Embryonic Cultures Research Articles

Analyzing Cell-free Genomic DNA in Spent Culture Media: Noninvasive Insight into the Blastocysts.

A commonly accepted standard protocol for noninvasive techniques for the genetic evaluation of an embryo remains elusive due to inconclusiveness regarding the volume of spent media to be acquired and the possibility of acquiring the same for subsequent analysis. Single embryo culture is imperative for standardizing noninvasive preimplantation testing using cell-free DNA (cf-DNA) released by individual developing embryos. This study aims to compare the development dynamics of single-drop embryonic culture against with group embryonic culture to establish a standardized protocol for noninvasive Preimplantation Genetic Testing (PGT) in bovine. A total of 239 cumulus-oocyte complexes were aspirated and subjected to in vitro maturation and fertilization. Among these, 120 embryos of day 3 were transferred to single-drop culture until the blastocyst stage. The single-drop culture drops were prepared using microdrops of 30 μL. At the blastocyst stage, spent media from all single-drop embryos were utilized for extracting cell-free genomic DNA to standardize the protocol. The blastocyst rate indicates no significant difference between the two culture methods, suggesting that single-drop culture is suitable for the process. Additionally, the extracted spent media yielded sufficient quantities of cf-DNA, supporting its potential use for PGT ( p < 0.05). These findings support the hypothesis that single-drop embryo culture is a viable method for cf-DNA extraction and confirm the potential of using DNA fragments from spent media as a reliable source for noninvasive PGT.

Larval development of Holothuria tubulosa, a new tractable system for evo-devo

To explore animal diversity, new experimentally tractable organisms must be established. Echinoderms include five groups of marine animals that have been used as developmental models for over a century thanks to their low costs, high fecundity, optically clear larvae and genetic tractability. An additional advantage of echinoderms is that their larval forms display diverse morphologies. This rich diversity enables comparative studies to investigate the evolutionary relationships among cell types, tissues, and organs. However, reproducible protocols to obtain gametes, detailed information on embryogenesis, and genomic tools have been optimized only for selected species of sea urchins and sea stars. To address this gap, we established the abundant Mediterranean sea cucumber Holothuria tubulosa as a new experimental system. Here we describe a method to reliably obtain gametes and make embryonic cultures multiple times from the same animal and characterize unique larval tissues combining immunohistochemistry and high-resolution microscopy. This work represents a step forward in our understanding of holothurian development and establishes H. tubulosa as an emerging experimental system for evo-devo and other biological disciplines.

Perspectives on chick embryo models in developmental and reproductive toxicity screening

Teratology, the study of congenital anomalies and their causative factors intersects with developmental and reproductive toxicology, employing innovative methodologies. Evaluating the potential impacts of teratogens on fetal development and assessing human risk is an essential prerequisite in preclinical research. The chicken embryo model has emerged as a powerful tool for understanding human embryonic development due to its remarkable resemblance to humans. This model offers a unique platform for investigating the effects of substances on developing embryos, employing techniques such as ex ovo and in ovo assays, chorioallantoic membrane assays, and embryonic culture techniques. The advantages of chicken embryonic models include their accessibility, cost-effectiveness, and biological relevance to vertebrate development, enabling efficient screening of developmental toxicity. However, these models have limitations, such as the absence of a placenta and maternal metabolism, impacting the study of nutrient exchange and hormone regulation. Despite these limitations, understanding and mitigating the challenges posed by the absence of a placenta and maternal metabolism are critical for maximizing the utility of the chick embryo model in developmental toxicity testing. Indeed, the insights gained from utilizing these assays and their constraints can significantly contribute to our understanding of the developmental impacts of various agents. This review underscores the utilization of chicken embryonic models in developmental toxicity testing, highlighting their advantages and disadvantages by addressing the challenges posed by their physiological differences from mammalian systems.

