Preimplantation genetic testing for more than one genetic condition: clinical and ethical considerations and dilemmas.

STUDY QUESTION: Which clinical and ethical aspects of preimplantation genetic testing for monogenic disorders or structural rearrangements (PGT-M, PGT-SR) should be considered when accepting requests and counselling couples for PGT when applied for more than one condition (combination-PGT; cPGT-M/SR)? SUMMARY ANSWER: cPGT is a feasible extension of the practice of PGT-M/SR that may require adapting the criteria many countries have in place with regard to indications-setting for PGT-M/SR, while leading to complex choices that require timely counselling and information. WHAT IS KNOWN ALREADY: Although PGT-M/SR is usually performed to prevent transmission of one disorder, requests for PGT-M/SR for more than one condition (cPGT-M/SR) are becoming less exceptional. However, knowledge about implications for a responsible application of such treatments is lacking. STUDY DESIGN, SIZE, DURATION: Retrospective review of all (40) PGT-M/SR applications concerning more than one genetic condition over the period 1995-2018 in the files of the Dutch national PGT centre. This comprises all relevant national data since the start of PGT in the Netherlands. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Data regarding cPGT-M/SR cases were collected by means of reviewing medical files of couples applying for cPGT-M/SR. Ethical challenges arising with cPGT-M/SR were explored against the background of PGT-M/SR regulations in several European countries, as well as of relevant ESHRE-guidance regarding both indications-setting and transfer-decisions. MAIN RESULTS AND THE ROLE OF CHANCE: We report 40 couples applying for cPGT-M/SR of which 16 couples started their IVF treatment. Together they underwent 39 IVF cycles leading to the birth of five healthy children. Of the couples applying for cPGT, 45% differentiated between a primary and secondary condition in terms of perceived severity. In the light of an altered balance of benefits and drawbacks, we argue the 'high risk of a serious condition' standard that many countries uphold as governing indications-setting, should be lowered for secondary conditions in couples who already have an indication for PGT-M/SR. As a consequence of cPGT, professionals will more often be confronted with requests for transferring embryos known to be affected with a condition that they were tested for. In line with ESHRE guidance, such transfers may well be acceptable, on the condition of avoiding a high risk of a child with a seriously diminished quality of life. LIMITATIONS, REASONS FOR CAUTION: We are the first to give an overview of cPGT-M/SR treatments. Retrospective analysis was performed using national data, possibly not reflecting current trends worldwide. WIDER IMPLICATIONS OF THE FINDINGS: Our observations have led to recommendations for cPGT-M/SR that may add to centre policy making and to the formulation of professional guidelines. Given that the introduction of generic methods for genomic analysis in PGT will regularly yield incidental findings leading to transfer requests with these same challenges, the importance of our discussion exceeds the present discussion of cPGT. STUDY FUNDING/COMPETING INTEREST(S): The research for this publication was funded by the Dutch Organization for Health Research and Development (ZonMw), project number: 141111002 (Long term safety, quality and ethics of Preimplantation Genetic Diagnosis). None of the authors has any competing interests to declare.

ESHRE PGD Consortium data collection XIV-XV: cycles from January 2011 to December 2012 with pregnancy follow-up to October 2013.

STUDY QUESTION: How does the data collection XIV-XV of the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium compare with the cumulative data for data collections I-XIII? SUMMARY ANSWER: The 14th and 15th retrospective collection represents valuable data on PGD/PGS cycles, pregnancies and children: the main trend observed is the increased application of array technology at the cost of FISH testing in PGS cycles and in PGD cycles for chromosomal abnormalities. WHAT IS KNOWN ALREADY: Since 1999, the PGD Consortium has collected, analysed and published 13 previous data sets and an overview of the first 10 years of data collections. STUDY DESIGN, SIZE, DURATION: Data were collected from each participating centre using a FileMaker Pro database (versions 5-12). Separate predesigned FileMaker Pro files were used for the cycles, pregnancies and baby records. The study documented cycles performed during the calendar years 2011 and 2012 and follow-up of the pregnancies and babies born which resulted from these cycles (until October 2013). PARTICIPANTS/MATERIALS, SETTINGS, METHOD: Data were submitted by 71 centres (full PGD Consortium members). Records with incomplete or inconsistent data were excluded from the calculations. Corrections, calculations and tables were made by expert co-authors. MAIN RESULTS AND THE ROLE OF CHANCE: For data collection XIV-XV, 71 centres reported data for 11 637 cycles with oocyte retrieval (OR), along with details of the follow-up on 2147 pregnancies and 1755 babies born. A total of 1953 cycles to OR were reported for chromosomal abnormalities, 144 cycles to OR for sexing for X-linked diseases, 3445 cycles to OR for monogenic diseases, 6095 cycles to OR for PGS and 38 cycles to OR for social sexing. From 2010 until 2012, the use of arrays for genetic testing increased from 4% to 20% in PGS and from 6% to 13% in PGD cycles for chromosomal abnormalities; the uptake of biopsy at the blastocyst stage (from <1% up to 7%) was only observed in cycles for structural chromosomal abnormalities, alongside the application of array comparative genomic hybridization. LIMITATIONS, REASONS FOR CAUTION: The findings apply to the 71 participating centres and may not represent worldwide trends in PGD. WIDER IMPLICATIONS OF THE FINDINGS: The annual data collections provide an important resource for data mining and for following trends in PGD/PGS practice. STUDY FUNDING/COMPETING INTEREST(S): None.

