Embryo Culture Methods Research Articles

Surveillance of avian influenza viruses in Hebei Province of China from 2021 to 2023: Identification of a novel reassortant H3N3

Avian influenza remains a global public health concern for its well-known point mutation and genomic segment reassortment, through which plenty of serum serotypes are generated to escape existing immune protection in animal and human populations. Some occasional cases of human infection of avian influenza viruses (AIVs) since 2020 posed a potential pandemic risk through human-to-human transmission. Both east-west and north-south migratory birds fly through and linger in the Hebei Province of China as a stopover habitat, providing an opportunity for imported AIVs to infect the local poultry and for viral gene reassortment to generate novel stains. In this study, we collected more than 6000 environmental samples (mostly feces) in Hebei Province from 2021 to 2023. Samples were screened using real-time RT-PCR, and virus isolation was performed using the chick embryo culture method. We identified 10 AIV isolates, including a novel reassortant H3N3 isolate. Sequencing analysis revealed these AIVs are highly homologous to those isolated in the Yellow River Basin. Our findings supported that AIVs keep evolving to generate new isolates, necessitating a continuous risk assessment of local avian influenza in wild waterfowl in Hebei, China.

Development of the first equine blastocyst produced by conventional IVF and in vitro culture in Europe resulting in the birth of a foal

In this case report, the production of the first equine blastocyst using conventional in vitro fertilization (IVF) and in vitro embryo culture methods in Europe is described. A healthy foal was born after transfer of a blastocyst using a complete in vitro production process, including in vitro oocyte maturation, in vitro sperm capacitation, fertilization in vitro and culture to the blastocyst stage in vitro. Oocytes were recovered from ovaries of slaughtered mares. After in vitro maturation, the oocytes were fertilized by conventional IVF and cultured for nine days. One embryo reached the blastocyst stage and was vitrified. After the selection of a suitable recipient mare that had ovulated four days earlier, the blastocyst was thawed and transferred. Five days after embryo transfer, a single embryonic vesicle was detected by transrectal ultrasonography. After a normal pregnancy of 323 days, a healthy colt was born. Parentage testing via microsatellite genotyping confirmed that the recipient was excluded as the foal’s dam and that the stallion whose semen was used, qualified as the sire.

Impact of Group vs Individual Embryo Culture Strategies on Blastocyst and Clinical Outcomes.

Embryo culture is one of the most important steps in an assisted reproduction laboratory. Embryos can be cultured individually, one embryo per media drop, or in groups, culturing several embryos in the same media drop. Due to the controversy generated on this subject, we wondered which embryo culture method would have the best results in terms of quality and blastocyst formation rate. We designed a prospective randomized study comparing two different embryo culture strategies: group and individual embryo culture. The data were obtained from 830 embryos from 103 egg donation treatments. The zygotes were randomized into two groups: individual culture (group 1) or group culture (group 2). The embryos were cultured in 35-µl drops until day 5 when they were classified morphologically. We observed a significant increase in the blastocyst formation rate and in the usable embryo rate in individual culture on day 5 compared to group culture. However, good embryo quality (A/B blastocysts), implantation, and pregnancy rates were similar regardless of the type of embryo-culture. As a conclusion, individual culture may increase blastocyst formation rate and may benefit embryo quality on day 5. Our results support previous reports suggesting that individual culture could improve embryo development.

Developmental Toxicity Using the Rat Whole Embryo Culture.

In vivo and in vitro experiments have been used to investigate the effect of drugs, chemical agents, growth factors, vitamins, etc., on embryonic development. Alternative tests have been developed to determine whether drugs or other compounds have toxic or teratogenic effects on a particular organ or tissue. The rat whole embryo culture method is useful for studying the mechanism of normal embryo development. The explanted rat conceptuses grow in the culture environment at the same rate as in utero development. Furthermore, the considerable advantage of explanting rat embryos is that it allows direct observation of embryogenesis. The rat whole embryo culture is run by explanting rat embryos on gestation day 9.5 for rats. The conceptuses are then cultured on a rotating platform in a mixture of culture medium with appropriate gassing for 48hours. The maternal serum is used as the culture medium, and chemicals or other agents to be evaluated separately are added to this medium. At the end of the culture period, growth and development of the embryos are evaluated morphologically.

