Affect Embryo Viability Research Articles

96 The effect of embryo stage and biopsy on embryo viability

Abstract The importance of embryo biopsy is running a 50K genomic test will be completed on the DNA biopsy, giving us the ability to collect genetic prediction data before the calf is even born. The objective of this study was to compare the viability of bovine embryos (n = 24) at different stages of embryo maturation, after microblade biopsy and embryo transfer. This process was achieved by taking a small biopsy of each embryo. The results of aspirating oocytes from packing house ovaries and in vitro fertilization (IVF), within a 48 h time period marked d 0. After a 7-d incubation period, the results consisted of 14 blastocysts and 10 early blastocysts. The final sample size, of 24 embryos, differing in stages between early blastocyst (TRT 6) and blastocyst (TRT 7). All the embryos, except for one, was a Grade 1 embryo. On d 7 the embryos were grouped by stage of embryo. Each embryo was washed, and biopsied with a microblade by a single technician. After the biopsy, the embryos were incubated for a 24 h period. Immediately after the incubation period, each embryo was analyzed for viability. On d 8, TRT 7 embryos had a 50% viability rate (n = 7), whereas TRT 6 embryos had a 70% viability rate (n = 7). Data were analyzed using GLIMMIX procedure of SAS 9.4. The viability did not differ between either treatment (P = 0.58). However, the biopsy of each embryo resulted in a significant effect on embryo viability (P < 0.01). In conclusion, using embryo biopsy and the stage of the embryo may affect embryo viability.

Advancing rhodium nanoparticle-based photodynamic cancer therapy: quantitative proteomics and in vivo assessment reveal mechanisms targeting tumor metabolism, progression and drug resistance.

Rhodium nanoparticles have been recently discovered as good photosensitizers with great potential in cancer photodynamic therapy by effectively inducing cytotoxicity in cancer cells under near-infrared laser. This study evaluates the molecular mechanisms underlying such antitumoral effect through quantitative proteomics. The results revealed that rhodium nanoparticle-based photodynamic therapy disrupts tumor metabolism by downregulating key proteins involved in ATP synthesis and mitochondrial function, leading to compromised energy production. The treatment also induces oxidative stress and apoptosis while targeting the invasion capacity of cancer cells. Additionally, key proteins involved in drug resistance are also affected, demonstrating the efficacy of the treatment in a multi-drug resistant cell line. In vivo evaluation using a chicken embryo model also confirmed the effectiveness of the proposed therapy in reducing tumor growth without affecting embryo viability.

USE OF A MODIFIED SPECIFIC GRAVITY DEVICE TO DETERMINE VIABILITY AND QUALITY OF HUMAN CRYOPRESERVED EMBRYOS: A PILOT TRIAL

Cryopreservation is currently the only effective means of long-term embryo storage, transport, and genetic testing of human and animal embryos. It also provides for significant flexibility in ART practices. However, cryopreservation can also lead to embryo damage, affecting embryo viability and post-thaw development. Previous research from this lab has demonstrated that a Modified Specific Gravity Device (MSGD) can be used to determine the viability status and future development potential of embryos in various animal species. The current pilot trial describes the use of the MSGD to predict post-thaw survival of donated cryopreserved human blastocysts.

Profiling the Influence of Gene Variants Related to Folate-Mediated One-Carbon Metabolism on the Outcome of In Vitro Fertilization (IVF) with Donor Oocytes in Recipients Receiving Folic Acid Fortification.

