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STUDY QUESTION How should ART/preimplantation genetic testing (PGT) centres manage the detection of chromosomal mosaicism following PGT? SUMMARY ANSWER Thirty good practice recommendations were formulated that can be used by ART/PGT centres as a basis for their own policy with regards to the management of ‘mosaic’ embryos. WHAT IS KNOWN ALREADY The use of comprehensive chromosome screening technologies has provided a variety of data on the incidence of chromosomal mosaicism at the preimplantation stage of development and evidence is accumulating that clarifies the clinical outcomes after transfer of embryos with putative mosaic results, with regards to implantation, miscarriage and live birth rates, and neonatal outcomes. STUDY DESIGN, SIZE, DURATION This document was developed according to a predefined methodology for ESHRE good practice recommendations. Recommendations are supported by data from the literature, a large survey evaluating current practice and published guidance documents. The literature search was performed using PubMed and focused on studies published between 2010 and 2022. The survey was performed through a web-based questionnaire distributed to members of the ESHRE special interest groups (SIG) Reproductive Genetics and Embryology, and the ESHRE PGT Consortium members. It included questions on ART and PGT, reporting, embryo transfer policy and follow-up of transfers. The final dataset represents 239 centres. PARTICIPANTS/MATERIALS, SETTING, METHODS The working group (WG) included 16 members with expertise on the ART/PGT process and chromosomal mosaicism. The recommendations for clinical practice were formulated based on the expert opinion of the WG, while taking into consideration the published data and results of the survey. MAIN RESULTS AND THE ROLE OF CHANCE Eighty percent of centres that biopsy three or more cells report mosaicism, even though only 66.9% of all centres have validated their technology and only 61.8% of these have validated specifically for the calling of chromosomal mosaicism. The criteria for designating mosaicism, reporting and transfer policies vary significantly across the centres replying to the survey. The WG formulated recommendations on how to manage the detection of chromosomal mosaicism in clinical practice, considering validation, risk assessment, designating and reporting mosaicism, embryo transfer policies, prenatal testing and follow-up. Guidance is also provided on the essential elements that should constitute the consent forms and the genetic report, and that should be covered in genetic counselling. As there are several unknowns in chromosomal mosaicism, it is recommended that PGT centres monitor emerging data on the topic and adapt or refine their policy whenever new insights are available from evidence. LIMITATIONS, REASONS FOR CAUTION Rather than providing instant standardized advice, the recommendations should help ART/PGT centres in developing their own policy towards the management of putative mosaic embryos in clinical practice. WIDER IMPLICATIONS OF THE FINDINGS This document will help facilitate a more knowledge-based approach for dealing with chromosomal mosaicism in different centres. In addition to recommendations for clinical practice, recommendations for future research were formulated. Following up on these will direct research towards existing research gaps with direct translation to clinical practice. Emerging data will help in improving guidance, and a more evidence-based approach of managing chromosomal mosaicism. STUDY FUNDING/COMPETING INTEREST(S) The WG received technical support from ESHRE. M.D.R. participated in the EQA special advisory group, outside the submitted work, and is the chair of the PGT WG of the Belgian society for human genetics. D.W. declared receiving salary from Juno Genetics, UK. A.C. is an employee of Igenomix, Italy and C.R. is an employee of Igenomix, Spain. C.S. received a research grant from FWO, Belgium, not related to the submitted work. I.S. declared being a Co-founder of IVFvision Ltd, UK. J.R.V. declared patents related to ‘Methods for haplotyping single-cells’ and ‘Haplotyping and copy number typing using polymorphic variant allelic frequencies’, and being a board member of Preimplantation Genetic Diagnosis International Society (PGDIS) and International Society for Prenatal Diagnosis (ISPD). K.S. reported being Chair-elect of ESHRE. The other authors had nothing to disclose. DISCLAIMER This Good Practice Recommendations (GPR) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation. ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type. Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE.

Thirty years of rapid technological advances in the field of genetic testing and assisted reproduction have reshaped the procedure of preimplantation genetic testing (PGT). The development of whole genome amplification and genome-wide testing tools together with the implementation of optimal hormonal stimulation protocols and more efficient cryopreservation methods have led to more accurate diagnoses and improved clinical outcomes. In addition, the shift towards embryo biopsy at day 5/6 has changed the timeline of a typical PGT clinical procedure. The Brussels' PGT centre started more than 25 years ago, offering PGT within a healthcare system funded by the government. In this paper, we have summarized our experience with PGT, presenting an up-to-date overview of the different steps in PGT from patient referral to baby follow-up.