Mitochondrial respiration of a primary mixed culture of neurons from hippocampus at various stages of differentiation

BACKGROUND: Primary mixed culture of neurons is often used to evaluate the molecular mechanisms underlying neurodegenerative disorders. However, isolating cells from the brain is accompanied by profound changes in cell morphology, behavior, and metabolic rate due to enzymatic disintegration and microenvironmental changes in cells. In this regard, the mitochondrial basal respiration of neurons should be considered as an indicator of the formation of functional activity of the cell.&#x0D; AIM: To evaluate mitochondrial respiration in a primary mixed culture of hippocampal neurons at various stages of differentiation.&#x0D; METHODS: This study included mice of line CD-1 and was conducted in two series: the first studied the primary mixed embryonic culture on the 18th day of gestation (E18) and the second used the postnatal culture on the 2nd day after birth (P2). A Seahorse XF HS Mini (Agilent, USA) was used to measure the functional parameters of cell metabolism. The oxygen consumption rate was calculated based on the results of the mitochondrial respiration profile of the primary mixed culture built during its differentiation.&#x0D; RESULTS: On the 5th day of neuronal differentiation in the culture, maximum mitochondrial respiration was established both in the hippocampal culture obtained from embryos on the 18th day of gestation and in the culture taken from mice on the 2nd day after birth. As cells differentiated in culture from days 2 to 11, the substrate oxidation rate increased almost twofold in the culture of hippocampal neurons obtained from embryos, which increased the metabolic potential.&#x0D; CONCLUSION: The study results prove that the hippocampus can be used to study the role of mitochondria in neurogenesis.

Can the addition of Interleukin-13 affect the cryosurvival of bovine embryos?

In this study, we investigated the impact of incorporating Interleukin-13 (IL-13) into the embryonic culture medium and its influence on cryotolerance and cellular viability of vitrified bovine embryos. Two distinct time points for IL-13 supplementation were explored: during the final hours of culture prior to cryopreservation and during the period of recultivation following cryopreservation and warming. Cryosurvival rates, total cell count, and cell viability were assessed using the TUNEL technique to determine the apoptotic percentage. Re-expansion and hatching rates did not show differences among all groups (P > 0.05), and the total cell number was comparable between the treated and control groups (P > 0.05). However, the group that received IL-13 before vitrification exhibited a higher apoptotic percentage (P < 0.05). This suggests that the anti-inflammatory effect of IL-13 may have impacted the embryo's defense capacity against the stress induced by cryopreservation, leading to an increased percentage of apoptosis, although it did not influence the developmental resumption capability.

Ethical Issues of Artificial Intelligence &amp; Assisted Reproductive Technologies

The downward trend in fertility rates has not surprisingly led to an increase in assisted reproduction techniques (ART), and assisted reproduction today is characterised by high-tech, high-end investment and international fertility companies, with the global fertility services market expected to grow beyond US$25 billion by 2026. Much has happened since the birth of the first test tube baby in 1978, including personalised ovarian stimulation, extended embryonic culture, intra cytoplasmic sperm injection (ICSI), pre-implantation genetic diagnosis, embryo selection (leading to elective single embryo transfer) and oocyte vitrification. Nevertheless, the success rate of IVF currently stands at around 30%, even though this depends heavily on a number of factors, such as age and changes in the physical and psychosocial environments. Many women, therefore, have to go through multiple rounds of IVF such that the process can be time-consuming, and challenges patients both financially and emotionally. Research is thus increasingly focused on how to improve treatments and outcomes, and rapid advancements in Artificial Intelligence look promising and are increasingly being utilised in fertility clinics around the world.

Effects of fetal bovine serum on trophectoderm and primitive endoderm cell allocation of in vitro-produced bovine embryos.

