Search

Hypothermic storage of gametes and embryos at 4 °C can be used as an alternative to cryopreservation, but hypothermic preservation can maintain embryo viability for a short duration only. This study investigated the effect of insulin-transferrin-sodium selenite (ITS) in embryo culture medium on hypothermic storage of bovine embryos at 4 °C. Day 7 bovine embryos were subjected to hypothermic storage in tissue culture medium 199 supplemented with 50% fetal bovine serum and 25 mM HEPES for different time durations. After recovery, the embryos were assessed for survival and hatching rate and gene and protein expression levels. Supplementation of embryo culture medium with ITS significantly increased (P 0.05) the survival and hatching ability of blastocysts stored at 4 °C for 72 h compared to the control group (100% and 76.3% vs 68.5% and 40.5%, respectively). Furthermore, the beneficial effects of ITS on embryos were associated with greater (P 0.05) total cell number per blastocyst and lesser apoptotic cells number. Moreover, embryos cultured in ITS had lower intracellular lipid content. The protein expression of sirt1 was greater (P 0.05) in the ITS group, however, caspase3 protein expression was significantly lesser (P 0.05) in the ITS group. Quantitative reverse transcription PCR indicated that the mRNA levels of SIRT1 and HSP70 were (P 0.05) increased upon culture with ITS; however, the mRNA levels of the pro-apoptotic genes BAX and CASP3 were reduced (P 0.05). Taken together, these data suggest that supplementation of embryo culture medium with ITS improves in vitro bovine embryo quality and survival following hypothermic storage.

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

For over a century, 2D cell culture has been extensively used for all the different research fields. However, this in vitro system does not allow to reproduce the natural structures of the original tissue, causing several changes and, in most cases, the loss of cell-to-cell communications and cell-to-extracellular matrix interactions. Based on this, during the last years, novel 3D platforms, able to mimic the in vivo milieu, are being developed. The advantages of the use of 3D models are: the reduction of the gap between cell culture and physiological environment; imitation of the specific architecture; partially maintenance of the mechanical and biochemical cues of the original tissue. Currently, 3D systems are used in a broad range of studies, including the field of reproduction, where they have been applied to promote maturation of follicles and oocytes and embryo culture. Here, we review 2D and 3D cell culture methods, discussing advantages and limitations of these techniques. We report the fundamental mechanisms involved in cell ability to perceive and respond to mechanical cues and their role in transmitting signals to and between cells and in regulating intracellular signaling pathways. In particular, we focus on the main effectors of the Hippo pathway, Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (TAZ), describing their behavior and function in oocytes and embryos. Lastly, we provide an overall perspective of the most recent 3D technologies developed in the field of reproduction, describing how their use may revolutionize the understanding of cellular behavior and provide novel tools, useful in reproductive technologies and livestock production.

Zearalenone (ZEA), which is produced by several fusarium mycotoxins, is found in animal feed and food products, and can exert estrogen-like activity. Melatonin (MT) is emerging as a supplement that can fight the toxic effects of mycotoxins. With a variety of physiological functions that play crucial roles in the development of animal germ cells and embryos, melatonin regulates circadian rhythms and has an anti-inflammatory and anti-oxidative role. This study investigated the protective effects of melatonin against ZEA in porcine early embryonic development. Our results showed that ZEA adversely affected this development, while melatonin supplementation ameliorated the toxic effects. ZEA exposure increased oxidative stress and impaired mitochondrial function, which may affect blastocyst formation. Moreover, we found that ZEA exposure promotes apoptosis, DNA damage, and autophagy in porcine blastocysts. The toxic effects of ZEA on early embryos may be the result of oxidative stress-mediated early apoptosis, while melatonin treatment significantly improved these phenotypes in ZEA-exposed porcine early embryos. Taken together, our results indicate that melatonin has a protective effect on defects caused by ZEA during early porcine embryonic development.

Gastrulation is a phase in early mammalian development when the three germ layers are generated and body plan is formed. Although well studied in mice, much less is known about gastrulation in humans. Owing to the lack of access to primary human tissue for study and experimental manipulation, as well as legal and ethical constraints surrounding the use of human embryos, a dissection of the molecular and cellular mechanisms that underlie this process in humans has proven elusive. Nonhuman primates, owing to their relatedness to human species, comprise a tantalizing alternative model system for understanding human biology. Two recent studies have established novel systems to study monkey embryos for 20 days, demonstrating landmark events of early primate embryogenesis with possible relevance to human development. Most strikingly, cells grown in the dish closely resembled cells in in vivo embryos, suggesting that embryo development in a dish might actually be equivalent to that which occurs in vivo. In this piece, the author discusses the tremendous potential of these new methods to unveil insights into mechanisms that mediate primate embryo development. Moreover, repurposing the extended monkey embryo culture methods to create human-monkey embryonic chimeras would aid the development of strategies to create human organs inside livestock species. Finally, the ethical and regulatory issues that emerge from reconsideration of extending time limits for human embryo culture beyond 14 days or primitive streak formation are also briefly considered.

The purpose of this study was to explore the application of glucose to improve embryo development potential when fertilization is delayed in mice. After recovery, mouse oocytes were cultured alone for 6 h before fertilization in three fertilization media: G-IVF PLUS, G-IVF PLUS with 5 mM and 10 mM glucose. G-IVF PLUS group was used as the control group. Then

The presence of serum in embryo culture medium has been implicated for increased embryo’s sensitivity to cryopreservation, compromised viability, abnormal embryo and fetal development. Hence, designing a serum free culture system is indispensable. The present study aims to compare the efficiency of the serum and granulosa cells monolayer free commercial culture system (SFCS) with the conventional serum supplemented co-culture system (SSCS) and optimized culture system (OCS). Generally, SFCS is designed explicitly for bovine oocyte maturation and embryo culture (SF-IVM and SF-IVC), and SSCS (based on M199, SS-IVM, and SS-IVC) is utilized for buffalo in vitro embryo production. However, OCS is a newly designed culture system in which oocyte maturation is performed in serum supplemented maturation medium, and the subsequent embryos are co-cultured with granulosa cells in serum free culture medium. To evaluate the effect of serum on buffalo embryo production, buffalo oocytes, and their subsequent embryos were cultured in SSCS, SFCS, and OCS, simultaneously. The percentage of cleaved embryos cultured in SSCS and OCS was approximately 4% higher as compared to SFCS. However, OCS significantly showed the maximum proportion of embryos that developed to the blastocyst stage (7d) and hatched (6d) as compared to the SFCS and SSCS. Additionally, OCS promoted the expression of developmentally important genes (BCL2-L1 and VEGF-A), cell number, and cryo-survival ability of blastocysts in comparison with SSCS. Taken together, OCS is more suitable for the oocyte maturation and culture of buffalo embryos. However, to design the serum free culture system, it is recommended to find suitable serum alternatives for in vitro oocyte maturation.

In the present study, four experimental groups were used: fresh embryos, cultured during in vitro maturation and in vitro culture in media supplemented with bovine serum albumin (BSA) (fresh BSA) o...

Improving the quality and the developmental competence of in vitro produced (IVP) embryos is an indispensable goal for assisted reproductive technology. Autophagy is a major protective mechanism for intracellular degradation of unnecessary cytoplasmic components. Autophagy ends by the fusion between autophagic vacuoles and lysosomes, allowing the degradation of the cargo by lysosomal enzymes, especially the cathepsins (CTSs). However, it is still unclear how autophagy and cathepsin K (CTSK) relate to embryo development. This study evaluated (1.) the activities of autophagy and CTSK in relation to bovine embryo quality and (2.) the effect of autophagy induction and/or CTSK inhibition on preimplantation embryo development and quality. We show here that good-quality embryos exhibited a greater autophagic activity and less CTSK activity compared to poor-quality embryos. Blastomeres of an individual embryo may vary in their quality. Good quality blastomeres showed an increased autophagic activity and decreased CTSK activity compared to poor-quality blastomeres within the same embryo at different developmental stages. Importantly, induction of autophagy and/or inhibition of CTSK improved the developmental rate (increased blastocyst and hatching rates) and the quality (increased total cell number and decreased the percentage of apoptotic cells) of IVP bovine embryos. These results demonstrate a promising approach to selectively isolate good-quality embryos and improve the efficiency of IVEP of cattle embryos.

