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PURPOSE: We aimed to evaluate the link between the GDF9 concentration in day 3 human embryo culture medium and embryo quality and viability. METHODS: Two independent, prospective, observational studies were conducted. In study 1, a total of 280 embryos from 70 patients who obtained at least 4 embryos with 6–10 blastomeres (2 transferable and 2 non-transferable embryos) at day 3 were enrolled. In study 2, a total of 119 embryos from 61 patients (29 fully implanted and 32 non-implanted patients) were enrolled. The corresponding GDF9 concentrations in spent culture medium of embryos were quantified by ELISA assay. The expression pattern of GDF9 in human embryos was investigated using Q-PCR and immunofluorescence. RESULTS: GDF9 mRNA and protein were detected from human oocytes to eight-cell embryos and displayed a slow decreasing trend. In study 1, GDF9 concentration in culture medium is lower for transferable embryos compared with non-transferable embryos (331 pg/mL (quartiles: 442, 664 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P

PURPOSE: This study was conducted to verify if the cfDNA integrity (cfDI) in follicular fluid and subsequent spent embryo medium (SEM) could serve as potential non-invasive biomarker for high-grade embryo selection during IVF/ICSI. METHODS: Thirty-two follicular fluids, 32 subsequent corresponding cleavage embryo SEM, and 23 subsequent blastocyst SEM were collected from 11 patients undergoing IVF/ICSI. CfDI was measured by ALU gene amplicons with different sizes by qPCR, as the ratio of long to short fragments. RESULTS: CfDI in follicular fluid corresponding to subsequent high-grade cleavage embryos and blastocysts was significantly lower than that related to low-grade embryos (p = 0.018). Conversely, cfDI in SEM was significantly and positively correlated with high-grade embryos at both stages (p = 0.009). ROC curves of the analysis of cfDI in follicular fluid showed great potential in predicting subsequent embryogenesis and embryo grade (AUC > 0.927). Regardless of the cleavage embryo grade by morphology, cfDI in day 3 SEM could predict if the cleavage embryo could develop to a high-grade blastocyst (AUC = 0.820). A concordant shift pattern of cfDI from follicular fluid to subsequent day 3 SEM and day 5 SEM was found in 81.82% participants featured by various clinical characteristics. CONCLUSION: CfDI in follicular fluid and SEM was significantly correlated with embryogenesis and embryo grade and could serve as a potential non-invasive biomarker in high-grade embryo selection. Direct qPCR was proved as a labor-saving and sensitive method for the analysis of cfDI in low volume of SEM. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02357-0.

OBJECTIVES: Using time-lapse microscopy, previous research has shown that IVF mouse embryos that cleave earlier at the first division (‘fast’) develop into blastocysts with increased glucose consumption and lower likelihood of post-implantation loss as compared to slower cleaving embryos (‘slow’). Further, metabolomics analysis employing LC–MS conducted on groups of ‘fast’ blastocysts revealed that more aspartate was consumed. With the worldwide adoption of single blastocyst transfer as the standard of care, the need for quantifiable biomarkers of viability, such as metabolism of specific nutrients, would greatly assist in embryo selection for transfer. METHODS: Here we describe the development of a targeted enzymatic assay to quantitate aspartate uptake of single blastocysts. RESULTS: Results demonstrate that the rates of aspartate and glucose consumption were significantly higher in individual ‘fast’ blastocysts. Blastocysts, together with placental and fetal liver tissue collected following transfer, were analysed for the expression of genes involved in aspartate and carbohydrate metabolism. In ‘fast’ blastocysts, expressions of B3gnt5, Slc2a1, Slc2a3, Got1 and Pkm2 were found to be significantly higher. In placental tissue derived from ‘fast’ blastocysts, expression of Slc2a1, Got1 and Pkm2 were significantly higher, while levels of Got1 and Pkm2 were lower in fetal liver tissue compared to tissue from ‘slow’ blastocysts. CONCLUSIONS: Importantly, this study shows that genes regulating aspartate and glucose metabolism were increased in blastocysts that have higher viability, with differences maintained in resultant placentae and fetuses. Consequently, the analysis of aspartate uptake in combination with glucose represents biomarkers of development and may improve embryo selection efficacy and pregnancy rates.

PURPOSE: The few studies that examined the effect of male and/or female features on early embryo development, notably using the time-lapse system (TL), reported conflicting results. This can be explained by the small number of studies using an adapted model. METHODS: We used two original designs to study the female and male effects on embryo development: (1) based on embryos from donor oocytes (TL-DO), and (2) from donor sperm (TL-DS). Firstly, we analyzed the female and male similarities using an ad hoc intraclass correlation coefficient (ICC), then we completed the analysis with a multivariable model to assess the association between both male and female factors, and early embryo kinetics. A total of 572 mature oocytes (TL-DO: 293; TL-DS: 279), fertilized by intracytoplasmic sperm injection (ICSI) and incubated in a TL (Embryoscope®) were included from March 2013 to April 2019; 429 fertilized oocytes (TL-DO: 212; TL-DS: 217) were assessed. The timings of the first 48 h have been analyzed. RESULTS: The similarities in the timings thought to be related to the female component were significant: (ICC in both DO-DS designs respectively: tPB2: 9–18%; tPNa: 16–21%; tPNf: 40–26%; t2: 38–24%; t3: 15–20%; t4: 21–32%). Comparatively, those related to male were lower. Surprisingly after multivariable analyses, no intrinsic female factors were clearly identified. However, in TL-DO design, oligozoospermia was associated with a tendency to longer timings, notably for tPB2 (p = 0.026). CONCLUSION: This study quantifies the role of the oocyte in the first embryo cleavages, but without identified specific female factors. However, it also highlights that sperm may have an early embryonic effect. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02336-5.

Purpose To evaluate whether ionophore application at the oocyte stage changes the morphokinetics of the associated embryos in cases of artificial oocyte activation. Methods In a prospective sibling oocyte approach, 78 ICSI patients with suspected fertilization problems had half of their MII-oocytes treated with a ready-to-use ionophore (calcimycin) immediately following ICSI (study group). Untreated ICSI eggs served as the control group. Primary analyses focused on morphokinetic behavior and the presence of irregular cleavages. The rates of fertilization, utilization, pregnancy, and live birth rate were also evaluated. Results Ionophore-treated oocytes showed a significantly earlier formation of pronuclei (t2PNa) and a better synchronized third cell cycle (s3) (P P > .05). Ionophore treatment significantly improved the overall rates of fertilization (P P Conclusion Ionophore application does not negatively affect cleavage timing nor is it associated with irregular cleavage.

PURPOSE: During fertilisation, female and male pronuclei (PNs) migrate to the centre of the ooplasm, juxtapose, and break down synchronously in preparation for the first mitosis. While PN non-juxtaposition and PN breakdown (PNBD) asynchrony are occasionally observed, their developmental implications remain uncertain. This study investigated the possible relationships among the two phenomena, preimplantation development patterns, and live birth rates in single blastocyst transfers. METHODS: A total of 1455 fertilised oocytes cultured in a time-lapse incubator were retrospectively analysed. Fertilised oocytes were divided into four groups according to the presence of PN juxtaposition and breakdown synchrony. The relationships of abnormal PN behaviour with embryo morphokinetics, blastocyst formation, and live birth were evaluated. RESULTS: PN non-juxtaposition and asynchrony were observed in 1.9% and 1.0% of fertilised oocytes, respectively. The blastocyst cryopreservation rates in the synchronous–non-juxtaposed and asynchronous–non-juxtaposed groups were significantly lower than that in the synchronous–juxtaposed group. The rates of clinical pregnancy, ongoing pregnancy, and live birth were comparable among the groups. Non-juxtaposition was significantly associated with increased trichotomous cleavage at the first cytokinesis (P < 0.0001) and an increase in the time interval from PNBD to first cleavage (P < 0.0001). Furthermore, asynchronous PNBD was significantly correlated with increased rapid cleavage at the first cytokinesis (P = 0.0100). CONCLUSION: Non-juxtaposition and asynchronous PNBD is associated with abnormal mitosis at the first cleavage and impaired preimplantation development. However, embryos displaying abnormal PNBD may develop to blastocyst stage and produce live births, suggesting blastocyst transfer as a more appropriate culture strategy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02335-6.