Nutritional status and gene polymorphisms of one-carbon metabolism confer a well-known interaction that in pregnant women may affect embryo viability and the health of the newborn. Folate metabolism directly impacts nucleotide synthesis and methylation, which is of increasing interest in the reproductive medicine field. Studies assessing the genetic influence of folate metabolism on IVF treatments have currently been performed in women using their own oocytes. Most of these patients seeking to have a child or undergoing IVF treatments are advised to preventively intake folate supplies that restore known metabolic imbalances, but the treatments could lead to the promotion of specific enzymes in specific women, depending on their genetic variance. In the present study, we assess the influence of candidate gene variants related to folate metabolism, such as Serine Hydroxymethyltransferase 1 SHMT1 (rs1979276 and rs1979277), Betaine-Homocysteine S-Methyltransferase BHMT (rs3733890), Methionine synthase reductase MTRR (rs1801394), Methylenetetrahydrofolate reductase MTHFR (rs1801131 and rs1801133), methionine synthase MTR (rs12749581), ATP Binding Cassette Subfamily B Member 1 ABCB1 (rs1045642) and folate receptor alpha FOLR1 (rs2071010) on the success of IVF treatment performed in women being recipients of donated oocytes. The implication of such gene variants seems to have no direct impact on pregnancy consecution after IVF; however, several gene variants could influence pregnancy loss events or pregnancy maintenance, as consequence of folic acid fortification.

The human embryo following biopsy on day 5 versus day 3: viability, ultrastructure and spindle/chromosome configurations

Research questionAre there any differences in viability, spindle abnormalities and mitochondrial and other organelle structures amongst embryos biopsied on day 3 versus day 5 before and after vitrification? DesignA total of 240 day 3 biopsied embryos that developed to blastocysts but were rejected for transfer following preimplantation genetic testing for monogenic/single gene defects (PGT-M) (n = 115) or for aneuploidies (PGT-A) (n = 125) were divided into two groups: (i) 120 blastocysts treated for viability, spindle/chromosome configuration (SCC) analysis and transmission electron microscopy (TEM) analysis (fresh n = 20, n = 20, n = 20 and following vitrification/warming n = 20, n = 20, n = 20); (ii) 120 embryos were re-biopsied at the blastocyst stage and treated for viability, SCC and TEM analysis (fresh n = 20, n = 20, n = 20 and following vitrification/warming n = 20, n = 20, n = 20). Also, 60 vitrified blastocysts biopsied only on day 5 that were rejected for transfer following PGT-M (n = 6) or PGT-A (n = 54) were treated following warming for viability (n = 20), SCC (n = 20) and TEM analysis (n = 20). ResultsNo differences were observed in SCC and ultrastructure between embryos biopsied on day 5 and day 3 but following vitrification higher numbers of abnormal spindles, distension of mitochondria, multivesicular bodies, lipofuscin droplets, altered cell junctions and occasionally excessive accumulation of glycogen granules were evident. The fresh day 3 biopsied group also had a lower incidence of damaged (propidium iodide-stained) cells compared with the fresh day 3+5 (P = 0.02) and the vitrified day 5 (P = 0.001) biopsied groups. ConclusionsBiopsies on day 5 and day 3 do not adversely affect embryo viability, SCC or ultrastructure, although following vitrification minimal embryo quality-dependent increases in spindle abnormalities and cell damage are observed.

P–518 Assessment of mitochondrial DNA viability ratio in day–4 biopsied embryos as an add-in to select euploid embryos for single embryo transfer

