Equivalency of culture media for human in vitro fertilization formulated to have the same pH under an atmosphere containing 5% or 6% carbon dioxide

This review highlights the role of the oviduct in early embryo development, which has to fulfil many aligned and well-tuned tasks during early embryogenesis. The oviductal lining is subjected to dynamic changes to timely accomplish gamete transport, fertilization and embryo development and to deliver a competent and healthy conceptus to the endometrium which can implant and develop to term. Although knowledge about the role of the oviduct is limited, we know that embryos are very sensitive to the environment in which they develop. The success of in vitro embryo production techniques demonstrates that it is possible to bypass the oviduct during early development and, to a certain extent, replicate the conditions in vitro. However, comparative studies show that embryos developed in vivo are superior to their in vitro produced counterparts, underlining our relatively poor knowledge of the biology of the oviduct. Oviduct activity is orchestrated by various factors, depending on cyclic dynamics, which crucially affect the success of tubal transfer and/or (re-)collection of embryos in embryo transfer studies. This paper reviews data which demonstrate that in vivo culture of embryos in the bovine oviduct is a useful tool for the assessment of embryos developed under various conditions (e.g. superovulation vs single ovulation, lactating dairy cows vs non-lactating cows). It is concluded that more work in the field of early embryo development within the oviduct would contribute to improved ART protocols leading to healthy pregnancies and offspring.

Study question How valid is the Mouse Embryo Assay (MEA) for certifying plastic labware primarily used in gamete processing for IUI? Summary answer This study showed discrepancies between MEA certified consumables and their toxicity assay performance. Type of test and exposure impact the certification success. What is known already Toxicity testing through MEA is standard to ensure the safety of consumables in assisted reproductive technologies (ART), including those used in gamete processing. Human Sperm Motility Assay (HSMA) is less frequently performed, and no products undergo additional mouse IVF. MEA focuses on embryonic development and does not involve direct gamete exposure, whereas mouse IVF does. HSMA assesses gamete toxicity via sperm motility but does not reflect fertilizing capacity. Furthermore, discrepancies exist between company-reported testing conditions and actual lab practices, questioning reliability of these certificates. Study design, size, duration Three plastic products used in sperm processing (sperm container, 15ml centrifuge tube and 5ml tube) from two companies were evaluated in-house for toxicity by MEA, mouse IVF and HSMA. For MEA and mouse IVF, exposures were set as over-exposures to a real lab situation: sperm container (polypropylene): 2h at 37 °C; 15 ml centrifuge tube (polypropylene): 1h at room temperature (RT); 5 ml tube (polystyrene): 2h at RT. Progressive motility was assessed in the HSMA test. Participants/materials, setting, methods B6CBAF1 mice were stimulated with 2.5IU FSH and 5.0IU hCG for MEA and mouse IVF assays (two repeats with at least 10 zygotes). In mouse IVF, sperm was exposed to labware followed by fertilization and culture in control medium. MEA involved culturing zygotes in product-exposed medium until day 6. The HSMA involved triplicate tests with washed semen exposed to labware at room temperature for up to 120 hours. Main results and the role of chance All individually tested labware passed the internal mouse IVF with ≥75% blastulation rates on day 6. Although MEA certified, three products failed our internal MEA. Sperm containers from both companies showed ≤ 80% blastulation rates on day 6 (35% and 70%, respectively).The 15 ml centrifuge tube of company 2 demonstrated blastulation rates of only 5% on day 6. Both companies use day 5 blastocyst development rates as their pass criterion; based on this benchmark, none of the three failed products (both sperm containers and 15 ml centrifuge tube of company 2) met the required blastulation rate in our study (15%, 30% and 0%, respectively). Despite a 100% blastulation on day 6, the 15 ml centrifuge tube from company 1 demonstrated a blastulation of only 70% on day 5. In the HSMA, a survival index (SI) was calculated based on progressive motility. All labware from both companies served as mutual controls, allowing a comparative assessment without requiring an additional product with an unknown HSMA outcome. With SI averages close to 1.0 (range: 0.79 - 1.27) most products passed all timepoints. One exception was the sperm container from company 2, which failed at 120 hours, where progressive motility dropped to 0%. Limitations, reasons for caution The small sample size may limit the generalizability of the findings. Testing exposure conditions differed from those reported by the companies and may also vary between labs. Products manufactured with polypropylene appear to be more embryo toxic compared to polystyrene consumables. Wider implications of the findings MEA alone may not provide sufficient assurance of labware safety, particularly for consumables exposed to gametes. Mouse IVF and/or HSMA offer complementary insights and should be considered when certifying labware safety in ART. Trial registration number No

Human embryonic development: from peri-implantation to gastrulation.