PURPOSE: To investigate whether inhibition of LINE-1 affects telomere reprogramming during 2-cell embryo development. METHODS: Mouse zygotes were cultured with or without 1 µM azidothymidine (AZT) for up to 15 h (early 2-cell, G1/S) or 24 h (late 2-cell, S/G2). Gene expression and DNA copy number were determined by RT-qPCR and qPCR respectively. Immunostaining and telomeric PNA-FISH were performed for co-localization between telomeres and ZSCAN4 or LINE-1-Orf1p. RESULTS: LINE-1 copy number was remarkably reduced in later 2-cell embryos by exposure to 1 µM AZT, and telomere lengths in late 2-cell embryos with AZT were significantly shorter compared to control embryos (P = 0.0002). Additionally, in the absence of LINE-1 inhibition, Dux, Zscan4, and LINE-1 were highly transcribed in early 2-cell embryos, as compared to late 2-cell embryos (P

Embryo selection within in vitro fertilization (IVF) is the process of evaluating qualities of fertilized oocytes (embryos) and selecting the best embryo(s) available within a patient cohort for subsequent transfer or cryopreservation. In recent years, artificial intelligence (AI) has been used extensively to improve and automate the embryo ranking and selection procedure by extracting relevant information from embryo microscopy images. The AI models are evaluated based on their ability to identify the embryo(s) with the highest chance(s) of achieving a successful pregnancy. Whether such evaluations should be based on ranking performance or pregnancy prediction, however, seems to divide studies. As such, a variety of performance metrics are reported, and comparisons between studies are often made on different outcomes and data foundations. Moreover, superiority of AI methods over manual human evaluation is often claimed based on retrospective data, without any mentions of potential bias. In this paper, we provide a technical view on some of the major topics that divide how current AI models are trained, evaluated and compared. We explain and discuss the most common evaluation metrics and relate them to the two separate evaluation objectives, ranking and prediction. We also discuss when and how to compare AI models across studies and explain in detail how a selection bias is inevitable when comparing AI models against current embryo selection practice in retrospective cohort studies.

PURPOSE: To determine if euploidy rates and embryo development differ when blastocysts are cultured in CCM or SCM. METHOD: A single-center retrospective observational study was performed from September 2018 to March 2019. Patients [23–46 years] with at least four fresh mature oocytes (MII) without severe male factor infertility were included. Sibling MII were injected and cultured in Global®Total®LP (CCM) or Sage Quinn’s Advantage® Cleavage and Blastocyst media (SCM) under 6% CO(2), 5% O(2), and 89% N(2). Fertilization, cleavage, day (D) 5 blastulation, usable blastocyst (blastocysts biopsied/normally fertilized oocytes), and euploidy rates were recorded. Blastocysts were graded prior to trophectoderm (TE) biopsy on D5, 6, or 7 for genetic testing and mitochondrial DNA (mtDNA) quantification. RESULTS: According to clinical practice, 1452 MII were randomly distributed: 751 in CCM and 701 in SCM. No differences were observed in fertilization and cleavages rates for CCM and SCM (77.4% vs 75.5%, p = 0.429 and 97.6% vs 99.1%, p = 0.094, respectively). Blastulation rate on D5 was higher in CCM (70.6% vs 62.2, p = 0.009); however, usable blastocyst rates were comparable (CCM: 58.3% vs SCM: 56.7%, p = 0.625). From a Poisson regression model adjusted for confounding factors, euploidy rates were not different between media (aOR = 1.18, [0.94–1.48], p = 0.157). Euploid blastocyst’s mtDNA values were similar (CCM: 32.2, [30.5, 34.1] and SCM: 33.5, [31.8, 35.2], p = 0.345) and top-quality blastocysts (AA/BA) were increased in SCM (OR=1.04, [1.00–1.09], p = 0.037). CONCLUSION: Under controlled in vitro conditions, euploidy rates and embryo development are comparable when embryos are cultured in CCM or SCM. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02187-0.

PURPOSE: This study aims to know what proportion of culture day 5 pre-blastocyst-stage embryos develop into blastocysts by culture day 6 and what patient and cycle characteristics are associated with delayed blastocyst formation. METHODS: A retrospective observational cohort analysis was performed including a total of 9886 embryos from 1008 IVF cycles in 835 patients, who underwent treatment between January 1, 2016, and December 31, 2018. Autologous fresh in vitro fertilization (IVF) cycles at a single academic center were included in the analysis. Embryos were group-cultured using single-step culture media. Blastulation was defined as the presence of a new blastocyst. Usable blastulation was defined as the presence of a new good or excellent quality, expanded, hatching, or hatched blastocysts. RESULTS: The mean blastulation rate between days 5 and 6 of extended embryo culture was 30.9%. The mean percentage of embryos developing into usable blastocyst-stage embryos was 19.8%. The factors associated with blastulation on day 6 included the total number of embryos and the number of pre-blastocysts on day 5, as well as the use of ICSI. Age, the number of total embryos, those remained in culture and pre-blastocysts, as well as the blastulation rate on day 5 were associated with usable blastulation. CONCLUSION: It is important to know the usable blastocyst development rate between culture days 5 and 6 in order to adequately counsel patients debating whether to proceed with fresh ET on day 5 or forego ET with the expectation that embryos will be biopsied for PGT and/or cryopreserved on culture day 6. Our findings provide evidence to help guide patients in this difficult decision.

PURPOSE: To study whether the application of femtosecond laser pulses for zona pellucida (ZP) drilling of blastocysts at the embryonic or abembryonic poles can promote hatching to start immediately through the hole formed and ensure high hatching rates and embryo viability. METHODS: Mouse blastocyst (E3.5) ZP were microdissected with femtosecond laser pulses (514-nm wavelength, 280-fs pulse duration, 2.5-kHz repetition rate) close to the trophoblast or inner cell mass (ICM). The sizes of the holes formed were in the range of 4.5–8.5 μm. Additional longitudinal incisions (5–7-μm long) on either side of the hole were created to determine whether hatching had started at the correct position. Embryos post-laser-assisted ZP drilling and intact embryos were cultured under standard conditions for 2 days; embryo quality was assessed twice daily. The hatching rates and in vitro and in vivo implantation rates (only for embryos with ZP dissected close to the ICM) were estimated. RESULTS: Femtosecond laser–assisted ZP drilling at the early blastocyst stage facilitated embryo hatching to start at the artificial opening with probability approaching 100%. Despite the artificial opening’s small size, no embryo trapping during hatching was observed. Both experimental groups had higher hatching rates than the control groups (93.3–94.7% vs. 83.3–85.7%, respectively). The in vitro implantation rate was comparable with that of the control group (92.3% vs. 95.4%). No statistically significant differences were obtained in the in vivo implantation rates between the experimental and control groups. CONCLUSIONS: Blastocyst-stage femtosecond laser microsurgery of ZP is fast and delicate and enables the hatching process to be initiated in a controlled manner through a relatively small opening, with no embryo trapping.

PURPOSE: Reduction in methylenetetrahydrofolate reductase (MTHFR) activity due to genetic variations in the MTHFR gene has been controversially implicated in subfertility in human in vitro fertilization. However, there is no direct gene-knockdown study of embryonic MTHFR to assess its involvement in mammalian preimplantation development. The purpose of this study is to investigate expression profiles and functional roles of MTHFR in bovine preimplantation development. METHODS: Reverse transcription-quantitative PCR (RT-qPCR) and analysis of publicly available RNA-seq data were performed to reveal expression levels of MTHFR during bovine preimplantation development. We knocked down MTHFR by siRNA-mediated RNA interference from the 8- to 16-cell stage and assessed the effects on preimplantation development. RESULTS: The RT-qPCR analysis showed relatively high MTHFR expression at the GV oocyte stage, which was decreased toward the 8- to 16-cell stage and then slightly restored at the blastocyst stage. Public data–based analysis also showed the similar pattern of expression with substantial embryonic expression at the blastocyst stage. MTHFR knockdown reduced the blastocyst rate (P

PURPOSE: This study used noninvasive, fluorescence lifetime imaging microscopy (FLIM)-based imaging of NADH and FAD to characterize the metabolic response of mouse embryos to short-term oxygen deprivation. We investigated the response to hypoxia at various preimplantation stages. METHODS: Mouse oocytes and embryos were exposed to transient hypoxia by dropping the oxygen concentration in media from 5–0% over the course of ~1.5 h, then 5% O(2) was restored. During this time, FLIM-based metabolic imaging measurements of oocyte/embryo cohorts were taken every 3 minutes. Experiments were performed in triplicate for oocytes and embryos at the 1- to 8-cell, morula, and blastocyst stages. Maximum hypoxia response for each of eight measured quantitative FLIM parameters was taken from the time points immediately before oxygen restoration. RESULTS: Metabolic profiles showed significant changes in response to hypoxia for all stages of embryo development. The response of the eight measured FLIM parameters to hypoxia was highly stage-dependent. Of the eight FLIM parameters measured, NADH and FAD intensity showed the most dramatic metabolic responses in early developmental stages. At later stages, however, other parameters, such as NADH fraction engaged and FAD lifetimes, showed greater changes. Metabolic parameter values generally returned to baseline with the restoration of 5% oxygen. CONCLUSIONS: Quantitative FLIM-based metabolic imaging was highly sensitive to metabolic changes induced by hypoxia. Metabolic response profiles to oxygen deprivation were distinct at different stages, reflecting differences in metabolic plasticity as preimplantation embryos develop. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01872-w) contains supplementary material, which is available to authorized users.