Abstract Study question Is mitochondrial DNA viability ratio of day–4 biopsied embryos associated with embryo implantation potential? Summary answer The mitochondrial DNA viability ratio is significantly higher in embryos that implant. The score might help to select euploid embryos for single embryo transfer. What is known already Embryo euploidy is a critical factor for successful pregnancy outcomes. However, transfer of euploid embryos does not invariably result in implantation, thus indicating that other factors may play a role. Metabolic rates and adenosine triphosphate content vary significantly in oocytes and embryos and might affect embryo viability. Embryo function, indirectly measured by mitochondrial DNA viability ratio (mtV) has emerged as a potential quantitative biomarker for embryonic selection before the transfer, but clinical data remains limited. The purpose of this study is to characterize and compare mtV in euploidy day 4 embryos. Study design, size, duration Retrospective cohort study carried out between Jan. 2017 to Jan. 2020, involving 75 infertile couples undergoing IVF-ICSI with PGT-A and single embryo transfer (SET) of day 4 euploid embryos. Participants/materials, setting, methods We compared the mtV ratios of 34 non-pregnant patients with those of 41 patients who achieved clinical pregnancy after SET. The mtV ratio was obtained from a cohort of 75 euploidy embryos. The embryos were biopsied 80–85 hours post–ICSI and subjected to next-generation sequencing (NGS). The mtV was determined using Multiple of Mean (MoM) values, obtained by dividing the mtV ratio of individual embryos by the mean mtV value of all implanted embryos. Main results and the role of chance The mean mtV ratio (1.51; 95% confidence interval [CI] 1.25–1.77) of non-pregnant patients was significantly lower than those of pregnancy counterparts (2.5; 95% CI 1.82–2.68; p < 0.01). At a 0.5 MoM cutoff, the sensitivity and specificity of mtV ratio to discriminate between implanted embryos versus non-implanted embryos were 35.3% and 78.2%, respectively., with a positive predictive value (PPV) of 41.4%. Limitations, reasons for caution Our study is limited by the small sample size and lack of stratification by causes of female/male infertility. Endometrial receptivity issues, which could have contributed to implantation failure, was not evaluated. Wider implications of the findings: Assessment of mtV ratio could provide additional prognostic information for selecting euploid embryos for transfer in SET programs. Further research is warranted to establish the clinical utility of routine application of mtV evaluation in PGT programs. Trial registration number N/A

Embryos are exposed to a significant drop in temperature during the embryo transfer procedure: a pilot study

Research questionDuring the embryo transfer procedure, to what degree of temperature drop are embryos exposed to between loading the transfer catheter and placing it into the uterus? DesignTwenty-nine simulated embryo transfer procedures were carried out across five clinics. A thermocouple probe was used for standardized measurements inside the embryo transfer catheter to investigate the change in temperature that occurred in the time period between loading and placing the catheter in the uterus. ResultsIn all cases, the temperature at the loaded catheter tip fell rapidly to ambient temperature during transit from the embryo transfer workstation in the laboratory to the procedure room, even though embryo transfer procedures, ambient temperatures and embryo transfer catheter temperatures at loading varied between clinics. ConclusionsGiven the sensitivity of the pre-implantation embryo to its immediate environment, the rapid and profound drop in temperature observed at the catheter tip that houses the embryo during transit from laboratory to the uterus may affect embryo viability and health. This issue should be addressed to ensure that the tight temperature control aimed for by IVF laboratories continues throughout the embryo transfer procedure, and could improve clinical outcomes.

Reduced effect of mineral oil toxicity using four-well culture dish in sheep embryo production

Abstract Mineral oil (MO) as a barrier can minimize temperature, osmolality and pH fluctuation in the oocyte/embryo culture medium. Regardless of these advantages, MO acts as a potential source of embryo-toxic compounds which can affect embryo viability. This study was conducted to assess the signs and effects of the four-well culture dish (FW) and microdrop culture systems (MD) on reducing the harmful effects of mineral oil during the in-vitro production of sheep embryos. Oocytes retrieved from abattoir-derived ovary were in-vitro matured, fertilized and cultured in MD and FW, overlaid with two different lots of mouse embryo tested mineral oils (SMO1; nMD = 133, nFW = 103, and SMO2; nMD = 137, nFW = 144) and a clinically tested mineral oil (OMO; n = 107) as a control group. The results showed that using SMO1 and SMO2 in the MD can dramatically decrease cleavage and blastocyst rates. Although they did not show any membrane damage after IVM and 24 h after IVF, the sign of damage was appeared at 48 h after fertilization and completely destroyed the blastomere membrane at 72 h after fertilization, while the FW did not affect membrane integrity. In conclusion, FW compares to the MD, can reduce the deleterious effects of mineral oil on sheep embryos.

