Embryo Grade Research Articles

A comparison of culture and cooling for the short term preservation of in vivo derived dromedary camel embryos of varying morphological quality

Despite recent advancements in the cryopreservation of dromedary camel embryos, widespread application of the technique is still limited by the need for specialised vitrification equipment and supplies. Temporary, liquid-phase embryo storage methods provide a useful tool for short-term preservation of camel embryos.In the current study, we compared the use of in vitro embryo culture with cold liquid storage in order to maintain both high- (Grade 1- Excellent and 2-Good) and low- (Grade 3- Moderate and 4-Poor) morphological grade Day-7 dromedary camel embryos in vitro for up to 3 days. Embryos were either cooled and placed in Hams-F10 medium supplemented with HEPES and 10% FBS and then kept at 4 °C; or placed in Hams-F10 supplemented with sodium bicarbonate and 10% FBS and then cultured in a humidified atmosphere of 6% CO2 at 37 °C before being assessed for viability at 24 h. In high-morphological grade embryos, both cold storage and culture supported 100% viability (maintenance of normal morphology) over this period (Cooled n = 22, Cultured n = 20). In low-morphological grade embryos, culture supported higher viability (16/18, 88.9%) than did cooling (4/18, 22.2%). We then evaluated the effect of up to 3 days of cold storage or culture on embryo morphological grade, diameter, and developmental competence following embryo transfer. High-grade embryos were divided between culture and cold storage; low-grade embryos were evaluated only after culture. Over 3 days of culture, both high- and low-grade embryos tended to either maintain or improve upon their initial morphological score (P < 0.05) and increased in diameter (P < 0.001). Embryos subjected to cooling tended to have reduced morphological scores by 48 h of storage and decreased in diameter by 72 h (P < 0.05). No significant influence of storage method (cooling vs. culture), duration (24–72 h), or embryo grade (high vs low) was observed on pregnancy establishment at Day-60 (22.2%–57.2% pregnancy rates for all treatments). Overall, rates of pregnancy establishment were similar for transferred cultured (n = 45) and cooled (n = 45) embryos (pregnancy rates at Day 18, 48% vs 51.1%; at Day 60, 37.7% vs 37.7%). Rates of embryonic loss also were similar (22.7% vs 26%).In conclusion, whilst similar rates of pregnancy and pregnancy loss were observed following the transfer of both cooled and cultured embryos held in vitro for up to 3 days, amongst the two methods, only embryo culture appears to provide a means of effectively preserving Day- 7 dromedary camel embryos with reduced morphological values in vitro. Considering these embryos appear to show poor tolerance to the cooling procedure and are unlikely candidates for vitrification, embryo culture may provide an effective method for deriving pregnancies from low-morphological grade embryos when immediate transfer is not possible on the day of flushing.

P-162 Numeric Embryo Quality Scoring Index (NEQsi) is Predictive of IVF Cycle Outcome at any Endometrial Thickness

Abstract Study question Is it possible to use NEQsi to determine the statistical contributions of embryo quality and endometrial thickness (ET) to IVF cycle outcome? Summary answer Yes. Analytics revealed that the likelihood of pregnancy is reliably predicted by the quality of the embryo at any endometrial thickness. What is known already ET is widely utilized to assess the quality of endometrial preparation in IVF cycles. The connection between endometrial thickness and cycle outcome is divided in the literature; studies report correlation between ET and cycle outcome while others find no link between the two. Regardless, the statistical interactions between ET and embryo quality remain understudied. NEQsi was recently developed as a method for converting Gardner embryo grades to interval variables for use in statistical modeling. The combination of NEQsi scores and endometrial thickness measurements provides an opportunity to assess the contributions of embryo quality and ET on IVF cycle outcomes. Study design, size, duration We conducted a retrospective analysis including IVF cycles conducted at a Canadian multi-clinician fertility care centre (n = 1623). Data were acquired from a chart review of deidentified patient files from 2014 – 2021. Participants/materials, setting, methods Participants were all patients who underwent single embryo transfer and had endometrial thickness measurements, Gardner embryo grading, and ultrasound based endometrial receptivity testing (usER) as part of their care program. No interventions were made. Gardner embryo grades on file were created using EmbryoScope (Vitrolife, Göteborg Sweden) and converted to NEQsi scores. We conducted multivariate statistical modelling analysis to determine the combined contribution of embryo grade and endometrial thickness on IVF cycle outcome. Main results and the role of chance NEQsi scores ranged from 3 – 11 (mean 8.32 ± 1.78 SD). Endometrial thicknesses ranged from 3.1 mm to 20.2 mm (mean 9.69 ± 2.23 SD). A positive linear relationship was observed between NEQsi score and probability of pregnancy. Multivariate logistical regression determined that embryo grade was a highly significant predictor of cycle outcome (p &amp;lt; 0.0001). Endometrial thickness was not a statistically significant predictor of cycle outcome (p = 0.585). Limitations, reasons for caution We acknowledge that the single centre design is a limitation of this study. Future investigation with more cycles and multiple centres will strengthen the study and interpretation of the data. Wider implications of the findings The application of NEQsi was simple and this work confirms the utility of the NEQsi in conducting statistical analyses with embryo quality as a covariate. In addition, our findings imply that cancellation of embryo transfers based on ET measurements may not be warranted. Trial registration number Not applicable

P-144 An artificial intelligence algorithm demonstrates optimal performance for evaluating embryo genetic status at 120 hours post-fertilization

Abstract Study question What is the effect of time-point on performance of a non-invasive artificial intelligence (AI) algorithm for evaluating embryo genetic status? Summary answer While predictive ability was maintained across different time-points on day 5, optimal performance for ranking and selecting euploid embryos was observed at 120 hours post-fertilization. What is known already Studies have shown that it is possible to develop computer vision-based AI algorithms capable of predicting embryo ploidy status using single images of blastocyst-stage embryos. The genetic status of embryos is linked to morphokinetic development, with aneuploidy generally resulting in earlier arrest. Given the dynamic nature of embryo development, it might be expected that the time-point selected for analysis could influence AI performance. The key questions remaining to be answered are to what extent is AI analysis affected by expansion grade, and what does this mean for selection of a time-point for evaluation? Study design, size, duration 2,683 images of day 5 blastocyst-stage embryos with matched ploidy outcomes from pre-implantation genetic testing for aneuploidies (PGT-A) were provided by 10 IVF clinics in the USA, Australia, Malaysia, and India. A subset of 182 embryos had images provided at 110, 115, and 120 hour time points (GERI and EmbryoScope time lapse systems). Participants/materials, setting, methods Images were analysed by a previously developed AI algorithm which evaluates the likelihood of an embryo being euploid according to PGT-A (Diakiw et al, 2022. Hum Reprod, Jul 30;37(8):1746-1759). Evaluation was performed on embryos of each expansion grade, and at three time-points on day 5. Correlations were assessed using Chi-squared test for trend, with pair-wise comparisons conducted using Student’s t-test. Performance was evaluated using ROC-AUC, and a simulated cohort ranking analysis method. Main results and the role of chance AI scores positively correlated with expansion grade, and expansion grade likewise correlated with an increasing proportion of euploid embryos. AI scores also increased over time on day 5, consistent with continued embryo expansion. Scores for grade 4 (expanded) embryos increased more than for grade 5 (hatching) embryos (+2.2-fold and +0.8-fold, respectively), indicative of continued expansion becoming limited at later stages. Despite the valid association of AI scores with expansion grade, results showed the AI could predict euploidy even amongst embryos of the same expansion grade (ROC-AUC ranging from 0.61−0.69). While predictive ability was maintained at each time-point on day 5, ROC-AUC values were highest at 120 hours (0.64, 0.64, and 0.68 for 110, 115, and 120 hours, respectively). Simulated cohort ranking analyses also showed that the AI performed best at 120 hours, selecting a euploid embryo as the top-ranked embryo in 77.1% of patient cohorts (71.4%, 75.1%, and 77.1% for 110, 115, and 120 hours, respectively). These results suggest that regardless of expansion grade, all embryos should be assessed at the same time-point on day 5, preferably closer to the 120 hour time-point. Limitations, reasons for caution The time-point analyses were conducted on a relatively small dataset, and therefore findings should be validated on a larger dataset including embryos of other expansion grades. The analysis was limited to day 5 post-fertilization, however it would be interesting to extend the study to embryos on day 6 also. Wider implications of the findings These results suggest that the AI is providing additional information regarding embryo genetic status, over and above that provided by known morphological parameters. The dynamic nature of AI score related to expansion is of interest as it relates to the optimal time-point for conducting analyses for selection of euploid embryos. Trial registration number Not applicable

