Cleavage-stage human embryo arrest, is it embryo genetic composition or others?

Embryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle.

MicroRNAs secreted by human embryos could be potential biomarkers for clinical outcomes of assisted reproductive technology.

Introduction: MicroRNAs (miRNAs) are important regulators of many biological functions, including embryo implantation and development. Recently, it has been reported that miRNAs in biofluids are predictive for physiological and pathological processes. Objectives: In this study, we aim to investigate whether the miRNAs secreted by human embryos in culture medium can be used as embryonic biomarkers. Methods: The culture media were prospectively collected from embryos of patients at reproductive medicine center with informed consent. A high-throughput miRNA sequencing method was applied to detect the miRNA profiles in the human embryo culture media. After bioinformatics analysis and screening of differentially expressed miRNAs, quantitative real-time polymerase chain reaction (qRT-PCR) assay was subsequently performed to further confirm the sequencing results with mixed samples. Furthermore, we performed droplet digital PCR (ddPCR) to verify the target miRNAs at single sample level. Receiver operating characteristic (ROC) analyses were performed for differentially expressed miRNAs. Results: Compared with embryos with failed pregnancy, the embryos with successful pregnancy secreted different miRNA profiles into the culture media, which were predicted to be involved in multiple biological processes. Validated by droplet digital polymerase chain reaction (ddPCR), the expression of hsa-miR-26b-5p and hsa-miR-21-5p in the culture media of cleavage embryos with successful pregnancy was significantly lower than that of embryos with failed pregnancy. Moreover, the Receiver Operating Characteristic (ROC) curve analysis indicated that hsa-miR-26b-5p and hsa-miR-21-5p could serve as potential biomarkers for reproductive outcomes. Conclusion: Together, our findings highlight the important predictive potential of miRNAs secreted by human embryos in culture media, which is meaningful for non-invasive embryo selection in assisted reproductive technology.

Constitutive Expression of TERT Enhances ß-Klotho Expression and Improves Age-Related Deterioration in Early Bovine Embryos.

Age-associated decline in oocyte quality is one of the dominant factors of low fertility. Aging alters several key processes, such as telomere lengthening, cell senescence, and cellular longevity of granulosa cells surrounding oocyte. To investigate the age-dependent molecular changes, we examined the expression, localization, and correlation of telomerase reverse transcriptase (TERT) and ß-Klotho (KLB) in bovine granulosa cells, oocytes, and early embryos during the aging process. Herein, cumulus-oocyte complexes (COCs) obtained from aged cows (>120 months) via ovum pick-up (OPU) showed reduced expression of ß-Klotho and its co-receptor fibroblast growth factor receptor 1 (FGFR1). TERT plasmid injection into pronuclear zygotes not only markedly enhanced day-8 blastocysts' development competence (39.1 ± 0.8%) compared to the control (31.1 ± 0.5%) and D-galactose (17.9 ± 1.0%) treatment groups but also enhanced KLB and FGFR1 expression. In addition, plasmid-injected zygotes displayed a considerable enhancement in blastocyst quality and implantation potential. Cycloastragenol (CAG), an extract of saponins, stimulates telomerase enzymes and enhances KLB expression and alleviates age-related deterioration in cultured primary bovine granulosa cells. In conclusion, telomerase activation or constitutive expression will increase KLB expression and activate the FGFR1/ß-Klotho pathway in bovine granulosa cells and early embryos, inhibiting age-related malfunctioning.

Ovarian Hyperstimulation Syndrome Is Associated with a High Secondary Sex Ratio in Fresh IVF Cycles with Cleavage-Stage Embryo Transfer: Results for a Cohort Study.

