Inner Cell Mass Numbers Research Articles

P-128 Non-invasive morphometric assessment of embryo quality: Effectiveness of the number of trophectoderm cells at the equatorial region of expanded blastocysts

Abstract Study question Does the number of trophectoderm cells at the equatorial region of expanded blastocysts (eTEs) affects pregnancy rate in single vitrified blastocyst transfer (SVBT)? Summary answer The number of eTEs highly correlated with the total number of TEs. The greater number of eTEs was higher pregnancy rate became. What is known already The increased use of blastocyst transfer has emphasized the importance of selecting high-quality embryos. Morphological grading: Gardner’s criteria, or time-lapse imaging of embryo morpho kinetics is established a tool for selection of high-quality blastocysts. The Gardner’s criteria is one of the most used evaluation according to grading scores of developmental stage, trophectoderm (TE) and inner cell mass (ICM). Although, it cannot completely exclude subjectivity, and it varies from observer to observer. A quantitative evaluation method for blastocyst selection is necessary to solve these problems and to improve the pregnancy rate of SVBT. Study design, size, duration First, we checked the correlation between the number of eTEs and the total number of TEs using 23 discarded blastocysts by immunostaining of CDX2. Then, we analyzed 1103 vitrified-warmed blastocysts from July 2017 to June 2021 for pregnancy rates. Expanded blastocysts which reached the blastocoel cavity of 160 µm or more with obvious ICM were vitrified and cryopreserved. Relationship between the number of eTEs and pregnancy rate after SVBT was analyzed by logistic regression. Participants/materials, setting, methods CDX2-positive cells were defined as TE, and the difference between total cells by Hoechst staining and CDX2-positive cells as ICM. The correlation between the number of eTEs and the total number of TEs was examined. In SVBT, all embryos were monitored by a time-lapse system. eTEs were counted in the time-lapse image just before cryopreservation. Pregnancy and abortion rates were examined for each eTEs. Also, logistic regression analysis was performed on eTEs and pregnancy. Main results and the role of chance There was a strong positive correlation between the number of eTEs and the total number of TEs (CDX2-positive cells) in the whole blastocysts (r = 0.945). In addition, there was a significant correlation between the number of eTEs and the total cell number of blastocysts (r = 0.73). On the other hand, there was no correlation between the number of eTEs and the cell number of ICM (r = 0.008). The number of eTEs was presented as quartiles: Pregnancy rates for <5, 5-6, 6-7 and ≥7 eTEs were 25.9, 29.4, 38.4, and 64.2% (P < 0.05), respectively, and the miscarriage rates were 50.0, 28.3, 14.1 and 19.2% (P < 0.05), respectively. Pregnancy rate for ≥7 eTEs was significantly higher than that for the other groups (P < 0.05), and miscarriage rate was lower than that for <6 eTEs. That is, increased eTEs significantly increased the pregnancy rate and decreased the miscarriage rate. Significant relationship between the number of eTEs and pregnancy was shown by logistic regression analysis (P < 0.0001), with an AUC of 0.68 and a cutoff value of 6.5. Limitations, reasons for caution In this study, expanded blastocysts at 160 µm or more in diameter were analyzed and cryopreserved. The different size of blastocysts may be different number of eTEs, and the relationship between the number of eTEs and pregnancy rate will be needed to confirm in individual clinic. Wider implications of the findings Quantitative analysis of eTEs makes it possible to select highly fertile blastocysts. This assessment is simply and easy. It can be the additional possible evaluation before cryopreservation to allows for quantitative treatment of continuous variables by classifying them in more detail than Gardner’s criteria. Trial registration number N/A

Single-cell multi-omics sequencing reveals chromosome copy number inconsistency between trophectoderm and inner cell mass in human reconstituted embryos after spindle transfer.

Is the chromosome copy number of the trophectoderm (TE) of a human reconstituted embryos after spindle transfer (ST) representative of the inner cell mass (ICM)? Single-cell multi-omics sequencing revealed that ST blastocysts have a higher proportion of cell lineages exhibiting intermediate mosaicism than conventional ICSI blastocysts, and that the TE of ST blastocysts does not represent the chromosome copy number of ICM. Preimplantation genetic testing for aneuploidy (PGT-A) assumes that TE biopsies are representative of the ICM, but the TE and ICM originate from different cell lineages, and concordance between TE and ICM is not well-studied, especially in ST embryos. We recruited 30 infertile women who received treatment at our clinic and obtained 45 usable blastocysts (22 from conventional ICSI and 23 reconstituted embryos after ST). We performed single-cell multi-omics sequencing on all blastocysts to predict and verify copy number variations (CNVs) in each cell. We determined the chromosome copy number of each embryo by analysing the proportion of abnormal cells in each blastocyst. We used the Bland-Altman concordance and the Kappa test to evaluate the concordance between TE and ICM in the both groups. The study was conducted at a public tertiary hospital in China, where all the embryo operations, including oocytes retrieval, ST, and ICSI, were performed in the embryo laboratory. We utilized single-cell multi-omics sequencing technology at the Biomedical Pioneering Innovation Center, School of Life Sciences, Peking University, to analyse the blastocysts. Transcriptome sequencing was used to predict the CNV of each cell through bioinformatics analysis, and the results were validated using the DNA methylation library of each cell to confirm chromosomal normalcy. We conducted statistical analysis and graphical plotting using R 4.2.1, SPSS 27, and GraphPad Prism 9.3. Mean age of the volunteers, the blastocyst morphology, and the developmental ratewere similar in ST and ICSI groups. The blastocysts in the ST group had some additional chromosomal types that were prone to variations beyond those enriched in the blastocysts of the ICSI group. Finally, both Bland-Altman concordance test and kappa concordancetest showed good chromosomal concordance between TE and ICM in the ICSI blastocysts (kappa = 0.659, P < 0.05), but not in ST blastocysts (P = 1.000), suggesting that the TE in reconstituted embryos is not representative of ICM. Gene functional annotation (GO and KEGG analyses) suggests that there may be new or additional pathways for CNV generation in ST embryos compared to ICSI embryos. This study was mainly limited by the small sample size and the limitations of single-cell multi-omics sequencing technology. To select eligible single cells, some cells of the embryos were eliminated or not labelled, resulting in a loss of information about them. The findings of this study are innovative and exploratory. A larger sample size of human embryos (especially ST embryos) and more accurate molecular genetics techniques for detecting CNV in single cells are needed to validate our results. Our study justifies the routine clinical use of PGT-A in ICSI blastocysts, as we found that the TE is a good substitute for ICM in predicting chromosomal abnormalities. While PGT-A is not entirely accurate, our data demonstrate good clinical feasibility. This trial was able to provide correct genetic counselling to patients regarding the reliability of PGT-A. Regarding ST blastocysts, the increased mosaicism rate and the inability of the TE to represent the chromosomal copy number of the ICM are both biological characteristics that differentiate them from ICSI blastocysts. Currently, ST is not used clinically on a large scale to produce blastocysts. However, if ST becomes more widely used in the future, our study will be the first to demonstrate that the use of PGT-A in ST blastocysts may not be as accurate as PGT-A for ICSI blastocysts. This study was supported by grants from the National Key R&D Program of China (2018YFA0107601) and the National Key R&D Program of China (2018YFC1003003). The authors declare no conflict of interest. N/A.

