Embryo Damage Research Articles

P-530 Precise quantification of DNA in spent culture media sheds light on associations between DNA quantity, embryonic ploidy status and morphological quality

Abstract Study question Is it possible to accurately measure cell-free DNA (cfDNA) quantity in embryo spent culture media, and are amounts correlated with embryo morphological and/or chromosomal status? Summary answer A sensitive DNA quantification method was created, demonstrating that cfDNA quantity is not associated with embryo ploidy, but increases when embryos are of poor morphology. What is known already Preimplantation genetic testing involves embryo biopsy, necessitating specialist equipment and highly trained staff, increasing costs. Additionally, there have been concerns over potential embryo damage to the embryo resulting from biopsy. The discovery of cfDNA in medium in which embryos were cultured, led to suggestions that non-invasive PGT (niPGT) might be possible. However, the amount of cfDNA in media samples, and potential correlations with other aspects of embryo biology, require clarification. We aimed to provide the first accurate measurement of the amount of DNA in spent culture medium, providing insights into whether niPGT is likely to provide acceptable accuracy rates. Study design, size, duration This prospective analysis involved media samples from 151 fresh embryos, which were produced at a single IVF clinic over the course of one year. Embryos were cultured individually, with a medium change on day-3 of development. Trophectoderm biopsy was undertaken on day-5/-6, followed by PGT-A. Embryo morphology was assessed according to ASEBIR criteria (ranked A-C). Participants/materials, setting, methods The culture and biopsy protocols used were standard for the clinic. Media was collected prior to biopsy and underwent a novel quantitative whole genome amplification method, allowing precise measurement of DNA with respect to a standard curve generated using samples containing known amounts of DNA. PGT-A involved the use of a highly validated method, based upon next generation sequencing. Correlation between DNA quantity and embryo morphological quality was also studied. Data were analyzed using ANOVA. Main results and the role of chance There was no difference in the quantity of DNA in media samples associated with euploid and aneuploid embryos (mean DNA quantity and standard deviations were 1.72 ± 3.23 pg and 1.54 ± 3.66 pg for aneuploid and euploid embryos, respectively; p = 0.75). Overall, 91% of media samples contained less than the equivalent of one cell (<5pg of DNA) and almost two-thirds of samples (65%) contained less than 1pg of DNA. Given such low quantities of embryonic DNA, achieving accurate genetic testing is likely to be challenging. Drops that held embryos until day-6 had significantly higher levels of DNA than those associated with culture to day-5 (P < 0.0001), suggesting cfDNA accumulates with time. Thus, longer culture periods are likely to yield more DNA for analysis. Comparing the amount of cfDNA with morphological status revealed a significant association. Embryos of fair quality (C) tended to have higher quantities of DNA in spent culture medium, whereas top-quality embryos (classified as A or B) typically had less (mean and standard deviation of DNA quantity: 1.23 ± 3.14 pg for class A and B versus 2.60 ± 3.85 pg for class C; p < 0.05). This may reflect increased cell death and DNA release in poorer quality embryos. Limitations, reasons for caution This project is limited by its single-centre nature and relatively small sample size. A large-scale, multicentre study would be necessary to confirm these results. Wider implications of the findings It has been hypothesised that cfDNA quantity in culture medium may be predictive of embryo chromosomal status. We found no evidence to support this. Although DNA quantities were higher after culture to day-6, levels remained low, calling into question the ability of niPGT to deliver high accuracy diagnosis of embryos. Trial registration number not applicable

External bending of cryodevice during vitrification leads to cryoprotectant cracks and damage to embryo blastomeres