The sex ratio at birth is defined as the secondary sex ratio (SSR). Ovarian hyperstimulation syndrome (OHSS) is a serious and iatrogenic complication associated with controlled ovarian stimulation (COS) during assisted reproductive technology (ART) treatments. It has been hypothesized that the human SSR is partially controlled by parental hormone levels around the time of conception. Given the aberrant hormonal profiles observed in patients with OHSS, this retrospective study was designed to evaluate the impact of OHSS on the SSR. In this study, all included patients were divided into 3 groups: non-OHSS (n=2777), mild OHSS (n=644), and moderate OHSS (n=334). Our results showed that the overall SSR for the study population was 1.033. The SSR was significantly increased in patients with moderate OHSS (1.336) compared to non-OHSS patients (1.002) (p=0.048). Subgroup analyses showed that increases in the SSR in patients with moderate OHSS were observed in the IVF group (1.323 vs 1.052; p=0.043), but not in the ICSI groups (1.021 vs 0.866; p=0.732). In addition, the elevated serum estradiol (E2) and progesterone (P4) levels in OHSS patients were not associated with SSR. In this study, for the first time, we report that a high SSR is associated with OHSS in patients who received fresh IVF treatments. The increases in SSR in OHSS patients are not attributed to the high serum E2 and P4 levels. Our findings may make both ART clinicians and patients more aware of the influences of ART treatments on the SSR and allow clinicians to counsel patients more appropriately.

Transcription factor OTX2 silences the expression of cleavage embryo genes and transposable elements.

Upon mammalian fertilization, zygotic genome activation (ZGA) and activation of transposable elements (TEs) occur in early embryos to establish totipotency and support embryogenesis. However, the molecular mechanisms controlling the expression of these genes in mammals remain poorly understood. The 2-cell-like population of mouse embryonic stem cells (mESCs) mimics cleavage-stage embryos with transient Dux activation. In this study, we demonstrated that deficiency of the transcription factor OTX2 stimulates the expression of ZGA genes in mESCs. Further analysis revealed that OTX2 is incorporated at the Dux locus with corepressors for transcriptional inhibition. We also found that OTX2 associates with TEs and silences the subtypes of TEs. Therefore, OTX2 protein plays an important role in ZGA and TE expression in mESCs to orchestrate the transcriptional network.

Analysis of Biochemical and Clinical Pregnancy Loss Between Frozen-Thawed Embryo Transfer of Blastocysts and Day 3 Cleavage Embryos in Young Women: A Comprehensive Comparison.

Background: To determine whether the embryo developmental stage affects biochemical or clinical pregnancy loss in young women undergoing frozen-thawed embryo transfer (FET) and to investigate the underlying mechanism. Methods: This was a retrospective study including a total of 18,34 ß-HCG (human chorionic gonadotropin)-positive FET cycles. According to the morphological appearance (MA) of transferred blastocysts, FET cycles with blastocysts were divided into two groups: Group A: morphologically good (MG) blastocysts only, and Group B: at least one morphologically non-good (MNG) blastocyst. FET cycles with day 3 cleavage embryos were assigned as Group C. Biochemical and clinical pregnancy loss were the main outcome measures. Results: We predicted 78% in vivo-formed MG and 53.9% in vivo-formed day 5 blastocysts in Group C. (a) Including cases in Group A and Group B for binary logistic regression, we showed that Group B and day 6 blastocysts had significantly higher rates of BPL and CPL than Group A and day 5 blastocysts, respectively. (b) Including cases in Group A, Group B, and Group C for binary logistic regression, we showed that Group C had a significantly higher rate of BPL than Group A and day 5 blastocysts and a similar rate of BPL as Group B and day 6 blastocysts. Group C had a higher rate of CPL than Group A (p=0.071) and day 5 blastocysts (p=0.039), and a lower rate of CPL than Group B (p=0.199) and day 6 blastocysts (p=0.234). Conclusions: (1) MA and days of usable blastocysts could serve as independent factors affecting the occurrence of BPL and CPL. (2) Transfer of day 3 cleavage embryos may produce "unusable blastocysts" in vivo, which significantly increased the rate of BPL. (3) The rate of CPL resulting from the transfer of day 3 embryos may depend on the rate of in vivo-formed MG or day 5 blastocysts. Our study indicated that the difference in the BPL or CPL between transfer of blastocysts and day 3 cleavage embryos may largely depend on the quality of embryos transferred.

Dynamics of telomeric repeat-containing RNA expression in early embryonic cleavage stages with regards to maternal age.