After fertilization, the mammalian embryo undergoes epigenetic reprogramming with genome-wide DNA demethylation and subsequent remethylation. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) was suggested to be an intermediate step in the DNA demethylation pathway. Other evidence, such as the stability of 5hmC in specific tissues, suggests that 5hmC constitutes a new epigenetic modification with its own biological function. Since few studies have been conducted on human material compared to animal models and species-specific epigenetic differences have been reported, we studied global DNA methylation and hydroxymethylation patterns in human in vitro preimplantation embryos using immunocytochemistry, comparing these patterns in good-quality and abnormally developing embryos. Our data showed that DNA methylation and hydroxymethylation modifications co-exist. 5mC and 5hmC signals were found in oocytes and in paternal and maternal pronuclei of zygotes, present in non-reciprocal patterns-which contrasts published data for the mouse. These two epigenetic modifications are present between Days 1 and 7 of in vitro development, with 5mC levels declining over cell divisions without noticeable remethylation during this period. A main decline in 5mC and 5hmC occurred as the embryo progressed from compaction to the blastocyst stage. No difference in (hydroxy)methylation was found between the inner cell mass and trophectoderm. When comparing normally and abnormally developing embryos, DNA (hydroxy)methylation reprogramming was abnormal in poor-quality embryos, especially during the first cleavages. Mol. Reprod. Dev. 83: 594-605, 2016 © 2016 Wiley Periodicals, Inc.

Study question: To assess ovarian response to conventional ovarian stimulation (COS) as part of a pre-implantation genetic diagnostic (PGD) treatment in BRCA1 mutation carriers. Summary answer: BRCA1 mutation is not associated with a reduced response to ovarian stimulation. What is known already: Loss-of-function mutations in the tumour suppressor genes BRCA1/2 are associated with an increased lifetime risk of developing breast- and/or ovarian cancer. In more recent years it has been hypothesized that germline mutations in BRCA-genes may be associated with in fertility-related problems. This is because BRCA genes participate in repair and maintenance of chromosome telomeres and the integrity of those is central in reproductive wellbeing. Study design, size, duration: A retrospective, case-control, non-randomised and mono-centre study. In the study group and both control groups 39 first treatment cycles were included (13:13:13). They were matched (1:1) for ovarian stimulation protocol, starting dose of gonadotrophins, for age ±3 years and pick-up date ±1 year. The study period covered 2006-2013. Participants/materials, setting, methods: Academic PGD reference centre. 13 BRCA1 cases (group A) stimulated in an antagonistic scheme with starting dose of 150 units of gonadotrophins, matched to two control groups, group B, patients for PGD for monogenic disorders not associated with infertility, and group C patients for ICSI and blastocyst transfer. Main results and the role of chance: Statistical analysis was performed using SPSS software (version 22.0). Numerical variables were compared using Mann- Whitney U-test, continuous variables were analysed by Fisher's exact test. Compared with the control groups, B1 and B2, BRCA patients do not produce a significant lower amount of mature oocytes (A: 10.077 (SD:5.6); B: 11.62 (SD:5.1); C:9.308 (SD:5.9)) and no significant difference was found in the total dose of gonadotrophins used in the three groups to achieve this result (A: 1431 (SD:257) U; B:1450 (SD:303) U; C:1315(SD:264) U). No difference in pregnancy rate was found; the pregnancy rate in all groups was 23.1%. Overall there are no significant differences in outcome parameters between the groups compared. Limitations, reason for caution: Although this series is larger than those previously reported, the numbers remain small and more extensive studies are required. The retrospective character of the study reflects clinical practice, however does not exclude selection bias. Wider implications of the findings: Contrary to recent studies that have suggested that BRCA1 mutations are associated with an earlier menopause and occult primary ovarian insufficiency, our study did not confirm reduced ovarian response. This implies that BRCA patients presenting for IVF and/or PGD can use conventional stimulation schedules. Study funding/competing interest(s): Funding by hospital/clinic(s), Universitair Ziekenhuis Brussel. Trial registration number: N/A.