Research questionEmbryo blastomeres and the zona pellucida are occasionally damaged during vitrification; is this a result of crack-induced mechanical damage in the glass state, caused by external bending of the device? DesignA stereomicroscope was used to observe external bending-induced cracks in a cryoprotectant. Thereafter, 309 human cleavage-stage embryos derived from abnormally fertilized eggs were used to assess embryo damage under two external bending conditions: forward bending and backward bending, with three bending degrees applied. Three distinct embryo positions were used to examine the correlation between bending and embryo damage. Damage was assessed by looking at blastomere lysis rates, and overall rates of damaged and surviving embryos. ResultsA series of parallel cracks were identified in the cryoprotectant used for external bending, which led to damage to the embryo blastomeres. Compared with forward bending and control, the embryos were found to be more easily damaged by backward bending, indicated by significantly higher blastomere lysis and embryo damage rates, and lower embryo survival rate of backward bending than forward bending (P < 0.001). The degree of embryo damage also increased as the degree of external forces increased. Embryo position correlated with degree of embryo damage. ConclusionsCryoprotectant crack-induced damage was identified as the cause of embryo damage. Mechanical damage to the glass state occurs because of improper external bending of the cryodevice strip in liquid nitrogen during vitrification. To prevent damage, bending of the strip should be avoided and the embryos should be placed near the tip of the strip.

Melatonin improves the vitrification of sheep morulae by modulating transcriptome.

Embryo vitrification technology is widely used in livestock production, but freezing injury has been a key factor hindering the efficiency of embryo production. There is an urgent need to further analyze the molecular mechanism of embryo damage by the vitrification process. In the study, morulae were collected from Hu sheep uterine horns after superovulation and sperm transfusion. Morulae were Cryotop vitrified and warmed. Nine morulae were in the vitrified control group (frozen), and seven morulae were vitrified and warmed with 10-5 M melatonin (melatonin). Eleven non-frozen morulae were used as controls (fresh). After warming, each embryo was sequenced separately for library construction and gene expression analysis. p < 0.05 was used to differentiate differentially expressed genes (DEG). The results showed that differentiated differentially expressed genes (DEG) in vitrified morulae were mainly enriched in protein kinase activity, adhesion processes, calcium signaling pathways and Wnt, PI3K/AKT, Ras, ErbB, and MAPK signaling pathways compared to controls. Importantly, melatonin treatment upregulated the expression of key pathways that increase the resistance of morulae against vitrification induced damage. These pathways include kinase activity pathway, ErbB, and PI3K/Akt signaling pathway. It is worth mentioning that melatonin upregulates the expression of XPA, which is a key transcription factor for DNA repair. In conclusion, vitrification affected the transcriptome of in vivo-derived Hu sheep morulae, and melatonin had a protective effect on the vitrification process. For the first time, the transcriptome profiles caused by vitrification and melatonin in sheep morulae were analyzed in single embryo level. These data obtained from the single embryo level provide an important molecular mechanism for further optimizing the cryopreservation of embryos or other cells.

Effect of different manual puncture methods on donkey embryo before vitrification

The application of embryo recovery and transfer technology in the donkey industry is far lower than that of horses and cattle. Sometimes the recovered embryos could not be transferred in time, which required embryo cryopreservation. The embryo cryopreservation technology is more conducive to the preservation and transportation of recovered embryos with excellent genetic traits. However, this technique for donkey embryos is not efficient and needs further optimization. The objective of this study was to evaluate the effect of different manual puncture methods on the viability and pregnancy rates of vitrified-thawed donkey embryos. A total of 117 donkey embryos were recovered on day 7–8 post-ovulation and were divided into four groups by random assortment. There were 28 embryos without puncture or cryopreservation (Control). 58 embryos were manually punctured using a 29G needle (VG, n = 29) or microneedle with a sharp tip of <10 μm (VM, n = 29), then vitrified in 15% EG + 15% DMSO + 0.5 M sucrose. Another 31 embryos were punctured with a microneedle and vitrified with 10% EG + 10% DMSO +0.5 M sucrose +2 mol/L proline (VMP). Both fresh embryos and vitrified-thawed embryos were incubated for 3 h (38.5 °C, 5% CO2 in air) before embryo transfer. The results showed that the embryo recovery rates on day 7.5 and 8 were higher than day 7 (P < 0.05). After incubation, dead cells were assessed and the percentages of dead cells in VM and VMP were lower than that in VG (P < 0.05), although both were higher than those in Control (P < 0.05). The pregnancy rates after 23 days post transfer were assessed and the results showed that the pregnancy rate in VG (8.0%) was lower than that in Control (41.7%), VM (24.0%) and VMP (29.6%) (P < 0.05). No pregnancies resulted from the 10 embryos with diameters ≤650 μm in VG, which lower than either VM (33.3%) or VMP (38.9%) (P < 0.05). While, there was no difference in pregnancy rates among all vitrification groups when embryos were >650 μm in diameter (P > 0.05). In conclusion, the embryo recovery rate on day 7 after ovulation was relatively low, and it was more appropriate to extend it to day 8. Microneedle puncture could reduce embryo damage and achieve a higher pregnancy rate compared with 29G needles. Proline has the potential to improve donkey embryo cryopreservation.

