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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 INTEREST(S) The study has no external funding, and all costs are covered by ESHRE. There are no competing interests declared. TRIAL REGISTRATION NUMBER N/A.

Study question Which are the trends shown in data collection XXII of the European Society of Human Reproduction and Embryology (ESHRE) PGT Consortium compared with previous years? Summary answer Data collection XXII, year 2020, represents valuable data on PGT activity in (mainly) Europe and reports on the main trends observed, being the further expansion of comprehensive testing technology in PGT-SR and PGT-A. What is known already The ESHRE PGT Consortium was set up in 1997 and from that time has been collecting data on PGT and PGT-A. The PGT database comprises the world’s largest collection of PGT / PGT-A data providing a valuable resource for data mining and for following trends in PGT practice. So far, up to the year 2015, data collections were carried out in a retrospective data way, from 2016 onwards a prospective cycle-by-cycle data collection was in place. Study design, size, duration As the nature of PGT/ PGT-A treatments has changed significantly over the last years and IVF cycle management and genetic analysis techniques are getting more complex, ESHRE uses an online data collection system in which data are collected prospectively from oocyte retrieval to analysis, embryo transfer and pregnancy / live birth. Data are collected cycle by cycle on a voluntary basis. Participants/materials, settings, method For the 2020 data, individual centres (37) from 20 countries directly entered the data into the PGT database through software developed by ESHRE. Data were analysed at ESHRE headquarters and include all aspects of PGT/PGT-A cycles. Main results and the role of chance The Consortium has analysed the PGT analyses (n = 2809) performed in 2020. The indications for PGT included inherited chromosomal abnormalities (n = 331 analyses), monogenic disorders (n = 987 analyses), aneuploidy testing for infertility (n = 1417 analyses) or combinations of the above (n = 74 analyses). In addition, 704 clinical pregnancies and 335 deliveries have been analysed in detail. The methods used for biopsy were polar body (2%), cleavage stage biopsy (20%) and blastocyst biopsy (78%), showing a further increase of blastocyst biopsy compared to 2019. The methodology used for diagnosis is what is evolving most over the last years, with data set XXII (2020) showing around 4% of FISH, 28% of PCR and 68% of WGA. Within WGA 95% of the analysis were done using NGS, in 4% of the cases SNP arrays were used and in 1% array-CGH was used. The overall clinical pregnancy rate is about 25% per analysis. The baby data show that it is difficult for most centres to have a detailed follow-up. Limitations, reasons for caution The findings apply to the 37 participating centres and may not represent worldwide trends in PGT. Data were collected prospectively, but details of the follow-up on PGT pregnancies and babies born were limited. Wider implications of the findings The ESHRE PGD Consortium continues its activities as an important forum for PGT practitioners to share data and exchange experiences. The information extracted from the data collections helps to monitor quality issues in PGT and survey the introduction and effectiveness of new PGT technologies and methods. Trial registration number

The implementation of high-resolution genome-wide methods, usually next-generation sequencing-based, in preimplantation genetic testing (PGT), has led to the frequent detection of embryos with chromosomal mosaicism (whole chromosome and/or segmental aberrations). Despite a growing series of papers showing the birth of healthy babies following the transfer of embryos indicating mosaicism on PGT-A analysis - albeit with lower implantation rates and higher miscarriage rates in comparison with euploid embryo transfer - many questions remain, making it difficult to decide on how to handle chromosomally mosaic embryos in the clinic. A dedicated ESHRE working group developed good practice recommendations on how to manage chromosomal mosaicism in clinical practice. The recommendations were formulated based on the expert opinion of the working group while taking into consideration the published data and outcomes of a survey on current practices in 239 PGT laboratories and ART clinics, mostly within Europe, Asia and America. The recommendations with regards to detection and management of chromosomal mosaicism were developed following the manual for development of ESHRE good practice recommendations with a stakeholder review of the paper on the ESHRE website. In addition to the recommendations, the working group identified missing information and scientific questions, which should guide further research in PGT, with relevance to the occurrence, detection and impact of chromosomal mosaicism. Trial registration number

STUDY QUESTION What is the likelihood of success of a single cycle of preimplantation genetic testing for monogenic disorders (PGT-M), measured as the cumulative live birth rate (CLBR) and based on various patient demographics? SUMMARY ANSWER For all women aged ≤40 years, the CLBR was at least 10% when the number of oocytes was ≥7 (range 10–30%) or was at least 5% when the number of oocytes was ≥3 (range 5–17%). WHAT IS KNOWN ALREADY The number of oocytes is significantly associated with the number of embryos for genetic testing and the clinical outcome in PGT-M. Embryos diagnosed as affected or embryos that remain without diagnosis cannot be used for embryo transfer. The size of the group of embryos non-suitable for transfer varies between 25% and 81%, depending on the indication. Thus, PGT-M is more likely to be more severely impacted by suboptimal ovarian response, poor fertilization and suboptimal embryo development than conventional IVF/ICSI schemes without PGT. STUDY DESIGN, SIZE, DURATION This was a single-centre retrospective comparative cohort study, of cycles between January 2011 and December 2015. A total number of 2265 PGT-M cycles were compared to 2833 conventional ICSI cycles. The principal aim of our study was the identification of the parameters of poor CLBR in couples undergoing PGT-M using multiplex short tandem repeat (STR) markers on blastomere biopsy DNA. The secondary aim was to compare the parameters of poor CLBR of the PGT-M population to those of couples undergoing ICSI without PGT. PARTICIPANTS/MATERIALS, SETTING, METHODS The baseline characteristics of the PGT-M group were compared to the conventional ICSI group. A multiple regression analysis was applied to account for the following potential confounding factors: female age, number of previous ART cycles, number of oocytes/suitable embryos for transfer and dosage of gonadotrophins used for ovarian stimulation. MAIN RESULTS AND THE ROLE OF CHANCE The PGT-M group was younger (female age 32.0 vs 34.5 years), had a higher number of previous ART cycles (1.1 vs 0.9 cycles) and used more gonadotrophins (2367 vs 1984 IU). Per cycle, the PGT-M group had more retrieved oocytes (11.8 vs 8.3 oocytes), fewer suitable embryos for transfer (1.7 vs 2.8 embryos) and a lower CLBR (29.4% vs 35.0%). Multiple regression analysis showed that the CLBR in the PGT-M group was significantly influenced by female age, the number of previous ART cycles, the number of oocytes and the dose of ovarian stimulation. In both groups, the predicted CLBR increased with increasing numbers of oocytes and suitable embryos. At least two retrieved oocytes or one embryo per single PGT-M cycle could confer an estimated CLBR above 10%. By assessing female age and the number of retrieved oocytes together, it was shown that for all women aged ≤40 years, the predicted CLBR per single PGT-M cycle was ≥10% when the number of oocytes was ≥7 or was ≥5% when the number of oocytes was ≥3. LIMITATIONS, REASONS FOR CAUTION Despite the large sample size, the findings are confined by limited confounder adjustment and the lack of specific PGT-M comparators. WIDER IMPLICATIONS OF THE FINDINGS This study aimed to describe the likelihood of success of PGT-M treatment, measured as CLBR, based on various patient demographics. In a PGT-M program, couples need to be informed of the prognosis more specifically when it is futile. The table of predicted CLBRs presented in this study is a useful tool in counselling PGT-M couples for making reproductive choices. STUDY FUNDING/COMPETING INTEREST(S) No funding was required and there are no competing interests. TRIAL REGISTRATION NUMBER N/A.

