Quantitative systems pharmacology modeling sheds light into the dose response relationship of a trispecific T cell engager in multiple myeloma.

In relapsed and refractory multiple myeloma (RRMM), there are few treatment options once patients progress from the established standard of care. Several bispecific T-cell engagers (TCE) are in clinical development for multiple myeloma (MM), designed to promote T-cell activation and tumor killing by binding a T-cell receptor and a myeloma target. In this study we employ both computational and experimental tools to investigate how a novel trispecific TCE improves activation, proliferation, and cytolytic activity of T-cells against MM cells. In addition to binding CD3 on T-cells and CD38 on tumor cells, the trispecific binds CD28, which serves as both co-stimulation for T-cell activation and an additional tumor target. We have established a robust rule-based quantitative systems pharmacology (QSP) model trained against T-cell activation, cytotoxicity, and cytokine data, and used it to gain insight into the complex dose response of this drug. We predict that CD3-CD28-CD38 killing capacity increases rapidly in low dose levels, and with higher doses, killing plateaus rather than following the bell-shaped curve typical of bispecific TCEs. We further predict that dose-response curves are driven by the ability of tumor cells to form synapses with activated T-cells. When competition between cells limits tumor engagement with active T-cells, response to therapy may be diminished. We finally suggest a metric related to drug efficacy in our analysis-"effective" receptor occupancy, or the proportion of receptors engaged in synapses. Overall, this study predicts that the CD28 arm on the trispecific antibody improves efficacy, and identifies metrics to inform potency of novel TCEs.

Expanding the time interval between ovulation triggering and oocyte injection: does it affect the embryological and clinical outcome?

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.

Follicular-phase endometrial scratching: a truncated randomized controlled trial.

STUDY QUESTION: Does intentional endometrial injury (scratching) during the follicular phase of ovarian stimulation (OS) increase the clinical pregnancy rate (CPR) in ART? SUMMARY ANSWER: CPR did not vary between the endometrial injury and the control group, but the trial was underpowered due to early termination because of a higher clinical miscarriage rate observed in the endometrial injury arm after a prespecified interim analysis. WHAT IS KNOWN ALREADY: Intentional endometrial injury has been put forward as an inexpensive clinical tool capable of enhancing endometrial receptivity. However, despite its widespread use, the benefit of endometrial scratching remains controversial, with several recent randomized controlled trials (RCTs) being unable to confirm its added value. So far, most research has focused on endometrial scratching during the luteal phase of the cycle preceding the one with embryo transfer (ET), while only a few studies investigated in-cycle injury during the follicular phase of OS. Also, the persistence of a scratch effect in subsequent treatment cycles remains unclear and possible harms have been insufficiently studied. STUDY DESIGN, SIZE, DURATION: This RCT was performed in a tertiary hospital setting between 3 April 2014 and 8 October 2017. A total of 200 women (100 per study arm) undergoing IVF/ICSI in a GnRH antagonist suppressed cycle followed by fresh ET were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were randomized with a 1:1 allocation ratio to either undergo a pipelle endometrial biopsy between Days 6 and 8 of OS or to be in the control group.The primary outcome was CPR. Secondary outcomes included biochemical pregnancy rate, live birth rate (LBR), early pregnancy loss (biochemical pregnancy losses and clinical miscarriages), excessive procedure pain/bleeding and cumulative reproductive outcomes within 6 months of the study cycle. MAIN RESULTS AND THE ROLE OF CHANCE: The RCT was stopped prematurely by the trial team after the second prespecified interim analysis raised safety concerns, namely a higher clinical miscarriage rate in the intervention group. The intention-to-treat CPR was similar between the biopsy and the control arm (respectively, 44 versus 40%, P = 0.61, risk difference = 3.6 with 95% confidence interval = -10.1;17.3), as was the LBR (respectively, 32 versus 36%, P = 0.52). The incidence of a biochemical pregnancy loss was comparable between both groups (10% in the intervention group versus 15% in the control, P = 0.49), but clinical miscarriages occurred significantly more frequent in the biopsy group (25% versus 8%, P = 0.032). In the intervention group, 3% of the patients experienced excessive procedure pain and 5% bleeding. The cumulative LBR taking into account all conceptions (spontaneous or following ART) within 6 months of randomization was not significantly different between the biopsy and the control group (54% versus 60%, respectively, P = 0.43). LIMITATIONS, REASONS FOR CAUTION: The trial was stopped prematurely due to safety concerns after the inclusion of 200 of the required 360 patients. Not reaching the predefined sample size implies that definite conclusions on the outcome parameters cannot be drawn. Furthermore, the pragmatic design of the study may have limited the detection of specific subgroups of women who may benefit from endometrial scratching. WIDER IMPLICATIONS OF THE FINDINGS: Intentional endometrial injury during the follicular phase of OS warrants further attention in future research, as it may be harmful. These findings should be taken in consideration together with the growing evidence from other RCTs that scratching may not be beneficial. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by 'Fonds Wetenschappelijk Onderzoek' (FWO, Flanders, Belgium, 11M9415N, 1524417N). None of the authors have a conflict of interest to declare with regard to this study.