P-266 Study on the mechanisms of ovol1/2 during the morula-to-blastocyst transition

Abstract Study question Screen the key gene groups that determine the development of blastocyst. Explore the effect of ovol1/2 gene on early embryonic development. Summary answer We founded that there were insufficient activation of the Major ZGA genes in the AE group,and focused on candidate gene ovol1/2. What is known already At present, the clinical success rate of assisted reproductive technology (ART) is less than 50%. A large number of embryos cultured in vitro can not develop to blastocyst, and there are cleavage or morula stage arrest. The developmental arrest of preimplantation embryos is an important reason for treatment failure of infertile patients. However, the cause of early embryonic development arrest is not clear, and there is also a lack of effective methods to improve embryo quality. Study design, size, duration In this study, fertilized embryos were obtained through ICSI and a single cell from eight-cell embryo was biopsied through micromanipulation for single-cell RNA sequencing (scRNA-seq). Subsequently, we tracked the developmental potential of remaining cells and divided them into high-quality blastocyst(Bla) group and high-quality eight-cell failed blastocyst formation(arrest embryo, AE) group. In addition,we employed a RNAi approach to study the function of candidate gene ovol1/2, by injecting ovol1/2-targeting small interfering RNAs (siovol1/2) into wild-type (WT) zygotes. Participants/materials, setting, methods The study collected 21 human blastocysts and 5 arrest embryos. Human immature oocytes from ICSI treatments were clinically discarded and donated by women after signing informed consent by donors. C57B6 background mouse strains were used in this study. Mice were maintained under specific pathogen free conditions in a controlled environment of 20–22 °C,with a 12/12 h light/dark cycle, 50–70% humidity, and food and water provided. Main results and the role of chance The results showed that the gene expression of embryos in AE group was significantly different from that of Bla group: 79.5% of differential expression genes were down-regulated, including 48.3% of the Major ZGA gene. Then we focused on the candidate gene ovol1/2. When ovol1/2 were knocked down alone, there was no significant difference in the development rate of embryos from 2-cell to blastocyst between knockdown group and normal control group. When ovol1/2 were knocked down at the same time, there was no significant difference in the development rate of embryos among three groups before the morula stage. But in the morula developed to the blastocyst stage, there were significant differences among three groups of embryos: about 73% of embryos in the normal control group formed blastocyst; about 39% of the embryos in the low-dose knockdown group; the high-dose knockdown group embryos were all arrested in the morula stage. Further investigation revealed that the number of inner cell mass(ICM) cells in low-dose knockdown group did not change significantly, while the number of trophoblastic ectoderm(TE) cells significantly decreased. In addition, the TE functions of low-dose knockdown group such as blastocyst cavity size, paved area, hatching and adhesion rate were damaged. Limitations, reasons for caution Further research of the mechanisms is needed. Wider implications of the findings This is significant to understand molecular mechanisms of early embryonic development, and will provide important theoretical basis for clinical diagnosis and intervention of embryonic development arrest. Our study provides clues for the causes of abnormal blastocyst formation and new ideas for possible clinical improvements in the success rate of ART. Trial registration number not applicable

Impact of mono(2-ethylhexyl) phthalate (MEHP) on the development of mouse embryo in vitro

Mono(2-ethylhexyl) phthalate (MEHP) is the most studied metabolite of di(2-ethylhexyl) phthalate (DEHP), a phthalate found in cosmetics, flooring, paints, and plastics products, including toys and medical tubing. Humans are frequently exposed to this compound due to its ubiquitous presence in our environment. DEHP and MEHP are known to be endocrine-disrupting chemicals and exposure levels have been associated to decreased reproductive success. However, few studies have focused on the direct effects of MEHP on embryos. The present study investigated effects of MEHP (0.1, 1, 10, 100 and 1000 µM) on mice preimplantation embryonic development, evaluating percentage of blastocyst formation, hatching from zona pellucida, methylation-related genes, cell lineage commitment, micronucleation, and adherens junction marker at different stages of development during in vitro culture for 6 days. We show MEHP negatively impacts embryo competence by reducing blastocyst formation and hatching at 100 and 1000 µM. In addition, 100 µM MEHP increases the expression of Tet3 gene in blastocysts, which is related to a reduction of DNA methylation, an important mechanism regulating gene expression. Exposed embryos that completed the hatching process in groups 0.1, 1 and 10 µM MEHP had similar number of inner cell mass and trophectoderm cells compared to the control, while micronucleation occurrence and E-cadherin expression was not affected in exposed morulae by MEHP at 10 or 100 µM. Our results showed that high concentrations of MEHP can negatively impact embryo development. New studies unveiling the mechanism of toxicity involved and encompassing further developmental stages are warranted for further understanding.