text Study question Which are the trends shown in data collection XXI of the European Society of Human Reproduction and Embryology (ESHRE) PGT Consortium compared with previous years? Summary answer Data collection XXI, year 2019, represents valuable data on PGT activity in (mainly) Europe and reports on the main trends observed, being the further expansion of comprehensive testing technology in PGT-SR and PGT-A. What is known already The ESHRE PGT Consortium was set up in 1997 and from that time has been collecting data on PGT and PGT-A. The PGT database comprises the world’s largest collection of PGT / PGT-A data providing a valuable resource for data mining and for following trends in PGT practice. So far, up to the year 2015, data collections were carried out in a retrospective data way, from 2016 onwards a prospective data collection was in place. Study design, size, duration As the nature of PGT/ PGT-A treatments has changed significantly over the last years and IVF cycle management and genetic analysis techniques are getting more complex, ESHRE uses an online data collection system in which data are collected prospectively from oocyte retrieval to analysis, embryo transfer and pregnancy / live birth. Data are collected cycle by cycle on a voluntary basis. Participants/materials, settings, method For the 2019 data, individual centres (31) from 19 countries directly entered the data into the PGT database through software developed by ESHRE. Data were analysed at ESHRE headquarters and include all aspects of PGT/PGT-A cycles. Main results and the role of chance The Consortium has analysed the PGT analyses (n = 2735) performed in 2019. The indications for PGT included inherited chromosomal abnormalities (n = 253 analyses), monogenic disorders (n = 1105 analyses), aneuploidy testing for infertility (n = 1111 analyses) or combinations of the above (n = 266 analyses). In addition, 662 clinical pregnancies and 216 deliveries have been analysed in detail. The methods used for biopsy were polar body (2%), cleavage stage biopsy (35%) and blastocyst biopsy (61%; comparable with data from 2018). The methodology used for diagnosis is what is evolving most over the last years, with data set XXI (2019) showing around 7% of FISH, 37% of PCR and 55% of WGA. Within WGA 90.6% of the analysis were done using NGS, in 4.4% cases SNP arrays were used and in 2.4% array-CGH was used. The overall clinical pregnancy rate is about 24% per analysis. The baby data show that it is difficult for most centres to have a detailed follow-up. Limitations, reasons for caution The findings apply to the 31 participating centres and may not represent worldwide trends in PGT. Data were collected prospectively, but details of the follow-up on PGT pregnancies and babies born were limited. Wider implications of the findings The ESHRE PGD Consortium continues its activities as an important forum for PGT practitioners to share data and exchange experiences. The information extracted from the data collections helps to monitor quality issues in PGT and survey the introduction and effectiveness of new PGT technologies and methods.

STUDY QUESTION: What are the factors influencing the success rate for couples undergoing preimplantation genetic testing (PGT) for polycystic kidney disease (PKD)? SUMMARY ANSWER: In our study cohort, the live birth delivery rate is significantly associated with female age while the male infertility accompanying autosomal dominant PKD (ADPKD) does not substantially affect the clinical outcome. WHAT IS KNOWN ALREADY: While women with ADPKD have no specific fertility problems, male ADPKD patients may present with reproductive system abnormalities and infertility. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study involves 91 PGT cycles for PKD for 43 couples (33 couples for PKD1, 2 couples for PKD2 and 8 couples for autosomal recessive PKD (ARPKD)) from January 2005 until December 2016 with follow-up of transfers until end of 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sixteen single-cell clinical tests for PKD based on multiplex PCR of short tandem repeat markers, with or without a specific mutation were developed and applied for diagnosis of 584 Day 3 cleavage stage embryos. In 18 couples, the male partner was affected with ADPKD (=Group A) and 12 of them had a documented infertility status. Group A underwent 52 cycles to oocyte retrieval. For 18 other couples, the female partner was affected with ADPKD (=Group B) and four male partners from this group had a documented history of infertility. This group underwent 31 cycles to OR. MAIN RESULTS AND THE ROLE OF CHANCE: Genetic analysis resulted in 545 embryos (93.3%) with a diagnosis, of which 215 (36.8%) were genetically transferable. Transfer of 74 embryos in 53 fresh cycles and of 34 cryopreserved embryos in 33 frozen-warmed embryo transfer cycles resulted in a live birth delivery rate of 38.4% per transfer with 31 singleton live births, two twin live births and one ongoing pregnancy. The observed cumulative delivery rate was 57.8% per couple after five treatment cycles. Thirty cryopreserved embryos still remain available for transfer. The clinical pregnancy rate per transfer (fresh + frozen; 45.9% in group A versus 60.0% in group B, P < 0.05) and the live birth delivery rate per transfer (fresh + frozen; 27.0% in group A versus 42.9% in group B, P < 0.05) was significantly lower for couples with the male partner affected with ADPKD compared with couples with the female partner affected with ADPKD. However, a multivariate logistic regression analysis showed that only female age was associated with live birth delivery rate (odds ratio = 0.87; 95% CI: 0.77-0.99; P = 0.032). LIMITATIONS, REASONS FOR CAUTION: This study is based on retrospective data from a single centre with Day 3 one-cell and two-cell biopsy. Further analysis of a larger cohort of PKD patients undergoing PGT is required to determine the impact of male infertility associated with ADPKD on the cumulative results. WIDER IMPLICATIONS OF THE FINDINGS: Knowledge about factors affecting the clinical outcome after PGT can be a valuable tool for physicians to counsel PKD patients about their reproductive options. Males affected with ADPKD who suffer from infertility should be advised to seek treatment in time to improve their chances of conceiving a child. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained. There are no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

RESEARCH QUESTION: Can (mosaic) aneuploidy be reliably detected in preimplantation embryos after multiple displacement amplification and single nucleotide polymorphism detection, independent of haplotyping and copy number detection, with a new method 'analysis of parental contribution for aneuploidy detection' or 'APCAD'? DESIGN: This method is based on the maternal contribution, a parameter that reflects the proportion of DNA that is of maternal origin for a given chromosome or chromosome segment. A maternal contribution deviating from 50% for autosomes is strongly indicative of a (mosaic) chromosomal anomaly. The method was optimized using cell mixtures with varying ratios of euploid and aneuploid (47,XY,+21) lymphocytes. Next, the maternal contribution was retrospectively measured for all chromosomes from 349 Karyomapping samples. RESULTS: Retrospective analysis showed a skewed maternal contribution (63.6%) in 57 out of 59 autosome meiotic trisomies and all autosome monosomies (n = 57), with values close to theoretical expectation. Thirty-two out of 7436 chromosomes, for which no anomalies had been observed with Karyomapping, showed a similarly skewed maternal contribution. CONCLUSIONS: APCAD was used to measure the maternal contribution, which is an intuitive parameter independent of copy number detection. This method is useful for detecting copy number neutral anomalies and can confirm diagnosis of (mosaic) aneuploidy detected based on copy number. Mosaic and complete aneuploidy can be distinguished and the parent of origin for (mosaic) chromosome anomalies can be determined. Because of these benefits, the APCAD method has the potential to improve aneuploidy detection carried out by comprehensive preimplantation genetic testing methods.