The proliferative phase endometrium in IVF/ICSI: an in-cycle molecular analysis predictive of the outcome following fresh embryo transfer.

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.

BMP4 plays a role in apoptosis during human preimplantation development.

Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra-embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4-treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4-treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.

Influence of ultra-low oxygen (2%) tension on in-vitro human embryo development.

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.

Expression of adhesion and extracellular matrix genes in human blastocysts upon attachment in a 2D co-culture system.

STUDY QUESTION: What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? SUMMARY ANSWER: Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. WHAT IS KNOWN ALREADY: Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in vitro implantation model. STUDY DESIGN, SIZE, DURATION: Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. PARTICIPANTS/MATERIALS, SETTING, METHODS: Six days post-fertilization (6dpf) human embryos were co-cultured with Ishikawa cells for 12, 24 (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 145 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. MAIN RESULTS AND THE ROLE OF CHANCE: The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expressions were validated at the protein level in firmly attached 7dpf embryos. Thrombospondin 1 (THBS1) resided in the cytoplasm of embryonic cells whereas laminin subunit alpha 3 (LAMA3) and tenascin C (TNC) were expressed on the cell surface of trophectoderm cells. Incubation with a neutralizing TNC antibody did not affect the rate of embryo attachment or hCG secretion. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This in vitro study made use of an endometrial adenocarcinoma cell line to mimic receptive luminal epithelium. Also, the number of embryos was limited. Contamination of recovered embryos with Ishikawa cells was unlikely based on their differential gene expression profiles. WIDER IMPLICATIONS OF THE FINDINGS: Taken together, we provide a 'proof of concept' that initiation of the implantation process coincides with the induction of specific embryonic genes. Genome-wide expression profiling of a larger sample set may provide insights into the molecular embryonic pathways underlying successful or failed implantation. STUDY FUNDING AND COMPETING INTEREST(S): A.A. was supported by a grant from the 'Instituut voor Innovatie door Wetenschap en Technologie' (IWT, 121716, Flanders, Belgium). This work was supported by the 'Wetenschappelijk Fonds Willy Gepts' (WFWG G142 and G170, Universitair Ziekenhuis Brussel). The authors declare no conflict of interest.

Mitotic spindle disruption in human preimplantation embryos activates the spindle assembly checkpoint but not apoptosis until Day 5 of development.