A successful pregnancy requires a synchronous dialogue between endometrium and embryo within the endometrial milieu. The aim of this study was to assess the role in the implantation of mediators in the endometrial milieu. Total secretome (TS), soluble secretome (SS) and small extracellular vesicles (containing exosomes) were generated from hormonally primed human endometrial epithelial cell culture medium. Human trophectoderm stem cell-derived spheroids were cultured with TS, SS or exosomes (30 µg/ml) on hormonally primed epithelial cells, with exosomes significantly increasing cell adhesion and outgrowth. Furthermore, F1 mouse 2-cell embryos were cultured in groups for 48 h followed by culture with each secretome fraction (30 µg/ml) for 48 h. Blastocyst cell number and hatching were quantified. In addition, blastocysts were further cultured on a fibronectin matrix for 72 h or transferred to recipient mice (with corresponding secretomes) with embryo implantation assessed after 6 days. Exosomes significantly increased total cell number in mouse embryos and complete hatching from zona pellucida, with both exosomes and SS significantly enhancing mouse embryo outgrowth. Importantly, exosomes increased the embryo implantation rate in comparison to other secretome fractions (normalized based on treatment amount) from the endometrial epithelia. These data indicate that endometrial epithelial exosomes support embryo growth, development and implantation while the SS has selective involvement specifically on mouse embryo outgrowth. This finding provides new insights into the molecular differences of endometrial secretome components in implantation and early embryo development and may implicate endometrial exosomes in the pathophysiology of implantation failure in infertility.

PURPOSE: To evaluate whether mtDNA content at the blastocyst stage differs between embryos derived from fresh or vitrified sibling oocytes. MATERIAL AND METHODS: A retrospective analysis was performed between March 2017 and September 2018, including 504 blastocysts from 94 couples undergoing preimplantation genetic testing for aneuploidies (PGT-A), using fresh oocytes together with previously vitrified oocytes. Trophectoderm biopsies were performed and subjected to next generation sequencing. RESULTS: On average, 1.8 ± 1.0 oocyte vitrification cycles were performed per patient. Between fresh and vitrified cycles, no difference was observed between the number of fertilized oocytes (5.3 ± 4.2 versus 5.5 ± 3.0). Blastulation rate on day 5 per fertilized oocyte was significantly higher in the fresh group (62% ± 29% versus 44% ± 31%; p

Does Embryogen®/BlastGen™ culture medium improve live birth rates compared with standard culture medium for women undergoing IVF and intracytoplasmic sperm injection (ICSI) with poor prognosis.Randomized clinical trial. A total of 100 couples undergoing IVF/ICSI were randomly allocated to having their inseminated oocytes incubated in either Embryogen®/BlastGen™ sequential culture media or standard Cleavage/Blastocyst sequential culture media for 5 days (ClinicalTrials.gov Identifier: NCT02305420).No statistically significant difference in live birth rate was found between the control group and the Embryogen®/BlastGen™ group (17 [34%] versus 11 [22%], respectively) (OR 0.55; 95% CI 0.22 to 1.32; P = 0.18). After adjustment for maternal age, body mass index and fertilization procedure, the blastulation rate reduced (40.6 ± 26.5 versus 24.6 ± 26.7; RR 0.70, CI 0.52 to 0.95; P0.05), and grade of the embryo transferred (OR 0.35, CI 0.16 to 0.77; P0.01) when Embryogen®/BlastGen™ medium was used.A significant reduction in day-5 embryo outcome parameters was found using Embryogen®/BlastGen™ compared with standard medium, and insufficient evidence of a difference in pregnancy outcomes. Taking into consideration the small samples size, study limitations and strict inclusion criteria of this single-centre study, further research is needed to determine the efficacy of Embryogen®/BlastGen™ medium in couples undergoing IVF/ICSI.

Many domestic donkey breeds are at risk of extinction, there is a critical urgency for genome resource banking. In the present study, we examined whether the use of Ficoll 70 added to the vitrification medium containing ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose improves the cryotolerance of donkey in vivo derived embryos. Day 7-8, grade 1-2 donkey embryos were measured and morphologically evaluated and then vitrified-warmed using the Cryotop technique. Before vitrification, embryos were randomly distributed into two groups: (i) VS1 (n = 14): vitrified using 15% EG + 15% DMSO + 0.5 M sucrose; and (ii) VS2 (n = 10): vitrified in the same medium supplemented also with 18% of Ficoll 70. After 24 h of warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). Post-warming survival was numerically higher but not significantly different (P 0.05) when embryos were vitrified in VS2 (70%) compared to VS1 (57.1%). Embryo rupture was only observed in the VS1 group (21.4%, 3/14). Higher embryo diameter was observed in all groups after 24 h culture (P 0.05). No significant differences (P 0.05) were observed among treatments in terms of percentages of cell death. These results demonstrate that the addition of Ficoll 70 to the vitrification medium is not a pre-requisite for successful vitrification of donkey embryos. However, its addition seems to enhance some of the post-warming embryo quality characteristics. Since no statistically significant evidence was found, further studies should be conducted in order to confirm our findings.

In somatic cell nuclear transfer (SCNT) embryos, developmental defects first appear at the time of zygotic genome activation (ZGA), a process that is under the control of DNA and histone methylation. However, dynamics of 5-mC and 5-hmC during ZGA differ between porcine and bovine SCNT embryos, and histone methylation during ZGA in goat SCNT embryos remains poorly understood. Therefore, in the present study, we investigated the dynamic changes of 5-mC, 5-hmC, H3K4me2/3, and H3K9me3, as well as the expression of key genes related to these epigenetic modifications, during ZGA in goat cloned embryos. Compared with the IVF embryos, the 5-mC signal intensity was significantly increased at the 2- and 4-cell stage SCNT embryos, and the H3K4me3 and H3K9me3 signal intensity was significantly increased at 2- to 8-cell stage SCNT embryos, while the 5-hmC and H3K4me2 signal intensity was significantly lower at the 4- and 8-cell stage SCNT embryos. Of note, the H3K9me3 level was also significantly higher, whereas H3K4me3 signal intensity showed no statistical difference in the pronuclear stage SCNT embryos. Moreover, the expression of TET2, DNMT3B, KDM4A, SUV39H1, G9A, and SETDB1 was significantly increased, while the expression of UHRF1, PCNA, KDM4B, KDM4D, KDM5A, KDM5B, and KDM5C was significantly decreased at the 8-cell stage SCNT embryos. Our data revealed aberrant DNA and histone methylation during ZGA in goat cloned embryos. We further inferred that the abnormally higher level of 5-mC, H3K4me3, and H3K9me3 might serve as epigenetic barriers of the reprogramming and modifying these aberrant modifications might be a promising strategy to improve cloning efficiency in goat.

The cytokine platelet factor 4 (PF4) enhances differentiation and cell viability of different stem cells lines in vitro. This study investigated whether PF4 addition to customary pig embryo semi-defined culture media can improve their developmental outcome (Experiment 1) and ultimately replace the need for bovine serum albumin (BSA, Experiment 2). Experiment 1 added PF4 (100-1000 ng/mL, 0 = control) to NCSU-23 with 0.4 mg/mL BSA culturing 3430 presumptive zygotes. Experiment 2 added PF4 (100 -1000 ng/mL, 0 = Control-PVA) to a BSA-free medium (NCSU-23 with 0.3 mg/mL PVA) culturing 3820 presumptive zygotes. Zygote culture in NCSU-23 with 0.4 mg/mL BSA was used as overall control. All groups of Experiment 1 displayed similar rates of day 2-cleavage (range: 65.0 +/- 10.9 to 70.0 +/- 5.8%); of day 7-blastocyst rates (range: 46.6 +/- 10.0 to 56.4 +/- 8.2%) and of total day 7-blastocyst efficiency (range: 32.3 +/- 8.3 to 37.2 +/- 7.3%). Addition of PF4 did not affect total cell numbers of day 7 blastocysts (range: 44.1 +/- 23.2 to 50.5 +/- 26.4). In Experiment 2, PF4 accelerated embryo development, increasing (P < 0.01) blastocyst yield compared to 0-PF4, and blastocyst formation by day 5 adding PF4 100-500 ng/mL (range: 29.9 +/- 7.8 to 31.8 +/- 5.5%; P < 0.05) compared with BSA-control (17.2 +/- 8.2%) and PF4 1000 ng/mL (15.5 +/- 7.9%); showing similar blastocyst rates (range: 42.0 +/- 11.5 to 49.3 +/- 10.0%), total efficiency (28.0 +/- 8.2 to 32.3 +/- 7.1%) total cell numbers (range: 42.6 +/- 19.3 to 45.7 +/- 23.9) as BSA-controls. In conclusion, although PF4 did not show additive improvement under usual semi-defined, BSA-supplemented embryo media, it successfully replaced BSA sustaining porcine blastocyst production in chemically defined conditions. (C) 2019 Elsevier Inc. All rights reserved. Funding Agencies|Ministry of Science, Innovation and Universities-ERDF, Madrid, Spain [RTI2018-093525-B-I00]; Seneca Foundation, Murcia, SpainFundacion Seneca [19892/GERM/15]; Research Council FORMAS, Stockholm, Sweden [2017e00946]