PURPOSE: Embryo genotyping in IVF clinics aims to identify aneuploid embryos, and current methodologies rely on costly, invasive and time-consuming approaches such as PGT-A screening. MALDI-ToF-based mass spectral analysis of embryo culture has been demonstrated to be a non-invasive, affordable and accurate technique that is able to capture secretome profiles from embryo culture media extremely quick. Thus, aneuploid embryo genotypes can be distinguished from euploids from these profiles towards the development of novel embryo selection tools. METHODS: A retrospective cohort study, including 292 spent media samples from embryo cultures collected from a single IVF clinic in USA. There were 149 euploid and 165 aneuploid embryos previously analysed by PGT-A next-generation sequencing techniques. Secretome mass spectra of embryos were generated using MALDI-ToF mass spectrometry in the UK. Data was systematically analysed using a fully automated and ultra-fast bioinformatic pipeline developed for the identification of mass spectral signatures. RESULTS: Distinct spectral patterns were found for euploid and aneuploid genotypes in embryo culture media. We identified 12 characteristic peak signatures for euploid and 17 for aneuploid embryos. Data analysis also revealed a high degree of complementarity among regions showing that 22 regions are required to differentiate between genotypes with a sensitivity of 84% and a false positive rate of 18%. CONCLUSION: Ultra-fast and fully automated screening of an embryo genotype is possible based on multiple combinations of specific mass spectral peak signatures. This constitutes a breakthrough towards the implementation of non-invasive and ultra-fast tools for embryo selection immediately prior to transfer.

PURPOSE: The aim of the study is to investigate presence and role of the gene encoding the maternally contributed nucleotide-binding oligomerization domain (NOD)-like receptors with a pyrin domain (PYD)-containing protein 9 (NLRP9) in human and mouse ovaries, respectively, and in preimplantation mouse embryo development by knocking down Nlrp9b.METHODS: Expression levels of NLRP9 mRNA in human follicles were extracted from RNA sequencing data from previous studies. In this study, we performed a qPCR analysis of Nlpr9b mRNA in mouse oocytes and found it present. Intracellular ovarian distribution of NLRP9B protein was accomplished using immunohistochemistry. The distribution of NLRP9B was explored using a reporter gene approach, fusing NLRP9B to green fluorescent protein and microinjection of in vitro-generated mRNA. Nlrp9b mRNA function was knocked down by microinjection of short interference (si) RNA targeting Nlrp9b, into mouse pronuclear zygotes. Knockdown of the Nlrp9b mRNA transcript was confirmed by qPCR.RESULT: We found that the human NLRP9 gene and its corresponding protein are highly expressed in human primordial and primary follicles. The NLRP9B protein is localized to the cytoplasm in the blastomeres of a 2-cell embryo in mice. SiRNA-mediated knockdown of Nlrp9b caused rapid elimination of endogenous Nlrp9b mRNA and premature embryo arrest at the 2- to 4-cell stages compared with that of the siRNA-scrambled control group.CONCLUSIONS: These results suggest that mouse Nlrp9b, as a maternal effect gene, could contribute to mouse preimplantation embryo development. It remains to investigate whether NLRP9 have a crucial role in human preimplantation embryo and infertility.

PURPOSE: Metformin is the most commonly prescribed drug in the management of metabolic disorders such as polycystic ovarian syndrome (PCOS) and gestational diabetes in women of reproductive age. Insulin-sensitizing effect of metformin helps in improving from PCOS features such as hyperandrogenism, anovulation, and infertility. However, its ability to cross placental barrier raises concern about safety of the drug on early embryonic development. In this study, we evaluated the effect of metformin on the ovarian function and embryo development. METHODS: Adult Swiss albino female mice were administered with metformin (0, 50, 100, and 200 mg/kg body weight) for 4 weeks and assessed for reproductive function and preimplantation embryo development. Further, effect of metformin (0, 10, 25, 50, 100, 250, and 500 μg/mL) exposure to 2-cell-stage embryos was tested under in vitro conditions. RESULTS: Metformin did not alter the body weight, blood glucose, ovarian weight, and follicular reserve. However, the early embryo development was significantly affected in mice treated with metformin in vivo at highest dose. Moreover, embryos which were exposed to metformin in vitro showed dose-dependent decline in blastocyst rate and hatching rate. Furthermore, at highest concentration of metformin (500 μg/mL), all the embryos were arrested at compaction stage. CONCLUSION: The study revealed that metformin affects the early embryonic development and raises concern about its use during conception. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01709-6) contains supplementary material, which is available to authorized users.

PURPOSE: The aim of this study was to compare the levels of hyperglycosylated human chorionic gonadotropin (hCG-H) secreted from balanced and unbalanced human embryos. METHODS: Single-step culture media samples from 155 good quality embryos, derived from 90 good prognosis patients undergoing intracytoplasmic sperm injection (ICSI), were collected on the fifth day of embryo cultivation. All embryos were tested by next-generation sequencing (NGS) technique. The hCG-H levels in the culture media were evaluated by ELISA kit (Cusabio Biotech, CBS-E15803h) according to the manufacturer’s instructions. Statistical analysis was performed using SPSS v.21 (IBM Corp., Armonk, NY, USA). RESULTS: The NGS analysis revealed that 36% of the embryos (n = 56) were balanced, and 64% of the embryos were unbalanced (n = 99). The presence of hCG-H was confirmed in all embryo culture media samples but was absent in the negative control. In addition, hCG-H concentration was significantly higher in the culture media from unbalanced embryos compared with the balanced ones (0.72 ± 0.30 mIU/ml vs. 0.62 ± 0.12 mIU/ml, p = 0.02, respectively). Furthermore, the mean levels of hCG-H were significantly increased in the samples from embryos with multiple abnormalities. Finally, the highest levels of hCG-H were expressed from embryos with monosomy of chromosome 11 (1.28 ± 0.04 mIU/ml) and those with trisomies of chromosomes 21 (2.23 mIU/ml) and 4 (1.02 ± 0.35 mIU/ml). CONCLUSION: Our results suggest that chromosomal aberrations in human embryos are associated with an increased secretion of hCG-H. However, hCG-H concentration in embryo culture media as a single biomarker is not sufficient for an accurate selection of balanced embryos.

PURPOSE: The aim of this study was to establish a new method of decreasing cytoplasmic fragmentation in early-stage human embryos. METHODS: The zona pellucida (ZP) of abnormally-fertilized oocytes (zygotes with three pronuclei (3PN)), which were donated by patients, was removed at the pronuclear stage. ZP-free embryos were observed in a time-lapse imaging and culturing system in order to examine developmental morphology and embryonic quality. RESULTS: Based on a modification of Veeck’s criteria, 47 of 69 ZP-free 3PN embryos (68.1%) showed fragmentation of less than 20% of the total volume of cytoplasm at the first cleavage (grades 1 and 2), 17 (24.6%) showed 20–40% cytoplasmic fragments (grade 3), and only 5 (7.2%) showed more than 40% fragments (grade 4). These results suggest that the rate of fragmentation is decreased by ZP removal before the first cleavage, compared with normal (ZP-intact) 3PN and 2-pronuclear/2-polar body embryos. CONCLUSIONS: This study revealed that the ZP is not always necessary for normal development after the pronuclear stage because the ZP-free embryos studied herein developed normally, maintained their cell adhesion well, and showed a decreased rate of fragmentation. This innovative culture system might provide the major breakthrough needed for patients who have difficulty obtaining good-quality embryos.

PURPOSE: DNA methylation is one of the epigenetic mechanisms that plays critical roles in preimplantation embryo development executed by DNA methyltransferase (Dnmt) enzymes. Dnmt1, responsible for the maintenance of methylation, and Dnmt3a, for de novo methylation, are gradually erased from the zygote in succeeding stages and then reestablished in the blastocyst. This study was designed to address the vital role of Dnmt1 and Dnmt3a enzymes by silencing their gene expressions in embryonic development in mice. METHODS: Groups were (i) control, (ii) Dnmt1-siRNA, (iii) Dnmt3a-siRNA, and (iv) non-targeted (NT) siRNA. Knockdown of Dnmt genes using siRNAs was confirmed by measuring the targeted proteins using Western blot and immunofluorescence. Following knockdown of Dnmt1 and Dnmt3a in zygotes, the developmental competence and global DNA methylation levels were analyzed after 96 h in embryo cultures. RESULTS: A significant number of embryos arrested at the 2-cell stage or had undergone degeneration in the Dnmt1 and Dnmt3a knocked-down groups. By 3D observations in super-resolution microscopy, we noted that Dnmt1 was exclusively found in juxtanuclear cytoplasm, while the Dnmt3a signal was preferentially localized in the nucleus, both in trophoblasts (TBs) and embryoblasts (EBs). Interestingly, the global DNA methylation level decreased in the Dnmt1 knockdown group, while it increased in the Dnmt3a knockdown group. CONCLUSION: Precisely aligned expression of Dnmt genes is highly essential for the fate of an embryo in the early developmental period. Our data indicates that further analysis is mandatory to designate the specific targets of these methylation/demethylation processes in mouse and human preimplantation embryos.