ESHRE PGD Consortium data collection XIII: cycles from January to December 2010 with pregnancy follow-up to October 2011.

STUDY QUESTION: How do data in the 13th annual data collection (Data XIII) of the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium compare with the cumulative data for collections I-XII? SUMMARY ANSWER: The 13th retrospective collection represents valuable data on PGD/PGS cycles, pregnancies and children: the main trend observed is the decrease in the routine implementation of PGS. WHAT IS KNOWN ALREADY: Since 1999, the PGD Consortium has collected, analysed and published 12 data sets and an overview of the first 10 years of data collections. STUDY DESIGN, SIZE, DURATION: Data were collected from each participating centre using a FileMaker Pro database (versions 5-11). Separate predesigned FileMaker Pro files were used for the cycles, pregnancies and baby records. The study documented cycles performed during the calendar year 2010 and follow-up of the pregnancies and babies born which resulted from these cycles (until October 2011). PARTICIPANTS/MATERIALS, SETTING, METHODS: Data were submitted by 62 centres (full PGD Consortium members). The submitted data were thoroughly analysed to identify incomplete data entries and corrections were requested from the participating centres. Records remaining with incomplete or inconsistent data were excluded from the calculations. Corrections, calculations and tables were made by expert co-authors. MAIN RESULTS AND THE ROLE OF CHANCE: For data collection XIII, 62 centres reported data for 5780 cycles with oocyte retrieval (OR), along with details of the follow-up on 1503 pregnancies and 1152 babies born. A total of 1071 OR were reported for chromosomal abnormalities, 108 OR for sexing for X-linked diseases, 1574 OR for monogenic diseases, 2979 OR for preimplantation genetic screening and 48 OR for social sexing. LIMITATIONS, REASONS FOR CAUTION: The findings apply to the 62 participating centres and may not represent worldwide trends in PGD. WIDER IMPLICATIONS OF THE FINDINGS: The annual data collections provide an important resource for data mining and for following trends in PGD practice. STUDY FUNDING/COMPETING INTERESTS: None.

Recurrent miscarriage in translocation carriers: no differences in clinical characteristics between couples who accept and couples who decline PGD.