Preimplantation genetic testing-human leukocyte antigen '(PGT-HLA) only' refers to the HLA typing of single or few cells biopsied from in vitro fertilized preimplantation embryos. The aim of the procedure is to establish a pregnancy, in which the fetus is HLA compatible with an affected sibling in need of a hematopoietic stem cell transplantation (HSCT). During PGT-M-HLA, the identification of a HLA-compatible embryo is combined with the detection of mutation(s) underlying immunodeficiencies and hemoglobinopathies. We report a combined retrospective and prospective cohort analysis of PGT-(M-)HLA procedures carried out from 1998 until 2017, with follow-up of transplantations to 2019. During the study period, 234 couples from 22 countries were invited for a multidisciplinary consultation. Two couples were rejected and 70 couples declined (various reasons), leaving 162 couples for which 414 clinical cycles were carried out. Cleavage stage biopsy followed by single-cell multiplex PCR for short tandem repeat-based haplotyping was applied in most cases (98.7%). The diagnostic efficiency was high (94.8%) but only 16.5% of the embryos was genetically suitable for transfer. Fresh and frozen-thawed embryo transfer resulted in 67 clinical pregnancies, 63 deliveries, and 74 live births, of which 60 children were HLA compatible. This yielded a live birth delivery rate of 30.3% per transfer. Information on neonatal characteristics of the matching PGT-(M-)HLA children showed reassuring outcomes. So far, HSCT was carried out successfully for 25 out of 26 cases. In conclusion, our data show that PGT-(M-)HLA is a valuable procedure: the high complexity and limited delivery rate are balanced by the successful HSCT outcome and the positive impact on families.

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 INTEREST(S) The study has no external funding and all costs are covered by ESHRE. There are no competing interests declared. TRIAL REGISTRATION NUMBER N/A.

STUDY QUESTION Does Day 3 embryo biopsy for pre-implantation genetic testing for monogenic (PGT-M) and structural chromosomal aberrations (PGT-SR) affect body composition and blood pressure readings of 6-year-old singletons? SUMMARY ANSWER This study of 87 PGT-M and PGT-SR conceived singletons showed no differences in anthropometric measurements and blood pressure readings in comparison with a matched cohort of peers born after ICSI without embryo biopsy. WHAT IS KNOWN ALREADY While neonatal outcomes after PGT conception have been found comparable to those after ICSI without embryo biopsy, only a few studies have reported outcomes after PGT at older ages. Moreover, embryo biopsy is also applied in couples who opt for PGT-M and PGT-SR and hence are not necessarily infertile. Health parameters and in particular body composition data in this group of children are lacking. STUDY DESIGN, SIZE, DURATION This single-centre matched-pair cohort study evaluated body composition of 6-year-old children born after fresh blastocyst embryo transfer with or without embryo biopsy performed at Day 3 for the purpose of PGT-M and PGT-SR. For each child born after embryo biopsy, a singleton born after transfer of a fresh ICSI embryo at the blastocyst stage and reaching the age of 6 years between May 2011 and June 2017 was matched as closely as possible for gender, age, maternal educational level and birth order. PARTICIPANTS/MATERIALS, SETTING, METHODS Anthropometry (weight, height, BMI, skinfold thickness, waist and mid-upper arm circumference) and blood pressure readings in a longitudinally followed cohort of 87 singletons conceived by PGT-M and PGT-SR and a pairwise matched sample of 87 children conceived by ICSI are described. Results are adjusted for current, neonatal and parental characteristics. MAIN RESULTS AND THE ROLE OF CHANCE From the 124 eligible PGT-M and PGT-SR families, 110 could be reached of whom 23 refused and 87 (87/110 = 79%) participated. All anthropometric measurements, including z-scores of BMI, waist and mid-upper arm circumference, were comparable between the PGT-M and PGT-SR (−0.23; 0.27; 0.17, respectively) and ICSI (−0.29; 0.11; 0.11, respectively) groups (all P > 0.05). Furthermore, indices of peripheral (triceps) and central (subscapular) adiposity derived from skinfold thickness were comparable (PGT-M and PGT-SR: 14.7 mm; 11.6 mm and ICSI: 15.5 mm; 11.5 mm) as well as the percentage total body fat mass derived from these (PGT-M and PGT-SR: 13.7% and ICSI: 13.9%) (all P > 0.05). Z-scores for blood pressure were also comparable between the PGT and ICSI groups (all P > 0.05). Results did not change when adjusted for neonatal (birthweight, birth order), current (age) and parental (smoking during pregnancy, parental BMI) characteristics. Hospitalization rate and surgical intervention rate were not different for PGT-M and PGT-SR children compared to matched peers born after ICSI. LIMITATIONS, REASONS FOR CAUTION Although our study describes the largest cohort of singletons born after embryo biopsy worldwide, we were only able to detect moderate differences in anthropometrics and blood pressure with our sample size. WIDER IMPLICATIONS OF THE FINDINGS Although Day 3 embryo biopsy followed by blastocyst transfer is not associated with adverse outcomes regarding anthropometry and blood pressure, future studies should focus on outcomes in children born after trophectoderm biopsy and/or transfer of warmed embryos after vitrification. STUDY FUNDING/COMPETING INTEREST(s) This study was supported by Methusalem grants and by grants from Wetenschappelijk Fonds Willy Gepts; all issued by the Vrije Universiteit Brussel (VUB). All co-authors, except M.B. declared no conflict of interest. M.B. has received consultancy fees from MSD, Serono Symposia and Merck. The Universitair Ziekenhuis Brussel (UZ Brussel) and the Centre for Medical Genetics have received several educational grants from IBSA, Ferring, Organon, Shering-Plough, Merck for establishing the database for follow-up research and organizing the data collection.