O-004 Male and female blastocysts display differences in development such that embryos assigned an identical morphological grade may have differing viability dependent on their sex

Abstract Study question Embryo selection methods based upon morphological or morphokinetic evaluation assume that male and female embryos have identical rates of preimplantation development, but is this true? Summary answer The distribution of trophectoderm grades differs for male and female blastocysts. Furthermore, male and female embryos of identical grade may have different probabilities of viability. What is known already Previous studies have suggested that male and female embryos may have subtle differences in their rates of preimplantation development. However, this possibility remains controversial. Apart from being of scientific interest, the question of whether the sex of an embryo can affect its growth trajectory is of clinical importance. Morphological grading, the primary method used by most IVF clinics when deciding which embryo to prioritise for transfer, assumes that developmental rates are independent of sex. Similarly, morphokinetic strategies for embryo evaluation, using data gathered from time-lapse incubators, are likely to be compromised if male and female embryos have differing developmental behaviour. Study design, size, duration 1,241 blastocysts underwent PGT-A and were shown to be euploid. Chromosome analysis also revealed the sex of the embryos, although this was not disclosed to patients. Standard morphological grading was carried out blindly with respect to the sex of the embryo. Information on clinical outcomes following embryo transfer was available for a subset of 336 embryos. Data was evaluated using various statistical methods to reveal any associations between embryo sex, morphology and clinical outcome. Participants/materials, setting, methods Embryos included in this study were derived from patients undergoing routine IVF with PGT-A. The only embryos excluded were those derived from patients carrying a monogenic disease mutation or a chromosome rearrangement. Embryos underwent PGT-A at the blastocyst stage on either day-5 or day-6, using a highly validated method. The euploid embryos were divided into male and female groups and blastocyst morphological grades were considered with respect to rates of implantation, miscarriage and ongoing pregnancy/birth. Main results and the role of chance The proportion of embryos biopsied on day-5 versus day-6 was identical for males and females (68% day-5 for both). No difference was observed in blastocyst expansion or inner cell mass grading on day-5. However, a highly significant difference was noted in the distribution of trophectoderm grades (A, B, C, D according to the system of Gardner and Schoolcraft, 1999) (P &amp;lt; 0.0001). This was characterised by a disproportionate representation of the highest grade amongst male embryos. 21.6% of male blastocysts had trophectoderm graded ‘A’, compared to 14.9% of females. Interestingly, euploid male embryos with a ‘B’ grade trophectoderm were associated with significantly higher implantation rates than females of the same morphological grade (82.1% vs. 58.7%; P = 0.0002), but the males of this grade also experienced a higher incidence of biochemical losses (16.7% vs. 4.2%; P = 0.017). As a group, male embryos do not have greater viability than female embryos, which implies that the greater proportion of high trophectoderm grades amongst these embryos is not an indicator of superior potential. In turn, this suggests that different criteria should be used for grading trophectoderm in males and females. Limitations, reasons for caution It may be desirable to introduce parallel morphological grading systems, one for male embryos and another for female. However, this would only be applicable in cycles involving PGT-A. The observation that embryos of equal grade can have different outcomes depending on their sex should be confirmed in a prospective study. Wider implications of the findings These results suggest the development of sex-specific morphological grading strategies might provide more reliable insights into embryonic potential, increasing the likelihood of selecting a viable embryo for transfer. Consideration of differences in the development of male and female embryos will also be important when developing morphokinetic algorithms for embryo selection. Trial registration number Not applicable

P-667 The attenuated response to elevated serum progesterone levels on pregnancy outcomes of fresh IVF/ICSI cycles in older patients

Abstract Study question Whether there are different impacts of serum progesterone (P) levels on the day of trigger on pregnancy outcomes in patients of different age groups. Summary answer Our results suggest that the pregnancy outcome was less sensitive to progesterone elevation in older patients. What is known already It is now generally believed that the P elevation at the late follicular phase reduces the pregnancy rate by opening the implantation window prematurely and disrupt the embryo-endometrial synchrony. Previous studies further investigated the effect of developmental stages of embryo, number of oocytes retrieved, cause of infertility and different stimulation regimens on the association of P level and the likelihood of pregnancy. However, the study of maternal age on such association is scarce. A previous study found that LH receptor (LHCGR) and progesterone receptor (PGR) were up regulated in advanced age group. Study design, size, duration This was a non-interventional, retrospective cohort study of patients undergoing routine practice in a university-based fertility center between 2015 and 2021. One thousand five hundred and seventeen IVF/ICSI cycles among 1305 patients were included in the analysis. Participants/materials, setting, methods Fresh ET using autologous oocytes with serum P level measured on the day of hCG administration were included. Exclusion criteria include cycles without P measurement, using frozen oocytes, PGT cycles, freeze-all cycles, oocyte donation and no embryo transfer cycles. The patient age was divided into 3 groups: group 1 included patients at age 35 or below, group 2 included patients at age 36 to 39, group 3 included those at 40 years old or above. Main results and the role of chance We performed logistic regression analysis for each group to determine the contributing factors to the live birth outcome. In group 1, age, serum P level, number of usable embryos, embryo grading and endometrial thickness were found to be significantly associated with live birth. In group 2, age and number of usable embryos were significantly associated with live birth. However, total dose of gonadotropin was found to be the sole contributing factor to live birth in group 3. We also divided the P levels into low (≤0.50 ng/mL), median (0.51-1.00 ng/mL) and high (&amp;gt;1.00 ng/mL) and compared the live birth rate in each age groups. The live birth rate was found to be significantly reduced with increasing P levels (40.3% vs. 35.5% vs. 23.6%, p = 0.005). No significant difference in live birth rate between different P levels was found in age group 2 and 3. Limitations, reasons for caution The limitation of the study is its retrospective nature which may have led to bias in the interpretation of the data. There were also significantly higher proportion of cleavage-stage embryo to blastocyst transfer in the older group. Wider implications of the findings The negative association of premature progesterone rise at pre-ovulatory phase to the IVF fresh ET outcome was more prominent for younger patients. This may be one of the contributing factors that accounts for the great heterogeneity in the cut-off of P and its predicting power in different studies. Trial registration number Not applicable

