The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis: toward an international consensus

Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented.

# Introduction
Cystic fibrosis (CF, OMIM #219700) is one of the most common life-shortening autosomal recessive disorders in populations of European descent, affecting about 1 in 4000 individuals. 1 It is also present in most other ethnicities, but the disease frequency varies greatly. CF is caused by sequence variations in the Cystic fibrosis transmembrane conductance regulator (CFTR, OMIM #602421) gene on 7q31.2, which lead to absent or decreased chloride and bicarbonate transports across the apical membranes of secretory epithelial cells, elevated intracellular sodium and decreased extracellular water. This results in thickened secretions in affected structures (airways, pancreatic and biliary ducts, intestines and vas deferens). Although the severity of symptoms is variable, most patients with CF typically present progressive obstructive lung disease with persistent airway colonization and bronchiectasis, pancreatic insufficiency, gastrointestinal and nutritional anomalies, abnormally high sweat chloride levels and male infertility due to absent or occluded vasa deferentia. Early detection of CF through newborn screening programs is of major importance for the improvement of both survival and quality of life of patients. [bib_ref] European best practice guidelines for cystic fibrosis neonatal screening, Castellani [/bib_ref] [bib_ref] Lessons learned from 20 years of newborn screening for cystic fibrosis, Massie [/bib_ref] [bib_ref] Newborn screening for cystic fibrosis, Wagener [/bib_ref] Close to 2000 unique variations have been identified so far in the CFTR gene (http://www.genet.sickkids.on.ca), either in CF, in CFTRrelated disorders (CFTR-RD), or in asymptomatic individuals. The term CFTR-RD refers to a distinct spectrum of nonlethal diseases that do not fulfill diagnostic criteria for CF but show evidence of CFTR dysfunction, [bib_ref] Recommendations for the classification of CFTR-related disorders, Bombieri [/bib_ref] including congenital bilateral absence of vas deferens (CBAVD), 6-12 recurrent pancreatitis, 13,14 disseminated bronchiectasis, [bib_ref] CFTR gene mutations in adults with disseminated bronchiectasis, Girodon [/bib_ref] or CFTR-related metabolic syndrome [bib_ref] Cystic Fibrosis Foundation practice guidelines for the management of infants with cystic..., Borowitz [/bib_ref] (Box 1, [fig_ref] Table 1: Examples of common CF-causing, indetermined, and non CF-causing variants [/fig_ref]. Besides the most common CF disease-causing variant, a 3-bp deletion c.1521_1523delCTT known as p.Phe508del, that accounts for approximately 67% of CFTR mutant alleles in Europe, another 130 variants have been clinically, genetically and experimentally classified as CF-causing. [bib_ref] Defining the disease liability of variants in the cystic fibrosis transmembrane conductance..., Sosnay [/bib_ref] The clinical consequences of many other variants, particularly those that change an amino acid in the protein (missense variants) are largely uncharacterized. In addition, some pathogenic variants are not necessarily fully penetrant [bib_ref] The very low penetrance of cystic fibrosis for the R117H mutation: a..., Thauvin-Robinet [/bib_ref] (eg their deleterious effect depends on other factors). The number of variants of unknown (clinical) significance (VUCS or VUS) is predicted to increase with the expansion of next generation sequencing methods for CF carrier screening, which is among the first general population genetic screening tests, with worldwide probably more than 10 millions tests each year. Assessing disease causality for CFTR variants is extremely challenging [bib_ref] The very low penetrance of cystic fibrosis for the R117H mutation: a..., Thauvin-Robinet [/bib_ref] [bib_ref] and p.D1270N CF causing mutations, Claustres [/bib_ref] [bib_ref] Genotype-phenotype correlation and frequency of the 3199del6 cystic fibrosis mutation among I148T..., Monaghan [/bib_ref] owing to their number and variety [bib_ref] A classification model relative to splicing for variants of unknown clinical significance:..., Raynal [/bib_ref] and the complexity of genotype-phenotype relationship in CF or CFTR-RDs (some identical genotypes may be associated with different phenotypes). Interpreting CFTR variations as 'pathogenic' or 'neutral' and, beyond, as 'CF-causing' or 'non CF-causing', has evident impact on diagnosis, genetic counseling and reproductive choices of patients and families.
Evidence from practice indicates that whilst natural conception followed by prenatal diagnostic (PND) was in the past the most common reproductive choice for many couples at risk of passing on CF, preimplantation genetic diagnosis (PGD) is increasingly being seen as a preferred choice. So far, the most used approach for PGD for CF involves the biopsy of one blastomere from 3 days-old embryos following ICSI (intracytoplasmic sperm injection) at the 6-10 cell stage. [bib_ref] Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification, Handyside [/bib_ref] Blastomeres are then analyzed and embryos shown to be free of the disease are transferred to the maternal uterus. This procedure Box 1 The spectrum of CFTR-associated diseases  Classical or typical cystic fibrosis (CF) includes the following features:
Chronic sinopulmonary disease with persistent colonization with typical CF pathogens, Specific gastrointestinal or nutritional abnormalities, Salt-loss syndromes, Obstructive azoospermia, Abnormal sweat test (chloride concentration 60 mmol/l), Two CF-causing variants in trans.
Over 85% of these patients have two severe CF-causing variants and are pancreatic insufficient (CF-PI). A minority of CF patients carry at least one 'moderate' variant, which confers some residual CFTR channel function maintaining sufficient exocrine pancreatic function to allow normal digestion of nutrients (pancreatic sufficiency, CF-PS).