STUDY QUESTION: Is the spindle assembly checkpoint (SAC) active during human preimplantation development? SUMMARY ANSWER: Mitotic spindle disruption during mitosis activates the SAC from at least Day 3 of human preimplantation development, but this does not lead to apoptosis until Day 5. WHAT IS KNOWN ALREADY: Human preimplantation embryos frequently acquire chromosomal abnormalities, but the mechanisms behind this are poorly understood. It has been speculated that a dysfunctional SAC could be responsible. Although research has shown that the SAC components are present during early human development, functional studies are lacking. STUDY DESIGN, SIZE, DURATION: In vitro study using human preimplantation embryos in a university research laboratory. We studied a total of 38 Day-3, 38 Day-4, 29 Day-5 and 21 Day-6 human preimplantation embryos, donated for research, during 16 h of incubation. PARTICIPANT/MATERIALS, SETTING, METHODS: We cultured human preimplantation embryos overnight in a time-lapse imaging system, in control or in a nocodazole-containing medium that prevents the formation of a proper mitotic spindle. The embryos were subsequently fixed and analysed by immunocytochemistry for tubulin or mitotic and apoptotic markers, or by FISH. MAIN RESULTS AND THE ROLE OF CHANCE: All embryos showed an increase in M-phase cells from 4.1-8.8% to 21.4-53.5% when exposed to nocodazole (P < 0.05; two-way ANOVA for all groups except Day-4 embryos, P = 0.128) suggesting SAC functionality. Apoptosis, which was rarely detected between Day 3 and Day 6 in good-quality control embryos, increased from Day 5 onwards in nocodazole-treated embryos and became statistically different from Day 6 (P < 0.01; two-way ANOVA). The FISH data suggest that in compacted Day-4 embryos, approximately one in six cells started a polyploid new cell cycle rather than to go in apoptosis after the failure to maintain the SAC-mediated M-phase arrest. These results suggest that during early embryo development, blastomeres with unresolved chromosome misalignments during M-phase can escape SAC-mediated apoptosis, continue cell division which can then result in aneuploid daughter cells. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: This study used nocodazole to inhibit microtubule polymerization, a drug that is regularly used to induce metaphase arrest and SAC activation. Results should be extrapolated to naturally occurring chromosome misalignments with care. WIDER IMPLICATIONS OF THE FINDINGS: Our results provide functional data that can help explain the high aneuploidy rates seen in human cleavage-stage embryos and suggest that this is due to their unusual cell cycle control. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Fund for Scientific Research Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen) and the Methusalem grant to Karen Sermon of the Research Council of the Vrije Universiteit Brussel. The authors declare no competing financial interests.

The role of methylation, DNA polymorphisms and microRNAs on HLA-G expression in human embryonic stem cells.

The human leukocyte antigen (HLA)-G gene seems to play a pivotal role in maternal tolerance to the fetus. Little is known about HLA-G expression and its molecular control during in vivo human embryogenesis. Human embryonic stem cells (hESC) provide an interesting in vitro model to study early human development. Different studies reported discrepant findings on whether HLA-G mRNA and protein are present or absent in hESC. Several lines of evidence indicate that promoter CpG methylation and 3' untranslated region (3'UTR) polymorphisms may influence HLA-G expression. We investigated how HLA-G expression is linked to the patterns of promoter methylation and explored the role of the 3'UTR polymorphic sites and their binding microRNAs on the post-transcriptional regulation of HLA-G in eight hESC lines. We showed that, while the gross expression levels of HLA-G are controlled by promoter methylation, the genetic constitution of the HLA-G 3'UTR, more specifically the 14bp insertion in combination with the +3187A/A and +3142G/G SNP, plays a major role in HLA-G mRNA regulation in hESC. Our findings provide a solid first step towards future work using hESC as tools for the study of early human developmental processes in normal and pregnancy-related disorders such as preeclampsia.

Do ARTs affect the incidence of monozygotic twinning?

STUDY QUESTION: Does the manipulation of gametes or embryos during ARTs increase the risk for monozygotic twinning (MZT)? SUMMARY ANSWER: Frozen embryo transfer (ET) is associated with a lower MZT rate, while blastocyst culture is associated with an increased risk of monozygotic pregnancy. WHAT IS KNOWN ALREADY: Monozygotic twins have a higher risk for perinatal complications. Although an increased incidence of monozygotic pregnancies after ART has been previously reported, data regarding the possible impact of different laboratory procedures are conflicting. STUDY DESIGN, SIZE, DURATION: All clinical pregnancies after single ET carried out in our centre between 2004 and 2013 (n = 6096) were retrospectively analysed for the incidence of MZT. The effect of different laboratory procedures on the incidence of MZT was evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS: The following ART risk factors were assessed: maternal age, type of ET (fresh versus frozen), zona pellucida (ZP) manipulation (specifically, ICSI, embryo biopsy and assisted hatching), use of donor oocytes, embryo stage at time of ET (cleavage, compaction, early or advanced blastocyst) and culture media. MAIN RESULTS AND THE ROLE OF CHANCE: The overall MZT rate was 2.2% (136/6096). Frozen ET was associated with a significant reduction in MZT incidence (adjusted odds ratio (aOR) 0.48, 95% CI 0.29-0.80), while blastocyst transfer (early or advanced blastocyst) was associated with a significant increase in MZT risk (aOR 2.70, 95% CI 1.36-5.34; aOR 2.05, 95% CI 1.29-3.26, respectively). No significant differences were found between the MZT and singleton (non-MZT) groups regarding maternal age, the use of different ZP manipulation techniques, not type of culture media used. LIMITATION, REASONS FOR CAUTION: This study is limited by its retrospective nature and the fact that monozygosity was not confirmed by genetic testing. Furthermore, since monozygotic pregnancy is a rare event, other ART parameters that may influence its incidence could not be assessed during our analysis. WIDER IMPLICATION OF THE FINDINGS: Our findings warrant future studies designed to investigate the association between specific ART procedures and MZT, namely the potential risk of blastocyst transfer to increase MZT. STUDY FUNDING/COMPETING INTERESTS: No external funding was used for this study. There are no conflicts of interest.

