_REF]. Conversely, two authors didn't observe a significant risk of obstetrical complications in pregnant women with endometriomas [START_REF] Benaglia | Pregnancy outcome in women with endometriomas achieving pregnancy through IVF[END_REF][START_REF] Roberti Maggiore | A systematic review on endometriosis during pregnancy: diagnosis, misdiagnosis, complications and outcomes[END_REF]. In our study, there was no difference between fresh and frozen ET in the two groups (p = 0.42). However, in a large Nordic Study, Wennerholm et al. (2013) showed that singletons conceived after frozen ET had reduced low birth weight, more preterm birth, and SGA [START_REF] Wennerholm | Perinatal outcomes of children born after frozen-thawed embryo transfer: a Nordic cohort study from the CoNARTaS group[END_REF]. Despite the absence of any suitable randomized trials without bias, a systematic review of observational studies suggested that singleton pregnancies resulting from frozen ET were associated with lower obstetric and perinatal morbidity. The meta-analysis of Maheshwari et al. showed that singleton pregnancies after transfer of frozen embryos were associated with better perinatal outcomes compared with those after fresh IVF embryos [START_REF] Maheshwari | Obstetric and perinatal outcomes in singleton pregnancies resulting from the transfer of frozen thawed versus fresh embryos generated through in vitro fertilization treatment: a systematic review and meta-analysis[END_REF]. The study of Rombauts et al. showed that perinatal outcome wasn't linked to the type of embryo transfer (fresh/frozen) but to the type of endometrial preparation, and suggested that the risk of placenta praevia in ART could be reduced by considering an elective frozen embryo transfer in a natural cycle, supported by growing evidence that this may provide several maternal and neonatal benefits [START_REF] Rombauts | Risk of placenta praevia is linked to endometrial thickness in a retrospective cohort study of 4537 singleton assisted reproduction technology births[END_REF].

Recently, the French longitudinal national study of Epelboin et al. showed that endometriosis is an independent risk factor for several obstetrical and neonatal pathologies, for which ART may additionally affect endometriosis by increasing risk of placenta previa, prematurity and small for gestational age [START_REF] Epelboin | Endometriosis and assisted reproductive techniques independently related to mother-child morbidities: a French longitudinal national study[END_REF]. Our study, contributes to the understanding of pregnancy complications in this population of women with endometriosis. Our results seem to confirm that women with moderate and severe endometriosis have increased adverse pregnancy outcomes like placenta previa and cesarean section even if we didn't find a higher rate of PPH in endometriosis group. It would be interesting to explore specific endometriosis risk factors and outcomes by conducting a large cohort prospective study taking into account rASRM stage and endometriosis localization, but also ART characteristics (fresh or frozen ET, Day 2-3 or blastocyst stage).

Table 1

 1 Pregnancy outcomes after IVF for moderate and severe endometriosis: women's characteristics.Values are presented as Mean § SD or numbers (%). P-value less than or equal to 0.05 was considered as statistically significant. BMI, body mass index; WG, weeks' gestation. ART, assisted reproductive technology. IVF, In vitro fertilization. ICSI, intra cytoplasmic sperm injection.

		Group A IVF for	Group B IVF without	p-Value
		severe	endometriosis
		endometriosis	n = 160
		n = 80
	Age (years)	32.8 § 3.8	32.8 § 3.8	1.00
	BMI (kg/m2)	21.9 § 3.6	21.9 § 3.6	0.98
	Active smoking hab-	30 (37.5)	60 (37.5)	1.00
	its (%)
	Type of ART	66 (82.5)	88 (55.0)	<0.001
	-IVF (%)	14 (17.5)	72 (45.0)
	-ICSI (%)
	-Fresh embryo	64 (80.0)	120 (75.0)	0.42
	transfer (%)
	-Frozen embryo	16 (20.0)	40 (25.0)
	transfer (%)
	Gestational age at	39.0 § 2.6	39.1 § 2.1	0.94
	delivery (WG)
	Birth weight (g)	3050.6 § 641.4	3114.3 § 559.9	0.43

Table 2

 2 Obstetric and neonatal outcomes after IVF for moderate and severe endometriosis: main results.

		Group A IVF for severe endometriosis (n = 80)	Group B IVF without endometriosis (n = 160)	p-value	Odds ratio	95% CI
	Placenta praevia	10	(12.5)	3	(1.9)	0.001	7.4	1.9-28.0
	Placenta abruption	2	(2.5)	3	(1.9)	1.00	1.3	0.2-8.2
	Postpartum hemorrhage	9	(11.2)	12	(7.5)	0.47	1.5	0.6-3.9
	Cesarean section	39	(49.4)	47	(29.6)	0.004	2.3	1.3-4.0
	Neonatal hospitalization	17	(22.1)	21	(14.7)	0.19	1.6	0.8-3.3
	Complications (PPH, PP, RPH, CS)	44	(5.0)	57	(35.6)	0.004	2.2	1.27-3.81

Author's roles E.R. and B.C. have conceived and designed the study. E.R., J.P., J.B., B.C. and A.A. made substantial contributions to acquisition, analysis and interpretation of data, in addition to drafting the article, reviewing the content and final manuscript approval. J.B. performed the analysis and interpretation of data, revising it critically for content and final approval of the version to be published.

Acknowledgments

There was no funding.

Ethics statement

Study was approved by Ethical Committee of Aix-Marseille University (2017-05-04-005).

Study funding/competing interest(s)

The authors have no competing interests to declare.

Declaration of competing interest

None declared.