IVF Laboratory Research Articles

Self-reparation signs in embryo development

Background and Aim: Time-lapse systems allow IVF laboratories to perform in-depth analysis of the embryo development thanks to the continuous monitorization. Some events impossible to detect with conventional morphologic evaluation, such as reverse cleavage or multinucleation, can be detected using time-lapse. Even though the low scientific evidence, the presence of these events is considered as negative factor when the embryo quality assessment is performed.

Studying the IVF Laboratory Performance Indicators the Vienna Consensus 2017 for the High Institute According to Diagnosis and Assisted Reproductive for Infertility Technologies, Al-Nahrain University, Iraq

Background: Performance indicators are used to assess patient safety, efficacy, equity, patient-centeredness, punctuality, and efficiency. The benchmark values for each Key Performance Indicator are aspirational values, and the minimum performance level values are the number of fertilized oocytes on Day 1 and the Normal Fertilization Rate, respectively (presence of 2Pro Nucleus and 2Polar Body measured at 17 h post-injection) as a Failed fertilization rate is calculated as the proportion of IVF cycles (excluding cycles with intracytoplasmic injection) on Day 1 (17 hours after insemination) with no signs of pregnancy.
 During fertilization (i.e., 0 oocytes with 2Pro Nucleus). The percentage of zygotes on Day 2 (44 hours after insemination) is known as the cleavage rate, and it can suggest an issue with sperm quality (sperm function, oocyte activation, and gamete receptors), sperm processing, or the quantity of spermatozoa used for insemination. which cleaves to create embryos. The percentage of cleaved embryos per successfully fertilized egg that are at the 4-cell stage on Day 2 (44 hours post-insemination) or at the 8-cell stage on Day 3 (68 hours post-insemination) is known as the embryo development rate. This evaluates the viability and quality of the embryos as well as the capacity of the culture system to promote cleavage at the necessary stages. The critical factor is the proportion of blastocysts observed at 116 hours after insemination as a function of the number of correctly fertilized oocytes. Measures of performance blastocyst development rate. The viability of the embryo as well as the culture system's capacity to support blastocyst formation from fertilized oocytes (i.e., the formation of an intracellular mass and a blastocoele cavity) are both determined by this factor. It should be noted that this phrase only considers blastocyst formation and not blastocyst stage or quality. The damage rate is the proportion of oocytes that are injured or have deteriorated by the time of fertilization assessment on Day 1 as a result of the intracytoplasmic injection. The percentage of biopsied and tubed/fixed samples where DNA is found represents the success rate of the biopsy. It serves as a gauge of the embryologists' ability to transfer biopsied samples to test tubes, as shown by successful DNA amplification. The number of gestational sacs divided by the total number of transplanted embryos is how one calculates the implantation rate, which is dependent on the cleavage stage. By dividing the number of gestational sacs by the total number of transplanted blastocysts, the implantation KPI (blastocyst stage) is calculated.

AI, Time-lapse and Biomarkers: What Can We Expect in the Next Decade?

For 40 years we have relied upon morphological assessment of the human embryo for its selection for transfer. With the advent of time-lapse microscopy (TLM), we have been able examine the timings of key developmental events and create algorithms to further advance embryo selection. However, such algorithms do not utilise all available data. Artificial intelligence (AI) can utilise the hundreds of successive images of each embryo as it develops to produce highly predictive rankings. This approach increases the speed of assessment, while decreasing variation typically associated with the subjective analysis of individual embryologists. Therefore, AI represents a means to increase both accuracy and standardisation of embryo selection. In the IVF laboratory, beyond its current function in embryo selection, AI will be used for gamete selection prior to fertilisation, and to increase laboratory function by its integration into Quality Control and Management systems. Although TLM combined with AI holds great potential, it does not quantitate the physiological status of the embryo. Analysis of spent culture medium has revealed that metabolism (glucose uptake and the utilisation of amino acids) of the human blastocyst is related to pregnancy. Currently, analysis of metabolic function by individual embryos requires highly specialised technologies, but with the advent of microfluidics and microfabrication we are entering an era of novel benchtop technologies capable of rapid single embryo analysis. Subsequently, it is envisaged that AI will be used in combination with TLM and the analysis of spent culture medium (to quantitate metabolism and plausibly cell-free DNA), to identify non-invasively the healthiest embryo for transfer.

Improving the Accuracy of Constant Temperature Monitoring of Open-Air Stereo Stages

Background: Temperature regulation and monitoring is critical in IVF laboratories. Constant temperature monitoring of open-air stereo stages has proven to be a difficult feat, as probes are exposed to ambient air, making readings irreflective of actual temperature within media. Aim: To determine the substrate that most accurately reflects the temperature of culture media on an open stereo-stage. Method: Media under oil was temperature logged on a stereo stage simultaneously with six different dish setups, each fitted with a temperature probe: empty dish, large steel spheres (5mm), small steel spheres (2mm), ultrasound gel, silicone mold and ceramic balls. The dish set up that most consistently reflected media temperature was further assessed to determine how closely it mimicked the warming rate of media, measured in 1-minute intervals over a 20-minute period. Results: The media logged an average temperature of 36.99°C with a tight range of 36.9°C−37.1°C. The commonly used empty dish recorded an average temperature of 35.44°C with a range of 35.06°C−35.81°C. The tested substrates, listed in ascending order of efficacy, measured as follows: ceramic balls −33.65°C (33.01°C−33.97°C), large steel spheres −33.16°C (35.03°C−35.31°C), silicone mold −35.67°C (35.40°C−36.01°C) and ultrasound gel −36.24°C (36.5°C−36.41°C). The small steel spheres replicated the media temperature most accurately, with an average temperature of 36.48°C and the least variation (36.38−36.60°C). When warmed from room temperature to 37.0°C, the small steel spheres were closely comparable to the warming rate of the media, with rates of 0.94°C per minute and 1.1°C per minute, respectively. Both reached range within 12-minutes. Conclusion: Small steel spheres can substitute for culture media during temperature monitoring, allowing accurate constant monitoring of open-air stage temperature, and are closely comparable to the warming rate of media.

