Blastocyst Stage Of Development Research Articles

O-030 The impact of aneuploidy on human peri-implantation development: findings from the extended in vitro culture of 191 human embryos

Abstract Study question How does aneuploidy affect lineage specification and the developmental potential of human embryos cultured up to 12 days post fertilization (D12)? Summary answer While distinct types of aberrations carry their own risk of embryonic arrest, prevailing aneuploidies appear to compromise lineage specification beyond the blastocyst stage of development. What is known already Chromosomal instability is a hallmark of human preimplantation development, with up to 50% of embryos diagnosed as aneuploid following preimplantation genetic testing (PGT). Aneuploidies are a major cause of implantation failure, pregnancy loss and human congenital defects. Yet despite their clinical relevance, the impact of distinct aberrations on early human development remains poorly understood. Innovative platforms for culturing human embryos up to D12 provide an important opportunity for investigating chromosomal instability. Understanding the effects of specific aneuploidies on human peri-implantation development will be key for advancing human embryo research and improving the interpretation of findings from in vitro embryo models. Study design, size, duration A total of 191 PGT blastocysts obtained from 103 patients were included in the study. Blastocysts were selected based on their original diagnosis, including embryos with single whole-chromosome aneuploidies (single trisomies, n = 37 and single monosomies, n = 71), single segmental aberrations (n = 22), embryos with complex chromosomal constitutions (two or more abnormalities, n = 44), as well as control euploid embryos (n = 17). Participants/materials, setting, methods Vitrified blastocysts were warmed and cultured up to D12 using an optimized protocol to generate embryo outgrowths. We correlated the original PGT diagnoses to developmental outcomes, including outgrowth viability, morphology and outgrowth size. We evaluated blastocyst quality using the Gardner grading system. Following culture, we immunostained 38 viable outgrowths for lineage-specific markers, including OCT4 (epiblast), GATA6 (hypoblast) and CK-7 (trophoblast). Fisher’s exact test was used for statistical analysis (p < 0.05 was considered significant). Main results and the role of chance Following extended culture, 43.5% of embryos remained viable and attached, while 56.5% degenerated and detached. No significant correlation was found between embryo grade and attachment rate. Euploid embryos exhibited the highest attachment rate, with 88.2% (15/17) forming viable outgrowths. Euploid embryos also generated significantly larger outgrowths than trisomic and monosomic embryos (p = 0.01). Embryos diagnosed with trisomies and segmental aberrations exhibited attachment rates comparable to euploid embryos, at 70.3% (p = 0.18) and 68.2% (p = 0.25), respectively. However, embryos with monosomies and complex chromosomal constitutions arrested more readily (28.2% and 15.9% viable, <0.00001, respectively). All euploid embryos (6/6) retained a discernible OCT4+ epiblast-like structure. This was similar to embryos with segmental aberrations and trisomies, with 75% (3/4) and 53.8% (7/13) of outgrowths containing OCT4+ cells, respectively. Nonetheless, distinct trisomies exerted varying effects on peri-implantation development in vitro. For instance, viable trisomies (13, 18, 21) developed similarly to euploid embryos, while outgrowths carrying a trisomy 22 had fewer OCT4+ cells (p = 0.02) and a higher number of GATA6+ cells (p < 0.01), suggesting a potential lineage proliferation defect. Monosomies had the most profound effects on development. Compared to euploid embryos, monosomic embryos formed significantly fewer OCT4+ outgrowths (28.6%, p = 0.02) and contained fewer epiblast (p < 0.0001) and hypoblast-like (p = 0.003) cells. Limitations, reasons for caution The limited number of embryos in certain groups warrants careful interpretation. The central drawback of the models is that embryo outgrowths are predominately flattened, which confounds identification of three-dimensional structures formed during normal embryogenesis. Future studies should encompass additional lineage specification markers and potential culture refinements to enhance our interpretation. Wider implications of the findings Our findings underscore the critical importance of reporting the chromosomal composition of human embryos utilized for research. This practice will enable a more nuanced interpretation of results from in vitro embryo models and ultimately advance our capabilities in human embryo research. Trial registration number not applicable

Absence of a molecular circadian clock in the preimplantation embryo is a conserved characteristic in the mammal.

It is not known when a functional circadian clock is established in the developing embryo. Lack of expression of key genes involved in the clock mechanism is indicative that a functional circadian clock mechanism is absent in the mammalian preimplantation embryo through the blastocyst stage of development. An embryonic circadian clock could conceivably organize cellular and developmental events temporally and in synchrony with other circadian rhythms in the mother. The hypothesis that a functional molecular clock exists in the preimplantation bovine, pig, human, and mouse embryo was tested by using publicly available RNAseq datasets to examine developmental changes in expression of the core genes responsible for the circadian clock - CLOCK, ARNTL, PER1, PER2, CRY1, and CRY2. In general, the transcript abundance of each gene decreased as development advanced to the blastocyst stage. The most notable exception was for CRY2, where transcript abundance was low and constant from the two-cell or four-cell to the blastocyst stage. Developmental patterns were generally the same for all species although there were some species-specific patterns such as an absence of PER1 expression in the pig, an increase in ARNTL expression at the four-cell stage in human, and an increase in expression of Clock and Per1 from the zygote to two-cell stage in the mouse. Analysis of intronic reads (indicative of embryonic transcription) for bovine embryos indicated an absence of embryonic transcription. Immunoreactive CRY1 was not detected in the bovine blastocyst. Results indicate that the preimplantation mammalian embryo lacks a functional intrinsic clock although specific components of the clock mechanism could conceivably play a role in other functions in the embryo.

