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Study Question: Is the time interval between ovulation triggering and oocyte denudation/injection associated with embryological and clinical outcome after ICSI?Summary Answer: Expanding the time interval between ovulation triggering and oocyte denudation/injection is not associated with any clinically relevant impact on embryological or clinical outcome.What Is Known Already: The optimal time interval between ovulation triggering and insemination/injection appears to be 38-39 h and most authors agree that an interval of >41 h has a negative influence on embryological and clinical pregnancy outcomes. However, in ART centres with a heavy workload, respecting these exact time intervals is frequently challenging. Therefore, we questioned to what extent a wider time interval between ovulation triggering and oocyte injection would affect embryological and clinical outcome in ICSI cycles.Study Design, Size, Duration: A single-centre retrospective cohort analysis was performed including 8811 ICSI cycles from 2010 until 2015. Regarding the time interval between ovulation triggering and oocyte injection, seven categories were considered: <36 h, 36 h, 37 h, 38 h, 39 h, 40 h and ≥41 h. In all cases, denudation was performed immediately prior to injection. The main outcome measures were oocyte maturation, fertilization and embryo utilization rate (embryos adequate for transfer or cryopreservation) per fertilized oocyte. Clinical pregnancy rate (CPR) and live birth rate (LBR) were considered as secondary outcomes. Utilization rate, CPR and LBR were subdivided into two groups according to the day of embryo transfer: Day 3 or Day 5.Participants/materials, Setting, Methods: During the study period, oocyte retrieval was routinely performed 36 h post-triggering except in the <36 h group. The interval of <36 h occurred only if OR was carried out before the planned 36 h trigger interval and was followed by immediate injection. Only cycles with fresh autologous gametes were included. The exclusion criteria were: injection with testicular/epididymal sperm, managed natural cycles, conventional IVF, combined conventional IVF/ICSI, preimplantation genetic testing and IVM cycles. Female age, number of oocytes, pre-preparation sperm concentration, post-preparation sperm concentration and motility, day of transfer, number of embryos transferred and quality of the best embryo transferred were identified as potential confounders.Main Results and the Role Of Chance: Among the seven interval groups, adjusted mean maturation rates ranged from 76.4% to 83.2% and differed significantly (P < 0.001). Similarly, there was a significant difference in adjusted mean fertilization rates (range 69.2-79.3%; P < 0.001). The adjusted maturation and fertilization rates were significantly higher when denudation/injection was performed >41 h post-triggering compared to 38 h post-triggering (reference group). Oocyte denudation/injection at <36 h post-triggering had no significant effect on maturation, fertilization or embryo utilization rates compared to injection at 38 h. No effect of the time interval was observed on CPRs and LBRs, after adjusting for potential confounders. When oocyte injection was performed before 36 h the adjusted analysis showed that compared to 38 h after ovulation triggering the chance of having a live birth tends to be lower although the difference was not statistically significant (odds ratio 0.533, 95% CI: 0.252-1.126; P = 0.099). Injection ≥41 h post-triggering did not affect LBR compared to injection at 38 h post-ovulation.Limitations, Reasons For Caution: As this is a large retrospective study, the influence of uncontrolled variables cannot be excluded. These results should not be extrapolated to other ART procedures such as IVM, conventional IVF or injection with testicular/epididymal sperm.Wider Implications Of the Findings: Our results indicate that the optimal injection time window may be less stringent than previously thought as both embryological and clinical outcome parameters were not significantly affected in our analysis. This is reassuring for busy ART centres that might not always be able to follow strict time intervals.Study Funding/competing Interest(s): No funding. The authors declare no conflict of interest related to the present study.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Does an early proliferative phase endometrial biopsy harvested during ovarian stimulation harbour information predictive of the outcome following fresh embryo transfer (ET) in that same cycle?Summary Answer: Transcriptome analysis of the whole-tissue endometrium did not reveal significant differential gene expression (DGE) in relation to the outcome; however, the secretome profile of isolated, cultured and in vitro decidualized endometrial stromal cells (EnSCs) varied significantly between patients who had a live birth compared to those with an implantation failure following fresh ET in the same cycle as the biopsy.What Is Known Already: In the majority of endometrial receptivity research protocols, biopsies are harvested during the window of implantation (WOI). This, however, precludes ET in that same cycle, which is preferable as the endometrium has been shown to adapt over time. Endometrial biopsies taken during ovarian stimulation have been reported not to harm the chances of implantation, and in such biopsies DGE has been observed between women who achieve pregnancy versus those who do not. The impact of the endometrial proliferative phase on human embryo implantation remains unclear, but deserves further attention, especially since in luteal phase endometrial biopsies, a transcriptional signature predictive for repeated implantation failure has been associated with reduced cell proliferation, possibly indicating proliferative phase involvement. Isolation, culture and in vitro decidualization (IVD) of EnSCs is a frequently applied basic research technique to assess endometrial functioning, and a disordered EnSC secretome has previously been linked with failed implantation.Study Design, Size, Duration: This study was nested in a randomized controlled trial (RCT) investigating the effect of endometrial scratching during the early follicular phase of ovarian stimulation on clinical pregnancy rates after IVF/ICSI. Of the 96 endometrial biopsies available, after eliminating those without fresh ET and