SummaryThe process of embryonic development is crucial and radically influences preimplantation embryo competence. It involves oocyte maturation, fertilization, cell division and blastulation and is characterized by different key phases that have major influences on embryo quality. Each stage of the process of preimplantation embryonic development is led by important signalling pathways that include very many regulatory molecules, such as primary and secondary messengers. Many studies, bothin vivoandin vitro, have shown the importance of the contribution of reactive oxygen species (ROS) as important second messengers in embryo development. ROS may originate from embryo metabolism and/or oocyte/embryo surroundings, and their effect on embryonic development is highly variable, depending on the needs of the embryo at each stage of development and on their environment (in vivoor underin vitroculture conditions). Other studies have also shown the deleterious effects of ROS in embryo development, when cellular tissue production overwhelms antioxidant production, leading to oxidative stress. This stress is known to be the cause of many cellular alterations, such as protein, lipid, and DNA damage. Considering that the same ROS level can have a deleterious effect on the fertilizing oocyte or embryo at certain stages, and a positive effect at another stage of the development process, further studies need to be carried out to determine the rate of ROS that benefits the embryo and from what rate it starts to be harmful, this measured at each key phase of embryonic development.

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

Cryopreservation of in vitro-derived bovine embryos is a crucial step for the widespread reproduction and conservation of valuable high-merit animals. Given the current popularity of bovine in vitro embryo production (IVP), there is a demand for a highly efficient ultra-low temperature storage method in order to maximize donor ovum pickup (OPU) turn-over, recipient availability/utilization and domestic/overseas commercial trading opportunities. However, IVP bovine embryos are still very sensitive to chilling and cryopreservation, and despite recent progress, a convenient (simple and robust) protocol has not yet been developed. At the moment, there are two methods for bovine IVP embryo cryopreservation: slow programmable freezing and vitrification. Both of the aforementioned techniques have pros and cons. While controlled-rate slow cooling can easily be adapted for direct transfer (DT), ice crystal formation remains an issue. On the other hand, vitrification solved this problem but the possibility of successful DT commercial incorporation remains to be determined. Moreover, simplification of the vitrification protocol (including warming) through the use of an in-straw dilution without the use of a microscope is a prerequisite for its use under farm conditions. This review summarizes the bovine IVP embryo cryopreservation achievements, strengths and limitations of both freezing systems and prospective improvements to enhance cryosurvival, as well as perspectives on future directions of this assisted reproductive technology.

application/pdf, Theriogenology. 2020, 147 (15), P.71-76

We aimed to evaluate whether or not time lapse selection was beneficial for the cleavage-stage embryo transfers. The study included 838 infertile women with good ovarian reserve (obtaining more than 8 oocytes) from January 2018 to August 2019. Based on the transferred embryos with different grades (grade I, II and III), the patients were divided into day 3 selection with conventional morphology (CM) and day 3 selection with time lapse (TL) groups. For the grade I and II embryos, we observed that CM and TL had similar implantation, clinical pregnancy and ongoing pregnancy (

SummaryThis study aimed to describe outcomes in four women aged 28–34 years with central cytoplasmic granulation (CCG) of the oocytes who underwentin vitrofertilization/intracytoplasmic sperm injection (ICSI) using gonadotrophin-releasing hormone (GnRH) agonist to replace human chorionic gonadotrophin (hCG) as a trigger of final oocyte maturation. The initial ICSI procedure showed that all four women had CCG of the ooplasm and poor quality embryos. Subsequent ICSI used an antagonist protocol with a GnRH agonist trigger replacing the agonist protocol, plus hCG triggered ovulation. Ooplasm and embryo quality were improved in all four patients. All four became pregnant and gave birth to live infants. This study provides GnRH agonist triggering that may improve ooplasm granularity and embryo quality.

Aim To compare the pregnancy outcomes after day 5 blastocyst-stage embryo transfers (BET) versus day 6 BET following vitrified-warmed cycle and to evaluate whether the number of embryos transferred and the chromosomal status of embryo influence effect estimates. Methods A literature search (PubMed, Embase and MEDLINE) up to January 2019 was conducted to identify studies where women with day 6 BET were compared to women with day 5 BET. Only studies published in English language, on peer-reviewed journal were considered eligible. The following subgroup analyses were performed: (i) number of embryos transferred and (ii) chromosomal status of embryo. Results From a total of 1956 articles identified, 23 observational studies were included in the meta-analysis. We observed that day 6 BET were associated with lower implantation rate (risk ratio, RR: 1.17, 95% confidence interval, CI: 1.10-1.24), clinical pregnancy rate (RR: 1.17, 95% CI: 1.10-1.24), ongoing pregnancy rate (RR: 1.15, 95% CI: 1.07-1.24) and live birth rate (RR: 1.22, 95% CI: 1.11-1.33) than day 5 BET following vitrified-warmed cycle. The subgroup analysis found that the superiority of day 5 BET compared with day 6 BET is influenced by the number of embryos transferred and chromosomal status of embryos. Conclusion Current evidence shows that day 5 BET is superior to day 6 BET following vitrified-warmed cycle in clinical practice. Due to the overall low quality of available evidence, more larger and well-conducted studies are needed to compare the pregnancy outcomes between day 5 and day 6 BET before drawing a clear conclusion.

Direct transfer (DT) of cryopreserved embryos to recipients facilitates on-farm application. We analyzed a new freezing/thawing (F/T) procedure for in vitro produced (IVP) embryos, integrating: 1) an ethylene-glycol based system; 2) a culture step without protein; and 3) a synthetic protein substitute (CRYO3) in cryopreservation medium. IVP embryos from abattoir ovaries were cultured in groups in BSA-containing synthetic oviduct fluid with or without 0.1% fetal calf serum (FCS) until Day-6. Morulae and early blastocysts were subsequently cultured without protein from Day-6 onwards. Day 7 and Day 8 expanded blastocysts (EXB) were subjected to F/T or vitrification/warming (V/W). Thawed and warmed EXB were cultured in vitro, and development rates, cell counts and dead cells were analyzed in surviving embryos. V/W improved survival over F/T (live and hatching rates at 2 h, 24 h and 48 h) (P 0.0001), and FCS before Day 6 did not affect in vitro survival. After F/T, embryos had lower cell counts in the ICM, TE and total cells than after V/W. Day-7 embryos after F/T showed % apoptotic, % pycnotic and % total dead cells higher (p 0.05) than their Day-8 counterparts, probably because F/T reduced the numbers of ICM cells within Day-8 embryos. Thereafter, Day-7 blastocysts were transferred to heifers in an experimental herd. There were no differences in birth rates with frozen (-FCS [n = 40]: 45%; +FCS [n = 14]: 28%), vitrified (-FCS [n = 47]: 53%; +FCS [n = 11]: 36%) and fresh (-FCS [n = 30]: 47%; +FCS [n = 17]: 53%) embryos. However, frozen embryos produced with FCS showed 5/9 miscarriages after Day-40. Calves born from frozen (n = 22), vitrified (n = 29) and fresh (n = 22) transfers did not differ in birth weight, gestation length and daily gain weight (P 0.10). Subsequently, transfer of frozen embryos (n = 29) derived from oocytes collected from live, hormonally stimulated cows in experimental herd, led to pregnancy rates of 57% (heifers) and 40% (dry cows). with EXB on Day-62 Finally, embryos produced with BSA were transferred to cows in an on-field trial (frozen [n = 80]; fresh [n = 58]), with no differences in pregnancy rates (days 30-40). Pregnancy and birth rates could not be predicted from in vitro approaches. The new F/T system yields pregnancy and birth rates comparable to vitrified and fresh embryos without birth overweight. The absence of products of animal origin, defined chemical composition, and direct transfer entail sanitary, manufacturing and application advantages.

Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus-oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/μl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/μl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non-CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/μl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage.

Resveratrol (Resv; 3,4,5-trihydroxy-trans-stilbene) is a phytoalexin with antioxidant activity that modulates redox homeostasis in oocytes and improves in vitro embryo production. Cold storage of cat ovaries for a period longer than 24 h alters oxidative status of oocytes after in vitro maturation and reduces their developmental competence. The aim of this study was to evaluate the effect of resveratrol supplementation to the maturation medium on embryo development of oocytes after storage of domestic cat ovaries at 4 °C for 24 h or 48 h. Cumulus-oocyte complexes (COCs) were recovered from ovaries of domestic queens and cultured in maturation medium supplemented with (+) or without (-) 5 μM resveratrol for 24 h. COCs collected from fresh ovaries were matured in vitro (IVM) in standard conditions as control. After IVM, oocytes were in vitro fertilized (IVF) and presumptive zygotes cultured for 7 days. Oocyte nuclear maturation, reactive oxygen species (ROS) and glutathione (GSH) levels as well as cleavage, blastocyst formation and blastocyst cell number were determined. There were no differences in the maturation rates of oocytes between the control and stored groups, irrespective of resveratrol supplementation. Resveratrol treatment during IVM significantly increased the level of GSH and reduced the level of ROS of oocytes recovered from ovaries stored for 48 h as compared to the non-treated group (48 h-). The rate of blastocyst formation from oocytes recovered from ovaries after 48 h storage that underwent IVM with resveratrol was higher (P 0.05) than that of oocytes matured without resveratrol and similar to that of control oocytes. Resveratrol treatment increased (P 0.05) cell number in blastocysts from 24 h + and 48 h + groups as compared to their respective counterparts. In conclusion, our results demonstrated that resveratrol supplementation during IVM can reverse the adverse effect of oxidative stress on oocytes, and enhances embryo development after ovary storage at 4 °C for 48 h. These results may provide a basis for improving culture conditions and extend the possibility of storage of cat ovaries for more than 24 h thus ensuring successful in vitro embryo production.

Targeted knock‐in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)‐assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR‐assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS‐1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single‐stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS‐1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS‐1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS‐1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos., RS‐1 biases DNA repair towards homologous directed repair (HDR). Enhancing HDR increases the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)‐mediated targeted KI. Following transient RS‐1 exposure half of the injected bovine embryos contain the intended DNA insertion.

A great interest surrounds the development of nanoparticles (NPs) for biomedical applications such as drug delivery and cancer therapy. However, the interplay between nanoscale materials and biological systems and the associated hazards have not been completely clarified yet. In this study, bovine oviductal epithelial cells (BOECs) and embryos were used as in vitro models to investigate whether cell mitosis and early mammalian embryo development could be affected by the exposure to polystyrene (PS) nanoparticles. Analysis of the karyotype performed on BOECs exposed to PS-NPs did not show chromosomal anomalies compared to the control, although more tetraploid metaphase plates were observed in the former. In vitro fertilization experiments designed to understand whether exposure to PS-NPs could affect pre-implantation development showed that incubation with PS-NPs decreased 8-cell embryo and blastocyst rate in dose-dependent fashion. The quality of the blastocysts in terms of mean cell percent blastomeres with fragmented DNA was the same in exposed blastocysts compared to controls. These results show that the exposure to PS-NPs may impair development. In turn, this may affect the rate of mitosis in embryos and yield a lower developmental competence to reach the blastocyst stage. This suggests that release in the environment and the subsequent accumulation of PS-NPs into living organisms should be carefully monitored to prevent cytotoxic effects that may compromise their reproduction rates.

Capacitation-like changes affect sperm of several species, such as ram, reducing cell survival and fertilizing competence. Proteins from seminal plasma stabilize sperm plasma membranes, being an interesting focus to develop strategies for improving cryopreserved ram semen performance. To date, biotechnologies are focused to reduce damage in frozen-thawed ram spermatozoa through the addition of bioactives. Serine Protease Inhibitor Kazal-type 3 (SPINK3) is a little protein synthesized by mouse seminal vesicle and secreted to seminal plasma. While attached to the sperm, this protein binds to non-capacitated sperm and blocks calcium entry, avoiding a premature physiological capacitation and consequently, acrosome reaction. Due to these characteristics, SPINK3 has been proposed as a decapacitating factor. The aim of this work was to assess whether heterologous SPINK3 is able to protect ram sperm from the well-known cell damages produced by freezing/thawing and to understand the mechanisms by which it is acting. Sperm were supplemented with 13 μM SPINK3 before freezing in an egg yolk-based extender or after thawing and selection. Under both conditions, SPINK3 decreased intracellular calcium content (p < 0.05) and reduced the 25 kDa tyrosine phosphorylated protein demonstrating a decapacitating effect, although the addition of the protein before cryopreservation was not enough to improve other sperm parameters. However, the addition of SPINK3 post thawing was able to significantly ameliorate viability, motility, mitochondrial status and to avoid the increase of lipid peroxidation (p < 0.05). Moreover, sperm treated with SPINK3 was not only still capable to fertilize, but also improved it, as evidenced by an increase in the oocyte cleavage rate (p < 0.05) although, the embryo development and embryo quality were not affected. Our findings would contribute to develop a strategy for improving sperm quality by using decapacitating proteins. In fact, the outcomes of this work demonstrate that SPINK3 is able to reduce sperm cryo-injuries when is added after thawing, improving functionality and thus in vitro fertilization results., This research was supported by the National Agency for Scientific and Technological Promotion (ANPCyT, grant PICT-2015-3682 awarded to A.C), M.I.C was supported by Ministry of Economy and Competitiveness scholarship and the BeCAR program that facilitates the L.Z′ s stage in the UCLM, Spain.

In vitro fertilization (IVF) is an established technology that is widely used in reproductive engineering. However, in rats, successful application of IVF is difficult to achieve, and it has had poor reproducibility. In a previous study on the critical issues associated with successful IVF in Wistar rats, we investigated the influence of oocyte collection duration on fertilization rates by dividing the procedure into three steps (oviduct extraction from euthanized animals, oocyte collection from the ampullae of oviducts, and oocyte preincubation until insemination), and identified the appropriate times for each. Here we show that use of the same defined duration for oviduct extraction from superovulated Wistar rats and for oocyte collection from the oviducts also produced highly reproducible fertilization rates of more than 90% in other rat strains. Furthermore, the versatility of these criteria was demonstrated using another IVF protocol. Thus, this simple procedure has enabled the standardization of IVF in rats and will enhance further experimental studies.

PURPOSE: To investigate the associations of previous pregnancy failures, including implantation failures (IFs), biochemical pregnancy losses (BPLs), and early (EMs) and late miscarriages (LMs), with blastocyst aneuploidy and pregnancy outcomes after PGT-A. METHODS: This study included 792 couples who underwent PGT-A after multiple pregnancy failures. Subgroup analyses were used to compare the blastocyst aneuploidy rate (BAR), implantation rate (IR), early miscarriage rate (EMR), and live birth rate (LBR). Multiple linear and logistic regression models were used to evaluate the associations. The control group comprised couples with ≤ 2 IFs, ≤ 1 BPL, ≤ 1 EM, and no LM. RESULTS: Notably, a history of ≥ 4 IFs was significantly associated with an increase in aneuploid blastocysts (42.86% vs. 33.05%, P = 0.044, B = 10.23 for 4 IFs; 48.80% vs. 33.05%, P = 0.002, B = 14.43 for ≥ 5 IFs). Women with ≥ 4 prior EMs also harbored more aneuploid blastocysts (41.00% vs. 33.05%, P = 0.048; B = 9.23). Compared with the control group, women with ≥ 4 prior EMs had a significantly higher EMR (6.58% vs. 31.11%, P

Culturing of human embryos in optimal conditions is crucial for a successful in vitro fertilisation (IVF) programme. In addition, the capacity to assess and rank embryos correctly for quality will allow for transfer of the potentially ‘best’ embryo first, thereby shortening the time to pregnancy, although not improving cumulative pregnancy and live birth rates. It will also encourage and facilitate the implementation of single embryo transfers, thereby increasing safety for mother and offspring. Time-lapse technology introduces the concept of stable culture conditions, in connection with the possibility of continuous viewing and documenting of the embryo throughout development. However, so far, even when embryo quality scoring is based on large datasets, or when using the time-lapse technology, the morphokinetic scores are still mainly based on subjective and intermittent annotations of morphology and timings. Also, the construction of powerful algorithms for widespread use is hampered by large variations in culture conditions between individual IVF laboratories. New methodology, involving machine learning, where every image from the time-lapse documentation is analysed by a computer programme, looking for patterns that link to outcome, may in the future provide a more accurate and non-biased embryo selection.