104 SOFaaci-HEPES or holding media can be used for embryo loading without changes in pregnancies per embryo transfer nor pregnancy loss in an invitro-produced embryo transfer program

Time elapsed between removal from culture and embryo transfer (ET) can have a profound effect on the success of an invitro-produced (IVP) ET program. The embryo culture medium provides the necessary nutrients for embryo development and the use of media with a different nutrient composition to load embryos into straws could negatively affect embryo viability. The objective of the present study was to evaluate the effect of type of media used for embryo loading on pregnancy establishment and maintenance. Holstein heifers (n=800) were synchronized using a modified 5-day CO-Synch + controlled internal drug release (CIDR) as follows: Day −8: CIDR inserted, Day −3: CIDR removed, prostaglandin F2α treatment (500μg cloprostenol sodium), Day 0: gonadotrophin-releasing hormone (GnRH; 100μg of gonadorelin acetate). Five days after GnRH, heifers were evaluated by ultrasonography to determine presence of a corpus luteum (CL). Embryos were removed from culture on Day 7 (Day 0=fertilization), placed into tubes containing SOFaaci, and transported in an incubator (LabMix, WTA) to the transfer facility within 1.5h. Upon arrival embryos were removed from transport tubes and randomly assigned to be loaded into 0.25-mL straws containing either holding media (Vigro Holding Plus) or SOFaaci-HEPES. After loading into straws, embryos were placed in an ET gun and AI gun warmers set at 35°C until transfer by 1 of 5 technicians. Heifers with a CL were randomised for transfer of a fresh IVP embryo loaded into a straw containing either holding media or SOFaaci-Hepes on Day 7±1. Interval from embryo loading to transfer ranged from 1 to 3h. Pregnancy was determined by ultrasonography on Days 32 and 60. Data were analysed by logistic regression and included the fixed effects of loading media, embryo stage, embryo quality, interval between GnRH and ET, and biologically relevant interactions. Pregnancies per ET (P/ET) on Day 32 were not different between the groups in which embryos were loaded using holding media and those which used SOFaaci-Hepes, nor there were interactions between loading medium and embryo stage, embryo quality, or interval from GnRH to ET (P>0.10; Table 1). Pregnancies per ET (P/ET) on Day 60 were not different between the loading media groups, nor were there interactions between loading medium, embryo stage, and embryo quality, or interval from GnRH to ET (P>0.10). Pregnancy loss between Days 32 and 60 was not different between groups, nor there were interactions between loading media groups and any other factor (P>0.10). In conclusion, the use of either holding medium or SOFaaci-HEPES for fresh IVP embryo loading does not affect fertility; thus, both are suitable alternatives for loading of embryos into transfer straws. Table 1.Pregnancies per embryo transfer (P/ET) and pregnancy loss in recipient heifers transferred with fresh invitro-produced embryos, using either holding medium or SOFaaci-HEPES medium for loading Item P/ET Day 32 (n) P/ET Day 60 (n) Pregnancy loss (n) Loading medium Holding 47.0% (186/396) 41.3% (163/395) 11.9% (22/185) SOFaaci-HEPES 48.8% (197/404) 43.1% (174/404) 11.7% (23/197) P-value 0.77 0.22 0.84

Embryo responses to stress induced by assisted reproductive technologies.

Assisted reproductive technology (ART) has led to the birth of millions of babies. In cattle, thousands of embryos are produced annually. However, since the introduction and widespread use of ART, negative effects on embryos and offspring are starting to emerge. Knowledge so far, mostly provided by animal models, indicates that suboptimal conditions during ART can affect embryo viability and quality, and may induce embryonic stress responses. These stress responses take the form of severe gene expression alterations or modifications in critical epigenetic marks established during early developmental stages that can persist after birth. Unfortunately, while developmental plasticity allows the embryo to survive these stressful conditions, such insult may lead to adult health problems and to long-term effects on offspring that could be transmitted to subsequent generations. In this review, we describe how in mice, livestock, and humans, besides affecting the development of the embryo itself, ART stressors may also have significant repercussions on offspring health and physiology. Finally, we argue the case that better control of stressors during ART will help improve embryo quality and offspring health.