Closed oocyte vitrification and storage in an oocyte donation programme: obstetric and neonatal outcome.

STUDY QUESTION: Does closed oocyte vitrification in an oocyte donation programme have an impact on obstetric and neonatal outcome? SUMMARY ANSWER: Obstetric and neonatal outcomes after closed system vitrification of donor oocytes appear to be reassuring. WHAT IS KNOWN ALREADY: The use of fresh oocytes has not been proved to be superior to the use of vitrified donor oocytes in terms of survival, embryo development and clinical pregnancies. Those studies used open devices to prove the non-superiority. Very limited information is available on the comparison of open and closed devices, and the results for survival, embryo development and pregnancy outcomes are conflicting. Data on obstetric and neonatal outcome from vitrified oocytes are scarce. Only one large report is available after the use of donor oocytes vitrified with an open device. STUDY DESIGN, SIZE, DURATION: Retrospective observational study performed at the Centre for Reproductive Medicine, UZ Brussel, Belgium. All 117 oocyte recipient cycles between March 2010 and August 2014 with the use of a closed vitrification device and leading to a pregnancy beyond 20 weeks were included in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS: All recipient warming cycles with a pregnancy beyond 20 weeks from vitrified donor oocytes: results from the fresh embryo transfers. MAIN RESULTS AND THE ROLE OF CHANCE: For 117 recipient cycles, a total of 793 oocytes were warmed of which 657 (82.8%) survived and 499 (76.0%) were fertilized. Nineteen single and 98 double embryo transfers led to 95 singleton and 22 twin pregnancies. Hypertensive disorders, haemorrhages and gestational diabetes were reported in 22/112 (19.6%), 30/112 (26.8%) and 13/112 (11.6%) of the pregnancies, respectively. No major adverse neonatal outcomes were observed. Congenital malformations were observed in 11 out of 139 children; for one an elective termination was performed at 25 weeks. LIMITATIONS, REASONS FOR CAUTION: Since March 2010, almost all oocytes for donation are vitrified in our centre. Therefore, no recent data are available to control the outcomes of fresh oocyte donations. WIDER IMPLICATIONS OF THE FINDINGS: The reassuring results obtained in the current study show that closed system vitrification devices for donor oocytes may be used as an alternative to open devices which have been linked to possible cross-contamination issues. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: N/A.

Open versus closed oocyte vitrification in an oocyte donation programme: a prospective randomized sibling oocyte study.