In many countries, CF is now diagnosed through newborn screening detecting abnormal values of immunoreactive trypsinogen in the blood, on the basis of abnormal sweat test and two CF-causing variants.  Nonclassical or atypical CF A small proportion of patients have symptoms characteristic of CF in at least one organ but a negative (o30 mmol/l) or borderline (30-60 mmol/l) sweat test. They are CF-PS and exhibit a milder and late lung disease. The diagnosis is confirmed by the identification of variants (either one mild in trans with a severe one, or two mild variants), and by abnormal nasal potential difference measurement or intestinal current measurement using rectal biopsy. Individuals in this group have an increased risk of pancreatitis.  CFTR-related disorders (CFTR-RD) [bib_ref] Recommendations for the classification of CFTR-related disorders, Bombieri [/bib_ref] A CFTR-RD is defined as a clinical entity with evident CFTR dysfunction that does not fulfill the CF diagnostic criteria. Three distinct subgroups must be distinguished: congenital bilateral absence of the vas deferens, acute recurrent or chronic pancreatitis, and disseminated bronchiectasis.
Congenital bilateral absence of the vas deferens (CBAVD) [bib_ref] Abnormal distribution of CF deltaF508 allele in azoospermic men with congenital aplasia..., Dumur [/bib_ref] [bib_ref] Mutations in the cystic fibrosis gene in patients with congenital absence of..., Chillon [/bib_ref] [bib_ref] Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of..., Claustres [/bib_ref] [bib_ref] Variation in a repeat sequence determines whether a common variant of the..., Groman [/bib_ref] [bib_ref] Molecular pathology of the CFTR locus in male fertility, Claustres [/bib_ref] [bib_ref] Arg117H is associated with CBAVD and other CFTR-related disorders, Thauvin-Robinet [/bib_ref] [bib_ref] Preimplantation genetic diagnosis for cystic fibrosis: the Montpellier center's 10-year experience, Girardet [/bib_ref] CBAVD is the most common cause of infertility in otherwise healthy males, with an incidence of 1:1000 men, and is present in all ethnicities. The male reproductive tract is the most sensitive tissue to CFTR dysfunction, so that CFTR variations not severe enough to cause CF can be responsible for vas deferens aplasia. Up to 80% of CBAVD men have two CFTR variants in compound heterozygosity, whereas 20% have either only one or no variant. None of CBAVD men carry two severe CFTR variants: the vast majority carry a mild in trans with a severe variant, and a minor proportion has two mild variants. In CBAVD men from European descent, the two most common genotypes are p.Phe508del in trans either with the '5T allele' in intron 8 (now designated as c.1210-12T [bib_ref] Recommendations for the classification of CFTR-related disorders, Bombieri [/bib_ref] in intron 9) (30%) or with the variants R117H-7T (p. Arg117His associated with a 7T allele (c.1210-12T [bib_ref] Mutations in the cystic fibrosis gene in patients with congenital absence of..., Chillon [/bib_ref] (6%). In humans, the variant 5T, also present in the general population at a frequency of 0.05, alters the splicing signals and leads to the skipping of exon 9 (10 in the HGVS nomenclature) in a proportion of transcripts. The skipping varies among tissues and individuals, which generates a proportion of incomplete and nonfunctional CFTR proteins; the result is a reduced quantity of normal CFTR protein. The disease penetrance of 5T is incomplete (some men carrying a 5T in trans with a severe variant are fertile), and is influenced by another polymorphic sequence immediately upstream, the 'TG tract': the longer the TGn and the shorter the Tn repeats are, the greater is the reduction in full-length CFTR transcripts. CBAVD is more likely to occur in males carrying a 5T associated in cis with 12TG (c.[1210-34TG [bib_ref] Preimplantation genetic diagnosis for cystic fibrosis: the Montpellier center's 10-year experience, Girardet [/bib_ref] ;1210-12T [bib_ref] Recommendations for the classification of CFTR-related disorders, Bombieri [/bib_ref] ] or 13TG (c.[1210-34TG [bib_ref] A conservative assessment of the major genetic causes of idiopathic chronic pancreatitis:..., Masson [/bib_ref] ;1210-12T [bib_ref] Recommendations for the classification of CFTR-related disorders, Bombieri [/bib_ref] ] than 11TG (c.[1210-34TG [bib_ref] Arg117H is associated with CBAVD and other CFTR-related disorders, Thauvin-Robinet [/bib_ref] ;1210-12T [bib_ref] Recommendations for the classification of CFTR-related disorders, Bombieri [/bib_ref] ] repeats. Thus the determination of TG repeat number is predictive of the pathogenicity of the 5T alleles.
Spermatogenesis in CBAVD males is normal, thus patients can be treated by sperm extraction followed by ICSI. If the female partner also carries a CF-causing variant, the offspring has an increased risk (25%) of CF (the CF variants of both partners), and male offspring have, depending on the penetrance of mild variants, up to 25% additional chance of having CBAVD (the mild variant of the father and the CF variant of the mother). Genetic counselling may be particularly difficult in couples with a CBAVD partner as they often carry variants with a wide range of effects from normal to CFTR-RD. [bib_ref] Arg117H is associated with CBAVD and other CFTR-related disorders, Thauvin-Robinet [/bib_ref] [bib_ref] Preimplantation genetic diagnosis for cystic fibrosis: the Montpellier center's 10-year experience, Girardet [/bib_ref] Idiopathic chronic pancreatitis and recurrent acute pancreatitis: approximately 30% of individuals in this group who do not fulfill the diagnostic criteria for CF are found with at least one CFTR variant and sweat chloride values in the intermediate range. [bib_ref] A conservative assessment of the major genetic causes of idiopathic chronic pancreatitis:..., Masson [/bib_ref] They include a small proportion with compound heterozygosity for a CF-causing and a mild CFTR variant (including the 5T allele) and a majority of carriers of a single CF or CFTR-RD variant with increased susceptibility to pancreas dysfunction. Recent studies suggest that certain missense variants could selectively alter the bicarbonate transport while chloride function is preserved, thus affecting only tissues that necessitate CFTR for bicarbonate secretion such as pancreas. 