Different incubation patterns affect selective antimicrobial properties of the egg interior: experimental evidence from eggs of precocial and altricial birds.

Avian eggs contend with omnipresent microorganisms entering the egg interior, where they affect embryo viability and hatchling phenotype. The incubation behaviour and deposition of egg white antimicrobial proteins (AMPs) vary highly across the avian altricial-precocial spectrum. Experimental evidence of how these alterations in avian reproductive strategies affect the antimicrobial properties of the precocial and altricial egg interior is lacking, however. Here, we tested the egg white antimicrobial activity in eggs of two representative model species, from each end of the avian altricial-precocial spectrum, against potentially pathogenic and beneficial probiotic microorganisms. Eggs were experimentally treated to mimic un-incubated eggs in the nest, partial incubation during the egg-laying period, the onset of full incubation and the increased deposition of two main egg white AMPs, lysozyme and ovotransferrin. We moreover assessed to what extent egg antimicrobial components, egg white pH and AMP concentrations varied as a result of different incubation patterns. Fully incubated precocial and altricial eggs decreased their antimicrobial activity against a potentially pathogenic microorganism, whereas partial incubation significantly enhanced the persistence of a beneficial probiotic microorganism in precocial eggs. These effects were most probably conditioned by temperature-dependent alterations in egg white pH and AMP concentrations. While lysozyme concentration and pH decreased in fully incubated precocial but not altricial eggs, egg white ovotransferrin increased along with the intensity of incubation in both precocial and altricial eggs. This study is the first to experimentally demonstrate that different incubation patterns may have selective antimicrobial potential mediated by species-specific effects on antimicrobial components in the egg white.

Sperm-mediated DNA lesions alter metabolite levels in spent embryo culture medium

Paternal genetic alterations may affect embryo viability and reproductive outcomes. Currently it is unknown whether embryo metabolism is affected by sperm-mediated abnormalities. Hence, using a mouse model, this study investigated the response to paternally transmitted DNA lesions on genetic integrity and metabolism in preimplantation embryos. Spent embryo culture media were analysed for metabolites by nuclear magnetic resonance spectroscopy and embryonic genetic integrity was determined by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay on embryonic Day 4.5 (E4.5). Metabolic signatures were compared between normally derived embryos (control) and embryos derived from spermatozoa carrying induced DNA lesions (SDL). SDL embryos showed a significant reduction in blastocyst formation on E3.5 and E4.5 (P<0.0001) and had an approximately 2-fold increase in TUNEL-positive cells (P<0.01). A cohort of SDL embryos showing delayed development on E4.5 had increased uptake of pyruvate (P<0.05) and released significantly less alanine (P<0.05) to the medium compared with the corresponding control embryos. On the other hand, normally developed SDL embryos had a reduced (P<0.001) pyruvate-to-alanine ratio compared with normally developed embryos from the control group. Hence, the difference in the metabolic behaviour of SDL embryos may be attributed to paternally transmitted DNA lesions in SDL embryos.

Genome-wide screening of DNA methylation in bovine blastocysts with different kinetics of development

Background The timing of the first cell divisions may predict the developmental potential of an embryo, including its ability to establish pregnancy. Besides differences related to metabolism, stress, and survival, embryos with different speeds of development present distinct patterns of gene expression, mainly related to energy and lipid metabolism. As gene expression is regulated by epigenetic factors, and that includes DNA methylation patterns, in this study we compared the global DNA methylation profile of embryos with different kinetics of development in order to identify general pathways and regions that are most influenced by this phenotype. For this purpose, bovine embryos were in vitro produced using sexed semen (female), classified as fast (four or more cells) or slow (two cells) at 40 hpi and cultured until blastocyst stage, when they were analyzed.ResultsGenome-wide DNA methylation analysis identified 11,584 differently methylated regions (DMRs) (7976 hypermethylated regions in fast and 3608 hypermethylated regions in slow embryos). Fast embryos presented more regions classified as hypermethylated distributed throughout the genome, as in introns, exons, promoters, and repeat elements while in slow embryos, hypermethylated regions were more present in CpG islands. DMRs were clustered by means of biological processes, and the most affected pathways were related to cell survival/differentiation and energy/lipid metabolism. Transcripts profiles from DM genes connected with these pathways were also assessed, and the most part disclosed changes in relative quantitation.ConclusionThe kinetics of the first cleavages influences the DNA methylation and expression profiles of genes related to metabolism and differentiation pathways and may affect embryo viability.