Freezing and thawing of Bos indicus embryos affect their quality for embryo transfer. The objective of this study was to compare the levels of reactive oxygen species between Bos indicus and Bos taurus embryos produced in vivo, before and after conventional freezing, as well as to analyze damage caused by apoptosis and lipid peroxidation. Bos indicus has higher levels of reactive oxygen species than Bos taurus embryos, both fresh (14.32 ± 1.41 auf vs 8.07 ± 1.15 auf (arbitrary units of fluorescence), P

To study the origin and temporal behaviour of cytoplasmic strings spanning the blastocoel (main objective) and their influence on treatment outcome (secondary objective).This retrospective analysis of prospectively collected data was set up in a university medical centre. Patients who either underwent fresh (n = 95) or vitrified-warmed (n = 55) single blastocyst transfer were included. Time-lapse sequences of in-vitro developed blastocysts were screened for the presence of cytoplasmic strings. Pregnancies in string-positive and string-negative transfers were followed up to live birth.A total of 387 blastocysts were obtained in the fresh cycles of 100 patients, corresponding to a blastocyst formation rate of 62.4%. Cytoplasmic strings were first detected around full stage (108.5 ± 6.4 h) in 170 blastocysts (43.9%). The number of strings varied (range: 1-7) and the duration of visibility was 5.2 ± 3.5 h. The occurrence of cytoplasmic strings was significantly associated with the presence of blastocoelic collapses (P 0.001) but not with any of the annotated morphokinetic parameters. Live birth and neonatal outcome were the same for both string-positive and string-negative pregnancies. Moreover, collapses did not affect treatment outcome.Time-lapse analysis of cytoplasmic strings at the blastocyst stage revealed that this morphological feature was not a negative predictor as previously reported. Although physiologically normal, at least some of the cytoplasmic strings are an artefact, possibly associated with blastocoelic collapses.

Aging oocytes undergo various molecular, cellular, and biochemical changes. Aging of oocytes results in reduced embryo developmental capacity and blastocyst quality, which is thought to be caused partly by the accumulation of reactive oxygen species (ROS). This study aimed to determine the effect of l -carnitine (LC) on the development of embryos formed from aged oocytes in vitro. The development and quality of the blastocysts in the LC-treated group were significantly higher than those in the untreated aged group after in vitro fertilization (IVF). In addition, after LC treatment, the level of intracellular ROS in aged group significantly decreased, and glutathione (GSH) levels significantly increased compared with those in the untreated aged group. There was no significant difference in the mitochondrial membrane potential among the three groups. Moreover, ROS could induce autophagy and LC3 antibody was widely used as a marker for detecting autophagy. The fluorescence intensity of LC3 in the aged group was significantly higher than that of LC3 in the LC-treated group. Furthermore, Real-time reverse transcriptase-polymerase chain reaction showed that the mRNA levels of antioxidation genes GPX4 and SOD1 were significantly higher in embryos from LC-treated group than in those from the untreated aged group. In summary, our results indicated that LC can improve the developmental capacity of embryos from aging oocytes in vitro by reducing oxidative stress.

STUDY QUESTION Does the quality of a single transferred blastocyst affect singleton birthweight in frozen-embryo transfer (FET) cycles? SUMMARY ANSWER The transfer of a poor-quality blastocyst was associated with lower mean birthweight and gestation-adjusted birthweight (Z-scores) when compared with the transfer of an excellent-quality blastocyst during FET cycles. WHAT IS KNOWN ALREADY Embryo quality is a strong predictor of IVF success rates. However, very few studies have examined the effect of embryo quality on singleton birthweight. STUDY DESIGN, SIZE, DURATION This retrospective study involved singleton live births born to women undergoing frozen-thawed single blastocyst transfers during the period from January 2010 to December 2017 at a tertiary care centre. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 1207 women who fulfilled the inclusion criteria were included and were grouped into four groups depending on the blastocyst quality: excellent, good, average and poor. The primary outcome measure was singleton birthweight. The Z-score was employed to calculate the birthweight adjusted for gestational age and newborn gender. Multiple linear regression analysis was performed to investigate the relationship between embryo quality and neonatal birthweight after adjustment for some potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE In the primary multivariable model, singletons from the poor-quality blastocyst group weighed 183.5 g less than those from the excellent-quality blastocyst group (95% CI: −295.1 to −71.9 g, P = 0.001) in terms of mean birthweight after accounting for patient characteristics, IVF treatment parameters, the year of treatment and newborn gender. Likewise, poor-quality blastocyst transfer was associated with lower gestation-adjusted Z-scores than the transfer of excellent-quality blastocysts (β = −0.35, 95% CI: −0.59 to −0.12, P = 0.003). LIMITATIONS AND REASONS FOR CAUTION The current study was limited by its retrospective design and the fact that our analysis was restricted to women with singleton births from single blastocyst transfers. Future prospective studies are required to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS Our findings provide new insight into the relationship between embryo quality and neonatal outcomes by showing that poor-quality blastocyst transfer was associated with a decrease in singleton birthweight. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the National Key Research and Development Program of China (grant no. 2018YFC1003000), the National Natural Science Foundation of China (grant nos. 81771533, 81571397 and 31770989), and the China Postdoctoral Science Foundation (Grant no. 2018M630456). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER Not applicable.

Purpose To evaluate the relationship between elective blastocyst transfer, freezing strategy, and the abandonment of frozen embryos with a storage time limit of 10 years as specified in the National Assisted Reproduction Act of Taiwan. Methods This two-phase retrospective cohort study was conducted at a single tertiary center, Kaohsiung Chang Gung Memorial Hospital (KCGMH), in 2019. Participants were selected from a data registry containing 4167 fresh IVF cycles, including phase 1 cycles from 1999 to 2009 and phase 2 cycles from 2010 to 2014, at KCGMH. Results In phase 1, embryo abandonment was associated with the production of more mature oocytes and embryos, the freezing of more embryos, young female age, blastocyst transfer, and positive pregnancy results. After adjustment for confounding factors, only positive pregnancy results (adjusted odds ratio [aOR] 4.38, 95% confidence interval [CI] 3.17, 6.04), the freezing of ≥ 2 embryos (aOR 3.68, 95% CI 3.10, 4.38), the production of ≥ 6 embryos (aOR 1.68, 95% CI 1.03, 2.73), and the use blastocyst transfer (aOR 2.46, 95% CI 1.64, 3.69) remained significantly associated with embryo abandonment. The factors associated with embryo abandonment or possible abandonment were similar in phase 2. Conclusion For elective blastocyst stage transfer and a freezing strategy performed according to the Taiwan National Assisted Reproduction Act, a young female age ≤ 35 with positive pregnancy status due to the original IVF treatment, the production of ≥ 6 embryos, and the cryopreservation of ≥ 2 blastocysts may increase the likelihood of abandoning embryos in the future.

PURPOSE: To evaluate clinical outcomes after frozen-thawed blastocyst transfer (TBT) with blastocysts which were derived from different cell numbers on day 3. METHODS: The study included 1444 patients undergoing single autologous frozen-thawed blastocyst transfer cycles, which were allocated to five groups according to the cell numbers on day 3 of the transferred blastocysts: ≤ 6-cell (n = 109), 7-cell (n = 169), 8-cell (n = 811), 9-cell (n = 136), and ≥ 10-cell (n = 219). RESULTS: The LBR of the ≤ 6-cell group was found to be statistically lower than that of the 8-cell group in single TBT cycles which had been transferred with fair quality blastocysts (defined as 4BB according to Gardner’s grading scale) (41.28% vs 55.73%, P = 0.004), while the miscarriage rate was significantly higher for the ≤ 6-cell group compared with the 8-cell group (25.00% vs 13.74%, P = 0.02). No differences were found between the two groups in terms of cPR (P = 0.06). However, for blastocysts categorized as high quality according to Gardner’s classification (defined as 4AA/4AB/4BA), cPR, LBR, and early miscarriage rates did not differ between the two groups (P = 0.76, P = 0.44, P = 0.40, respectively). CONCLUSIONS: When transferring blastocysts, an evaluation of the cleavage stage should be performed along with blastocyst morphology to shorten the time of conceiving. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-019-01664-x) contains supplementary material, which is available to authorized users.

