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To compare assisted reproductive technology (ART) outcomes and preimplantation embryo development between underweight and normal-weight women.This retrospective cohort study included 26 underweight women (body mass index [BMI] 18.50 kg/mThe mean ± standard deviation age of the underweight and normal-weight women was similar (31.6 ± 4.17 vs 32.4 ± 3.59 years;Underweight status has no adverse impacts on the outcomes of IVF/ICSI with either fresh or frozen ET or on preimplantation embryo development and quality.

Research question In women at advanced age of 43–45 years undergoing repeated IVF cycles with autologous oocytes, who has the highest chance for birth and who should be referred early to receive donor oocytes? Design A retrospective cohort study was conducted at a university hospital reproductive center. The computerized database of 394 women aged 43–45 undergoing 1528 non-donor IVF or ICSI cycles between 2010 and 2019 was analyzed. a decision tree was developed, enabling a comprehensive study of a set of clinical parameters and the expected outcomes. Results The analysis included the outcomes of 394 women undergoing a total of 1528 cycles. The cumulative clinical pregnancy rate was 15% (59/394) and the cumulative live birth rate was 8.4% (33/394). The decision tree developed to predict women that should be offered egg donation included age, poor ovarian response to stimulation, the number of top-quality embryos, dominant follicles, previous pregnancy or live birth, fertilized oocytes and BMI. The model showed that a good ovarian response in the first cycle was the best predictor for live birth (13.3% gave birth). However, among women with poor responses, 7.1% of those who were younger than 43.5 years gave birth, and none of the women who were older than 43.5 years did. Conclusion Women over 43.5 years old with less than four oocytes collected in their first IVF cycle should be offered ovum donation, since their live birth rate in subsequent cycles is negligible.

To analyze whether cleavage stage at compaction, and not only kinetics, can serve as a reliable predictor for clinical outcome.A retrospective cohort study including 1194 embryos, classified by compaction initiation stage (Group 1: compaction at fewer than eight cells, Group 2: compaction at eight cells, Group 3: compaction at more than eight cells). Of these, 815 embryos were evaluated for morphokinetic preimplantation parameters, and 379 embryos were analyzed for clinical implantation following thawing and transfer of single blastocysts during the same period.In total, 1194 embryos were analyzed. Embryos that underwent compaction from more than eight cells (Group 3) exhibited more synchronous cleavage compared with Groups 1 and 2 (at both S2 and S3; P 0.001), and displayed a significantly lower fragmentation rate. The likelihood of obtaining top-quality blastocysts decreased by 73% and 44% when comparing Group 3 embryos with those of Groups 1 and 2, respectively, (P 0.03). Clinical validation of the results shows that while compaction from fewer than eight cells barely produced blastocysts for transfer, compaction at eight or more cells is crucial for implantation and birth (birth rates 11.1% and 18.5% for Groups 2 and 3, respectively).Cleavage stage at compaction has a direct effect on blastocyst quality and subsequent pregnancy, so can be included in newly developed deep learning models for embryo selection.

Research question Can patient selection for successful preimplantation genetic testing for women who are fragile X (FMR1) premutation carriers be optimized using a decision tree analysis? This decision support tool enables a comprehensive study of a set of clinical parameters and the expected outcomes. Design A retrospective case–control study analysing the results of 264 fresh and 21 frozen preimplantation genetic testing for monogenic disorders/single gene defects (PGT-M) cycles in 64 FMR1 premutation carriers. Primary outcome was live birth per cycle start. Live birth rate was calculated for the start of the ovarian stimulation cycle. Fresh and frozen embryo transfers from the same cycle were included. Results The decision tree model showed that the number of cytosine guanine (CGG) repeats was only a moderate predictor for live birth, whereas an age younger than 36 years was the best predictor for live birth, followed by a collection of 14 or more oocytes. These findings were supported by the results of the logistic regression, which found that only age and oocyte number were significantly associated with live birth (P = 0.005 and 0.017, respectively). Conclusions The number of CGG repeats is a relatively poor predictor for live birth in PGT-M cycles. FMR1 premutation carriers are no different from non-carriers. Age is the best identifier of live birth, followed by the number of retrieved oocytes.

The purpose of this research is to study the efficacy of GnRH-a versus r-hCG triggering in patients who go through fertility preservation cycles. This retrospective cohort study was performed in a tertiary university-affiliated medical center. It includes 191 patients undergoing fertility preservation cycles between May 2013 and September 2018, in which ovulation was induced by either GnRH-a or r-hCG. Main outcome measures were number and rate of mature oocyte. Among treatment cycles with medical indication, GnRH agonist significantly increases the odds for high mature rate by 3.55 (1.30-9.66), while in treatment cycles with social indication, there is no significant effect of the triggering agent. An advantage for GnRH-a triggering was observed in medically indicated preservation cycles.