14 Disseminated bronchiectasis: 15-25% of individuals with abnormal and irreversible dilatation of thick-walled bronchi are found to carry CFTR variants, mostly with residual CFTR function (including a high proportion of allele 5T) and mostly in the heterozygous state. CFTR variants may act as risk alleles for multigenic disorders in the general population. [bib_ref] CFTR gene mutations in adults with disseminated bronchiectasis, Girodon [/bib_ref] CFTR-related metabolic syndrome (CRMS): Asymptomatic newborns with an inconclusive diagnosis following a positive NBS test (abnormal immunoreactive trypsinogen levels but nondiagnostic sweat chloride or genetic analysis). They may either remain symptom free or develop mild CF or CFTR-RD later in life, and therefore need regular follow-up. [bib_ref] Cystic Fibrosis Foundation practice guidelines for the management of infants with cystic..., Borowitz [/bib_ref] avoids the dilemma of therapeutic abortion following PND by chorionic villus sampling or amniocentesis in the first or second trimester of pregnancy. As the blastomere biopsy procedure on day 3 can be traumatic to the embryo, reducing its developmental potential, there is an increasing tendency to move to the day 5 or 6 trophectoderm biopsy at the blastocyst stage. [bib_ref] Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does..., Scott [/bib_ref] However, blastocyst biopsy is more challenging than blastomere biopsy, and only some centers (especially those that test PGD embryos for aneuploidy [bib_ref] First systematic experience of preimplantation genetic diagnosis for single-gene disorders, and/or preimplantation..., Retchisky [/bib_ref] have changed their protocols so far. CF is one of the most common indications of PGD worldwide, accounting for almost 10% of all indications for monogenic disorders, as recorded in the European Society of Human Reproduction and Embryology (ESHRE) PGD consortium data collection XII. [bib_ref] with pregnancy follow-up to, Moutou [/bib_ref] Best practice guidelines for amplification-based PGD dealing with infrastructures, equipment and materials as well as molecular procedures (pre-examination, examination and post-examinations processes) have been established and updated by the ESHRE consortium. [bib_ref] ESHRE PGD Consortium "best practice guidelines for clinical preimplantation genetic diagnosis (PGD)..., Thornhill [/bib_ref] [bib_ref] ESHRE PGD consortium best practice guidelines for amplification-based PGD, Harton [/bib_ref] These guidelines describe general recommendations on genetic practices for PGD programs, in order to build a consensus opinion on best practices within the PGD community and to achieve a minimum standard across all centers. However, due to the absence of a universal, standardized PGD assay, each center develops its own inhouse methods. Such methods are based on direct screening of specific variants, [bib_ref] Assessment of the reliability of single blastomere analysis for preimplantation diagnosis of..., Ray [/bib_ref] [bib_ref] Clinical application of preimplantation genetic diagnosis for cystic fibrosis, Goossens [/bib_ref] linkage analysis using informative microsatellites, [bib_ref] Multiplex PCR of polymorphic markers flanking the CFTR gene; a general approach..., Dreesen [/bib_ref] [bib_ref] Duplex, triplex and quadruplex PCR for the preimplantation genetic diagnosis (PGD) of..., Moutou [/bib_ref] or a combination of direct and indirect testing. [bib_ref] The minisequencing method, an alternative strategy for preimplantation genetic diagnosis of single..., Fiorentino [/bib_ref] [bib_ref] Clinical outcome of preimplantation genetic diagnosis for cystic fibrosis: the Brussels'experience, Keymolen [/bib_ref] Some centers use fluorescent multiplex PCR protocols, others perform nested PCRs, and a few centers use Whole Genome Amplification techniques (mainly Multiple Displacement Amplification, MDA) to generate larger quantities of DNA template, followed by haplotyping of several polymorphic markers. [bib_ref] Preimplantation genetic haplotyping: 127 diagnostic cycles demonstrating a robust, efficient alternative to..., Renwick [/bib_ref] [bib_ref] Clinical application of multiple displacement amplification in preimplantation genetic diagnosis, Hellani [/bib_ref] Many health practitioners, even among highly trained specialists in academic medical institutions and including those in reproductive medicine, wish more information about genetic testing and counseling. [bib_ref] Cystic fibrosis carrier screening in obstetric clinical practice: knowledge, practices, and barriers,..., Darcy [/bib_ref] The guidelines presented here are the result of a meeting held in December 2011 with the partnership of EuroGentest (www. eurogentest.org). The main purpose of the meeting was to formulate more specific PGD guidelines for CF-PGD, in line with the general guidelines for amplification-based PGD, [bib_ref] ESHRE PGD consortium best practice guidelines for amplification-based PGD, Harton [/bib_ref] for organization of a PGD center for PGD/PGS 37 and for polar body and embryo biopsy [bib_ref] Consortium/Embryology Special Interest Group -best practice guidelines for polar body and embryo..., Harton [/bib_ref] previously published by ESHRE. The specific guidelines are based on experience with CFTR genetic testing, and will contribute to the harmonization and quality of procedures, methods and protocols and to the standardization of reporting results for cystic fibrosis. We also present updated information on CFTR variation including nomenclature, classification of variants, and genotype-phenotype correlations.