Is Cleavage Stage Multinucleation Indicative of Lower Pregnancy Potential in Euploid Blastocysts?

Background: Cleavage stage multinucleation is a morphological assessment IVF labs may use to rank embryos for transfer, as blastocysts that were multinucleated at the 2-cell or 4-cell stage have a lower live birth rate (LBR) compared to blastocysts that were mononucleated 1 . However, when these blastocysts are PGT-A tested, the euploidy rate is similar 2 , indicating that the poorer pregnancy outcomes may not be due to aneuploidy. It is unknown if multinucleation is indicative of a lower likelihood of pregnancy for euploid blastocysts. Aim: To determine if LBR following transfer of euploid blastocysts is lower when blastocysts were multinucleated at the 2-cell or 4-cell stages. Method: Retrospective study on 360 euploid blastocysts cultured in the EmbryoScope or EmbryoScope+ between March 2018 and October 2019, and individually transferred. Time-lapse images were retrospectively assessed for multinucleation at the 2-cell and 4-cell stage. A multinucleated embryo was defined as having [Formula: see text] 1 cell with [Formula: see text] 2 nuclei. Results: Blastocysts that were multinucleated at the 2-cell stage (n=139) had a similar LBR to mononucleated embryos (40.3% vs 41.6%). Multinucleation at the 4-cell stage (n=43) also did not significantly affect LBR (37.2% vs 43.6%). Multivariate logistic regression analysis found no significant effect of multinucleation on LBR. Biochemical pregnancy, clinical pregnancy (fetal heart), and miscarriage rates were also similar between multinucleated and mononucleated embryos. Conclusion: This study found no evidence that multinucleation at the 2-cell or 4-cell stage influences pregnancy rate in euploid blastocysts. As such, the assessment of cleavage stage multinucleation for PGT-A tested embryos may not be useful in ranking PGT-A tested embryos for transfer. Further work is required with larger numbers of embryos to confirm how the degree of multinucleation may influence LBR.

The Impact of Covid-19 Travel Restrictions on Gamete Shipping from Australian IVF Clinics

Background: Cross-border surrogacy and egg donor arrangements are an increasingly common means to family building. Establishing patterns of use has always been difficult in relation to Australian patients. Accurate data is stymied by lack of documentation of international third-party reproductive care available to Australian authorities. Since Australia’s international travel ban came into effect, it is hypothesised that those planning to use cross-border reproductive care have had to rely significantly more on local IVF clinics for services such as sperm freezing, embryo creation and gamete release procedures. Establishing the increase in gamete shipping as a result of the ban on non-essential travel will improve understanding of the resource impact on Australian IVF laboratories. Such data can also assist policymakers assess the utility of state-based regulations relevant to CBRC. Aim: Quantify and characterise the impact of the Covid-19-related travel ban on Cross-Border Reproductive Care amongst patients of Australian IVF clinics. Method: All Australian IVF clinics were invited to provide de-identified data on interstate and international gamete export applications from two 12 month time periods pre and during Covid-19 related international travel lockdowns. Each dataset included: patient age; type of gametes exported; destination country or state; and date gamete release was approved. Results: Results showed a very significant increase in gamete shipping in the twelve months after Covid-19 travel restrictions were enforced. They illustrate the comparative prevalence of shipping to established and newer destinations during the periods March 2019–February 2020 and June 2020–May 2021. The volume of shipping to the commonest destinations is quantified. Conclusion: The inability of involuntarily infertile patients to travel internationally, rather than halt cross-border reproductive care, has led to a significant increase in update of gamete shipping. This is likely associated with increased utilization of Australian IVF clinics for embryo creation purposes. The high proportion of internationally-shipped gametes going to the US and Ukraine is likely a reflection of the availability of surrogates and donors and the legal frameworks.

Impact of Adoption of Radio Frequency Identification (RFID) Witnessing System on Workflow, Organization, and Quality Management in the IVF Laboratory

Impact of Adoption of Radio Frequency Identification (RFID) Witnessing System on Workflow, Organization, and Quality Management in the IVF Laboratory

Impact of the Use of Radio Frequency Identification (RFID) Electronic Witnessing System on the IVF Laboratory Staff and Patient Experience

Impact of the Use of Radio Frequency Identification (RFID) Electronic Witnessing System on the IVF Laboratory Staff and Patient Experience

P-230 Senescence in mammalian embryos as a novel assay for quality control in the IVF laboratory