STUDY QUESTION: Do clinical characteristics of recurrent miscarriage couples with a chromosomal abnormality and who opt for PGD differ from couples that decline PGD after extensive genetic counselling? SUMMARY ANSWER: No differences in clinical characteristics are identified between recurrent miscarriage couples carrying a structural chromosomal abnormality who opt for PGD compared with those that decline PGD after extensive genetic counselling. WHAT IS KNOWN ALREADY: Couples who have experienced two or more miscarriages (recurrent miscarriage) are at increased recurrence risk if one of the partners carries a structural chromosomal abnormality. PGD can be offered to avoid (another) miscarriage or pregnancy termination when (invasive) prenatal diagnosis shows an abnormal result. To date, no reports are available that describe reproductive decision-making after genetic counselling on PGD in these specific couples. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study of 294 couples carrying a structural chromosomal abnormality seeking genetic counselling on PGD between 1996 and 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were recurrent miscarriage couples carrying a structural chromosomal abnormality. They had been referred for genetic counselling to the only national licensed PGD centre. Clinical characteristics analysed included couple associated characteristics, characteristics concerning reproductive history and external characteristics such as type of physician that referred the couple for genetic counselling and the clinical geneticist performing the counselling on PGD. MAIN RESULTS AND THE ROLE OF CHANCE: Of 294 couples referred for counselling on PGD, 26 were not accepted because they did not meet the criteria for IVF-PGD. The remaining cohort of 268 couples consisted of two-thirds female and one-third male carriers. Main PGD indications were reciprocal translocations (83.9%) and Robertsonian translocations (16.7%). Following genetic counselling, 76.9% of included couples chose PGD as their reproductive option, the others declined PGD. Reproductive choice is not influenced by sex of the translocation carrier (P = 0.499), type of chromosomal abnormality (P = 0.346), number of previous miscarriages (P = 0.882), history of termination of pregnancy (TOP) because of an unbalanced fetal karyotype (P = 0.800), referring physician (P = 0.208) or geneticist who performed the counselling (P = 0.410). LIMITATIONS, REASONS FOR CAUTION: This study only included recurrent miscarriage couples carrying a structural chromosomal abnormality, who were actually referred to a PGD clinic for genetic counselling. We lack information on couples who were not referred for PGD. Some of these patients may not have been informed on PGD at all, while others were not referred for counselling because they did not opt for PGD to start with. WIDER IMPLICATIONS OF THE FINDINGS: This study shows that reproductive choices in couples with recurrent miscarriage on the basis of a structural chromosomal abnormality are not influenced by characteristics of the couple itself, nor by their obstetric history or external characteristics. These findings suggest that a couples' intrinsic attitude towards PGD treatment is a major factor influencing their reproductive choice. Future research will focus on these personal motives that seem to push reproductive decision-making following genetic counselling in a given direction.

ESHRE PGD Consortium data collection XII: cycles from January to December 2009 with pregnancy follow-up to October 2010.

STUDY QUESTION: How do data in the 12th annual data collection (Data XII) of the European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis (PGD) Consortium compare with the cumulative data for collections I-XI? SUMMARY ANSWER: Since the beginning of the data collections, there has been a steady increase in the number of cycles, pregnancies and babies reported annually. WHAT IS KNOWN ALREADY: The PGD Consortium has collected, analysed and published 11 previous data sets since 1997. STUDY DESIGN, SIZE, DURATION: Data were collected from each participating centre using a pre-designed FileMaker Pro database (versions 5-10). Separate FileMaker Pro files were used for the cycles, pregnancies and baby records. The study documented cycles performed during the calendar year 2009 and follow-up of the pregnancies and babies born which resulted from these cycles (until October 2010). PARTICIPANTS/MATERIALS, SETTING, METHODS: Data were submitted by 60 centres (full PGD Consortium members), and the blank files were distributed to each PGD Consortium member centre at the end of 2008. The submitted data were thoroughly analysed to identify incomplete data entries and corrections were requested from the participating centres. Records remaining with incomplete data were excluded from the calculations. Corrections, tables and calculations were made by expert co-authors. MAIN RESULTS AND THE ROLE OF CHANCE: For data collection XII, 60 centres reported data for 6160 cycles with oocyte retrieval (OR), along with details of the follow-up on 1607 pregnancies and 1238 babies born. A total of 870 OR were reported for chromosomal abnormalities, 113 OR for sexing for X-linked diseases, 1597 OR for monogenic diseases, 3551 OR for preimplantation genetic screening and 29 OR for social sexing. LIMITATIONS, REASONS FOR CAUTION: These data cannot include every PGD cycle performed annually, and only indicate the trends in PGD worldwide. WIDER IMPLICATION OF THE FINDINGS: The annual data collections provide an extremely valuable resource for data mining and for following trends in PGD practice. STUDY FUNDING/COMPETING INTEREST(S): None.

Preimplantation genetic diagnosis for X;autosome translocations: lessons from a case of misdiagnosis.