To compare clinical pregnancy rate (CPR) and live birth rate (LBR) after frozen-thawed embryo transfer (FET) of day (D-) 6 blastocysts on D-5 versus D-6. A retrospective cohort study. A university-affiliated single-center tertiary hospital. Women undergoing FET of D-6 blastocysts between August 2015 and March 2019 were included in the study. Exclusion criteria were endometriosis and maternal age ≥ 42. Cycles involving embryo transfer (ET) at D-6 were compared to cycles involving ET on D-5. Primary outcomes assessed were CPR and LBR, and the secondary outcomes were spontaneous abortion and chemical pregnancy rates. Forty-two cycles were assessed, 21 in which ET occurred on D-6 and 21 in which ET occurred on D-5. There were no significant differences between groups regarding age, body mass index (BMI), etiology of infertility, number of oocytes aspirated and blastocysts cryopreserved in the fresh cycle, reason for freezing on D-6, endometrial thickness before ET, and blastocyst grade. A comparison of outcomes of ET on D-5 with those involving ET on D-6 revealed that D-5 transfer produced significantly higher CPR (8, 38% vs. 2, 8.5%; P = 0.030) and LBR (6, 28.6% vs. 1, 4.8%; P = 0.038), respectively. FET of D-6 embryos on D-5 compared with D-6 is associated with increased CPR and LBR values. These findings might be related to the limited time window for optimal rates of implantation and indicate that transferring embryos on D-6 of a FET cycle is likely too late.

Research question In-vitro maturation (IVM) of oocytes recovered during ovarian tissue cryopreservation (OTC) is often practised, although it is still considered experimental. To date, only a few studies have examined the success of this maturation process in pre-menarche girls. The aim of this study was to examine the outcomes of IVM of oocytes recovered during OTC in pre-menarche patients scheduled for onco-therapy. Design A retrospective cohort study in a tertiary university-affiliated hospital. A total of 93 patients aged 0–25 years who underwent OTC as part of onco-fertility preservation between 2007 and 2019 were included in the study. Oocytes were recovered from the medium after OTC and matured over 48 h. Oocyte development and maturation rate were recorded and compared between different age groups. Results Patient's age was not correlated linearly with the total number of mature oocytes R = 0.2. The absolute maturation rate in post-menarche and pre-menarche patients differed significantly (38.0% versus 25.3%, respectively; P > 0.001), whereas the degeneration rate of the oocytes did not (39.8% versus 33.5%; P = 0.167). The pre-menarche group had significantly lower mean number of metaphase II oocytes compared with the post-menarche group (2.8 [±2.3] versus 5.6 [±4.6]; P = 0.01; 95% CI –4.62 to –0.46). Oocytes recovered from patients aged 1–5 years demonstrated low maturation rate. Conclusions Oocytes recovered from pre-menarche girls, and especially those younger than the age of 5 years who undergo fertility preservation, have a lower chance of reaching maturity in IVM compared with older women. This may indicate a need for alternative methods for preserving fertility in these young patients.

What are the effects of testosterone treatment on oocyte fertilization and preimplantation embryo development among transgender men who have undergone fertility preservation?A retrospective study was undertaken in a university-affiliated tertiary hospital between April 2016 and November 2021. Embryos were divided into three groups by source: 210 embryos from 7 testosterone-exposed transgender men, 135 from 10 cisgender women who cryopreserved embryos, and 276 from 24 cisgender women who underwent fertility treatment. Statistical analyses compared assisted reproductive technology outcomes between the group of transgender men and both groups of cisgender women. Morphokinetic and morphological parameters were compared between the embryos derived from these three groups.The transgender men (30.2 ± 3.5 years of age) were significantly younger than the cisgender women who cryopreserved embryos (35.1 ± 1.8 years; P = 0.005) and the cisgender women who underwent fertility treatment (33.8 ± 3.2 years; P = 0.017). After adjusting for participant age, the fertilization rate was comparable between the transgender men and both groups of cisgender women (P = 0.391 and 0.659). There were no significant differences between the transgender men and the cisgender women who preserved fertility in terms of number of cryopreserved embryos (7.2 ± 5.1 and 3.5 ± 2.6; P = 0.473) or the distribution of embryo age at cryopreservation (P = 0.576). All morphokinetic parameters evaluated by time-lapse imaging, as well as the morphological characteristics, were comparable for the embryos in all three groups.Testosterone exposure among transgender men has no adverse impact upon fertilization rates or preimplantation embryo development and quality.

Does inheritance of the fragile X mental retardation 1 (FMR1) premutation allele affect embryo morphokinetic development?A retrospective cohort analysis of 529 embryos from 126 IVF cycles of 39 FMR1 premutation female carriers undergoing preimplantation genetic testing for monogenic/single gene defects (PGT-M). Morphological and morphokinetic parameters obtained using a time-lapse monitoring system were compared between embryos that inherited the FMR1 premutation allele (FMR1 group, n = 271) and those who received the normal allele (normal group, n = 258). The following embryo outcome measures were compared: morphokinetic parameters up to day 3, start of blastulation time (tSB) for day 5 embryos and the rate of top-quality embryos on days 3 and 5.No differences were found in morphokinetic parameters between the groups from the time of intracytoplasmic sperm injection (ICSI) until a biopsy on day 3. The blastulation rate in the two groups was comparable. However, the start of blastulation was delayed in FMR1 embryos compared to that in the genetically normal embryos (median tSB: 104.2 h [99.3-110.3] versus 101.6 h [94.5-106.7], P = 0.01). In addition, the rate of top-quality FMR1 embryos was lower than that of genetically normal embryos (25.6% versus 38.8%, P = 0.04).Embryos that inherit the FMR1 premutation allele are of lower quality at the blastocyst stage compared with those that do not inherit the mutated allele.