Abstract Study question Can senescence biomarkers be used as a more sensitive assay for IVF laboratory quality control than a conventional mouse embryo assay (MEA)? Summary answer Senescence biomarkers are more sensitive than a MEA and should be considered as a novel quality control assay in assessing overall IVF laboratory environment. What is known already An MEA is a bioassay that is utilized to detect toxicity and suboptimal conditions, but only assesses the ability of the embryos to develop to the blastocyst stage. The majority of human embryos created during IVF do not result in a live birth, most either arrest or degenerate. Suboptimal culture conditions and accompanying environmental stressors may be responsible or some of this lack of embryo development. Senescence or developmental arrest, like apoptosis, is a defensive mechanism that halts further development of stress, poor quality or abnormal embryos. Senescence should be considered a possible reason for a high embryos loss rate. Study design, size, duration Murine zygotes from the strain B6C3F1 x B6D2F1 (N = 263) were exposed to 12.5nM Doxorubicin (DOX), a known inducer of senescence as a positive control, either 250nM or 500nM H2O2 or atmospheric oxygen conditions (20%) to mimic oxidative stress. These treatment groups were compared to embryos cultured without H2O2 in a low oxygen (5% 02) environment. The zygotes were cultured for 72 hours, and biomarkers of senescence were assessed. Participants/materials, setting, methods Embryos were assessed at 72 hours with a confocal microscope (Zeiss) and blastocyst conversion rate and number of cells/blastocyst was calculated. The following senescence markers were examined, SAβ-gal (SPiDER-β-Gal), IL-6 mRNA (RT-qPCR) and CellRox Deep Red, a fluorogenic reactive oxygen species (ROS) indicator was also evaluated. Fiji Image J software was used to calculate Corrected Total Cell Fluorescence (CTCF) as a percentage of the low oxygen group and GraphPad Prism was used for data analysis. Main results and the role of chance When comparing the blastocyst conversion rate, all treatment groups showed a blastocyst conversion rate of > 80% after 72 hours of exposure, indicating that all would have “passed” assessment with a conventional MEA (p > 0.05). However, when the average number of cells per blastocyst was compared to the low oxygen group, all groups had a significantly lower average number of cells per blastocyst (112±26.65 for low oxygen, 74±7.95 for high oxygen, 82± 24.54 for 500 nM H2O2, 89 ± 34 for 250 nM H2O2 and 63± 20.01 for 12.5 nM DOX, p < 0.05). All of the treatment groups had a significant increase in the relative fold change of IL-6 mRNA compared to the low oxygen environment (2.2 for the high oxygen, 2.5 for 500 nM H2O2, 2.9 for 250 nM H2O2, and 7.4 for 12.5 nM DOX, p < 0.05). The CTCF for SAβ-Gal using the SPIDER-β- Gal assay was significantly elevated in all treatment groups (145% for high oxygen, 162% for H2O2250 nM, 193% for 12.5 nM DOX and 196% for 500 nM H2O2, p < 0.05). A significant increase in the CTCF for CellRox Deep Red was also observed (289% for 12.5 nM DOX, 492% for 250 nM H2O2, 763% for 500 nM H2O2, and 925% for high oxygen, p < 0.05). Limitations, reasons for caution This analysis is currently limited by the lack of a specific senescence marker. This approach is also invasive, destroying the embryo, not lending itself to clinical human embryo selection. Although the mouse model has advantages, translating these results into human embryos is a limitation of this study. Wider implications of the findings This optimized novel assay can be used to provide great insight about the subtle laboratory culture/environmental conditions in the IVF laboratory. The results support the hypothesis that environmental stressors can induce senescence biomarkers and implicate their participation in the degeneration of previously viable embryos. Trial registration number not applicable

P-709 Fine-tuning IVF laboratory key performance indicators of the Vienna consensus according to female age

Abstract Study question Is the Vienna consensus appropriate to monitor IVF laboratory outcomes of treatments involving women of different age ranges? Summary answer Most IVF laboratory key performance indicators(KPIs) are reliably applicable irrespective of female age, while total good blastocyst rate should be adjusted according to female age What is known already The IVF laboratory is central to ART treatments. This demands methodical and precise monitoring of the performance. To assess laboratory efficiency, the Vienna consensus identified a set of relevant performance indicators(PIs) and KPIs applicable to a “reference population” defined by female age <40years, exclusion of PGT cases and use of own fresh oocytes and ejaculated sperm. It is known that women older than 39years – whose treatment outcome is affected by reduced oocyte quality–represent an increasingly large proportion of IVF patients. Thus, in this subgroup of patients the relevance of the Vienna indicators to treatments of remains to be demonstrated. Study design, size, duration Reported data concern a retrospective, single-center cohort analysis of 862 ART cycles carried out between January 2014 and May 2021. Inclusion criteria were indication for IVF/ICSI, blastocyst culture of all embryos formed in each cohort, use of own ejaculated spermatozoa (fresh or frozen), and complete cycles, i.e. those whose all embryos were transferred, cryopreserved or disposed of. Cancelled and PGT cycles were not included Participants/materials, setting, methods The overall population was divided into two groups according to female age: Vienna consensus (≤39 years) and older female age(≥40 years). We measured a selection of the Vienna performance indicators and KPIs, with a focus on measures relevant to embryo cleavage and blastocyst formation. Assessment of fertilization, cleavage and blastocyst rates was carried out. To assess more comprehensively blastocyst quality and quantity, we estimated the total usable blastocyst rate(TBUR). Finally, blastocyst cryosurvival rate was assessed. Main results and the role of chance No differences were observed in fertilization and embryo cleavage KPIs between the Vienna consensus and the older female age groups (standard IVF fertilization, 67.2 vs. 67.3; ICSI fertilization, 72.3 vs. 75.3; Day 2 development, 57.6% vs 58.7%; Day 3 development, 52.4% vs. 50.7 %, respectively). TBUR was lower in the older female age group (45.5% vs. 33.4% p < 0.001). This outcome decreased steadily with increasing female age. Clinical outcome significantly decreased with increasing female age. Implantation rate decreased from 34.3% in the Vienna consensus group to 16.9% in the older female age group (p<.001). Cumulative CPR showed a similar trend decreasing from 52.4% to 23.9% (p<.001). In the two populations, TBUR was further assessed after normalization of the number of retrieved oocytes. Rates were comparable in cycles with only few (1- 5 oocytes) collected oocytes (51.9% and 45.5%, p=ns). In cycles with 6-10, 11-15 and>15 collected oocytes TBUR was lower in older patient groups(p < 0.01). Univariate and multivariate logistic regression analysis showed female age as a factors independently associated with TBUR. Higher female age was associated with a reduced probability to achieve a TBUR greater than 40%. Limitations, reasons for caution The study design is retrospective and requires further refinement to control for factors that may impact clinical outcome Wider implications of the findings The study confirms the general validity of the Vienna Consensus but also indicate a need for fine-tuning in relation to factors intrinsic to gamete quality that can impact laboratory outcomes. Trial registration number Not applicable

P-758 Evaluating mismatch categories and true errors using an electronic witnessing system