Comparison the effects of 5-Aza-2'-deoxycytidine and zebularine on the in vitro development, blastocyst quality, methylation pattern and conception rate on handmade cloned buffalo embryos.

In this study we treated the handmade cloned (HMC) buffalo embryos with the DNA methylation inhibitors; 5-aza-2'-deoxycytidine (AzadC) or Zebularine individually after post-fusion and during in vitro culture till eighth day. The blastocysts production rate significantly improved (p < .01) after treating embryos independently with 5nM AzadC and 5nM zebularine compared with 2 and 10nM AzadC or zebularine groups, respectively. The highest cleavage rates were obtained for 5nM treatment of AzadC and zebularine compared with other treatments and untreated control group. Quality of blastocysts were evaluated using total cell number (TCN) and the ratio of number of inner cell mass (ICM) cells/total cell number (ICM/TCN). Zebularine treatments (2/5/10nM) significantly improved both TCN and ICM/TCN ratio compared with AzadC treatments (2/5/10nM); however, control group TCN and ICM/TCN ratio was found lower. The methylation percentage of pDS4.1 and B. bubalis satellite DNA were comparatively more attenuated with 5nM zebularine than 5nM AzadC treatment. The increased in vitro development rates of the treated embryos were correlated with the decreased level of DNA methylation and the improved blastocyst quality. Following transfer of 5nM zebularine treated embryos to 6 recipients, 4 were found to be pregnant, though the pregnancies were not carried to full term.

Comparison of the Morphology and Developmental Potential of Oocytes Obtained from Prepubertal and Adult Domestic and Wild Cats.

Simple SummaryThis study was conducted with the aim of determining the morphological similarities and developmental potential of oocytes obtained from adult and prepubertal domestic cats (Felis catus) and wild cats. The results of our research showed that ovaries obtained from prepubertal felids may be a rich source of good quality oocytes that are competent for in vitro maturation and able to reach blastocyst stage after in vitro fertilization. The results are important in the context of the possibility of using oocytes from prepubertal felids threatened with extinction in conservation programs based on assisted reproductive techniques (ART).The aim of the study was to compare the morphology and developmental potential of oocytes obtained from adult and prepubertal domestic cats (Felis catus) and wild cats (Lynx lynx, Leptailurus serval, Felis manul, Panthera tigris altaica). The average number of oocytes obtained from an adult domestic cat was 23 ± 11, which was significantly lower than from kittens (43 ± 29). A similar number of oocytes was derived from adult Pallas’s cats (28 ± 8), and serval (30). The lowest number of oocytes was collected from the lynx (5 ± 3). No oocytes were obtained from newborn Amur tiger while in the case of older domestic and Pallas’s cat and lynx kittens (1–3 months) 43, 48 and 41 oocytes were collected, respectively. Significant differences (p < 0.001) were observed between the number of oocytes with dark cytoplasm from adult and prepubertal animals of all analyzed species. The diameter of oocytes from adult and prepubertal animals was similar in all species, and was on average 161 ± 4 µm for oocytes with dark cytoplasm and 150 ± 18 µm for oocytes with light cytoplasm. In all species, oocytes with light cytoplasm were significantly smaller (p < 0.05) than dark ones, and their population was more diverse. Results of in vitro maturation of the domestic and wild cat′s oocytes obtained from adult and prepubertal females were similar (47–52%). The cleavage rate after in vitro fertilization (IVF) was lower for prepubertal than adult domestic cats (42 vs. 51%; p < 0.05%). Moreover, we observed differences in the quantity (28 vs. 39%; p < 0.05) and quality of blastocysts and even greater problems with hatching blastocysts from prepubertal kittens (8 vs. 19%; p < 0.001). More blastomeres were detected in blastocysts of adult cats. They also demonstrated significantly higher number of inner cell mass (ICM) (p < 0.001) and higher number of trophoblast cells (TE) (p < 0.05).

Melatonin enhances in vitro developmental competence of cumulus-oocyte complexes collected by ovum pick-up in prepubertal and adult dairy cattle

