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

Anàlisi citogenètica i monogènica en cèl·lula única: Diagnòstic Genètic Preimplantacional de Doble Factor

El diagnòstic genètic preimplantacional (PGD) permet diagnosticar embrions obtinguts mitjançant tècniques de reproducció assistida. L’objectiu principal del PGD és diagnosticar els embrions de parelles portadores de malalties hereditàries greus, per tal d’aconseguir el naixement d’individus sans. S’ha postulat que les aneuploïdies són, en molts casos, responsables de la infertilitat o del fracàs de les metodologies de fecundació in vitro (FIV) per això, s’ha desenvolupat una variant del PGD, el PGS (Preimplantational Genetic Screening). Aquest té com a objectiu estudiar alteracions cromosòmiques, ja siguin numèriques o estructurals. Fins a l’actualitat la tècnica més emprada per al diagnòstic d’aquest tipus d’alteracions ha estat la FISH (Hibridació in situ Fluorescent). Rutinàriament amb aquesta metodologia s’estudien 9 cromosomes dels 24 totals del complement. Una altra metodologia per a l’estudi d’aneuploïdies és la CGH (Hibridació Genòmica Comparada), aquesta permet analitzar citogenèticament tot el complement cromosòmic de la cèl·lula d’estudi. L’anàlisi conjunta de malalties monogèniques familiars i d’aneuploïdies de tot el complement cromosòmic conformen el diagnòstic genètic preimplantacional de doble factor (DF-DGP). El primer objectiu d’aquesta tesi ha estat dur a terme un estudi bàsic que permetés aprofundir en el coneixement de les aneuploïdies pre-meiòtiques existents en l’oòcit utilitzant la tècnica de CGH. Estudiant 157 oòcits immadurs de dones de diferents edats i indicacions, s’ha determinat que el 15,9% dels oòcits estudiats presentaven aquest tipus d’anomalies. Aquestes alteracions cromosòmiques d’origen pre-meiòtic es troben en un nivell basal en totes les dones, sense veure’s afectades per l’edat materna o per problemes de fertilitat, i s’han trobat en un 33,9% de les dones estudiades. El segon objectiu ha estat posar a punt una nova aproximació per al DF-DGP, que permetés diagnosticar monogènicament i citogenèticament els embrions a Dia+3, per tal d’obtenir els resultats de l’anàlisi monogènica i citogenètica poc més de 30h després de la biòpsia i poder transferir els embrions sans i euploides dins del mateix cicle de FIV. S’han posat a punt dues aproximacions diferents de DF-DGP. La primera consisteix en biopsiar dos blastòmers dels embrions evolutius per fer-ne servir un per a l’anàlisi monogènica i l’altre per a la citogenètica. La segona aproximació consisteix en biopsiar un sol blastòmer i realitzar el doble diagnòstic a partir d’una sola cèl·lula. Ambdues aproximacions s’han aplicat clínicament en diferents parelles portadores de malalties monogèniques greus, obtenint embarassos i naixements d’individus sans. Algunes d’aquestes aplicacions per a determinades malalties monogèniques com per exemple la síndrome de Lynch, han estat pioneres a l’estat espanyol aconseguint per primera vegada el naixement d’individus sans per la malaltia. En conclusió, durant aquesta tesi doctoral s’ha demostrat la rellevància dels desequilibris cromosòmics en els oòcits i embrions estudiats, amb orígens molt diversos. És per això que les aproximacions del DF-DGP proposades prenen importància, ja que permeten seleccionar els embrions sans i que alhora són euploides, per tant, tindran més probabilitats d’implantar i de donar lloc al naixement d’individus sans. Cal destacar també, que tots aquests procediments diagnòstics es duen a terme durant el mateix cicle de FIV, i es pot realitzar transferència embrionària a final del dia+4 o dia+5, sense haver de criopreservar els embrions, fet que podria reduir la seva viabilitat.Preimplantation Genetic Diagnosis (PGD) enables genetic analysis of embryos produced by assisted reproductive technologies. The aim of PGD is to diagnose embryos from couples carrying monogenic hereditary diseases, and to achieve birth of healthy individuals. It has been described that aneuploidies are the main causes of infertility or failure of IVF techniques (in vitro fertilisation), that is the reason why PGS (Preimplantational Genetic Screening) was developed. The most frequent technique to perform PGS is FISH (Fluorescent in situ hybridisation) where usually 9 chromosomes are analysed. Another methodology to screen aneuploidies is CGH (Comparative Genomic Hybridisation), which permits the assessment of the whole chromosome complement of the cell. Double Factor Preimplantation Genetic Diagnosis (DF-PGD) is based on the analysis of single gene diseases together with comprehensive aneuploidy screening of the embryos. The first objective of this work was to perform a basic study to have a better knowledge of the pre-meiotic aneuploidies found in oocytes using CGH. Different female groups were studied according to age and fertility problems. A total of 157 immature oocytes were analysed, and 15,9% of them showed aneuploidies originated before entering meiosis. The differences between the studied groups showed no significant differences. These type of abnormalities have been found in 33,9% of the females. The second aim of the study was to set up a new methodology for DF-PGD, that permits not only monogenic but also cytogenetic diagnosis of cleavage-stage embryos. Two different approaches were applied, the first one consists in biopsying two blastomeres from each evolved embryo at day+3, and use one of them for monogenic analysis, and the other for comprehensive aneuploidy screening by CGH. The other strategy was to biopsy a single blastomere to perform both analyses from the single biopsied cell. These two methodologies have been applied clinically in different diseases and birth of healthy individuals was achieved. Certain monogenic diseases such as Lynch syndrome, had never been analysed before by PGD in our country, but in this work were applied for the first time and birth of healthy offspring was achieved. In conclusion, this work has demonstrated the importance of chromosomal abnormalities originated in different stages either in oocytes or blastomeres. That is the reason why DF-PGD approaches catch importance, because they permit the selection of not only healthy but also euploid embryos for transfer. Therefore, theses embryos would have more implantation potential. All these DF-PGD procedures can be performed within approximately 30h, which permits embryo transfer in the same IVF cycle, avoiding cryopreservation steps that might be detrimental for the embryos