Telomeres are transcribed into long non-coding RNAs known as Telomeric Repeat-Containing RNA (TERRA). They have been shown to be essential regulators of telomeres and to act as epigenomic modulators at extra-telomeric sites. However the role of TERRA during early embryonic development has never been investigated. Here, we show that TERRA is expressed in murine and bovine early development following a wave pattern. It starts at 4-cell stage, reaching a maximum at the 16-cell followed by a decline at the morula and blastocyst stages. Moreover, TERRA expression is not affected by increasing oocyte donor age whereas telomere length does. This indicates that TERRA expression is independent of the telomere length in early development. Our findings anticipate an essential role of TERRA in early stages of development and this might be useful in the future for a better understanding of age related female infertility.

High three pro-nuclei (3PN) zygotes proportion associated with normal embryo multinucleation at the two-cell stage: two cases report.

Objective: Blastomere multinucleation at the two-cell stage (MN2) is a common nuclear abnormality observed in early human embryos and known to decrease the implantation rate. The aim of this study is to explore whether or not there is a link between high 3PN zygotes proportion and MN2 incidence.Methods: For embryo culture in the conventional incubator, the evaluation of nuclear status of two-cell stage is usually not performed. Therefore, the MN2 phenomenon is easily ignored. The time-lapse monitoring system (TLS) offers a promising new method to evaluate embryo development. We reported two cases who had single 2PN zygote and more than four 3PN zygotes in the conventional in-vitro fertilization (c-IVF) cycle.Results: We observed the MN2 incidence in the single normal embryo by TLS which suggested that it might be resulted from high 3PN zygotes proportion incidence. No available embryo was obtained in the first c-IVF cycle and the intracytoplasmic sperm injection (ICSI) treatment was performed in the second cycle. In subsequent ICSI cycles, we observed no 3PN zygotes incidence and transferred two day 3 embryos for the patients. Finally, the two couples successfully obtained healthy babies.Conclusions: High 3PN zygotes proportion might be associated with the MN2 incidence.

Morphokinetic analysis of cleavage stage embryos and assessment of specific gene expression in cumulus cells independently predict human embryo development to expanded blastocyst: a preliminary study.

To assess whether morphokinetic features at the cleavage stage together with specific gene expression in cumulus cells (CCs) may be used to predict whether human embryos are able to achieve the expanded blastocyst stage on day 5. Eighty-one embryos were cultured using the Geri plus® time-lapse system. Twenty-seven embryos progressing to the expanded blastocyst stage (BL group) were compared with thirty-five embryos showing developmental arrest (AR group) and nineteen reaching the stage of early or not fully expanded blastocyst (nBL group). The analyzed morphokinetic variables were pronuclear appearance (tPNa), pronuclear fading (tPNf), and completion of cleavage to two, three, four, and eight cells (t2, t3, t4, and t8). CCs were analyzed by RT-qPCR for bone morphogenetic protein 15 (BMP15), cytochrome c oxidase subunit II (COXII), ATP synthase subunit 6 (MT-ATP6), connexin 43 (Cx43), and heme oxygenase-1 (HO-1). Embryos of BL group showed a significantly faster kinetic. BMP15, COXII, and MT-ATP6 mRNA expression was significantly higher in CCs of BL group embryos, whereas Cx43 and HO-1 mRNA levels were higher in AR group. Kinetic parameters and gene expression were not significantly different between either the BL and nBL groups or the AR and nBL groups. ROC curves showed that the most predictive cut-offs were t2 < 26.25 for morphokinetics and COXII > 0.3 for gene expression. Multivariable logistic regression analysis showed that morphokinetic variables and gene expression were both valuable, independent predictors of embryo development to expanded blastocyst. Our results suggest the possibility of developing integrated prediction models for early embryo selection at the cleavage stage.

Pre-implantation genetic testing alters the sex ratio: an analysis of 91,805 embryo transfer cycles.