Chrysoeriol Improves In Vitro Porcine Embryo Development by Reducing Oxidative Stress and Autophagy

Chrysoeriol (CHE) is a flavonoid substance that exists in many plants. It has various physiological and pharmacological effects, including anti-inflammatory, antioxidant, anti-tumor, and protective activity, especially for the cardiovascular system and liver. Among common livestock embryos, porcine embryos are often considered high-quality objects for studying the antioxidant mechanisms of oocytes. Because porcine embryos contain high levels of lipids, they are more vulnerable to external stimuli, which affect development. Our study explored the influence of CHE supplementation on oxidative stress in porcine oocytes and its possible mechanisms. Different concentrations of CHE (0, 0.1, 1, and 3 µM) were supplemented in the in vitro culture medium of the porcine oocytes. The results showed that supplementation with 1 µM CHE significantly increased the blastocyst rate and total cell number of embryos in vitro. After finding the beneficial effects of CHE, we measured reactive oxygen species (ROS), glutathione (GSH), and mitochondrial membrane potential (MMP) when the oocytes reached the 4-cell stage of development and determined the levels of apoptosis, cell proliferation, and autophagy at the blastocyst stage of development. The expression levels of some related genes were preliminarily detected by qRT-PCR. The results showed that the apoptosis of blastocysts in the CHE-treated culture also decreased compared with the untreated culture. Furthermore, CHE downregulated intracellular ROS and increased GSH in the embryos. CHE was also shown to improve the activity of mitochondria and inhibit the occurrence of autophagy. In addition, antioxidant-related genes (SOD1, SOD2, and CAT) and cell pluripotency-related genes (SOX2, OCT4, and NANOG) were upregulated. At the same time, apoptosis-related (Caspase 3) and autophagy-related (LC3B) genes showed a downward trend after supplementation with CHE. These results indicate that CHE improved the development of porcine embryos in vitro by reducing oxidative stress and autophagy levels.

Actions of DKK1 on the preimplantation bovine embryo to affect pregnancy establishment, placental function, and postnatal phenotype†.

One mechanism by which the maternal environment regulates the early embryo is by secretion of cell-signaling molecules. One of these is dickkopf WNT signaling pathway inhibitor 1. Objectives were to (A) resolve discrepancies in the literature regarding effects of dickkopf WNT signaling pathway inhibitor 1 in the bovine embryo on development of trophectoderm and competence to establish pregnancy after embryo transfer and (B) determine whether there are long-term consequences of dickkopf WNT signaling pathway inhibitor 1 on placental function and postnatal phenotype. Embryos produced in vitro were cultured with vehicle or 100ng/mL recombinant human dickkopf WNT signaling pathway inhibitor 1 from Days 5 to 7.5 of development (i.e., the morula and blastocyst stages of development). dickkopf WNT signaling pathway inhibitor 1 increased the number of cells positive for the trophectoderm marker CDX2 at Day 7.5 of development while having no effect on number of cells positive for the inner cell mass marker SOX2. There was no effect of dickkopf WNT signaling pathway inhibitor 1 on pregnancy or calving rate after transfer of blastocysts produced with Y-sorted semen to either lactating dairy cows or suckling beef cows. Treatment with dickkopf WNT signaling pathway inhibitor 1 at the morula-to-blastocyst stages programmed placental function, as measured by an effect of dickkopf WNT signaling pathway inhibitor 1 on plasma concentrations of pregnancy associated glycoproteins and placental lactogen at Day 160 of gestation (although not on other days examined). dickkopf WNT signaling pathway inhibitor 1 treatment also resulted in calves that were heavier at birth as compared to calves derived from control embryos. After birth, dickkopf WNT signaling pathway inhibitor 1 calves grew slower than controls. Results confirm that dickkopf WNT signaling pathway inhibitor 1 alters the developmental program of the bovine embryo to affect both prenatal and postnatal phenotypes.

The Role of Mitochondria in Human Fertility and Early Embryo Development: What Can We Learn for Clinical Application of Assessing and Improving Mitochondrial DNA?

Mitochondria are well known as ‘the powerhouses of the cell’. Indeed, their major role is cellular energy production driven by both mitochondrial and nuclear DNA. Such a feature makes these organelles essential for successful fertilisation and proper embryo implantation and development. Generally, mitochondrial DNA is exclusively maternally inherited; oocyte’s mitochondrial DNA level is crucial to provide sufficient ATP content for the developing embryo until the blastocyst stage of development. Additionally, human fertility and early embryogenesis may be affected by either point mutations or deletions in mitochondrial DNA. It was suggested that their accumulation may be associated with ovarian ageing. If so, is mitochondrial dysfunction the cause or consequence of ovarian ageing? Moreover, such an obvious relationship of mitochondria and mitochondrial genome with human fertility and early embryo development gives the field of mitochondrial research a great potential to be of use in clinical application. However, even now, the area of assessing and improving DNA quantity and function in reproductive medicine drives many questions and uncertainties. This review summarises the role of mitochondria and mitochondrial DNA in human reproduction and gives an insight into the utility of their clinical use.