Bovine oocytes from prepubertal donors have been used for in vitro embryo production to decrease the generation interval. However, reduced cumulus-oocyte competence, mainly attributed to increased apoptosis, has been observed in oocytes/embryos collected from prepubertal donors. Here, we investigated the effects of the potent antioxidative molecule melatonin on cumulus-oocyte competence and embryo development in prepubertal and adult dairy cattle in vitro. A total of fifteen Holstein Friesian calves, six to ten months old (7.6 ± 1.34 months of age). And fifteen adult cows with one to four calvings (2.3 ± 0.96 calvings) were enrolled as ovum pick up (OPU) donors in this study. Cumulus-oocyte complexes (COCs) were cultured either in the presence or absence of melatonin (0.01 nM). The proportion of cleavage stages, blastocysts, and advanced blastocysts was determined. Embryo quality was assessed via differential staining to determine the total embryonic cells and allocation to the inner cell mass (ICM) and trophectoderm (TE) cells. Melatonin treatment yielded a greater percentage of blastocysts compared to the control group, i.e. oocytes from both adult cows (P = 0.0485; 24.8 ± 3.5% vs. 16.0 ± 3.4%, respectively), and from prepubertal donors (P = 0.0007; Melatonin 23.1 ± 5.1% vs. Control: 11.1 ± 3.5%). Adult cows had significantly (P = 0.0370) greater advanced blastocyst rates than those found in the prepubertal group (13.9%± vs. 7.0±%, respectively). Additionally, the number of ICM, total cells, and the ratios ICM: Total, ICM: TE, respectively, were greater (P < 0.05) after melatonin treatment compared with the control group (39.1 ± 2.8, 98.6 ± 5.7, 0.4 ± 0.01, and 0.7 ± 0.04 vs. 27.3 ± 2.9, 81.2 ± 5.8, 0.34 ± 0.01, and 0.52 ± 0.04, respectively). Blastocysts derived from adult cows had a greater number of TE (P = 0.01) and total embryonic cells (P = 0.0095) compared to the prepubertal donor group (63.5 ± 3.2 and 101.05 ± 4.8 vs. 48.9 ± 4.3 and 78.8 ± 6.5, respectively). Nevertheless, embryonic cell counting in embryos derived from prepubertal COCs equated to that observed from adult donors after melatonin exposure. In conclusion, these results indicate that the presence of melatonin during in vitro maturation improves cumulus-oocyte competence, embryo development, and quality by increasing the allocation of embryonic cells to the ICM compartment and the total number of embryonic cells in both adult and prepubertal bovine donors.

Alpha-ketoglutarate affects murine embryo development through metabolic and epigenetic modulations.

α-Ketoglutarate (α-KG) is an intermediary metabolite in the tricarboxylic acid (TCA) cycle and functions to inhibit ATPase and maintain the pluripotency of embryonic stem cells (ESCs); however, little is known regarding the effects of α-KG on the development of preimplantation embryos. Herein, we report that α-KG (150 μM) treatment significantly promoted the blastocyst rate, the number of inner cell mass (ICM) cells and foetal growth after embryo transfer. Mechanistic studies revealed two important pathways involved in the α-KG effects on embryo development. First, α-KG modulates mitochondria function by inducing relatively low ATP production without modification of mitochondrial copy number. The relatively low energy metabolism preserves the pluripotency and competence of the ICM. Second, α-KG modifies epigenetics in embryos cultured in vitro by affecting the activity of the DNA demethylation enzyme TET and the DNA methylation gene Dnmt3a to increase the ratio of 5hmC/5mC ratio. Elevation of the 5hmC/5mC ratio not only promotes the pluripotency of the ICM but also leads to a methylation level in an in vitro embryo close to that in an in vivo embryo. All these functions of α-KG collectively contribute to an increase in the number of ICM cells, leading to greater adaptation of cultured embryos to in vitro conditions and promoting foetal growth after embryo transfer. Our findings provide basic knowledge regarding the mechanisms by which α-KG affects embryo development and cell differentiation.

Antioxidant activity of oily extract obtained from Lippia origanoides improves the quality of bovine embryos produced in vitro

ABSTRACT The aim of this study was to evaluate the supplementation of embryo culture medium with antioxidant obtained from oily extract of Lippia origanoides on in vitro blastocyst development and quality. Oocytes collected from slaughterhouse ovaries were matured and fertilized in vitro following standard laboratory procedures. Zygotes were cultured in SOF medium supplemented according to the following treatments: T1 embryo culture medium without antioxidant supplementation; T2)50μM/mL Cysteamine; T3)2.5μg/mL; T4)5.0μg/mL and T5)10.0μg/mL of antioxidant obtained from oily extract of Lippia origanoides. On the seventh day of culture, the blastocysts were fixed and evaluated for apoptosis rates, number of total cell and inner cell mass cells by means of the TUNEL Test. The use of antioxidants during cultivation did not increase (P&gt; 0.05) the final blastocyst production rate. The treatments T2, T3, T4 and T5 had the lowest (P&lt; 0.05) apoptotic indexes (4.5±1.1%, 8.4±2.5%, 3.4±1.1% and 5.5±0.9%, respectively) when compared to T1 treatment (10.0±1.4%). The number of inner cell mass did not differ (P&gt; 0.05) among embryos from different treatments. The addition of antioxidant obtained from oily extract of Lippia origanoides reduces the apoptosis rate and improves the quality without increasing the total in vitro production of bovine embryos.

Human frozen-thawed blastocyst morphokinetics observed using time-lapse cinematography reflects the number of trophectoderm cells.

Recent studies reported morphokinetic indices for optimal selection of embryos in assisted reproductive technology (ART). The morphokinetics in blastocyst stage include the collapse and re-expansion rates after thawing. However, evaluation methods using these morphokinetics have not been established, mainly because the underlying molecular mechanisms remain unclarified. In this study, we focused on the relationship between these morphokinetic observation of the blastocyst behaviour and the number of cells constituting the blastocyst. We evaluated 38 surplus human frozen-thawed blastocysts using time-lapse cinematography and recorded their expansion, contraction, and hatching. A total of 28 blastocysts expanded in culture (cross-sectional area ≥ 5,000 π μm2). In comparison to the ones that did not, the expanded group presented significantly more number of inner cell mass (ICM) and trophectoderm (TE) cells, which eventually develop into the fetus and placenta, respectively (ICM: Expanded 10.2 ± 6.3 vs. Non-Expanded 6.0 ± 12.3, p < 0.05; TE: Expanded 165.7 ± 74.8 vs. Non-Expanded 57.0 ± 29.4, p < 0.05). Moreover, a positive correlation was found between the expansion rate (up to 4 h) and the number of TE cells (r = 0.558, p = 0.0021). Additionally, blastocysts that hatched had a significantly higher number of TE cells than those that did not (hatching 225.2 ± 61.2 vs. no hatching 121.1 ± 48.6, p < 0.0001). The number of TE cells per unit of cross-sectional area correlated negatively with the contraction time (r = –0.601, p = 0.0007). No correlation between the number of ICM cells and these morphokinetics was detected. In conclusion, our study demonstrates that different morphokinetics of frozen-thawed blastocysts reflect the number of TE cells. The differentiation of blastocysts containing sufficient TE cells would be beneficial for implantation and prognosis of a subsequent pregnancy. Thus, evaluation of these morphokinetics can be an effective method to screen good embryos for ART.