Oocyte vitrification has been introduced into clinical settings without extensive pre-clinical safety testing. In this study, we analysed major safety aspects of human oocyte vitrification in a high security closed system: (i) chromosomal meiotic segregation, (ii) embryonic developmental kinetics and (iii) DNA (hydroxy)methylation status. Fresh and vitrified sibling oocytes from young donors after intracytoplasmic sperm injection (ICSI) were compared in three different assays. Firstly, the chromosomal constitution of the fertilized zygotes was deduced from array comparative genomic hybridization results obtained from both polar bodies biopsied at Day 1. Secondly, embryo development up to Day 3 was analysed by time-lapse imaging. Ten specific time points, six morphokinetic time intervals and the average cell number on Day 3 were recorded. Thirdly, global DNA methylation and hydroxymethylation patterns were analysed by immunostaining on Day 3 embryos. The nuclear fluorescence intensity was measured by Volocity imaging software. Comprehensive chromosomal screening of the polar bodies demonstrated that at least half of the zygotes obtained after ICSI of fresh and vitrified oocytes were euploid. Time-lapse analysis showed that there was no significant difference in cleavage timings, the predictive morphokinetic time intervals nor the average cell number between embryos developed from fresh and vitrified oocytes. Finally, global DNA (hydroxy)methylation patterns were not significantly different between Day 3 embryos obtained from fresh and from vitrified oocytes. Our data further consolidate the safety of the oocyte vitrification technique. Nevertheless, additional testing in young and older sub-fertile/infertile patients and sound follow-up studies of children born after oocyte cryopreservation remain mandatory.

Background: The use of GnRH analogue medication is essential in reproductive medicine to avoid premature ovulation by pituitary suppression for the duration of ovarian stimulation by gonadotrophins. The type of pituitary suppression by either GnRH agonist analogues versus GnRH antagonist analogues may result in different embryological hence clinical results. Preimplantation genetic diagnosis is a subtype of IVF in which embryos are created for genetic diagnosis of hereditary disorders in order to avoid genetically affected children. Embryological quality hence ovarian stimulation in preimplantation genetic diagnosis is crucial as genetic selection will reduce the number of available embryos to a fraction of the total. Objective: The aim of this study was to assess the efficiency of GnRH antagonist versus GnRH agonist treatment for pituitary suppression in ovarian stimulation for PGD, by proxy of number and quality of embryos at cleavage stage available for biopsy. Method: We conducted a prospective randomisedcontrolled trial comparing pituitary suppression by GnRH antagonist versus GnRH agonist in ovarian stimulation for PGD. The primary outcome measure was the number of embryos of sufficient quality for biopsy at cleavage stage. Secondary outcome parameters were the number of blastocysts available of top quality, and clinical pregnancy rate. Results: There was no difference in number of oocytes retrieved, embryos at cleavage stage available for biopsy or embryo quality. The clinical pregnancy rate was higher in the GnRH agonist group; however the sample size was insufficient to allow conclusions. Conclusion: The use of GnRH agonist versus antagonist treatment does not result in differences in a number of oocytes, embryos or embryo quality in ovarian stimulation for preimplantation genetic diagnosis.

Purpose The aim of this study was to determine whether BRCA1/2 mutation carriers produce fewer mature oocytes after ovarian stimulation for in vitro fertilization (IVF) with preimplantation genetic diagnosis (PGD), in comparison to a PGD control group. Methods A retrospective, international, multicenter cohort study was performed on data of first PGD cycles performed between January 2006 and September 2015. Data were extracted from medical files. The study was performed in one PGD center and three affiliated IVF centers in the Netherlands and one PGD center in Belgium. Exposed couples underwent PGD because of a pathogenic BRCA1/2 mutation, controls for other monogenic conditions. Only couples treated in a long gonadotropin-releasing hormone (GnRH) agonist-suppressive protocol, stimulated with at least 150 IU follicle stimulating hormone (FSH), were included. Women suspected to have a diminished ovarian reserve status due to chemotherapy, auto-immune disorders, or genetic conditions (other than BRCA1/2 mutations) were excluded. A total of 106 BRCA1/2 mutation carriers underwent PGD in this period, of which 43 (20 BRCA1 and 23 BRCA2 mutation carriers) met the inclusion criteria. They were compared to 174 controls selected by frequency matching. Results Thirty-eight BRCA1/2 mutation carriers (18 BRCA1 and 20 BRCA2 mutation carriers) and 154 controls proceeded to oocyte pickup. The median number of mature oocytes was 7.0 (interquartile range (IQR) 4.0–9.0) in the BRCA group as a whole, 6.5 (IQR 4.0–8.0) in BRCA1 mutation carriers, 7.5 (IQR 5.5–9.0) in BRCA2 mutation carriers, and 8.0 (IQR 6.0–11.0) in controls. Multiple linear regression analysis with the number of mature oocytes as a dependent variable and adjustment for treatment center, female age, female body mass index (BMI), type of gonadotropin used, and the total dose of gonadotropins administered revealed a significantly lower yield of mature oocytes in the BRCA group as compared to controls (p = 0.04). This finding could be fully accounted for by the BRCA1 subgroup (BRCA1 mutation carriers versus controls p = 0.02, BRCA2 mutation carriers versus controls p = 0.50). Conclusions Ovarian response to stimulation, expressed as the number of mature oocytes, was reduced in BRCA1 but not in BRCA2 mutation carriers. Although oocyte yield was in correspondence to a normal response in all subgroups, this finding points to a possible negative influence of the BRCA1 gene on ovarian reserve. Electronic supplementary material The online version of this article (doi:10.1007/s10815-017-1014-3) contains supplementary material, which is available to authorized users.

Preimplantation genetic diagnosis (PGD) involves the testing of a single or a few cells biopsied from oocytes and/or embryos generated in vitro. As only embryos unaffected for a given genetic condition are transferred to the uterus, prenatal diagnosis and the termination of a pregnancy are avoided. PGD was first performed in 1990, and it is now an established alternative to prenatal diagnosis with expanding uses and a broad range of applications. Continuous technical progress in single cell testing has led to high levels of efficiency and accuracy. The gold standard methods of single cell multiplex-polymerase chain reactions for monogenic diseases and fluorescent in situ hybridization for chromosomal aberrations are being replaced by whole-genome amplification and genome-wide technologies. These generic methods substantially reduce the preclinical workload. Follow-up data from PGD pregnancies, deliveries, and children show an acceptable live birth rate, and so far, no detrimental effects of the procedure have been observed.

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 IXI? Since the beginning of the data collections, there has been a steady increase in the number of cycles, pregnancies and babies reported annually. The PGD Consortium has collected, analysed and published 11 previous data sets since 1997. Data were collected from each participating centre using a pre-designed FileMaker Pro database (versions 510). 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). 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. 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. These data cannot include every PGD cycle performed annually, and only indicate the trends in PGD worldwide. The annual data collections provide an extremely valuable resource for data mining and for following trends in PGD practice. None.