PURPOSE: Studies on rodents have shown that assisted reproductive technologies (ARTs) are associated with perturbation of genomic imprinting in blastocyst-stage embryos. However, the vulnerable developmental window for ART influence on the genomic imprinting of embryos is still undetermined. The purpose of this study was to establish the specific embryonic development stage at which the loss of methylation of H19 imprinting control regions (ICRs) was caused by ART occurrence. Additionally, we explored protocols to safeguard against possible negative impacts of ART on embryo H19 imprinting. METHODS: Mouse embryos were generated under four different experimental conditions, divided into four groups: control, in vitro culture (IVC), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). The methylation levels of H19 ICR of the grouped or individual embryos were analyzed by bisulfite-sequencing PCR. RESULTS: Our data showed that the loss of methylation of H19 ICR in mouse blastocysts was inflicted to a similar extent by IVC, IVF, and ICSI. Specifically, we observed a significant loss of methylation of H19 ICR between the mouse 8-cell and morula stages. In addition, we revealed that the transfer of mouse embryos generated by ARTs in the uterus at the 8-cell stage induced the occurrence of methylation patterns in the blastocysts closer to the in vivo ones. CONCLUSIONS: Our findings indicate that the loss of methylation of H19 ICR caused by ARTs occurs between the 8-cell and the morula stages, and the transfer of cleavage embryos to the uterus mitigates the loss methylation of H19 derived by mice ARTs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-019-01575-x) contains supplementary material, which is available to authorized users.

Purpose Selecting an embryo at the transfer stage with the best chance of a successful pregnancy is still largely dependent on preceding subjective evaluation of morphokinetics. Expensive prenatal genomic profiling has been so far proved ineffective. Proteomics and metabolomics are promising new approaches to assess embryo viability, but methodologies are often complex and do not lend themselves to rapid analysis in the critical time between blastocyst formation and embryo transfer. Here, we used matrix-assisted laser desorption ionization time-of-flight (MALDI ToF) mass spectrometry to assess the secretome of blastocysts in the minutes prior to embryo transfer and correlated spectral features with pregnancy outcome. Methods Four hundred one samples of spent blastocyst culture media were collected from embryo cultures at the time of embryo transfer, of which 136 were used to construct the predictive model. The media samples were frozen at − 20 °C and stored for analysis. Sample analysis was conducted in batches using 1 μl of spent embryo in direct MALDI ToF mass spectral analysis. Quantitative characteristics within this mass range (2000–17,000 m/z) were used to generate a score for selected mass regions (bins) in order to predict pregnancy outcome for each sample. Results With a simple algorithm based on nine mass bins within the 2000–10,000 m/z region, it was possible to identify samples with the best chance of becoming an ongoing pregnancy (positive predictive value of 82.9%, p = 0.0018). Conclusion A simple, direct and rapid analysis of spent culture fluid from blastocysts at the point of embryo transfer can quickly identify optimal embryos with the best chance of achieving ongoing pregnancy. Methods like this, which take less than 20 min to perform, could dramatically improve the approach to embryo selection and live births.

Propose The presence of metaphase II (MII) spindle together with the polar body (PB) indicates completion of oocyte maturation. This study was designed to explore if spindle imaging can be used to optimize timing of intracytoplasmic sperm injection (ICSI). Methods The study involved 916 oocytes from 234 conventionally stimulated ICSI cycles with an unexpectedly poor ovarian response. All PB-displaying oocytes were subjected to polarized light microscopy (PLM) prior to ICSI. When MII spindle was absent in the majority of oocytes, ICSI was postponed and performed after additional spindle imaging. Fertilization, embryo development, and clinical outcome were evaluated with respect to the observed spindle pattern. Results The visible spindle was absent in 32.64% of PB-displaying oocytes. The late-maturing oocytes extruding PB in vitro were less likely to exhibit a spindle signal than in vivo matured MII oocytes (38.86% vs. 89.84%). When fertilization was postponed, 59.39% of initially spindle-negative oocytes developed detectable MII spindle. Spindled eggs had significantly higher developmental potential, and the presence of the spindle has been identified as an independent measure for predicting the formation of the blastocyst. Embryos derived from spindle-positive oocytes also showed a higher chance to implant and develop to term. Notably, 11 children were conceived by finely timed fertilization of late-maturing oocytes which are normally discarded. Conclusions The study confirms the prognostic value of spindle imaging and demonstrates that immature oocytes can be clinically utilized and give rise to live births when the timing of ICSI is adjusted to their developmental stage. Electronic supplementary material The online version of this article (10.1007/s10815-018-1393-0) contains supplementary material, which is available to authorized users.

PURPOSE: To study the relationship between the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN) and human embryo development during assisted reproduction. METHODS: The migration speed of 263 NPBs from 47 zygotes was quantitated, and embryonic development was observed until the blastocyst stage. The central coordinates of mPN, fPN, and NPBs were noted at multiple timepoints. Then, the distance traveled by the NPBs between two sequential images was measured, and migration speed was calculated. Additionally, we investigated the relationship between NPB migration speed and ploidy status (N = 33) or live birth/ongoing pregnancy (LB/OP) (N = 60) after assisted reproduction. RESULTS: The NPB migration speed in both mPN and fPN was significantly faster in the zygotes that developed into blastocysts (N = 25) than that in the zygotes that arrested (N = 22). The timing of blastulation was negatively correlated with NPB migration speed in the mPN. Faster NPB migration was significantly correlated with LB/OP. In multivariate logistic analysis, NPB migration speed in the mPN was the only morphokinetic parameter associated with LB/OP. In a receiver-operating characteristic curve analysis of LB/OP by the NPB migration speed in the mPN, the cut-off value was 4.56 μm/h. When this cut-off value was applied to blastocysts with preimplantation genetic testing for aneuploidy, 100% of the blastocysts faster than or equal to the cut-off value were euploid. CONCLUSION: The NPBs migrated faster in zygotes having the potential to develop into a blastocyst, and eventually into a baby. This predictor could be an attractive marker for non-invasive embryo selection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02172-7.

PURPOSE: The aim of this study was to analyze the metabolic profiles of blastocoel fluid (BF) obtained from bovine embryos produced in vivo and in vitro. METHODS: Expanded blastocysts (20/group) that were in vitro and in vivo derived at day 7 were used. BF was collected and analyzed under direct infusion conditions using a microTOF-Q(®) mass spectrometer with electrospray ionization and a mass range of 50–650 m/z. RESULTS: The spectrometry showed an evident difference in the metabolic profiles of BF from in vivo and in vitro produced embryos. These differences were very consistent between the samples of each group suggesting that embryo fluids can be used to identify the origin of the embryo. Ions 453.15 m/z, 437.18 m/z, and 398.06 m/z were identified as biomarkers for the embryo’s origin with 100% sensitivity and specificity. Although it was not possible to unveil the molecular identity of the differential ions, the resulting spectrometric profiles provide a phenotype capable of differentiating embryos and hence constitute a potential parameter for embryo selection. CONCLUSION: To the best of our knowledge, our results showed, for the first time, an evident difference between the spectrometric profiles of the BF from bovine embryos produced in vivo and in vitro.

PURPOSE: To study embryo morphokinetics in relation to release in spent media of molecules with possible roles in development and implantation (miR-20a, miR-30c, and sHLA-G). METHODS: Data were obtained from embryos generated in standard IVF and ICSI cycles. The Eeva system was used for embryo assessment, based on early morphokinetic parameters and producing a score (1–5, best–worst) corresponding to higher/medium/lower chances of development to blastocyst. miRNAs — mm miR-20a-5p and miR-30c-5p — and sHLA-G were quantified in 25 μl of spent blastocyst media (SBM) collected before vitrification or transfer. Statistical analyses were performed applying Kolmogorov-Smirnov, Shapiro-Wilk, and Spearman’s correlation coefficient tests, where appropriate. RESULTS: SBM were collected from a total of 172 viable blastocysts. Their analysis showed that concentration of miR-20a was progressively lower as Eeva score increased and probability of development to blastocyst decreased (P = 0.016). The opposite trend was observed in the case of miR-30c, i.e., concentration was higher as score increased and chances of development to blastocyst decreased (P = 0.004). Analysis of sHLA-G revealed a negative correlation with Eeva score, i.e., levels were progressively lower as Eeva score increased and probability of development to blastocyst decreased (R = − 0.388, N = 141, P = 0.001). CONCLUSION: Our data suggest that morphokinetic algorithms that predict development to blastocyst stage, in fact, also identify embryos with molecular and cellular profiles more consistent with developmental functions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02162-9.