STUDY QUESTION: Is the survival of donor oocytes with the CryotopSC device superior to the survival with the closed CBSvit device? SUMMARY ANSWER: The CryotopSC device and the CBSvit device showed similar survival rates. WHAT IS KNOWN ALREADY: Health authorities are cautious about possible cross contamination during liquid nitrogen storage or handling when working with open vitrification devices. At present, the use of open devices is still allowed since little information is available on the efficiency of closed devices. STUDY DESIGN, SIZE, DURATION: A prospective randomized sibling oocyte study was performed in the Centre for Reproductive Medicine (UZBrussel) between January 2014 and July 2015. The survival after warming and the embryological outcome of donor oocytes vitrified using two devices was compared: the CBSvit device (closed vitrification and closed storage) and the CryotopSC device (open vitrification and closed storage). A difference of 10% was defined to prove the superiority of the CryotopSC device. In total, 250 warmed oocytes were needed in each arm. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocytes from 48 donors were included in the study: 253 vitrified with the CBSvit device and 257 with the CryotopSC device. Equal numbers of oocytes from both devices and originated from the same donor cycle were allocated to each of 78 recipients, in order to exclude donor and recipient (male factor) effects. MAIN RESULTS AND THE ROLE OF CHANCE: There were no differences found between the CBSvit and the CryotopSC in terms of survival after warming (93.7 versus 89.9%) or fertilization per injected oocyte (74.3 versus 81.4%). The degeneration rate after ICSI was significantly higher for the CBSvit device: 11.4 versus 6.1% (P = 0.041). A significantly higher number of zygotes in the CryotopSC group finished their first mitosis 25-27 h post-injection (34.1 versus 52.1%, P = 0.001). On Day 3, the overall embryo quality distribution did not vary between groups, but a significantly higher cell number was obtained in the CryotopSC device: 6.8 ± 2.8 versus 7.6 ± 2.8 (P = 0.01). The utilization rate per mature oocyte, per surviving oocyte or per fertilized oocyte did not differ. The embryos with the highest quality were selected for transfer on Day 3. The clinical pregnancy rate per transfer cycle was 36.5%. LIMITATIONS, REASONS FOR CAUTION: The results of this study should not be extrapolated to other female groups, since oocytes from young fertile donors were used in this study. WIDER IMPLICATIONS OF THE FINDINGS: In many countries, the use of open devices is still allowed due to the limited reports on the efficiency of closed devices. Knowing the caution of health authorities about the use of open devices, there is an urgent need for efficiency studies with closed devices. The results obtained in the current study shows the efficiency of a safe closed vitrification device, leaving behind any concern about possible cross contamination during handling or storage. STUDY FUNDING/COMPETING INTERESTS: No funding was obtained. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: NCT01952184. TRIAL REGISTRATION DATE: 24 September 2013. DATE OF FIRST PATIENT'S ENROLMENT: 23 January 2014.

A prospective randomized controlled trial investigating the effect of artificial shrinkage (collapse) on the implantation potential of vitrified blastocysts.

STUDY QUESTION: What is the effect of artificial shrinkage by laser-induced collapse before vitrification on the implantation potential after transfer of vitrified-warmed blastocysts? SUMMARY ANSWER: The artificial shrinkage by laser-induced collapse did not significantly increase the implantation rate per transferred collapsed blastocyst (37.6%) compared with non-collapsed blastocysts (28.9%) [odds ratio (OR): 1.48, 95% confidence interval (CI): 0.78-2.83]. WHAT IS KNOWN ALREADY: Retrospective studies have demonstrated that artificial shrinkage of the blastocyst prior to vitrification can have a positive effect on blastocyst survival after warming. A recent study found a similar survival rate but higher implantation rate for collapsed blastocysts. So far, no randomized controlled trial has been conducted to investigate the implantation potential of collapsed blastocysts. STUDY DESIGN, SIZE, DURATION: Prospective randomized trial. Patients were recruited from December 2011 until April 2014 and warming cycles were included until July 2014. Patients were randomized in the fresh cycle if blastocysts were available for vitrification and were allocated to the study or control arm according to a computer-generated list. In the study group, blastocysts underwent laser-induced collapse before vitrification. In the control group, blastocysts were vitrified without collapsing. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 443 patients signed informed consent and 270 patients had blastocysts vitrified. One-hundred and thirty-five patients were allocated to the study group and 135 to the control group. Sixty-nine patients from the study group and 69 from the control group returned for at least one warming cycle in which 85 and 93 blastocysts were warmed in the first cycle, respectively. Primary outcome was implantation rate per embryo transferred in the first warming cycle. Secondary outcomes were survival and transfer rates, blastocyst quality after warming, clinical pregnancy rate and implantation rate per warmed blastocyst. Blastocysts were vitrified-warmed one by one using closed vitrification and one or two blastocysts were transferred per warming cycle. MAIN RESULTS AND THE ROLE OF CHANCE: We calculated that the group sample sizes of 80 embryos in the collapse group and 80 embryos in the control group were needed to achieve 80% power to detect a difference between the group proportions of +20% with P < 0.05. In the study group, 69 first warming cycles resulted in 69 transfers with 1.2 blastocysts (n = 85) transferred. In the control group, an average of 1.3 blastocysts (n = 83) were transferred in 67 out of 69 warming cycles. Implantation rates per embryo transferred in the first warming cycle were not different between both groups (38 versus 29%, OR: 1.48; 95% CI: 0.78-2.83), neither was the implantation rate per warmed embryo (38 versus 26%, OR: 1.74; 95% CI: 0.92-3.29). When all warming cycles were considered (n = 135 in each group), survival rate after collapse was significantly higher compared with the control group (98.0 versus 92.0%, OR: 4.25; 95% CI: 1.19-15.21). Furthermore, a higher percentage of high-quality blastocysts (36.3 versus 23.5%, OR: 1.86; 95% CI: 1.12-3.08) and hatching blastocysts (19.2 versus 5.4%, OR: 4.18; 95% CI: 1.84-9.52) were found compared with the control group. LIMITATIONS, REASONS FOR CAUTION: The study lasted more than 2.5 years since fewer patients than expected returned for a warming cycle because of the high ongoing pregnancy rates in the fresh IVF/ICSI cycle. WIDER IMPLICATIONS OF THE FINDINGS: Although no significant higher implantation rate was found after collapse, the better survival and post-warm embryo quality convinced us to recognize a clinical benefit of artificial shrinkage and to implement it in routine vitrification practice. TRIAL REGISTRATION NUMBER: NCT01980225, www.clinicaltrials.gov. The first patient was included November 2011 and the study was registered October 2013.