P-681 Embryo utilisation rate and blastocyst formation rate correlate negatively with the oocyte yield and positively with live birth rate: an analysis from HFEA’s national database

Abstract Study question Do embryo utilisation rate (EUR) and blastocyst formation rate (BFR) correlate with oocyte yield and livebirth rate (LBR) in fresh IVF/ ICSI cycles? Summary answer EUR and BFR correlate positively with livebirth rate (LBR) but decline with increasing number of oocytes to a nadir when the fresh cycle LBRs plateaus. What is known already LBR in fresh IVF cycles plateaus once a certain number of oocytes are retrieved. While impaired endometrial receptivity with high ovarian response appears to be an underlying cause, other factors, such as decreasing competency of oocytes or embryos cannot be ruled out. Cytoplasmic dysmorphism of oocytes and chromosomal disorders have been shown to rise with increasing number of oocytes. Consequently, cumulative livebirth rate per oocytes has been reported to decline with increasing oocyte yield. When obvious male factor is excluded, the competency of oocytes can be reflected in the embryo grading. Study design, size, duration Retrospective analysis of 8015 fresh IVF/ ICSI cycles that met the inclusion criteria from the national database published by Human Fertilisation and Embryology Authority (HFEA) in the United Kingdom. We analysed 2 years of published data between January 2015 and December 2016. Participants/materials, setting, methods Population included couples at their first IVF treatment with single fresh blastocyst transfer, due to tubal or unexplained infertility among women aged &amp;lt;40 years. Only cycles that produced at least 1 transferable blastocyst were included to determine the blastocyst formation rate. Inclusion criteria were designed to minimise other co-factors that may influence embryo quality. Linear regression analysis was done to find the correlation between EUR/ BFR and oocyte yield; multiple regression to predict livebirth Main results and the role of chance EUR (correlation coefficient = -0.156605, p &amp;lt; 0.0001) and transferable BFR correlation coefficient = -0.156605, p &amp;lt; 0.0001) declined with increasing number of oocytes. The findings were no different whether IVF or ICSI was performed. Both EUR (OR 1.002, CI 1.001- 1,004) and (transferable) BFR (OR 1.005, CI 1.003-1.006) correlated positively with LBR (p &amp;lt; 0.0001, for both), after adjusting for other confounders including age and method of insemination. Both (transferable) BFR and EUR declined with the increasing number of oocytes falling to a nadir when around 8 oocytes were retrieved then remained static with higher oocyte yield. LBR in fresh cycles also reaches its peak at around 8 oocytes then plateaus. Limitations, reasons for caution Information on patient’s BMI or stimulation dose is not available in the HFEA database. However, it might have little impact in our findings given large sample size. Cycles with no blastocyst formation through extended culture were not included, as this intention to treat information was not available Wider implications of the findings Declining proportion of competent embryos may result in flattening of LBR graph in fresh cycles after certain number of oocytes retrieved. Increasing absolute number of competent embryos fails to boost fresh cycle LBR after a saturation point, We have to improve embryo selection than increasing ovarian stimulationto improve fresh LBR Trial registration number NA

P-522 Embryo transfer procedural parameters do not predict IVF cycle outcome

Abstract Study question Do embryo transfer (ET) procedural parameters affect the clinical outcome, in terms of positive human chorionic gonadotropin (hCG), clinical pregnancy and live-birth? Summary answer Observation of ET procedural parameters, either as single parameters or combined in a model, does not seem to have a predictive value on cycle outcome. What is known already It has been reported that several ET procedural parameters of technical nature may influence IVF outcome. Observational studies on variables during and post ET from mucus presence to catheter reload, have been published and data hitherto is conflicting leaving practitioners confounded. It is essential to evaluate the effect of the reported ET performance parameters, clarifying the degree of effect on a singular or combinatorial level. This will assist practitioners in recording and analysing strictly factors that hold high-significance predictive value, instead of redundantly recording moot parameters that may be unfit to serve as key performance indicators post ET. Study design, size, duration A prospective observational study was performed between 3/2018 and 9/2022 in a single IVF center. ET procedures for a total of 1417 women were assessed. The performance parameters studied and associated with clinical outcomes were the following: time of ET, presence of cervical mucus in the catheter, presence of blood, catheter reload, employment of tenaculum and stylet, resistance during transfer, ease of transfer according to the practitioner and discomfort as experienced by the patient. Participants/materials, setting, methods Only women of normal endometrial and uterine anatomy and function were included. Genetic and endocrine abnormalities, azoospermia and PGT cycles were excluded. Embryo grading was performed according to Veeck’s and Gardner’s system depending on developmental stage. All ETs were performed employing the same type of soft catheter. The clinical outcomes evaluated were positive hCG, clinical pregnancy, and live-birth. Associations were evaluated on a singular or combinatorial level between the ET performance parameters and clinical outcomes. Main results and the role of chance A univariate logistic regression was employed to evaluate the possible effect of each of the ET procedural parameters. None of the ET parameters presented with statistical significance. The employment of ultrasound, the number, and the quality of transferred embryos, maternal age as well as the physician and embryologist performing the transfer were evaluated as covariates in a univariate regression model. Number (p = 0.01) and quality of transferred embryos (p = 0.003), as well as maternal age (p = 0.004) presented with statistical significance. The multivariate approach yielded similar results. To evaluate the possibility that interactions between the ET procedural parameters could affect cycle outcome a model was created evaluating all possible interactions and combination of the ET parameters. To adjust for possible multiple comparison bias, the Bonferroni correction was employed. Only the presence of mucus along with significant resistance seemed to present with statistical significance (p &amp;lt; 0.0001). However, when adjusting for the covariates the combination of cervical mucus and significant resistance did not provide a statistically significant effect on clinical outcomes and thus cannot be considered as an independent predictor. When subgrouping only for women below the age of 35 with at least one top quality embryo, no statistically significant association was observed. Limitations, reasons for caution The sample size could be a reason for caution when interpreting the results of the present study as a larger number of women presenting with each combination may be required to reach the threshold of statistical significance. The single-center nature of the study may present as another limitation. Wider implications of the findings Data indicates that ET procedural parameters hold no predictive value singularly or combinatorially. The only robust predictive factors are embryo quality, number, and maternal age. While further studies are required to cement this finding, it may be the case that the time-consuming recording and analysis of such parameters is redundant. Trial registration number Not applicable

O-121 Temporal development of scores from a time-lapse based artificial intelligence provides no additional benefit compared to the latest score