UNLABELLED: Preimplantation genetic diagnosis (PGD) is offered to couples carrying a reciprocal translocation in an attempt to increase their chance of phenotypically normal offspring. For the selection of embryos that are balanced for the translocation chromosomes, it is critical to use a combination of DNA probes that can take account of all the segregation patterns of the particular translocation. The frequency of the different segregation types differs depending on the chromosomes involved, the location of the breakpoints and the number of chiasmata and the sex of the carrier. We report on a case of misdiagnosis after PGD-fluorescence in situ hybridization in a female translocation 46,X,t(X;5)(q13;p14) carrier. Transfer of two embryos diagnosed as balanced for the translocation chromosomes resulted in a singleton pregnancy that miscarried at 8 weeks' gestational age. The unbalanced karyotype of the fetus was consistent with 3:1 segregation resulting in tertiary trisomy for the derivative chromosome 5: 47,XX,+der(5)t(X;5)(q13;p14)mat. Based on additional molecular cytogenetic studies of fetal tissue and the initially investigated blastomeres, we concluded that the misdiagnosis was most probably due to a technical error, i.e. a partial hybridization failure or co-localization of the Xq/Yq subtelomere probe signals. No evidence for a normal cell line (mosaicism) was found in the fetus, which could have explained the discrepancy. This case demonstrates the importance of using two diagnostic probes or testing 2 cells to detect translocation products with potentially viable imbalance. X;autosome translocations are a special case due to the added complication of X chromosome inactivation and particular caution is advised when designing a PGD strategy. TRIAL REGISTRATION NUMBER: not applicable.

ESHRE PGT Consortium data collection XXI: PGT analyses in 2018.

STUDY QUESTION: What are the trends and developments in preimplantation genetic testing (PGT) in 2018 as compared to previous years? SUMMARY ANSWER: The main trends observed in this 21st dataset on PGT are that the implementation of trophectoderm biopsy with comprehensive whole-genome testing is most often applied for PGT-A and concurrent PGT-M/SR/A, while for PGT-M and PGT-SR, single-cell testing with PCR and FISH still prevail. WHAT IS KNOWN ALREADY: Since it was established in 1997, the ESHRE PGT Consortium has been collecting and analysing data from mainly European PGT centres. To date, 20 datasets and an overview of the first 10 years of data collections have been published. STUDY DESIGN SIZE DURATION: The data for PGT analyses performed between 1 January 2018 and 31 December 2018 with a 2-year follow-up after analysis were provided by participating centres on a voluntary basis. Data were collected using an online platform, which is based on genetic analysis and has been in use since 2016. PARTICIPANTS/MATERIALS SETTING METHODS: Data on biopsy method, diagnostic technology, and clinical outcome were submitted by 44 centres. Records with analyses for more than one PGT for monogenic disorders (PGT-M) and/or PGT for chromosomal structural rearrangements (PGT-SR), or with inconsistent data regarding the PGT modality, were excluded. All transfers performed within 2 years after the analysis were included, enabling the calculation of cumulative pregnancy rates. Data analysis, calculations, and preparation of figures and tables were carried out by expert co-authors. MAIN RESULTS AND THE ROLE OF CHANCE: The current data collection from 2018 covers a total of 1388 analyses for PGT-M, 462 analyses for PGT-SR, 3003 analyses for PGT for aneuploidies (PGT-A), and 338 analyses for concurrent PGT-M/SR with PGT-A.The application of blastocyst biopsy is gradually rising for PGT-M (from 19% in 2016-2017 to 33% in 2018), is status quo for PGT-SR (from 30% in 2016-2017 to 33% in 2018) and has become the most used biopsy stage for PGT-A (from 87% in 2016-2017 to 98% in 2018) and for concurrent PGT-M/SR with PGT-A (96%). The use of comprehensive, whole-genome amplification (WGA)-based diagnostic technology showed a small decrease for PGT-M (from 15% in 2016-2017 to 12% in 2018) and for PGT-SR (from 50% in 2016-2017 to 44% in 2018). Comprehensive testing was, however, the main technology for PGT-A (from 93% in 2016-2017 to 98% in 2018). WGA-based testing was also widely used for concurrent PGT-M/SR with PGT-A, as a standalone technique (74%) or in combination with PCR or FISH (24%). Trophectoderm biopsy and comprehensive testing strategies are linked with higher diagnostic efficiencies and improved clinical outcomes per embryo transfer. LIMITATIONS REASONS FOR CAUTION: The findings apply to the data submitted by 44 participating centres and do not represent worldwide trends in PGT. Details on the health of babies born were not provided in this manuscript. WIDER IMPLICATIONS OF THE FINDINGS: The Consortium datasets provide a valuable resource for following trends in PGT practice. STUDY FUNDING/COMPETING INTERESTS: The study has no external funding, and all costs are covered by ESHRE. There are no competing interests declared. TRIAL REGISTRATION NUMBER: N/A.

ESHRE PGD Consortium data collection XI: cycles from January to December 2008 with pregnancy follow-up to October 2009.