Supplementing embryonic culture medium with fetal bovine serum (FBS) renders this medium undefined. Glucose and growth factors present in FBS may affect the results of cell differentiation studies. This study tested the hypothesis that FBS supplementation during in vitro culture (IVC) alters cell differentiation in early bovine embryo development. Bovine embryos were produced in vitro and randomly distributed into three experimental groups at 90 h post insemination (90 hpi): the KSOM-FBS group, which consisted of a 5% (v/v) FBS supplementation; the KSOM33 group, with the renewal of 33% of medium volume; and the KSOM-Zero group, without FBS supplementation nor renewal of the culture medium. The results showed that the blastocyst rate (blastocyst/oocytes) at 210 hpi in the KSOM-FBS group was higher than in the KSOM-Zero group but not different from the KSOM33 group. There were no significant changes in metabolism-related aspects, such as fluorescence intensities of CellROX Green and MitoTracker Red or reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+). Immunofluorescence analysis of CDX2 revealed that the lack of FBS or medium supplementation reduced the number of trophectoderm (TE) cells and total cells. Immunofluorescence analysis revealed a reduction of SOX17-positive cell numbers after FBS supplementation compared with the KSOM33 group. Therefore, we concluded that FBS absence reduced blastocyst rates; however, no reduction occurred when there was a 33% volume renewal of the medium at 90 hpi. We also concluded that FBS supplementation altered TE and primitive endoderm cell allocation during early bovine embryo development.

Differential proMMP-2 and proMMP-9 secretion in human pre-implantation embryos at day 5 of development.

Morphological development is the most common non-invasive criterion used to select in vitro human embryos for implantation. With this criterion, however, embryos in cellular arrest go unnoticed. A more accurate criterion is needed to improve the success rate of implantation. Extracellular matrix metalloproteases type 2 (MMP-2) and MMP-9 are key markers of embryonic development and the implantation process, according to various animal studies. The first objective of this study was to examine proMMP-2 and proMMP-9 activity in the culture media of human embryos with good morphological development. Secondly, the results of proMMP-2 and proMMP-9 activity in the culture medium were compared between pregnant and non-pregnant. Forty-two patients were approved by the Ethics and Research Committees of the Instituto Nacional de Perinatología in México City hospital, based on institutional inclusion criteria for in vitro fertilization. On day 5 of development, embryos were transferred to patients, and the culture media secretion profile of proMMP-2 and proMMP-9 activity was determined by substrate gel zymography. After analysis of embryo sac development, each patient was assigned to the pregnant (n=17) or non-pregnant (n=25) group. Our results demonstrate that proMMP-2 was active in the culture media corresponding to all 17 women achieving full-term pregnancy and proMMP-9 in the media corresponding to 11 of these women. Contrarily proMMP-2 and proMMP-9 were active in the culture media corresponding to 3 and 11 of the 25 non-pregnant patients, respectively. The clinical implications of this study suggest the activity evaluation of proMMP-2 and proMMP-9 in embryonic culture media on day 5 of development appears to be a reliable indicator of the quality of embryos and their capacity to establish a pregnancy.

Melatonin accelerates the developmental competence and telomere elongation in ovine SCNT embryos.

SCNT embryos suffer from poor developmental competence (both in vitro and in vivo) due to various defects such as oxidative stress, incomplete epigenetic reprogramming, and flaws in telomere rejuvenation. It is very promising to ameliorate all these defects in SCNT embryos by supplementing the culture medium with a single compound. It has been demonstrated that melatonin, as a multitasking molecule, can improve the development of SCNT embryos, but its function during ovine SCNT embryos is unclear. We observed that supplementation of embryonic culture medium with 10 nM melatonin for 7 days accelerated the rate of blastocyst formation in ovine SCNT embryos. In addition, the quality of blastocysts increased in the melatonin-treated group compared with the SCNT control groups in terms of ICM, TE, total cell number, and mRNA expression of NANOG. Mechanistic studies in this study revealed that the melatonin-treated group had significantly lower ROS level, apoptotic cell ratio, and mRNA expression of CASPASE-3 and BAX/BCL2 ratio. In addition, melatonin promotes mitochondrial membrane potential and autophagy status (higher number of LC3B dots). Our results indicate that melatonin decreased the global level of 5mC and increased the level of H3K9ac in the treated blastocyst group compared with the blastocysts in the control group. More importantly, we demonstrated for the first time that melatonin treatment promoted telomere elongation in ovine SCNT embryos. This result offers the possibility of better development of ovine SCNT embryos after implantation. We concluded that melatonin can accelerate the reprogramming of telomere length in sheep SCNT embryos, in addition to its various beneficial effects such as increasing antioxidant capacity, reducing DNA damage, and improving the quality of derived blastocysts, all of which led to a higher in vitro development rate.