Abstract Study question Does the temporal development of scores from an artificial intelligence (AI) based embryo selection model provide additional predictive power compared to the latest score? Summary answer Earlier predictions provide no additional predictive power given the latest prediction. Knowing the development of AI scores is thus not beneficial for embryo selection. What is known already Traditionally, embryo grading takes into consideration the final morphology score together with the history of embryo development events. Recent publications have shown that time-lapse based AI models trained on clinical outcomes can automatically rank embryos by the likelihood of implantation that equal or surpass traditional methods, without the necessity for manual evaluation. AI-based methods based on time-lapse data may account for the impact of the full temporal development on likelihood of implantation. In this study, we examine if the addition of previous scores adds to the performance of an embryo selection model. Study design, size, duration A retrospective multicenter study of transferred embryos (n = 2422) with known implantation data (KID) between 2012-2020 from 21 international clinics. Embryos were cultured in EmbryoScope time-lapse incubators for at least 5 days. Both single and multi-embryo transfers in fresh and warmed cycles were included. Embryos were classified as positive (KIDp) or negative (KIDn) by the presence of fetal heartbeat. Implantation likelihood was evaluated at cleavage- and blastocyst stages by a 3D CNN model; iDAScore v2.0. Participants/materials, setting, methods A kernel-based conditional independence test was used to evaluate if a score at x hours post insemination (hpi) provide extra information compared to a score at y hpi. The test estimates the likelihood that a prediction after x hpi does not provide any additional information with regards to predicting KIDp when a prediction after y hpi is available. These likelihoods (p-values) were Bonferroni corrected to adjust for multiple comparisons. Main results and the role of chance KIDp predictions were computed for all embryos at 40, 44, 64, 68, 112, and 116 hpi corresponding to early and median time of transfer for day 2, 3, and 5. For all time points, the conditional independence test showed that there was no added information from knowing predictions at an earlier time point (p &amp;gt; 0.21). Similarly, it was tested if a known later prediction will improve model performance for an earlier prediction. This was always the case (p &amp;lt; 0.01) except for predictions close in time (68 vs 64 hpi; p = 1.0) and (44 vs 40 hpi; p = 1.0). This may reflect that there was little additional embryo development information during a 4-hour period at this development stage. The above shows that knowledge of the temporal score development does not improve model performance compared to using latest available score. This means that an AI model based on time-lapse videos likely considers earlier embryo development events in the context of the entire development history. Limitations, reasons for caution This study is a retrospective study that only looks at AI based on time-lapse videos. There is a potential bias as the embryos were likely of higher quality having been chosen for transfer. It is possible that the results do not generalize to simpler non-3D CNNs. Wider implications of the findings Knowing the temporal score development can introduce subjectivity that might lower implantation rates as the earlier scores were shown to not provide any significant prediction improvement. Trial registration number not applicable

P-309 High-viscosity oil overlay provides less protection against media evaporation in extended dry incubation conditions compared with conventional light oil but a reduced heat loss

Abstract Study question Does high-viscosity oil (HVO) improve stability on osmolality, temperature, pH, Na+ concentration, and mouse embryo development compared with conventional light oil (CLO)? Summary answer In dry incubation condition, osmolality and Na+ concentration were significantly higher in embryo culture media overlaid with either oils, but HVO was worst than CLO. What is known already Suboptimal in-vitro culture conditions affect embryo quality and viability. Overlaying culture media with oil is used to maintain a stable optimal culture environment, including reduction of evaporation. Despite dry incubators are known to increase evaporation, they have become more common, especially with the widespread use of time-lapse imaging. The risk of excessive evaporation with the consequent impact on osmolality is even more significant with the promoted usage of undisturbed extended embryo culturing. Novel products (HVO) therefore been developed and recently commercialised with the aim of reducing such effect. Nonetheless, no independent data are available to determine their superiority over CLO. Study design, size, duration Culture media dishes overlaid with CLO or HVO were incubated in dry and humidified conditions while mimicking extended culture conditions in four independent experimental groups. Changes in osmolality, pH and Na+ concentrations over three days were measured. Lipid peroxidation and mouse embryo development were recorded to assess toxicity. Additionally, the ability of different oils to maintain the temperature in the culture drops when culture dishes were removed from the incubator was compared. Participants/materials, setting, methods Standard embryo culture media drops overlayered with HVO or CLO were incubated in two equally calibrated, identical incubators providing dry or humid conditions. Osmolality (Day1, D3, D5 measured by freezing-point depression osmometer), temperature drop rate at room temperature (T-type thermocouple sensor probe), pH and [Na+] (hand-held blood gas analyser), mouse embryo assay (MEA, 75 embryos from naturally mated CD1 female mice (N = 5), and lipid peroxidation end-product levels (MDA, TBARS assay) were compared among groups. Main results and the role of chance The osmolality of the media incubated in humidified conditions did not show a significant change throughout the 5-day experiment, regardless of the type of oil used. However, in dry conditions, a significant daily increase in osmolality was evident; changes were more prominent under HVO (D1, 278.13 ± 0.82; D3, 286.00 ± 1.51; D5 289.86 ± 1.17 mOsm/kg H2O ± SEM. p ≤ 0.001). The osmolality of the media overlaid by the same type of oil raised between different incubators, and reached a significantly higher value on D5. [Na+] values were consistent with the osmolality changes. The recovery time of the temperature on the heated stage to 37 °C was similar in CLO- and HLO-overlaid media (1:07 ± 1:02 min and 2:00 ± 0.7 min, respectively). The temperature drop rate after media were kept at ambient temperature was similar in the first 4 min. By the end, the temperature difference reached a significance between CLO and HVO overlays (32.4 ± 0.4 °C and 32.7 ± 0.4 °C respectively, p = 0.045). No significant differences were found in pH, lipid peroxidation or mouse embryo development between the study experimental groups. Limitations, reasons for caution The embryos for MEA were obtained without hyperstimulation, resulting in a small sample size for the study. The exact timing of fertilisation was impossible to detect after mice mating, which caused inconsistency in embryo grading. Wider implications of the findings Dry incubators may determine suboptimal embryo culture conditions. A newly developed heavy oil proved to be inferior to conventional light oil in maintaining osmolality stable. Further research should be carried out in engineering systems that limit culture media evaporation or support undisturbed embryo culture in humidified conditions. Trial registration number not applicable

P-230 Does laser-assisted hatching improve pregnancy rates : a retrospective analysis