Dynamics of small non-coding RNAs in bovine scNT embryos through the maternal-to-embryonic transition†.

The efficiency of somatic cell nuclear transfer (scNT) for production of viable offspring is relatively low as compared to in vitro fertilization (IVF), presumably due to deficiencies in epigenetic reprogramming of the donor cell genome. Such defects may also involve the population of small non-coding RNAs (sncRNAs), which are important during early embryonic development. The objective of this study was to examine dynamic changes in relative abundance of sncRNAs during the maternal-to-embryonic transition (MET) in bovine embryos produced by scNT as compared to IVF by using RNA sequencing. When comparing populations of miRNA in scNT versus IVF embryos, only miR-2340, miR-345, and miR34a were differentially expressed in morulae, though many more miRNAs were differentially expressed when comparing across developmental stages. Also of interest, distinct populations of piwi-interacting like RNAs (pilRNAs) were identified in bovine embryos prior to and during embryonic genome activation (EGA) as compared bovine embryos post-EGA and differentiated cells. Overall, sncRNA sequencing analysis of preimplantation embryos revealed largely similar profiles of sncRNAs for IVF and scNT embryos at the 2-cell, 8-cell, morula, and blastocyst stages of development. However, these sncRNA profiles, including miRNA, piRNA, and tRNA fragments, were notably distinct prior to and after completion of the MET.

Does miRNA Expression in the Spent Media Change During Early Embryo Development?

Distinct miRNA populations have been detected in the spent media of in-vitro culture systems. However, profiling has been limited to media conditioned with blastocyst-stage embryos. Therefore, the aim of the study was to profile extracellular miRNAs throughout the pre-implantation period in bovine embryos. To achieve this, cumulus oocyte complexes were aspirated from ovaries, in-vitro matured, fertilized, and cultured under standard laboratory procedures to the 2-cell, 8-cell, or blastocyst stage of development. At each developmental stage, 25 μl of spent in-vitro culture media was collected, pooled to 300 μl, and processed for total RNA extraction. In-vitro culture media, which never came in contact with any embryos, were additionally processed for total RNA extraction to serve as a negative control. Following hybridization on a GeneChip miRNA 4.0 array, comparative analysis was conducted between spent media and control samples. In total, 111 miRNAs were detected in the spent media samples, with 56 miRNAs identified in blastocyst spent media, 14 miRNAs shared between 8-cell and blastocyst spent media, 7 miRNAs shared between all 3 conditions, and 6 miRNAs exclusive to 2-cell spent media. miRNA-mRNA target prediction analysis revealed that the majority of genes predicted to be regulated by the miRNAs identified in the study have roles in cellular process, metabolic process, and biological regulation. Overall, the study suggest that miRNAs can be detected in the spent media of in-vitro culture system throughout the pre-implantation period and the detected miRNAs may influence genes impacting early embryo development.

84 Characterization of agouti-signalling protein expression within the bovine ovary and early embryo

Factors present in the oocyte and surrounding follicular cells aid in the attainment of oocyte competence. Agouti-signalling protein (ASIP) is a known regulator of melanocyte function through binding to melanocortin receptors including MC1R and MC4R. Additionally, ASIP has been classified as an adipokine due to a link with insulin resistance and obesity in humans. In mice, expression is limited to hair follicles where ASIP regulates hair pigmentation. In cattle, however, ASIP mRNA has been detected in a variety of tissues, including adipose, skin, heart, testis, and the ovary. Despite ovarian expression, the role of ASIP in reproduction remains undetermined. Bovine ASIP is a secreted protein consisting of 133 amino acids. The aim of this experiment was to provide a detailed description of the ASIP expression profile within the bovine ovary and during early embryonic development. Reverse transcription PCR (RT-PCR) was conducted to analyse ASIP, MC1R, and MC4R mRNA expression. Samples examined included fetal ovaries from gestational day 90 to 250, adult ovary, fetal testis, adult testis, and 12 somatic tissues including adrenal, cerebral cortex, gut, heart, intestine, kidney, liver, lung, muscle, pituitary, stomach, and thymus. Amplification of ribosomal protein L19 (RPL19) served as a positive control for all samples. Expression of ASIP was detected in the fetal testis, 9 somatic tissues, and the fetal and adult ovary. In the fetal ovary, ASIP was detected as early as 90 days of gestation and continued throughout gestation. Expression of the ASIP receptors, MC1R and MC4R, were detected exclusively in the fetal ovary. To further characterise ASIP expression, quantitative real-time PCR (RT-qPCR) was utilised to examine samples including germinal vesicle and MII oocytes (pool of 10 oocytes), invitro-produced embryos ranging from the 2-cell to blastocyst stages (pool of 10 embryos), and cumulus and granulosa cells collected from a pool from 5 cumulus–oocyte complexes (COCs) and follicles, respectively. Theca cells from a single follicle were analysed. Samples with cycle threshold values below 35 were considered to express the gene of interest. Of the follicular cells examined, ASIP expression was present in theca, granulosa, and cumulus cells. ASIP expression was detected in both GV and MII oocytes. Early embryonic expression of ASIP was detected in the 2-cell embryo and continued to the blastocyst stage of development. In conclusion, ASIP is present in the bovine adult and fetal ovary, follicular cells including cumulus, granulosa, and theca cells, GV and MII oocytes, and invitro-produced embryos from the 2-cell to blastocyst stages. Future research will focus on identifying the function of ovarian and early embryonic ASIP in cattle.

