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Research question What temperature is optimal for human embryo development up to day 5 or 6? Design Two prospective sibling oocyte studies on culture temperature were conducted in a university-based tertiary referral centre. Eligibility critera for both studies: Study 1: 50 cycles between August and October 2015, with culture at a stable temperature (37.0°C ± 0.3°C) or culture using a circadian temperature rhythm (CTR) (1 am to 6 am: 36.6°C, gradual increase to 37.5°C; 11 am to 9 pm: 37.5°C; gradual decrease to 36.6°C); study 2: 99 cycles between April and November 2016, with stable culture at 36.6°C or 37.1°C. Primary outcome measures: fertilization and embryo development (top and good quality) up to day 5 or 6, and utilization rate (number of embryos transferred and cryopreserved per zygote). Secondary outcome measure: clinical pregnancy (number of pregnancies with at least one gestational sac). Results An incubator with CTR was used for culture. An effect was found on embryo development (utilization rate: 42.1% versus 32.6%; P = 0.014), but not on clinical pregnancy rate (60.0% versus 45.5%; P = 0.670). Stable culture at 36.6°C or 37.1°C did not affect embryo development (utilization rate: 40.0% versus 40.4%; P = 0.905); clinical pregnancy rate was improved by culture at 37.1°C (46.4% versus 74.2%; P = 0.036). Conclusion Culture in an incubator with CTR does not improve fertilization rate or embryo quality. Embryo culture at 36.6°C or 37.1°C showed similar embryo development.

Study question Is it possible to detect and quantify low frequency heteroplasmic variants, large deletions and single nucleotide variants (SNV), in the mtDNA of single cells? Summary answer Our method detects heteroplasmic SNVs and large deletions in single cells and data on single oocytes provides interesting insight on SNV segregation during oogenesis. What is known already The advent of MPS has opened new possibilities for the analysis of low frequency heteroplasmies in the mtDNA, but no methods are currently available to accurately detect and quantify low frequency SNVs and large deletions. Mutations in the mtDNA have been previously reported in ART-derived oocytes and embryos, as well as methods for the analysis of individual mutations in preimplantation genetic diagnosis. Conversely, up to now, it has not been possible to identify all mutations in one single cell simultaneously, nor to establish their mutation load. Study design, size, duration We developed a method to analyze large deletions and SNVs in DNA samples and downscaled it to single cells. We sequenced six single fibroblasts from a control individual, two muscle fibers from a patient with mtDNA deletions, and 11 oocytes from three different patients (5GV, 3 MI, 3 MII). Participant/materials, setting, methods Single cells were lysed in alkaline lysis buffer. MtDNA was enriched by long-range PCR. Sequencing was done at 6000x. SNVs called with CLCBio Genomic Workbench and large deletions were identified using BLAST. Mutation load was established using the sequencing depth. The lower detection limits are linked to the number of PCR cycles needed, down to loads for deletions and SNVs of 0,5% and 1,5% at 35 cycles, and 0,5% and 2% at 45 cycles, respectively. Main results and role of chance The single fibroblasts revealed somatic mosacism, with two variants appearing in 1/6 cells at high loads, and not present in the other cells. The muscle fibers showed a low number of large deletions at a high frequency, and most of them were also identified in the blood sample of the same patient. In the oocytes, one large deletion at 0,51% was found in the cohort analyzed. We detected SNVs in 9/11 oocytes, at frequencies ranging from 1,5% to 20%. The same SNVs were found recurrently in the oocytes from the same donor but often at different frequencies. In one patient we observed a variant in 3/ 4 oocytes, at loads of 5%, 15% and 20%. In the same patient, another variant was detected in 2/4 oocytes at frequencies of 5% and 15%. In 2/4 oocytes from a different patient, a third SNV was detected at frequencies of 2,5% and 3%. Half of the detected variants mapped to the hypervariable region (12/24), and only 1 variant at load of 4% was predicted to be pathogenic. There was no correlation between maturation stage and presence of variants. Limitations, reasons for caution The results we obtained with the oocytes must be matched with the variants in somatic tissues of the same patient to have a full validation of the variants called, and to follow the variants segregation Wider implications of the findings. Our method for low frequency detection can be used to study mtDNA variants in single cells with high accuracy. This tool can be applied also to the diagnostic of mitochondrial diseases involving large deletions or SNVs. Finally, our data provides interesting preliminary information about heteroplasmy shifts during gametogenesis. Funding Trial n° Not applicable

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.