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.

IS PGT-A BENEFICIAL FOR PATIENTS WITH ONLY ONE BLASTOCYST? A 2014-2018 NATIONAL STUDY OF OVER 21,000 FREEZE-ALL CYCLES

Women with only one blastocyst have been largely excluded from studies on preimplantation genetic testing for aneuploidy (PGT-A) due to cost and theoretical risk of embryo damage. Existing data on the benefit of PGT-A in this specific context are therefore sparse. Our objective was to use a large, national database to assess the association between PGT-A and live birth rate among women with only one embryo achieving blastocyst stage.

Development of an artificial intelligence model for predicting the likelihood of human embryo euploidy based on blastocyst images from multiple imaging systems during IVF.

Development of an artificial intelligence model for predicting the likelihood of human embryo euploidy based on blastocyst images from multiple imaging systems during IVF.

Seed Maturity and Its In Vitro Initiation of Chilean Endemic Geophyte Alstroemeriapelegrina L.

Alstroemeria pelegrina (A. pelegrina), a Chilean endemic, is considered vulnerable as its natural habitat is currently threatened. The decline in the reproductive capacity of the species due to anthropogenic impacts and climate change has made it imperative to address the problem by developing large-scale propagation methods. The objective of this study was to establish protocols for breaking the dormancy and in vitro germination of A. pelegrina seeds to speed up the germination and seedling production processes. The research began with morphological observations of the reproductive process, followed by in vitro sowing. The results showed that the seeds reached full maturity in 51 days, and physiological maturity in 41 days, at which point the seeds could be harvested for in vitro germination. The mechanical scarification pretreatment improves the in vitro germination rate to 96% and the germination time to 7 days, showing that the species is characterized by physical seed dormancy. On the other hand, if the seed coat incisions are deeper than 0.5 mm, 30% of the potential plants are lost due to embryo damage. The study provides scientific evidence for the feasibility of large-scale in vitro propagation of the species and establishes an efficient method of seedling production.

Berberine alleviates LPS-induced apoptosis, oxidation, and skewed lineages during mouse preimplantation development†.

Female infertility is a heterogeneous disorder with a variety of complex causes, including inflammation and oxidative stress, which are also closely associated with the pathogenesis of polycystic ovary syndrome (PCOS). As a new treatment for PCOS, berberine (BER), a natural compound from Berberis, has been clinically applied recently. However, the mechanisms underlying the association between BER and embryogenesis are still largely unknown. In this study, effects of BER on preimplantation development were evaluated under both normal and inflammatory culture conditions induced by lipopolysaccharide (LPS) in mice. Our data first suggest that BER itself (25nM) does not affect embryo quality or future developmental potency; however, it can effectively alleviate LPS-induced embryo damage by mitigating apoptosis via reactive oxygen species (ROS)-/caspase-3-dependent pathways and by suppressing proinflammatory cytokines via inhibition of the NF-κB signaling pathway during preimplantation embryonic development. In addition, skewed cell lineage specification in the inner cell mass (ICM) and primitive endoderm (PE) caused by LPS can also be successfully rescued with BER. In summary, these findings for the first time demonstrate the nontoxicity of low doses of BER and its antiapoptotic and antioxidative properties on embryonic cells during mammalian preimplantation development.

Exogenous melatonin protects preimplantation embryo development from decabromodiphenyl ethane-induced circadian rhythm disorder and endogenous melatonin reduction