Cyclin E1 plays a key role in balancing between totipotency and differentiation in human embryonic cells.

STUDY HYPOTHESIS: We aimed to investigate if Cyclin E1 (CCNE1) plays a role in human embryogenesis, in particular during the early developmental stages characterized by a short cell cycle. STUDY FINDING: CCNE1 is expressed in plenipotent human embryonic cells and plays a critical role during hESC derivation via the naïve state and, potentially, normal embryo development. WHAT IS KNOWN ALREADY: A short cell cycle due to a truncated G1 phase has been associated with the high developmental capacity of embryonic cells. CCNE1 is a critical G1/S transition regulator. CCNE1 overexpression can cause shortening of the cell cycle and it is constitutively expressed in mouse embryonic stem cells and cancer cells. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We investigated expression of CCNE1 in human preimplantation embryo development and embryonic stem cells (hESC). Functional studies included CCNE1 overexpression in hESC and CCNE1 downregulation in the outgrowths formed by plated human blastocysts. Analysis was performed by immunocytochemistry and quantitative real-time PCR. Mann-Whitney statistical test was applied. MAIN RESULTS AND THE ROLE OF CHANCE: The CCNE1 protein was ubiquitously and constitutively expressed in the plenipotent cells of the embryo from the 4-cell stage up to and including the full blastocyst. During blastocyst expansion, CCNE1 was downregulated in the trophectoderm (TE) cells. CCNE1 shortly co-localized with NANOG in the inner cell mass (ICM) of expanding blastocysts, mimicking the situation in naïve hESC. In the ICM of expanded blastocysts, which corresponds with primed hESC, CCNE1 defined a subpopulation of cells different from NANOG/POU5F1-expressing pluripotent epiblast (EPI) cells and GATA4/SOX17-expressing primitive endoderm (PrE) cells. This CCNE1-positive cell population was associated with visceral endoderm based on transthyretin expression and marked the third cell lineage within the ICM, besides EPI and PrE, which had never been described before. We also investigated the role of CCNE1 by plating expanded blastocysts for hESC derivation. As a result, all the cells including TE cells re-gained CCNE1 and, consequently, NANOG expression, resembling the phenotype of naïve hESC. The inhibition of CCNE1 expression with siRNA blocked proliferation and caused degeneration of those plated cells. LIMITATIONS, REASONS FOR CAUTION: The study is based on a limited number of good-quality human embryos donated to research. WIDER IMPLICATIONS OF THE FINDINGS: Our study sheds light on the processes underlying the high developmental potential of early human embryonic cells. The CCNE1-positive plenipotent cell type corresponds with a phenotype that enables early human embryos to recover after fragmentation, cryodamage or (single cell) biopsy on day 3 for preimplantation genetic diagnosis. Knowledge on the expression and function of genes responsible for this flexibility will help us to better understand the undifferentiated state in stem cell biology and might enable us to improve technologies in assisted reproduction. LARGE SCALE DATA: NA STUDY FUNDING AND COMPETING INTERESTS: This research is supported by grants from the Fund for Scientific Research - Flanders (FWO-Vlaanderen), the Methusalem (METH) of the VUB and Scientific Research Fond Willy Gepts of UZ Brussel. There are no competing interests.