Effect of alpha-lipoic acid during preimplantation development of cattle embryos when there were different in vitro culture conditions

In many species, alpha-lipoic acid (ALA) is essential for embryo development. There, therefore, was investigation of effects of ALA supplementation to culture media for in vitro development of cattle embryos. In Experiment I, there were assessments of embryo production and oxidative status of cattle embryos derived by in vitro maturation and fertilization (IVM/IVF)that were cultured until the blastocyst stage of development using different ALA concentrations (5, 25 and 100 μM), fetal bovine serum (FBS) and amino acids (aa) as well as 20 % oxygen (O2) in the culture atmosphere. In Experiment II, embryos were cultured without FBS, at different ALA concentrations (2.5, 5 and 7.5 μM) and in the presence or absence of aa when there was a 7 % O2 atmosphere. Embryo development rates and blastocyst quality were evaluated. With 20 % O2 concentration, treatment with 100 μM ALA resulted in lesser hatching rates and development to the blastocyst stage (P < 0.01), while with supplementation with 5 μM ALA there were lesser (P = 0.04) glutathione concentrations and greater protein contents of embryos (P < 0.01). Culturing in the 7 % O2 atmosphere, combined with supplementation with 2.5 μM ALA with FBS and aa resulted in a greater blastocyst cell number (P = 0.03) and lesser hatching rates (P = 0.04). Taken together, results indicate supplementation with the greater ALA concentrations resulted in impairment of embryo development, regardless of the O2 concentration imposed during the culture period, while the relatively lesser supplementation-concentrations with ALA led to improvements in embryo quality.

49 Expression and actions of the dickkopf-1 receptors KREMEN1 and KREMEN2 in the bovine pre-implantation embryo

Dickkopf-1 (DKK1) is a secreted inhibitor of canonical WNT signaling expressed in the endometrium that can increase the competence of bovine blastocysts to establish pregnancy after transfer into recipients. The mechanism by which DKK1 regulates embryo function is not known. KREMEN1 and KREMEN2 are paralogs that function as DKK1 transmembrane receptors. Binding of DKK1 to KREMEN leads to internalization of the LRP5/6 WNT co-receptor and inhibition of β-catentin (CTNNB1)-dependent WNT signaling. Here we evaluated whether the bovine pre-implantation embryo expresses KREMEN1 and KREMEN2 and whether knockdown of KREMEN1 would alter embryonic development and accumulation of CTNNB1. In the first experiment, expression of KREMEN1 and KREMEN2 was determined in individual matured oocytes and embryos at the 2-cell, 3-4-cell, 5-8-cell, 9-16-cell, morula, and blastocyst stages of development (n=4) using RT-qPCR with GAPDH and YWHAZ as internal controls. Data were analysed by analysis of variance using GLM procedure of SAS (SAS Institute Inc.; stage, replicate in model), and Tukey test for multiple comparisons. Expression of both KREMEN1 (P&amp;lt;0.0001) and KREMEN2 (P=0.003) varied between stages of development. There was a large decline in transcript abundance for KREMEN1 after the 9-16-cell stage. Expression (fold-change) relative to housekeeping genes was 0.29, 0.47, 0.27, 0.47, 0.34, 0.05, and 0.01 (standard error=0.08) for oocyte, 2-cell, 4-cell, 5-8 cell, 9-16-cell, morula, and blastocyst stages, respectively. Expression of KREMEN2 was low at early stages of development, increased at the 8-16 cell and morula stages (i.e. after embryonic genome activation), and then declined. Expression (fold-change) relative to housekeeping genes was 0.00, 0.04, 0.03, 0.05, 0.12, 0.15, and 0.01 (standard error=0.04) for oocyte, 2-cell, 4-cell, 5-8-cell, 9-16-cell, morula, and blastocyst stages, respectively. In the second experiment, we determined effects of a GapmeR antisense oligonucleotide directed against KREMEN1 on development to the blastocyst stage and amounts of immunoreactive CTNNB1 in the blastocyst. The experiment was performed in 3 replicates using 217 putative zygotes per treatment. The GapmeR treatment did not affect cleavage rate (78.5±0.02% for GapmeR vs. 71.7%±0.02% for vehicle; P=0.21), percent putative zygotes becoming blastocysts (23.4±0.02% vs. 20.0±0.02%; P=0.57), or the percent of cleaved embryos becoming blastocysts (30.6±0.03% vs. 28.8±0.03%; P=0.88). Amounts of immunoreactive CTNNB1 were determined in blastocysts collected at 168h after insemination (n=7/treatment). Treatment with GapmeR tended (P=0.08) to increase the fluorescence intensity of CTNNB1 (1761±68 vs. 1583±72 units). In summary, KREMEN1 is expressed before genome embryonic activation, whereas KREMEN2 is expressed in 8-16 cells and morula. Knocking down KREMEN1 does not compromise embryonic development, but preliminary studies indicate KREMEN1 can regulate CTNNB1 abundance. Support was provided by USDA-NIFA 2017-67015-26452.