The 11th report of the European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium is presented, documenting cycles collected for the calendar year 2008 and follow-up of the pregnancies and babies born until October 2009 which resulted from these cycles. Since the beginning of the data collections, there has been a steady increase in the number of cycles, pregnancies and babies reported annually. For data collection XI, 53 centres have participated, reporting on 5641 cycles to oocyte retrieval (OR), along with details of the follow-up on 1418 pregnancies and 1169 babies born. A total of 774 OR were reported for chromosomal abnormalities, 96 OR for sexing for X-linked diseases, 1363 OR for monogenic diseases, 3401 OR for preimplantation genetic screening and 5 OR for social sexing. Data XI is compared with the cumulative data for data collections I-X.

ESHRE PGD consortium best practice guidelines for fluorescence in situ hybridization-based PGD.

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. The subsequent years have seen the introduction of new technologies as well as evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD, the new guidelines are separated into four new documents that apply to different aspects of a PGD programme, i.e. organization of a PGD centre, fluorescence in situ hybridization (FISH)-based testing, amplification-based testing and polar body and embryo biopsy for PGD/preimplantation genetic screening (PGS). Here, we have updated the sections that pertain to FISH-based PGD. PGS has become a highly controversial technique. Opinions of laboratory specialists and clinicians interested in PGD and PGS have been taken into account here. Whereas some believe that PGS does not have a place in clinical medicine, others disagree; therefore, PGS has been included. This document should assist everyone interested in PGD/PGS in developing the best laboratory and clinical practice possible. Topics covered in this guideline include inclusion/exclusion criteria for FISH-based PGD testing, referrals and genetic counselling, preclinical validation of tests, FISH-based testing methods, spreading of cells for analysis, set-up of local IVF centre and transport PGD centres, quality control/ quality assurance and diagnostic confirmation of untransferred embryos.

ESHRE PGT Consortium data collection XIX-XX: PGT analyses from 2016 to 2017†.

STUDY QUESTION: What are the trends and developments in pre-implantation genetic testing (PGT) in 2016-2017 as compared to previous years? SUMMARY ANSWER: The main trends observed in this 19th and 20th data set on PGT are that trophectoderm biopsy has become the main biopsy stage for PGT for aneuploidies (PGT-A) and that the implementation of comprehensive testing technologies is the most advanced with PGT-A. WHAT IS KNOWN ALREADY: Since it was established in 1997, the ESHRE PGT Consortium has been collecting and analysing data from mainly European PGT centres. To date, 18 data sets and an overview of the first 10 years of data collections have been published. STUDY DESIGN SIZE DURATION: The data for PGT analyses performed between 1 January 2016 and 31 December 2017 with a 2-year follow-up after analysis were provided by participating centres on a voluntary basis. Data were collected using a new online platform, which is based on genetic analysis as opposed to the former cycle-based format. PARTICIPANTS/MATERIALS SETTING METHODS: Data on biopsy method, diagnostic technology and clinical outcome were submitted by 61 centres. Records with analyses for more than one PGT for monogenic/single gene defects (PGT-M) and/or PGT for chromosomal structural rearrangements (PGT-SR) indication or with inconsistent data regarding the PGT modality were excluded. All transfers performed within 2 years after the analysis were included enabling the calculation of cumulative pregnancy rates. Data analysis, calculations, figures and tables were made by expert co-authors. MAIN RESULTS AND THE ROLE OF CHANCE: The current data collection from 2016 to 2017 covers a total of 3098 analyses for PGT-M, 1018 analyses for PGT-SR, 4033 analyses for PGT-A and 654 analyses for concurrent PGT-M/SR with PGT-A.The application of blastocyst biopsy is gradually rising for PGT-M (from 8-12% in 2013-2015 to 19% in 2016-2017), is status quo for PGT-R (from 22-36% in 2013-2015 to 30% in 2016-2017) and has become the preferential biopsy stage for PGT-A (from 23-36% in 2013-2015 to 87% in 2016-2017). For concurrent PGT-M/SR with PGT-A, biopsy was primarily performed at the blastocyst stage (93%). The use of comprehensive diagnostic technology showed a similar trend with a small increased use for PGT-M (from 9-12% in 2013-2015 to 15% in 2016-2017) and a status quo for PGT-SR (from 36-58% in 2013-2015 to 50% in 2016-2017). Comprehensive testing was the main technology for PGT-A (from 66-75% in 2013-2015 to 93% in 2016-2017) and for concurrent PGT-M/SR with PGT-A (93%). LIMITATIONS REASONS FOR CAUTION: The findings apply to the data submitted by 61 participating centres and do not represent worldwide trends in PGT. Details on the health of babies born were not provided in this manuscript. WIDER IMPLICATIONS OF THE FINDINGS: Being the largest data collection on PGT in Europe/worldwide, the data sets provide a valuable resource for following trends in PGT practice. STUDY FUNDING/COMPETING INTERESTS: The study has no external funding and all costs are covered by ESHRE. There are no competing interests declared. TRIAL REGISTRATION NUMBER: N/A.