Abstract Study question What are the types and frequency of errors occurring in the laboratory identified by an electronic witnessing system (EWS)? Summary answer Several types of mismatch categories were identified, and the rate of a potentially catastrophic true mismatch error was < 1%. What is known already Mix-ups in the IVF laboratory have been recently highlighted in the media. These errors can have devastating long-term consequences for the fertility clinics and families involved. Prevention of these errors should be paramount in the laboratory. Study design, size, duration A retrospective analysis was conducted using 15 months of electronic witnessing data from two IVF laboratories in the United States. A total of 11,357 witnessed procedures were evaluated to determine the type and frequency of mismatches that occurred. Participants/materials, setting, methods All procedure match and mismatch data was recorded by an EWS (Matcher, IMT International). The mismatch data from two laboratories was evaluated to determine the category and occurrence of mismatches taking place in the assisted reproductive technology (ART) laboratory. Main results and the role of chance The overall mismatch rate was 0.78% (89/11,357). The types of mismatches were identified as: 1) Program: duplicate barcodes scanned, barcode misread by program, non-Matcher barcode picked up during scan, connectivity issues; 2) Wrong patient selected; 3) Transcription error; 4) Cycle set up: treatment cycle not set up correctly, donor not linked to treatment cycle; 5) True error: dishes and/or tubes from two different patients were in the same workstation at the same time. Program error happened most frequently at 0.30% (34/11,357). The wrong patient selected and cycle set up errors occurred 0.18% (20/11,357), followed by transcription error at 0.11% (12/11,357). A true error occurred 0.03% (3/11,357) and was rectified in < 10 seconds meaning there was no time for actual mixing of gametes to occur. These findings agree with previously published reports worldwide where the occurrence of a true mismatch is < 1%. Limitations, reasons for caution The present study retrospectively analyzed mismatch data gathered over a 15-month period after implementation of EWS. Further investigation is warranted to determine if mismatches occur more or less frequently after long term use of an EWS. Wider implications of the findings Failure to identify a true mismatch error may lead to a catastrophic error. Continued evaluation of EWS may provide insight into how and why a true error occurs. This information can then be utilized to improve laboratory protocols and procedures for enhanced patient protection. Trial registration number Not applicable

P-527 Comparison of implantation rate and preimplantation genetic testing between hatching and hatched blastocysts

Abstract Study question Does embryo stage, hatching versus hatched blastocyst, affect ploidy status and implantation rates (IR)? Summary answer The embryo stage may influence ploidy status, but no correlation was made between embryo stage and IR. What is known already It is generally accepted that hatched embryos present increased vulnerability when handled and are often selected against. The literature on this varies with some reporting that the transfer of hatched blastocysts resulted in lower pregnancy rates compared to hatching or expanded blastocysts. Other studies have found no difference in clinical outcome with the transfer of hatched blastocysts compared to other stages. Reports defining the relationship between hatched or hatching blastocysts and ploidy rates are limited. Study design, size, duration Retrospective analysis of IR of 224 transferred euploid embryos and ploidy rate (P-Rt) of 1431 embryos produced in two IVF labs in the United States between 2019-2021. Embryos underwent assisted hatching on Day 3 and were subjected to preimplantation genetic testing (PGT) on Day 5 or Day 6 followed by vitrification. IR was defined as the presence of an intrauterine implantation sac with the detection of a fetal heartbeat on ultrasound. Participants/materials, setting, methods Trophectoderm biopsies were evaluated by NextGen Sequencing at the same genetics laboratory (Ovation Genetics, Nashville, TN). P-Rt included the following categories: Normal (N) or euploid, Abnormal (ABN) or aneuploid, Low-Mosaic (LM), and High-Mosaic (HM). Euploid embryos were transferred in subsequent frozen embryo transfer (FET) cycles and monitored by clinical ultrasound. Chi-square statistical analysis was performed to identify potential differences (p < 0.05). Main results and the role of chance No significant difference was observed in IR after transferring euploid hatched blastocysts compared to euploid hatching blastocysts. The IR of hatched blastocysts was 40.3% compared to 50.3% for hatching blastocysts (p = 0.21). A significant difference in ABN and HM embryos was observed according to embryo stage (p = 0.02). The ABN rate was 21.2% for hatched blastocysts compared to 28.0% for hatching blastocyst and the HM rate of hatched blastocysts was 10.3% versus hatching blastocyst at 7.5%. No significant difference in Normal and LM ploidy rates were observed according to embryo stage. The N ploidy rate of hatched blastocysts was 58.1% versus 54.4% in hatching blastocysts, and LM rate for hatched blastocysts was 10.3% compared to 10.1% in hatching blastocysts. Limitations, reasons for caution The limited data set did not include fresh embryo transfers or the transfer of untested embryos. The inclusion of increased embryo numbers could strengthen the power of the evaluation and allow for more robust statistical analysis. Wider implications of the findings Embryo selection is a multifactorial process consisting of a wide range of parameters. Numerous advances in embryo production and evaluation have resulted in the need for an algorithm or guide to rank embryos for transfer. Further investigation is warranted to elucidate comprehensive recommendations for embryo selection. Trial registration number not applicable