Decabromodiphenyl ethane (DBDPE) is a novel flame retardant that is widely used in plastics, electronic products, building materials and textiles. Our previous studies have revealed the oocyte toxicity of DBDPE, but the effect of DBDPE on preimplantation embryo development has not been reported. Here, we investigated whether and how DBDPE exposure affects preimplantation embryo development. Adult female mice were orally exposed to DBDPE (0, 5, 50, 500 μg/kg bw/day) for 14 days. First, we found that after DBDPE exposure, mice showed obvious circadian rhythm disorder. Moreover, the development of preimplantation embryos was inhibited in DBDPE-exposed mice after pregnancy. Then, we further explored and revealed that DBDPE exposure reduced the endogenous melatonin (MLT) level during pregnancy, thereby inhibiting the development of preimplantation embryos. Furthermore, we discovered that exogenous MLT supplementation (15 mg/kg bw/day) rescued the inhibition of preimplantation embryo development induced by DBDPE, and a mechanistic study demonstrated that exogenous MLT inhibited the overexpression of ROS and DNA methylation at the 5-position of cytosine (5-mC) in DBDPE-exposed preimplantation embryos. Simultaneously, MLT ameliorated the DBDPE-induced mitochondrial dysfunction by increasing the mitochondrial membrane potential (MMP), ATP, and Trp1 expression. Additionally, MLT restored DBDPE-induced changes in zona pellucida (ZP) hardness and trophectoderm (TE) cortical tension. Finally, the protective effect of MLT on embryos ameliorated the adverse reproductive outcomes (dead fetus, fetus with abnormal liver, fetal weight loss) induced by DBDPE. Collectively, DBDPE induced preimplantation embryo damage leading to adverse reproductive outcomes, and MLT has emerged as a potential tool to rescue adverse reproductive outcomes induced by DBDPE.

P–168 RCT comparing the effect of Continuous ( Single Step ) embryo culture system versus a Sequential embryo culture system on the outcome of IVF/ICSI cycles

Abstract Study question Is the outcome of IVF/ICSI cycles done with continuous (single step ) embryo culture system different from that with sequential embryo culture system ? Summary answer Yes the outcome of IVF / ICSI cycles done with continuous (single step ) embryo-culture system is better than that with sequential embryo-culture system . What is known already Embryo culture media are important factors in IVF, which can significantly influence the clinical outcome of IVF/ICSI cycles. However it is not clear which formulation is most optimal and whether sequential or continuous media (single step) should be favored. Sequential media complies with embryo demands based on developmental stage , taking into account metabolic changes embryos undergo in-vivo, while moving from the oviduct to the uterus. The embryos in the early cleavage stage prefer to use pyruvate to produce energy, whereas once development nears the blastocyst stage , the embryos start using glucose in the process of glycolysis . Study design, size, duration A prospective RCT was carried out at our centre between 2018–2019 and IVF-ICSI patients meeting inclusion criteria (at least six normal MII - Oocytes) were included in this study. The aim of study was to compare blastocyst formation rates after embryo-culture in two different culture media systems. 436 metaphase II Oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media or single-step media. Participants/materials, setting, methods In this prospective trial with sibling oocytes, 436 metaphase II oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media ( n = 218 MII oocytes) or a single medium ( n = 218 MII oocytes). In both groups, embryos were cultured in an interrupted fashion with media changes on day 3. Embryo transfer was performed on day 5. Main results and the role of chance Blastocyst formation rates on day 5 were significantly higher following culture in single step media 60.55% (132 / 218 ) as compared to sequential media 34.86% ( 76 / 218) . The percentage of good quality blastocysts was also significantly higher in single step media. In conclusion, culture in single step media was associated with higher blastocyst formation rates compared to sequential media , suggesting that the single medium may provide better support to the developing embryo. The proportion of poor quality embryos was significantly higher in the sequential media group. Results indicate that embryo culture in continuous media could be as efficient as embryo culture in sequential media. A significant difference observed was the proportion of poor quality embryos on day 5 , which was significantly higher when the embryos were cultured in sequential media. Our results suggest that the type of embryo culture media can influence the quality of embryos both at the cleavage stage and blastocyst stage. The use of continuous embryo culture media does not seem to cause an adverse effect; in fact, their use can lower the workload in busy IVF labs and lower the stress that embryos are exposed to during handling. Limitations, reasons for caution Although single-step-medium for extended culture has practical advantages and blastocyst formation rates appear to be higher, there is insufficient evidence to recommend either sequential or single-step media as being superior for the embryo-culture to days 5/6. Further studies comparing these two media systems in well-designed trials should be performed. Wider implications of the findings: When employing sequential media for embryo culture , it is necessary to transfer the embryos from one medium to another ( cleavage stage medium to blastocyst stage medium) which increases stress related embryo damage . Therefore, single-step media is beneficial as the embryos can develop undisturbed till blastocyst stage. Trial registration number Not applicable