136 DNA Methylation Status and Transcript Profile of Genes Associated with Lipid Metabolism, Cell Survival, and Pluripotency in Bovine Blastocysts with Different Kinetics of Development

During in vitro production (IVP), blastocysts can be differentiated based on the kinetics of early cleavage. These groups present distinct patterns of global DNA methylation, an epigenetic characteristic generally responsible for suppression (presence of methylation) or activation (absence of methylation) of genes from different biological pathways. This work investigated the DNA methylation and mRNA levels of genes related to embryo development and viability. For this purpose, bovine embryos underwent IVP using conventional protocols. After 40 h of insemination, embryos were classified as FBL (fast cleavage: 4 cells or more) or SBL (slow cleavage: 2 or 3 cells), remaining in culture until blastocyst stage. Sexed semen was used to prevent differences due to sex, even without statistical differences in male:female ratio already reported. Blastocysts (40 per group) were analysed by EmbryoGENE Methylation DNA Array (Ispada et al. 2016 Proc. 49th SSR: 181) and later analysed through BioMark™HD (Fluidigm Corp., South San Francisco, CA, USA) for the transcripts profile. The PPIA gene was used as endogenous control for ΔCt calculation and submitted to Student’s t-test. Genome-wide DNA methylation analysis identified 47,713 methylated regions (7976 hypermethylated in FBL and 3608 hypermethylated in SBL). Fast embryos presented more hypermethylations distributed throughout the genome, such as introns, exons, promoter and repeat elements, whereas hypermethylation were more present in CpG islands in slow embryos. Differentially methylated regions were clustered by means of biological processes and the most affected pathways were related to lipid metabolism and cell differentiation and survival. Regarding the gene expression analysis, all results are presented in FBL in relation to SBL. Of genes involved in lipid metabolism, ACSL3, ELOVL6, PPARA, and FADS, previously identified as hypermethylated genes, were down-regulated, whereas PPARG and PTGS2 showed no statistical difference; SCD and FASN, although hypomethylated, were also down-regulated, and ACSL6, which did not differ in DNA methylation status, was down-regulated. Of genes involved in survival/death, BAX, HSPA1A, BID, NFE2L2, and GPX1 were hypermethylated; however, the first 2 were up-regulated, BID was down-regulated, and the last 2 were not statistically different. Although CASP9, TXNRD1, and FOXO3 were all hypomethylated, only CASP9 was up-regulated. Also, DDIT3 was down-regulated and NOS2 was up-regulated, although they did not differ in DNA methylation between groups. Of genes involved in cell differentiation, POU5F1 and SALL4 were both hypermethylated, but only the POU5F1 was down-regulated; NANOG, which did not differ in DNA methylation status between groups, was also down-regulated. In conclusion, although we did not find correlation in DNA methylation and RNA levels for all genes evaluated, the chosen pathways were indeed different between groups, which could lead to their potential suppression/activation and affect embryo viability. Also, this lower correlation may be a result of the influence of other epigenetic mechanisms differently activated between groups.