# Materials and methods
In order to discuss relevant technical and genetic issues in PGD for cystic fibrosis, a 'Best practice guidelines for CF-PGD meeting' was held in Montpellier, France, on 14 December 2011. The meeting brought together eighteen experts in the fields of PGD and molecular diagnosis of CF and CFTR-RDs from seven countries (Spain, Belgium, Italy, USA, UK, Greece and France). Presentations and subsequent discussions formed the basis for establishing specific guidelines.

## Nomenclature
Since the discovery of the CFTR gene in 1989, the CF scientific community developed its own nomenclature system (Cystic Fibrosis Mutation Database, (http://www.genet.sickkids.on.ca/)). Despite the introduction of a worldwide standardized Human Genome Variation Society (HGVS) nomenclature, 39,40 the original ('legacy') names are still commonly used for CFTR variants and polymorphisms in clinical services, whereas diagnostic laboratories use both nomenclatures simultaneously to ensure compatibility with previous clinical reports and understanding by clinicians and couples. The HGVS DNA variant numbering system is based on cDNA, defining position '+1' as the A of the ATG translation initiation codon in the CFTR reference sequence (NM_000492.3), with initiation codon as codon 1 and exons numbered 1-27. In contrast, in the legacy (traditional) annotation, '+1' was the first base of the mRNA major transcript, and the A of the ATG was consequently nucleotide 133. These two different nomenclatures can lead to confusion and serious erroneous interpretation and reporting of the location of variants. For example, the well-known 1717-1G4A and 3120+1G4A variants (legacy names) are designated as c.1585-1G4A and c.2988+1G4A, respectively, according to the HGVS nomenclature. Not being aware of nomenclature changes and testing a variant at a wrong position in the gene will ultimately lead to erroneous PND or PGD. Variants nomenclature is a complex and evolving process, therefore constant vigilance is needed for accurate reporting. [bib_ref] Mutation nomenclature in practice: findings and recommendations from the cystic fibrosis external..., Berwouts [/bib_ref] It is strongly recommended to obtain the original molecular genetic reports including the description of identified variants in both the HGVS and legacy nomenclatures. Then, it is recommended to verify the reported variants on new DNA samples from the couples, using standard molecular techniques (Box 2).
Traditional and HGVS nomenclatures for the most common CF-causing, non CF-causing and variants with varying clinical consequences, and examples of common CFTR genotypes nomenclatures, are presented in Tables 1 and 2, respectively. Useful Internet resources are compiled in Box 3.