Background Since it was established in 1997, the ESHRE PGD Consortium has been collecting data from international preimplantation genetic diagnosis (PGD) centres. Ten papers have been published, including data from January 1997 to December 2007. Methods The data collection originally used a hard-copy format, then an excel database and finally a FileMaker Pro database. The indications are divided into five categories: PGD for chromosome abnormalities, sexing for X-linked disease, PGD for single gene defects, preimplantation genetic screening (PGS) and PGD for social sexing. The main end-points are pregnancy outcome and follow-up of deliveries. Results In data collection I, 16 centres contributed data, which increased to 57 centres by data X (average of 39 centres per data collection). These centres contributed data on over 27 000 cycles that reached oocyte retrieval. Of these cycles, 61% were for aneuploidy screening, 17% for single gene disorders, 16% for chromosomal abnormalities, 4% for sexing of X-linked disease and 2% for social sexing. Cumulatively, 5187 clinical pregnancies gave rise to 4140 deliveries and 5135 newborns (singletons: 3182, twins: 921, triplets: 37). Conclusions In this paper, we present an overview of the first 10 years of PGD data, highlighting trends. These include the introduction of laser-assisted biopsy, an increase in polar body and trophectoderm biopsy, new strategies, methodologies and technologies for diagnosis, including recently arrays, and the more frequent use of freezing biopsied embryos. The Consortium data reports represent a valuable resource for information about the practice of PGD.

To compare low-field magnetic resonance arthrography (MRA) and computed tomographic arthrography (CTA) for identification of intra-articular ligaments and joint cartilage in the normal canine shoulder with the aid of cross-sectional anatomy.Normal adult Foxhounds (n = 3) and 4 Foxhound cadaver shoulders.CTA of 3 dogs was performed after intra-articular administration of contrast medium in both shoulders. Transverse slices and reformatted sagittal and dorsal images were obtained. Later, an MRA study of all 6 shoulder joints was performed using a low-field system. T1- and T2-weighted images were made in 3 planes, arthrography performed, and T1-weighted images repeated. Unrelated cadaver shoulder joints were injected with red methylmethacrylate, frozen, and sectioned into dorsal, sagittal, and transverse sections. Representative anatomic images were compared with the CTA, MRA, and T2-weighted images.The joint cartilage was identified on all CTA images. The glenohumeral ligaments were clearly visible on the transverse and dorsal CTA and MRA images but were not detected on the T2-weighted sequences. The biceps tendon could be evaluated on all images. The supraspinatus, infraspinatus, and subscapularis muscle tendons were most easily identified on the MRA and T2-weighted MR images.CTA was useful for seeing articular cartilage and both CTA and MRA were useful for seeing the normal glenohumeral ligaments; however, further research is needed to examine each technique's sensitivity for diagnosing the pathology of these ligaments and any associated lesions of the capsule, tendon, and bone, and for determining the clinical importance of CTA for evaluating loose fragments or osteochondral lesions.

Feeder-free human embryonic stem cell (hESC) culture is associated with the presence of mesenchymal-like cells appearing at the periphery of the colonies. The aim of this study was to identify this early differentiation process. Long-term feeder-free hESC cultures using matrigel and conditioned medium from mouse and from human origin revealed that the appearance of mesenchymal-like cells was similar regardless of the conditioned medium used. Standard characterization confirmed the preservation of hESC properties, but the feeder-free cultures could not be maintained longer than 37 passages. The early differentiation process was characterized in the short term after switching hESCs cultured on feeders to feeder-free conditions. Transmission electron microscopy showed an epithelium-like structure inside the hESC colonies, whereas the peripheral cells revealed the acquisition of a rather mesenchymal-like phenotype. Immunochemistry analysis showed that cells at the periphery of the colonies had a negative E-cadherin expression and a positive Vimentin expression, suggesting an epithelial-mesenchymal transition (EMT). Nuclear staining of beta-catenin, positive N-cadherin and negative Connexin 43 expression were also found in the mesenchymal-like cell population. After RT-PCR analysis, Slug and Snail, both EMT-related transcription factors, were detected as up-regulated in the mesenchymal-like cell population. Taken together, our data suggest that culturing hESCs in feeder-free conditions enhances an early differentiation process identified as an EMT.

PGD is an alternative to prenatal diagnosis that circumvents therapeutic abortion. Diagnosis is carried out on single cells obtained from three-day-old embryos, and only those that are free of the disease under consideration are transferred to the mother. Neurofibromatosis type 1 (NF1) is a common neurocutaneous disorder, inherited as an autosomal dominant trait and caused by mutations in the NF1 gene. For some patients, PGD may be the only acceptable manner to ensure the birth of unaffected children. Because of the large number of known NF1 mutations, the development of mutation-specific single-cell protocols is impractical, labour-intensive and expensive. This paper discusses the development of five PGD protocols, three of which are based on multiplex PCR for microsatellite-markers linked to the NF1 gene. After a linkage study, the diagnosis can be established through the markers, thereby obviating the need to detect the mutation itself. This not only ensures the accurate diagnosis of the embryos, but also a prompt acceptance of PGD referrals since one protocol can be useful for several couples. In addition, two mutation-specific PCRs were developed for two couples where a marker-based protocol was not applicable. In total, 16 PGD cycles were carried out for six couples, which resulted in one ongoing pregnancy and the delivery of a healthy unaffected boy.

Background Beta-Thalassaemia results from co-inheritance of two mutant beta-globin alleles. Allogeneic cord blood cell transplantation (CBT) from an HLA-identical sibling donor is an excellent treatment option for beta-thalassaemia. In families with an affected child and willing to have another child, IVF followed by preimplantation genetic diagnosis (PGD) can be applied to exclude affected embryos. Furthermore, healthy embryos could be HLA matched with the affected child so that cord blood from the future newborn can be used to transplant the affected sibling. Methods We developed an indirect single-cell HLA typing technique based on the use of a bank of seven microsatellite markers within the HLA locus from which four informative and evenly distributed markers were selected. Results The methodology was validated in three beta-thalassaemia families having six ovarian stimulation cycles in view of IVF and PGD. Six PGD cycles were performed in two families. On 58 embryos tested, the combined PCR was successful in 54 (93%). Two transfers were done and one clinical pregnancy was obtained. Using confirmatory analysis on 50 embryos, the accuracy for HLA typing was 100%. Conclusion This strategy offers a new therapeutic option for patients with beta-thalassaemia and other monogenic diseases that can be cured with CBT.