The “14‐day rule”—broadly construed—is used in science policy and regulation to limit research on human embryos to a maximum period of 14 days after their creation or to the equivalent stage of development that is normally attributed to a 14‐day‐old embryo (Hyun et al, 2016; Nuffield Council on Bioethics, 2017). For several decades, the 14‐day rule has been a shining example of how science policy and regulation can be developed with interdisciplinary consensus and applied across a number of countries to help fulfil an ethical and practical purpose: to facilitate efficient and ethical embryo research. However, advances in embryology and biomedical research have led to suggestions that the 14‐day rule is no longer adequate (Deglincerti et al, 2016; Shahbazi et al, 2016; Hurlbut et al, 2017). Therefore, should the 14‐day rule be extended and, if so, where should we draw a new line for permissible embryo research? Here, we provide scientific, regulatory and ethical arguments that the 14‐day rule should be extended to 28 days (or the developmental equivalent stage of a 28‐day‐old embryo).

Study question Does the type of culture medium and its supplementation with protein affect the sensitivity of the Mouse Embryo Assay (MEA) to detect Triton X-100 (TX-100)? Summary answer HTF medium showed a higher sensitivity than KSOM’s complex formulation, whilst the concentration of protein tested did not have a significant impact on MEA results. What is known already TX-100 is a detergent commonly used in the cleaning of production material. Occasionally, small TX-100 traces can remain in the final products, having sublethal effects on embryo development, potentially affecting their viability and implantation potential. The effectiveness of the MEA in detecting low-toxicity levels is not well described due to the lack of methodological standardization or regulation on the appropriateness of different testing protocols. It is unknown whether media formulation modifies the sensitivity of the MEA. Similarly, media supplementation with albumin is suspected to chelate toxins and decrease the sensitivity of the assay, but no clear relation has been described. Study design, size, duration KSOM and HTF were selected and compared as media with complex vs. simple-formulation, respectively. The effect of protein concentration was assessed with both media at 0.5 and 5mg/ml HSA. Both variables were combined to culture embryos in the presence of sublethal concentrations of TX-100, previously established at 0.0005% and 0.0010%. Sensitivity was established by comparing blastocyst formation rates and total cell counts, which were statistically compared between groups to determine the conditions of greatest sensitivity. Participants/materials, setting, methods Experiments were performed with freshly-collected mouse zygotes (n = 783). Each TX-100 concentration (0% [control], 0.0005%, 0.0010%) was tested with all the proposed medium-protein combinations: KSOM 0.5mg/ml HSA, KSOM 5mg/ml HSA, HTF 0.5 HSA and HTF 5mg/ml HSA. All tests were performed in triplicate in a big-box humidified trigas incubator. Blastocyst formation was assessed after 96h of culture, and the obtained blastocysts were fixed and stained for cell counting. Main results and the role of chance No difference was observed in the blastocyst formation rate, nor the total cell counts between any of the control groups, showing that both studied media and protein concentrations were able to successfully support embryo development in vitro. Based on the expanded blastocyst formation rates, HTF conferred greater sensitivity than KSOM with 5mg/ml HSA at 0.0010% TX-100 (p = 0.0024), and a similar but non-significant trend was observed with 0.5mg/ml (p = 0.0525). Interestingly, increasing HSA to 5mg/ml in HTF reported a higher sensitivity than 0.5mg/ml (p = 0.0399), which was not observed in KSOM. No medium-protein combination was able to detect TX-100 at 0.0005%. Regarding the cell counting results, each test group cultured with 0.0005% or 0.0010% TX-100 was compared to its own medium-protein combination control. A statistically lower cell number (p < 0.05) was obtained with 0.0010% TX-100 compared to the control groups, regardless of the culture medium or protein concentration used. By contrast, no statistical differences were detected in any of the groups when reducing TX-100 to 0.0005%. Interestingly, with both media and all TX-100 concentrations, the mean cell number per blastocyst was higher when increasing the concentration of HSA from 0.5 to 5mg/ml, whilst this had no effect on the result of the assays. Limitations, reasons for caution HTF proved more sensitive than KSOM with 5mg/ml HSA. The limited number of embryos/group could not confirm the statistical significance of this difference with 0.5mg/ml, but a clear trend was observed (p = 0.0525), which might be confirmed by additional replicas. Moreover, the results applicability to other toxicity sources requires further study. Wider implications of the findings Numerous culture variables can influence MEA’s sensitivity. Our results suggest that the type of culture medium can have a direct impact on the ability of the MEA to detect certain types of toxins. This information could help detect sublethal amounts of embryotoxic substances, avoiding potential negative clinical events. Trial registration number Not applicable