PURPOSE: To determine if pre-implantation genetic testing (PGT) shifts the sex ratio (SER), the ratio of male to female births in a population normalized to 100 and typically stable at 105, following in vitro fertilization (IVF). METHODS: Data from 2014 to 2016 was requested from the Society for Assisted Reproductive Technologies (SART) database including fresh and frozen transfer cycles. Women with a singleton live birth following a fresh or frozen autologous embryo transfer of a PGT blastocyst, non-PGT blastocyst, or non-PGT cleavage stage embryo were included. The SER between groups was compared using chi-square tests. Modified Poisson regression modeled the relative risk (RR) of having a male compared to a female among PGT blastocyst transfers versus non-PGT cleavage and blastocyst transfers adjusting for age, BMI, smoking status, race, parity, number of oocytes retrieved, and clinic region. RESULTS: The SER was 110 among PGT blastocyst offspring, 107 among non-PGT blastocyst offspring (p = 0.005), and 99 among non-PGT cleavage offspring (p < 0.001). The risk of having a male infant was 2% higher among PGT blastocyst transfers compared to non-PGT blastocyst transfers (RR 1.02; 95% CI: 1.01, 1.04). The risk was 5% higher among PGT blastocyst transfers compared to non-PGT cleavage transfers (RR 1.05; 95% CI: 1.02, 1.07). The association between PGT and infant gender did not significantly differ by region (p = 0.57) or parity (p = 0.59). CONCLUSION: Utilizing PGT shifts the SER in the IVF population from the standard of 105 to 110, increasing the probability of a male offspring.

Par-1 controls the composition and growth of cortical actin caps during Drosophila embryo cleavage.

Cell structure depends on the cortex, a thin network of actin polymers and additional proteins underlying the plasma membrane. The cell polarity kinase Par-1 is required for cells to form following syncytial Drosophila embryo development. This requirement stems from Par-1 promoting cortical actin caps that grow into dome-like metaphase compartments for dividing syncytial nuclei. We find the actin caps to be a composite material of Diaphanous (Dia)-based actin bundles interspersed with independently formed, Arp2/3-based actin puncta. Par-1 and Dia colocalize along extended regions of the bundles, and both are required for the bundles and for each other's bundle-like localization, consistent with an actin-dependent self-reinforcement mechanism. Par-1 helps establish or maintain these bundles in a cortical domain with relatively low levels of the canonical formin activator Rho1-GTP. Arp2/3 is required for displacing the bundles away from each other and toward the cap circumference, suggesting interactions between these cytoskeletal components could contribute to the growth of the cap into a metaphase compartment.

Crosslinking activity of non-muscle myosin II is not sufficient for embryonic cytokinesis in C. elegans.

Cytokinesis in animal cells requires the assembly and constriction of a contractile actomyosin ring. Non-muscle myosin II is essential for cytokinesis, but the role of its motor activity remains unclear. Here, we examine cytokinesis in C. elegans embryos expressing non-muscle myosin motor mutants generated by genome editing. Two non-muscle motor-dead myosins capable of binding F-actin do not support cytokinesis in the one-cell embryo, and two partially motor-impaired myosins delay cytokinesis and render rings more sensitive to reduced myosin levels. Further analysis of myosin mutants suggests that it is myosin motor activity, and not the ability of myosin to crosslink F-actin, that drives the alignment and compaction of F-actin bundles during contractile ring assembly, and that myosin motor activity sets the pace of contractile ring constriction. We conclude that myosin motor activity is required at all stages of cytokinesis. Finally, characterization of the corresponding motor mutations in C. elegans major muscle myosin shows that motor activity is required for muscle contraction but is dispensable for F-actin organization in adult muscles.This article has an associated 'The people behind the papers' interview.

Localisation of phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (WBP2 N-terminal like) on equine spermatozoa and flow cytometry quantification of PLCZ1 and association with cleavage in vitro.

Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal piece of the tail. Anti-WBP2NL antibody identified two WBP2NL bands (~28 and ~32kDa) in the postacrosomal region, midpiece and principal piece of the tail. PLCZ1 and WBP2NL expression was positively correlated (P=0.04) in sperm heads. Flow cytometry evaluation of PLCZ1 revealed large variations in fluorescence intensity and the percentage of positively labelled spermatozoa among stallions. PLCZ1 expression was significantly higher in viable than non-viable spermatozoa, and DNA fragmentation was negatively correlated with PLCZ1 expression and the percentage of positively labelled spermatozoa (P<0.05). The use of equine sperm populations considered to have high versus low PLCZ1 content resulted in significantly higher cleavage rates after ICSI of bovine and equine oocytes, supporting the importance of PLCZ1 for oocyte activation.