P-780 Ten years electronic witnessing in the IVF laboratory

Abstract Study question What did we learn after 10 years of electronic witnessing? Summary answer Only when applied correctly, electronic witnessing can prevent sample mix-up. What is known already In the early days of IVF, manual double witnessing was used to reduce the misidentification of gametes for insemination. However, risk for human errors due to fatigue, automatism, distraction or work load make this a mistake-prone approach. Hence, there is increasing interest in using electronic witnessing systems for patient and sample identification. Although not always mandatory, such systems have been implemented in many fertility laboratories to avoid identification errors. A mismatch is generated when non-matching samples are simultaneously present in a single workstation. Yet, when the system is incorrectly used, corrective intervention by an administrator (administrator assign) may be required. Study design, size, duration This evaluation investigates the mismatch (MM) rate and administrator assigns (AA) over a 10-year period (March 2011- December 2021) with the use of RI Witness (RIW) (CooperSurgical). Radio frequency identification tags (RFID) were used for patient and sample identification. Conventional IVF and ICSI cycles, and frozen embryo cycles (FET) were included since 10 years, IUI cycles since 8 years. Participants/materials, setting, methods The total number of tags and witness points (WP) were recorded. Witness points represent all the actions in RIW that have been performed during the entire process. MM and AA were collected and stratified by procedure (sperm preparation, oocyte retrieval, IVF/ICSI, (embryo)biopsy, vitrification/warming, embryo transfer, medium changeover and IUI). Critical MM (such as mis-labeling or non-matching samples within one work area) and critical AA (such as non-RIW-identified samples and unconfirmed WP) were selected. Main results and the role of chance A total of 109,655 cycles were included: 53,023 IVF/ICSI, 36,347 FET, and 20,285 IUI cycles. The 724,096 used tags, led to a total of 849,650 WP. The overall MM rate per WP was 0.25% (2,132/849,650), 1.9% per cycle, of which 144 critical mismatches occurred in the different procedures: sperm preparation: 66, oocyte retrieval: 5, IVF/ICSI: 33, (embryo)biopsy: 1, vitrification/warming: 26, embryo transfer: 8, medium changeover: 3 and IUI: 2. The mean critical MM rate per WP per year was 0.017% ± 0.007% and 0.13 ± 0.05% per cycle. The overall AA rate per WP was 0.11% (940/849,650), 0.86% per cycle, including 320 critical assigns: sperm preparation: 83, oocyte retrieval: 34, IVF/ICSI: 173, (embryo)biopsy: 3, vitrification/warming: 6, embryo transfer: 0, medium changeover: 21. Critical AA rate per year was on average 0.041%/WP ± 0.01% and 0.30 ± 0.07% per cycle. All MM and AA rates remained stable during this 10-year period, except for sperm preparation, where the removal of a single witness point led to a huge increase in AA. RIW should still be used in combination with the manual labelling of bottom and lid of dishes and tubes to guarantee correct assignment in case of RFID malfunction or AA. Limitations, reasons for caution The procedures and method of integration of RIW may vary from one laboratory to another and result in differences in the potential risks. RIW cannot (yet) identify individual embryos, making double manual witnessing indispensable at certain critical steps, where potential errors are not recorded. Wider implications of the findings Using an electronic witnessing is considered to be the ultimate tool to safeguard correct identification of gametes and embryos. But this is only possible when used correctly and requires proper training and attention of the staff. It may also induce new risks, i.e. blind witnessing of samples by the operator. Trial registration number not applicable

O-268 Cumulative toxicity of plastic disposable devices used during IVF procedures

Abstract Study question Is there a cumulative toxicity from the different plastic consumables used during IVF procedures? Summary answer A toxicity may be detected when IVF plastic consumables are cumulatively used, while no toxicity is detected when the same consumables are tested individually. What is known already Many components used in IVF laboratories, such as culture media and plastic consumables may impair human embryonic development. Consequently, it is necessary to screen all reagents and materials which will be in contact with gametes and embryos. Toxicity tests such as the Mouse Embryo Assay (MEA) and the Human Sperm Survival Assay (HSSA) are used by manufacturers as quality control tools to demonstrate the safety of their products. This evaluation is individually performed for each consumable, while in IVF several plastic devices are used, potentially creating a cumulative exposure to chemical contaminants which could not be detected for single consumables. Study design, size, duration The objective of this observational, prospective study was to evaluate the cumulative toxicity from the plastic consumables used during an IVF procedure. First, each consumable was tested individually using HSSA. Then, different associations of 3, 4 and 5 consumables, previously validated as non-embryotoxic, were tested. A total of 36 references of IVF plastic consumables were individually tested and 7 associations were tested. Participants/materials, setting, methods After obtaining the patients’consent, the leftovers of semen qualified with normal parameters according to the WHO 2010 criteria was used to perform the HSSAs according to the methodology described by Crichtlow et al. 1989. The items or associations were tested 3 times with 3 different semen to ensure the repeatability of the results. The toxicity threshold was defined by a Sperm Motility Index (SMI) less than 0.85 observed on at least 2 of 3 tests. Main results and the role of chance A total of 32 IVF plastic consumables from different manufacturers (Vitrolife, CCD, ThermoFisher Scientific, SparMED, Cryo Bio System, Apex Medical, VWR, Starstedt, Corning Science Mexico, Ellios Bio Tek, Sempermed, Cardinal Health, Codan Medical and Henke-Sass Wolf) were individually tested and revealed no toxicity: 200- and 1000-µl cones, condoms, 3-ml Pasteur pipets, 2- and 5-ml serological pipets, 35- and 100-mm dishes, 4 well dishes, examination nitrile powder-free gloves and latex powder-free nitrile coating microsurgical gloves, 1-ml syringes, soft embryo transfer catheters, oocyte pick-up needles, 5-, 14- and 15- ml tubes, cryotubes, sperm collection receptacles, sperm freezing straws, embryo vitrification straws). Four IVF consumables tested individually revealed toxicity: latex powder-free nitrile coating microsurgical gloves, 1-ml ethylene oxide sterilized syringes, 80-ml sperm collection cups, and 25-ml flasks. Three associations of 4 and 5 IVF disposables mimicking the chain of sperm preparation were found no toxic. However, 4 associations of 4 and 5 IVF disposables mimicking the chain of oocyte pick-up were found toxic. Further detailed results of this study will be presented at the conference. Limitations, reasons for caution The methodology of HSSA (extraction and incubation times, type of medium, temperature, chosen toxicity thresholds…) can affect the results. Thus, the HSSA may lack sensitivity for detecting toxicity. Wider implications of the findings Further studies are needed to quantify the cumulative levels of toxins (e.g. bisphenols) released from associated plastic devices during an IVF procedure. Also, professionals should rationalize and minimize the number of plastic consumables used during an IVF procedure to improve the environment in which the embryos develop. Trial registration number Not applicable