Spermatozoa DNA methylation patterns differ due to peripubertal age in bulls

In the dairy industry, using semen as soon as the bull is mature enough to produce it is advantageous for breeding purposes. Mammalian spermatogenesis is a hormone-dependent developmental program in which a complex cascade of events must take place to ensure that germ cells reach the proper stage of development at the proper time. Conventional indicators of semen quality such as sperm cell motility and viability usually improve as bulls mature, meeting quality criteria satisfactorily at around 16 months. Using semen before that age may affect embryo viability, but other changes occurring during the peripubertal period should be considered. Although it is known that establishment of these patterns begins during foetal life, the extent to which sperm cell DNA methylation changes during puberty has not been studied. The aim of this study is to correlate the age of a young bull with the overall DNA methylation pattern of its spermatozoa.Spermatozoa were collected from bulls at the ages of 10 months (early pubertal), 12 months (late pubertal) and 16 months (pubertal). Each animal (n = 4) was compared to itself with 16 months as control. Genome-wide DNA methylation was analyzed by microarray using the EmbryoGENE DNA Methylation Analysis platform. Using a fold change over 1.5 and a 5% FDR p-value correction, a total of 2602 differently methylated regions were found in common between 10 months of age and 16 months of age. No differently methylated regions between 12 months and 16 months of age were found at the same level of statistical significance. We conclude that spermatozoa from bulls aged 10 months have a different epigenetic profile, which could compromise their value.

Development of bovine and rabbit preimplantation embryos in vitro after cryostorage

Evaluation of quality and viability of bovine and rabbit preimplantation embryos following cryopreservation was the objective of this study. Bovine embryos of Holstein breed (n= 88) at the morula-early blastocyst stage (on the 7 th day after the first insemination) and rabbit embryos (n= 135) from New Zealand breed at the morula stage (90–92 hpc) were cryopreserved by the two-step vitrification procedure. Following thawing the embryos of both cattle and rabbit were cultured for 48 hours in order to reach expanded blastocyst stage, and then were analyzed for the developmental rate and viability (embryo cell number and incidence of the dead cells). The results demonstrate that cryopreservation can only slightly affect embryo viability and quality do not compromising their further development. Therefore, vitrification techniques tested in our study can be used for cryopreservation of embryos of national cattle and rabbit breeds for the purpose of long-term storage of embryos in the animal gene bank.

Factors affecting embryo viability and uterine receptivity: insights from an analysis of the UK registry data

Many studies have identified prognostic factors for IVF treatment outcome; however, little information is available on the mechanism of their action. Embryo–uterus models have the potential to distinguish between factors acting on the embryo directly and those acting through the uterine environment. Here we apply embryo–uterus models to comprehensive UK registry data from two periods, 2000–2005 and 2007–2011, containing 139,444 and 226,542 embryo transfer cycles, respectively. Given this large dataset, the embryo–uterus model is capable of distinguishing between uterine and embryo effects. Maternal age is the predominant predictor of live birth and acts on both the embryo and uterine components, but with larger effects on the embryo. Prolonged embryo culture is associated with greater embryo viability, reflecting the greater degree of selection, but is also associated with greater uterine receptivity. Cryopreserved embryos are less viable and were associated with poorer uterine receptivity. This work suggests that, in addition to the direct effects of in-vitro culture on the embryonic environment during the first few days of the embryo's life, the delay in transfer after extended culture or cryopreservation can lead to an altered uterine environment for the embryo after transfer.

Oestrogen reporter transgenic medaka for non-invasive evaluation of aromatase activity

Vertebrate reproduction involves complex steroid hormone interplay and inter-conversion. A critical element in maintaining sex steroid levels is the enzyme aromatase (cytochrome P450 19A1) which converts androgens to oestrogens. In turn oestrogen signalling is targeted by numerous chemicals, from pharmaceuticals to agricultural chemicals, both frequent sources of contamination in waste waters and consequently rivers. Although many models are now available to address disruption of oestrogen signalling, there are currently no published protocols allowing discrimination between alterations in testosterone metabolism and in oestrogenic signalling. It was with this limitation in mind that we optimised this protocol. We show using a 48h protocol that pre-feeding fry of the choriogenin h-gfp (chgh-gfp) medaka line are sensitive to 0.05nM EE2 (15ng/L), within the range of the lowest published observable physiological effect concentrations for medaka. In addition, co-treatment with testosterone can reveal potential effects of test substances on aromatase enzymatic activity. As the measurements are visualised in real-time without affecting embryo viability, repeated measures are possible. We demonstrate the ability of this model to detect oestrogen receptor agonists, aromatisable androgens, P450 aromatase activity modulators and selective oestrogen response modulators. Importantly, the range of this assay is physiologically relevant.