The effect of cell penetrating peptide-conjugated coactivator-associated arginine methyltransferase 1 (CPP-CARM1) on the cloned mouse embryonic development

Abnormalities in gene expression that negatively affect embryonic development are frequently observed in cloned embryos generated by somatic cell nuclear transfer (SCNT). In the present study, we successfully produced a cell-penetrating peptide (CPP)-conjugated with coactivator-associated arginine methyltransferase 1 (CARM1) protein from mammalian cells and confirmed introduction into donor somatic cells and cloned 8-cell embryos within 3 hours after addition to culture medium. In addition, H3R17 dimethylation and embryonic development up to the blastocyst stage were increased in the group treated with exogenous CPP-CARM1 protein compared with the untreated group (control). Interestingly, the number of total cells and trophectoderm in blastocysts as well as implantation rate were significantly increased in the CPP-CARM1 protein-treated group. However, the cell number of inner cell mass (ICM) was not changed compared with the control group; similarly, expression of pluripotency-related genes Oct4 and Nanog (ICM markers) was not significantly different between groups. On the other hand, expression of the implantation-related gene Cdx2 (trophectoderm marker) was transiently increased after treatment with CPP-CARM1 protein. On the basis of these results, we conclude that supplementation with exogenous CPP-CARM1 protein improves embryonic development of cloned embryos through regulation of histone methylation and gene expression. In addition, our results suggest that CPP-CARM1 protein may be a useful tool for strengthening implantation of mammalian embryos.

Enhancing developmental rate and quality of mouse single blastomeres into blastocysts using a microplatform.

The present work reports the beneficial effects of using a microplatform on the development of mouse single blastomeres (SBs) to the blastocyst stage. Development of blastocysts from SBs separated from two- and four-cell stage embryos (two- and four-cell SBs) can provide a valuable supply both for couples who use fertility-assisted techniques and farm animals. As a step forward, we introduce three chips that provide the possibility of culturing SBs separately, in groups, and in the vicinity of the intact embryo(co-culture), while each well of the chips is assigned to an isolated SB. Two- and four-cell SBs co-cultured with intact embryos showed 97.1% and 76.6% developmental rates and up to 34.1% and 49.1% growth relative to the microdroplet method (control). We examined the quality of developed blastocysts by assessing the total cell number, the number of inner cell mass (ICM) according to the octamer-binding transcription factor 4 marker (OCT4), and trophectoderm (TE). Co-culture of SBs with an intact embryo in a chip with nanoscale culture medium volume also increased the cell population of the developed embryo. The ICM:TE ratio, which is the most important blastocyst quality parameter, also indicated that developed two-cell SBs have a higher degree of similarity to intact embryos despite fewer numbers of total cells.

Antioxidants improve IVF outcome and subsequent embryo development in the mouse

What is the effect of a combination of three antioxidants (Acetyl-L-Carnitine, N-Acetyl-L-Cysteine and α-Lipoic Acid), present in IVF medium during mouse oocyte and sperm collection, on fertilization and subsequent IVF embryo development? A combination of antioxidants resulted in faster developmental times from the 2-cell stage through to expanded blastocyst stage, accompanied by a significant increase in blastocyst cell number and a reduction of intracellular hydrogen peroxide (H2O2) levels. The antioxidant combination Acetyl-L-Carnitine, N-Acetyl-L-Cysteine and α-Lipoic Acid, when present in embryo culture media, has a significant beneficial effect on in vitro fertilized mouse pronucleate oocyte development, especially under oxidative stress. IVF was conducted with combined antioxidants supplemented in IVF medium that was used for mouse oocyte collection and fertilization (oocyte IVF medium, 4 h exposure) and sperm collection and preparation (sperm IVF medium, 1 h exposure). IVF was conducted under 20% oxygen, in the presence or absence of a combination of antioxidants (10 μM Acetyl-L-Carnitine, 10 μM N-Acetyl-L-Cysteine, 5 μM α-Lipoic Acid) and resultant embryos cultured with and without antioxidants under 20% oxygen. Subsequently, the effects of antioxidants on either oocytes or sperm was evaluated. Embryo development was analysed through time-lapse microscopy followed by differential nuclear staining to determine cell allocation in the blastocyst. Intracellular levels of H2O2 were assessed using an aryl boronate probe after 4 h of incubation with antioxidants. Controls were gametes and embryos that had no antioxidants in the medium. In a separate series of experiments, pronucleate oocytes were collected in handling medium with and without antioxidants for 20 min and subsequent cell numbers analysed. Antioxidant treatment during both IVF and culture resulted in significantly faster development times to two cell cleavage (P < 0.01), which continued through to the expanded blastocyst stage (P < 0.05). Resultant blastocysts had a significant increase in both trophectoderm (TE) cell numbers, inner cell mass (ICM) and total cell numbers (P < 0.001). The addition of antioxidants to IVF medium or embryo culture media exclusively also resulted in a significant increase in both blastocyst TE and ICM numbers leading to an increase in total cell numbers (P < 0.001). Antioxidant supplementation of either oocyte IVF medium alone, or in both oocyte and sperm IVF medium, lead to significantly faster times to two cell cleavage, which continued through to the expanded blastocyst stage. Blastocyst cell number in both these groups had significantly higher TE cell numbers resulting in an increase in total cell numbers. In contrast, there were no differences in embryo developmental rates and blastocyst cell number when antioxidants were present only in the sperm IVF medium. Levels of H2O2 were significantly reduced in pronucleate oocytes that were cultured in the presence of antioxidants (P < 0.001) compared to control, untreated embryos. Similarly, pronucleate oocytes treated with the combined antioxidants during pronucleate oocyte collection resulted in significantly increased blastocyst ICM numbers compared with controls (P < 0.05). Embryo development was only examined in the mouse. These findings suggest that supplementation of antioxidants to the IVF medium, as well as to embryo culture media, may further assist in maintaining the viability of human embryos in ART, conceivably through the reduction of oxidative stress. This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.