P-412 3D live-imaging reconstruction of the human embryo implantation ex vivo

Abstract Study question How do human embryos implant in physiological conditions and develop beyond blastocyst stage? Summary answer Human embryos apply forces during invasion of the matrix, the mural trophectoderm undergoes a quick compaction leading to a fast radial expansion of polar side What is known already Human embryos can be cultured beyond blastocyst stage using supplements of animal origin, such as fetal bovine serum. However, two main issues limit our understanding of the implantation of the human embryo. First, human embryos do not express fluorescent proteins, severely limiting the access to advanced live-imaging tools. Second, the animal serums may not represent the physiological peri-implantation conditions of the human embryo, thus limiting the reproducibility of the in vitro experiments. Here we combine label-free multiphoton imaging with media formulations including clinical-grade protein supplements from human plasma unlocking the observation of the human embryo in 3D and physiological conditions Study design, size, duration We have cultured 150 human embryos from D3 until blastocyst stage and also more than 600 mouse embryos from zygote until blastocyst stage and then transferred them into a 3D implantation platform. All embryos were supplemented with clinical-grade human-derived serum, which is rich in globulins and growth factors, and commercial basal media. The implantation was monitored for 3-to-5 days up until day 11. Traction-force microscopy and molecular imaging was employed to quantify implantation markers. Participants/materials, setting, methods Implantation was measured using our novel version of traction-force microscopy, which reveals the force applied by entire embryos during live-imaging experiments. Multiphoton illumination of autofluorescent molecules allows to reconstruct images of live human embryos on 3D at unprecedented resolution. Key molecular markers for the formation of the epiblast rosette (OCT4), extra-embryonic visceral endoderm (GATA4), extra-embryonic ectoderm and trophoblast (CDX2) and primary yolk sac and pre-amniotic cavity were reconstructed in 3D using high resolution confocal imaging. Main results and the role of chance The implantation of human embryos was visualized in 3D movies at high resolution, during time-resolved experiments and compared to implantation in mouse embryos. Human embryos undergo a remarkable compaction upon attachment of the polar trophectoderm and thereafter they implant by engaging the matrix through many different points (>8). The post-implantation compaction leads to a strong invasion of the polar trophectoderm, digging large holes in the matrix and expanding radially. Some trophoblast cells escape the embryo colonizing the matrix. Human embryos expressed the correct pattern of molecular markers by day 11 of culture. Actin staining and OCT4 expression revealed the formation of a central rosette defining the pre-amniotic cavity. GATA6 expression defined the visceral endoderm and the formation of a primordial yolk sac. Mouse embryos conversely, compact upon implantation of the mural trophectoderm and are less invasive, typically anchoring to the matrix through 2-to-3 points. Post-implantation culture of mouse embryos starting at zygote was possible until the formation and large expansion of the amniotic cavity, which constituted 2/3 of the whole embryo size, corresponding with the initial steps of gastrulation, longer than any previous protocol Transfer of mouse blastocysts back to receptive mothers resulted in birth of viable litter. Limitations, reasons for caution This study has been performed with human embryos ex vivo using bioengineering technology to implant embryos outside the mother uterus. The bio-compatibility of our serum supplements was validated by live birth of transferred embryos in animal models, but would require further evaluation in clinical trials. Wider implications of the findings Our work stresses the safety and efficiency of supplementing embryo culture and particularly embryo transfer, with human plasma-derived serums of clinical-grade. The correct expression of main molecular markers indicates that this type of supplementation promotes implantation physiologically, which opens the possibility to employ these serums in the IVF laboratory. Trial registration number Not applicable

P-206 Could day 7 embryo culture become standard practice to improve patients’ treatment and outcome?

Abstract Study question What are the clinical outcomes following transfer of day 7 embryos; could day 7 embryo culture become part of standard embryology practice? Summary answer Although clinical outcomes remain low, success has been seen following transfer of day 7 embryos. Extended culture plays an important role in standard embryology practice. What is known already In vitro culture conditions and cryopreservation techniques have improved greatly in recent years, allowing for extended culture, and freezing of embryos at the blastocyst stage. Optimally, embryos should develop to the blastocyst stage on day 5, however deviation from normal development can sometimes occur due to problems in either the embryonic intrinsic factors or imprinting timings. Live births have been reported from slower developing day 7 embryos. Extended culture to day 6 is standard practice in most IVF laboratories, however the aim of this study is to investigate the outcomes following prolonged embryo culture to day 7. Study design, size, duration This is a retrospective analysis study of 47 patients who underwent frozen embryo transfers (FETs) with a cryopreserved day 7 embryo at a UK licensed centre between 2017-2021. Patients were split into two groups: those transferring untested (non-PGT-A) day 7 embryos, and those transferring euploid PGT-A tested day 7 embryos. Clinical pregnancy (CPR) was analysed to study the clinical outcome of day 7 embryo culture followed by a frozen transfer. Participants/materials, setting, methods All patients who had FET with a day 7 embryo between 2017-2021 were included in this study. A total of 49 FETs (48 eSET,1 DET) involving 47 patients took place: 38 with untested embryos and 11 with euploid embryos. Clinical pregnancy rate (CPR) and live birth (LBR) was analysed to study the efficacy in transfer of day 7 embryos and the two groups of patients (untested and PGT-A tested) were compared. Main results and the role of chance The overall CPR per embryo transfer was 8% (47 patients, 49 ETs, 50 embryos transferred) and the LBR per embryo transfer was 4%. In the PGT-A group, a total of 201 day 7 embryos were biopsied over the 5-year study period; 28 were euploid following testing (14% euploidy rate). Of these, 11 have been transferred; 4 resulted in a pregnancy and 2 in a live birth. (LBR 18%) There is a significant difference in CPR between the PGT-A group (11 patients, 11 FETs; 11 embryos transferred, 4 foetal hearts seen, CPR=36%) and the untested group (36 patients, 38 ETs; 39 embryos transferred, CPR=0%) Chi square test performed and confirmed (p < 0.001). The mean age of the patient at the time of embryo transfer was 38.6 ± 0.5 and the mean age of the embryo transferred was 35.6± 0.5 (this was the patient’s age at the time of embryo freezing). Limitations, reasons for caution Limiting factors include the retrospective study design and the small sample size. It is also worth noting the high number of day 7 embryos required to undergo PGT-A testing to result in a low number of euploid embryos suitable for embryo transfer. Wider implications of the findings This study indicates the value of day 7 embryo culture as it has resulted in some patients achieving an ongoing pregnancy and a live birth. However, care must be taken to counsel patients appropriately around the chance of success when transferring day 7 embryos, particularly patients using untested embryos. Trial registration number Not Applicable