## Inclusion criteria
Multiple occasions arise to detect individuals carrying CFTR variants [fig_ref] Table 3: Main indications for CFTR testing [/fig_ref] , who may become future candidates for PGD. Some variants are not causing CF but have been included in commercially available population-based carrier screening panels. These kits may be used by accredited and non-accredited laboratories, thus accreditation does not guarantee the non-detection of clinically non-significant variants. Couples may not have been correctly counseled about the risks. [bib_ref] The dangers of including nonclassical cystic fibrosis variants in population-based screening panels:..., Strom [/bib_ref] [bib_ref] Is there evidence of correct diagnosis in cystic fibrosis registries?, Thomas [/bib_ref] Before enrolling a couple for a PGD procedure for CF, it is recommended to evaluate within a multidisciplinary team whether the variants identified in both partners should be considered for PND or PGD. Variants may be grouped in four categories: (i) loss-of-Box 2 Importance of verifying the presence of variants initially reported in each member of the couple requesting a CF-PGD Analysing the DNA of each member of the couple will confirm the initial result and will confirm that both parents carry at least one CF-causing variant.
For example, inability to confirm the variants in both parents may be found in cases with apparent homozygosity for a common or a rare variant: Nonpaternity [bib_ref] Defining the disease liability of variants in the cystic fibrosis transmembrane conductance..., Sosnay [/bib_ref] However, the disease penetrance of the vast majority of hundreds of rare missense variants identified so far remains unknown. It is therefore recommended to work in close collaboration with specialized clinical geneticists and reference laboratories that have gained considerable knowledge in the interpretation of CFTR variants. Otherwise, with respect to variations in local or national regulations and specific laboratory practices, the Montpellier meeting revealed some discrepancies in the way that couples are included or not in CF-PGD programs [fig_ref] Table 4: Inclusion and exclusion criteria for CF-PGD according to countries [/fig_ref]. In several countries, when at least one [bib_ref] and p.D1270N CF causing mutations, Claustres [/bib_ref] [bib_ref] Genotype-phenotype correlation and frequency of the 3199del6 cystic fibrosis mutation among I148T..., Monaghan [/bib_ref] G576A is found in cis with R668C and R668C can be found alone or in cis with G576A. I1027T is usually found in cis with F508del: Notes: (i) Some missense variants classified as either indeterminate or non CF-causing (R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R and D1270N) can selectively alter the bicarbonate permeation of the CFTR channel (but not the chloride channel), thus affecting primarily the organs that utilize CFTR for bicarbonate secretion (pancreas, nasal sinus, or vas deferens) and, consequently, they could be involved in the pathogenic mechanisms of CFTR-RDs. 14 (ii) In [fig_ref] Table 1: Examples of common CF-causing, indetermined, and non CF-causing variants [/fig_ref] , the traditional name of common CFTR variants is referenced alongside the HGVS version in order to ensure compatibility with clinical reports and understanding by clinicians and couples. In the associated text, for reasons of space, some CFTR variants may be referred using traditional names.
severe CF-causing or large spectrum variant is identified in one of the members of the couple, PGD is accepted even though a mild or benign variant is found in the other partner. This is an exclusion criterion in France where PGD is restricted to couples 'having a high probability to give birth to a child affected by a particularly severe genetic disease and recognized as being incurable at the time of diagnosis'. Consequently, only the risk of transmitting CF (and not milder phenotypes such as CFTR-RD) is considered for PGD. In the UK, when one couple member is heterozygous for a variant of unproven or uncertain clinical relevance, PGD is accepted only in the context of a family history of CF. As a precaution, in all participating centres, novel rare variants of unproven or uncertain clinical significance may be considered as potentially CF-causing upon advice of specialists of CFTR alterations.