PURPOSE: To compare a single-step medium with a sequential medium on human blastocyst development rates, aneuploidy rates, and clinical outcomes. METHODS: Retrospective cohort study of IVF cycles that used Sage advantage sequential medium (n = 347) and uninterrupted Sage 1-step medium (n = 519) from July 1, 2016, to December 31, 2017, in an academic fertility center. Main outcome measures are blastocyst formation rates per two-pronuclear (2PN) oocyte and aneuploidy rates per biopsy. RESULTS: Of all IVF cycles, single-step medium yielded higher blastocyst formation rate (51.7% vs 43.4%) but higher aneuploidy rate (54.0% vs 45.8%) compared with sequential medium. When stratified by maternal age, women under age 38 had no difference in blastocyst formation (52.2% vs 50.2%) but a higher aneuploidy rate (44.5% vs 36.4%) resulting in a lower number of euploid blastocysts per cycle (2.6 vs 3.3) when using single-step medium compared to sequential medium. In cycles used single-step medium, patients ≥ age 38 had higher blastocyst rate (48.0% vs 33.6%), but no difference in aneuploidy rate (68.8% vs 66.0%) or number of euploid embryos (0.8 vs 1.1). For patients reaching euploid embryo transfer, there was no difference in clinical pregnancy rates, miscarriage rates, or live birth rates between two culture media systems. CONCLUSIONS: Our study demonstrates an increase in aneuploidy in young women whose embryos were cultured in a single-step medium compared to sequential medium. This study highlights the importance of culture conditions on embryo ploidy and the need to stratify by patient age when examining the impact of culture conditions on overall cycle potential. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-019-01621-8) contains supplementary material, which is available to authorized users.

The safety of assisted reproductive technology (ART) is of frequent concern. Unfortunately, animal models for studying the safety of intracytoplasmic sperm injection (ICSI) have limitations in mimicking human ICSI manipulations. As reported herein, we invented a modified holding pipette for mouse oocyte injection that resulted in the delivery of live pups. A modified holding pipette was prepared for mouse oocyte injection and was compared with the conventional pipette for human use and a trumpet-shaped pipette. After ICSI, the oocytes were cultured to cleavage embryos until fallopian transfer. The use of the trumpet-shaped holding pipette and the new modified holding pipette for mouse oocyte injection achieved comparable and satisfactory oocyte survival rates (83.44% and 85.71%, respectively) and embryo cleavage rates (41.98% and 42.42%, respectively), which were significantly higher than those obtained with the human egg-holding pipette (oocyte survival rate: 65.85%; embryo cleavage rate: 27.78%). After 13 embryos were transferred using each type of pipette, three live pups were produced with the new modified holding pipette, one was produced with the holding pipette for human use, and none were produced with the trumpet-shaped holding pipette. The modified holding pipette for oocyte injection is effective and very easy to prepare. Moreover, using this new method, we produced live pups, which will contribute to a useful animal model for safety studies of ICSI in the future.

Autophagy is a critical process in early mammalian embryogenesis. Mammalian target of rapamycin (mTOR) inhibitors are major regulators of autophagy. However, mTOR plays a vital role in major signaling pathways controlling cell growth and metabolism; thus, more secure autophagy activation methods should be considered. The present study investigated the effects of supplementary trehalose, a novel mTOR-independent autophagy enhancer, on oocyte maturation and embryonic development after parthenogenetic activation (PA). Trehalose treatment during in vitro maturation (IVM) did not affect the nuclear maturation rates of oocytes. Oocytes treated with 25 mM trehalose during IVM had a significantly higher (P 0.05) blastocyst formation rate (64.2%) after PA compared to that in control oocytes (52.0%). Blastocyst quality was also improved in the trehalose-treated group. The total cell numbers for blastocyst formation and expanded blastocyst formation were significantly increased in the trehalose-treated group (52.2% and 27.7%, respectively) compared to those in the control group (36.9% and 11.0%, respectively). Trehalose treatment led to the increased expression of LC3, an autophagy marker, in metaphase II oocytes and 4-cell stage embryos. Gene expression analysis revealed that the expression of several autophagy related genes (LAMP2, pATG5, and LC3) increased, while the Bax/Bcl2 ratio and pro-apoptotic Bak transcript levels were decreased in the trehalose-treated group. In conclusion, these results indicate that treatment with trehalose during IVM improved the developmental potential of porcine embryos by down-regulation of pro-apoptotic genes and up-regulation of autophagy-related genes and marker. Trehalose may be useful for the large-scale production of high-quality porcine blastocysts in vitro.

Research question Is there a difference in blastocyst formation between fresh and vitrified-warmed sibling oocytes and can this difference be attributed to changes in embryo morphokinetics? Design Between February 2016 and December 2017, 472 metaphase II (MII) oocytes in 67 donor-recipient cycles from 27 different healthy anonymous oocyte donors were allocated for fresh transfer (FSHO) (n = 220) to a synchronous recipient (n = 36) or vitrified (VITO) (n = 252) to be warmed and transferred to another recipient (n = 31). Embryos derived from the FSHO and their sibling VITO were analysed for morphokinetic development using time-lapse imaging, blastocyst formation and clinical outcome. Results Time-lapse analysis showed an overall delay in cleavage rate from the time of pronuclei disappearance up to the time of blastulation in the VITO compared with their sibling FSHO. Twelve morphokinetic variables were significantly different between the groups. On Day 5 significantly more FSHO embryos developed to blastocyst (expansion 1-6) and reached the full blastocyst stage (expansion 3-6) compared with the VITO embryos [53.2% (84/158) versus 40.0% (64/160); P = 0.0244 and 48.1% (76/158) versus 31.3% (50/160); P = 0.0028, respectively]. The embryo utilization rate was similar in both groups at the time of cryopreservation; 51.3% (FSHO) versus 45.0% (VITO) (P = 0.3124). The pregnancy rate per cycle was 47.2% (17/36) in FSHO patients and 48.4% (15/31) in VITO patients (P = 1). Limitations in this study: non-randomized, small study size and not powered to detect differences in clinical outcomes. Conclusions Timing of development is altered and blastocyst formation is delayed in embryos derived from vitrified-warmed donor oocytes compared with their fresh sibling counterparts. Although preliminary results suggest that the clinical impact of this delay may be limited, this needs further investigation in larger randomized studies.

Asiatic acid is a pentacyclic triterpene enriched in the medicinal herb Centella asiatica, and it has been suggested to possess free radical scavenging and anti-apoptotic properties. The purpose of the current study was to explore the effects of asiatic acid on porcine early-stage embryonic development and the potential mechanisms for any observed effects. The results showed that 10 μM asiatic acid supplementation during the in vitro culture period dramatically improved developmental competence in porcine embryos derived from parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). Further analysis revealed that asiatic acid attenuated H2O2-induced intracellular reactive oxygen species (ROS) generation. Notably, asiatic acid not only enhanced intracellular GSH levels but also attenuated mitochondrial dysfunction. Gene expression analysis revealed that asiatic acid upregulated expression of the antioxidant-related gene Sod-1 and the blastocyst formation related gene Cox-2, while downregulating expression of the apoptosis-related gene Caspase-9 in SCNT blastocysts. These results suggest that asiatic acid exerts beneficial effects on early embryonic development in porcine embryos and that asiatic acid may be useful for improving the in vitro production of porcine embryos.

Male and female embryos are known to be different in developmental kinetics, metabolism, gene expression, and epigenetic patterns. Therefore, the objective of this study was to clarify whether the morphological criteria used to select embryos for cryopreservation lead to a deviation in the male:female ratio, and whether vitrification effects vary according to embryo sex. Initially, five sires were tested to evaluate the effect of the bull on embryo development, sex ratio, speed of development, and response to cryopreservation. Results showed that bulls affected (P 0.05) embryo production, response to cryopreservation, and sex ratio. Then, one bull was selected, and used to produce embryos in vitro to characterize the responses of male and female embryos to vitrification. Results suggested that male and female embryos have the same morphological responses to vitrification, as no differences (P 0.05) were observed between the two sexes in post-warming survival and re-expansion rates. However, their molecular responses as evaluated by gene expression (FOSL1, HSPB1, CASP3, CASP8, HSPA5, HSPA1A, G6PD, and PGK1) analysis indicated an effect of sex on vitrification; vitrified female embryos exhibited higher mRNA levels of HSPA1A, CASP3, and G6PD compared to their male counterparts. In conclusion, bulls affected embryo production, speed of development, sex ratio, and response to cryopreservation. Male and female embryos differed in their molecular responses to vitrification; and also, deviations in the male:female ratio when selecting embryos for cryopreservation were confirmed.