PURPOSE: The goal is to determine if variations exist between male and female blastocysts in preimplantation measurements of quality and ploidy and in vitro fertilization elective single-embryo transfer (eSET) outcomes. METHODS: A retrospective chart review was conducted from a private fertility center’s database of blastocysts undergoing preimplantation genetic testing for aneuploidy, along with details of eSET from this screened cohort. Main outcomes included preimplantation embryo quality and sex-specific eSET outcomes. RESULTS: A total of 3708 embryos from 578 women were evaluated, with 45.9% male and 54.1% female. The majority were High grade. No difference existed between embryo sex and overall morphological grade, inner cell mass or trophectoderm grade, or blastocyst transformation day. Female blastocysts had a higher aneuploidy rate than male blastocysts (P < 0.001). Five hundred thirty-nine eSETs from 392 women were evaluated, with High grade embryos more likely to have implantation (P < 0.001), clinical pregnancy (P < 0.001), and ongoing pregnancy (P = 0.018) than Mid or Low grade embryos. Day 5 blastocysts were more likely to have implantation (P = 0.018), clinical pregnancy (P = 0.005), and ongoing pregnancy (P = 0.018) than day 6 blastocysts. Male and female embryos had similar transfer outcomes, although female day 5 blastocysts were more likely to result in clinical pregnancy (P = 0.012), but not ongoing pregnancy, than female day 6 blastocysts. Male eSET outcomes did not differ by blastocyst transformation day. CONCLUSION: Male and female embryos have comparable grade and quality; however, female embryos were more likely to be aneuploid. Ongoing pregnancy rates did not differ by embryo sex. Day 5 embryos had more favorable transfer outcomes than day 6 embryos.

PURPOSE: Different commercial human embryo culture mediums can alter embryo quality and change birthweight. One component that could be contributing to variations but is not widely investigated is human serum albumin (HSA). HSA plays a multitude of roles during embryo culture and is a carrier for molecules including lipids. It remains unclear if lipid composition of HSA varies among commercial products and its effects on embryo quality, implantation, and fetal outcomes are relatively unknown. METHODS: Utilizing a mouse model of embryo culture, we cultured zygotes until the blastocyst stage (72-h culture) in G1/G2 containing either Vitrolife HSA, Sage HSA, or Recombinant HSA at 10%. Blastocyst quality (development, total cell number, superoxide generation), blastocyst lipid content (neutral lipids, non-esterified fatty acids, phospholipids, and triglycerides), implantation, and fetal lengths and weights were assessed. Fatty acid quantification of HSA source was assessed by standard thin-layer chromatography. RESULTS: Sage HSA had the greatest fatty acid composition, with an eightfold increase in saturated fatty acids. This coincided with reduced blastocyst development, increased superoxide generation, neutral lipids and triglycerides levels of blastocysts, and decreased implantation rates (p < 0.05). Unexpectedly, while Recombinant HSA had the lowest overall lipids it had 70-fold increase in palmitoleic acid and the lowest fetal weights (p < 0.05). CONCLUSION: Indicates the importance of a balance between different types/amount of lipids, and an “optimal ratio” required for embryo and fetal development. Therefore, the lipid content of HSA should be considered when choosing a suitable HSA source for use in clinical IVF. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02255-5.

PURPOSE: Implantation is essential for a successful pregnancy. Despite the increasing number of studies, implantation is still an unknown process. This study aimed to determine whether sirtuin-1 has a role in embryo implantation in oxidative stress–induced mice. METHODS: Pregnant mice were separated into 5 groups: control, vehicle, paraquat, SRT1720, and SRT1720+Paraquat. Paraquat is a herbicide and is used to induce oxidative stress. SRT1720 is a specific sirtuin-1 activator. Implantation and inter-implantation sites were removed in the morning of the 5th day of pregnancy after Chicago blue injection was performed. Sirtuin-1 and Forkhead box O1 (FoxO1) were detected by immunohistochemistry and Western blot while acetylated lysine was evaluated by Western blot analysis. Reactive oxygen and nitrogen species (ROS/RNS) and superoxide dismutase (SOD) activity were determined by fluorometric and spectrometric methods, respectively. RESULTS: Although there was no embryo implantation in paraquat-treated mice, 5 out of 9 SRT1720+Paraquat-treated mice had implantation sites which were significantly higher compared to the paraquat-treated group. Sirtuin-1 and FoxO1 expressions were increased at implantation sites of SRT1720-treated mice. ROS/RNS levels were decreased, while deacetylated FoxO1 levels and SOD activity were increased in SRT1720-treated mice. CONCLUSION: Our findings suggest that sirtuin-1 may play a role in embryo implantation against oxidative stress through FoxO1-SOD signaling.

PURPOSE: To verify which genetic abnormalities prevent embryos to blastulate in a stage-specific time. METHODS: A single center retrospective study was performed between April 2016 and January 2017. Patients requiring Preimplantation Genetic Testing for Aneuploidies (PGT-A) by Next Generation Sequencing (NGS) were included. All embryos were cultured in a time-lapse imaging system and single blastomere biopsy was performed on day 3 of development. Segmental duplications and deletions as well as whole chromosome monosomies and trisomies were registered. Embryo arrest was defined if the embryo failed to blastulate 118 h post-injection. A logistic regression model was applied using the time to blastulate as the response variable and the different mutations as explanatory variables. A p value < 0.05 was considered significant. RESULTS: Of the 285 biopsied cleavage stage embryos, 103 (36.1%) were euploid, and 182 (63.9%) were aneuploid. There was a significant difference in the developmental arrest between euploid and aneuploid embryos (8.7% versus 42.9%; p = 0.0001). Segmental duplications and whole chromosome monosomies were found to have a significant effect on developmental arrest (p = 0.0163 and p = 0.0075), while trisomies and segmental deletions had no effect on developmental arrest. In case of segmental duplications, an increase of one extra segmental duplication increases the odd of arrest by 159%. For whole chromosome monosomies, the odd will only increase by 29% for every extra chromosomal monosomy. Both chromosomal abnormalities remained significant after adding age as an explanatory variable to the model (p = 0.014 and p = 0.009). CONCLUSION: Day 3 cleavage stage embryos with segmental duplications or monosomies have a significantly decreased chance to reach the blastocyst stage.

PURPOSE: This study aimed to determine the effects of drilling and thinning treatment of laser-assisted hatching on the expression and methylation of imprinted gene IGF2/H19 in embryos and offspring. METHODS: The prehatching blastocysts with treatment of drilling or thinning, or control prehatching blastocysts, were transplanted in surrogate uteri. The DNA methylation of IGF2/H19 imprinting control region (ICR) and the expression of IGF2 and H19 were respectively evaluated using bisulfite conversion-mediated sequencing and real-time PCR. RESULTS: The drilling group showed a significant increase in the development rate of hatched blastocysts in comparison with the control and thinning group. DNA methylation level of IGF2/H19 ICR of hatched blastocysts in the thinning group was 27.33% in comparison with the 38.67% and 36% observed in the control and drilling group. The thinning treatment increased the DNA methylation level of IGF2/H19 ICR in the placenta in comparison with the control and drilling group. The drilling and thinning treatment decreased the expression level of H19 mRNA in prehatching and hatched blastocysts as well as placenta, while a significant increase in the expression level of H19 mRNA of offspring was observed in the thinning group. The thinning treatment increased the expression level of IGF2 mRNA of prehatching blastocysts and offspring and a significant decrease in placenta, while the drilling treatment resulted in a significant increase in the expression level of IGF2 mRNA of hatched blastocysts and placenta. CONCLUSION: These observations suggested that drilling used for hatching of in vitro cultured mouse blastocysts may improve the production of offspring. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01975-4) contains supplementary material, which is available to authorized users.