Abstract Study question Is there any effect of laser-assisted zona drilling in improving pregnancy outcomes in women undergoing vitrified-warmed embryo transfers? Summary answer Our data suggests that there is evidence of possible improvement in pregnancy rates with laser-assisted hatching of embryos among women &amp;gt;40 years of age. What is known already Mammalian embryo is surrounded by a glycoprotein shell called zona pellucida. The embryo must hatch out of this shell for it to implant in the uterus. However, certain factors such as sub-optimal in vitro culture conditions, advanced maternal age, zona-hardening due to vitrification and warming, among others, negatively impact the ability of the embryo to escape from the matrix surrounding it. Laser-assisted hatching is the safest and easiest technique available to overcome the problem of hatching by artificially disrupting the zona, though there isn’t sufficient data available on whether this has any actual benefit or is just a fancy add-on. Study design, size, duration Retrospective cohort study comparing embryo transfer results of 88 women whose embryos underwent LAH prior to transfer with that of a control group of women. Data was collected from embryo transfers that took place between March 2021 to December 2022. Participants/materials, setting, methods Beta-hCG results of women (N = 88) whose embryos underwent LAH prior to transfer was compared with the results of transfer without LAH in women (N = 88) of similar age and clinical history. Patient age, clinical indications and embryo grades were comparable across both groups. LAH was performed immediately post-warming of blastocysts using a 1.48 µm diode to drill the zona at a point opposite the ICM. Data was analyzed using independent t test and chi square test. Main results and the role of chance Women from both test and control groups were further stratified into the following sub-groups based on age – A (25-29 years), B (30-34 years), C (35-39) and D ( &amp;gt;40 years). Beta-hCG results were assessed and was found to be lower in sub-groups A (57.1% vs. 78.5%) and B (46.4% vs. 67.7%) of the test group as compared to control. It was marginally higher in test group C as compared to that of control (50% vs. 47.3%). However, we found an increase in pregnancy outcomes of test sub-group D in comparison to control (83.3% vs. 20%). Limitations, reasons for caution This is a retrospective, preliminary study to assess initial results of effect of LAH on vitrified-warmed embryo transfer outcomes. A prospective study with a larger sample size is needed to confirm our results in both frozen and fresh embryos. Wider implications of the findings Our results indicate that laser-assisted hatching does not provide any advantage except possibly in older women above the age of 40 years. Thus we can select the exact group of patients who would benefit with this add-on and offer it to them routinely to improve their pregnancy rates. Trial registration number Not applicable

P-201 Developmental analysis of blastocysts with blastomere exclusion during compaction and its relationship to pregnancy outcomes and preimplantation genetic testing for aneuploidy results

Abstract Study question What effects do blastocysts with blastomere exclusion during compaction have on embryo morphokinetics, morphology grade, genetic testing for aneuploidy (PGT-A) results, and pregnancy outcomes? Summary answer Blastocysts with blastomere exclusion leads to delay blastulation and poor morphology grade, but does not affect pregnancy outcomes and euploidy in PGT-A. What is known already It has been reported that aneuploid blastomeres are excluded during compaction, which is a mechanism for chromosome repair in blastocysts through the exclusion of aneuploid cells.Furthermore, in this phenomenon, it is known that the first cleavage of embryos is often abnormal, in which three or more cells originate from a single cell division event.However, this phenomenon has not been reported in the evaluation of embryo morphokinetics, artificial intelligence (AI) scores, or PGT-A results. Study design, size, duration In this retrospective study, we analyzed clinical medical reports at the Takahashi Women’s Clinic in Japan. We included 934 blastocysts cultured in a time-lapse incubator (EmbryoScope) after intracytoplasmic sperm injection (ICSI) from January 2018 to June 2021. Participants/materials, setting, methods Blastocysts cultured using a time-lapse incubator (EmbryoScope) after ICSI were classified into a control group (N = 515) with blastomere compaction or a BE group (N = 419) with blastomere exclusion during compaction. The first type of cleavage, AI score (IDAScore), embryo morphokinetics, and single frozen-thawed blastocyst transfer outcome were evaluated.Logistic regression analysis was performed to compare the two groups, considering patient age, blastocyst grade (Gardner classification), culture days, body mass index, and basal anti-Müllerian hormone level. Main results and the role of chance Comparing the control and BE groups, the first cleavage abnormality rate was 16.9% vs. 67.3% (P &amp;lt; 0.001), the percentage with good embryo grade (Gardner criteria ≧BB) was 93.4% vs. 61.0% (P &amp;lt; 0.001), and the mean score by AI analysis was 8.3 ± 1.3 vs. 6.4 ± 1.9 (P &amp;lt; 0.001), respectively. The BE group had a higher rate of abnormal cleavage and a significantly worse embryo grade and AI score than the control group.In the evaluation of embryo morphokinetics, the time to morula was 84.2 ± 7.0 h vs. 89.0 ± 8.5 h (P &amp;lt; 0.05) and time to blastocyst was 105.9 ± 9.4 h vs. 114.7 ± 11.4 h (P &amp;lt; 0.05). The BE group significantly delayed blastulation compared to the control group.The euploidy, mosaicism, and aneuploidy rates of PGT-A were 13.6% (9/66), 9.1% (6/66), and 77.3% (51/66) in the control group vs. 13.2% (5/38), 13.2% (5/38), and 73.7% (28/38) in the BE group, respectively. There was no significant difference in the PGT-A results between the two groups. Furthermore, the single-blastocyst transfer pregnancy rates (gestational sac with heart activity) in the control and BE groups were 47.1% (147/312) and 32.6% (60/184), respectively. Pregnancy rate was lower in the BE group, but was not significantly different from that of the control group. Limitations, reasons for caution The study was conducted at a single in vitro fertilization center. Embryo transfer results were based on ongoing pregnancies, while live birth data for all pregnancies are not yet available. Wider implications of the findings Although the BE group was graded worse, statistical analysis considering the grade showed no significant difference in PGT-A and pregnancy rates between the two groups.These results suggest that blastocysts with blastomere exclusion can be useful as transferable embryos if they grow to a transferable grade. Trial registration number not applicable

O-063 Accurate mitochondrial DNA quantification clarifies the clinical value of measuring mtDNA in trophectoderm biopsy specimens