181 The effects of aging in the oocyte induced by trichostatin A

Successful fertilization and embryonic development decreases for an aged oocyte that is not fertilized during the optimal window of time after ovulation. If fertilization of an aged oocyte does occur, the developing embryo is prone to have a delay through meiosis and a decrease in cleavage and blastocyst formation. Trichostatin A (TSA) prohibits the breakdown of the germinal vesicle during meiosis. The objective of this study was to create an invitro model to study the effects of aging in pig oocytes. Oocytes (n=1489) were matured with or without TSA (100ngmL−1) for 24 or 48h followed by an additional 16h of maturation without TSA. A portion of the oocytes were evaluated for stage of meiosis (MI and MII) before maturation (n=95), after the first part of maturation (OMI; 24 or 48h, n=363), or after completing maturation (OMII; 16h, n=230). The remaining oocytes (n=801) were fertilized for 6 to 8h and potential embryos developed. A portion of the embryos were evaluated for fertilization characteristics 12h after IVF (n=400). Remaining embryos (n=401) were evaluated for cleavage by 48h and blastocyst formation by 144h, after IVF. Data were analysed using ANOVA and Tukey's test. Oocytes matured without TSA for 64h had a significantly higher (P&amp;lt;0.05) percentage of oocytes at the MI stage of meiosis compared with all other treatment groups (16.00±6.80), and oocytes matured without TSA for 40h had a significantly higher (P&amp;lt;0.05) percentage of oocytes at the MII stage of meiosis compared with all other treatment groups (14.7±11.30). The results (Table 1) indicate that supplementation of TSA to OMI significantly decreased fertilization penetration rates compared with not supplementing TSA for 24h. However, the percent of embryos cleaved by 48h after IVF was significantly higher in oocytes matured for 40h compared with those matured for 64h. The percent of embryos reaching the blastocyst stage of development by 144h after IVF was significantly lower in oocytes matured for 64h compared with those matured for 40h without TSA. In conclusion, these results suggest that supplementation of TSA during early maturation delays meiosis and decreases fertilization, and increasing maturation time causes a decrease in early embryonic development. By supplementing TSA during early maturation and increasing the length of maturation by 24h, we have created an invitro model to study the effects of aging in pig oocytes. Table 1.Fertilization and developmental characteristics of oocytes1 Amount of TSA added during OMI (ngmL−1) Length of OMI (h) Oocytes penetrated (%) Polyspermicoocytes2 (%) Oocytes with MPN2 (%) Cleavage (%) Day 7 blastocyst (%) 0 24 83.00±3.80a 15.70±4.00 75.90±4.70 77.20±4.20a 27.70±4.20a 0 48 72.00±4.50ab 20.80±4.80 76.40±5.00 58.00±5.00bc 12.00±5.00b 100 24 65.00±4.80b 30.80±5.80 73.80±5.50 68.00±4.70ab 21.00±4.70ab 100 48 56.00±5.00b 30.40±6.20 62.50±6.50 46.00±5.00c 8.00±5.00b a,bMeans with different superscripts in the same column differ (P&amp;lt;0.05). 1TSA, trichostatin A; OMI, oocyte maturation 1. 2Percent of the number of oocytes penetrated.

Importance of prostate androgen-regulated mucin-like protein 1 in development of the bovine blastocyst

BackgroundProstate androgen-regulated mucin-like protein 1 (PARM1) is a pro-proliferative and anti-apoptotic glycoprotein involved in the endoplasmic reticulum (ER) stress response. A single nucleotide polymorphism in the coding region of PARM1 has been associated with competence of bovine embryos to develop to the blastocyst stage. Here we tested the importance of PARM1 for development by evaluating consequences of reducing PARM1 mRNA abundance on embryonic development and differentiation, gene expression and resistance to ER stress.ResultsKnockdown of PARM1 using an anti-PARM1 GapmeR did not affect competence of embryos to develop into blastocysts but decreased the number of trophectoderm (TE) cells in the blastocyst and tended to increase the number of cells in the blastocyst inner cell mass (ICM). Treatment of embryos with anti-PARM1 GapmeR affected expression of 4 and 3 of 90 genes evaluated at the compact-morula and blastocyst stage of development at days 5.5 and 7.5 after fertilization, respectively. In morulae, treatment increased expression of DAB2, INADL, and STAT3 and decreased expression of CCR2. At the blastocyst stage, knockdown of PARM1 increased expression of PECAM and TEAD4 and decreased expression of CCR7. The potential role of PARM1 in ER stress response was determined by evaluating effects of knockdown of PARM1 on development of embryos after exposure to heat shock or tunicamycin and on expression of ATF6, DDIT3 and EIF2AK3 at the compact morula and blastocyst stages. Both heat shock and tunicamycin reduced the percent of embryos becoming a blastocyst but response was unaffected by PARM1 knockdown. Similarly, there was no effect of knockdown on steady-state amounts of ATF6, DDIT3 or EIF2AK3.ConclusionPARM1 participates in formation of TE and ICM cells in early embryonic development but there is no evidence for the role of PARM1 in the ER stress response.