PURPOSE: To provide a comprehensive analysis of mtDNA quantity in D5 and D6 blastocysts, as well as a further insight to the origin of delayed blastocyst development. METHODS: A retrospective cohort analysis of 829 D5 and 472 D6 blastocysts from 460 patients who underwent in vitro fertilization (IVF) with next-generation sequencing (NGS)–based preimplantation genetic testing for aneuploidy (PGT-A). The quantity of trophectoderm mtDNA was extrapolated from the NGS data, followed by the analysis of mean mtDNA levels between D5 and D6 blastocysts of the same ploidy (aneuploid/euploid) and transfer outcomes (positive/negative clinical pregnancy). RESULTS: D5 blastocysts had significantly higher euploidy rate and clinical pregnancy rate when compared with D6 blastocysts. The proportion of blastocysts derived from patients ≧ 40 years old were similar between the D5 and D6 cohorts. When blastocysts with identical ploidy were analyzed, the D5 cohorts all had significantly higher mean mtDNA levels than their D6 counterparts. Similarly, when embryo transfers with identical outcome were analyzed, the D5 cohorts also had significantly higher mean mtDNA levels than the D6 cohorts. Trophectoderm mtDNA level was independent of maternal age and blastocyst morphology grades. CONCLUSIONS: Our data provided further evidence D5 blastocysts contained significantly greater mtDNA quantity than D6 blastocysts, and mtDNA quantity could be a key factor that affects the development rate of blastocysts. Furthermore, one must avoid using an arbitrary threshold when incorporating mtDNA quantity into the embryo selection criteria, as the observed value may have vastly different clinical implication when blastulation rate is also considered.

PURPOSE: The fertility of women decreases with age because of factors such as an increased incidence of aneuploidies and—possibly—decreased mitochondrial activity in oocytes. However, the relationship between maternal aging and mitochondrial function of their embryos remains unknown. Here, we assessed the relationship between maternal age and mitochondrial functions in their oocytes and embryos METHODS: The relationships between maternal age and oxygen consumption rates (OCRs), mitochondrial DNA (mtDNA) copy numbers, or blastocyst development was investigated using 81 embryos donated from 63 infertility couples. The developmental rates from morulae to blastocysts were retrospectively analyzed using data of 105 patients. RESULTS: The OCRs of morulae decreased with maternal age (r(2) = 0.48, P < 0.05) although there were no relationships between maternal age and mtDNA copy number in any stages. The more oxygen consumed at the morula stage, the shorter time was required for embryo development to the mid-stage blastocyst (r(2) = 0.236, P < 0.05). According to the clinical data analysis, the developmental rate from morulae to blastocysts decreased with maternal age (P < 0.05, < 37 years, 81.1%, vs. ≥ 37 years, 64.1%). CONCLUSIONS: The data of the present study revealed that mitochondrial function at the morula stage of human embryos decreased with their maternal age and a decrease of mitochondrial function led to slow-paced development and impaired developmental rate from morulae to blastocysts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01869-5) contains supplementary material, which is available to authorized users.

OBJECTIVE: Assisted oocyte activation (AOA) can restore fertilization rates after IVF/ICSI cycles with fertilization failure. AOA is an experimental technique, and its downstream effects remain poorly characterized. Clarifying the relationship between AOA and embryo, morphokinetics could offer complementary insights into the quality and viability of the embryos obtained with this technique. The aim of this study is to compare the preimplantation morphokinetic development of embryos derived from ICSI-AOA (experimental group) vs. ICSI cycles (control group). METHODS: A retrospective cohort study was carried out with 141 embryos from fresh oocyte donation cycles performed between 2013 and 2017; 41 embryos were derived from 7 ICSI-AOA cycles and 100 embryos from 18 ICSI cycles. Morphokinetic development of all embryos was followed using a time-lapse system. RESULTS: We show that embryos from both groups develop similarly for most milestones, with the exception of the time of second polar body extrusion (tPB2) and the time to second cell division (t3). CONCLUSIONS: We conclude that ionomycin mediated AOA does not seem to affect the morphokinetic pattern of preimplantation embryo development, despite the alterations found in tPB2 and t3, which could directly reflect the use of a Ca(2+) ionophore as a transient and quick non-physiologic increase of free intracytoplasmic Ca(2+). SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-020-02025-9.

PURPOSE: Laevo (l)-carnitine plays important roles in reducing the cytotoxic effects of free fatty acids by forming acyl-carnitine and promoting beta-oxidation, leading to alleviation of cell damage. Recently, the mitochondrial functions in morula has been shown to decrease with the maternal age. Here, we assessed the effect of l-carnitine on mitochondrial function in human embryos and embryo development. METHODS: To examine the effect of l-carnitine on mitochondrial function in morulae, 38 vitrified–thawed embryos at the 6–11-cell stage on day 3 after ICSI were donated from 19 couples. Each couple donated two embryos. Two siblings from each couple were divided randomly into two groups and were cultured in medium with or without 1 mM l-carnitine. The oxygen consumption rates (OCRs) were measured at morula stage. The development of 1029 zygotes cultured in medium with or without l-carnitine was prospectively analyzed. RESULTS: Addition of l-carnitine to the culture medium significantly increased the OCRs of morulae and improved the morphologically-good blastocyst formation rate per zygote compared with sibling embryos. Twenty healthy babies were born from embryos cultured in l-carnitine-supplemented medium after single embryo transfers. CONCLUSION(S): l-carnitine is a promising culture medium supplement that might be able to counteract the decreased mitochondrial function in human morula stage embryos.

PURPOSE: The purpose was to investigate the association between embryonic development or implantation and the content of interleukin-6 and 10 (IL-6, IL-10) and tumor necrosis factor-α (TNF-α) in single-blastocyst conditioned medium (SBCM). METHODS: Thirty-eight SBCM samples (SBCMs) were collected from blastocysts with different morphological scores. IL-6, IL-10, and TNF-α concentration in 38 SBCMs was detected by Single Molecule Array and compared according to the blastocyst quality: top-quality (TQ) and non-top quality (NTQ), or blastulation time: day 5 (D5) and day 6 (D6). In another experiment, 61 SBCMs were collected from TQ blastocyst transplanted on D5, and IL-6 concentration in SBCM was compared based on whether embryos are implanted or not (implanted and non-implanted). RESULTS: In the first experiment, IL-6, IL-10, and TNF-α concentration was not significantly different between the TQ-SBCM and NTQ-SBCM. The D6-SBCM had a higher IL-6 concentration compared with the D5-SBCM, while IL-10 and TNF-α concentration was not significantly different between the D5-SBCM and D6-SBCM. The IL-6 concentration in D5-NTQ or D6-TQ SBCM was higher than that in D5-TQ or D6-NTQ SBCM (P < 0.05), respectively. Furthermore, the spearman analysis demonstrated that IL-6 concentration in SBCM was negatively correlated with the blastocyst quality on D5 and positively correlated with the blastocyst quality on D6. In the second experiment, no significant difference in IL-6 concentration was found between SBCM from implanted and non-implanted blastocyst. CONCLUSION: IL-6 concentration in SBCM was associated with embryo quality depending on the blastulation time, although it might not be associated with the blastocyst implantation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01805-7) contains supplementary material, which is available to authorized users.

Purpose: miRNAs have been suggested as biomarkers of embryo viability; however, findings from preliminary studies are divergent. Furthermore, the presence of other types of small RNA molecules remains to be investigated. The purpose of this study was to perform a comprehensive analysis of small non-coding RNA levels in spent and unconditioned embryo culture media, along with miRNA levels in blastocoelic fluid samples from human embryos. Methods: miRNAs in unconditioned culture medium from 3 different manufacturers, along with miRNA from day 5 conditioned culture medium, control medium, and corresponding blastocoel fluid from 10 human blastocysts were analyzed with array-based q-PCR analysis. Subsequently, deep sequencing of total and small RNA in day 5 spent culture medium from 5 human blastocysts and corresponding controls was performed. Results: In spite of using state-of-the-art sensitive detection methods, no miRNAs were found to be reliably present in the spent culture medium or the blastocoel fluid. Ct values were above the recommended limit for detection in the array-based analysis, a finding that was confirmed by deep sequencing. The majority of miRNAs identified by deep sequencing were expressed in all samples including control media and seem to originate from sources other than conditioned IVF media. Conclusions: Our findings question the use of miRNAs as a reliable biomarker and highlight the need for a critical methodological approach in miRNA studies. Interestingly, tiRNA fragments appear to be overexpressed in conditioned IVF media samples and could potentially be a novel biomarker worthy of investigation.

PURPOSE: To describe an interesting not previously described morphokinetic finding. METHODS: Retrospective case report of a couple undergoing controlled ovarian stimulation (COS) followed by in vitro fertilization and blastocyst transfer. RESULTS: We identified a unique finding of blastulation of a fertilized human zygote after conventional in vitro fertilization. The fertilized zygote did not show any clear cytokinesis until approximately 107 h post insemination, when it started dividing into a blastocyst. By 113 h post insemination, inner cell mass and trophectoderm cells could be clearly distinguished and the blastocyst was completely hatched by 136 h post insemination. CONCLUSION: Time-lapse systems offer more detailed observations of embryonic development. Here, we report an atypical development of an embryo that was not described previously. We hope to become an insightful discussion among peers and incentive the publication of such findings in the future. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01704-x) contains supplementary material, which is available to authorized users.