after extensive matching in order to minimize the risk of potential confounding, 18 samples were retained to study two clinical groups: nine biopsies of patients with a live birth versus nine biopsies of patients with an implantation failure, both following fresh ET performed in the same cycle as the biopsy. We studied the proliferative endometrium by analysing its transcriptome and by isolating, culturing and decidualizing EnSCs in vitro. We applied this latter technique for the first time on proliferative endometrial biopsies obtained during ovarian stimulation for in-cycle outcome prediction, in an attempt to overcome inter-cycle variability.Participants/materials, Setting, Methods: RNA-sequencing was performed for 18 individual whole-tissue endometrial biopsies on an Illumina HiSeq1500 machine. DGE was analysed three times using different approaches (DESeq2, EdgeR and the Wilcoxon rank-sum test, all in R). EnSC isolation and IVD was performed (for 2 and 4 days) for a subset of nine samples, after which media from undifferentiated and decidualized cultures were harvested, stored at -80°C and later assayed for 45 cytokines using a multiplex suspension bead immunoassay. The analysis was performed by partial least squares regression modelling.Main Results and the Role Of Chance: After correction for multiple hypothesis testing, DGE analysis revealed no significant differences between endometrial samples from patients who had a live birth and those with an implantation failure following fresh ET. However secretome analysis after EnSC isolation and culture, showed two distinct clusters that clearly corresponded to the two clinical groups. Upon IVD, the secretome profiles shifted from that of undifferentiated cells but the difference between the two clinical groups remained yet were muted, suggesting convergence of cytokine profiles after decidualization.Limitations, Reasons For Caution: Caution is warranted due to the limited sample size of the study and the in vitro nature of the EnSC experiment. Validation on a larger scale is necessary, however, hard to fulfil given the very limited availability of in-cycle proliferative endometrial biopsies outside a RCT setting.Wider Implications Of the Findings: These data support the hypothesis that the endometrium should be assessed not only during the WOI and that certain endometrial dysfunctionalities can probably be detected early in a cycle by making use of the proliferative phase. This insight opens new horizons for the development of endometrial tests, whether diagnostic or predictive of IVF/ICSI treatment outcome.Study Funding/competing Interest(s): This study was supported by Fonds Wetenschappelijk Onderzoek (FWO, Flanders, Belgium, 11M9415N, 1 524 417N), Wetenschappelijk Fonds Willy Gepts (WFWG G160, Universitair Ziekenhuis Brussel, Belgium) and the National Medicine Research Council (NMRC/CG/M003/2017, Singapore). There are no conflicts of interests.Trial Registration Number: NCT02061228. [ABSTRACT FROM AUTHOR]

Study Question: Is a reduction in the oxygen tension from 5 to 2% during extended culture from Day 3 onwards beneficial for human blastocyst development in vitro?Summary Answer: A reduction in oxygen concentration from 5 to 2% O2 after Day 3 did not improve embryo development, quality and utilization rate.What Is Known Already: The human embryo leaves the fallopian tube to reach the uterine cavity around Day 3-4 post-ovulation. As the oxygen concentration ranges from 5 to 7% in the fallopian tube and decreases to 2% in the uterus, reducing the oxygen tension during extended culture from Day 3 onwards seems more physiological. We aim to mimic the in-vivo environment during in-vitro embryo culture. Therefore, we compared the effect of extended culture performed at 5% (control arm) or 2% oxygen (O2; study arm) tension on blastocyst formation and quality.Study Design, Size, Duration: Between December 2016 and September 2017, in two prospective studies, sibling embryos were randomized on Day 3 to either 5% O2 (control) or 2% O2 (study) for extended culture. In the control arms of both studies 1 and 2, the dishes with blastocyst medium were pre-equilibrated overnight in 5% O2, 6% CO2 and 89% N2 at 37°C. In the 2% study groups, the overnight pre-equilibration of blastocyst media was performed in either 2% O2 (study 1, 99 cycles) or 5% O2 (study 2, 126 cycles). The latter provides a gradual transition from 5 to 2% O2 environment for the study arm.Participants/materials, Settings, Methods: Embryo culture until Day 3 was always performed in 5% O2; if at least four embryos of moderate to excellent quality were obtained on Day 3, the sibling embryos were randomized to either 5% O2 or 2% O2 for extended culture. The endpoints were embryo development and quality on Day 5/6 and the utilization rate (embryos transferred and cryopreserved). Statistical analysis was performed using the chi-square test, a P-value of <0.05 was considered significantly different.Main Results and the Role Of Chance: In study 1, 811 embryos were randomized on Day 3: 405 to the 2% O2 and 406 to the 5% O2 condition. No differences were observed in the blastulation rate (68.6 versus 71.9%; P = 0.319) and the proportion of good quality blastocysts on Day 5 (55.8 versus 55.2%; P = 0.888), nor in the utilization rate (53.1 versus 53.2%; P = 1.000). In study 2, 1144 embryos were randomized: 572 in each arm. Similarly, no significant difference was demonstrated in terms of the blastulation rate (63.6 versus 64.7%; P = 0.758), the proportion of good quality blastocysts (46.9 versus 48.8%; P = 0.554) or the utilization rate (49.8 versus 48.1%; P = 0.953).Limitations, Reason For Caution: This study evaluated embryo development only until Day 5/6. The effect of oxidative stress on the developing embryo may only become evident at later stages (i.e. during implantation) and should therefore be studied in an RCT. The question also remains as to whether the switch to ultra-low oxygen tension from Day 4 onwards, when the embryo arrives in the uterus in vivo, would be preferential.Wider Implications Of the Findings: Based on the present study results, there is no benefit in lowering the oxygen tension from 5 to 2% from Day 3 onwards during extended human embryo culture.Study Funding/competing Interest(s): No funding was received for this study and the authors have no conflicts of interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]