Morphological phenotyping after mouse whole embryo culture.

Morphological phenotyping of the mouse embryo is described at neurulation stages, primarily as a guide to evaluating the outcome of whole embryo cultures between embryonic days 8.5 and 9.5. During this period, neural tube closure is initiated and progresses to completion in the cranial region. Spinal closure is still underway at the end of the culture period. The focus of this article is particularly on phenotyping that can be performed at the bench, using a stereomicroscope. This involves assessment of embryonic health, through observation and scoring of yolk sac blood circulation, measurement of developmental stage by somite counting, and determination of crown-rump length as a measure of growth. Axial rotation ("turning") can also be assessed using a simple scoring system. Neural tube closure assessment includes: 1) determining whether closure has been initiated at the Closure 1 site; 2) evaluating the complex steps of cranial neurulation including initiation at Closure sites 2 and 3, and completion of closure at the anterior and hindbrain neuropores; 3) assessment of spinal closure by measurement of posterior neuropore length. Interpretation of defects in neural tube closure requires an appreciation of, first, the stages that particular events are expected to be completed and, second, the correspondence between embryonic landmarks, for example, somite position, and the resulting adult axial levels. Detailed embryonic phenotyping, as described in this article, when combined with the versatile method of whole embryo culture, can form the basis for a wide range of experimental studies in early mouse neural development.

Current Applications and Controversies in Preimplantation Genetic Testing for Aneuploidies (PGT-A) in In Vitro Fertilization.

Preimplantation genetic testing for aneuploidy (PGT-A) has evolved over recent years, including improvements in embryo culture, biopsy, transfer, and genetic testing. The application of new comprehensive chromosome screening analysis has improved the accuracy in determining the chromosomal status of the analyzed sample, but it has brought new challenges such as the management of partial aneuploidies and mosaicisms. For the past two decades, PGT-A has been involved in a controversy regarding its efficiency in improving IVF outcomes, despite its widespread worldwide implementation. Understanding the impact of embryo aneuploidy in IVF (in vitro fertilization) should theoretically allow improving reproductive outcomes. This review of the literature aims to describe the impact of aneuploidy in human reproduction and how PGT-A was introduced to overcome this obstacle in IVF (in vitro fertilization). The article will try to analyze and summarize the evolution of the PGT-A in the recent years, and its current applications and limitations, as well as the controversy it generates. Conflicting published data could indicate the lacking value of a single biopsied sample to determine embryo chromosomal status and/or the lack of standardized methods for embryo culture and management and genetic analysis among other factors. It has to be considered that PGT-A may not be a universal test to improve the reproductive potential in IVF patients, rather each clinic should evaluate the efficacy of PGT-A in their IVF program based on their population, skills, and limitations.

P-469 Body mass index impacts ectopic pregnancy during in vitro fertilization: are we jumping the gun? An analysis of 42,362 clinical pregnancy cycles