Follicle-stimulating hormone administration affects amino acid metabolism in mammalian oocytes†.

Culture media used in assisted reproduction are commonly supplemented with gonadotropin hormones to support the nuclear and cytoplasmic maturation of in vitro matured oocytes. However, the effect of gonadotropins on protein synthesis in oocytes is yet to be fully understood. As published data have previously documented a positive in vitro effect of follicle-stimulating hormone (FSH) on cytoplasmic maturation, we exposed mouse denuded oocytes to FSH in order to evaluate the changes in global protein synthesis. We found that dose-dependent administration of FSH resulted in a decrease of methionine incorporation into de novo synthesized proteins in denuded mouse oocytes and oocytes cultured in cumulus-oocyte complexes. Similarly, FSH influenced methionine incorporation in additional mammalian species including human. Furthermore, we showed the expression of FSH-receptor protein in oocytes. We found that major translational regulators were not affected by FSH treatment; however, the amino acid uptake became impaired. We propose that the effect of FSH treatment on amino acid uptake is influenced by FSH receptor with the effect on oocyte metabolism and physiology.

SIRT6 participates in the quality control of aged oocytes via modulating telomere function.

It has been well recognized that oocyte quality declines in aging animals. However, to date, the underlying mechanism remains to be explored. In the present study, we report that oocytes and embryos from aged mice (42-45 weeks old) display the reduced expression of SIRT6 protein, accompanying with telomere shortening and DNA lesions. Moreover, we demonstrate that specific depletion of SIRT6 in oocytes induces dysfunctional telomeres and apoptosis of the resultant early embryos, leading to the developmental delay and cytoplasmic fragmentation. Importantly, we further find that overexpression of SIRT6 in aged oocytes promotes the telomere elongation in 2-cell embryos and lowers the incidence of apoptotic blastomeres. In summary, our data indicate a role for SIRT6 in modulating telomere function during oocyte maturation and embryonic development, and discover that SIRT6 reduction is an important point connecting maternal aging and quality control of oocyte/embryos.

Direct Single-Cell Analysis of Human Polar Bodies and Cleavage-Stage Embryos Reveals No Evidence of the Telomere Theory of Reproductive Ageing in Relation to Aneuploidy Generation.

Reproductive ageing in women, particularly after the age of 35, is associated with an exponential increase in the proportion of chromosomally abnormal oocytes produced. Several hypotheses have attempted to explain this observation, including the 'limited oocyte pool' hypothesis and the 'two-hit' hypothesis, the latter explaining that a depletion in oocyte quality with age results from the multiple opportune stages for errors to occur in meiosis. Recently however, the telomere theory of reproductive ageing in women has been proposed. This suggests that shortened telomeres in oocytes of women of advanced maternal age render oocytes unable to support fertilization and embryogenesis. Despite a credible rationale for the telomere theory of reproductive ageing in women, very few studies have assessed telomere length directly in human oocytes or preimplantation embryos. Therefore, we directly assessed relative telomere length in first polar bodies and blastomeres from cleavage stage (day 3) embryos. In both cell types we tested the hypothesis that (1) older women have shorter telomeres and (2) chromosomally abnormal (aneuploid) gametes/embryos have shorter telomeres. In all cases, we found no evidence of altered telomere length associated with age-related aneuploidy.

p66Shc is associated with hydrogen peroxide-induced oxidative stress in preimplantation sheep embryos.

The low efficiency of in vitro embryo production is associated with oxidative stress induced by suboptimal culture conditions. p66Shc is a 66-kDa protein of the ShcA (Src homologous-collagen homolog) adaptor protein family, which is involved in signaling pathways involved in oxidative stress regulation, apoptosis induction, and aging. However, the functional role of p66Shc during the preimplantation development of sheep embryos is not understood. Our results showed that early-cleavage (≤28 hr) embryos had a higher developmental potential than late-cleavage (>28 hr) embryos. The poor quality of these late-cleavage embryos was associated with increased the transcripts and protein of p66Shc and decreased mitochondrial activity. In addition, exogenous hydrogen peroxide-induced oxidative stress significantly increased p66Shc protein abundance and suppressed embryonic development, which was ameliorated by antioxidant treatment. Notably, oxidative stress induced the nuclear localization of p66Shc and phosphorylated (Ser-36) p66Shc. Collectively, these observations suggest that p66Shc may be playing an important role in the regulation of oxidative stress during the preimplantation development of sheep embryos.