146 Quercetin supplementation during boar semen thawing and incubation improves the in vitro production of pig embryos

Elevated levels of reactive oxygen species in the in vitro environment cause oxidative stress, which leads to membrane damage, decreased fertility, and morphological deformities of spermatozoa. Antioxidants, such as quercetin (a polyphenol flavonoid), are often supplemented to reduce the effects of oxidative stress on spermatozoa. Supplementing frozen-thawed boar semen with quercetin improves sperm forward progressive motility, viability and lipid peroxidation up to 10h after thawing. However, the effects of fertilizing with quercetin-supplemented sperm are unknown. Therefore, the objective of this study was to determine the effects of supplementing quercetin (0.25, 0.50, 0.75mM) during the thawing and incubation of frozen-thawed boar semen on oocyte fertilization characteristics (n=400) and subsequent embryonic development (n=1340) at 48 and 144h for cleavage and blastocyst formation, respectively. Oocytes from aspired aspirated mature follicles (3-6mm diameter) were obtained from a local abattoir and matured in medium 199 for 40 to 44h at 38.5°C in an atmosphere of 5% CO2. Fertilization was performed using pooled frozen-thawed semen from 3 different boars, and co-incubation of the sperm (2×105 sperm mL−1) and oocytes (30 oocytes/well) lasted for 6 to 8h at 38.5°C in an atmosphere of 5% CO2. Data were analysed using ANOVA with the main effects including treatment, well and replicate. Chi-squared analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no differences in penetration rates and male pronuclear formation between treatment groups; however, supplementation of 0.25 (18.18±10.63%), 0.50 (20.93±9.89%) and 0.75mM (18.07±12.02%) quercetin significantly decreased (P&amp;lt;0.05) polyspermic penetration rates compared with no supplementation (40.00±11.34%). Embryos produced from frozen-thawed boar sperm supplemented with 0.25 and 0.50mM quercetin had a significantly higher percentage (P&amp;lt;0.05) of embryos reaching the 2-cell stage of development by 48h after IVF (75.00±7.89%, 68.75±2.23%, respectively) compared with 0.75mM quercetin supplementation (64.62±3.88%) and no supplementation (62.97±4.11%). Supplementation of 0.25 (44.12±6.23%), 0.50 (43.75±7.02%) and 0.75mM (43.08±2.98%) quercetin to the sperm significantly increased (P&amp;lt;0.05) the percentage of embryos reaching the blastocyst stage of development by 144h after IVF compared with no supplementation (28.27±8.07%). These results indicate that supplementing frozen-thawed boar semen with quercetin decreases the incidence of polyspermic penetration and improves early embryonic development in pigs.

Identification of Beta Human Chorionic Gonadotrophin (bHCG) Expression in Frozen Cleavage Embryo of In Vitro Fertilization (IVF) Patients: An Essential Approach for Better Outcome towards Human Being Inheritance in Line with Maqasid Syari’ah Concept

Expression of beta-human chorionic gonadotropin (bHCG) mRNA in early cleavage cell stages is important in predicting embryo viability at blastocyst stage of development. Embryo viability is crucial for in vitro fertilization (IVF) treatment to be successful in terms of increment of implantation and pregnancy rate. In order to establish fertilization outside the human body, optimum condition mimicking the natural body environment like hormones and growth factors as well as impeccable timing must be established. Imam Muslim narrated from Hudhayfa ibn Asad that the Prophet Muhammad (SAW) said: “After the sperm-andovum drop (nut.fa) has been [in the uterus] forty-two days, Allah sends it an angel that gives it form and fashions its hearing, sight, skin, flesh, and skeleton”. Therefore the aim of this study is to find the significant association of bHCG expression with early cell stage cleavages and its relationship as predictive marker for potential embryo implantation. Our study focuses on leftover frozen embryos from eight patients consists of six pregnant patients and two non-pregnant patients. We assessed the human bHCG mRNA expression at different cell cleavage stages in these frozen embryos using reverse transcriptasepolymerase chain reaction (RT-PCR). Our results have shown three out of eight patients with five to ten cells of blastomeres were expressed with bHCG. This study indicated that bHCG was expressed on frozethawed late cleavage stage of embryos in IVF patients.

Specification of trophoblast from embryonic stem cells exposed to BMP4.