Abstract Study question Does female body mass index (BMI) have impact on ectopic pregnancy (EP) in women undergoing in vitro fertilization (IVF) and embryo transfer (ET) treatment? Summary answer EP rate is higher in underweight patients in fresh cycles. However, BMI is not a risk factor for EP after controlling confounders. What is known already It has been shown that tubal factor infertility, stage of embryos transferred (cleavage stage or blastocyst stage), high estrogen levels during ovarian stimulation, and endometrial thickness, have impact on EP rate in IVF cycles. In addition, other potential factors including ovarian stimulation protocols, number of embryos transferred, and type of embryos (fresh or frozen thawed) are also seemed to be associated with EP rate. However, BMI has interactions with these confounding factors mentioned above. Study design, size, duration This was a retrospective cohort study. From January 2010 to June 2022, patients between the ages of 20 and 45 years who had their first fresh/frozen thawed ET cycles, and reported as clinical pregnant were included. BMI was categorized into three groups: underweight (< 18.5 Kg/m2), normal weight (18.5–23.9 Kg/m2), overweight and obesity (≥ 24 Kg/m2). Participants/materials, setting, methods A total of 42,362 cycles were included in the final analysis. The overall ectopic pregnancy rate was 2.57% (1,089/42,362). Firstly, possible factors affecting ectopic pregnancy were screened in both fresh and frozen thawed ET cycles. Then, multivariate logistic regression analysis was used to adjust confounding risk factors. Main results and the role of chance EP rates in fresh, and frozen thawed transfer cycles were 2.43% (672/27,600) and 2.82% (417/14,762), respectively. In fresh ET cycles, the ectopic pregnancy rate was significantly higher in underweight patients when compared with that in normal and overweight/obesity patients (3.29% vs. 2.29% vs. 2.54%; P = 0.029). However, the ectopic pregnancy rate was not differ among patients with different BMI (2.72% vs 2.76% vs 2.96%; P = 0.782) in frozen thawed cycles. In fresh ET cycles, secondary infertility, tubal infertility, elevated estrogen level, thin endometrial thickness, and cleavage stage embryo transfer were risk factors with ectopic pregnancy. However, female BMI was not associated with ectopic pregnancy (adjusted OR: 0.98, P = 0.894, for BMI 18.5-23.9 Kg/m2; adjusted OR: 0.89, P = 0.205, for BMI ≥ 24 Kg/m2. Reference = BMI < 18.5 Kg/m2). In frozen thawed embryo transfer cycles, only thin endometrial thickness, and cleavage stage embryo transfer were risk factors with ectopic pregnancy. Female BMI was not predictable for ectopic pregnancy, either (adjusted OR: 1.15, P = 0.513, for BMI 18.5-23.9 Kg/m2; adjusted OR: 1.24, P = 0.367, for BMI ≥ 24 Kg/m2. Reference = BMI < 18.5 Kg/m2). Limitations, reasons for caution This is a retrospective study with 12 years period. New embryo culture methods and embryo transfer strategy emerged. It is possible that not all confounders have been controlled. Meanwhile, the definition of tubal infertility and pelvic disease need more caution as the lack of laparoscopic examination in some cases. Wider implications of the findings Understanding the impact of BMI on EP during IVF treatment may be useful for predicting results. At the first glance, underweight patients are more likely to encounter with EP during fresh ET. However, it is the high estrogen level, but not low BMI, that contributes to this phenomenon. Trial registration number N/A

O-075 Deficiency of DNA double-strand break repair in human preimplantation embryos revealed by CRISPR-Cas9

Abstract Study question Is double-strand DNA break repair functional in human preimplantation embryos and what implications might a deficiency have for genome editing performed at early developmental stages? Summary answer CRISPR-Cas9 succeeds in generating DNA breaks with high efficiency, but these often remain unresolved, indicating that early human embryos are deficient in DNA repair. What is known already Genome editing (GE) technologies, such as CRISPR-Cas9, raise the possibility of avoiding transmission of inherited disorders by converting mutant genes back to wild-type (normal). The therapeutic application of CRISPR-Cas9 at the preimplantation stage is controversial due to uncertainty over safety and concerns about the alteration of the human germline. However, from a technical perspective it remains highly attractive, since it is the only developmental stage where delivery of CRISPR-Cas9 to every cell of the individual can be guaranteed. Currently, information on safety aspects and efficacy are lacking. Therefore, evaluation of the cellular response to CRISPR-Cas9 in human embryos is needed. Study design, size, duration 84 embryos were generated for research in an IRB approved study. For this purpose, donor oocytes were fertilised with donor sperm using ICSI. 51 of the resulting embryos served as controls, while in the other 33 double strand DNA breaks (DSBs) were created in a highly controlled fashion, directed at specific genomic sites using CRISPR-Cas9 technology. Successfully fertilised oocytes underwent culture in a time-lapse incubator and the duration of key developmental events were carefully timed. Participants/materials, setting, methods CRISPR-Cas9 components targeted several non-coding sites in the genome. An artificial DNA fragment was provided, which cells could potentially use for homology directed repair (HDR). Embryos were grown for 60 hours, then disaggregated. Blastomeres underwent whole genome amplification, followed by next generation sequencing to detect segmental aneuploidy related to failure of DNA repair. Additionally, sites targeted by CRISPR-Cas9 were amplified and sequenced to confirm whether repair had occurred and to distinguish the specific mechanism employed. Main results and the role of chance The study successfully validated and utilised three CRISPR ‘guides’ that targeted sites on chromosomes 2, 3 and 12. Alterations at the targeted sites were detected in 24/25 embryos (96% targeting efficiency). Repair of DSBs created by CRISPR-Cas9 was considered successful when an embryo contained at least one blastomere with changes to the DNA sequence at the targeted site consistent with nonhomologous end joining (NHEJ) or HDR. In total, 53 double-stranded breaks were generated by CRISPR-Cas9 during this study. Of these, 51% (27) were repaired by NHEJ, 9% (5) repaired by HDR and 40% (21) remained unresolved, the persistent breakage eventually causing segmental aneuploidy with a breakpoint at the targeted side. Segmental abnormalities are known to be detrimental to embryonic viability and risk congenital abnormalities in offspring. This study shows that human embryos, prior to activation of the genome, have a DNA damage repair deficiency. Timings of morphokinetic milestones were altered in embryos with unresolved DNA damage, confirming that checkpoint control is active. However, continued mitotic division, despite the presence of DSBs, shows cellular mechanisms that usually preserve genetic integrity lack stringency, ultimately failing to ensure repair. The results provide a warning against the therapeutic use of CRISPR-Cas9 in human embryos. Limitations, reasons for caution While targeting the embryonic genome using CRISPR-Cas9 was remarkably efficient (DSBs introduced in 96% of embryos), the majority of cells utilised NHEJ for repair, a process allowing introduction of additional mutations rather than correcting existing ones. Only <10% of cells used the HDR repair mechanism necessary for removal of mutations. Wider implications of the findings These findings reveal that cells of early human embryos are less able to process DSBs than other cell types. This suggests that embryo culture methods should give consideration to protecting the genome from damage. The results caution against clinical application of genome editing technologies that create DSBs, such as CRISPR-Cas9. Trial registration number NA