Anàlisi citogenètica i monogènica en cèl·lula única : diagnòstic genètic preimplantacional de doble factor /

El diagnòstic genètic preimplantacional (PGD) permet diagnosticar embrions obtinguts mitjançant tècniques de reproducció assistida. L'objectiu principal del PGD és diagnosticar els embrions de parelles portadores de malalties hereditàries greus, per tal d'aconseguir el naixement d'individus sans. S'ha postulat que les aneuploïdies són, en molts casos, responsables de la infertilitat o del fracàs de les metodologies de fecundació in vitro (FIV) per això, s'ha desenvolupat una variant del PGD, el PGS (Preimplantational Genetic Screening). Aquest té com a objectiu estudiar alteracions cromosòmiques, ja siguin numèriques o estructurals. Fins a l'actualitat la tècnica més emprada per al diagnòstic d'aquest tipus d'alteracions ha estat la FISH (Hibridació in situ Fluorescent). Rutinàriament amb aquesta metodologia s'estudien 9 cromosomes dels 24 totals del complement. Una altra metodologia per a l'estudi d'aneuploïdies és la CGH (Hibridació Genòmica Comparada), aquesta permet analitzar citogenèticament tot el complement cromosòmic de la cèl·lula d'estudi. L'anàlisi conjunta de malalties monogèniques familiars i d'aneuploïdies de tot el complement cromosòmic conformen el diagnòstic genètic preimplantacional de doble factor (DF-DGP). El primer objectiu d'aquesta tesi ha estat dur a terme un estudi bàsic que permetés aprofundir en el coneixement de les aneuploïdies pre-meiòtiques existents en l'oòcit utilitzant la tècnica de CGH. Estudiant 157 oòcits immadurs de dones de diferents edats i indicacions, s'ha determinat que el 15,9% dels oòcits estudiats presentaven aquest tipus d'anomalies. Aquestes alteracions cromosòmiques d'origen pre-meiòtic es troben en un nivell basal en totes les dones, sense veure's afectades per l'edat materna o per problemes de fertilitat, i s'han trobat en un 33,9% de les dones estudiades. El segon objectiu ha estat posar a punt una nova aproximació per al DF-DGP, que permetés diagnosticar monogènicament i citogenèticament els embrions a Dia+3, per tal d'obtenir els resultats de l'anàlisi monogènica i citogenètica poc més de 30h després de la biòpsia i poder transferir els embrions sans i euploides dins del mateix cicle de FIV. S'han posat a punt dues aproximacions diferents de DF-DGP. La primera consisteix en biopsiar dos blastòmers dels embrions evolutius per fer-ne servir un per a l'anàlisi monogènica i l'altre per a la citogenètica. La segona aproximació consisteix en biopsiar un sol blastòmer i realitzar el doble diagnòstic a partir d'una sola cèl·lula. Ambdues aproximacions s'han aplicat clínicament en diferents parelles portadores de malalties monogèniques greus, obtenint embarassos i naixements d'individus sans. Algunes d'aquestes aplicacions per a determinades malalties monogèniques com per exemple la síndrome de Lynch, han estat pioneres a l'estat espanyol aconseguint per primera vegada el naixement d'individus sans per la malaltia. En conclusió, durant aquesta tesi doctoral s'ha demostrat la rellevància dels desequilibris cromosòmics en els oòcits i embrions estudiats, amb orígens molt diversos. És per això que les aproximacions del DF-DGP proposades prenen importància, ja que permeten seleccionar els embrions sans i que alhora són euploides, per tant, tindran més probabilitats d'implantar i de donar lloc al naixement d'individus sans. Cal destacar també, que tots aquests procediments diagnòstics es duen a terme durant el mateix cicle de FIV, i es pot realitzar transferència embrionària a final del dia+4 o dia+5, sense haver de criopreservar els embrions, fet que podria reduir la seva viabilitat.Preimplantation Genetic Diagnosis (PGD) enables genetic analysis of embryos produced by assisted reproductive technologies. The aim of PGD is to diagnose embryos from couples carrying monogenic hereditary diseases, and to achieve birth of healthy individuals. It has been described that aneuploidies are the main causes of infertility or failure of IVF techniques (in vitro fertilisation), that is the reason why PGS (Preimplantational Genetic Screening) was developed. The most frequent technique to perform PGS is FISH (Fluorescent in situ hybridisation) where usually 9 chromosomes are analysed. Another methodology to screen aneuploidies is CGH (Comparative Genomic Hybridisation), which permits the assessment of the whole chromosome complement of the cell. Double Factor Preimplantation Genetic Diagnosis (DF-PGD) is based on the analysis of single gene diseases together with comprehensive aneuploidy screening of the embryos. The first objective of this work was to perform a basic study to have a better knowledge of the pre-meiotic aneuploidies found in oocytes using CGH. Different female groups were studied according to age and fertility problems. A total of 157 immature oocytes were analysed, and 15,9% of them showed aneuploidies originated before entering meiosis. The differences between the studied groups showed no significant differences. These type of abnormalities have been found in 33,9% of the females. The second aim of the study was to set up a new methodology for DF-PGD, that permits not only monogenic but also cytogenetic diagnosis of cleavage-stage embryos. Two different approaches were applied, the first one consists in biopsying two blastomeres from each evolved embryo at day+3, and use one of them for monogenic analysis, and the other for comprehensive aneuploidy screening by CGH. The other strategy was to biopsy a single blastomere to perform both analyses from the single biopsied cell. These two methodologies have been applied clinically in different diseases and birth of healthy individuals was achieved. Certain monogenic diseases such as Lynch syndrome, had never been analysed before by PGD in our country, but in this work were applied for the first time and birth of healthy offspring was achieved. In conclusion, this work has demonstrated the importance of chromosomal abnormalities originated in different stages either in oocytes or blastomeres. That is the reason why DF-PGD approaches catch importance, because they permit the selection of not only healthy but also euploid embryos for transfer. Therefore, theses embryos would have more implantation potential. All these DF-PGD procedures can be performed within approximately 30h, which permits embryo transfer in the same IVF cycle, avoiding cryopreservation steps that might be detrimental for the embryos