Trophoblast (TB) comprises the outer cell layers of the mammalian placenta that make direct contact with the maternal uterus and, in species with a highly invasive placenta, maternal blood. It has its origin as trophectoderm, a single epithelial layer of extra-embryonic ectoderm that surrounds the embryo proper at the blastocyst stage of development. Here, we briefly compare the features of TB specification and determination in the mouse and the human. We then review research on a model system that has been increasingly employed to study TB emergence, namely the BMP4 (bone morphogenetic protein-4)-directed differentiation of human embryonic stem cells (ESCd), and discuss why outcomes using it have proved so uneven. We also examine the controversial aspects of this model, particularly the issue of whether or not the ESCd represents TB at all. Our focus here has been to explore similarities and potential differences between the phenotypes of ESCd, trophectoderm, placental villous TB, and human TB stem cells. We then explore the role of BMP4 in the differentiation of human pluripotent cells to TB and suggest that it converts the ESC into a totipotent state that is primed for TB differentiation when self-renewal is blocked. Finally we speculate that the TB formed from ESC is homologous to the trophectoderm-derived, invasive TB that envelopes the implanting conceptus during the second week of pregnancy.

5 Live Offspring Produced from Reproductive Material Recovered During the Annual Cull of Bison from Yellowstone National Park

Bison from Yellowstone National Park (YNP) have a unique genetic history that makes them a conservation priority for the species. Unfortunately, there is a high prevalence of the zoonotic disease brucellosis in the herd. Part of the management strategy for controlling the disease and herd size in YNP is an annual culling process. This culling may result in loss of valuable genetic material and, until now, has been a missed opportunity to recover and preserve those genetics. The goal of this project was to demonstrate the feasibility of producing healthy offspring from reproductive material collected during the nonbreeding season from bison postmortem and to determine effects of donor parameters, including sexual maturity and pregnancy status on IVF outcomes. Oocytes were collected from ovaries harvested from bison within 2 h of slaughter during winters of 2014 to 2017 for use in in vitro embryo production (IVP). Frozen-thawed semen from 7 YNP bulls collected via electroejaculation in vivo or from epididymal flushes postmortem was used for IVF. Embryos were produced using standard cattle IVP procedures. On Days 7, 7.5, and 8 of in vitro culture, embryos were assessed for developmental stage and quality, and Grade 1 and 2 embryos were vitrified on Cryotops® (Kitazato, Tokyo, Japan) in a two-step equilibration process. Embryos were then stored until the breeding season when they were warmed, cultured for 6 h, evaluated for survival, and transferred to healthy bison recipients. Data were analysed using a Student’s t-test. Age did not affect the mean number of oocytes collected from a mature female (n = 254) v. juvenile (n = 27; 24 ± 16 v. 24 ± 13), embryo cleavage rates (54 ± 24% v. 45 ± 24%), or the rate of blastocyst development (8 ± 12% v. 7 ± 8%, respectively). Pregnancy status did have a significant effect on the number of oocytes collected per female (pregnant 22 ± 15%, n = 258; nonpregnant 26 ± 20%, n = 82; P = 0.04) and the percent of oocytes that cleaved (pregnant 56 ± 23%, n = 189; nonpregnant 47 ± 24, n = 61; P = 0.005). No difference was detected in the percent of embryos that reached the blastocyst stage of development (pregnant 8 ± 10%, nonpregnant 8 ± 15%). In August 2016, 12 embryos were transferred singly or in pairs to 10 recipient bison synchronized using a standard synch protocol. Ten days following removal of the CIDR, embryos were transferred to recipient bison with a palpable corpus luteum. In spring of 2017, a healthy female calf was born to a recipient who had received 2 Grade-2 blastocysts as graded at the end of the 6-h incubation period. The genetic dam of the calf was a pregnant adult bison cow. This calf demonstrates that live offspring can be generated from oocytes collected from bison postmortem in the nonbreeding season. Although cleavage rates were higher with oocytes from pregnant bison, viable embryos were generated from nonpregnant adults and juvenile females, warranting continued collection of their oocytes and banking of resulting embryos. To our knowledge, this is the first bison calf produced by in vitro production using postmortem reproductive material.

156 OVIDUCTAL FLUID SUPPLEMENTATION DURING MATURATION AND FERTILIZATION IMPROVES IN VITRO EMBRYONIC DEVELOPMENT IN PIGS

Oviducal fluid has a major role in the maturation of gametes and the process of fertilization. The objective of this study was to determine the effects of oviducal fluid supplementation in vitro, during oocyte maturation and IVF on fertilization characteristics and early embryonic development rates. Oocytes from aspired aspirated mature follicles (3–6 mm diameter) were obtained from a local abattoir. During the last 24 h of maturation, oocytes (n = 1303) were placed into maturation media supplemented either 1% (vol/vol) or 5% (vol/vol) thawed snap-frozen oviducal fluid. Fertilization was performed using pooled frozen-thawed semen from 3 different boars. During IVF, the fertilization medium was supplemented with 1% (vol/vol) or 5% (vol/vol) oviducal fluid. Fertilization characteristics were evaluated 12 h after IVF and rates of embryonic cleavage and blastocyst development were observed at 48 and 144 h after IVF, respectively. Data were analysed using ANOVA with the main effects including treatment, well, and replicate. Chi-square analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no significant differences in the percentages of oocytes that reached metaphase II by the end of maturation or in sperm penetration rates after IVF. However, oocytes treated with 1% (vol/vol) oviducal fluid during the end of maturation and IVF (33.33 ± 2.61) and 5% (vol/vol) oviducal fluid during maturation (33.33 ± 2.66) or IVF (39.53 ± 3.78) had significantly less (P &lt; 0.05) incidence of polyspermic penetrations and a significantly higher (P &lt; 0.05) incidence of male pronuclear formation (87.50 ± 4.01; 86.67 ± 4.83; 86.05 ± 3.19, respectively) compared with no oviducal fluid supplementation. Oocytes supplemented with 5% (vol/vol) oviducal fluid during maturation and IVF had significantly lower (P &lt; 0.05) incidences of polyspermic penetration (27.91 ± 2.50) and significantly higher (P &lt; 0.05) percentages of embryos reaching the 2-cell stage (81.76 ± 3.72) and blastocyst stage of development (37.74 ± 1.09) by 48 and 144 h, respectively, compared with all other groups. The results of this study suggest that supplementing 5% (vol/vol) oviducal fluid during maturation and IVF improves the success rates of in vitro embryo development in pigs.