Abstract Study question Can accurate mitochondrial DNA (mtDNA) quantification of trophectoderm (TE) biopsy specimens provide insights into the biology and viability of blastocyst-stage human embryos? Summary answer mtDNA quantity in TE cells is correlated with embryo morphology and shows alterations associated with aneuploidy. However, measurement does not significantly improve embryo viability assessment. What is known already Mitochondria are essential organelles, responsible for producing ATP. Changes in the amount of mtDNA in blastocysts biopsy specimens have been reported in association with embryo implantation potential, leading to proposals that mtDNA quantification might serve as a useful biomarker of embryo viability. However, results from clinical studies to explore this possibility have yielded contradictory data, due in part to deficiencies of the molecular methods used for mtDNA measurement. We sought to clarify what quantification of mtDNA can tell us about embryo biology and viability by developing and applying a method that we believe to be the most accurate ever devised. Study design, size, duration This study involved the analysis of samples collected during the course of routine preimplantation genetic testing for aneuploidy (PGT-A). The IVF treatments and embryo biopsies were undertaken at two different clinics, while chromosomal analyses were carried out at a single reference laboratory. Mitochondrial data was subsequently analysed in a university setting. The embryos analysed were derived from a broad population of patients referred for PGT-A (average age 38.7 years; range 25-47). Participants/materials, setting, methods 651 blastocysts from 133 couples underwent trophectoderm biopsy on day-5 or day-6. The specimens were analysed using a highly validated real-time PCR method, which was used to quantify three distinct sites in the mtDNA and 198 loci in the nuclear genome. The measurement of multiple independent loci provided outstanding sensitivity and accuracy. The nuclear loci were used to normalise the mtDNA data, adjusting for differences in the number of cells in the biopsy specimens. Main results and the role of chance The method developed displayed extraordinary sensitivity and accuracy when quantifying mtDNA. Lower mtDNA quantities were associated with day-6 biopsy (p &amp;lt; 0.0001), extent of blastocyst expansion (p &amp;lt; 0.0001), and superior TE morphology, although the latter was not statistically significant (p = 0.09). mtDNA levels were higher in aneuploid embryos (p &amp;lt; 0.0001), independent on patient age. However, the difference was not sufficient to be considered diagnostic. There was no correlation between mtDNA level and the chances of blastocyst implantation in this dataset. Lower mtDNA levels, previously reported to be associated with higher probabilities of embryo implantation, were most often observed in embryos of excellent morphological grade and likely reflect the increased TE cell numbers of such embryos. Little if any mtDNA replication occurs during preimplantation development and consequently the mtDNA content is divided amongst an ever-growing number of cells, meaning less mtDNA per cell. In this context, mtDNA quantification of blastocyst biopsy specimens provides a highly sensitive measure of TE cellularity, but probably provides little additional benefit for embryo selection beyond conventional morphological grading. However, the fact that higher mtDNA quantities were observed in aneuploid embryos, may indicate that subtle differences in TE cellularity exist in abnormal embryos, which are not fully captured by traditional morphological assessment. Limitations, reasons for caution Previous studies suggested that some blastocysts have greatly elevated mtDNA levels and that such embryos are not viable. In this study, only 5% of embryos were considered outliers in terms of mtDNA quantity. Unfortunately, none of these embryos were transferred, so the potential of these embryos could not be assessed. Wider implications of the findings The quantification of mtDNA in trophectoderm biopsies has sometimes been used for the prioritisation of embryos for transfer. While our results confirm existence of biologically interesting associations between mtDNA and aneuploidy, and a relationship with certain aspects of embryo morphology, measurement of mtDNA seems unlikely to significantly improve embryo selection. Trial registration number not applicable

O-099 The inner cell mass (ICM) regions can be used to predict pregnancy with a similar accuracy as the original images of the entire embryo

Abstract Study question Can clinical pregnancy be accurately predicted using only ICM and TE regions in an embryo microscopy image? Summary answer The AI model accurately predicted pregnancy using ICM segmented images with a similar accuracy as using original embryo images. What is known already Previous research suggests that ICM and TE are key factors in determining embryo grade and clinical pregnancy. Grad-CAM images on the CNN models in various literature also highlight the significance of ICM and TE regions. However, there have been no studies to compare and assess the importance of the two regions in pregnancy prediction. This study aims to test the significance of each region by evaluating the performance of models after segmenting ICM and TE regions. Study design, size, duration We performed a retrospective study of single static images of 2,555 Day 5 blastocysts from seven in vitro fertilization (IVF) clinics between June 2011 and May 2022. The images were collected from standard optical light microscopes and matched with metadata such as pregnancy outcomes. ICM and TE regions were manually labeled and examined by 5 embryologists with over 10 years of experience. Participants/materials, setting, methods We defined a positive pregnancy indication as the presence of a gestational sac (G-SAC). We built 4 CNN models using original embryo images (“Original model”), ICM segmented images (“ICM model”), TE segmented images (“TE model”) and ICM &amp; TE segmented images (“ICM &amp; TE model”). The performances were measured through 3-fold cross validation. Main results and the role of chance In 3-fold cross validation, the mean and the standard deviation of AUROCs were 0.753±0.005 for Original model, 0.733±0.003 for ICM model, 0.724±0.016 for ICM &amp; TE model and 0.690±0.011 for TE model, respectively. The DeLong test showed significant differences between the AUCs of Original model and ICM model (p = 0.01), and ICM model and ICM &amp; TE model (p = 0.0003). Positive correlations were found between prediction values with pearson correlation [Confidence interval] for Original image and ICM model (0.426 [0.343, 0.495]), Original model and ICM &amp; TE model (0.345 [0.266, 0.419]) and Original model and TE model (0.223 [0.140, 0.304]). Original model showed the highest accuracy and TE model was the least accurate as expected. However, it is noteworthy that ICM model outperformed ICM &amp; TE model. Although it is well known that TE plays a significant role in implantation, forcing the model to utilize both ICM and TE to predict pregnancy could have muddled the analysis. This study proved that pregnancy can be successfully predicted using ICM regions only as well as original embryo images. Limitations, reasons for caution This study has limitations due to its retrospective nature, using embryo images from seven IVF centers. Further study with a larger dataset is warranted to offset the differences in focus, magnification and color of embryo images. Wider implications of the findings We showed that the AI model using ICM segmented images predicted pregnancy with a similarly high accuracy as using original embryo images. However, the model using TE segmented images showed the lowest performances. Utilizing well-focused ICM images may enhance the performance of pregnancy prediction models. Trial registration number not applicable

P-214 The size of the embryo can be used as a single predictor for selecting embryo for transfer

Abstract Study question What is the performance of the blastocyst area feature compared to the state-of-art AI algorithms in the embryo ranking problem? Summary answer We study a simple embryo selection rule: the larger the better. This rule gives the same quality answers as other methods including AI. What is known already There are many existing methods for scoring embryos starting from the human executed rules, and ending with AI grading systems. Previous studies indicate systematic problems with comparing these methods. This is caused by the lack of a gold standard algorithm, i.e., the repeatable, easy and objective test that would not be susceptible to human interpretation. Common benchmarks typically lead to speeds-up in development of ML methods. We show that the embryo area, a single, interpretable feature, can be used as such a gold standard. It delivers the same quality results (aucroc = 0.66) as existing state-of-art AI algorithms. Study design, size, duration The data-set for retrospective study was collected from a single-center - Kriobank, Bialystok. The data-set includes 1550 time-lapse videos of embryos cultured in an incubator for up to 140 hours post-fertilization with no hatching. All these embryos were transferred with known implantation outcome (beta-hCG). All the videos have been recorded using the same optical magnification setting. Participants/materials, setting, methods Two methods of embryo ranking are compared: - In the first method, the score was equal to the embryo area on the last frame of time-lapse. The area was computed using an AI segmentation algorithm that was trained on hundreds of manually annotated images/frames. - In the second method, state-of-art AI algorithms were designed and trained to grade embryos directly from image data. AUC ROC was used to compare the ranking performance. Main results and the role of chance The proposed gold standard, i.e., the embryo area, requires no training process. It induces the total ranking of all embryos, which should correlate with the treatment outcome, and the way to test it is ROC AUC score. For the whole data-set available (1550 cases) ROC AUC = 0.659 (CI 0.630-0.688), which is relatively high value and compares to the one reported for state-of-art AI algorithms based on deep-learning. We have compared the area-based algorithms with state-of-art machine learning methods used in commercial solutions. In this test we have split the data-set into training (1236 cases) and test subsets (314 cases). The area-based algorithm resulted in ROC AUC = 0.657 (CI 0.597-0.717). For the deep-learning algorithm we have obtained ROC AUC = 0.659 (CI 0.599-0.719). We note that several previous studies have proven relatively high disagreement between embryologists and AI-algorithms. Thus revealing a need for standardization in this area of study, and the need of development of common grounds for tests. The above study strongly suggests that embryo area can serve as a gold standard for further comparison of the developed algorithms, and clear improvement against such gold standards shall be expected. Limitations, reasons for caution • Currently, the application of the embryo area rule is limited to devices with fixed optical magnification. • Further work is needed to compare the segmentation algorithm with manually annotated images. • Despite initial confirmation of the viability the algorithm should be verified and tested on a large, multi-centric data-set. Wider implications of the findings • While AI based tools have the highest potential of increasing the efficacy of embryo selection, there are simple methods that can support embryologists without or with very simple software. • There is a strong need for gold standards that will be further improved. Trial registration number not applicable