Does Embryonic Culture Environment Affect Ploidy Rates in ART Cycles: A Single Center Study in UK.

The purpose of the current study was to assess whether embryonic culture conditions has an impact on embryo ploidy in a preimplantation genetic testing for aneuploidy (PGT-A) cycle. In this retrospective single center cohort study, a total of 1099 blastocysts from 278 PGT-A cycles were analyzed. The generated blastocysts were biopsied on days 5 and 6. Inseminated oocytes were allocated in different incubators (benchtop and time lapse) and assisted zona hatching was performed on day 3 of embryo development to facilitate the biopsy process which was performed on days 5 and 6 (blastocyst stage). The average age across the groups was 38.7±3.6 years and the total number of mature eggs was 2912 which were randomly distributed across both incubators. The euploidy rate obtained from both groups showed a higher proportion of euploid embryos in the TLM incubator (37.03%, 95% CI 31.9-42.1) compared to those cultured in the BT incubator (30.4%, 95% CI 23.1-37.7). Regression analysis showed that female age remains to be the key variable driving euploidy rates (0.85, 95% CI 0.82-0.88) although incubator type could be an important covariable (0.54, 95% CI 0.45-0.59). A subgroup analysis of 74 single euploid embryo transfers showed comparable pregnancy and live birth rates. This large cohort study demonstrates that uninterrupted controlled culture environment provides increased probability to develop euploid embryo in a PGT-A cycle. However, further evaluation is required to assess how environmental culture conditions at a cellular level could affect epigenetic mechanisms in embryo development and higher aneuploidy rate.

Embryonic analysis and larval culture of Ucides occidentalis

The purpose of this work is to disseminate the findings related to the reproduction trials of the species Ucides occidentalis, which are especially linked to the basic infrastructure and equipment for obtaining seeds. It is necessary to add that bibliographical information on this species is scarce, which is why it was associated with other types of organisms. It is explained from the capture of the females to the obtaining of the larval stage, remaining to complete the investigation, through future trials and new reproduction attempts. The monitoring of the females was for 90 days, and at this same time the development of larvae and water quality, microbiological and pathological analyzes were carried out. The main results show that the reproduction of Ucides occidentalis requires a study related to the management of the mothers and the larval development where several factors of great importance influence.&#x0D; Keywords: Red crab, embryo culture, larva.

P-204 Transcriptome analysis of cell-free RNAs in embryonic culture medium reveals their potentials on non-invasive testing