WNT3 and membrane-associated ß-catenin regulate trophectoderm lineage differentiation in human blastocysts.

WNT/ß-catenin signaling has been described as a crucial regulator of embryonic stem cells and embryogenesis. However, little is known on its role during human preimplantation embryo development, besides the RNA expression of its multiple players. In this study, we performed ß-catenin loss- and gain-of-function studies on human preimplantation embryos by adding either Cardamonin or GSK3 inhibitor, 1-Azakenpaullone, to the embryo culture medium from the cleavage until blastocyst stages (Days 3-5/6). ß-Catenin was displayed in the cortical region underneath the membrane during all stages, but it only showed nuclear localization at cleavage stages after stabilization with 1-Azakenpaullone. We did not observe any effects on the inner cell mass markers NANOG, POU5F1, SOX2 and SALL4 in these functional experiments. However, both ß-catenin degradation and stabilization caused inhibition of the trophectoderm (TE) fate, illustrated by KRT18 and GATA3 RNA, and CDX2 protein expression. Based on the TE-specific WNT3 protein expression in blastocysts, we postulated that this protein may be an upstream regulator for the observed membrane ß-catenin function. The addition of either WNT3 or 1-Azakenpaullone to the culture medium promoted EOMES expression specific for trophoblast development. In both studies, the canonical WNT pathway target gene, TCF1, was not affected. Therefore, we conclude that WNT3 and membrane-associated ß-catenin promote progenitor trophoblast development in human blastocysts. These results have important implications in assisted reproduction and stem cell biology.

Deposition of the spermatozoon in the human oocyte at ICSI: impact on oocyte survival, fertilization and blastocyst formation.

PURPOSE: To evaluate whether the deposition of the spermatozoon in the human oocyte at ICSI has any effect on oocyte survival, fertilization, blastocyst development and quality. METHODS: In a prospective study, including 78 ICSI cycles, sibling oocytes were injected with "no intention" (group A, standard ICSI, n = 393) or "intention" to deposit the spermatozoon under the cortex (group B, n = 354). Outcome parameters were oocyte survival and fertilization, as well as blastocyst formation and quality. RESULTS: Depositing the sperm under the cortex of the oocyte was not always successful for its final position, therefore, group B was divided into three subgroups: B1 successful deposition (119 oocytes, 33.6 % of oocytes in group B); B2 initially successful but spermatozoon spontaneously relocated after 2 min (136 oocytes, 38.4 %); and B3 unsuccessful deposition (99 oocytes, 28.0 %). Group A and B were compared on an intention-to-treat basis. Additionally, A, B1, B2 and B3 were also compared. The oocyte survival and fertilization, blastocyst and top-quality blastocyst developmental rates were not significantly different. CONCLUSIONS: The procedure of depositing the spermatozoon intentionally under the oocyte cortex demanded high technical skills. Successful positioning was only obtained in 34 % of the attempts. We obtained no evidence of improved oocyte survival and fertilization, blastocyst formation and quality when the spermatozoon was permanently positioned under the oocyte cortex. Taken together, depositing the spermatozoon under the oocyte cortex is not recommended for routine ICSI application.

Chromosomal meiotic segregation, embryonic developmental kinetics and DNA (hydroxy)methylation analysis consolidate the safety of human oocyte vitrification.

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.

The type of culture medium and the duration of in vitro culture do not influence birthweight of ART singletons.