Objective To investigate the effect of the different fertilization time after human chorionic gonadotropin (hCG) injection on the early embryonic development and clinical outcome of patients during in vitro fertilization-embryo transfer (IVF-ET). Methods A total of 200 patients undergoing IVF-ET were devided into 4 groups according to the different fertilization time after hCG injection. Group A: fertilization happened in 38.0-39.0 h after hCG injection, group B: 39.1-40.0 h after hCG injection, group C: 40.1-41.0 h after hCG injection, group D: 41.1-42.0 h after hCG injection, 50 cases in each group. The normal fertilization rate, cleavage rate, the high quality embryo rate, the embryo utilization rate, the clinical pregnancy rate, the implantation rate, and the spontaneous abortion rate were evaluated among the 4 groups. Results The embryo utiliazation rate in group B (65.7%), group C (63.3%), group D (66.8%) was higher than that of group A (55.5%) (P 0.05). Conclusion The different fertilization time after hCG injection in IVF-ET have effects on high-quality embryo rate. During a certain time range (38-42 h), as the increase of delayed fertilization time after hCG injection, high-quality embryo rate is also increasing. The best fertilization time of patients in IVF-ET was 40.1-41.0 h after hCG injection in our reproductive center, which can improve the clinical pregnancy rate, reduce the early abortion rate and get better clinical pregnancy outcome. Key words: Fertilization time; In vitro fertilization-embryo transfer (IVF-ET); Human chorionic gonadotropin (hCG); Clinical pregnancy rate

Insulin-like growth factor-II (IGF-II), IGF-binding protein-3 (IGFBP-3), and IGFBP-4 in follicular fluid are associated with oocyte maturation and embryo development

The effect of sperm miR-34c on human embryonic development kinetics and clinical outcomes

The molecular basis of the lymphatic development remains largely unknown. Using zebrafish as a model, we discovered a novel role for the Ras guanine-releasing protein 1 (RasGRP1), a protein involved in Ras activation in lymphangiogenesis. Secondary lymphatic sprouts from the posterior cardinal vein give rise to thoracic duct which is the first lymphatic vessel in zebrafish. Knockdown of rasgrp1 by injecting morpholino in zebrafish embryos impaired formation of thoracic duct accompanied by pericardial and truck edema, whereas blood vessel development of the embryos was largely unaffected. In rasgrp1-knockdown embryos, the number of sprouts producing the string of parachordal lymphangioblast cells was reduced. Meanwhile the total number of the secondary sprouts was not changed. As a result, the number of venous intersegmental vessels was increased, whereas the number of lymphatic vessel was reduced at a later stage. The lymphatic developmental defects caused by rasgrp1 knockdown could be rescued by ectopic expression of a constitutively active HRas. Further analysis revealed that RasGRP1 knockdown could synergize with flt4/vegfr3 knockdown to induce defects in lymphangiogenesis. Taken together, this finding demonstrates a critical role for RasGRP1 in lymphatic development in zebrafish.

What is the relationship between the metabolism of preimplantation embryos and their developmental competence?