The objective of this research was to develop a system for piglet production by transvaginal ultrasound-guided ovum pick-up (OPU), in vitro production (IVP) of embryos and embryo transfer. First, to establish a culture system for a small number of oocytes or embryos, we evaluated the effect of different incubation volumes and culture densities on fertilizing ability and developmental competence in vitro. Porcine oocytes derived from slaughterhouse ovaries were matured, fertilized and then cultured in vitro in groups as follows: 50 oocytes in 500 μL medium for IVM, 20 oocytes in 100 μL medium for IVF and 20 embryos in 40 μL medium for IVC (Group I); 20 in 100 μL for IVM, 20 in 100 μL for IVF and 20 in 40 μL for IVC (Group II); and 10 in 100 μL for IVM, 10 in 100 μL for IVF and 10 in 40 μL for IVC (Group III). Percentages of sperm penetration, cleavage and blastocyst formation did not differ among the groups. Second, to increase the collection efficiency of porcine oocytes by transvaginal ultrasound-guided OPU, the effects of aspiration pressure on follicular oocyte collection were assessed. Oocytes were aspirated from ovaries of live sows using 80 or 100 mmHg. The recovered oocytes were divided into four categories according to the surrounding cumulus cells and quality of oocytes. The number of oocytes recovered using 100 mmHg pressure was significantly higher than with 80 mmHg pressure. However, there were no significant differences in the population of oocytes grouped by the morphological criteria, number of blastocysts per session and the total cell number in blastocysts between the two vacuum pressures. Finally, 81 oocytes obtained by OPU from five donor sows were subjected to IVP and 47 transferable embryos (9.4 ± 4.0 [mean ± SD] morulae/blastocysts per session) were obtained at 5 days after IVF. When they were transferred into five recipient gilts (5–16 embryos per recipient), three of five recipients became pregnant and farrowed a total of 12 live piglets. The present results demonstrate that porcine blastocysts can be produced by OPU-IVP and develop to full term after embryo transfer.

We examined the effect of ploidy on mitochondrial DNA (mtDNA) copy number in embryos and the amount of cell-free mitochondrial and nucleic DNA content (cf-mtDNA and cf-nDNA) in spent culture medium (SCM). Oocytes collected from the ovaries were matured, activated, incubated in medium containing cycloheximide (CHX) or CHX and cytochalasin B (CB) for 4.5 h to produce haploid or diploid embryos (H-group and D-group embryos). These embryos were cultured for 7 days, and the blastocysts and SCM were examined. The amount of mtDNA and nDNA was determined by real-time PCR. The rate of development to the blastocyst stage was higher for the D-group than for the H-group. Moreover, D-group blastocysts had less mtDNA compared to the H-group blastocysts. After activation, the mitochondrial content was constant before the blastocyst stage in D-group embryos, but increased earlier in H-group embryos. The amount of cf-mtDNA in the SCM of D-group blastocysts was greater than that of H-group blastocysts. However, when the cf-mtDNA in the SCM of 2 cell-stage embryos (day 2 post-activation) was examined, the amount of cf-mtDNA was greater in the H-group than in the D-group embryos. When D-group embryos were cultured for 7 days, a significant correlation was observed between the total cell number of blastocysts and cf-nDNA content in the SCM. Hence, although careful consideration is needed regarding the time point for evaluating mtDNA content in the embryos and SCM, this study demonstrates that mtDNA in the embryos and SCM was affected by the ploidy of the embryos.

Endoplasmic reticulum (ER) stress, a dysfunction in protein folding capacity of the ER, is involved in many physiological responses including mammalian reproductive systems. Studies have shown that ER stress interferes with the developmental process of in vitro oocyte maturation and embryo development; however, little is known about its effects on bovine preimplantation embryonic development. In this study, we examined the effects of ER stress during IVC on developmental competency and cryo-tolerance in bovine embryos. IVF-derived zygotes were cultured in CR1aa medium supplemented with tauroursodeoxycholic acid (TUDCA) and/or tunicamycin (TM), which are ER stress-inhibitory and stress-inducing agents, respectively, for 8 days. TM treatment decreased the blastocyst developmental rate and increased the percentage of apoptotic cells compared to that in the control group (10.2 ± 2.3% vs. 39.75 ± 1.3% and 17.8 ± 1.2% vs. 3.6 ± 1.1%, respectively; P 0.01). However, the blastocyst developmental rate was increased and the percentage of apoptotic cells was decreased by addition of TUDCA in IVC medium compared to that in the control group (50.9 ± 0.9% vs. 39.75 ± 1.3% and 1.13 ± 1.0% vs. 3.6 ± 1.1%, respectively; P 0.01). Importantly, in the group treated with TM plus TUDCA, the developmental rate and the percentage of apoptotic cells in blastocysts were similar to that in the control group, indicating that TUDCA ameliorates the adverse effects of TM alone on embryo development. In addition, TUDCA treatment significantly reduced the reactive oxygen species, expression of ER stress (GRP78, ATF4, ATF6, IER1, and sXBP1) and pro-apoptotic (CHOP and BAX) genes, while it increased anti-apoptotic BCL2 gene expression and glutathione levels. Moreover, TUDCA improved blastocyst cryo-tolerance as marked by a significantly increased hatching rate and decreased the number of apoptotic cells recorded at 48 h after a post-warming. Therefore, in concordance with a previous report in mice or pig, we showed that TUDCA supplementation during IVC increases the developmental competency of bovine in vitro-derived embryos. Additionally, we found that the presence of TUDCA in IVC medium improves the cryo-tolerance of bovine embryos. These results suggest that modulation of ER stress during IVC contributes to the production of high-quality bovine embryos in terms of cryo-tolerance.

