Preimplantation Genetic Diagnosis Research Articles

P-188 Cleavage stage morphokinetic timings can predict the likelihood of aneuploidy in blastocysts

Abstract Study question Can morphokinetic analysis be used as a tool to predict the ploidy status of blastocysts? Summary answer A blastocyst is more likely to be aneuploid when it divides slowly to two cells and eight cells but quickly to three cells. What is known already Existing data suggests morphokinetic analysis and PGT-A both have advantages and limitations when used independently. However, the hope is that if strong associations can be found linking morphokinetic qualities of embryos and their ploidy status, then this can be used as a predictive tool. Currently, morphokinetic analysis cannot be used as a predictive tool for euploidy. However, there is some existing data that suggests that clinics can correlate morphokinetics and ploidy status using their in-house data. Study design, size, duration The study was retrospective and involved the assessment of blastocysts that had undergone PGT-A with next generation sequencing (NGS) and had a confirmed outcome of aneuploid or euploid between January 2021 and June 2022. Only patients who obtained genetic results showing they had at least one aneuploid embryo and at least one euploid embryo were included to allow for a matched case-control study – this resulted in 89 aneuploid and 89 euploid embryos being analysed. Participants/materials, setting, methods All embryos were cultured in the same conditions, including the same incubators, media (both from Vitrolife, SWE), and standard operating procedures. Morphokinetic data was exported and analysed from annotations made on the EmbryoScope annotation software. Embryos that could not be fully annotated were excluded from analysis. A conditional logistic regression was performed in order to ascertain the effects of each morphokinetic parameter on the likelihood that the embryo will be aneuploid or euploid. Main results and the role of chance From 89 patients, 178 embryos were analysed, of which half were euploid and half were aneuploid. The morphokinetic time points assessed were time to pronuclei fading (tPNF), time to 2-cell (t2), time to 3-cell (t3), time to 4-cell (t4), time to 5-cell (t5), time to 8-cell (t8), time to start of blastulation (tSB) and time to blastulation (tB). The duration of the second cell cycle (cc2) and its synchrony (s2) were also analysed. When euploid and aneuploid case-control matched embryos were compared, t2, t3 and t8 were the best predictors of aneuploidy when analysed together (P = 0.06, P = 0.074, P = 0.071 respectively). There are higher odds of a blastocyst being aneuploid if a later t2 (OR = 1.361), an earlier t3 (OR = 0.784) and a later t8 (OR = 1.047) have occurred. A later t2 (P = 0.044, OR = 1.361) is statistically significant when alongside an earlier t3 (P = 0.122, OR = 0.828). A later t8 is statistically significant, irrespective of t2 and t3 (P = 0.05, OR = 1.043). In addition to these results, there is a general trend for aneuploid embryos to develop to each time point slower than euploid embryos. Furthermore, there is more variation in the range of values for each time point in aneuploid embryos compared to less variation in euploid embryos. Limitations, reasons for caution There was a small number of embryos included in the study, so a larger cohort would increase the statistical power. A multi-centre study would also be beneficial to incorporate factors such as larger patient demographics, multiple embryo culture environments and variable clinical protocols. Wider implications of the findings Utilising morphokinetic data to predict aneuploidy can help us to counsel the patient on selecting embryos for biopsy, and to make a predictive tool that would be applicable within our laboratory and patient settings. A secondary impact could be supporting the use of non-invasive methods to predict embryo ploidy. Trial registration number Not applicable

O-299 Sperm DNA fragmentation by TUNEL influences live birth in egg recipients after euploid embryo transfer