Desiccation tolerance of embryos of Syagrus oleracea , a cerrado native bitter palm heart

There is great interest in seedling production of plant species with ornamental and food potential; however, there is great difficulty in propagating this species. A method of fruit drying was proposed with the goal of evaluating the desiccation tolerance of embryos of guariroba palm Syagrus oleracea. Tissue culture was used to test the effect of different fruit drying times (0, 4, 8 and 12 days) on embryo viability and in vitro germination. Desiccation tolerance of guariroba palm embryos was determined, and a methodology for their in vitro establishment was proposed. The seed water content was estimated from the fruit water content without extracting the seed. Drying at 37±2°C did not negatively affect embryo viability or germination. In addition, contamination of zygotic embryos decreased with increasing drying time. Fruit drying at 37±2°C for 8 days is recommended because it made the embryo extraction easier without embryo viability loss or contamination during the in vitro establishment of guariroba palm. Key words: Desiccation, guariroba, propagation, ornamentation, physiological quality, Arecaceae.

Effects of ulipristal acetate on human embryo attachment and endometrial cell gene expression in an in vitro co-culture system.

Does ulipristal acetate (UPA) used for emergency contraception (EC) interfere with the human embryo implantation process? UPA, at the dosage used for EC, does not affect human embryo implantation process, in vitro. A single pre-ovulatory dose of UPA (30 mg) acts by delaying or inhibiting ovulation and is recommended as first choice among emergency contraceptive pills due to its efficacy. The compound has also been demonstrated to have a dose-dependent effect on the endometrium, which theoretically could impair endometrial receptivity but its direct action on human embryo implantation has not yet been studied. Effect of UPA on embryo implantation process was studied in an in vitro endometrial construct. Human embryos were randomly added to the cultures and cultured for 5 more days with UPA (n = 10) or with vehicle alone (n = 10) to record the attachment of embryos. Endometrial biopsies were obtained from healthy, fertile women on cycle day LH+4 and stromal and epithelial cells were isolated. A three-dimensional in vitro endometrial co-culture system was constructed by mixing stromal cells with collagen covered with a layer of epithelial cells and cultured in progesterone containing medium until confluence. The treatment group received 200 ng/ml of UPA. Healthy, viable human embryos were placed on both control and treatment cultures. Five days later the cultures were tested for the attachment of embryos and the 3D endometrial constructs were analysed for endometrial receptivity markers by real-time PCR. There was no significant difference in the embryo attachment rate between the UPA treated group and the control group as 5 out of 10 human embryos exposed to UPA and 7 out of 10 embryos in the control group attached to the endometrial cell surface (P = 0.650). Out of 17 known receptivity genes studied here, only 2 genes, HBEGF (P = 0.009) and IL6 (P = 0.025) had a significant up-regulation and 4 genes, namely HAND2 (P = 0.003), OPN (P = 0.003), CALCR (P = 0.016) and FGF2 (P = 0.023) were down-regulated with the exposure of UPA, compared with control group. This proof of concept study was conducted with a few human embryos, as their availability was limited. Although the 3D model used for this study is well established and the artificial endometrial luminal epithelium shown to express progesterone regulated markers of endometrial receptivity it is still an in vitro model, lacking all cell types that constitute the receptive endometrium in vivo. This study provides new insights on the mechanism of action of UPA on human embryo implantation, demonstrating that UPA in a dosage used for EC does not affect embryo viability and the implantation process of embryo. Progesterone receptor modulators (PRMs) hold the potential to be attractive estrogen- and gestagen-free contraceptives and thus may be made available to a larger proportion of women globally due to these findings. Swedish Research Council (K2010-54X-14212-09-3) and support provided through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska University Hospital.