Consequences of endogenous and exogenous WNT signaling for development of the preimplantation bovine embryo.

The specific role of WNT signaling during preimplantation development remains unclear. Here, we evaluated consequences of activation and inhibition of β-catenin (CTNNB1)-dependent and -independent WNT signaling in the bovine preimplantation embryo. Activation of CTNNB1-mediated WNT signaling by the agonist 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and a glycogen synthase kinase 3 inhibitor reduced development to the blastocyst stage. Moreover, the antagonist of WNT signaling, dickkopf-related protein 1 (DKK1), alleviated the negative effect of AMBMP on development via reduction of CTNNB1. Based on labeling for phospho c-Jun N-terminal kinase, there was no evidence that DKK1 activated the planar cell polarity (PCP) pathway. Inhibition of secretion of endogenous WNTs did not affect development but increased number of cells in the inner cell mass (ICM). In contrast, DKK1 did not affect number of ICM or trophectoderm (TE) cells, suggesting that embryo-derived WNTs regulate ICM proliferation through a mechanism independent of CTNNB1. In addition, DKK1 did not affect the number of cells positive for the transcription factor yes-associated protein 1 (YAP1) involved in TE formation. In fact, DKK1 decreased YAP1. In contrast, exposure of embryos to WNT family member 7A (WNT7A) improved blastocyst development, inhibited the PCP pathway, and did not affect amounts of CTNNB1. Results indicate that embryo-derived WNTs are dispensable for blastocyst formation but participate in regulation of ICM proliferation, likely through a mechanism independent of CTNNB1. The response to AMBMP and WNT7A leads to the hypothesis that maternally derived WNTs can play a positive or negative role in regulation of preimplantation development.

82 HOW LOW CAN YOU GO? DEFINING THE MINIMAL NUTRIENT REQUIREMENTS FOR BOVINE EMBRYOS IN CULTURE

Recent metabolomic studies in our laboratory have indicated that bovine embryos only use a small amount of the nutrients provided to them in culture media. Our objective was to determine the effects of reducing nutrient concentrations in our media to 75, 50, and 25% (experiment 1) or 25, 12.5, and 6.25% (experiment 2) of those in our control medium (100%) on the development of in vitro-matured/IVF bovine embryos in a serum-free medium. Cumulus–oocyte complexes were matured in a defined maturation medium (0.1 IU mL−1 recombinant human FSH, 50 ng mL−1 recombinant mouse epidermal growth factor, and 2.5 mg mL−1 recombinant human serum albumin) and co-incubated with frozen–thawed spermatozoa (2 × 106 mL−1, 20 h). Embryos were cultured (7.5% CO2, 6.5% O2, 38.7°C) in a sequential media system (0–72 h and 72–168 h). Concentrations of salts, bicarbonate, and protein (2.5 mg mL−1 fatty acid–free BSA) were the same in all treatments. All nutrients (glucose, lactate, pyruvate, amino acids, and vitamins) were diluted to the same extent (e.g. 25%) relative to the control medium for each culture period. Blastocyst formation and hatching (per cleaved embryo) were evaluated on Day 7 of culture. Hatching blastocysts were stained to determine the number of inner cell mass (ICM; SOX2+), trophectoderm (TE; CDX2+), and total cells (ICM+TE) in the embryo. All data were analysed by ANOVA. The proportion of zygotes that cleaved on Day 3 was not affected (P &gt; 0.05) by the concentration of nutrients present. In experiment 1, dilution of nutrients to 25% did not affect (P &gt; 0.05) blastocyst development (40.1 ± 3.7%) or hatching (16.3 ± 2.8%) compared with 100% (45.2 ± 3.8% blastocyst and 24.9 ± 3.3% hatching). In experiment 2, dilution of nutrients to 12.5% tended (P = 0.08) to reduce hatching (12.9 ± 2.6%) compared with 100% (20.0 ± 3.1%) but did not affect (P &gt; 0.05) blastocysts formation (12.5%, 41.7 ± 3.9% v. 100%, 40.0 ± 3.8%). It was not until nutrient concentrations were reduced to 6.25% that blastocyst formation (18.3 ± 3.0%) and hatching (3.0 ± 1.3%) were inhibited (P &lt; 0.05). Hatching blastocysts cultured with 25 or 12.5% nutrients had fewer total (P &lt; 0.05; 150.7 ± 9.7 and 121.6 ± 7.6, respectively), TE (P &lt; 0.05; 124.1 ± 8.5 and 90.5 ± 7.1), and ICM (P = 0.06; 26.6 ± 3.4 and 30.7 ± 4.0) cells compared with control embryos (195.2 ± 15.9 total, 156.1 ± 14.1 TE, and 39.1 ± 4.0 ICM). To determine whether the embryo’s ability to develop with reduced concentrations of nutrients was dependent on lipid metabolism, embryos were cultured with 50, 25, 12.5, and 6.25% nutrients in the presence or absence of an inhibitor of fatty acid oxidation (50 μM etomoxir). The presence of etomoxir reduced (P &lt; 0.05) blastocyst development at all nutrient concentrations, but this effect was more pronounced when nutrients were limited (≤25% nutrients, 28.7 to 40.9% reduction) compared with 50% (12.5% reduction). Although blastocyst cell numbers decrease when nutrient concentrations are reduced to 25% of those in control media, the proportion of embryos reaching the blastocyst stage is not affected until nutrients are reduced to 6.25%. The ability to develop under nutrient-restricted conditions appears to be related to fatty acid metabolism.