## Genetic counseling and embryo transfer
Similarly, discrepancies in the regulations about embryo transfer may exist from one country to another. It is recommended that all couples are individually counseled before the clinical cycle and the expected embryo genotypes and corresponding phenotypes are clearly explained.
(i) Rare variants or variants with incomplete penetrance in disease Couples should be aware that in case of a genotype that includes rare variants with incomplete penetrance, individual outcomes cannot be reliably predicted. For instance, an embryo with p.Phe508del in trans with the 5T allele (genotype c.[1210-12 T [bib_ref] Recommendations for the classification of CFTR-related disorders, Bombieri [/bib_ref] ]; [1521_1523delCTT]) may be considered as suitable for transfer in some countries, while in others the parents' wish to preferentially transfer carrier embryos without the IVS8-5 T allele is taken into account. In countries in which embryos compound heterozygous for a severe and a mild/benign variant may be transferred, couples should be informed of possible risks for their children, ranging from no clinical signs to moderate and/or late-onset phenotypes. Some common or rare CFTR variants are associated with a wide phenotypic spectrum and updated information should be provided in light of current knowledge [fig_ref] Table 1: Examples of common CF-causing, indetermined, and non CF-causing variants [/fig_ref].
(ii) Selection of embryos for transfer Couples should be informed that following the identification of genetically transferable embryos a subsequent selection is made based upon the morphology of the embryo and its potential to implant. Thus morphology rather than distinction between carrier embryos and embryos free of both parental germ-line variants, forms the basis of the final selection of the embryo for transfer. Some centres may also include aneuploidy screening in the PGD protocol in order to achieve higher implantation and live birth rates. In these cases, couples need to be appropriately counseled as the identification of aneuploidy may exclude embryos that had been considered transferable on the basis of CFTR analysis alone. Couples should also be made aware that diagnosis may not be achieved for some embryos for various reasons including a failure of amplification or due to cumulus cell contamination. It is useful for the IVF unit to know the live birth rate per cycle started for CF-PGD at their center as well as the outcome following transfer after cryopreservation of surplus transferable embryos. Multiplex PCR protocols have proven to be robust, sensitive and highly accurate, 12,31 however it is recommended in most countries that the results should be confirmed with a prenatal diagnostic test.