P-737 Progress in non-invasive embryo assessment with cell-free nucleic acids in blastocoel fluid

Abstract Study question Can cell-free nucleic acids (cfDNA) from blastocoel fluid (BF) after a trophectoderm (TE) biopsy be used as a non-invasive sample type to assess embryos? Summary answer Both BF and spent media carry comparable, highly fragmented cfDNA and cell-free RNA (cfRNA) which may reveal more about the quality of the embryo. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) is commonly used by IVF clinics to screen embryos for any chromosomal abnormalities during the IVF process. Noninvasive preimplantation genetic testing (ni-PGT) is an emerging category of tools within the field of assisted reproductive technology. Currently, niPGT products have been developed for embryo aneuploidy detection using cfDNA released by the embryo into the spent media. Embryo spent media (ESM) carries cfDNA, which has the potential to be applied to evaluate the quality of the embryo. Previous studies have demonstrated that cfDNA in ESM is highly fragmented to the nucleosome level. Study design, size, duration The quality and quantity of cell free nucleic acid from forty BF-conditioned media (BFCM) samples were analyzed in this study. Embgenix™ Analysis Software was used to analyze and interpret sequencing data. For each sample, the concentration and fragmentation of cfDNA were examined and compared using generalized linear mixed models. To assess RNA quality at a picogram level, reverse-transcribed and amplified cDNA was used to represent the quality of cfRNA in BFCM. Participants/materials, setting, methods Embryos were washed and placed in individual biopsy media droplets for TE biopsy. After biopsy, blastocysts were collapsed, releasing BF into the droplet. The entire droplet was collected as BFCM. The Embgenix ESM Screen Kit was used to prepare sequencing libraries. The SMART-Seq® Single Cell Kit was used to evaluate the RNA quality. The BFCM cfDNA results were also compared to spent media profile that had already been obtained from a different cohort of embryos. Main results and the role of chance Amounts of cfDNA fragmentation detected in BFCM samples was comparable to ESM. Whole-genome amplification (WGA) yields correlated to the amount of cfDNA input but not the degree of cfDNA fragmentation. Informative sequencing reads and computed copy number variations (CNV) plot noise, as evaluated by distribution of log2 ratio spread (DLRS), also showed correlation and stable performance down to ∼2 pg of cfDNA input. We extracted detectable cfRNA from BFCM and created cDNA for RNA quality analysis. Our model demonstrates a significant correlation between cDNA length and RNA quality metrics, with a low DV200 of cfRNA from BFCM samples. The quantity and quality of cfDNA released into spent culture media by a developing embryo are comparable to the cfDNA released into BFCM upon TE biopsy, indicating that BFCM has potential as a noninvasive method for assessing the ploidy status of embryos. WGA yield, informative reads, and DLRS of BFCM samples were likewise comparable to data previously acquired using spent media samples. However, the severely fragmented cfRNA in these samples illustrate the inherent instability of RNA and the difficulty of utilizing RNA analysis to determine embryo health. Assays compatible with small RNA or microRNA (miRNA) are more appropriate for RNA-seq of BFCM. Limitations, reasons for caution Different clinics use varying methods for embryo culture and biopsy; the data reported here is from a single research center. The characteristic of samples may vary between clinics. Our assay estimates RNA quality using amplified cDNA; nonetheless, discrepancies between fragmented RNA and amplified cDNA may exist. Wider implications of the findings In PGT-A, BFCM samples are routinely discarded. Our findings imply BFCM samples contain considerable levels of cell-free nucleic acids which could also be used to screen embryo quality. BFCM samples, therefore, could be analyzed in conjunction with PGT-A to select and/or rank embryos for implantation. Trial registration number Not applicable