We developed single-cell polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples carrying mutations in the beta-globin gene. With PGD the genetic status of an embryo obtained after intracytoplasmic sperm injection (ICSI) is determined by PCR analysis in single blastomeres, allowing only healthy embryos to be transferred to the uterus. We carried out nine PGD cycles using fluorescent PCR for two couples in whom the partners carried sickle-cell trait. Both couples achieved pregnancies, one of which was spontaneously aborted. We have developed two beta-thalassemia PGD protocols: one for the analysis of the 25-26delAA and the IVS2+1G>A mutation, and the other for the simultaneous detection of the IVS1+6T>C and the IVS1+110G>A mutations. For the second protocol, both non-labelled PCR and later fluorescent PCR were used. Both protocols were applied in clinical cycles (two non-labelled PCR cycles and one fluorescent PCR cycle) for two couples. The patient with the fluorescent PCR-PGD cycle became pregnant. Overall, the three fluorescent PCR assays were accurate and reliable with amplification efficiencies of minimum 93% and allele dropout (ADO) rates between 0 and 12%.

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.

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 a number of new technologies as well as the 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, as in the original publication, these 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-based testing, Amplification-based testing and Polar Body and Embryo Biopsy for PGD/preimplantation genetic screening. Here, we have updated the sections that pertain to amplification-based PGD. Topics covered in this guideline include inclusion/exclusion criteria for amplification-based PGD testing, preclinical validation of tests, amplification-based testing methods, tubing of cells for analysis, set-up of local IVF centre and Transport PGD centres, quality control/quality assurance and diagnostic confirmation of untransferred embryos.

BACKGROUND Couples undergoing preimplantation genetic diagnosis (PGD) have a different background and set of treatment characteristics to couples undergoing regular IVF or ICSI. The aim of this study was to analyse the cumulative reproductive outcome of a large cohort of couples undergoing PGD in relation to a number of explanatory variables potentially affecting the prognosis. METHODS Prospective cohort study, Kaplan-Meier analysis was performed to estimate real (observed) and expected (calculated) cumulative delivery rates, and Cox proportional hazard regression analysis was used to assess the effect of age, number of cumulus oocyte complexes collected, fertility status, parity, genetic category and method of pituitary suppression. RESULTS Between 1993 and 2005, 2753 unselected consecutive cycles of ICSI and PGD were carried out in 1498 couples. The cumulative observed delivery rate overall per couple with a maximum of six treatment cycles of ICSI and PGD performed was 29%. The expected cumulative delivery rate (max six cycles) overall was 62%. There were no significant differences in cumulative delivery rates between the different genetic categories (i.e. availability of transferable embryos after PGD of 50 or 75%, chromosomal translocations or aneuploidy screening). The cumulative reproductive outcome in this PGD cohort was also not significantly affected by the fertility status of the couple, their parity or the method of pituitary suppression. However, the age of the patient and the number of oocytes contributed significantly to the reproductive results. CONCLUSION This prospective observational study demonstrates that age has a significantly negative effect on outcome of PGD, due to poor reproductive performance of female partners 40 years of age and older. The number of oocytes collected has a significant and independent effect. The other factors studied did not affect the cumulative reproductive outcome in this PGD cohort.

Background This study aimed to analyse the reproductive outcome of a large cohort of myotonic dystrophy type 1 (DM1) patients undergoing ICSI and PGD. The secondary outcome parameter of this study was ovarian response as a way to express gonadal function in female DM1 patients. Methods Prospective cohort study. Real and expected cumulative delivery rates are descriptive. The reproductive outcome per cycle was compared with that of a control group of patients with X-linked recessive disorders. The comparative analysis of ovarian stimulation parameters in the study group versus the control group was carried out using both bivariate (crude) and multivariate (linear regression) analysis. Results Between 1995 and 2005, 205 cycles of ICSI and PGD were carried out for DM1 in 78 couples. The real cumulative delivery rate (max 6 cycles) overall was 46%. The expected overall cumulative delivery rate was 72%. Multivariate analysis did not show a significant difference in total dose of gonadotrophins used for ovarian stimulation between Group A (in which the female partner was affected) and a control group. Conclusions This study shows that ICSI and PGD for DM1 offer good reproductive outcome, both in cumulative terms and per treatment cycle. There is no evidence of impaired gonadal function in female DM1 patients.

Feeder-free culture induces spontaneous differentiation of human embryonic stem cells (hESCs), identified as an epithelial to mesenchymal transition (EMT). The maintenance of pluripotency of hESCs in feeder-free cultures through the activation of the WNT pathway using a glycogen synthase kinase (GSK)-3-specific inhibitor (BIO) was reported. The aim of this study was to determine the effect of BIO on the EMT process. In contrast with those grown in feeder-free conditions with control medium, hESC colonies cultured with BIO-supplemented hESC medium did not show any fibroblast-like cells at the periphery. Transmission electron microscopy, relative quantitative real-time RT-PCR and immunostaining analyses showed the presence of epithelial features and a diminution of mesenchymal features in the BIO-treated hESCs such as a strong E-cadherin expression, the down-regulation of Vimentin, Snail and Slug expressions and a cytoplasmic beta-catenin expression. An up-regulation of matrix metalloproteinases (MMP) MMP-2, MMP-9, MT-1MMP (membrane-type 1 MMP) and EMMPRIN (extracellular MMP inducer) expression was also found associated with the EMT occurring in feeder-free hESCs cultures using mouse embryonic fibroblasts conditioned medium (MEF CM). The presence of BIO clearly down-regulated the expression of these MMPs. This study showed that BIO, a GSK-3-specific inhibitor, prevents the EMT process which is associated with the feeder-free hESC culture. Nevertheless, BIO was not sufficient to expand hESCs in a long-term culture system.

BACKGROUND: Human embryonic stem (hES) cells are pluripotent cells usually derived from the inner cell mass (ICM) of blastocysts. Because of their ability to differentiate into all three embryonic germ layers, hES cells represent an important material for studying developmental biology and cell replacement therapy. hES cell lines derived from blastocysts diagnosed as carrying a genetic disorder after PGD represent in vitro disease models. METHODS: ICMs isolated by immunosurgery from human blastocysts donated for research after IVF cycles and after PGD were plated in serum-free medium (except VUB01) on mouse feeder layers. RESULTS: Five hES cell lines were isolated, two from IVF embryos and three from PGD embryos. All lines behave similarly in culture and present a normal karyotype. The lines express all the markers considered characteristic of undifferentiated hES cells and were proven to be pluripotent both in vitro and in vivo (ongoing for VUB05_HD). CONCLUSIONS: We report here on the derivation of two hES cell lines presumed to be genetically normal (VUB01 and VUB02) and three hES cell lines carrying mutations for myotonic dystrophy type 1 (VUB03_DM1), cystic fibrosis (VUB04_CF) and Huntington disease (VUB05_HD).

A broad spectrum of assisted reproductive technologies has become available for couples with fertility problems. Follow-up studies of children born as a result of assisted reproduction have shown that neonatal outcome and malformation rates are not different from those of the general population, except for a low birthweight and a slight increase in chromosomal abnormalities. The safety aspect of assisted reproduction at the epigenetic level has not been well studied. Epigenetics refers to phenomena where modifications of DNA methylation and/or chromatin structure underlie changes in gene expression and phenotype characteristics. This article intends to analyse epigenetic risks related to assisted reproduction on the basis of an overview of epigenetic reprogramming events in the gamete and early embryo. Two epigenetic modifications, methylation and imprinting, are considered in more detail. The interference of in-vitro embryo culture, immature sperm cells and nuclear transfer with epigenetic reprogramming is discussed, as well as the possibility of epigenetic inheritance.