Sex differences in response of the bovine embryo to colony-stimulating factor 2.

We tested whether gene expression of the bovine morula is modified by CSF2 in a sex-dependent manner and if sex determines the effect of CSF2 on competence of embryos to become blastocysts. Embryos were produced in vitro using X- or Y-sorted semen and treated at Day 5 of culture with 10 ng/mL bovine CSF2 or control. In experiment 1, morulae were collected at Day 6 and biological replicates (n = 8) were evaluated for transcript abundance of 90 genes by RT-qPCR using the Fluidigm Delta Gene assay. Expression of more than one-third (33 of 90) of genes examined was affected by sex. The effect of CSF2 on gene expression was modified by sex (P < 0.05) for five genes (DDX3Y/DDX3X-like, NANOG, MYF6, POU5F1 and RIPK3) and tended (P < 0.10) to be modified by sex for five other genes (DAPK1, HOXA5, PPP2R3A, PTEN and TNFSF8). In experiment 2, embryos were treated at Day 5 with control or CSF2 and blastocysts were collected at Day 7 for immunolabeling to determine the number of inner cell mass (ICM) and trophectoderm (TE) cells. CSF2 increased the percent of putative zygotes that became blastocysts for females, but did not affect the development of males. There was no effect of CSF2 or interaction of CSF2 with sex on the total number of blastomeres in blastocysts or in the number of inner cell mass or trophectoderm cells. In conclusion, CSF2 exerted divergent responses on gene expression and development of female and male embryos. These results are evidence of sexually dimorphic responses of the preimplantation embryo to this embryokine.

Total Cell Number and its Allocation to Trophectoderm and Inner Cell Mass in In Vitro Obtained Cats' Blastocysts.

The aim of this study was to evaluate the developmental kinetics of cats' blastocysts in connection with their morphology and blastomeres allocation to trophoblast or embryoblast cells. We examined gross blastocyst morphology and the total number of blastomeres together with its allocation to inner cell mass (ICM) or trophectoderm (TE) cells in pre-implantation feline embryos obtained from 6th to 9th day of invitro culture. From all the investigated embryos, 61.8% developed to early blastocyst, 37.4% to full and 7.6% to hatching blastocyst stage. The total cell number (TCN) varied form 58 cells in early day 6 to 245 in hatching day 8 blastocyst, with the mean 84.9 cells in early, 156.7 in full, and 204.4 in hatching ones. Day 8 blastocyst had the highest number of total cells, together with the highest mean number of ICM regardless of its morphological assessment. Early blastocyst (apart from day 6) had the highest number of arrested cells, while dead cells were the highest in full day 9 blastocyst. More data about the relationship between blastocyst development and morphology would facilitate the selection of optimal blastocysts for further procedures.

93 DEVELOPMENT OF BOVINE PRE-IMPLANTATION EMBRYOS IS ALTERED BY ADDITION OF ACTIVIN AND CONNECTIVE TISSUE GROWTH FACTOR FROM DAYS 5 TO 7 AFTER FERTILIZATION

The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. Here we tested actions of two molecules that are highly expressed in the endometrium for actions to modify development of bovine embryos. The molecules tested were activin and connective tissue growth factor (CTGF), and endpoints were percentage of fertilized oocytes becoming blastocysts and number of inner cell mass (ICM) and trophectoderm (TE) cells in Day 7 blastocysts. Bovine embryos produced in vitro from slaughterhouse ovaries were cultured in a serum-free culture medium. On Day 5 of culture, culture drops were supplemented with vehicle (control), human recombinant activin or CTGF (10–11, 10–10, or 10–9 M). On Day 7, blastocysts (n = 202) were collected and labelled with a nuclear dye (Hoescht 33342) and a TE cell marker (anti-CDX2). Statistical analysis was performed using the GLM procedure of SAS. Results are shown as least squares means ± s.e.M. The percentage of putative zygotes becoming a blastocyst on Day 7 was 28.4 ± 1.5% for control embryos; 32.2 ± 1.5%, 30.7 ± 1.5%, and 33.1 ± 1.5% for embryos cultured with 10–11, 10–10, and 10–9 M activin, respectively; and 27.8 ± 1.5%, 25.1 ± 1.5%, and 28.4 ± 1.5% for embryos cultured with 10–11, 10–10, and 10–9 M CTGF, respectively. Activin increased the proportion of putative zygotes becoming a blastocyst when added at 10–9 M (P = 0.0321) and tended to have the same effect at 10–11 M (P = 0.0898). There was no effect of any concentration of CTGF on development of putative zygotes to blastocyst on Day 7. There was no effect of treatment on total cell number or number of TE or of activin on ICM cell number or TE : ICM ratio. However, CTGF increased (P = 0.0011) the number of ICM (40.1 ± 1.5, 46.2 ± 1.4, 46.5 ± 1.4, and 56.3 ± 1.4 for control and 10–11, 10–10, and 10–9 M CTGF, respectively). CTGF also reduced (P = 0.0319) the TE : ICM ratio. Values were 2.8 ± 0.2 for control and 2.1 ± 0.2, 1.9 ± 0.2, and 1.6 ± 0.2 for 10–11, 10–10, and 10–9 M CTGF, respectively. Results indicate that activin and CTGF can affect embryo development. In particular, activin increases competence of embryos to develop to the blastocyst stage and CTGF affects blastocyst differentiation. Support was provided by NIH R03 HD080855.