Study question What is the optimum ovarian stimulation protocol for a first treatment cycle of IVF/ICSI and preimplantation genetic diagnosis (PGD) in terms of i. number of good quality embryos for biopsy at cleavage stage, ii. clinical pregnancy rates per first treatment cycle and iii. incide nce of ovarian hyperstimulation syndrome (OHSS). Summary answer There is no difference in number of embryos for biopsy according to the type of ovarian stimulation protocol used. The clinical pregnancy rate (CPR) per started first-rank cycle is higher in the agonist group. The incidence of OHSS is low in both the agonist and the antagonist group. What is known already The most recent meta-analyses do not show a significant difference in pregnancy rates between agonist and antagonist stimulation protocols, although there is a consensus on a lower risk of ovarian hyperstimulation syndrome (OHSS) and a lower number of oocytes when using GnRH antagonist. There is a shift of focus in PGD in view of the necessity of an optimal number of embryos for genetic analysis. Data on optimal stimulation protocols for PGD are lacking. Study design, size, duration Prospective randomized (blocks of 6; concealed) trial with intention-to-treat analysis. A sample size of 120 subjects was required to allow 80% power with a significance level of 5% in order to detect a difference of 2 embryos for biopsy. The duration of the study was 18 months. Participants/materials, setting, methods Tertiary referral centre for PGD. Inclusion criteria: i. age Main results and the role of chance 132 patients were recruited; of these 129 were randomized; 5 patients dropped out of the study leaving 124 cases for analysis. There was no significant difference in background variables. There was no difference in number of oocytes (13.2 (SD 7.3) v 12.6 (SD 7.1)) and number of embryos for biopsy (6.7 (SD 4.8) v 6.4 (SD 4.2)(NS) in A versus B respectively. The transfer rate was lower in group B (62%) than in group A (84%)(p =0.006). The clinical pregnancy rate (CPR) per cycle was higher in group A (49.2%) than in group B (26.2%)(p = 0.008). The CPR per embryo transfer was not different (42.1% v 58.4% in B versus A respectively). The incidence of OHSS was 1.6% in each group; both cases were mild. Limitations, reason for caution Though the clinical pregnancy rate per first rank PGD cycle showed a significant difference, the results need to be interpreted with caution as the study was not powered for this analysis. The data apply exclusively to PGD for monogenic or chromosomal disorders and not for preimplantation genetic screening (PGS). Wider implications of the findings This is the first prospective randomised trial by intention-to-treat analysis comparing the two most common ovarian stimulation protocols and their effect on number of embryos for biopsy and clinical pregnancy rate in PGD. The number of embryos for biopsy is not affected by the type of ovarian stimulation protocol used, however the transfer rate is different indicating qualitative differences in embryogenesis. PGD patients, mostly (68%) fertile, may have a lower risk of OHSS. Study funding/competing interest(s) The study was supported by an unconditional grant by Ferring Pharmaceuticals Belgium. The authors have no competing interests. Trial registration number av

Introduction After fertilisation the mammalian embryo undergoes epigenetic reprogramming with genome-wide DNA demethylation and subsequent remethylation. This reprogramming is essential for proper embryonic development. It was suggested that the recently discovered oxidation of 5-methylcytosine (5meC) into 5-hydroxymethylcytosine (5hmeC) may be an intermediate in the pathway of DNA demethylation and/or 5hmeC may be a new epigenetic modification. 5meC and 5hmeC are likely to have distinct biological roles: 5mC is generally associated with gene repression while 5hmeC has been correlated with gene expression. As many methods do not allow distinguishing between 5meC and 5hmeC, many DNA methylation data require re-analysis. Therefore we investigated the DNA methylation and hydroxymethylation patterns in human in vitro preimplantation embryos using immunocytochemistry. Material & methods Spare human preimplantation embryos from infertility treatments at our centre were donated for research after informed consent of the patients and with the approval of the local and the Federal Ethical Committee for scientific research on human embryos in vitro. All embryos were cultured in vitro in sequential medium and only embryos of good morphological quality were analysed (n= 5, 2, 4, 5 and 3 respectively). The relative quantification of 5meC and 5hmeC levels was assessed at day 3, 4, 5, 6 and 7 of embryonic development in double staining experiments with antibodies against 5meC and 5hmeC. Fluorescence was visualized by confocal laser scanning and quantification of the signals was done with Volocity imaging software to overcome artefacts related to signal concentration in smaller nuclei. The nuclear fluorescence intensity of 5meC and 5hmeC was measured in all nuclei and expressed as the ratio of 5meC over 5hmeC. Ratios from nuclei of embryos at the same day of development were taken together to calculate mean ratios. The mean 5meC and 5hmeC levels per embryo, with the mean intensities at day 3 arbitrarily set at 1, were presented in another graph. In blastocysts, nuclei from the inner cell mass (ICM) and trophectoderm (TE) were studied separately. Results Both 5meC and 5hmeC signals were present in all studied preimplantation stages. The ratio of 5meC over 5hmeC was high at day 3 (ratio = 1.70) and remained unchanged at day 4. A decrease in the global methylation level was observed from the early blastocyst stage on, while the global hydroxymethylation signal first slightly decreased at day 5 (ratio = 1.0), but then became the stronger signal at day 6 and 7 (ratio = 0.80 and 0.77 respectively). No difference in 5meC or 5hmeC levels or ratio was found between ICM and TE. Conclusions The DNA methylation and hydroxymethylation modifications co-exist between day 3 and 7 of human in vitro embryo development but their ratio is changed between day 4 and 6. Initially DNA methylation is the more dominant modification but beyond day 5, hydroxymethylation becomes more prevalent. Earlier immunocytochemistry data described a decrease in methylation level during cleavage divisions from zygote to morula; but further decrease of 5meC in blastocysts as seen in our data has not been reported in humans or mice. Given that all 5hmeC is likely derived from pre-existing 5meC, this drop in methylation corresponds well with the accumulation of 5hmeC in blastocysts. High 5hmeC levels were found in embryonic stem cells and their progenitors. At day 7, 5hmeC remains the prevailing modification as we observe that remethylation starts slowly around the time of implantation after day 6. Our finding of similar methylation levels in ICM and TE is not in agreement with another dataset on human embryos, this may relate to limited sample numbers and/or differences in embryo quality.