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Modified Xenopus laevis approach (R-FETAX) as an alternative test for the evaluation of foetal valproate spectrum disorder

In compliance to animal welfare 3Rs principle there is a great demand for refined tests alternative to classical mammal teratogenicity tests. We propose a refined alternative amphibian method (R-FETAX) to evaluate chemical induced embryotoxicity. The human foetal valproate spectrum disorder (FVSD) characteristics are morphological defects (including cranio-facial, neural tube defects) and behavioural alterations due to valproate (VPA) exposure in pregnancy. Vertebrate assays to evaluate FVSD include classical and alternative mammal (implying adult sacrifice), and non-mammal developmental models (zebrafish, amphibians, chick). Among these latter only zebrafish assays report in the same test both morphological and behavioural examinations. Compared to zebrafish, the amphibian Xenopus laevis excels having a more comparable organ development and morphology to mammalian systems. We used X. laevis embryos exposed during developmental specific windows to VPA therapeutic concentrations. Different VPA effects were observed depending on the exposure window: concentration-related embryo-lethal and teratogenic effects (neural tube, facial, tail defects) were observed in groups exposed at the organogenetic phylotypic stages. Neurobehavioral deficits were described using a functional swimming test at the highest VPA concentration exposure during the phylotypic stages and at any concentration during neurocognitive competent stages. Malformations were compared to those obtained in a mammalian assay (the rat post-implantation whole embryo culture method, WEC), that we used in the past to evaluate VPA teratogenicity. R-FETAX and WEC data were modelled and their relative sensitivity was calculated. We suggest the amphibian R-FETAX as a refined windowed alternative test for the evaluation of chemicals inducing both morphological and behavioural anomalies, including VPA.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis.

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

P–216 Successful pregnancies and deliveries in patients with a recurrent failure of ART treatments following artificial removal of the zona pellucida (ZP) at the pronuclear stage