154 COMPARISON OF DIFFERENT CULTURE MEDIA AND INCUBATION METHODS ON CULTURING MURINE EMBRYOS IN VITRO USING STRAW AS A RECEPTACLE

In vitro fertilization in the straw system might increase the efficiency of fertilization and the quality of blastocyst formation as compared with micro-drops-IVF systems. The aim of the study was to in vitro fertilize mouse oocytes and culture the resulting zygotes in bi-gas incubator and in a goat vagina and compare the in vitro embryo developmental stages in TCM-199 and Ham’s F10 culture media until the blastocyst-stage of development. F1 generations (Balb C × C57) were used to harvest oocytes and spermatozoa. The fresh sperm were capacitated in different incubation methods (bi-gas incubator and in the vagina of a goat). A volume of 2–4 µL of Ham’s F10 containing capacitated sperm (~8 × 106 per mL) were placed into Ham’s F10 fertilization drops under the oil, containing 10 oocytes and penicillamine, hypotaurine, and epinephrine for enhancing sperm motility and penetration of oocytes. The same procedure was used with the TCM-199 medium and IVF drops without oil (both TCM-199 and Ham’s F10) for straw filling. The presumptive embryos in Ham’s F10 and TCM-199 were divided into different groups: first group were cultured in micro-drops, second group the embryos were aspirated in semen straws and placed in the incubator (incubator straws) for culture, and other straws were covered with a sponge and inserted in the vagina of a goat (vaginal straws) for culture. The resulted blastocysts were stained using Hoechst 33528 solution and blastomeres were counted on a fluorescent UV light inverted microscope at 400× magnification (Nikon Eclipse TI, Narishige Co., Ltd., Amityville, NY, USA). The results were analysed by 2 × 2 factorial designs and Student’s t-test was used to separate the mean. There was no statistical difference (P &gt; 0.05) between the media and incubators on the stage of murine embryo development. The overall fertilization rate was 94 to 99%. The incubator straws with Ham’s F10 (80.5%) had the highest rate of embryos that reached the blastocyst stage, followed by incubator straws with TCM-199 (77.0%), and vaginal straws with Ham’s F10 (60.0%) had the lowest rate of embryos that reached the blastocyst stage. The overall mean number of blastomeres in the blastocyst stage of the embryos ranged from 85 ± 9 to 90 ± 9 cells in all receptacles and incubators. It was concluded that the fertilization and culturing of murine embryos are possible in straws incubated in a bi-gas incubator and in the goat vagina as an alternative method of fertilizing oocytes and culturing murine embryos. In addition, Ham’s F10 and TCM-199 can both be used to fertilize oocytes and culture murine embryos until blastocyst formation embryo in vitro, incubated in a bi-gas incubator or in the vagina.

Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified

To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment. Prospective embryo cohort study. Academic center and private invitro fertilization (IVF) clinic. Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts. Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting. Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos. Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (∼4 copies) and mtDNA (∼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis. Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin.

Human Oocyte-Derived Methylation Differences Persist in the Placenta Revealing Widespread Transient Imprinting.

Thousands of regions in gametes have opposing methylation profiles that are largely resolved during the post-fertilization epigenetic reprogramming. However some specific sequences associated with imprinted loci survive this demethylation process. Here we present the data describing the fate of germline-derived methylation in humans. With the exception of a few known paternally methylated germline differentially methylated regions (DMRs) associated with known imprinted domains, we demonstrate that sperm-derived methylation is reprogrammed by the blastocyst stage of development. In contrast a large number of oocyte-derived methylation differences survive to the blastocyst stage and uniquely persist as transiently methylated DMRs only in the placenta. Furthermore, we demonstrate that this phenomenon is exclusive to primates, since no placenta-specific maternal methylation was observed in mouse. Utilizing single cell RNA-seq datasets from human preimplantation embryos we show that following embryonic genome activation the maternally methylated transient DMRs can orchestrate imprinted expression. However despite showing widespread imprinted expression of genes in placenta, allele-specific transcriptional profiling revealed that not all placenta-specific DMRs coordinate imprinted expression and that this maternal methylation may be absent in a minority of samples, suggestive of polymorphic imprinted methylation.