Objective—To use computed tomography to provide a detailed description of tarsal joint structures in clinically normal dogs. Animals—6 clinically normal adult mixed-breed dogs weighing 25 to 35 kg and one 12-month-old Bullmastiff weighing 65 kg. Procedure—To perform computed tomography (CT) of both tarsal regions, dogs were anesthetized and placed in ventral recumbency. One- and 2-mm contiguous slices were obtained, using a third generation CT scanner. Individual images were reviewed, using bone (window width = 3,500 Hounsfield units; window level = 500 Hounsfield units) and soft-tissue (window width = 400 Hounsfield units; window level = 66 Hounsfield units) settings. After euthanasia, the hind limbs from the Bullmastiff were removed and frozen at –18 C. Tarsal joints were sectioned into approximately 1-mmthick slab sections, using a cryomicrotome. Anatomic sections were photographed and compared with the corresponding CT images. Computed tomographic reconstructions of the tarsocrural joint were created in sagittal and dorsal planes. Results—Structures on the CT images were matched with structures in the corresponding anatomic sections. The entire tarsocrural joint surface could be evaluated on the reconstructed images in the sagittal and dorsal planes. Conclusions and Clinical Relevance—CT images provide full anatomic detail of the bony structures of the tarsal joint in dogs. Tendons and large blood vessels can also be evaluated. These results could be used as a basis for evaluation of CT images of the hind limbs of dogs with tarsal joint injuries. (Am J Vet Res 2001;62:1911–1915)

A natural monopoly is usually characterized by a property of the production technology represented by the cost function. If Q is the vector of outputs and C the total production cost, we denote by φ the cost function $$C = \varphi (Q),$$ which also depends on the input price vector and, in general, on some specific environmental variables. The subadditivity property which defines the natural monopoly, assumes that: for all p, for all $$for all p, for all {{Q}_{1}}, \ldots {{Q}_{p}} \varphi \left( {\sum\limits_{{j = 1}}^{p} {{{Q}_{j}}} } \right) \leqslant \sum\limits_{{j = 1}}^{p} {\varphi ({{Q}_{j}}).}$$ (1.1)

Autosomal dominant polycystic kidney disease (ADPKD) is primarily characterized by renal cysts and progression to renal failure. It is a genetically heterogeneous disease, with mutations in the PKD1 gene accounting for the majority of cases. Direct mutation detection for PKD1-linked ADPKD or type 1 is complicated by the large size and complex genomic structure of PKD1. This paper describes a microsatellite marker-based assay for PGD in couples at risk of transmitting ADPKD type 1. During PGD, genetic analysis is carried out on single blastomeres biopsied from preimplantation embryos obtained after IVF, and only embryos unaffected by the disease under investigation are selected for transfer. Single-cell genetic analysis relied on a fluorescent duplex-PCR of linked polymorphic markers followed by fragment length determination on an automated sequencer. The co-amplification of the intragenic KG8 and the extragenic D16S291 marker at the single-cell level was evaluated in pre-clinical tests on lymphoblasts and research blastomeres. The developed assay proved to be efficient (96.1% amplification) and accurate (1.4% allele drop-out and 4.3% contamination), and can be applied in all informative ADPKD type 1 couples. From five clinical cycles carried out for three couples, two pregnancies ensued, resulting in the birth of two healthy children. Mol Hum Reprod

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infection of the respiratory tract and pancreatic insufficiency. The first preimplantation genetic diagnosis (PGD) for CF was carried out in 1992. At our centre the first cycle was performed in 1993. However, the number of known CF mutations is >1000, so developing mutation-specific PCR protocols for PGD is unfeasible. This is why a number of marker-based duplex PCRs were developed at the single cell level. A duplex PCR of a mutation and one or two microsatellites is not only a diagnostic tool, but it can also be used as a control for allele drop-out and contamination. During PGD, embryos obtained in vitro are analysed for the presence or absence of a particular genetic disease, after which only embryos shown to be free of this disease are returned to the mother. In total, 22 PGD cycles with duplex PCR (IVS8CA/IVS17BTA, DeltaF508/IVS8CA, DeltaF508/IVS17BTA and D7S486/D7S490) were carried out in 16 couples, which resulted in four ongoing pregnancies and one miscarriage.

OK, The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minutequantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.

BACKGROUND Apart from its use in research, spermatogonial stem cell transplantation (SSCT) may have important clinical applications. This controlled study aimed at evaluating the safety of SSCT by analyzing the DNA methylation pattern of Igf2, Peg1 and alpha-Actin both in spermatozoa and live born offspring obtained after SSCT in mice. METHODS Testicular cell suspensions were transplanted to the testes of genetically sterile WW recipients. Transplanted males were mated with fertile females and their first and second generation offspring were examined and compared with controls with respect to weight, length and DNA methylation patterns. Sodium-bisulfite treated genomic DNA extracted from post-transplantation spermatozoa, liver, kidney and placenta of first and second generation offspring was PCR-amplified to obtain Igf2, Peg1 and alpha-Actin gene fragments. Pyrosequencing was used to individually quantify the resulting artificial C/T sequence variation at CpG sites. RESULTS First and second generation offspring developed normally with their length and weight not being different from controls. Also the DNA methylation patterns of Igf2, Peg1 and alpha-Actin were not different among controls and first and second generation offspring after SSCT. CONCLUSIONS SSCT between syngenic individuals was not associated with changes in fetal development nor with differences in the DNA methylation patterns of Igf2, Peg1 and alpha-Actin in spermatozoa or other tissues from two subsequent generations of offspring obtained after SSCT.