STUDY QUESTION: Does the type of in vitro culture medium or the duration of in vitro culture influence singleton birthweight after IVF/ICSI treatment? SUMMARY ANSWER: In a comparison of two culture media, neither the medium nor the duration of culture (Day 3 versus Day 5 blastocyst transfer) had any effect on mean singleton birthweight. WHAT IS KNOWN ALREADY: Previous studies indicated that in vitro culture of human embryos may affect birthweight of live born singletons. Both the type of culture medium and the duration of culture may be implicated. However, these studies are small and report conflicting results. STUDY DESIGN, SIZE, DURATION: A large retrospective analysis was performed including all singleton live births after transferring fresh Day 3 or Day 5 embryos. IVF and ICSI cycles performed between April 2004 and December 2009 at a tertiary care centre were included for analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 2098 singleton live births resulting from singleton pregnancies were included for analysis. Two different sequential embryo culture media were concurrently used in an alternating way: Medicult (n = 1388) and Vitrolife (n = 710). Maternal age, maternal and paternal BMI, maternal parity, maternal smoking, main cause of infertility, cycle rank, stimulation protocol, method of fertilization (IVF or ICSI), time in culture and number of embryos transferred were taken into account. Embryo transfers were performed either on Day 3 (n = 1234) or on Day 5 (n = 864). Singleton birthweight was the primary outcome parameter. Gestational age and gender of the newborn were accounted for in the multiple regression analysis. MAIN RESULTS AND THE ROLE OF CHANCE: No significant differences in mean singleton birthweight were observed between the two culture media: Medicult 3222 g (±15 SE) and Vitrolife 3251 g (±21 SE) (P = 0.264). The mean singleton birthweight was not different between Day 3 embryo transfers (3219 ± 16 g) and Day 5 blastocyst transfers (3250 ± 19 g; P = 0.209). Multiple regression analysis controlling for potential maternal, paternal, treatment and newborn confounders confirmed the non-significant differences in mean singleton birthweight between the two culture media. Likewise, the adjusted mean singleton birthweight was not different according to the duration of in vitro culture (P = 0.521). LIMITATIONS, REASONS FOR CAUTION: The conclusions are limited by its retrospective design; however, the two different sequential culture systems were used in an alternating way during the same time period. Pregnancy-associated factors possibly influencing birthweight (such as diabetes, hypertension, pre-eclampsia) were not included in the analysis. WIDER IMPLICATIONS OF THE FINDINGS: This large retrospective study does not support earlier concerns that both the type of culture medium and the duration of embryo culture influence singleton birthweight. However, a continuous surveillance of human embryo culture procedures (medium type, culture duration and other culture conditions) should remain a priority within assisted reproduction technology. STUDY FUNDING/COMPETING INTERESTS: None.

CAR expression in human embryos and hESC illustrates its role in pluripotency and tight junctions.

Coxsackie virus and adenovirus receptor, CXADR (CAR), is present during embryogenesis and is involved in tissue regeneration, cancer and intercellular adhesion. We investigated the expression of CAR in human preimplantation embryos and embryonic stem cells (hESC) to identify its role in early embryogenesis and differentiation. CAR protein was ubiquitously present during preimplantation development. It was localised in the nucleus of uncommitted cells, from the cleavage stage up to the precursor epiblast, and corresponded with the presence of soluble CXADR3/7 splice variant. CAR was displayed on the membrane, involving in the formation of tight junction at compaction and blastocyst stages in both outer and inner cells, and CAR corresponded with the full-length CAR-containing transmembrane domain. In trophectodermal cells of hatched blastocysts, CAR was reduced in the membrane and concentrated in the nucleus, which correlated with the switch in RNA expression to the CXADR4/7 and CXADR2/7 splice variants. The cells in the outer layer of hESC colonies contained CAR on the membrane and all the cells of the colony had CAR in the nucleus, corresponding with the transmembrane CXADR and CXADR4/7. Upon differentiation of hESC into cells representing the three germ layers and trophoblast lineage, the expression of CXADR was downregulated. We concluded that CXADR is differentially expressed during human preimplantation development. We described various CAR expressions: i) soluble CXADR marking undifferentiated blastomeres; ii) transmembrane CAR related with epithelial-like cell types, such as the trophectoderm (TE) and the outer layer of hESC colonies; and iii) soluble CAR present in TE nuclei after hatching. The functions of these distinct forms remain to be elucidated.