Accreditation of the PGD laboratory.

Accreditation according to an internationally recognized standard is increasingly acknowledged as the single most effective route to comprehensive laboratory quality assurance, and many countries are progressively moving towards compulsory accreditation of medical testing laboratories. The ESHRE PGD Consortium and some regulatory bodies recommend that all PGD laboratories should be accredited or working actively towards accreditation, according to the internationally recognized standard ISO 15189, 'Medical laboratories-Particular requirements for quality and competence'. ISO 15189 requires comprehensive quality assurance. Detailed management and technical requirements are defined in the two major chapters. The management requirements address quality management including the quality policy and manual, document control, non-conformities and corrective actions, continual improvement, auditing, management review, contracts, referrals and resolution of complaints. Technical requirements include personnel competence (both technical and medical), equipment, accommodation and environment, and pre-analytical, analytical and post-analytical processes. Emphasis is placed on the particular requirements of patient care: notably sample identification and traceability, test validation and interpretation and reporting of results. Quality indicators must be developed to monitor contributions to patient care and continual improvement. We discuss the implementation of ISO 15189 with a specific emphasis on the PGD laboratory, highlight elements of particular importance or difficulty and provide suggestions of effective and efficient ways to obtain accreditation. The focus is on the European environment although the principles are globally applicable.

ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008.

The 10th report of the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium is presented, documenting cycles collected for the calendar year 2007 and follow-up of the pregnancies and babies born until October 2008 which resulted from these cycles. Since the beginning of the data collections there has been a steady increase in the number of cycles, pregnancies and babies reported annually. For data collection X, 57 centres participated, reporting on 5887 cycles to oocyte retrieval (OR), along with details of the follow-up on 1516 pregnancies and 1206 babies born. A total of 729 OR were reported for chromosomal abnormalities, 110 OR for sexing for X-linked diseases, 1203 OR for monogenic diseases, 3753 OR for preimplantation genetic screening and 92 OR for social sexing. Data X is compared with the cumulative data for data collections I-IX.

ESHRE PGT Consortium data collection XVI-XVIII: cycles from 2013 to 2015.

STUDY QUESTION: What are the trends and developments in preimplantation genetic testing (PGT) in 2013-2015 as compared to previous years? SUMMARY ANSWER: The main trends observed in the retrospective data collections 2013-2015, representing valuable data on PGT activity in (mainly) Europe, are the increased application of trophectoderm biopsy at the cost of cleavage stage biopsy and the continuing expansion of comprehensive testing technology in PGT for chromosomal structural rearrangements and for aneuploidies (PGT-SR and PGT-A). WHAT IS KNOWN ALREADY: Since it was established in 1997, the ESHRE PGT Consortium has been collecting data from international PGT centres. To date, 15 data sets and an overview of the first 10 years of data collections have been published. STUDY DESIGN SIZE DURATION: Collection of (mainly) European data by the PGT Consortium for ESHRE. The data for PGT cycles performed between 1 January 2013 and 31 December 2015 were provided by participating centres on a voluntary basis. For the collection of cycle, pregnancy and baby data, separate, pre-designed MS Excel tables were used. PARTICIPANTS/MATERIALS SETTING METHODS: Data were submitted by 59, 60 and 59 centres respectively for 2013, 2014 and 2015 (full PGT Consortium members). Records with incomplete or inconsistent data were excluded from the calculations. Corrections, calculations, figures and tables were made by expert co-authors. MAIN RESULTS AND THE ROLE OF CHANCE: For data collection XVI/XVII/XVIII, 59/60/59 centres reported data on 8164/9769/11 120 cycles with oocyte retrieval: 5020/6278/7155 cycles for PGT-A, 2026/2243/2661 cycles for PGT for monogenic/single gene defects, 1039/1189/1231 cycles for PGT-SR and 79/59/73 cycles for sexing for X-linked diseases. From 2013 until 2015, the uptake of biopsy at the blastocyst stage was mainly observed in cycles for PGT-A (from 23% to 36%) and PGT-SR (from 22% to 36%), alongside the increased application of comprehensive testing technology (from 66% to 75% in PGT-A and from 36% to 58% in PGT-SR). LIMITATIONS REASONS FOR CAUTION: The findings apply to the 59/60/59 participating centres and may not represent worldwide trends in PGT. Data were collected retrospectively and no details of the follow-up on PGT pregnancies and babies born were provided. WIDER IMPLICATIONS OF THE FINDINGS: Being the largest data collection on PGT worldwide, detailed information about ongoing developments in the field is provided. STUDY FUNDING/COMPETING INTERESTS: The study has no external funding and all costs are covered by ESHRE. There are no competing interests declared. TRIAL REGISTRATION NUMBER: N/A.