Exogenous glutathione improves intracellular glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos.

Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0-32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.

What is the effect of embryo morphology on serum ß-hCG levels?

OBJECTIVE: To determine the effect of embryonic factors on serum beta human chorionic gonadotropin (ß-hCG) levels in pregnancy and live birth resulting after a single fresh cleavage embryo and blastocyst transfer. STUDY DESIGN: This was a retrospective cohort study conducted at a tertiary care hospital. All fresh single embryo transfers (sETs) between September 2011 and December 2016 were included. The correlation analysis was performed to determine the association of embryo morphological parameters on mean serum ß-hCG levels on day 12 after the transfer of a fresh single cleavage embryo and a fresh single blastocyst embryo. RESULTS: Out of a total of 455 fresh sETs, 60 positive ß-hCG results after the transfer of a single fresh cleavage-stage embryo and 82 after the transfer of a single fresh blastocyst. The mean ß-hCG level resulting from a single fresh blastocyst ET was 371.7 ± 52.7 IU/L, which was similar to the mean ß-hCG level resulting from a cleavage ET (314.5 ± 36.9 IU/L) (p = .70). Interestingly, serum ß-hCG levels resulting from a single fresh blastocyst ET showed a correlation with day 5 blastocoele expansion, trophectoderm cell number and blastocyst quality score in ongoing pregnancy (r = .33, p = .02; r = .29, p = .04; and r = .31, p = .03, respectively). Moreover, day 5 blastocoele expansion and blastocyst quality score showed a correlation with the serum ß-hCG levels resulting from a single fresh blastocyst ET in live birth (r = .36, p = .02; r = .31, p = .04, respectively). CONCLUSION: Our study suggests that serum ß-hCG levels resulting from a single fresh blastocyst ET showed a correlation with day 5 blastocoele expansion and blastocyst quality score in both ongoing pregnancy and live birth.

Retrospective analysis: reproducibility of interblastomere differences of mRNA expression in 2-cell stage mouse embryos is remarkably poor due to combinatorial mechanisms of blastomere diversification.