Purpose To compare assisted reproductive technology (ART) outcomes and preimplantation embryo development between underweight and normal weight women. Methods This retrospective cohort study included 26 underweight women (body mass index [BMI] 2) and 104 normal weight women (BMI > 20 and 2) who underwent a total of 204 in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles and 358 fresh/frozen embryo transfers (ET) in our institution between January 2016 and December 2018. Statistical analyses compared selected ART outcomes (ovarian stimulation, fertilization, and pregnancy) between both weight groups. Morphokinetic and morphological parameters were also compared between 346 and 1467 embryos of underweight and normal weight women, respectively. Results The mean ± standard deviation age of the underweight and normal weight women was similar (31.6 ± 4.17 vs 32.4 ± 3.59 years; p = 0.323). There were no differences in the peak estradiol levels, the number of retrieved oocytes, the number of metaphase II oocytes, and the oocyte maturity rates between the two groups. The IVF/ICSI fertilization rates and the number of embryos suitable for transfer or cryopreservation were similar for both groups. All morphokinetic parameters that were evaluated by means of time-lapse imaging as well as the morphological characteristics were comparable between low and normal BMI categories. There were no significant differences in pregnancy achievement, clinical pregnancy, live births, and miscarriage rates between the suboptimal and optimal weight women. Conclusion Underweight status has no adverse impacts on the outcomes of IVF/ICSI with either fresh or frozen ET or on preimplantation embryo development and quality.

The purpose of this research is to study the efficacy of GnRH-a versus r-hCG triggering in patients who go through fertility preservation cycles. This retrospective cohort study was performed in a tertiary university-affiliated medical center. It includes 191 patients undergoing fertility preservation cycles between May 2013 and September 2018, in which ovulation was induced by either GnRH-a or r-hCG. Main outcome measures were number and rate of mature oocyte. Among treatment cycles with medical indication, GnRH agonist significantly increases the odds for high mature rate by 3.55 (1.30-9.66), while in treatment cycles with social indication, there is no significant effect of the triggering agent. An advantage for GnRH-a triggering was observed in medically indicated preservation cycles.

A major challenge in clinical In-Vitro Fertilization (IVF) is selecting the highest quality embryo to transfer to the patient in the hopes of achieving a pregnancy. Time-lapse microscopy provides clinicians with a wealth of information for selecting embryos. However, the resulting movies of embryos are currently analyzed manually, which is time consuming and subjective. Here, we automate feature extraction of time-lapse microscopy of human embryos with a machine-learning pipeline of five convolutional neural networks (CNNs). Our pipeline consists of (1) semantic segmentation of the regions of the embryo, (2) regression predictions of fragment severity, (3) classification of the developmental stage, and object instance segmentation of (4) cells and (5) pronuclei. Our approach greatly speeds up the measurement of quantitative, biologically relevant features that may aid in embryo selection.

To compare clinical pregnancy rate (CPR) and live birth rate (LBR) after frozen-thawed embryo transfer (FET) of day (D-) 6 blastocysts on D-5 versus D-6. A retrospective cohort study. A university-affiliated single-center tertiary hospital. Women undergoing FET of D-6 blastocysts between August 2015 and March 2019 were included in the study. Exclusion criteria were endometriosis and maternal age ≥ 42. Cycles involving embryo transfer (ET) at D-6 were compared to cycles involving ET on D-5. Primary outcomes assessed were CPR and LBR, and the secondary outcomes were spontaneous abortion and chemical pregnancy rates. Forty-two cycles were assessed, 21 in which ET occurred on D-6 and 21 in which ET occurred on D-5. There were no significant differences between groups regarding age, body mass index (BMI), etiology of infertility, number of oocytes aspirated and blastocysts cryopreserved in the fresh cycle, reason for freezing on D-6, endometrial thickness before ET, and blastocyst grade. A comparison of outcomes of ET on D-5 with those involving ET on D-6 revealed that D-5 transfer produced significantly higher CPR (8, 38% vs. 2, 8.5%; P = 0.030) and LBR (6, 28.6% vs. 1, 4.8%; P = 0.038), respectively. FET of D-6 embryos on D-5 compared with D-6 is associated with increased CPR and LBR values. These findings might be related to the limited time window for optimal rates of implantation and indicate that transferring embryos on D-6 of a FET cycle is likely too late.