Validation of preimplantation genetic diagnosis by PCR analysis: genotype comparison of the blastomere and corresponding embryo, implications for clinical practice.

The aim of this study was to validate the overall preimplantation genetic diagnosis (PGD)-PCR procedure and to determine the diagnostic value. Genotyped embryos not selected for embryo transfer (ET) and unsuitable for cryopreservation after PGD were used for confirmatory analysis. The PGD genotyped blastomeres and corresponding embryos were compared, and morphology was scored on Day 4 post fertilization. To establish the validity of the PGD-PCR procedure and the diagnostic value, misdiagnosis rate, false-negative rate and negative predictive value were calculated. Moreover, comparison on the validity was made for the biopsy of one or two blastomeres. For the total embryo group (n = 422), a misdiagnosis rate of 7.1% and a false-negative rate of 3.1% were found. The negative predictive value was 96.1%. Poor morphology Day 4 embryos (Class 1) were over-represented in the embryo group in which the blastomere genotype was not confirmed by the whole embryo genotype. The misdiagnosis rate of Class 1 embryos was 12.5% and the false-negative rate 17.1%. Exclusion of these embryos resulted in a misdiagnosis rate of 6.1%, a false-negative rate of 0.5% and a negative predictive value of 99.3%. The two blastomere biopsies revealed a significant higher positive predictive value, lowering the misdiagnosis rate, whereas the negative predictive value remained the same. In conclusion, the PGD-PCR procedure is a valid diagnostic method to select unaffected embryos for ET. The misdiagnosis and false-negative rates decrease by rejecting Class 1 embryos for ET. The biopsy of a second blastomere improves the positive predictive value, lowering the misdiagnosis rate.

[Genetic diagnosis of IVF embryos: preliminary results from 'preimplantation genetic diagnoses' in the Netherlands]. / Genetische diagnostiek bij IVF-embryo's: eerste ervaringen met 'preïmplantatiegenetische diagnostiek' in Nederland.

Preimplantation genetic diagnosis (PGD) is a very early form of genetic testing. It involves testing one or two cells taken from a recent embryo of eight cells produced by in vitro fertilization, and selective transfer of genetically normal embryos. So far in the Academic Hospital Maastricht, the Netherlands, 20 couples have undergone PGD, resulting in 6 ongoing pregnancies (one twin pregnancy). In three women the indications for PGD were: cystic fibrosis, sex-linked Pelizaeus-Merzbacher disease and chromosomal translocation, respectively. In the Netherlands PGD is only allowed if there is a high risk of a serious genetic disease. PGD can be carried out in Maastricht for: cystic fibrosis, sex-linked diseases, chromosomal abnormalities, fragile X syndrome, spinal muscular atrophy and myotonic dystrophy. The advantage of PGD is that it excludes the necessity of a therapeutic abortion. Disadvantages ages are the requirement of in vitro fertilization, which has only a 15-20% pregnancy rate, and the experimental nature of the PGD procedure. To date, about 200 children have been born worldwide following PGD.

[Results from 10 years of preimplantation-genetic diagnostics in The Netherlands]. / Resultaten van 10 jaar preimplantatiegenetische diagnostiek in Nederland.