O-194 Blastulation timing in embryos is influenced by ploidy and is a predictor of transfer outcome

Abstract Study question Is there an association of the timing of blastulation (TE biopsy day) on embryo ploidy and pregnancy outcome after frozen embryo transfer? Summary answer Blastocysts expanded on day 5 are more likely to be euploid and result in an ongoing pregnancy after single euploid frozen embryo transfer (FET). What is known already Embryo selection for ET is commonly based on static morphological parameters for patients undergoing in vitro fertilization (IVF). Embryo quality on the day of biopsy in combination with preimplantation genetic testing (PGT) are considered important predictors for successful implantation. At present, coherent data regarding any association between the timing of blastulation with embryo ploidy and pregnancy outcomes following single FET are lacking. The aim of our study was to investigate the correlation of the culturing method and blastulation day (day 5/6/7) with embryo ploidy and outcomes of matched d5 vs d6 vs d7 FET cycles. Study design, size, duration This retrospective cohort study was performed at the CReATe Fertility Centre and its Reproductive Genetics Laboratory, between January 2019-December 2022. 29660 blastocysts from 5302 cycles (3645 patients) were analyzed using high resolution NGS PGT-A (10Mb resolution for copy number variations and 30%-70% for mosaicism). Pregnancy outcome data from 3942 single-FETs were analyzed. Average age of ovum provider was 32.9±5.8 and sperm provider 39.1±6.4. Clinical, laboratory and demographic data were obtained for statistical analysis. Participants/materials, setting, methods Embryos were allocated into four groups based on static morphological grading before biopsy on either d5 (n = 8312), d6 (n = 19183) or d7 (n = 2165): Good (&amp;gt;1/2BB), n = 5648, Average (1BB, 2BB), n = 10492, Poor (&amp;lt;1/2 BB) n = 12219 and Early blast, n = 1301. Embryo ploidy in each group and pregnancy outcomes from single euploid FET were compared between matched cycles of day 5 biopsy to days 6 and 7. Main results and the role of chance PGT-A analysis of 29660 embryos showed 53.1% were euploid, 33.9% were aneuploid and 13.0% were mosaic. There were significantly higher proportion of euploid embryos among those biopsied on d5 compared to d6 and d7 with average morphology grade (p = 0 O.R.=1.35979; p = 0.008098 O.R.=1.34442), poor (p = 0 O.R.=1.76481; p = 0 O.R.=2.73903) and early grades (p = 0 O.R.=2.71891; p = 0 O.R.=2.74271), but not among embryos with good grade (p = 0.244399 O.R.=1.07276; p = 0.182084 O.R.=1.38189). Ongoing pregnancy rates were significantly higher for d5 vs d6 biopsied embryos, for all morphology grade groups: good (p = 0.030036 O.R.=1.26598), average (p = 0 O.R.=1.80782), poor (p = 0.000303 O.R.=1.89235) and early blasts (p = 0.049501 O.R.=3.30556). D5 blasts had higher ongoing pregnancy rates compared to d7 among average and poor morphology grade as well as early blast (p = 0.000419 O.R.=8.93182, p = 0.000036 O.R.=7.29464, p = 0.010034, respectively), except among good grade embryos (p = 0.885598 O.R.=0.9028). Continuous culture of embryos in Embryoscope™ versus conventional incubator did not influence the ploidy (n = 24321; n = 4756, respectively) or the pregnancy outcome after FET (n = 3134; n = 752, respectively) while comparing between each day of biopsy. Limitations, reasons for caution Although this is the largest sample size study to investigate the association of blastulation with ploidy and pregnancy outcomes, the sample size for d7 embryos is relatively low and results should be confirmed in a bigger study. Wider implications of the findings Prioritization of euploid embryos for transfer should consider the timing to blastulation and embryos biopsied on d5 should be considered before those biopsied on d6 or d7. This data has potential to shed light on the impact of normal chromosomal content on preimplantation embryo development. Trial registration number not applicable

Effect of mare's age and embryo quality on pregnancy and foaling rate in a clinical ICSI program

There are few studies on the quality of in vitro produced (IVP) equine blastocysts related to the mare age. A retrospective study on the outcome of a clinical equine IVP program using donor mares of different ages and comparing their embryo quality and pregnancy potential is presented. Pregnancy, foaling, and pregnancy loss rates are reported for the transfer of fresh ICSI embryos derived from oocytes collected from mares aged 2 - 26 years old. ICSI blastocysts were classified as: Grade A – D6, 7, or 8, good quality; Grade B – D6, 7, or 8, moderate quality and D9 or D10, good quality; Grade C – D9 or D10, moderate quality (adapted from Lewis.et.al. Reproduction, Fertility and Development. 2023; 35: 338-351). Donor mares were divided on the basis of age: young (2-10y), mid-age (11-17y), and old (18-26y). Differences concerning groups were analyzed by Fisher's Exact Test (significance level of P<0.05). Results are presented in Table 1. For grade A embryos, pregnancy and foaling rates were similar among ages, but old mares showed higher pregnancy loss than young mares. For grade B embryos, old mares showed lower pregnancy rates than other groups. For grade C embryos, no difference in pregnancy, foaling, or pregnancy loss was seeing between ages. Embryo grades in each age interval were also analyzed. For young mares, pregnancy rate for different embryo grades was similar. However, foaling rate of grade A embryos was higher, and pregnancy loss of grade A embryos was lower than grade B. In mid-age mares,grade C embryos showed a lower pregnancy rate than grade A and B (tendency, P=0.09). Similarly, grade A embryos showed a higher foaling rate than the others. In old mares, grade A embryos showed higher pregnancy and foaling rates than grade B embryos. Thus, ICSI program outcomes for pregnancy, foaling, and pregnancy loss are related to the mare age. Special attention must be given to the quality of the embryo since pregnancy and foaling rates may vary within the same embryo grade, due to the mare's age.