Abstract Study question Can a novel embryo culture method that artificially removes the ZP at the pronuclear stage yield successful pregnancy in patients with poor-quality embryos and/or blastocysts? Summary answer A blastocyst transfer after ZP-free culture can result in pregnancy for patients who cannot obtain good quality blastocysts from conventional culture methods. What is known already Perivitelline threads are been associated with the formation of cytoplasmic fragments. We had previously observed perivitelline threads in the adhesive region between the ooplasm and the ZP at the first cleavage in human embryos. We removed the ZP at the pronuclear stage in 71 abnormally fertilized oocytes (zygotes with three pronuclei), donated after conventional IVF (c-IVF), and termed them ZP-free 3PN. We found ZP-free 3PN embryos could be cultured without losing blastomere adhesions. Furthermore, the rate of good quality embryos was significantly higher in ZP-free 3PN embryos compared with ZP-intact embryos (ZP-intact 2PN/2PB and 3PN embryos; P < 0.05). Study design, size, duration This study was conducted in two cases selected among patients who underwent ART treatment in our clinic between 2018 and 2019. Cases were selected if they lacked good quality blastocysts in previous c-IVF/Intracytoplasmic Sperm Injection (ICSI) cycles due to massive cytoplasmic fragmentation at the first and second cleavage. We performed a clinical trial of ZP-free culture from December 2019 to March 2020. Participants/materials, setting, methods Two cases were selected for this trial. Normally fertilized oocytes were grouped as ZP-free or ZP-intact. For the ZP-free group, 2PN embryos were placed in 0.125M sucrose-containing HEPES to reduce ooplasm size, then ooplasms were completely separated from ZPs by a laser and pipetting. ZP-free and ZP-intact embryos were cultured with time-lapse imaging for up to seven days. Resultant blastocysts were either transferred into uterus or cryopreserved on Day5/6/7 for future embryo transfer cycles. Main results and the role of chance The ZP-free culture method was applied to two patients (patient A and B) with recurrent failure of ART in our clinic due to poor-quality embryos and/or difficulties in obtaining good quality blastocysts. In both cases, blastocysts were successfully obtained and cryopreserved for all ZP-free culture cycles. In patient A, one good quality ZP-free blastocyst was freshly transferred five days after oocyte retrieval, and a live male baby (2925g) was delivered at 40 weeks of gestation by caesarean section). In patient B, a frozen/thawed ZP-free blastocyst transfer was conducted, and a live female baby (3225g) was delivered at 39 weeks of gestation by vaginal delivery. This shows ZP-free culturing may help obtain viable embryos in patients for which conventional in vitro culturing methods result in embryos characterized with severe cytoplasmic fragmentation and poor quality in the early cleavage stage. Limitations, reasons for caution Although successful pregnancies and deliveries were confirmed in two cases, postnatal evaluations will be absolutely necessary for infants derived from ZP-free culture. In addition, the number of trial cases needs to be expanded, however careful selection of suitable patients is necessary for this novel culture method. Wider implications of the findings: We found removing the ZP at the pronuclear stage improved embryo development and led to successful pregnancies and deliveries after blastocyst transfer. This indicates ZP-free culturing may be an effective method for decreasing cytoplasmic fragmentation caused by perivitelline threads or adhesion between the ooplasm and the zona pellucida. Trial registration number Not applicable

P–146 Differential impact of three embryo culture media for IVF on in vitro development and perinatal outcome: a single-center RCT