STUDY QUESTIONCan time-lapse analysis of cell division timings [morphokinetics (MK)] in mouse embryos detect toxins at concentrations that do not affect blastocyst formation?SUMMARY ANSWERAn MK algorithm enhances assay sensitivity while providing results 24–48 h sooner than the traditional mouse embryo assay (MEA).WHAT IS KNOWN ALREADYCurrent quality control testing methodology is sensitive but further improvements are needed to assure optimal culture conditions. MKs of embryo development may detect small variations in culture conditions.STUDY DESIGNCross sectional—control versus treatment. Mouse embryo development kinetics of 466 embryos were analyzed according to exposure to various concentrations of toxins and toxic mineral oil.MATERIALS, SETTING, METHODSCryopreserved 1-cell embryos from F1 hybrid mice were cultured with cumene hydroperoxide (CH) (0, 2, 4, 6 and 8 µM) and Triton X-100 (TX-100; 0, 0.0008, 0.0012, 0.0016 and 0.002%). Using the Embryoscope, time-lapse images were obtained every 20 min for 120 h in seven focal planes. End-points were timing and pattern of cell division and embryo development. The blastocyst rate (BR) was defined as the percentage of embryos that developed to the expanded blastocyst stage within 96 h.MAIN RESULTS AND THE ROLE OF CHANCEBR was not affected for embryos cultured in the three lowest concentrations of CH and the four lowest concentrations of TX-100. In contrast, a unique MK model detected all concentrations tested (P < 0.05). The MK model identified toxicity in two lots of toxic mineral oil that did not affect BR (P < 0.05).LIMITATIONS, REASONS FOR CAUTIONA limited number of toxins were used so that the results may not apply to all potential embryo toxins. A larger sample size may also demonstrate other statistically significant developmental kinetic parameters.WIDER IMPLICATIONS OF THE FINDINGSMKs in mouse embryos are a sensitive and efficient method for quality control testing of in vitro culture conditions. BR, the end-point of traditional quality control assays, did not detect sublethal concentrations of toxins in the culture milieu in our study. This study demonstrates that temporal variation at key developmental stages reflects the quality of the culture environment. An MEA that incorporates MK will provide enhanced sensitivity and faster turn-around times.STUDY FUNDING/COMPETING INTEREST(S)The study was supported by Mayo Clinic Department of Obstetrics and Gynecology Small Grant Program. The authors have no competing interests to declare.

Study question What type of disposables and solutions exhibit a higher frequency of Mouse Embryo Assay (MEA) failure and where should manufacturers put special attention during production? Summary answer Flexipet-pipette types, protein supplements, ovum-pick-up needles, catheters, steripettes, culture media, and mineral oils showed higher MEA-failure frequencies and/or lethality levels due to embryotoxicity among IVF-supplies. What is known already The MEA is considered the gold standard assay for quality control of IVF products. It is based on the analysis of in vitro mouse embryonic development under direct or indirect presence of a to-be-tested product, and it requires over 80% expanded blastocyst formation rates (EBFRs) for a product to be considered non-toxic and be released to the market. Recent episodes reported toxicity issues found in embryo culture supplies and IVF industry devices that were recalled by manufacturers. This underscores the necessity for more specific characterization of products, guidelines, and rigorous quality control protocols to prevent potentially adverse clinical outcomes. Study design, size, duration MEAs were conducted over 5 years on 6759 batches encompassing 16 categories of IVF products from various manufacturers. Results were retrospectively analysed based on MEA test outcomes for each category. A total of 217 dishes, 610 catheters, 1182 culture media, 135 steripettes, 1907 mineral oils, 167 glass-pasteur pipettes, 66 protein supplements, 207 flexipet-pipettes, 191 pipette tips, 96 PVPs, 68 syringes, 126 tubes, 1294 vitrification kits, 182 micropipettes, 98 disinfectants, and 213 needles lots were analysed. Participants/materials, setting, methods All procedures adhered to protocols approved by an Ethical Committee. Samples underwent classification as toxic or non-toxic based on MEAs results, and the proportion of samples that tested positive for embryotoxicity was determined for each category. Statistical comparisons were made among categories to identify those most prone to be identified as toxic. Additionally, in failed assays, mean EBFRs were categorized into 0-30%, 30%-70%, and 70%-80% ranges to assess extreme, mild and low sample-toxicity levels, respectively. Main results and the role of chance Out of 16 studied categories, only 5 of them exceeded a 5% of toxic lots. Flexipet-pipette types were reported as the highest toxic-samples rate category, with 72 out of the 207 tested batches (34.8%) failing the MEA. Protein supplements were found to be the second highest toxicity-ranked category, with 14/66 noxious lot samples (21.2%). They were followed by needles and catheters with 36/213 (16.9%) and 68/610 toxic lot samples (11.2%), respectively. Finally, a considerably lower toxicity rate was obtained with steripettes, with 10/135 samples reported as toxic (7.4%). Among these five top toxic-samples rate categories, protein supplements were found to possess a mild lethal effect on embryos, with a 43.6% mean EBFR in failed assays. However, extreme embryotoxic effects were reported for the remaining highest toxic-samples rate categories, based on their mean EBFR in failed assays: 20.8% for flexipet-pipette types, 16.2% for needles, 10.8% for steripettes, and 10.3% for catheters. Interestingly, although culture media and mineral oils were not among the top MEA-failure frequencies, as only 48/1182 (4.1%) and 23/1907 (1.2%) toxic lot samples were registered for each category, respectively, both categories showed a nearly-extreme lethality caused by their toxic-assessed samples, reflected in 31.7 and 35.1% mean EBFRs. Limitations, reasons for caution Increasing the number of sample-providing manufacturers per category might strengthen the wider market representativeness of the results. Moreover, it should be noted that the identification of specific sources of toxicity results highly difficult, hindering the implantation of corrector modifications within the manufacturing process. Wider implications of the findings These findings indicate that protein supplements, flexipet-pipette types, steripettes, catheters and needles exhibit a heightened toxicity tendency. Additionally, toxic mineral oils and culture media-linked high-embryotoxicity levels were observed. Therefore, special attention should be given to the production of these demonstrated potentially embryotoxic products to identify and mitigate clinical toxicity risks. Trial registration number Not applicable