PURPOSE: To assess the role of the genetic background, the culture medium supplements, and the presence of modulators of signaling pathways on mouse embryonic stem cell derivation from single blastomeres from 8-cell embryos. METHODS: Mice from permissive and non-permissive genetic backgrounds, different culture media supplements, knockout serum replacement (KSR) and N2B27, and the presence or absence of 2i treatment were used to derive mouse embryonic stem cells (mESC) from single blastomeres isolated from 8-cell embryos and from control embryos at the blastocyst stage. After the sixth passage, the putative mESC were analyzed by immunofluorescence to assess their pluripotency and, after in vitro differentiation induction, their ability to differentiate into derivatives of the three primary germ layers. Selected mESC lines derived from single blastomeres in the most efficient culture conditions were further characterized to validate their stemness. RESULTS: In control embryos, high mESC derivation efficiencies (70–96.9%) were obtained from permissive backgrounds or when embryos were cultured in medium complemented with 2i regardless of their genetic background. By contrast, only blastomeres isolated from embryos from permissive background cultured in KSR-containing medium complemented with 2i were moderately successful in the derivation of mESC lines (22.9–24.5%). Moreover, we report for the first time that B6CBAF2 embryos behave as permissive in terms of mESC derivation. CONCLUSIONS: Single blastomeres have higher requirements than whole blastocysts for pluripotency maintenance and mESC derivation. The need for 2i suggests that modulation of signaling pathways to recreate a commitment towards inner cell mass could be essential to efficiently derive mESC from single blastomeres.

Purpose Reactive oxygen species (ROS) are thought to play a critical role in the success of IVF. The relationships between oxidative stress parameters in culture media and IVF outcomes have not been extensively investigated. The objective of this study is to examine the relationships between early human embryonic parameters and levels of ROS in culture media. Methods This prospective study was conducted with 2633 spent culture media collected from patients undergoing conventional IVF (n = 101) and intracytoplasmic sperm injection (ICSI) (n = 60). Both fertilization and early culture were performed in universal IVF medium and G series medium. ROS levels were measured by chemiluminescence assays using luminol as the probe on days 1, 3, and 5 and determined the relationships of ROS levels with zygote condition, embryo quality, and clinical pregnancy rate. Results ROS levels per embryo in culture media on the corresponding days 1, 3, and 5 showed significant correlations between each pair in the total cohort. Similar results were observed in the IVF and ICSI groups, but day 1 and day 3 ROS levels were significantly higher in culture media of IVF than of ICSI embryos. ROS levels in culture medium were not significantly associated with embryo quality, blastocyst formation, or arrest. ROS levels on day 1 were similar in media of normally fertilized zygotes, unfertilized oocytes, and polyspermic zygotes and were not associated with delayed embryonic development, high fragmentation, blastocyst formation, or arrest after prolonged culture. ROS levels in media were not associated with the likelihood of conception. Conclusions ROS levels in culture media may not be an effective indicator of embryo selection for IVF.

Purpose To study whether members of the miR-290-295 cluster in spent culture medium (SCM) of embryos are correlated with morphokinetics and apoptosis. Methods Cryopreserved 1-cell stage mouse embryos were cultured to the blastocyst stage, development was monitored by time-lapse, 59 SCM were collected, and miR-291a and miR-294 were detected with polymerase chain reaction (PCR). Blastocysts were immuno-stained for sexing (H2AK119ub) and for apoptosis (TUNEL). Each embryo and SCM were individually processed. Correlations were run between the miRNAs and developmental events (t2, t3, t4, t5, t8, tSB, tB, ECC2, ECC3, s2, s3, dB) and apoptosis (apoptotic cells/total cell number %). MiR-294 SCM and cell levels were compared in 40 blastocysts. Apoptosis was induced in 15 blastocysts with UV radiation and SCM samples were analyzed for miR-294. Results MiR-291a and miR-294 are released in variable levels by mouse blastocysts. Their release is similar between male and female embryos. No significant correlations were found between these miRNAs and development. MiR-294 was significantly positively correlated with apoptosis (r = 0.560, p p p Conclusion(s) MicroRNAs are important regulators of preimplantation development. Apoptosis triggers the release of miR-294 by blastocysts which possibly serves a secretory role for embryo-maternal communication. SCM miRNA analysis is possible for individually cultured embryos and future studies can investigate miRNAs as noninvasive markers of embryo quality.

PURPOSE: A microwell culture system that facilitates group culture, such as well-of-the-well (WOW), improves embryonic development in an individual culture. We examined the effect of WOW on embryonic development in vitro with commercially available human single culture media. METHODS: Using four different commercial human single culture media, in vitro development and imprinted gene expression of bovine embryos cultured in WOW were compared to droplet culture (one zygote per drop). To determine the effects of microwell and group culture on embryonic development, different numbers of embryos were cultured in droplet or WOW. Diffusion simulation of accumulating metabolites was conducted using the finite volume method. RESULTS: WOW had a positive effect on bovine embryonic development, regardless of the type of single culture media. Imprinted gene expression was not different between droplet- and WOW-derived blastocysts. The microwell and group cultures in WOW showed a significant positive effect on the rate of total blastocysts and the rate of development to the expanded and hatching blastocyst stages. The assumed cumulative metabolite concentration of WOW with one embryo was 1.47 times higher than that of droplet culture with one embryo. Furthermore, the concentration of WOW with three embryos was 1.54 times higher than that of WOW with one embryo. CONCLUSIONS: In using human single culture media, a microwell culture system that allows group culture could be a powerful clinical tool for improving the success of assisted reproductive technologies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-018-1252-z) contains supplementary material, which is available to authorized users.

Purpose To study the outcomes of mouse preimplantation embryos irradiated with low doses of X-rays (≤ 1 Gy) and investigate apoptosis and pluripotency of the irradiated embryos. Methods Mouse embryos at the 2-cell stage were collected for in vitro culture. After reaching the 8-cell stage, embryos were irradiated with various low doses of X-rays (0–1 Gy). Blastocysts with a normal appearance were transferred into a pseudopregnant uterus. The developmental rate to blastocysts and the survival rate following embryo transfer were examined. Expression levels of p21, Smad2, Foxo1, Cdx2, Oct4, and Nanog genes were measured by RT-PCR. Apoptotic cells in mouse blastocysts were examined immunofluorescently by staining for cleaved caspase-3. Results More than 90% of non-irradiated and low-dose X-ray-irradiated preimplantation embryos developed to morphologically normal blastocysts that could be implanted and survive in the uterus. However, embryos irradiated with X-rays had more apoptotic cells in a dose-dependent manner. Expression of p21, Smad2, and Foxo1 genes in X-ray-irradiated embryos was increased significantly, while expression of Cdx2, Oct4, and Nanog genes was maintained in comparison with non-irradiated embryos. Conclusions Although irradiated embryos contained apoptotic cells, the low doses of irradiation did not disturb development of 8-cell stage embryos to blastocysts or their survival in utero. The underlying mechanisms might involve anti-apoptotic systems, including the Smad-p21 pathway, and preservation of pluripotency. Electronic supplementary material The online version of this article (10.1007/s10815-018-1156-y) contains supplementary material, which is available to authorized users.

PURPOSE: Preimplantation genetic screening (PGS) and assessment of mitochondrial content (MC) are current methods for selection of the best embryos for transfer. Studies suggest that time-lapse morphokinetics (TLM) may also be helpful for selecting embryos more likely to implant. In our study, we sought to examine the relationship between TLM parameters and MC to determine if they could be used adjunctively in embryo selection. We also examined the relationship between MC with ploidy and blastulation. METHODS: Cryopreserved human embryos at the zygote stage were thawed and cultured in a time-lapse system. Blastomere and trophectoderm biopsies were performed on days 3 and 6. Biopsied cells and all whole embryos from day 6 were analyzed for MC (ratio of mitochondrial to nuclear DNA) and ploidy using next-generation sequencing. RESULTS: In embryos, MC per cell declined between day 3 and day 6. While early cleavage parameters did not predict MC, embryos with longer blastulation timing had higher MC on day 6. Day 6 MC was lower in euploid vs. aneuploid embryos and lower in blastocysts vs. arrested embryos. CONCLUSIONS: A lower MC at the blastocyst stage was associated with euploid status and blastocyst formation, indicating better embryo quality compared to those with a higher MC. Higher MC in aneuploid and arrested embryos may be explained by slower cell division or degradation of genomic DNA over time. Blastulation timing may be helpful for selection of higher quality embryos. Combining blastulation timing and MC along with morphologic grading and euploid status may offer a new direction in embryo selection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-018-1113-9) contains supplementary material, which is available to authorized users.