Introduction: It has been shown that procedures associated with assisted reproductive technologies may interfere with epigenetic reprogramming of oocytes and embryos. After fertilisation the mammalian embryo undergoes a gradual genomewide DNA demethylation, which results in relatively low DNA methylation levels at the morula stage. Subsequently, DNA methylation is re-established from the blastocyst stage onward. This de novo DNA methylation is set up by de novo DNA methyltransferases, DNMT3a and DNMT3b. We investigated if the process of embryo cryopreservation on day 3 (using a slow-freezing DMSO protocol) affects the expression levels of DNMT3a and DNMT3b or global DNA methylation levels. Material and Methods: Spare human preimplantation embryos from infertility treatments at our centre were donated for research after informed consent of the patients and with the approval of the Local and the Federal Ethical Committee for scientific research on human embryos in vitro. The embryos had been cryopreserved on day 3 and were thawed using a slow-thawing DMSO protocol (n = 34). At day 6, this group was classified into a subgroup of good quality embryos (n = 15) and a subgroup of delayed and arrested embryos (n = 19). The control group consisted of 'fresh' embryos (n = 10) that were of good quality, but were affected after preimplantation genetic diagnosis, or embryos of suboptimal quality that did not fulfil the criteria for transfer or cryopreservation. All embryos were cultured in vitro for 6 days in sequential medium. We compared the global DNA methylation level as well as the expression levels of de novo DNA methyltransferases DNMT3a and 3b in cryopreserved and fresh embryos at day 6 of preimplantation development. The analysis was done by immunofluorescence with antibodies against 5'methyl-cytosine (Eurogentec) in single staining experiments or against the DNMT3a and DNMT3b protein (Santa Cruz) in double staining experiments. Embryos of similar stages were stained with normal serum of the same host species as an internal negative control for the primary antibody. Fluorescence was visualized by confocal laser scanning and further analysis was done with Volocity imaging software. The fluorescence intensity (FI) per embryo was measured in all embryonic cells and expressed as the sum of all intensities over the embryonic size. Finally the mean FI for the global DNA methylation or DNMT3a and DNMT3b levels was calculated per embryo (sub)group. Data of the fresh embryo group or the subgroup of delayed and arrested cryopreserved embryos were relatively quantified against the mean FI of the good quality cryopreserved embryo subgroup (mean FI = 1). Results: No difference was observed between the mean FI of DNA methylation levels (1.2 ± 0.1) in fresh embryos compared to levels in good quality cryopreserved embryos. Similar DNMT3a (1 ± 0.3) and DNMT3b (0.8 ± 0.2) levels were found in the fresh embryo group versus the good quality cryopreserved embryos. The mean FI of DNA methylation (1.1 ± 0.4) levels in delayed and arrested cryopreserved embryos was similar to the levels of good quality cryopreserved embryos as were the mean FI of DNMT3a (1 ± 0.3) and DNMT3b (0.8 ± 0.3). Conclusions: These results show that at day 6, the de novo DNA methylation levels and enzyme expression levels are reinstated in a timely manner in human blastocysts that developed from embryos cryopreserved on day 3 using a slow-freezing DMSO protocol. The cryopreservation process does not seem to interfere with DNA methylation epigenetic reprogramming.

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