P-210 Development of a new device for automatic vitrification of oocytes

Abstract Study question Could an automatic vitrification device prevent human errors and lead to better results and the same survival rates to avoid variability between reproductive centres? Summary answer Developing an automatic vitrification device using Kitazato vitrification/warming medium could optimize survival rates and outcome, regardless of the centre or user using the technique. What is known already Vitrification is the most common technique used daily in assisted reproduction clinics for oocytes and embryos cryopreservation. This procedure is becoming more popular since women are delaying motherhood and they want to preserve their fertility. The most used methodology worldwide is the Kitazato protocol, which has reported the best survival rates (>90%). Although these results are attractive, the procedure involves several manual steps, which requires highly skilled and trained embryologists to be as reproducible as possible. It exists variability between users according to their experience and between clinics so, a device that ensures reproducibility, safety and efficacy is the future. Study design, size, duration The automated device and the microfluidic chip were designed by our team. During 3 weeks, 210 mice zygotes were used in the study to mimic oocyte behaviour because of their greater availability, similar features and their use for trials of biocompatibility of materials for subsequent use in human samples. They were divided in 2 groups to compare manual and automated vitrification using Kitazato media. Both groups were warmed manually according to standard Kitazato protocol. Participants/materials, setting, methods Mice zygotes (210) were divided in 2 groups: A) Manual control (n = 103) with standard vitrification protocol by Kitazato; B) Automated group (n = 107), following a microfluidic protocol performed with Kitazato media. All the zygotes were stored with the same vitrification device: Cryotop by Kitazato. After their storage in liquid nitrogen, both groups were warmed manually following the standard Kitazato thawing protocol. Chi square test was performed to compare survival and blastocyst rates between groups. Main results and the role of chance The manual control, Group A (n = 103), showed 99% survival rate and 92.2% blastocyst rate vs. 99% and 85.8% respectively, when performing the automatic protocol, Group B (n = 107). Chi square test was performed in order to compare survival and blastocyst rates between protocols. No significant differences were found between groups A and B comparing survival rate (p = 0.9985) or blastocyst rate (p = 0.7257). Our device for the automation of oocyte vitrification offers promising results on survival and blastocyst rates, being similar to manual protocols routinely used in IVF laboratories. Limitations, reasons for caution This is the previous step before moving on to human studies to see if the same results can be reproduced. Subsequently, it will be necessary the approval of a clinical trial to verify that pregnancy and live birth rates are similar to those obtained with the non-automated manual protocol. Wider implications of the findings The use of an automated device that allows the adequate exposure to the different vitrification solutions, obtaining at least the same outcome rates, would let the development of a standardised and reproducible protocol that would reduce human error and variability between reproductive clinics and embryologists. Trial registration number Not applicable

O-173 Artificial intelligence blastocyst ploidy distinction through morphokinetics data

Abstract Study question Is it possible to estimate blastocyst ploidy with an artificial intelligence (AI) algorithm built using embryo morphokinetics data? Summary answer AI was able to estimate blastocysts status in euploid or aneuploid with an accuracy of 0.99 (training) and 0.71 and 0.70 AUC (blind-test), respectively. What is known already Morphokinetic parameters are well associated with implantation rates, thus ensuring time-lapse as a useful tool to enhance embryo ranking and selection. However, regarding embryo ploidy status, clinics still rely exclusively on molecular/genetic testing. Our previous studies has indicated that euploid blastocysts are faster at time of pronucleous fading (tPNf) and time to blastulation (tB). Considering the amount of data derived from the morphokinetic annotations and the promising results that AI approach is bringing to the field, this study aims to verify AI accuracy to distinguish euploid and aneuploidy blastocysts using 17 morphokinetics parameters and prospectively compared to embryo biopsy results. Study design, size, duration This is a prospective cohort study including 402 embryos (cultured in a time-lapse incubator, EmbryoscopePlus, Vitrolife) from 140 patients undergoing IVF treatment with preimplantation genetic testing for aneuploidy (NGS platform) after inform consent form signature between July 2019 and September 2021. Morphokinetics annotations were analyzed by AI in an association of the technique of artificial neural networks (ANN) and genetic algorithms (GA) for ploidy assessment. Participants/materials, setting, methods Morphokinetic parameters were manually annotated up to full-expanded blastocyst time. Time intervals and time ratios were calculated, resulting in 17 variables for the IA analysis. Of the 402 embryos data, 252 were randomly divided into training, validating and testing (70%, 15% and 15%, respectively) of the ANN. The remaining 150 data were used for the blind test. The area under the curve (AUC) of the receiver operating characteristic curve was measured to obtain predictive power. Main results and the role of chance From 402 blastocysts biopsied, 185 were euploid and 217 aneuploid. The AI algorithm was trained with 176 embryos (AUC for both euploid and aneuploid= 0.99), tested with 38 embryos (AUC for euploid=0.62 and aneuploid=0.61), validated with 38 embryos (AUC for euploid=0.82 and aneuploid=0.83). For the blind-test 150 embryos were used (AUC for euploid=0.70 and aneuploid=0.71). Blind-test database was checked only after AI algorithm was tested. Limitations, reasons for caution The development of this AI algorithm was built from a single IVF center database. The absence of an extent dataset for the blind-test does not allow us to transpose this algorithm for clinical use at this moment. Wider implications of the findings The use of artificial intelligence for embryo assessment is a promising tool in IVF laboratories. In our model, using only morphokinetics, a considerable predictive power to evaluate euploid (0.71 AUC) and aneuploid (0.70 AUC) embryos was achieved, indicating a potential use as a non-invasive approach for embryo ranking and selection. Trial registration number Not applicable

P-078 The role of male factor accompanying PGT-A as possible complementary strategies to control miscarriage