The mouse embryo assay (MEA) is the standard test used in assisted reproduction to evaluate the toxicity and effectiveness of culture media and consumables. However, the assay has been criticised for its limited sensitivity, inconsistencies between laboratories, and ethical concerns. Despite the 3Rs principles, over 111 million mice and rats were used in the USA in 2017, with an unknown proportion of these being used in the MEA. While the FDA has provided MEA guidelines, its aim is to phase out animal toxicity testing within 3-5years. This article explores the possibility of replacing the MEA with the bovine embryo assay (BEA), providing justifications based on ethics, science, practicality, and economics. Through a review of MEA applications, market data, regulatory frameworks and industry disclosures, the article estimates the current impact of the MEA. Incorporating the BEA into regulations could eliminate the need to breed mice for the MEA and greatly reduce the use of animals. Standardising and validating the BEA would provide a reliable and ethically preferable alternative that aligns with the growing demand from regulators and society for non-animal testing methods.

Successful in vitro fertilization (IVF) relies on sound laboratory methods and culture conditions which depend on sensitive quality control (QC) testing. This study aimed to improve the sensitivity of mouse embryo assays (MEA) for detection of mineral oil toxicity. Five experiments were conducted to study modifications of the standard mouse embryo assay (MEA) in order to improve sensitivity using clinical grade mineral oil with known peroxide concentrations. Assessment of blastocyst development at either 96h or in an extended MEA (eMEA) to 144h was tested in each experiment. In experiment 1, ability to detect peroxides in oil was compared in the MEA, eMEA, and cell number at 96h. In experiment 2, serial dilutions of peroxide in oil were used along with time-lapse imaging to compare sensitivity of the morphokinetic MEA to the eMEA. Culture conditions that may affect assay sensitivity were assessed in experiments 3-5, which examined the effect of group versus individual culture, oxygen concentration, and protein supplementation. Extended MEA and cell counts identified toxicity not detected by the routine endpoint of blastocyst rate at 96h. The eMEA was fourfold more sensitive than the standard MEA, and this sensitivity was similar to the morphokinetic MEA. Group culture had a protective effect against toxicity, while oxygen concentration did not affect blastocyst development. Protein supplementation with HSA had a protective effect on blastocyst development in eMEA. The standard MEA used by manufacturers does not detect potentially lethal toxicity of peroxides in mineral oil. While group culture may mask toxicity, protein supplementation and oxygen concentration have minimal effect on assay sensitivity. The eMEA and time-lapse morphokinetic assessment are equally effective in detection of peroxide toxicity and thus provide manufacturers and end-users a simple process modification that can be readily adopted into an existing QC program.

During early vertebrate development, a large number of noncoding RNAs are maternally inherited or expressed upon activation of zygotic transcription. The exact identity, expression levels, and function for most of these noncoding RNAs remain largely unknown. miRNAs (microRNAs) and piRNAs (piwi-interacting RNAs) are two classes of small noncoding RNAs that play important roles in gene regulation during early embryonic development. Here, we utilized next-generation sequencing technology to determine temporal expression patterns for both miRNAs and piRNAs during four distinct stages of early vertebrate development using zebrafish as a model system. For miRNAs, the expression patterns for 198 known miRNAs within 122 different miRNA families and eight novel miRNAs were determined. Significant sequence variation was observed at the 5' and 3'ends of miRNAs, with most extra nucleotides added at the 3' end in a nontemplate directed manner. For the miR-430 family, the addition of adenosine and uracil residues is developmentally regulated and may play a role in miRNA stability during the maternal zygotic transition. Similar modification at the 3' ends of a large number of miRNAs suggests widespread regulation of stability during early development. Beside miRNAs, we also identified a large and unexpectedly diverse set of piRNAs expressed during early development.