PURPOSE: To assess the effects of letrozole or tamoxifen coadministration on fertility preservation treatment outcomes. METHODS: Retrospective cohort study of 118 breast cancer patients undergoing fertility preservation treatment between 2008 and 2018. Patients who received letrozole (n = 36) or tamoxifen (n = 30) were compared to controls (n = 52) who underwent standard ovarian stimulation protocols. The primary outcome measures included the number of retrieved oocytes, mature oocytes (MII), fertilization, and top-quality embryo rates. The secondary outcome measures included duration of stimulation, gonadotropin dose and peak estradiol level. RESULTS: The number of oocytes retrieved, MII oocytes, fertilization rate, duration of stimulation, or gonadotropin dose were similar in the letrozole and tamoxifen groups, compared to controls. Top-quality embryo rate was lower in the tamoxifen group compared to controls (25% vs 39.4%, respectively, P = 0.034). The abnormal fertilization rate was higher in the letrozole group compared to controls (7.8% vs 3.60%, respectively, P = 0.015). A stepwise logistic regression analysis revealed that letrozole and peak estradiol were significantly associated with abnormal fertilization (OR 11.94; 95% CI 2.35–60.4, P = 0.003 for letrozole and OR 1.075; 95% CI 1.024–1.12, P = 0.004 per 100 unit change in estradiol). CONCLUSIONS: There may be a negative effect of letrozole or tamoxifen on fertilization and embryo quality, in fertility preservation cycles. Further studies are needed to confirm these findings. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-020-02030-y.

Summary Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/β-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency., Graphical abstract, Highlights • Inhibition of SRC, PKC, and WNT consolidates human naive pluripotency induction • Competitiveness of p53 depleted human PSCs in cross-species chimeric embryos • Opposing net effect for ACTIVIN and WNT on mouse versus human naive pluripotency • 2i and ERKi independent alternative human naive-like PSC conditions, Engineered systems were used to screen for conditions that enable robust induction of human naive PSCs without the obligation for exogenous transgenes or feeder cells. The latter allowed defining the signaling and transcriptional foundations of human naive PSCs with enhanced (epi)genetic stability and competence for differentiation into all lineages.

Different conditions have been devised to isolate MEK/ERK signalling independent human naïve pluripotent stem cells (PSCs) that are distinct from conventional primed PSCs and better correspond to pre-implantation developmental stages. While the naïve conditions described thus far endow human PSCs with different extents of naivety features, isolating human pluripotent cells that retain characteristics of ground state pluripotency while maintaining differentiation potential and genetic integrity, remains a major challenge. Here we engineer reporter systems that allow functional screening for conditions that can endow both the molecular and functional features expected from human naive pluripotency. We establish that simultaneous inhibition of SRC-NFκB, WNT/ßCATENIN and PKC signalling pathways is essential for enabling expansion of teratoma competent fully naïve human PSCs in defined or xeno-free conditions. Divergent signalling and transcriptional requirements for maintaining naïve pluripotency were found between mouse and human. Finally, we establish alternative naïve conditions in which MEK/ERK inhibition is substituted with inhibition for NOTCH/RBPj signalling, which allow obtaining alternative human naïve PSCs with diminished risk for loss of imprinting and deleterious global DNA hypomethylation. Our findings set a framework for the signalling foundations of human naïve pluripotency and may advance its utilization in future translational applications.Highlights of key findingsCombined inhibition of SRC, WNT and PKC signaling consolidates human naïve pluripotencyStable expansion of DNA/RNA methylation-independent and TGF/ACTIVIN-independent human naïve PSCsOpposing roles for ACTIVIN and WNT/ßCATENIN signaling on mouse vs. human naive pluripotency2i and MEK/ERKi independent alternative human naïve PSC conditions via inhibiting NOTCH/RBPj signaling

OBJECTIVE To investigate the developmental potential of oocytes and embryos derived from extremely small follicles (

Despite much research, our understanding of the architecture and cis-regulatory elements of human promoters is still lacking. Here, we devised a high-throughput assay to quantify the activity of approximately 15,000 fully designed sequences that we integrated and expressed from a fixed location within the human genome. We used this method to investigate thousands of native promoters and preinitiation complex (PIC) binding regions followed by in-depth characterization of the sequence motifs underlying promoter activity, including core promoter elements and TF binding sites. We find that core promoters drive transcription mostly unidirectionally and that sequences originating from promoters exhibit stronger activity than those originating from enhancers. By testing multiple synthetic configurations of core promoter elements, we dissect the motifs that positively and negatively regulate transcription as well as the effect of their combinations and distances, including a 10-bp periodicity in the optimal distance between the TATA and the initiator. By comprehensively screening 133 TF binding sites, we find that in contrast to core promoters, TF binding sites maintain similar activity levels in both orientations, supporting a model by which divergent transcription is driven by two distinct unidirectional core promoters sharing bidirectional TF binding sites. Finally, we find a striking agreement between the effect of binding site multiplicity of individual TFs in our assay and their tendency to appear in homotypic clusters throughout the genome. Overall, our study systematically assays the elements that drive expression in core and proximal promoter regions and sheds light on organization principles of regulatory regions in the human genome.