Abstract Study question Could cell-free RNA (cfRNA) in culture medium be non-invasive strategy to select in vitro fertilized (IVF) embryos? Summary answer CfRNAs in culture medium could be potential markers correlated with embryo qualities. What is known already Infertility is a critical component of reproductive health and couples with infertility may choose assisted reproductive technology (ART) to increase their chances of conception. However, the effectiveness remains low. One way to improve the outcome is to screen the embryo prior to implantation. Noninvasive testing opens new perspectives in analyzing embryo qualities and current pending includes microscopy images coupled with artificial intelligence and omics analysis of the spent blastocyst medium (SBM). Cell-free DNA in SBM could be used to identify the embryo ploidy and metabolomic/proteomic markers could be correlated with embryo potential, while the research of cfRNA is lacking. Study design, size, duration We developed a new library method of polyadenylation ligation-mediated sequencing (PALM-Seq) to capture the low amount of cfRNA and collected twelve culture mediums of in vitro embryos on Day 3 and five samples of embryos on Day 5. Participants/materials, setting, methods We employed PALM-Seq on culture medium of embryos generated by IVF or Intracytoplasmic sperm injection (ICSI). Owing to the new method, we could comprehensively profile the cfRNAs, including miRNA, tRNA, piRNA, lncRNA and mRNA. The morphology and fragmentation of the recruited embryos were also recorded. Main results and the role of chance Generally, thousands of genes could be detected in all culture mediums and the number greatly increased in SBM. We identified several reported miRNA markers in our study, such as miR-21-5p and miR-92a-3p, while highly expressed miRNAs varied across samples and stages. We indeed found piRNA in mediums and several specific piRNAs had large amount of expression. In contrast, detected number and expression of tRNA were stable across samples. Besides of reported mRNA markers, we could profile the expression of all detected mRNAs and no differences were found between embryos with and without fragmentation. We found that the majority of mRNA fragments were shorter than 30 nucleotides. In addition, some granular-specific expressed mRNAs could be detected in medium on Day 3 and they decreased of medium on Day 5. Limitations, reasons for caution We have only conducted preliminary exploration in the culture medium of pre-implantation embryos and studies of larger sample size are needed. Wider implications of the findings Our research proved the feasibility of performing high-throughput sequencing of cfRNAs in SBM. It opens new perspectives on defining non-invasive markers and selection of embryos with the most potential. Our results also contribute a new insight on improving the treatment of infertility. Trial registration number 2018YFC1004900

Modulating the lipid profile of blastocyst cell membrane with DPPC multilamellar vesicles

The aim of this study was to evaluate the effect of multilamellar vesicles (MLVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in co-culture with in vitro-produced bovine embryos (IVPEs). The stability of five concentrations of MLVs (1.0, 1.25, 1.5, 1.75, and 2.0 mM) produced using ultrapure water or embryonic culture medium with 24 or 48 h of incubation at 38.5 °C with 5% CO2 was assessed. In addition, the toxicity of MLVs and their modulation of the lipid profile of the plasma membrane of IVPEs were evaluated after 48 h of co-culture. Both media allowed the production of MLVs. Incubation (24 and 48 h) did not impair the MLV structure but affected the average diameter. The rate of blastocyst production was not reduced, demonstrating the nontoxicity of the MLVs even at 2.0 mmol/L. The lipid profile of the embryos was different depending on the MLV concentration. In comparison with control embryos, embryos cultured with MLVs at 2.0 mmol/L had a higher relative abundance of six lipid ions (m/z 720.6, 754.9, 759.0, 779.1, 781.2, and 797.3). This study sheds light on a new culture system in which the MLV concentration could change the lipid profile of the embryonic cell membrane in a dose-dependent manner.

Identification of N-methyl-D-aspartate receptor antagonists using the rat postnatal mixed cortical and hippocampal neurons

The goal of this study was to evaluate mixed cortical and hippocampal primary rat postnatal neuronal culture as in vitro tool for identification of N-methyl-D-aspartate receptor (NMDAR) antagonists and to find out, whether this model is comparable with other commonly used primary rat neuronal models differing in their origin (pure cortical vs. mixed cortical and hippocampal) and differentiation state (embryonal vs. postnatal). Induced pluripotent stem cell (iPSC) – derived human glutamatergic neurons have been included in this study as well. First, the cultures were characterized by their neuron/astrocyte composition, the mRNA expression of NR2B/NR2A NMDAR subunit ratios, and the expression of glutamate transporters (GLT1, GLAST). Then, selected endogenous steroids and synthetic neuroactive steroids that have been previously identified as negative allosteric modulators of recombinant GluN1/GluN2B NMDA receptors, were evaluated for their ability to prevent an NMDA or glutamate-induced Ca2+ influx (acute effect) and excitotoxicity over 24 h. Though the neuroprotective potential against excitotoxic stimuli varied among the models studied, postnatal mixed cortical and hippocampal culture proved to be a convenient and robust tool for NMDAR antagonist screening. The most widely used embryonal (E18) cultures offered higher cell yields but at the expense of a higher sensitivity to compounds’ cytotoxicity. iPSC-derived neurons were not found to be superior to rat cultures for screening purposes.