STUDY QUESTION: What is the prevalence, reproducibility and biological significance of transcriptomic differences between sister blastomeres of the mouse 2-cell embryo? SUMMARY ANSWER: Sister 2-cell stage blastomeres are distinguishable from each other by mRNA analysis, attesting to the fact that differentiation starts mostly early in the mouse embryo; however, the interblastomere differences are poorly reproducible and invoke the combinatorial effects of known and new mechanisms of blastomere diversification. WHAT IS KNOWN ALREADY: Transcriptomic datasets for single blastomeres in mice have been available for years but have never been systematically analysed together, although such an analysis may shed light onto some unclarified topics of early mammalian development. Two unknowns that remain are at which stage embryonic blastomeres start to diversify from each other and what is the molecular origin of that difference. At the earliest postzygotic stage, the 2-cell stage, opinions differ regarding the answer to these questions; one group claims that the first zygotic division yields two equal blastomeres capable of forming a full organism (totipotency) and another group claims evidence for interblastomere differences reminiscent of the prepatterning found in embryos of lower taxa. Regarding the molecular origin of interblastomere differences, there are four prevalent models which invoke (1) oocyte anisotropy, (2) sperm entry point, (3) partition errors of the transcript pool and (4) asynchronous embryonic genome activation in the two blastomeres. STUDY DESIGN, SIZE, DURATION: Seven transcriptomic studies published between 2011 and 2017 were eligible for retrospective analysis, since both blastomeres of the mouse 2-cell embryo had been analysed individually regarding the original pair associations and since the datasets were made available in public repositories. Five of these studies, encompassing a total of 43 pairs of sister blastomeres, were selected for further analyses based on high interblastomere correlations of mRNA levels. A double cut-off was used to select mRNAs that had robust interblastomere differences both within and between embryos (hits). The hits of each study were compared and contrasted with the hits of the other studies using Venn diagrams. The hits shared by at least four of five studies were analysed further by bioinformatics. PARTICIPANTS/MATERIALS, SETTING, METHODS: PubMed was systematically examined for mRNA expression profiles of single 2-cell stage blastomeres in addition to publicly available microarray datasets (GEO, ArrayExpress). Based on the original normalizations, data from seven studies were screened for pairwise sample correlation at the gene level (Spearman), and the top five datasets with the highest correlation were subjected to hierarchical cluster analysis. Interblastomere differences of gene expression were expressed as a ratio of the higher to the lower mRNA level for each pair of blastomeres. A double cut-off was used to make the call of interblastomere difference, accepting genes with mRNA ratios above 2 when observed in at least 50% of the pairs, and discarding the other genes. The proportion of interblastomere differences common to at least four of the five datasets was calculated. Finally, the corresponding gene, pathway and enrichment analyses were performed utilizing PANTHER and GORILLA platforms. MAIN RESULTS AND THE ROLE OF CHANCE: An average of 17% of genes within the datasets are differently expressed between sister blastomeres, a proportion which falls to 1% when considering the differences that are common to at least four of the five studies. Housekeeping mRNAs were not included in the 17% and 1% gene lists, suggesting that the interblastomere differences do not occur simply by chance. The 1% of shared interblastomere differences comprise 100 genes, of which 35 are consistent with at least one of the four prevalent models of sister blastomere diversification. Bioinformatics analysis of the remaining 65 genes that are not consistent with the four models suggests that at least one more mechanism is at play, potentially related to the endomembrane system. Although there are many dimensions to the issue of reproducibility (biological, experimental, analytical), we consider that the sister blastomeres are poised to escape high interblastomere correlations of mRNA levels, because at least five sources of diversity superimpose on each other, accounting for at least 25 = 32 different states. As a result, interblastomere mRNA differences of a given 2-cell embryo are necessarily difficult to reproduce in another 2-cell embryo. LARGE SCALE DATA: Data were as provided by the original studies (GSE21688, GSE22182, GSE27396, GSE45719, GSE57249, E-MTAB-3321, GSE94050). LIMITATIONS, REASONS FOR CAUTION: The original studies present similarities (e.g. fertilization in vivo after ovarian stimulation) as well as differences (e.g. mouse strains, method and timing of blastomere separation). We identified robust mRNA differences between the sister blastomeres, but these differences are underestimated because our double cut-off method works with thresholds and affords more protection against false positives than false negatives. Regarding the false negatives, transcriptome analysis may have captured only part of the interblastomere differences due to: (1) the 2-fold cut-off not being sensitive enough to detect the remaining part of the interblastomere differences, (2) the detection limit of the transcriptomic methods not being sufficient, or (3) interblastomere differences being oblivious to transcriptomic identification because transcriptional changes are oscillatory or because differences are mediated non-transcriptionally or post-transcriptionally. Regarding the false positives, it seems unlikely that a difference was found just by chance for the same group of transcripts due to the same technical error, given that different laboratories produced the data. WIDER IMPLICATIONS OF THE FINDINGS: It is clear that the sister blastomeres are distinguishable from each other by mRNA analysis even at the 2-cell stage; however, efforts to identify large stable patterns may be in vain. This elicits thoughts about the wisdom of adding new transcriptomic datasets to the ones that already exist; if all transcriptomic datasets produced so far show a reproducibility of 1%, then any future study would probably face the same issue again. Possibly, a solid identification of the 'large stable pattern that should be there but was not found' requires an even larger dataset than the sum of the seven datasets considered here. Conversely, small stable patterns may be easier to identify, but their biological relevance is less obvious. Alternatively, interblastomere differences may not be mediated by nucleic acids but by other cellular components. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Deutsche Forschungsgemeinschaft (grant DFG BO 2540-4-3 to M.B. and grant NO 413/3-3 to V.N.). The authors declare that they have no competing financial interests.