The purpose of the study was to explore the effect of blastomere biopsy for preimplantation genetic diagnosis (PGD) on the embryos’ dynamics, further cleavage, development, and implantation. The study group included 366 embryos from all PGD treatments (September 2012 to June 2014) cultured in the EmbryoScope™ time-lapse monitoring system. The control group included all intracytoplasmic sperm injection (ICSI) embryos cultured in EmbryoScope™ until day 5 during the same time period (385 embryos). Time points of key embryonic events were analyzed with an EmbryoViewer™. Most (88 %) of the embryos were biopsied at ≥8 cells. These results summarize the further dynamic development of the largest cohort of biopsied embryos and demonstrate that blastomere biopsy of cleavage-stage embryos significantly delayed compaction and blastulation compared to the control non-biopsied embryos. This delay in preimplanation developmental events also affected postimplantation development as observed when the dynamics of non-implanted embryos (known implantation data (KID) negative) were compared to those of implanted embryos (KID positive). Analysis of morphokinetic parameters enabled us to explore how blastomere biopsy interferes with the dynamic sequence of developmental events. Our results show that biopsy delays the compaction and the blastulation of the embryos, leading to a decrease in implantation.

Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs, we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences serving as promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences and allows the characterization of TF binding determinants within and outside of core binding sites.

Although traditionally viewed as universal stretches of DNA, core promoters are diverse regulatory sequences with a high impact on transcriptional activity in promoters and enhancers. However, our understanding of their function, architecture, and cis-regulatory elements is still lacking. Here, we devised a high-throughput assay to quantify the core promoter activity of ~15,000 fully designed sequences that we integrated and expressed from a fixed location within the human genome. By measuring ~500 Pre-Initiation Complex (PIC) binding sequences, we find functional differences between promoters and enhancers and that both exhibit unidirectional activity. Our systematic investigation of all possible combinations of core promoter elements reveals a positive effect on expression of TATA and Initiator, and a negative effect of BREu and BREd. Moreover, we observe a 10bp periodicity in the optimal distance between the TATA and the Inr. By comprehensively screening TF binding-sites, we show that site orientation has little effect, that the effect of binding site number on expression is factor-specific, and that there is a striking agreement between the effect of binding site multiplicity and TF appearance in endogenous homotypic clusters. Thus, we provide a systematic view of the core- and proximal- promoter regions and shed light on organization principles of regulatory regions in the human genome.

Study question What is the optimal timing for blastomere biopsy during the 8-cell stage, at which embryos will have the best implantation potential? Summary answer Fast-cleaving embryos that are biopsied during the last quarter (Q4) of the 8-cell stage and are less affected by the biopsy procedure, and their implantation potential is better than that of embryos biopsied earlier during the 8-cell stage (Q1-Q3). What is known already Blastomer biopsy from cleavage-stage embryos is usually performed on the morning of Day 3 when the embryos are at the 6- to 8-cell stage and is still the preferred biopsy method for preimplantation genetic diagnosis (PGD) for monogentic disorders or chromosomal translocations. Human embryos usually remain at the 8-cell stage for a relatively long 'arrest phase' in which cells grow, duplicate their DNA and synthesize various proteins in preparation for the subsequent division. Study design, size, duration This is a retrospective cohort study. The study group (195 embryos) included all 8-cell stage embryos that underwent blastomere biopsy for PGD for monogenetic disorders and chromosomal translocations in our unit between 2012-2014 and cultured in the EmbryoScope until transfer. The control group (115 embryos) included all embryos that underwent intracytoplasmic sperm injection without a biopsy during the same period. Participants/materials, setting, methods The 8-cell stage was divided into four quarters: the first 5 h post-t8 (Q1), 5-10 h post-t8 (Q2), 10-15 h post-t8 (Q3) and at 15-20 h post-t8 (Q4). Non-biopsied control embryos were divided into four equivalent quarters. Embryos were evaluated for timing of developmental events following biopsy including timing of first cleavge after biopsy, timing of comapction (tM) and start of blastulation (tSB). Timing of these events were compared between PGD and control embryos, as well as with 56 PGD implanted embryos with Known Implantation Data (PGD-KID-positive embryos). Main results and the role of chance Embryos that were biopsied during Q3 (10-15 h from entry into 8-cell stage) were delayed in all three subsequent developmental events, including first cleavage after biopsy, compaction and start of blastulation. In contrast, these events occurred exactly at the same time as in the control group, in embryos that were biopsied during Q1, Q2 or Q4 of the 8-cell stage. The results show also that embryos that were biopsied during Q1, Q2 or Q3 of the 8-cell stage demonstrated a significant delay from the biopsied implanted embryos already in t8 as well as in tM and tSB. However, embryos that were biopsied during Q4 demonstrated dynamics similar to those of the biopsied implanted embryos in t8 and tM, and a delay was noticed only in the last stage of tSB. Limitations, reasons for caution This is a retrospective study that is limited to the timing of biopsy that is routinely performed in the IVF lab. A prospective study in which biopsy will be performed at a desired timing is needed in order to differ between the effect of biopsy itself and the cleavage rate of the embryo. Wider implications of the findings Our findings showed that blastomere biopsy can be less harmful to further development if it is carried out during a critical period of embryonic growth, i.e during Q4 of the 8-cell stage. They also demonstrated the added value of time-lapse microscopy for determining the optimal timing for blastomere biopsy. Study funding/competing interest(s) The study was funded by the routine budget of our IVF unit. Trial registration number N/A.