Pre-implantation genetic testing for aneuploidy: motivations, concerns, and perceptions in a UK population

PurposePre-implantation genetic testing for aneuploidies (PGT-A) is a technique used as part of in vitro fertilisation to improve outcomes. Despite the upward trend in women utilising PGT-A, data on women’s motivations and concerns toward using the technology, and perceptions having undergone the process, remain scarce.MethodsThis cross-sectional survey, based at a fertility clinic in the UK, utilised an electronic questionnaire to assess the motivations of women who undergo PGT-A and their perceptions and attitudes toward PGT-A after using it.ResultsOne hundred sixty-one women responded. The most significant motivating factors to undergo PGT-A were to improve the probability of having a baby per cycle (9.0 ± 2.1) and enhance the chance of implantation (8.8 ± 2.5). The least important motivations were reducing the number of embryos transferred per cycle (2.7 ± 3.3) and saving money by reducing the number of procedures required (4.6 ± 3.4). The most significant concerning factors identified included not having embryos to transfer (5.7 ± 3.4) and the potential for embryo damage (5.2 ± 3.3). The least concerning factors included religious (0.6 ± 1.7) or moral (1 ± 2.2) concerns. The majority of women were satisfied/very satisfied following treatment (n = 109; 68%). The proportion of those who were satisfied/very satisfied increased to 94.2% (n = 81) following a successful outcome, and reduced to 43.5% (n = 27) in those who had an unsuccessful outcome or had not undergone embryo transfer (p < 0.001).ConclusionThis study highlights that perceptions amongst women who use PGT-A are mostly positive. We also demonstrate a significant association between satisfaction and reproductive outcomes, with those who achieve a live birth reporting more positive perceptions toward PGT-A.

139 Effect of lycopene supplementation in maturation medium on production of reactive oxygen species and post-vitrification quality of bovine blastocysts

Excessive production and accumulation of reactive oxygen species (ROS) may cause embryo damage associated with oxidative stress. Lycopene, a natural antioxidant, can scavenge singlet oxygen and is one of the most effective antioxidants among carotenoids. We evaluated the effects of supplementation of lycopene (antioxidant), menadione (prooxidant), and their combination during invitro oocyte maturation on ROS generation in matured oocytes and the quality of vitrified-warmed embryos. Cumulus–oocyte complexes, collected from the slaughterhouse, were matured in groups of 60 in 500μL of TCM-199 medium+50mg mL−1 gentamycin+20ng mL−1 epidermal growth factor, for 22h at 38.5°C in 5% CO2 in air and then supplemented with (1) 0.2μM lycopene, (2) 5μM menadione, (3) 0.2μM lycopene+5μM menadione (L+M), or (4) not supplemented (control). Fertilization and embryo culture were performed similarly for all the groups. In the first experiment, ROS measurement (n=236; via fluorescent microscopy) was performed in denuded, matured oocytes incubated in 5μM CellROX® Green (ThermoFisher Scientific) for 1h. Fluorescent intensity was measured in Image-J. In the second experiment, embryos in the blastocyst stage (n=143) were vitrified as previously described by Ortiz-Escribano et al. (2017 Biol. Reprod. 96, 288-301). Vitrified blastocysts were then warmed and washed in decreasing concentrations of sucrose and incubated for 2 days in culture medium [50µL of synthetic oviductal fluid (SOF)+(5g mL−1 insulin, 5g mL−1 transferrin, 5ng mL−1 selenium)]. The quality of vitrified-warmed blastocysts was assessed using a differential staining as described by Wydooghe et al. (2011 Anal. Biochem. 416, 228–230). The effects of pro- and antioxidant supplementation on oocyte fluorescent intensity and embryo quality parameters were fitted in linear mixed-effects models, and results are expressed as least squares means and standard errors. The fluorescent intensity for ROS was lower (P&amp;lt;0.05) in lycopene (10.06±2.92) than in menadione (16.8±2.92). No differences (P&amp;gt;0.05) in ROS intensity values were found among the other groups [control (13.5±2.92) and L+M (13.7±2.90)]. Total cell number (TCN) was similar (P&amp;gt;0.05) in lycopene (153±2.95), L+M (143±4.59), and control (145±3.67) but lower (P&amp;lt;0.05) in menadione (134±6.08). Lesser numbers of apoptotic cells (AC) and AC/TCN values (P&amp;lt;0.05) were recorded in lycopene (4.12±3.07 and 2.71±2.21) compared with control (6.18±3.82 and 4.31±2.75), L+M (6.00±4.79 and 4.22±3.45), and menadione (7.75±6.33 and 5.82±4.56). For the remaining embryo quality parameters, no differences were found (P&amp;gt;0.05). In conclusion, lycopene supplementation during invitro oocyte maturation effectively scavenged free radicals, lowering oxidative stress and improving embryo quality post-vitrification and warming.