## Pgd strategy
Recommended strategies and decision procedures for PGD are presented in the following section. In particularly difficult situations, it is essential that the diagnostic strategies be shared with the referring clinical geneticist.
As mentioned in the amplification-based PGD guidelines, [bib_ref] ESHRE PGD Consortium "best practice guidelines for clinical preimplantation genetic diagnosis (PGD)..., Thornhill [/bib_ref] [bib_ref] ESHRE PGD consortium best practice guidelines for amplification-based PGD, Harton [/bib_ref] it is recommended to perform both indirect (marker based) and direct (variant based) genetic diagnosis.
PGD diagnostic strategies based on polymorphic markers Several microsatellite markers within or close to the CFTR gene can be used in PGD for CF [fig_ref] Table 5: List of markers commonly used in PGD for Cystic FibrosisAbbreviation [/fig_ref]. Laboratories may have to search databases for previously unpublished microsatellite markers in order to obtain sufficient informative markers for a given couple.
It is recommended to offer PGD using at least two fully informative flanking markers within a 1-megabase (Mb, equivalent to one million base pairs) region on both sides of the gene and to include intragenic markers when available.
As recombination events within the CFTR gene have been very rarely described, it is acceptable to perform indirect genetic diagnosis using intragenic microsatellites located on the same side of the variant if markers flanking the CFTR gene are not informative or gave inconclusive results.
When MDA is performed, it is recommended to study at least four fully informative microsatellite markers (two each side of the gene) because of the higher Allele Drop Out (ADO) rates frequently reported. Furthermore, semi-informative or noninformative markers can still be used in an informative haplotype to detect the presence of contamination and/or chromosomal abnormality (presence of one or three haplotypes).
All customized protocols must be thoroughly validated before clinical use. [bib_ref] with pregnancy follow-up to, Moutou [/bib_ref] [bib_ref] ESHRE PGD Consortium "best practice guidelines for clinical preimplantation genetic diagnosis (PGD)..., Thornhill [/bib_ref] Guidelines for CF-PGD A Girardet et al

## Pgd strategies based on variants identified in couples
Case 1: p.Phe508del in one or both couple members. As the most frequent CF variant is the p.Phe508del in exon 11 of the CFTR gene (exon 10 in the traditional nomenclature), it is very common to identify this variant in at least one of the couple members (Box 4). Therefore, it is highly recommended to set up single-cell PCR conditions for the p.Phe508del variant in combination with indirect genetic diagnosis to monitor contamination and preclude misdiagnosis owing to ADO. For couples in whom one of the partners is heterozygous for a variant other than p.Phe508del, it is acceptable to perform a direct diagnosis for the p.Phe508del and an indirect diagnosis for the other variant, provided sufficient informative markers are available to trace inheritance of both chromosomes from the non-p.Phe508del carrier.
When there is a lack of informative markers in the parent carrier of the other variant, it is recommended to specifically study this causative variant (in addition to the p.Phe508del) and also to biopsy and independently analyze two cells instead of one, preferably at the blastocyst stage.
Case 2: Identical variant (other than p.Phe508del) in both couple members. It is recommended to develop PCR conditions for the unique CF-causing variant in addition to indirect genetic diagnosis. It is acceptable to perform only indirect genetic diagnosis for fully informative couples (at least two informative markers o1 Mb flanking the gene or one intragenic marker and one flanking marker).
Case 3: Different variants (other than p.Phe508del) in both couple members. It is acceptable to perform only indirect genetic diagnosis for fully informative couples (at least two informative markers o1 Mb flanking the gene or one intragenic marker and one flanking marker). In cases that lack informativity for markers within or near the gene, it is recommended to specifically study both causative variants and to search and add informative markers located further up-or downstream. The biopsy and analysis of trophectoderm cells at the blastocyst stage, should also help in reducing the misdiagnosis risk due to ADO.

## Reporting
There is currently no formal consensus regarding PGD reports. It also depends on legislation and ethical laws of each country. However, according to recommendations for reporting results of diagnostic genetic testing, [bib_ref] Recommendations for reporting results of diagnostic genetic testing (biochemical, cytogenetic and molecular..., Claustres [/bib_ref] guidelines for accreditation of the PGD laboratory, [bib_ref] Accreditation of the PGD laboratory, Harper [/bib_ref] and recommendations from OECD (Organization for Economic Co-operation and Development), some important items should be included in a PGD report. They are listed below.