Abstract Study question This report provides updated data from an RCT determining which embryo culture medium yields optimal IVF outcomes. Summary answer Embryo culture systems used for IVF differentially affected preimplantation development and resultant obstetric and perinatal outcomes, including birthweights of live-born singletons. What is known already Currently, multiple embryo culture medium systems are in use for IVF, raising questions regarding which is optimal. However, the ability of a medium to yield preimplantation embryos is not necessarily indicative of embryo viability. For example, supplementation of medium with serum was commonly used to increase animal blastocyst yields, but this impaired embryonic, fetal, and offspring health. In humans, medium composition and culture duration can influence IVF efficacy and offspring phenotype. Given the importance of culture systems in determining clinical outcomes, existing data regarding differential culture system impacts are insufficient and additional well-designed studies are required. Study design, size, duration Between February 2016 and August 2017, 795 couples undergoing their first autologous clinical IVF cycle and freeze-all strategy were recruited. Participants were randomized via computer-generated tables into three groups. Following standard oocyte retrieval and IVF/ICSI procedures, embryos were cultured using three different culture media, G1 Plus/G2 Plus (G1/G2; Vitrolife), Global Total (GT; LifeGlobal), or Sequential Cleav/Sequential Blast (SC/SB; Origio). Thirty-eight patients exhibiting no 2PN oocytes following insemination or those undergoing fresh embryo transfers were excluded. Participants/materials, setting, methods For patients yielding a single good-quality cleavage-stage (day–2 or day–3) embryo, that cleavage-stage embryo was vitrified. For patients yielding two or more good-quality cleavage-stage embryos, two or less good-quality cleavage-stage embryos were vitrified. The culture period of the remaining embryos was extended, and all good-quality blastocyst-stage (day–5 or day–6) embryos were vitrified. This report presents data for vitrified embryo transfer performed until the end of December 2020. Main results and the role of chance The mean per-cycle vitrified embryo yield (± SD) was comparable between groups for cleavage-stage embryos, but significantly different for blastocyst-stage embryos (G1/G2: 1.69 ± 2.2, GT: 2.53 ± 3.01, SC/SB: 2.04 ± 2.42; P = 0.001). Following vitrified cleavage- or blastocyst-stage embryo transfers, biochemical pregnancy rates were significantly different between groups (G1/G2: 55.6%, GT: 59.1%, SC/SB: 46.2%; P = 0.011). Furthermore, a between-group trend towards different live birth rates was observed (G1/G2: 41.7%, GT: 42.1%, SC/SB: 33.1%; P = 0.063). Of 382 live births, data for first-borns (n = 323; 295 singletons and 14 twin-pairs) are reported here. Perinatal data did not differ significantly between groups for both cleavage- and blastocyst-stage embryo transfers, including gestational age- and gender-adjusted singleton birthweight (z-score). Following multiple linear regression (including selected covariates), adjusted mean singleton birthweights were significantly lower in the G1/G2 and GT groups than in the SC/SB group (by 131 g; P = 0.011 and 110 g; P = 0.032, respectively) and tended to be lower for cleavage-stage embryo transfers than for blastocyst-stage embryo transfers (by 102 g; P = 0.053). Limitations, reasons for caution A larger cohort size and longer-term follow-up are required to verify and further elucidate the impact of embryo culture methods on child health. Such studies will raise awareness regarding the sensitivity of in vitro-cultured human embryos to their environment, ultimately resulting in practices that decrease IVF risks to offspring. Wider implications of the findings: Pregnancy outcome of the medium yielding fewer blastocysts was comparable or superior to that of other media, highlighting the importance of differentiating between the ability to support preimplantation development versus the ability to yield viable embryos. Embryo culture medium had a greater impact than embryo transfer stage on live birthweight. Trial registration number UMIN000020910

Ex Ovo Culture System for Avian Embryos and its Application.

Ex ovo culture of avian embryos can be applied not only to embryology but also to various fields of basic research such as embryo manipulation, toxicology, and regenerative medicine. The windowing method, which facilitates various manipulations and observations by opening a hole in one part of the eggshell, and culture systems using surrogate eggshells, are widely used. Despite this, biology lessons in high schools cover shell-less culture systems, which involve the development of avian embryos in artificial vessels, such as rice bowls, without using surrogate eggshells. However, as embryo development stops at its early stages in this method, it is not possible to continuously observe the development of the embryo. This led to attempts to develop an embryo culture method using a complete artificial culture vessel that does not use surrogate eggshells, and Kamihira et al. (1998) succeeded in hatching quail embryos in an artificial culture vessel using polytetrafluoroethylene membranes. In addition, Tahara succeeded in hatching chick embryos in artificial culture vessels that used cling film made of polymethylpentene and reported their detailed methodology (Tahara and Obara, 2014). These technologies are being applied not only to school education but also to various fields of research.

Ayçiçeğinde Olgunlaşmamış Embriyo Kültürünün Generasyon Atlatma Amaçlı Kullanımında Uygun Embriyo Yaşının Saptanması

Since sunflower is a highly cross-pollinated species, obtaining homozygous lines is important for both genetic studies and hybrid seed production.Traditional methods require at least six generations to obtain homozygous lines. Immature embryo culture method, which is one of the alternative methods, has the advantage of shortening the generation cycle by half. The response of immature sunflower embryos to embryo culture and the success of the method depends on the genotype of the donor plant, the growing conditions of the donor plant, the age of the embryo, the composition of the medium used in in vitro culture, the in vitro culture conditions and the culture time. In this study, it was aimed to determine the most appropriate embryo age in order to overcome rapid generation with immature embryo culture technique in sunflower breeding. The study was conducted in the Generation Shortening Center within the Eastern Mediterranean Agricultural Research Institute. A modified version of MS medium was preferred as the main medium in in vitro culture studies. In the study, immature embryos of 7 different ages (8, 10, 12, 14, 16, 18, 20 days after pollination) taken from plants grown in open field conditions of 5 oil restorer lines were tested 8-20 days after pollination. Plant formation rates of the cultured embryos, plant weight in regenerated plants, plant root length, plant height and number of leaves per plantlet were examined. Research findings showed that the optimum embryo age in the selected MS environment was 14 days. It has been shown that there is an opportunity to overcome a rapid generation by culturing 14-day-old embryos in the modified MS medium used in this study.