STUDY QUESTION Do different oxygen levels during human IVF embryo culture affect embryo utilization, cumulative IVF success rates per cycle and neonatal birthweight? SUMMARY ANSWER After 2 days of culture, a lower oxygen level (5%) leads to more good-quality embryos and more embryos that can be cryopreserved, and thereby to a higher cumulative live birth rate per cycle when compared to embryo culture in 20% oxygen, while birthweights are similar. WHAT IS KNOWN ALREADY Several studies have compared IVF outcome parameters after embryo culture in a more physiological level of 5% oxygen and the atmospheric level of 20%. Although there is consensus that embryo development improves in 5% oxygen, effects on pregnancy and live birth rates are mainly seen in blastocyst, but not cleavage-stage transfers. A major drawback of these studies is that only fresh embryo transfers were included, not taking additional frozen-thawed transfers from these cycles into account. This might have underestimated the effects of oxygen level, especially in cleavage-stage embryo transfers. Furthermore, little is known about the effect of oxygen level during culture on birthweight. STUDY DESIGN, SIZE, DURATION This is a cohort study in 871 consecutive patients who had an IVF cycle between January 2012 and December 2013, and 5–7 years follow-up to allow transfer of frozen-thawed embryos. Based on daily availability of positions in the incubators, all oocytes and embryos of one cycle were allocated to one of the three incubators with traditional ambient oxygen levels (6% CO2 and 20% O2 in air), or to a fourth incubator that was adjusted to have low oxygen levels of 5% (6% CO2, 5% O2 and 89% N2). Embryos were cultured under 5 or 20% oxygen until Day 2 or 3, when embryos were transferred or cryopreserved, respectively. Clinical and other laboratory procedures were similar in both groups. PARTICIPANTS/MATERIALS, SETTING, METHODS To compare embryo characteristics and (cumulative) pregnancy outcomes between the two oxygen groups, for each patient only the first cycle in the study period was included in the analysis, resulting in 195 cycles in the 5% group (1627 oocytes) and 676 in the 20% oxygen group (5448 oocytes). Embryo characteristics were analysed per cycle and per embryo and were corrected for maternal age, cycle rank order, fertilization method (IVF or ICSI) and cause of subfertility. Perinatal data from the resulting singletons (n = 124 after fresh and 45 after frozen-thawed embryo transfer) were collected from delivery reports from the hospitals or midwife practices. MAIN RESULTS AND THE ROLE OF CHANCE In the 5% oxygen group, there were significantly more embryos of good quality (45.8 versus 30.9% in the 20% group, adjusted odds ratio (OR) [95% CI] = 1.9 [1.6–2.4]). This did not result in higher live birth rates per cycle, but after fresh transfers more good-quality spare embryos could be cryopreserved (46.1 versus 29.7%, adjusted OR [95% CI] = 2.0 [1.7–2.5]). After a follow-up period of 5–7 years, in which 82.4% of the cryopreserved embryos from the 5% oxygen group and 85.4% from the 20% oxygen group were thawed, the percentage of patients with at least one live birth resulting from the study cycle was significantly higher in the low oxygen group (adjusted OR [95% CI] = 1.5 [1.01–2.2]). In 124 live born singletons from fresh embryo transfers and in 45 from transfers of cryopreserved embryos, birthweight was similar in both oxygen groups after correction for confounding factors. LIMITATIONS, REASONS FOR CAUTION This is a retrospective study, and treatment allocation was not randomised. The study was not powered for a predefined birthweight difference. With the number of live births in our study, small differences in birthweight might not have been detected. The selection of embryos to be cryopreserved was based on embryo morphology criteria that might be different in other clinics. WIDER IMPLICATIONS OF THE FINDINGS Improved embryo utilization by more cryopreservation leading to higher cumulative live birth rates per cycle favours the use of 5% instead of 20% oxygen during human IVF embryo culture. This study also demonstrates that for comparison of different IVF treatment regimens, the cumulative outcome, including transfers of fresh and frozen-thawed embryos, is to be preferred instead of analysis of fresh embryo transfers only. STUDY FUNDING/COMPETING INTEREST(S) No external funding was received for this study. None of the authors has a conflict of interest to declare. TRIAL REGISTRATION NUMBER NA

One of the major challenges of artificial reproductive technologies is to develop new methods for producing greater numbers of embryos. An oocyte fosters the ability to develop into an embryo before oocyte meiotic resumption. The aim of the present study was to assess the effect of adenosine (ADO), a purine nucleoside found in follicular fluid, on the inhibition of oocyte meiotic resumption and the production of blastocysts. The results showed the efficacy of ADO to inhibit oocyte meiotic resumption. The use of ADO (3 mM) during a pre-in vitro maturation (pre-IVM) culture period of 6 h resulted in a significant increase (p 0.05) of blastocysts compared to control conditions with no pre-IVM culture period. No effect on the percentage of cleavage was observed. The effect of adenosine on blastocyst yield was time- and concentration-dependent with an optimum effect at 3 mM for 6 h. Supplementing the ADO pre-IVM culture medium with estradiol, follicle-stimulating hormone, progesterone, epidermal growth factor, insulin-like growth factor-2 or reelin did not improve the blastocyst yield. Transcriptional analyses of ADO-treated cumulus cells revealed that NRP1, RELN, MAN1A1, THRA and GATM were up-regulated. Finally, bioinformatic analysis identified mitochondrial function as the top canonical pathway affected by ADO. This opens up new opportunities for further investigations.

This study evaluated embryo production after superovulation (SO) with a reduced number of FSH applications and increased eCG dose in 26 Bos taurus × Bos indicus donors. On Day 0, donors received an intravaginal device (CIDR) with 1.9 g of progesterone plus 2.5 mg of estradiol benzoate and 50 mg of progesterone via IM. On Day 4, donors were randomly allotted to one of three SO treatments: 1) 455 IU of Folltropin +400 IU of eCG (n = 9), 2) 350 IU of Folltropin +600 IU of eCG (n = 9), and 3) 500 IU of Pluset + 600 IU of eCG (n = 8). In treatment 455 IU of Folltropin +400 IU of eCG, donors received eight IM Folltropin injections in decreasing dose 12 h apart from Day 4 to Day 7. On Day 6, at the same time as the Folltropin, donors received via IM 25 mg of dinoprost tromethamine (PGF2a). On Day 7, the CIDR was removed, and together with the Folltropin, donors received 200 IU of eCG via IM. In treatment 350 IU of Folltropin +600 IU of eCG, donors received four IM Folltropin injections in decreasing dose 12 h apart on Days 4 and 5. On Day 6, donors received via IM 600 IU of eCG in the morning and two doses of 25 mg of PGF2a 12 h apart. On Day 7, the CIDR was removed. Donors from treatment 500 IU of Pluset +600 IU of eCG received four IM Pluset injections in decreasing dose 12 h apart on Days 4 and 5. On Day 6, donors received via IM 600 IU of eCG in the morning and two doses of 25 mg of PGF2a 12 h apart. On Day 7, the CIDR was removed. In the morning of Day 8, donors from the three treatments received 0.25 mg of GnRH via IM. Artificial insemination was performed on Day 8 (pm) and Day 9 (am). Embryos were collected on Day 15. Variables evaluated were number of CL before embryo collection, number of structures recovered, transferable embryos, degenerate embryos and unfertilized oocytes, recovery rate, and viability rate. There was no difference in any variable among treatments (P > 0.05). In conclusion, replacement of four Folltropin or Pluset injections from a conventional eight FSH-injection SO protocol, by a single injection of 600 IU of eCG, is a good alternative to reduce donor handling without decreasing yield of transferable embryos.

Made available in DSpace on 2020-12-12T02:26:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The effectiveness of the use of natriuretic peptide C (NPPC) in the blocking of meiosis has already been proven in several species. However, there are no reports on the use of NPPC in the activation of metabolic processes in embryos. Whereas modulations of cAMP concentrations alter the lipid metabolism of bovine oocytes, the present study aims to evaluate the effect of NPPC on the development, lipid content and transcript levels of genes related to lipid metabolism of IVP bovine embryos. For this purpose, ovaries were obtained from a slaughterhouse, and oocytes were fertilized in vitro (D0). From D5 of in vitro culture, embryos were treated with 100 nM NPPC (NPPC group) or with no NPPC (Control group) and evaluated in terms of Blastocyst (D7) and hatching rates (D10). For the assessment of the cytoplasmatic lipid amounts, blastocysts were stained with Sudan Black B dye. The embryonic lipid profile was investigated by electrospray ionization desorption-mass spectrometry (DESI-MS). The abundance of nine transcripts related to lipid metabolism were assessed using the Biomark HD system. For statistical analysis, blastocyst and hatching rates, lipid content by the Sudan Black B and variation of gene expression between groups were compared by Student t-test. For lipid profile analysis, principal component analysis (PCA) and fold-change were performed. The embryo lipid content was similar between NPPC (881 ± 3.7) and Control (883 ± 5.2) groups (p > 0.05). However, cholesteryl esters and TAGs were downregulated by NPPC at multiple levels according to the DESI-MS profiles. Of the analyzed genes, ELOVL6 and SREBF1 showed an up-regulation in the control group (p < 0.05), while CPT2 was observed to be up-regulated in the NPPC-treated embryos. There was no significant difference in the blastocyst production rate between NPPC (44.4%) and Control (42.4%), however the hatching rate at D10 was higher (p < 0.05) in the NPPC group (69.77%) when compared to the Control group (48.33%). These findings demonstrate that NPPC alters the mRNA expression of genes related to lipid metabolism and that it exerts a positive effect on the hatching rates of IVP Bos taurus indicus embryos. University of Londrina (UEL) Laboratory of Animal Reproduction São Paulo State University (Unesp) Institute of Biosciences Department of Pharmacology Center for Natural and Human Sciences University ABC Federal São Paulo State University (UNESP) School of Sciences Humanities and Languages Department of Biological Science Laboratório de Virologia Animal Departamento de Medicina Veterinária Preventiva Universidade Estadual de Londrina Department of Chemistry and Center for Analytical Instrumentation Development Purdue University Instituto de Ciências Biomédicas Universidade de São Paulo Centro de Ciências Naturais e Humanas Universidade Federal Do ABC São Paulo State University (Unesp) Institute of Biosciences Department of Pharmacology São Paulo State University (UNESP) School of Sciences Humanities and Languages Department of Biological Science CNPq: 403862/2016-7