OBJECTIVE: To report the data from couples who were referred for preimplantation-genetic diagnostics (PGD) and treatment due to a significantly increased risk of offspring with a serious genetic disorder. DESIGN: Descriptive, prospective. METHOD: Data were collected from couples that underwent PGD in the period 1993/'03 at Maastricht University Hospital. Embryos produced by means of in-vitro fertilisation (IVF) were subjected to genetic tests several days after fertilisation. Subsequently 1 or 2 unaffected embryos were transferred to the uterus. Where there was an increased risk of a male with an X-linked genetic disorder, the gender was determined using fluorescence in-situ hybridisation (FISH). This method was also used to detect structural chromosomal abnormalities. The polymerase chain reaction (PCR) method was used for mutation detection and/or marker analysis of monogenetic disorders. RESULTS: A total of 691 couples were referred for PGD. The most frequent indications were X-linked disorders (30%), in particular Fragile-X syndrome, Duchenne/Becker muscular dystrophy and haemophilia A/B. This was followed by autosomal dominant disorders (26%), such as Huntington's disease and myotonic dystrophy, and then structural chromosomal abnormalities (24%). A total of 120 women underwent 260 PGD cycles. An embryo transfer was possible in 158 of the cycles and this resulted in 45 successful pregnancies. The pregnancy rate was 17% per cycle initiated and 28% per cycle with embryo transfer. Up until december 2003 29 singletons, 8 sets of twins and 1 set of triplets were born. There were no misdiagnoses and none of the babies had congenital abnormalities. CONCLUSION: PGD was a reliable and successful method, with pregnancy rates similar to those of IVF or intracytoplasmatic sperm injection. PGD should be stated as an alternative during the preconception counselling of couples with an increased genetic risk, especially for disorders where PGD can be routinely applied, such as Huntington's disease, myotonic dystrophy, cystic fibrosis, spinal muscular atrophy, Fragile-X syndrome and structural chromosomal abnormalities.

Intermediate filament protein expression in early developmental stages of the mouse. A confocal scanning laser microscopy study of in vitro fertilized and in vitro cultured pre-implantation mouse embryos.

Expression patterns of intermediate filament proteins have been studied during early mouse embryo development. For this purpose, pre-implantation embryos at different stages of development after in vitro fertilization were studied using antibodies to cytokeratins, vimentin and lamins, using the indirect immunofluorescence assay. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies showed that, although the embryos grew in culture, vimentin could not be detected in a filamentous organization. Immunofluorescence for cytokeratins was only positive from the 8-cell stage onwards. In the morula stage an increased level of cytokeratin expression was observed with a transitional staining pattern, combining a filamentous and a diffuse occurrence. In the blastocyst stages profound cytokeratin filaments were seen in trophoblast cells but not in the inner cell mass. When the cytokeratin subtypes were analysed separately, it became apparent that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage and showing a filamentous distribution, were not found as prominently as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected in all developmental stages examined, as a faintly reactive nuclear lamina. In blastocysts both lamin types were detected in trophoblast as well as in inner cell mass.

Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescence in-situ hybridization.

A new method was developed to prepare and isolate interphase nuclei from murine preimplantation embryos for analysis by fluorescence in-situ hybridization. Embryos were washed with phosphate-buffered saline and disaggregated in a small drop of bidistilled water containing HCl and Tween 20. During the disaggregation procedure embryos were watched continuously with an inverted microscope. Embryonic nuclei were digested with pepsin to make them accessible for hybridization to the probes and to remove remnants of cytoplasm. Nuclear and probe DNA were denatured simultaneously and hybridization was done overnight, followed by immunocytochemical detection of the probes. Using this method we were able to perform successful in-situ hybridization on all preimplantation stages of the mouse embryo (pronuclei, 2-cell, 4-cell, morula and blastocyst). Probes for the X and Y chromosomes were applied for sex determination. From the results described in this paper we conclude that the preparation and isolation of interphase nuclei from murine embryos with acid and Tween 20 offers high reproducibility, good morphology of the cells and a high hybridization efficiency.

Application of in-situ hybridization techniques to study human preimplantation embryos: a review.

Although the first in-situ hybridization (ISH) experiments were performed more than two decades ago, it was not until 1987 that the technique was implemented in the study of preimplantation. Since then, several groups have explored the feasibility of using the ISH technique as a diagnostic tool to study the genomic constitution of human preimplantation embryos. Though a fair number of such ISH studies have been published and unravelling of the embryos' genomic secrets proceeds with great strides, there are still many challenges in this area of research. The scope of this review is to outline the research and diagnostic applications of single and multicolour (fluorescence) ISH to study the chromosomal content of human preimplantation embryos and to discuss the implications for human preimplantation genetic diagnosis. Current hybridization techniques are being discussed, including the capabilities and pitfalls of this approach when applied to human preimplantation embryos. Finally, future perspectives for the ISH technique in studying the genomic constitution of preimplantation embryos are reviewed.