Genetically identical cells exhibit large variability (noise) in gene expression, with important consequences for cellular function. Although the amount of noise decreases with and is thus partly determined by the mean expression level, the extent to which different promoter sequences can deviate away from this trend is not fully known. Here, we present a high-throughput method for measuring promoter-driven noise for thousands of designed synthetic promoters in parallel. We use it to investigate how promoters encode different noise levels and find that the noise levels of promoters with similar mean expression levels can vary more than one order of magnitude, with nucleosome-disfavoring sequences resulting in lower noise and more transcription factor binding sites resulting in higher noise. We propose a kinetic model of gene expression that takes into account the nonspecific DNA binding and one-dimensional sliding along the DNA, which occurs when transcription factors search for their target sites. We show that this assumption can improve the prediction of the mean-independent component of expression noise for our designed promoter sequences, suggesting that a transcription factor target search may affect gene expression noise. Consistent with our findings in designed promoters, we find that binding-site multiplicity in native promoters is associated with higher expression noise. Overall, our results demonstrate that small changes in promoter DNA sequence can tune noise levels in a manner that is predictable and partly decoupled from effects on the mean expression levels. These insights may assist in designing promoters with desired noise levels.

The aim of the study was to study whether the trophoblasts carrying unbalanced translocation 11,22 [t(11;12)] display abnormal expression of trophoblastic genes and impaired functional properties that may explain implantation failure. t(11;22) hESCs and control hESCs were differentiated in vitro into trophoblast cells in the presence of BMP4, and trophoblast vesicles (TBVs) were created in suspension. The expression pattern of extravillous trophoblast (EVT) genes was compared between translocated and control TBVs. The functional properties of the TBVs were evaluated by their attachment to endometrium cells (ECC1) and invasion through trans-well inserts. TBVs derived from control hESCs expressed EVT genes from functioning trophoblast cells. In contrast, TBVs differentiated from the translocated hESC line displayed impaired expression of EVT genes. Moreover, the number of TBVs that were attached to endometrium cells was significantly lower compared to the controls. Correspondingly, invasiveness of trophoblast-differentiated translocated cells was also significantly lower than that of the control cells. These results may explain the reason for implantation failure in couple carriers of t(11;22). They also demonstrate that translocated hESCs comprise a valuable in vitro human model for studying the mechanisms underlying implantation failure.

Despite extensive research, our understanding of the rules according to which cis-regulatory sequences are converted into gene expression is limited. We devised a method for obtaining parallel, highly accurate gene expression measurements from thousands of designed promoters and applied it to measure the effect of systematic changes in the location, number, orientation, affinity and organization of transcription-factor binding sites and nucleosome-disfavoring sequences. Our analyses reveal a clear relationship between expression and binding-site multiplicity, as well as dependencies of expression on the distance between transcription-factor binding sites and gene starts which are transcription-factor specific, including a striking ~10-bp periodic relationship between gene expression and binding-site location. We show how this approach can measure transcription-factor sequence specificities and the sensitivity of transcription-factor sites to the surrounding sequence context, and compare the activity of 75 yeast transcription factors. Our method can be used to study both cis and trans effects of genotype on transcriptional, post-transcriptional and translational control.

The E2F family of transcription factors plays a pivotal role in the regulation of cell proliferation in higher eukaryotes. We used DNA microarrays and cell lines containing either inducible E2F-1 or inducible E2F-3 to identify novel E2F target genes. Our data indicate that E2F up-regulates the expression of genes not previously described as E2F target genes. A number of these E2F-regulated genes are involved in DNA replication, DNA repair and mitosis. These results suggest that E2F affects cell cycle progression both at S phase and during mitosis. Furthermore, our findings indicate that E2F-dependent gene activation may contribute to the cellular response to DNA damage.

PURPOSE: The purpose of the study was to compare the morphokinetic parameters of embryos carrying balanced chromosomal translocations with those carrying unbalanced chromosomal translocations using time-lapse microscopy. METHODS: The study group included 270 embryos that underwent biopsies on day 3 for preimplantation genetic diagnosis (PGD) for chromosomal translocations in our unit between 2013 and 2015. All embryos were incubated under time-lapse microscopy and evaluated for timing of developmental events up to day 5. The timing of these events was compared between balanced and unbalanced embryos, potentially viable and nonviable variants, and maternal versus paternal inheritance of the translocation. RESULTS: The PGD analysis found that 209 (77%) of the 270 biopsied embryos carried an unbalanced translocation. Embryos carrying unbalanced translocations, which are expected to lead to implantation failure or miscarriage, cleaved less synchronously and were delayed in time of cleavage to the 4-cell stage (t4) and in time of start of blastulation (tSB) compared with balanced embryos (P

Precise gene expression patterns are established by transcription factor (TFs) binding to regulatory sequences. While these events occur in the context of chromatin, our understanding of how TF-nucleosome interplay affects gene expression is highly limited. Here, we present an assay for high-resolution measurements of both DNA occupancy and gene expression on large-scale libraries of systematically designed regulatory sequences. Our assay reveals occupancy patterns at the single-cell level. It provides an accurate quantification of the fraction of the population bound by a nucleosome and captures distinct, even adjacent, TF binding events. By applying this assay to over 1,500 promoter variants in yeast, we reveal pronounced differences in the dependency of TF activity on chromatin and classify TFs by their differential capacity to alter chromatin and promote expression. We further demonstrate how different regulatory sequences give rise to nucleosome-mediated TF collaborations that quantitatively account for the resulting expression.