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

Oligonucleotide Arrays <i>vs.</i> Metaphase-Comparative Genomic Hybridisation and BAC Arrays for Single-Cell Analysis: First Applications to Preimplantation Genetic Diagnosis for Robertsonian Translocation Carriers

<div><p>Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers.</p></div

Examples of a) totally coincident profiles and b) highly similar profiles, obtained with oligonucleotide aCGH and BAC aCGH.

<p>Oligonucleotide aCGH profiles are shown at the top of the figure and BAC-aCGH profiles are shown below.</p

Examples of a) oligonucleotide aCGH profiles and b) their corresponding mCGH profiles.

<p>The first profile from a) and b) correspond to one blastomere with totally coincident profiles between techniques, and the second profile from a) and b) correspond to one blastomere with highly similar cytogenetic results.</p

Cytogenetic results obtained from human single blastomeres analyzed by BAC aCGH and oligonucleotide aCGH.

<p>BL: Blastomere; SC: Sexual chromosomes, A: Aneuploidies; S: Segmental imbalances.</p><p>Cells 19-34: PGDs for six different couples with either male factor or repeated implantation failures.</p><p>Cytogenetic results obtained from human single blastomeres analyzed by BAC aCGH and oligonucleotide aCGH.</p

Cytogenetic results obtained from human single blastomeres analyzed by mCGH and oligonucleotide aCGH.

<p>BL: Blastomere; SC: Sexual chromosomes, A: Aneuploidies; S: Segmental imbalances.</p><p>Cells 10-13: Globozoospermia PGD case, cells 14-18: Robertsonian translocation carrier PGD case (45,XY,der(13;14)(q10;q10)).</p><p>Cytogenetic results obtained from human single blastomeres analyzed by mCGH and oligonucleotide aCGH.</p

Resolution, procedure duration and costs of mCGH, BAC aCGH and oligonucleotide aCGH (8x60K and 4x180K formats).

<p>One person performing analysis.</p><p>Resolution, procedure duration and costs of mCGH, BAC aCGH and oligonucleotide aCGH (8x60K and 4x180K formats).</p