ТЕХНІКО-ТЕХНОЛОГІЧНІ ПОКАЗНИКИ В УПРАВЛІННІ ПРОЦЕСАМИ ЗБИРАННЯ ТА ПІСЛЯЗБИРАЛЬНОЇ ОБРОБКИ НАСІННЯ ГІБРИДІВ КУКУРУДЗИ

The research result of technical and technological indicators, which largely characterize the seed condition and quality and allow to optimize the harvest and post-harvest processing of maize seeds, was presented. The harvest and post-harvest processing of seeds are particularly affected by such indicators as: size, uniformity, level of damage, the content of self-hulled seeds in the total mass. In order to optimize the high-quality seed preparation, it is recommended to use for sowing the coarse seeds with a uniformity of more than 80 %. It was found that a uniformity of 80% or less causes the decrease of field seed germination (A) by 2–6 %, the extension of "sowing – full sprouts" period (B) by 6–8 days, and the decrease of index A/B by 32.1–41.0 %. Thus, the low seed uniformity has the negative effect. The indicator of uniformity is controlled by means the seed separation, and it must be at least 81 %. It is not allowed a presence of self-hulled seeds with embryo damage level more than 10 % in the seed material. With a higher content of self-hulled seeds in the total mass, laboratory germination decreases by 5–7 %, field germination – by 10–17 %, growth rate by number of sprouts – by 6–12 %, by sprout weight – by 8.0–11.6 %. To reliably predict the moisture loss rate of seeds at drying, it is proposed to determine the coefficient of the specific seed surface by a ratio of outer surface to volume. We established that an increase in the coefficient of specific surface area from 1.053 to 1.167 mm results to increase the moisture loss rate of seeds by 19.1%, and ears – by 30.9 %. The effectiveness for determination of the seed quality, in particular, its viability was confirmed by the cold test (germination at a temperature of 6–10 ºC). Key words: maize hybrids, technical and technological indicators, seed quality, harvest and post-harvest processing, fraction, cold test.