Carriers of the balanced translocation t(11;22), the most common reciprocal translocation in humans, are at high risk of creating gametes with unbalanced translocation, leading to repeated miscarriages. Current research models for studying translocated embryos and the biological basis for their implantation failure are limited. The aim of this study was to elucidate whether human embryonic stem cells (hESCs) carrying the unbalanced chromosomal translocation t(11;22) can provide an explanation for repeated miscarriages of unbalanced translocated embryos. Fluorescent in situ hybridization and karyotype analysis were performed to analyze the t(11;22) in embryos during PGD and in the derived hESC line. The hESC line was characterized by RT-PCR and FACS analysis for pluripotent markers. Directed differentiation to trophoblasts was carried out by bone morphogenetic protein 4 (BMP4). Trophoblast development was analyzed by measuring β-hCG secretion, by β-hCG immunostaining and by gene expression of trophoblastic markers. We derived the first hESC line carrying unbalanced t(11;22), which showed the typical morphological and molecular characteristics of a hESC line. Control hESCs differentiated into trophoblasts secreted increasing levels of β-hCG and concomitantly expressed the trophoblast genes, CDX2, TP63, KRT7, ERVW1, CGA, GCM1, KLF4 and PPARG. In contrast, differentiated translocated hESCs displayed reduced and delayed secretion of β-hCG concomitant with impaired expression of the trophoblastic genes. The reduced activation of trophoblastic genes may be responsible for the impaired trophoblastic differentiation in t(11;22)-hESCs, associated with implantation failure in unbalanced t(11;22) embryos. Our t(11;22) hESCs are presented as a valuable human model for studying the mechanisms underlying implantation failure.

LAP2beta is an integral membrane protein of the nuclear envelope involved in chromatin and nuclear architecture. Using the yeast two-hybrid system, we have cloned a novel LAP2beta-binding protein, mGCL, which contains a BTB/POZ domain and is the mouse homologue of the Drosophila germ-cell-less (GCL) protein. In Drosophila embryos, GCL was shown to be essential for germ cell formation and was localized to the nuclear envelope. Here, we show that, in mammalian cells, GCL is co-localized with LAP2beta to the nuclear envelope. Nuclear fractionation studies reveal that mGCL acts as a nuclear matrix component and not as an integral protein of the nuclear envelope. Recently, mGCL was found to interact with the DP3alpha component of the E2F transcription factor. This interaction reduced the transcriptional activity of the E2F-DP heterodimer, probably by anchoring the complex to the nuclear envelope. We demonstrate here that LAP2beta is also capable of reducing the transcriptional activity of the E2F-DP complex and that it is more potent than mGCL in doing so. Co-expression of both LAP2beta and mGCL with the E2F-DP complex resulted in a reduced transcriptional activity equal to that exerted by the pRb protein.

The transcription factor E2F-1 plays a pivotal role in the regulation of G1/S transition in higher eukaryotes cell cycle. We used a cell line containing an inducible E2F-1 and oligonucleotide microarray analysis to identify novel E2F target genes. We show that E2F-1 up-regulates the expression of a number of genes coding for components of the DNA replication machinery. Among them is the gene coding for the 32 Kd subunit of replication protein A (RPA2). Replication protein A is the most abundant single strand DNA binding complex and it is essential for DNA replication. We demonstrate that RPA2 is a novel E2F target gene whose expression can be directly regulated by E2F-1 via E2F binding sites in its promoter. In addition, expression of Topoisomerase IIalpha and subunit IV of DNA polymerase alpha is also up-regulated upon E2F-1 induction. Taken together, these results provide novel links between components of the DNA replication machinery and the cell growth regulatory pathway involving the Rb tumor suppressor and E2F.

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

Nature 504, 282–286 (2013); doi:10.1038/nature12745 The reduced representation bisulphite sequencing (RRBS) data generated and analysed in this Letter were not originally uploaded to the Gene Expression Omnibus (GEO), but can now be found under accession number GSE52617 (within the GSE52824 super series).

The coordinated function of the different compartments of the follicle, the oocyte and the somatic cumulus/granulosa cells, is enabled by the presence of a network of cell-to-cell communication generated by gap junctions. Connexin 43 (Cx43) is the most abundant gap junction protein expressed by the ovarian follicle. The expression of Cx43 is subjected to the control of gonadotropins as follows: FSH up-regulates, whereas LH down-regulates its levels. The aim of this study was to explore the mechanism by which LH reduces the levels of Cx43 and to identify the signal transduction pathway involved in this process. The effect of LH was studied in vitro using isolated intact ovarian follicles. The possible mediators of LH-induced Cx43 down-regulation were examined by incubating the follicles with LH in the presence or absence of inhibitors of protein kinase A (PKA) and of MAPK signaling pathways. Our experiments revealed a 3-h half-life of Cx43 in both control and LH-treated follicles, suggesting that LH did not affect the rate of Cx43 degradation. We further demonstrated that the level of Cx43 mRNA was not significantly influenced by this gonadotropin. However, upon LH administration, [35S]methionine incorporation into Cx43 protein was remarkably reduced. The LH-induced arrest of Cx43 synthesis was counteracted by inhibitors of both the PKA and the MAPK cascades. We show herein that LH inhibits Cx43 expression by reducing its rate of translation and that this effect is mediated by both PKA and MAPK.