BACKGROUND: Preimplantation genetic diagnosis (PGD) and subsequently preimplantation genetic screening (PGS) have been introduced since I990. The difference from the already existing in vitro fertilization (IVF) technology, using intracytoplasmic sperm injection (ICSI), was the embryo biopsy at day 3 after fertilization. Although healthy children post-PGD/PGS have been born, the question of whether embryo biopsy could have any harmful effects has to be studied on large series in a prospective manner. METHODS: A prospective cohort study was undertaken from I992 until 2005, using the same approach as for the follow-up of IVF and ICSI children conceived in the same centre. Questionnaires were sent to physicians and parents at conception and at delivery. Children were examined at 2 months of age by trained clinical geneticists whenever possible. RESULTS: Data collected on 58I post-PGD/PGS children showed that term, birthweight and major malformation rates were not statistically different from that of 2889 ICSI children, with overall rates of major malformation among these post-PGD/PGS and ICSI children being 2.I3 and 3.38%, respectively (odds ratio [OR]: 0.62; exact 95% confidence limits [95% CL]: 0.31 - 1.15). However, the overall perinatal death rate was significantly higher among post-PGD/PGS children compared with ICSI children (4.64 versus I.87%; OR: 2.56; 95% CL: I.54―4.I8). When stratified for multiple births, perinatal death rates among PGD/PGS singleton and ICSI singleton children were similar (I.03 versus I.30%; OR: 0.83; 95% CL: 0.28―2.44), but significantly more perinatal deaths were seen in post-PGD/PGS multiple pregnancies compared with ICSI multiple pregnancies (II.73 versus 2.54%; OR: 5.09; 95% CL: 2.80-9.90). The overall misdiagnosis rate was below I%. CONCLUSIONS: Embryo biopsy does not add risk factors to the health of singleton children born after PGD or PGS. The perinatal death rate in multiple pregnancies is such that both caution and long-term follow-up are required.

background: Outcome data on children born after assisted reproduction treatments are important for both patients and health-care providers. The objective of this study was to determine whether embryo biopsy as performed in PGD has an impact on the health of infants up to 2 months of age. methods: A prospective comparative follow-up study of children born after PGD and children born after ICSI by collecting written reports and performing a physical examination at 2 months was performed. Auxological data at birth and physical findings up to 2 months of age were compared for 995 children consecutively live born after embryo biopsy (1994 –2009) and for a control group of 1507 children born after ICSI with embryo transfer on Day 5. results: No differences regarding mean term, prematurity (term ,32 w and ,37 w), mean birthweight, very low birthweight (,1500 g), perinatal death, major malformations and neonatal hospitalizations in singletons and multiples born following PGD versus ICSI were observed. Compared with ICSI, fewer multiples born following PGD presented a low birthweight (,2500 g) (P ¼ 0.005). conclusions: Embryo biopsy for PGD does not introduce extra risk to the overall medical condition of newborn children. Multiples born following embryo biopsy appear to be at lower risk for low birthweight compared with multiples born following ICSI.

Background Human embryo biopsy is performed for preimplantation genetic diagnosis (PGD). The impact of 1- or 2-cell removal at cleavage-stage on future embryo development and implantation capacity is highly debated. Methods In order to explore this issue further, a cohort of Day 5 single embryo transfers was analysed prospectively for embryological and clinical outcome. All transferred embryos resulted from 8-cell embryos on Day 3, from which subsequently either one cell (group I, n = 182) or two cells (group II, n = 259) were removed, or on which no invasion by means of embryo biopsy was performed (group III, control group, n = 702). RESULTS Blastocyst formation was significantly better in group III compared with group II, and similar to group I. Group I and group II did not differ in Day 3 nor in Day 5 embryo development. The overall live birth rate was significantly higher in group I (37.4%, CI 29.0-47.4%) than in group II (22.4%, CI 17.0-28.9%), and comparable to the reference ICSI population (35.0%, CI 30.8-39.7%). Conclusions The clinical outcome of 1-cell biopsy was significantly better than that of 2-cell biopsy, even when adjusted for availability of genetically transferable embryos.

Introduction: NANOG, SOX2, and SALL4 are transcription factors that play a key role in controlling stemness in embryonic stem cells (ESC) and are therefore candidate markers for developmental triggers in early embryos. KRT18, a trophoblast determining gene, may mark early differentiation in embryos. The expression pattern of NANOG, SOX2, SALL4 and KRT18 may identify the totipotent cells during early human development and determine when early embryonic cells lose their totipotent competence. Material and Methods: Thirtheen human oocytes and 121 human preimplantation embryos were examined for the presence of NANOG, SOX2, SALL4 or KRT18 proteins using immunostaining and confocal microscopy. Results: None of the stemness markers seemed to direct the dividing blastomeres towards the inner cell mass (ICM) or trophectoderm (TE) lineage, nor could they identify all and none other than the totipotent cells during human preimplantation development. NANOG proteins could at the earliest be detected at the blastocyststage and were consistently present in expanded blastocysts, when the two cell lineages were already visibly defined. Within the expanded blastocyst, we found NANOG proteins to be present in the nuclei of a subpopulation of the ICM. A sporadic NANOG expression was also detected in a minority of TE cells located near the ICM. A cytoplasmic SOX2 expression was detected in oocytes and throughout the whole preimplantation development. A nuclear expression appeared at day three of development and became prominent in all cells. After the ICM and the TE could clearly be distinguished, the nuclear staining increased in all cells of the ICM and disappeared in nearly all TE cells. SALL4 proteins were detected in the nucleus and the cytoplasm in oocytes and early cleavage stages until day 3 of development. During further development, the cytoplasmic staining was lost whereas the nuclear staining increased. In blastocysts, a strong expression was found in the nuclei of the ICM and TE. A joint expression of NANOG, SOX2 and SALL4 was only detected in the nuclei of a subpopulation of ICM cells. Expression of KRT18 was seen for the first time during compaction in some outer cells. During blastocyst expansion, KRT18 proteins were found in the cytoskeleton of the TE and the outer cells of the ICM adjacent to the blastocoel cavity, which will contribute to the yolk sac. Conclusions: The expression pattern of markers that define stemness in ESC could not tell us when and in which cells totipotency is lost during early human development. However, because stemness markers act in concert to keep the properties of ESC, these markers may maintain totipotency in a subpopulation of the ICM. Assessing the presence of KRT18 proteins implied that the outer cells of compacting embryos have lost their totipotent competence prior to any visible morphological differentiation. KRT18 may be used as an early marker of differentiation during human preimplantation development.

Objectives Mutations in the APC, NF2 and BRCA1 genes cause adult-onset cancer predisposition syndromes. Prenatal diagnosis (PND) and selective pregnancy termination for adult-onset disorders is emotionally difficult and, in some cases, socially not well accepted. Preimplantation genetic diagnosis (PGD) appears as an attractive alternative to PND, as it ensures the establishment of a pregnancy free of the mutation from the onset, circumventing the potentially difficult decision of termination of pregnancy. Methods Development of single-cell PCRs using Epstein-Barr virus transformed lymphoblasts as single-cell model, followed by clinical application in PGD. Results A total of five duplex-PCRs were developed, three for adenomatous polyposis of the colon (APC), one for neurofibromatosis type 2 (NF2) and one for inherited breast and ovarian cancer caused by BRCA1 mutations. Eleven clinical cycles were performed, resulting in the birth of an unaffected girl. For one of the couples undergoing PGD for NF2, a spontaneous pregnancy ensued after five unsuccessful PGD cycles. The couple underwent chorionic villus sampling (CVS) and the application of the same protocol as used during PGD showed an unaffected fetus. Conclusion In this work, we present the development and clinical application of PGD for three cancer predisposition syndromes. Copyright © 2007 John Wiley & Sons, Ltd.