The relationship between grade and metabolism for in vivo and ICSI derived embryos

The objective of this study was to describe the metabolism of glucose, pyruvate and lactate by in vitro- (ICSI derived) and in vivo-derived equine embryos and the relationship with embryo grade and sex. During the course of this study, a simplified embryo grading system was developed for both in vivo- and in vitro-derived embryos that proved to be significantly associated with both embryo metabolism and survival rates following transfer into a recipient mare. A total of 15 in vivo derived embryos and 32 ICSI-derived embryos were cultured in protein free synthetic oviduct fluid (SOF) for 24h between day 8 and day 9 of development. Aliquots of 10µL of media before culture (t=0) and spent media after culture (t=24h) were snap frozen for subsequent analysis by GC-Mass Spectrometry for glucose, lactate and pyruvate. The embryos were photographed and classified as grade 1, 2 or 3 based on the percentage of extruded debris between the trophoblast and the zona pellucida. In this study the sex of the embryos (in vivo n = 5, ICSI n = 25) was determined by PCR. At day 8 of development, the embryo metabolism of glucose, pyruvate and lactate in relation to embryo origin (in vivo vs in vitro), grade and sex wasevaluated following 24h of culture in protein free media. Overall, there was a positive correlation between embryo volume and lactate peak area (r2 = 88.2%, p ≤ 0.001) and a negative correlation with both glucose peak area (r2 = 29.8%, p =0.023) and pyruvate peak area (r2 = 46.2%, p =0.003) in the spent media. These results illustrated that as embryo volume increased, the amount of glucose and pyruvate in the media decreased and the production of lactate increased. After accounting for embryo volume, general linear model analysis revealed an effect of embryo grade (p =0.031), but no significant effect of embryo sex or origin on lactate consumption over 24h. Grade 2 embryos, however, consumed more lactate than Grade 1 embryos (p = 0.029). In conclusion, the metabolic relationships between glucose, lactate and pyruvate were described for both in vivo- and in vitro-derived equine embryos in this study. Understanding the optimal metabolic conditions for in vitro-derived embryos in comparison with in vivo-derived embryos will enable the development of models to investigate early embryonic death and to optimize pregnancy rates in the mare.

Study the Influence of Smoking in Male on Sperm Chromatin Immaturity and ICSI Outcomes

Cigarette smoke contains a substance which is a toxic mutagenic chemical, and carcinogenic compounds, which have a negative effect on male fertility. Smoking cigarettes is associated with noticeably higher levels of the oxidative stress-causing reactive oxygen species in the seminal fluid. Because of this, smoking may cause changes to the sperm plasma membrane and a significant amount of deoxyribonucleic acid fragmentation. To study the influence of smoking on sperm chromatin immaturity and ICSI outcomes (fertilization rate, embryo grading and pregnancy rate). At the laboratories of the High Institute of Infertility Diagnosis and Assisted Reproductive Technologies and after 1-7 days of sexual abstinence, 92 males’ sperm samples were taken. After liquefaction, macro- and microscopic studies were carried out, criteria applied by the WHO 2021. Masturbation was used to obtain the sample, which was then placed in sterile containers. The relationship between the smoking and chromatin immaturity and ICSI outcomes have been evaluated. A significant positive correlation between smoking status and sperm chromatin immaturity. Furthermore, there was a negative non-significant correlation between smoking status and fertilization rate. Weak and non-significant correlations between smoking status and embryo grade 2 as well as embryo grade 3. There was a positive association between smoking status and embryo grade 1. The sperm chromatin integrity was significantly lower in the non-smoker group compared to the smoker group.

19 Relationship Between pH in Vagina and a Successful Pregnancy in Small Ruminant Recipients

Abstract Reproductive efficiency and efficacy are key components of economic return in small ruminant production, therefore utilization of assisted reproductive technologies has increased. Despite advances in assisted reproductive technologies, pregnancy rates per embryo culture and transfer still remain low. The physio-chemical parameter of pH within the female reproductive tract and its potential implications in pregnancy success in caprine remains to be elucidated. The objective of this study was to evaluate vaginal pH and compare pregnancy rate for embryos produced in-vivo and in-vitro in goats. Spanish and Spanish Boer Cross females (n = 78) were estrous synchronized using a progesterone vaginal insert protocol and gonadotropin was administered at time of removal. Embryo grade and stage for conventional fresh embryo transfer and in-vitro fertilization were recorded. Vaginal pH was measured post transfer. At d 45 pregnancy status was determined via trans-abdominal ultrasonography. Successful embryo transfer (ET) was determined by kidding percentages. Data were analyzed using an ANOVA with a multiple linear regression model in GraphPad Prism 9.4. It was found that vaginal pH of females receiving cultured embryos did significantly predict pregnancy detection at d 45 and kidding (P = 0.02). However, the vaginal pH in females receiving fresh embryos had no effect of pregnancy and kidding rates (P = 0.451). While vaginal pH had a correlation with successful kidding of cultured embryos, further research is warranted to determine the relationship between physio-chemical parameters, environmental factors and pregnancy rates in goats.

Biochemical Pregnancy Outcomes among Day 3 Vs 5 Embryotransfer in the Fertility Reproductive Mecicine Center at Teritary Hospital, Addis Ababa, Ethiopia

Introduction: the burden of infertility is the worst in sub-Saharan countries with rare availability of assisted reproductive technology treatment and, even in the best set up where this service is available, generally it has a low implantation rate that could be partially explained by the days of embryo transferred after ovum pick up. however, the effects of a day of embryo transfer on pregnancy outcomes are an area of controversy in countries with advanced fertility treatment, on top of that we have very little research done on issues related to invitro fertilization Objective: The aim of this study was to see the effect of the day of (D3 vs D 5) embryos transfer on biochemical pregnancy outcome in the first and the only public IVF center in Ethiopia Method: it was a retrospective record review that was conducted in the center of fertility at saint Paul hospital millennium medical college, Addis Ababa, Ethiopia from January 2021-December 2021 G.C. with a total sample size of 201 Result: During the study period there were a total of 201 clients who has undergone embryo transfer fulfilling the inclusion criteria(age less than 35, at least one embryo transferred is grade one, fresh transfer), 94 of them were day 5 embryos and 107 were day 3. Both groups are comparable in baseline characteristics including the mean age, duration of infertility, cause of infertility, method of sperm insemination, level of physician transferring the embryo, number of embryos transferred, and grade of embryo transferred. Biochemical pregnancy between day 3 and day 5 embryo transfer was similar in both groups 67 (62.6%) and 59 (62.8%) respectively (p=0.98). However, the chance of having twin pregnancy was higher among day 5 embryo transfer 19 sets of twin pregnancy (36%) compare to day 3 embryo transfer 10 sets of twin pregnancy (17%) with p =0.053. Conclusion: In women aged less than 35 as long as we have available good quality embryos transferring day 3 embryo is an alternative option over blastocyst transfer with comparable biochemical pregnancy without increasing the chance of multiple pregnancies, this in turn decreases the risk of multiple pregnancies related complications. In addition, cleavage-stage embryo transfer carries fewer burdens and costs in a setup with low resources and busy environment and it has also low cost and stress on patients. Keywords: day 5 and day 3 embryo transfer, Ethiopia, public IVF center, biochemical pregnancy