59 Incubation of frozen/thawed bovine embryos

High-quality, properly developed bovine embryos are crucial to the embryo transfer industry and enable cryopreservation; however, ∼30% of invivo embryos do not develop to the appropriate stage and are discarded. Currently, frozen/thawed embryos are transferred directly into a recipient, and the quality of embryos post-thaw is seldom evaluated. The objective of this experiment was to evaluate frozen/thawed bovine embryos immediately post-thaw, and again after incubation in different environments. Frozen/thawed bovine embryos (n=30/treatment) processed for direct transfer were thawed (30°C water; 30s) and graded and staged in holding medium. Embryos were then placed into either holding medium, phosphate-buffered saline supplemented with 15% fetal bovine serum (v/v) and antibiotic/antimycotic (2μL mL−1) (PBS+FBS), or a commercially available invitro culture (IVC) medium for ∼18h. Embryos in holding medium and PBS+FBS were loaded into 0.25-mL straws with the appropriate medium, and the straws were sealed and submerged in 38.5°C water. The IVC embryos were placed individually into 25-μL culture drops on tissue-coated 60-mm plastic Petri dishes overlaid with mineral oil and incubated (18h) at 38.5°C in 5% CO2 and air at 100% humidity. Embryos were then collected, regraded, and staged, and comparisons among groups were analysed via the Kruskal–Wallis H-test. Quality score of all embryos decreased by at least one-third post-thaw; however, the developmental stage was unaffected by the freeze/thaw cycle. Following incubation, all embryos suffered a significant (P&amp;lt;0.05) decrease in embryo quality but the IVC group demonstrated less (P&amp;lt;0.05) of a decline in resulting quality (Table 1). The IVC group demonstrated significant development (P&amp;lt;0.05) during incubation compared to the Holding and PBS+FBS groups (Table 1) indicating that on average, viability was maintained during IVC. Regardless of group, the zona pellucida was damaged during the freeze/thaw process in 31.1% of embryos. These data illuminated embryo damage after cryopreservation, and demonstrated that short-term invitro incubation of frozen/thawed embryos (IVC) facilitated continued development and may be a practical mechanism to salvage poor quality or developmentally suppressed embryos. Future research will focus on salvaging fresh embryos that are classified as degenerate and may prove useful in the bovine embryo industry, and for cattle producers alike, by ultimately increasing the number of transferable embryos that would otherwise be discarded. Table 1. Descriptive statistics (mean±s.e.m.) of bovine embryos pre-freeze and post-thaw and incubation Item Media Pre-freeze Post-thaw Post-incubation Grade Holding 1.4±0.1 1.7±0.1a 3.3±0.1b,x PBS+FBS 1.2±0.1 1.6±0.1a 3.1±0.1b,x IVC1 1.2±0.1 1.6±0.1a 2.5±0.2b,y Stage Holding 4.0±0.1 4.0±0.1 3.9±0.1x PBS+FBS 4.7±0.2 4.4±0.2 4.5±0.2y IVC 4.3±0.1 4.3±0.1a 5.8±0.2b,z a,b, x–zDifferent superscripts within row (a, b) and column (x, y, z) indicate a significant difference (P&amp;lt;0.05, Kruskal-Wallis H-test). 1Invitro culture medium.

Expression and molecular mechanism of PCNA, Caspase-3, IL-6 and Survivin proteins in chorionic villi and decidual tissue of early embryo damage

Expression and molecular mechanism of PCNA, Caspase-3, IL-6 and Survivin proteins in chorionic villi and decidual tissue of early embryo damage

Studi Pendahuluan: Pengaruh Krioprotektan yang Berbeda terhadap Persentase Kerusakan dan Penetasan Embrio Ikan Lele Dumbo (Clarias gariepinus)

Tujuan penelitian ini adalah mengetahui pengaruh krioprotektan yang berbeda dalam kriopreservasi embrio terhadap persentase kerusakan dan penetasan embrio ikan lele dumbo (Clarias gariepinus). Embrio ikan lele pada fase gastrula diberi perlakuan larutan dimethyl sulfoxide (DMSO), propylene glycol (PG), madu, dan kombinasinya dengan konsentrasi 5% untuk masing-masing perlakuan. Embrio disimpan pada suhu 4 dan 0ºC, masing-masing selama 30 menit, 1, 2, 3, 4, 5, dan 6 jam. Thawing embrio dilakukan pada suhu 28ºC. Selanjutnya, embrio diinkubasi dalam akuarium pada suhu 28ºC. Hasil penelitian menunjukkan bahwa perbedaan krioprotektan dalam kriopreservasi embrio fase gastrula memberikan perbedaan kerusakan dan penetasan embrio ikan lele pada lama waktu penyimpanan yang berbeda. Persentase kerusakan embrio meningkat seiring dengan lama waktu penyimpanan dan suhu penyimpanan yang berbeda. Kombinasi larutan krioprotektan DMSO dan madu serta PG dan madu menunjukkan persentase kerusakan embrio yang lebih rendah dan persentase penetasan embrio yang lebih tinggi dibandingkan perlakuan krioprotektan lainnya (p&lt;0,05).

Reduced live birth rates in frozen versus fresh single cleavage stage embryo transfer cycles: A cross -sectional study.