## Feasibility report
Laboratory issuing results (affiliation, address, secretarial phone and fax numbers) Date of report Name of the clinician referring the couple Unequivocal identification of the couple (2 identifiers per individual, e.g. name and date of birth) Specimen informationtype of sample and date of sampling (also time if appropriate) Disease (OMIM number, inheritance mode) and gene (OMIM number) being tested for Methods performed and markers used Clearly presented results with appropriate interpretative comments Appropriate reference intervals Error rates clearly stated Variant nomenclature using Human Genome Variation Society (HGVS) recommendations Accession number of reference sequence (including version number) Identification of individual providing results and authorizer

## Pgd case report
Laboratory issuing results (affiliation, address, secretarial phone and fax numbers) Referring clinician: name and affiliation Date of report Title, that is 'PGD for cystic fibrosis' Disease (OMIM number and inheritance mode) and gene (OMIM number) being tested for Unequivocal identification of both couple members (2 identifiers per individual, e.g. name and date of birth) Specimen information: type of sample, date/time of sampling, date of arrival of samples in the lab Methods performed J List of DNA sequences studied J Names of the variants analyzed (using HGVS nomenclature) J Accession number of reference sequence (including version number)
J Limits of the test (risk of recombination, ADO rate if indicated) Results obtained (genotype may be given or not depending on local rules). Clearly presented results with appropriate interpretative comments with clear indication of whether embryos should or should not be transferred Error rates clearly stated Identification of individual providing results and authorizer The report must be signed (some labs sign all steps: experiments, reading, validation) Pagination to include the actual and total number of pages The reports must be sent (faxed or emailed, depending on legislation on confidentiality of medical information) to the referring physician and/or IVF team as described in the official test request form, in time for embryo transfer. Results should be reported only to persons authorized to receive and use this kind of medical information. In some jurisdictions, the lab report must also be sent to the patient (ie Italy).

## Box 4 genotype coverage in cf
With a frequency of p.Phe508del reaching~70% of CF alleles in populations from Northern European descent, almost half of individuals with CF have two copies of the p.Phe508del allele (p 2 =(0.7) 2 =0.49), 42% have one p.Phe508del in trans with another CFTR variant (2pq=20.70.3=0.42) and 9% have two non-p.Phe508del variants (q 2 =(0.3) 2 =0.09). Commercially available testing panels including the 30 most common CF variants detect about 87% of CF alleles, so that it can be predicted that 24% of patients with CF carry at least one rare variant not included in these panels (20.870.13+(0.13) 2 =0.24). However, these features can be very different in Southern-European and non-European populations, who exhibit a much larger proportion of rare variants and a decreased frequency of p.Phe508del.

## Guidelines for cf-pgd
A Girardet et al

## External quality assessment
To meet the ISO 15189 standards, accredited laboratories are required to participate in EQA schemes, which provide an independent evaluation of the quality of their service and inter-laboratory comparison, with an educational purpose. The United Kingdom National External Quality Assessment Service has developed a scheme specifically for molecular PGD, based on experience gained with a three-years pilot scheme for CF-PGD. [bib_ref] The experience of 3 years of external quality assessment of preimplantation genetic..., Deans [/bib_ref] The scheme examines (i) the ability of each participating lab to genotype genomic DNA and single cells (ii) the interpretation of results (including the determination of the limitations of the protocol that was applied for the testing of single cell samples supplied in the EQA), and (iii) the reporting of PGD results. The scheme recommendations were followed by the participating laboratories and led to the improvement in the content of the reports.

## Concluding remarks
This meeting organized on behalf of EuroGentest has highlighted important variation among the participating countries both in technical protocols and in the counseling of couples. It emphasized the need to pursue harmonization and quality assurance, preferably through some form of internationally or nationally recognized accreditation. Given the complexity of phenotype and genotype relationships, the extremely high number of variants found in the CFTR gene, and the reclassification of some variants as (non)pathogenic as more data become available, genetic counseling is mandatory, and less common variants should be reviewed with CFTR experts before acceptance of a PGD CF request.
[table] Table 1: Examples of common CF-causing, indetermined, and non CF-causing variants (modified from5,8,17 ) [/table]
[table] Table 2: Examples of CFTR genotypes in traditional (legacy) and HGVS nomenclatures (modified and updated from Berwouts et al., 2011)41 [/table]
[table] Table 3: Main indications for CFTR testing [/table]
[table] Table 4: Inclusion and exclusion criteria for CF-PGD according to countries [/table]
[table] Table 5: List of markers commonly used in PGD for Cystic FibrosisAbbreviation: NA, not applicable. a UniSTS has migrated to UCSC. b According to UCSC GRCh38/hg38, December 2013. c Exon numbering according to the HGVS nomenclature. Physical distances are taken from location along chromosome 7 according to the UCSC genome browser. [/table]