Abstract Study question Considering maternal influence or management of male factor, should only PGT-A be indicated to reduce miscarriage? Summary answer The finest cost-effective treatment for each patient to improve ART success, is a good, tailored, and individualized indication for choosing the best IVF technique. What is known already Advanced maternal age is related with fertility decline, oocyte quality and the increased risk of aneuploid embryos. Aneuploid embryos lead to a reduced implantation potential and higher miscarriage rate. PGT-A is a screening method to determine the chromosomal status of the embryos and it’s the most expanded technique used to reduce the miscarriage rates in most clinics. Common sperm capacitation techniques used in IVF laboratories could cause sperm DNA damage, leading to a smaller number of blastocysts obtained per cycle and a higher miscarriage rate. Microfluidics devices or sperm selection centrifugation-free (Swim-up modified techniques) could reduce sperm DNA fragmentation. Study design, size, duration This is a retrospective study from 2018-2020 including patients using their own oocytes fertilized by ICSI. No major differences on male factor were found between male patients. In all cycles at least one good quality blastocyst was obtained to biopsy on blastocyst stage in cycles with PGT-A or to transfer without PGT-A. Live-Birth Rate (LBR) per cycle is compared and analyzed by the t-Student and X2 test between groups. Participants/materials, setting, methods A total of 308 cycles were included in this study. Three groups of patients were formed: Group 1: 76 cycles with PGT-A; Group 2: 191 cycles without PGT-A and sperm samples processed by Swim-Up technique; and Group 3: 41 cycles without PGT-A and a centrifugation free sperm selection (IO-Lix) used for ICSI. The main indication for PGT-A was maternal age. No differences between male or other female infertility factors were found between groups. Main results and the role of chance When we compare the 3 groups, no statistically significant differences were obtained in fertilization rate (79.8%; 73.5%; 72.6%; p = 0.28); development to the blastocyst stage (48.7%; 43.1%; 44.9%; p = 0.11) and pregnancy rate per transfer (48.8%; 45.6%; 52.8%; p = 0.55). It seems that PGT-A doesn’t affect the viability, or the implantation potential of the biopsied embryos and a special sperm capacitation technique is useful for daily clinical use. If we include only patients with ages >38 years old to avoid age differences, comparing Group 1 (PGT-A) and Group 3 (no-PGT-A and IO-lix) (LBR=19.7% vs 50%), we obtain statistically significant differences between both groups (p = 0.01). No differences were found between Group 2 (LBR=26.4%; p = 0.25) and the Group 1. To summarize, the female age appears to be the most important factor for IVF success and the PGT-A can help to avoid a miscarriage, however when we compare women with advanced maternal age, the LBR is increased when PGT-A is non performed, and the sperm DNA is not damaged by the capacitation technique. Why not PGT-A to be complemented with efficient sperm selection? Limitations, reasons for caution Although sperm selection techniques to decrease sperm DNA damage could reduce miscarriage rates, PGT-A is the only used technique that allows us to ensure that euploid embryos are transferred. Wider implications of the findings A better understanding of male factor in IVF treatments can reduce miscarriage. Male factor assessment is a cost-effective technique to improve LBR in those patients without a clear indication for PGT-A, (advanced maternal age). Patient must be studied individually, and PGT-A shouldn’t be used indiscriminately to ensure a healthy new-born. Trial registration number Not Applicable

P-201 Identifying patients benefiting from delayed-matured oocytes insemination

Abstract Study question Are there any predictive factors supporting the decision of inseminating delayed-matured oocytes? Summary answer Patients with ≤59% mature oocytes at retrieval and/or Anti-Mullerian-Hormone (AMH) >2.52 ng/ml have increased chances of obtaining a euploid embryo from delayed-matured oocytes. What is known already Approximately 15% of oocytes retrieved after ovarian stimulation are immature, at metaphase I (MI) or germinal-vesicle (GV) stages at the time of oocyte denudation. Performing IVM in those oocytes could permit an increase on the number of usable embryos. Nevertheless, the utility of delayed-matured oocytes varies greatly among IVF laboratories with relatively low success rates, hence its practice in daily routine might be counter-productive. Determining which patient population could benefit from such strategy is valuable thereof to the clinical practice. Moreover, data comparing euploid rates of embryos derived from delayed-matured oocytes with its mature sibling oocytes are needed. Study design, size, duration This observational study was performed at ART Fertility Clinics, Abu Dhabi, UAE, between January 2019 and June 2021. A total of 5454 cumulus oocytes complexes (COC) were retrieved from 469 ovarian stimulation cycles. Out of the retrieved COCs, 3473 oocytes were immediate at metaphase II (MII-D0), and 915 were delayed-metaphase II oocytes (MII-D1). Participants/materials, setting, methods Patients with primary and secondary infertility undergoing Controlled ovarian stimulation (COS) in standardized protocols for IVF/ICSI treatment were included. Ovum pick up performed 34-36h post final oocyte maturation trigger shot (TS). Insemination was done 39-41h post TS for the MII-D0, while MII-D1 ICSI was performed 63-68h post TS. All cycles were planned for Preimplantation Genetic Testing for Aneuploidies (PGT-A) at blastocyst stage using Next Generation Sequencing (NGS). Main results and the role of chance Fertilization rates significantly differed between MII-D0 and MII-D1 oocytes (69.54% vs 55.96%, p < 0.001, respectively). Blastocyst utilization rates were significantly higher in MII-D0 group compared to MII-D1 group (59.47% vs 18.52%, p < 0.001). However, no difference was observed in the rate of euploid blastocysts between MII-D0 and MII-D1 (46.3% vs 39.0%, p = 0.163). As identified by univariant logistic regression analysis, the following parameters augmented the chances of obtaining at least 1 blastocyst for biopsy when MII-D1 were injected: AMH (OR 1.15, p < 0.001), number of COCs collected (OR: 1.03, P = 0.005), maturation rate on day0 (OR: 0.19, P = 0.001). When the multivariant analysis model was applied, AMH and maturation rate on day0 remained significant factors predicting the success of inseminating delayed-matured oocytes (OR:1.15, [CI:1.00-1.32], p = 0.045); OR:0.06, [CI:0.03-0.31], p < 0.001, respectively), with cut off values of AMH >2.52 ng/ml and maturity rate of ≤ 59%, being identified by ROC analysis. Limitations, reasons for caution ICSI of MII-D1 was performed with the fresh or frozen sperm samples from the previous day. Exact timing of polar body extrusion of delayed-matured MI/GV was not identified. Wider implications of the findings The results of this study might provide guidance to the IVF laboratories for targeting the patient population who would benefit from MII-D1 ICSI without adhering to unnecessary costs and workload. Trial registration number not applicable