Avian Embryonic Culture: A Perspective of In Ovo to Ex Ovo and In Vitro Studies.

The avian embryos growing outside the natural eggshell (ex ovo) were observed since the early 19th century, and since then chick embryonic structures have revealed reaching an in-depth view of external and internal anatomy, enabling us to understand conserved vertebrate development. However, the internal environment within an eggshell (in ovo) would still be the ideal place to perform various experiments to understand the nature of avian development and to apply other biotechnology techniques. With the advent of genetic manipulation and cell culture techniques, avian embryonic parts were dissected for explant culture to eventually generate expandable cell lines (in vitro cell culture). The expansion of embryonic cells allowed us to unravel the transcriptional network for understanding pluripotency and differentiation mechanism in the embryos and in combination with stem cell technology facilitated the applications of avian culture to the next levels in transgenesis and wildlife conservation. In this review, we provide a panoramic view of the relationship among different cultivation platforms from in ovo studies to ex ovo as well as in vitro culture of cell lines with recent advances in the stem cell fields.

Genome-wide analysis of the CCCH zinc finger family in longan: Characteristic identification and expression profiles in Dimocarpus longan Lour

CCCH (C3H) Zinc finger (Znf) transcription factors (TFs), as a novel type of Znf gene, regulate the expression of genes by binding to their mRNAs and play important roles in plant growth and development and abiotic stress resistance. Longan (Dimocarpous longan) is a tropical/subtropical fruit tree of great economic importance in Southeast Asia. However, genomic information on C3H and their functions in longan are still unknown. In this study, a comprehensive analysis of the longan C3H (DlC3H) gene family was carried out. A total of 49 DlC3H genes in three clades were identified from the longan genome database. Characteristics of the genes were analyzed with respect to gene structure, motif composition, phylogenetic tree and potential functions. The analysis of alternative splicing (AS) events suggested that AS events in DlC3H genes were related to the transformation from longan non-embryonic to embryonic cultures. Promoter analysis indicated that most of the DlC3H genes included cis-acting elements associated with hormones and stresses responses. Quantitative real-time PCR (qRT-PCR) analysis indicated that 26 of the 49 DlC3Hs, which possess methyl jasmonate (MeJA) and abscisic acid (ABA) responsive cis-acting elements, showed differential expression patterns under treatment with ABA, MeJA and their endogenous inhibitors, suggesting that DlC3Hs might be involved in the ABA and MeJA signaling pathways. The expression profiles of 17 of the 49 DlC3Hs in non-embryonic callus and three tissues of embryonic cultures showed that only five of the 17 DlC3Hs had the same expression trends as the FPKM trends in transcriptome data; the expression levels of DlC3H07/14/16/36/49 in embryogenic callus and DlC3H04/38 in globular embryos were high, suggesting that they have different functions in embryonic development. Further, we verified that DlC3H01/03/05/11/19/39 were regulated by sRNAs by a modified 5′ RLM-RACE method. This study provides the first systematic analysis of C3H genes in longan, and found that C3H genes may be involved in hormone and stress responses, and somatic embryogenesis. Our preliminary investigation may provide clues to further studies on the characteristics and functions of this family in longan.

The effects of supplemented sericin on in vitro maturation and preimplantation development of mouse embryos: An experimental study.