PURPOSE: The objective of this study was to evaluate the effects of multiple growth factors on the development of individually cultured murine embryos. METHODS: Embryos produced by in vitro fertilization using in vitro (IVM) or in vivo (IVO) matured oocytes from three strains of mice (CF1, Swiss Webster, B6D2F1) were cultured individually (10 μl) in the absence (control) or presence of growth factors (paf, epidermal growth factor [EGF], insulin-like growth factor 1 [IGF-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Blastocyst formation, hatching, and blastocyst cell numbers (trophectoderm, inner cell mass, and total) were evaluated on days 4 and 5 of culture. Post-hatching development of CF1 IVO embryos was also evaluated in vitro and in vivo. RESULTS: The presence of growth factors did not improve the proportion of embryos forming blastocysts or initiating hatching for any of the types of embryos tested. The only significant (P

This study tests whether metformin or diet supplement BR-DIM-induced AMP-activated protein kinase (AMPK) mediated effects on development are more pronounced in blastocysts or 2-cell mouse embryos. Culture mouse zygotes to two-cell embryos and test effects after 0.5–1 h AMPK agonists’ (e.g., Met, BR-DIM) exposure on AMPK-dependent ACCser79P phosphorylation and/or Oct4 by immunofluorescence. Culture morulae to blastocysts and test for increased ACCser79P, decreased Oct4 and for AMPK dependence by coculture with AMPK inhibitor compound C (CC). Test whether Met or BR-DIM decrease growth rates of morulae cultured to blastocyst by counting cells. Aspirin, metformin, and hyperosmotic sorbitol increased pACC ser79P ~ 20-fold, and BR-DIM caused a ~ 30-fold increase over two-cell embryos cultured for 1 h in KSOMaa but only 3- to 6-fold increase in blastocysts. We previously showed that these stimuli decreased Oct4 40–85% in two-cell embryos that was ~ 60–90% reversible by coculture with AMPK inhibitor CC. However, Oct4 decreased only 30–50% in blastocysts, although reversibility of loss by CC was similar at both embryo stages. Met and BR-DIM previously caused a near-complete cell proliferation arrest in two-cell embryos and here Met caused lower CC-reversible growth decrease and AMPK-independent BR-DIM-induced blastocyst growth decrease. Inducing drug or diet supplements decreased anabolism, growth, and stemness have a greater impact on AMPK-dependent processes in two-cell embryos compared to blastocysts.

The purpose of this study is to explore the factors associated with embryo multinucleation, particularly focused on the influence of parental chromosomal polymorphisms in embryo multinucleation. This is a retrospective case-control study involving 1260 infertile couples undergoing their first IVF/ICSI cycles. Couples were screened for abnormalities in their karyotype and were evaluated for blastomere persistence of multinucleation. Demographic characteristics, stimulation protocol, and pregnant outcomes were analyzed using logistic regression analysis. The level of basal FSH was lower in the multinucleated embryos group (5.37 vs 5.72 IU/L). The Multinucleated embryos group received less gonadotropins (1788.5 vs 1891.3 IU), and the level of LH on day of HCG triggering was lower (1.09 vs 1.30 IU/L). More oocytes were recovered in the multinucleated embryos group (11.51 vs 9.23). Chromosomal polymorphisms were seen in at least 1 out of 163 (12.9%) couples. Multivariate logistic regression analysis revealed that chromosomal polymorphisms were independently associated with an increase in the occurrence risk of multinucleated embryos (OR = 1.61, 95% CI, 1.06–2.44) in the first IVF/ICSI cycle. The miscarriage rate in the multinucleated embryos group was 10% higher than that of the control group. Chromosomal polymorphisms were independently associated with multinucleation embryo formation. A higher LH level on the day of HCG triggering was associated with a decreased chance of multinucleation.

This study aimed to test whether there is an association between embryo morphokinetic parameters and ploidy status.Patients with high risk of aneuploidy were analyzed by time-lapse microscopy combined with preimplantation genetic screening (PGS). Accordingly, 256 blastocysts from 75 patients were subjected to trophectoderm biopsy and microarray comparative genomic hybridization (array-CGH). Blastocyst development process was analyzed using time-lapse images.Morphokinetic parameters: tPNf, t2, t3, t4, t5, t8, t9, tcom, tM, tSB, tB, tEB, CC1, CC2, CC3, S2, S3, t5-t2, and tB-tSB showed no significant difference in euploid embryos compared to aneuploid counterparts. In addition, two risk models based on previously published morphokinetic parameters failed to segregate euploid from aneuploid embryos.Morphokinetic parameters subjected to investigation in the present study failed to improve the chance of selecting euploid embryos.

Implantation of the mammalian embryo in the uterus is preceded by escape from the zona pellucida. In some species, hatching from the zona occurs preferentially from one or the other poles of the embryo. The situation for the bovine embryo, in which hatching precedes attachment to the uterus by more than a week, is unclear. The purpose was to describe whether hatching of the bovine embryo from the zona pellucida occurs preferentially from the embryonic or abembryonic pole. Bovine blastocysts undergoing hatching were examined by light microscopy (n = 84) and epifluorescence imaging using antibodies for markers of epiblast, hypoblast, and trophectoderm (TE) (n = 26). The location of hatching was classified as being at the embryonic pole, if hatching occurred ipsilateral to the inner cell mass (ICM), or abembryonic, if hatching occurred contralateral to the ICM. A total of 55% of blastocysts exited the zona pellucida through an opening at the embryonic pole. In these cases, 68% of the cells emerging through the zona pellucida were derived from the ICM. The remainder of blastocysts hatched from an opening either contralateral or to the side of the ICM. In these cases, 87% of hatched cells were TE. For the bovine embryo, there is nearly equal probability of hatching from the embryonic or abembryonic poles. Given that the surface area of the zona pellucida in contact with the TE overlying the ICM is less than for the remainder of the blastocyst, there is some preference for hatching through the embryonic pole. Thus, the bovine embryo is distinct from the mouse and human, where hatching occurs preferentially at the abembryonic pole.

The purpose of this study is to explore which part of the trophectoderm best represents the inner cell mass after aCGH analysis. Fifty-one preimplantation genetic diagnosis/preimplantation genetic screening of abnormal blastocysts diagnosed by array comparative genomic hybridization were included in this study. Blastocysts were thawed, incubated for 3 to 4 h, and then biopsied. Four regions were biopsied per blastocyst, including the inner cell mass (ICM), trophectoderm (TE) cells opposite the ICM, TE cells at the upper right of the ICM, and TE cells at the lower right of the ICM. The biopsied pieces were processed through multiple annealing and looping-based amplification cycle sequenced for 24-chromosome aneuploidy screening. The aneuploidy results were compared among the ICM and the different regional trophectoderm cells from the same blastocyst. Fifty of 51 (98.04%) ICM samples were concordant with at least one of the TE biopsies derived from the same embryos. There were 43 blastocysts in which ICM and the other three TE pieces were consistent. Discordance among the four pieces occurred in eight blastocysts. Only one blastocyst was discordant between the ICM and the other three TE pieces, while seven blastocysts were discordant between one of TE and the other three biopsied pieces. There was no special region that the mosaic TE was located. Our findings indicate that TE aneuploidy is an excellent predictor of ICM aneuploidy. The blastocyst mosaic cells are inclined to be located in TE. Moreover, the mosaic TE was not limited to the special region.

This study was conducted to examine the dynamic distribution of polo-like 1 kinase (Plk1) and the possible role it plays in first mitotic division during early porcine embryo development. Indirect immunofluorescence and confocal microscopy imaging techniques combined with western blot analyses were used to study the dynamic expression and subcellular localization of Plk1 protein in pig parthenogenetic embryos. Finally, a selective Plk1 inhibitor, GSK461364, was used to evaluate the potential role of Plk1 during this special stage. The results showed that Plk1 upon expression exhibited specific dynamic intracellular localization, which closely correlated with the α-tubulin distribution during the first mitotic division. GSK461364 treatment resulted in cleavage failure, with majority of the GSK461364-treated embryos being arrested in prometaphase. Further results of the subcellular structure examination showed that GSK461364 treatment led to a significantly higher proportion of the treated embryos having abnormal spindles and misarranged chromosomes at the prometaphase stage. Thus, these results indicated that Plk1 is essential for porcine embryos to complete the first mitotic division. Furthermore, Plk1 regulation was associated with effects on spindle assembly and chromosome arrangement.