BackgroundStudies have suggested that embryo-endometrial developmental asynchrony caused by slow-growing embryos can be corrected by freezing the embryo and transferring it back in a subsequent cycle. Therefore, we hypothesized that live birth rates (LBR) would be higher in frozen embryo transfer (FET) compared with fresh embryo transfers.ObjectiveTo compare LBR between fresh and FET cycles.Materials and MethodsA cross-sectional analysis of 10,744 single autologous embryo transfer cycles that used a single cleavage stage embryo was performed. Multivariate analysis was performed to compare LBR between FET and fresh cycles, after correcting for various confounding factors. Sub-analysis was also performed in cycles using slow embryos.ResultsBoth LBR (19.13% vs 14.13%) and clinical pregnancy (22.48% vs 16.25%) rates (CPR) were higher in the fresh cycle group (p 0.00). Multivariate analysis for confounding factors also confirmed that women receiving a frozen-thawed embryo had a significantly lower LBR rate compared to those receiving a fresh embryo (OR 0.76, 95% CI 0.68-0.86, p 0.00). In the sub-analysis of 1,154 cycles using slow embryos, there was no statistical difference in LBR (6.40% vs 6.26%, p = 0.92) or CPR (8.10% vs 7.22%, p = 0.58) between the two groups.ConclusionThis study shows a lower LBR in FET cycles when compared to fresh cycles. Our results suggest that any potential gains in LBR due to improved embryo-endometrial synchrony following FET are lost, presumably due to freeze-thaw process-related embryo damage.

Systematic review of artemisinin embryotoxicity in animals: Implications for malaria control in human pregnancy

Pregnant women are one of the most susceptible and vulnerable groups to malaria, the most important parasitic disease worldwide. Artemisinin-based combination therapies (ACTs) are recommended for the treatment of uncomplicated malaria in all population groups including pregnant women. However, due to the embryotoxicity observed in animal studies, ACTs have long been contraindicated during the first trimester in pregnant women. Despite the safety concerns raised in pre-clinical studies, recent findings on ACTs's use in pregnant women appear to be reassuring regarding safety and have prompted a revision of malaria treatment guidelines for first trimester of pregnancy. To contribute to the risk-benefit assessment of ACTs, we conducted a systematic literature review of animal studies published between 2007 and 2019, which evaluated the embryotoxic effects of artemisinin and its derivatives among pregnant mammals. Eighteen experimental studies fitted the inclusion criteria. These studies confirmed and further characterized the severe embryolethal and embryotoxic dose-dependent effects of artemisinin and its derivatives when administered during the organogenesis period in rats, rabbits and monkeys. Timing of administration and dosage of the drug were found to be key factors in the appearance of embryo damage. Overall, the translation of the findings of artemisinin derivatives use in animal studies to pregnant women remains disturbing. Thus, a policy change in the use of ACTs during the first trimester in pregnant women for the treatment of uncomplicated malaria does not seem pertinent and if implemented, it should be accompanied by solid pharmacovigilance systems, which are challenging to establish in malaria endemic countries.

Glyphosate Induces Metaphase II Oocyte Deterioration and Embryo Damage by Zinc Depletion and Overproduction of Reactive Oxygen Species

Glyphosate is the most popular herbicide used in modern agriculture, and its use has been increasing substantially since its introduction. Accordingly, glyphosate exposure from food and water, the environment, and accidental and occupational venues has also increased. Recent studies have demonstrated a relationship between glyphosate exposure and a number of disorders such as cancer, immune and metabolic disorders, endocrine disruption, imbalance of intestinal flora, cardiovascular disease, and infertility; these results have given glyphosate a considerable amount of media and scientific attention. Notably, glyphosate is a powerful metal chelator, which could help explain some of its effects. Recently, our findings on 2,3-dimercapto-1-propanesulfonic acid, another metal chelator, showed deterioration of oocyte quality. Here, to generalize, we investigated the effects of glyphosate (0 – 300 μM) on metaphase II mouse oocyte quality and embryo damage to obtain insight on its mechanisms of cellular action and the tolerance of oocytes and embryos towards this chemical. Our work shows for the first time that glyphosate exposure impairs metaphase II mouse oocyte quality via two mechanisms: 1) disruption of the microtubule organizing center and chromosomes such as anomalous pericentrin formation, spindle fiber destruction and disappearance, and defective chromosomal alignment and 2) substantial depletion of intracellular zinc bioavailability and enhancement of reactive oxygen species accumulation. Similar effects were found in embryos. These results may help clarify the effects of glyphosate exposure on female fertility and provide counseling and preventative steps for excessive glyphosate intake and resulting oxidative stress and reduced zinc bioavailability.