BackgroundMouse embryo culture condition is an essential part of transgenic, reproductive and developmental biology laboratories. Mouse embryonic culture media may have a high risk of serum contamination with pathogens.ObjectiveTo investigate the effect of sericin as an embryo culture medium supplement on in vitro maturation (IVM), in vitro fertilization (IVF), and development of the preimplantation embryo in mice.Materials and Methods The effects of sericin at three concentrations (subgroups) of 0.1%, 0.5%, and 1% as a medium supplement on IVM, IVF, and in vitro development of mouse embryos were separately investigated and compared with a sericin-free (control) group. The cumulative effect of the three concentrations was evaluated for IVM + in vitro development and IVF + in vitro development as follow-up groups.ResultsIn the IVM group, compared to the control group, the number of oocysts reaching the MII stage was significantly higher when 1% sericin was used (161/208 = 77.4%). No significant results were observed in the IVF and in vitro development groups with different concentrations of sericin compared to the control group. Among the follow-up groups, in the IVM + in vitro development group, the number of oocytes was higher after passing the IVM and IVF and reaching the blastocysts stage when 1% sericin was used, compared with other sericin subgroups. A significant difference was also noted when compared with the control group (p = 0.048). The IVF + in vitro development study group, on the other hand, did not show any significant relationship.Conclusion It can be concluded that 1% sericin can be used as a supplement in mouse embryo cultures to improve the IVM rate. Also, based on the findings, sericin appears to be an effective supplement which can have a positive effect on the development of embryos derived from IVM.

COMPARATIVE STUDY BETWEEN ROTA VIRUSES ISOLATED FROM DIARRHEATIC INFANT BABIES AND NEWLY BORN CALVES

Rota viruses were isolated in vero continuous cell line from infant babies and newly borne calves affected by diarrhea after treatment in every passage with 10 mg/ml of trypsin and adding 0.5 mg/ml of trypsin in the maintenance media. The isolated viruses induced distinctive and progressive type of cytopatheic effect in infected cells and highest titer was 2x10 6. TCID,50/0.1 for bovine viral isolate. The isolated 50/0.1 for human viral isolate and 2x10 » viruses were identified by indirect fluorescent antibody technique by using reference calf rotavirus antisera. Comparative study were conducted on both human and bovine viral isolate including growth in different cell culture, vero cell line was very sensitive to support growth of both viruses than secondary embryonic calf kidney cell culture, lamb testes and primary embryonic chicken fibroblast cell culture. Both viruses induced morphologically similar kind or plaques in vero cell line but plaqes formed by human isolate were larger in size about 1.5 - 2.5 mm in diameter than those of bovine isolate 1.5 - 2 mm. Cross reactive viral antigens were detected between the isolated viruses in indirect fluorescent antibody technique but not in serum neutralization test by using reference bovine rotavirus antisera.

Preimplantation Genetic Testing for Monogenic Conditions: Is Cell-Free DNA Testing the Next Step?

Genetic assessment of an embryo via preimplantation genetic testing (PGT) represents an important reproductive option for couples wanting to try and improve success rates from in vitro fertilisation (IVF) cycles, as well as reduce their risk of having a child born with a genetic condition. Currently, biopsy of the developing embryo prior to transfer allows genetic assessment of an embryo for either chromosome copy number (aneuploidy [PGT-A] or segmental rearrangement [PGT-SR]) or to avoid the transmission of a single gene condition (monogenic conditions [PGT-M]). However, this technology is invasive and commands considerable resources. Non-invasive PGT (niPGT) offers a potential alternate mode of embryonic analysis. Whilst the utility of niPGT-A has been recently explored, there has been limited consideration of niPGT-M as an option for couples at risk of passing on a single gene or chromosomal condition. This review examines the historical and current clinical context of preimplantation embryonic analysis for monogenic conditions, in addition to important considerations surrounding the origin and analysis of cell-free deoxyribose nucleic acid (cfDNA), whether it is sourced via blastocentesis or spent embryonic culture medium (SCM). Future capabilities of this testing modality will almost certainly be enhanced by integration of whole genome sequencing into everyday practice. In addition, the increased utilisation of reproductive carrier screening as part of standard reproductive healthcare will likely result in the identification of a larger high-risk population. As a result, stratification of limited and highly specialised reproductive genetic resources will be required. Prospective parents should continue to be made aware of the limitations of this technology, with prenatal confirmatory testing remaining an essential part of antenatal care in these patients. However, niPGT-M poses an important alternate testing modality for high-risk couples, particularly in the setting of embryos that cannot be biopsied for traditional PGT-M and as demand for this treatment continues to grow.