Objective To explore the possibility that endometrial injury modulates the expression of specific genes that may increase uterine receptivity. Design Controlled clinical study. Setting Clinical IVF unit and academic research center. Patient(s) IVF patients with 28- to 30-day menstrual cycles. Intervention(s) Endometrial biopsies from two groups of patients were collected on days 20–21 of their spontaneous menstrual cycle. The experimental, but not the control, group underwent biopsies on days 11–13 and 21–24 of their preceding cycle. Main Outcome Measure(s) Global endometrial gene expression and specific analysis of uroplakin Ib (UPIb) mRNA level throughout the menstrual cycle. Result(s) Local injury modulated the expression of a wide variety of genes. One of the prominently up-regulated genes was the bladder transmembranal protein, UPIb, whose expression by the endometrium is shown here for the first time. Endometrial UPIb mRNA increases after biopsy in the same cycle wct 2with an additional elevation in the following cycle. Immunohistochemical analysis localized the UPIb protein to the glandular-epithelial cells. Genes encoding other membrane proteins such as adipose differentiation-related protein and mucin 1, transmembrane, were also up-regulated. Conclusion(s) The biopsy-induced increase in the expression of UPIb and other genes encoding membrane proteins supports the possible importance of the membrane structure and stability during implantation. The specific role of UPIb in uterine receptivity should be elucidated.

Advances in molecular cell biology, medical research, and drug development are driving a growing need for technologies that enable imaging the dynamics of molecular and physiological processes simultaneously in numerous non-adherent living cells. Here we describe a platform technology and software – the CKChip system – that enables continuous, fluorescence-based imaging of thousands of individual living cells, each held at a given position (“address”) on the chip. The system allows for sequential monitoring, manipulation and kinetic analyses of the effects of drugs, biological response modifiers and gene expression in both adherent and non-adherent cells held on the chip. Here we present four specific applications that demonstrate the utility of the system including monitoring kinetics of reactive oxygen species generation, assessing the intracellular enzymatic activity, measuring calcium flux and the dynamics of target cell killing induced by conjugated cytotoxic T-lymphocytes. We found large variations among individual cells in the overall amplitude of their response to stimuli, as well as in kinetic parameters such as time of onset, initial rate and decay of the response, and frequency and amplitude of oscillations. These variations probably reflect the heterogeneity of even cloned cell populations that would have gone undetected in bulk cell measurements. We demonstrate the utility of the system in providing kinetic parameters of complex cellular processes such as Ca++ influx, transients and oscillations in numerous individual cells. The CKChip opens up new opportunities in cell-based research, in particular for acquiring fluorescence-based, kinetic data from multiple, individual non-adherent cells.

Background Most cases of colorectal cancer (CRC) are initiated by inactivation mutations in the APC gene, which is a negative regulator of the Wnt-β-catenin pathway. Patients with familial adenomatous polyposis (FAP) inherit a germline mutation in one APC allele, and loss of the second allele leads to the development of polyps that will turn malignant if not removed. It is not fully understood which molecular mechanisms are activated by APC loss and when the loss of the second APC allele occurs. Methods Two FAP human embryonic stem cell (hESCs) lines were derived from APC mutated embryos following pre-implantation genetic diagnosis (PGD) for FAP. These FAP-hESCs were cultured in vitro and following extended culture: 1) β-catenin expression was analyzed by Western blot analysis; 2) Wnt-β-catenin/TCF-mediated transcription luciferase assay was performed; 3) cellular localization of β-catenin was evaluated by immunoflorecence confocal microscopy; and 4) DNA sequencing of the APC gene was performed. Results We have established a novel human in-vitro model for studying malignant transformation, using hESCs that carry a germline mutation in the APC gene following PGD for FAP. Extended culturing of FAP1 hESCs led to activation of the Wnt signaling pathway, as demonstrated by enhanced β-catenin/TCF-mediated activity. Additionally, β-catenin showed a distinct perinuclear distribution in most (91 %) of the FAP1 hESCs high passage colonies. DNA sequencing of the whole gene detected several polymorphisms in FAP1 hESCs, however, no somatic mutations were discovered in the APC gene. On the other hand, no changes in β-catenin were detected in the FAP2 hESCs, demonstrating the natural diversity of the human FAP population. Conclusions Our results describe the establishment of novel hESC lines from FAP patients with a predisposition for cancer mutation. These cells can be maintained in culture for long periods of time and may serve as a platform for studying the initial molecular and cellular changes that occur during early stages of malignant transformation. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2809-9) contains supplementary material, which is available to authorized users.