Cryotop vitrification of porcine parthenogenetic embryos at the early developmental stages

The objective of this study was to evaluate the effects of early developmental stages at which Cryotop vitrification is performed on subsequent survival and in vitro development of porcine parthenogenetic activation embryos. The zygotes that were cultured for 4, 8, and 18 hours post electric activation (h.p.a.) and two- and four-cell embryos were vitrified, warmed, and continuously cultured for the remaining period. The zygotes vitrified at 4, 8, and 18 h.p.a. showed similar percentages of survival, cleavage, and blastocyst formation. No difference in viability was observed after vitrification of two- and four-cell embryos, but the embryos vitrified at the two-cell stage exhibited significantly higher blastocyst formation rate than those vitrified at the four-cell stage. However, vitrifying embryos resulted in significantly decreased survival and development rates, regardless of the developmental stage of the embryos. In addition, the final developmental stage, diameter, apoptotic index, and the number of inner cell mass, trophectoderm, and total cells of blastocysts derived from embryos vitrified at any stage of the early culture were similar to those of fresh blastocysts. In conclusion, our data indicate that the early-stage porcine parthenogenetically activated embryos including the zygote, two cells, and four cells have a high ability to survive cryopreservation; these viable embryos after vitrification can produce respectable development rates and good-quality blastocysts.

Developmental kinetics of cleavage stage mouse embryos are related to their subsequent carbohydrate and amino acid utilization at the blastocyst stage.

What is the relationship between cleavage stage embryo kinetics, blastocyst metabolism and subsequent embryo viability? Embryos cleaving faster at the first cleavage division resulted in blastocysts with a larger inner cell mass (ICM), higher glucose consumption, lower glycolytic rate, higher aspartate uptake, lower global amino acid turnover and higher percentage of developing fetuses on E13.5 when compared with blastocysts that developed from slower cleaving embryos. Previous research has shown that morphokinetics, blastocyst carbohydrate metabolism and cleavage stage amino acid metabolism of the preimplantation embryo can be used independently as markers of its developmental competence and subsequent viability. Morphokinetics of in vitro fertilized mouse zygotes were observed using a time-lapse imaging system and they were identified as 'fast' or 'slow' cleaving embryos. Spent culture media from resultant blastocysts were analysed for carbohydrate and amino acid utilization. Blastocysts either had their ICM and trophectoderm (TE) cell number determined, were cultured further in an outgrowth assay or transferred to a recipient female to assess implantation and fetal development. Morphokinetics of in vitro fertilized C57BL/6xCBA (F1) zygotes individually cultured in 2 µl drops of G1/G2 media with HSA under Ovoil in 5% O2, 6% CO2 and 89% N2 were analysed using a time-lapse incubator. At 72 h post-insemination, blastocysts were separated into quartiles derived from timing of the first cleavage division. Blastocysts were cultured for a further 24 h and spent media samples, including controls containing no embryos, were frozen and subsequently analysed for amino acid utilization using liquid chromatography-mass spectrometry. These blastocysts were then analysed over a further 1.5 h period for carbohydrate utilization and subsequently stained to determine ICM and TE cells. To analyse implantation potential, fetal quality and viability, additional 'fast' and 'slow' blastocysts were cultured further in an outgrowth model or transferred to recipient females. Embryos cleaving faster at the time of first cleavage (first quartile, designated 'fast') were on average 2.5 h ahead of slower embryos (fourth quartile, designated 'slow', 15.1 ± 0.1 versus 17.6 ± 0.1 h, P < 0.001). On Day 5 of culture, blastocysts developed from 'fast' embryos had a larger ICM number (17.4 ± 2.1 versus 7.4 ± 2.0, P < 0.01), a higher glucose consumption (21.2 ± 1.2 versus 14.3 ± 1.0 pmol/embryo/h, P < 0.001) and a lower glycolytic rate (expressed as the percentage of glucose converted to lactate) (49.6 ± 2.8 versus 59.7 ± 2.8%, P < 0.05) compared with 'slow' embryos. Further non-invasive metabolomic analysis revealed that 'fast' blastocysts consumed more aspartate (2.2 ± 0.1 versus 1.8 ± 0.1 pmol/embryo/h, P < 0.05) and produced little or no glutamate compared with 'slow' blastocysts (0.02 ± 0.07 consumed versus 0.32 ± 0.11 pmol/embryo/h produced, P < 0.05). Transfer of 'fast' blastocysts to pseudo-pregnant recipients resulted in higher fetal survival post-implantation compared with 'slow' blastocysts (69.6 versus 40.4%, P < 0.01). The timing of the first cleavage division was used to classify blastocysts as 'fast' or 'slow' embryos; however, a combination of several developmental kinetic markers (cleavage time of 3- to 8-cell, duration between cleavage division times) may be used to more accurately determine an embryo as 'fast' or 'slow'. Only the fastest and slowest quartiles (those embryos with the fastest and slowest first cleavage division) were analysed in this study. These findings show that kinetically different embryos develop into blastocysts with different metabolic profiles and viability. Work is now being undertaken to determine if using these viability markers in combination will increase embryo selection efficacy and further improve implantation and pregnancy rates. The study was funded by the University of Melbourne. The authors have no conflicts of interest to declare.