P–037 Influence of sperm selection by microfluidic device on outcomes of in vitro fertilization cycles

Abstract Study question Does sperm selection by a microfluidic sperm-sorting device improve laboratory outcomes of intracytoplasmic sperm injection cycles? Summary answer The ZyMōtTMdevice seems a good alternative to the conventional swim-up technique, possibly increasing progressive sperm motility post processing and blastulation rate. What is known already It is well-known that high levels of sperm DNA fragmentation negatively impact not only early embryonic development but also embryo genome activation. Microfluidic technologies for processing sperm samples selection such as the ZyMōtTM device have been developed to sort sperm with lower rates of sperm DNA fragmentation in order to improve in vitro fertilization (IVF) outcomes. Moreover, it offers several advantages: working with very small volume samples, high sensitivity and low reaction times, automatic sample treatment and analysis, among others. However, the real benefits on the clinical and laboratory outcomes of IVF cycles is yet to be determined. Study design, size, duration We performed a retrospective study including 126 seminal samples processed between May 2019 and December 2020. These samples belong to 63 infertile couples that underwent 2 consecutive IVF cycles using the swim-up technique in the first cycle (n = 63) and the ZyMōtTM device in the second (n = 63). The oocytes of the female partners were recovered and inseminated, and the embryonic culture was carried out until the blastocyst stage, when the quality of these embryos was analyzed. Participants/materials, setting, methods: A total of 118 seminal samples from 59 couples were included. Four couples were excluded due to insufficient seminal volume / concentration (2) or absence of mature oocytes recovered (2). Laboratory outcomes were evaluated including semen parameters post processing, as well as fertilization, cleavage, blastulation (blastocyst among cleavage stage) and top-quality blastocyst rates. Continuous variables were analyzed by paired Student’s t-test or Wilcoxon signed-rank test, as appropriate. Differences were considered significant when p-value &amp;lt; 0.05. Main results and the role of chance We did not find any difference in the percentage of non-progressive sperm motility [0 (0–33%) vs. 0 (0–10%); p = 0.063] and immotile sperm [0 (0–70%) vs. 0 (0–80%); p = 0.095] post processing when comparing the swim-up technique with the ZyMōtTM device. Likewise, no significant differences were detected regarding fertilization [83% (0–100%) vs. 89% (0–100%); p = 0.104] and cleavage [100% (0–100%) vs. 100% (80–100%); p = 0.217] rates in both groups. Nevertheless, there was a significant difference in the percentage of progressive sperm motility [100% (20–100%) vs. 100% (10–100%); p = 0.012] post processing, as well as blastulation [37% ± 0.32 vs. 50% ± 0.3; p = 0.031] and top-quality blastocyst [13.5% (0–100%) vs. 33% (0–100%); p = 0.009] rates, favoring the ZyMōtTM device. Limitations, reasons for caution The retrospective design and the small sample size make the study prone to bias. Furthermore, we do not have data on sperm DNA fragmentation pre and post processing. Clinical outcomes were not evaluated due to insufficient statistical power, since embryo transfer has not yet been accomplished in several patients. Wider implications of the findings: Besides the ZyMōtTM device has been indicated as a new tool to potentially improve laboratory and clinical IVF outcomes, it offers the advantage of being safe, fast, easy-to-use and less influenced by human errors. Further well-designed prospective studies are needed to prove its cost-effectiveness. Trial registration number Not applicable