Calcium carbonate supplementation to chorioallantoic membranes improves hatchability in shell-less chick embryo culture

Developing chick embryos are a classical research tool in developmental biology. The whole embryo culture technique can be applied to various fields, such as embryo manipulation, toxicology, tumorigenesis, and basic research in regenerative medicine. When used for the generation of transgenic chickens, a high hatchability of genetically engineered embryos is essential to support normal embryonic development during culture. In this study, calcium carbonate, which is the main component of eggshells, was added as a calcium source in shell-less chick embryo cultures using a transparent plastic film as a culture vessel. In the absence of a calcium source in the shell-less culture system, embryogenesis ceased during culture, resulting in failed embryonic hatching. We found that the direct addition of calcium carbonate to the chorioallantoic membrane of the developing embryo was effective for the hatching of cultured chick embryos. The amount, timing, and location of calcium carbonate addition were investigated to maximize the hatchability of cultured embryos. Starting from the time of calcium carbonate supplementation, >40% hatchability was obtained with the optimal condition. This established method of shell-less chick embryo culture provides a useful tool in basic and applied fields of chick embryo manipulation.

Creation of new forms of feeding cereal grasses from A to Z

Aim. To create a new cultivars of perennial cereal grasses with the high feeding and seed productivity, longevity, drought resistance and freezing tolerance in order to increase the quality of livestock super-elite seeds production. DNA certification of economically valuable properties estimation. Methods. Hybridization, a partially known combination. Embryo culture method from immature caryopsis with completion of growing in a nutrient medium. Techniques of plant tissue culture (initiation, multiplication, rooting and adaptation in vitro plants to ex vitro conditions. DNA certification was performed using SSR (Simple Sequence Repeat) marker system. Results. The new competitively cultivar of Agropyron cristatum "Malarytsky" have been created. The system of the cultivar and parental forms registration in the form of molecular genetic passports is presented. Conclusions. The cultivar of Agropyron cristatum "Malarytsky" was turned over to the State Institution “The State Inspection for Testing and Protection of Plant Varieties” for state testing and for inclusion in the State Register of Varieties. The economic efficiency of this new feeding culture cultivating for Belarus and countries with sharply continental climate is presented.
 Keywords: Agropyron cristatum, hybridization, cul populations, in vitro, water-soluble carbohydrates, DNA certification, molecular genetic passports, cultivar.

Monkey Embryos Cultured to 20 Days.

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.

Media optimization to promote rat embryonic development to the blastocyst stage in vitro

Efficient model production in rats that incorporates newly developed genetic editing and embryo transfer tools, such as CRISPR/Cas9 technology and non-surgical embryo transfer, requires availability of an optimal embryo culture system. However, current technologies for in vitro manipulation of rat gametes, including embryo culture techniques, are less advanced compared to those in mice. In this study, we (1) identified a culture medium that was able to support optimal rat embryonic development by comparing two rat culture media: mR1ECM (modified rat 1-cell embryo culture medium) and KSOM-R (modified potassium simplex optimized medium for rats), and (2) evaluated the effect of glutamine dipeptides: alanyl-l-glutamine and glycyl-l-glutamine, on rat embryonic development. We also investigated the possibility of simplifying the KSOM-R culture procedure by increasing the volume of culture medium, reducing the need for daily medium changes. The results showed that rat embryos cultured in KSOM-R developed faster than those cultured in mR1ECM. Both alanyl-l-glutamine and glycyl-l-glutamine showed detrimental effects on rat embryonic development when supplemented in KSOM-R at the same concentration as glutamine. By increasing the volume of KSOM-R, rat zygotes were able to develop without daily medium refreshment at a similar rate and developmental competence as those in smaller volumes with daily medium changes. These results represent important improvements to rat embryo culture methods and will assist in more efficient production of rat models.