Abstract Study question Does sperm DNA fragmentation (SDF) by TUNEL affect reproductive success measured as live birth rate (LBR) in egg recipients after euploid embryo transfer? Summary answer SDF increases miscarriage rates in egg recipients with PGT-A. Therefore, live birth rates depends on sperm DNA damage in egg recipients after euploid embryo transfer. What is known already Sperm DNA integrity is important for optimal fertilisation, implantation and pregnancy. Although controversial, several studies have shown that sperm DNA fragmentation correlates with adverse pregnancy outcomes in infertile men. Ooplasmic sperm DNA fragmentation repair mechanisms may occur in female gametes, mainly in young women. However, research on the impact of DNA fragmentation on LBR are limited. Also, chromosomally normal embryos analysed by PGT-A offers opportunity to isolate female factor and study male factor. The aim of this study is to assess whether high level of semen DNA damage has an impact on reproductive outcomes in terms of live birth rate. Study design, size, duration This is a retrospective study of 467 PGT-A cycles in egg recipients. The study population consists of infertile couples for euploid embryo transfer (February 2017-November 2022). Semen samples were obtained to measure DNA damage from 447 men. PGT-A was performed in 1831 blastocysts. Trophectoderm biopsies (d5/d6 blastocysts) were analysed by NGS (Veriseq Illumina). Embryos were frozen and 568 blastocysts transferred in the subsequent cycle. Conventional hormone replacement was used for endometrial preparation in recipient patients. Participants/materials, setting, methods Cohort study with 447 infertile couples attending to private fertility clinic undergoing ART with their autologous semen, donated oocytes and PGT-A. SDF was measured by Terminal-deoxyribonucleotidyl-ARTSferase-mediated dUTP Nodule End Labelling (TUNEL) assay using the FITC-labelled-dUTP in situ cell death detection kit (Roche). The cohort was divided into two groups according the DNA fragmentation index (DFI): high SDF group (DFI>20%) and normal SDF group (DFI≤20%). To evaluate reproductive outcomes Chi-square and Linear regression tests were used. Main results and the role of chance Of the 447 couples included (average paternal age 41.29 + 7.32 years), 66.5% of male partners had normal semen parameters. We reported high sperm DNA fragmentation in 8% of patients. Overall, the embryo aneuploidy rate was 31.7%. For the further statistical analysis confounding factors such as, female and male age, embryo quality, day of embryo biopsy were included. PGT-A analysis showed an aneuploidy rate of 32.1% in the high DNA fragmentation group and 30.3% in the normal DNA fragmentation group (p = 0.453). Regarding clinical data, the overall implantation rate was 34.9%. There was no significant difference between elevate DNA fragmentation and normal DNA fragmentation with respect to biochemical miscarriage rate (4.3% vs 7.6%, p = 0.799). Although not significant differences between groups, a lower implantation rate near to reach significance was found in the high SDF group (20.0% vs 34.4%, p = 0.050). Pregnancy and clinical pregnancy rates were significantly lower in the high DNA fragmentation group (26.1% vs 44.9%, p = 0.042; 21.7% vs 31.3%, p = 0.044;), respectively. Also, miscarriage rate was significantly higher in the high SDF group (40% vs 12.5%, p = 0.014). Therefore, the live birth rate was significantly lower in the high SDF group (13.0% vs 32.6%, p = 0.024). Limitations, reasons for caution Larger studies including a higher number of samples are needed to confirm the correlation observed between LBR in eggs recipients with semen DNA fragmentation. Those patients with a DFI higher than control values should be treated with antioxidants. The pregnancy outcomes should be analyzed after treatment of DNA fragmentation. Wider implications of the findings Our findings suggest that autologous semen DNA fragmentation influence in live birth rate in egg recipients. Our data show that live birth rates decreased in patients with a high DFI after PGT-A. This study confirms published studies in the literature showing that ART outcomes are affected by sperm DNA fragmentation. Trial registration number Not Applicable

P-251 Differences in fertilization, blastocyst, and ploidy rates in intracytoplasmic sperm injection (ICSI) versus conventional insemination for patients undergoing in vitro fertilization (IVF)

Abstract Study question Are there differences in fertilization, blastocyst development, or euploidy rates when comparing ICSI versus conventional insemination among patients with male and non-male factor infertility? Summary answer Fertilization rate was lower for male factor ICSI. There were no differences in blastocyst development. Euploidy rate was lower for non-male factor ICSI. What is known already ICSI was developed for male factor infertility due to its requirement for a very small number of viable sperm. Its use has expanded beyond male factor due to some studies suggesting enhanced fertilization, and lower risks of failed fertilization or inadvertent contamination of embryo biopsy specimens. There are potential risks associated with ICSI, including imprinting disorders, birth defects, and significantly increased cost. It is also unclear whether ICSI contributes to genetic abnormalities by bypassing the egg’s natural sperm selection and potentially disrupting the meiosis apparatus. Study design, size, duration This retrospective cohort study of 576 patients was conducted at a single clinic from January 2021 to December 2021. Patients were grouped into (1) ICSI with male factor (n = 201), (2) ICSI with non-male factor (n = 160), and (3) conventional insemination with non-male factor (n = 215). Primary outcome was ploidy status, determined by calculating the percentage of euploid, aneuploid, and mosaic (both low and high mosaic) among the three groups. Secondary outcomes included fertilization and blastocyst development rates. Participants/materials, setting, methods Patients undergoing autologous IVF at a single clinic, with PGT-A performed at a single lab were included. Fertilization rate was defined as the percentage of 2PNs per number of mature eggs injected/inseminated. Blastocyst rate was defined as the percentage of blastocysts per 2PNs. Ploidy rate was defined as the percentage of euploid/aneuploid/mosaic blastocysts per total biopsied blastocysts for which a result was obtained. Percentage data was compared using N-1 Chi-squared test. Main results and the role of chance There were no significant differences in patient age, number of oocytes retrieved, or number of mature oocytes among the three groups. Fertilization rate for male factor ICSI was significantly lower compared to both non-male factor ICSI (74.2% vs 77.8%, p = 0.005) and conventional insemination (74.2% vs 76.8%, p = 0.018). There was no significant difference in fertilization rate between non-male factor ICSI and conventional insemination. There were no significant differences between blastocyst rates on Day 5, 6, or 7, or total blastocyst rate among the three groups. The euploidy rate for conventional insemination was significantly higher than that for non-male factor ICSI (53.4% vs 46.8%; p = 0.008). Non-male factor ICSI yielded lower euploidy rate than male factor ICSI (46.8% vs 52.4%, p = 0.034). Aneuploidy rate for non-male factor ICSI was significantly higher than both male factor ICSI (36.1% vs 31.0%, p = 0.041) and conventional insemination (36.1% vs 31.0%, p = 0.030). Mosaicism levels were similar between the three groups. There were no significant differences in number of biopsied embryos with no results: 5 for ICSI male factor group (0.59%), 6 for ICSI non-male factor (0.96%), and 10 for conventional insemination (0.89%). Limitations, reasons for caution This was a retrospective analysis of patients from a single clinic, with a relatively small sample size so results may not be generalizable to all populations. Extensive demographic information and baseline characteristics (including reason for ICSI performed among non-male factor patients) were not analyzed so there may be confounding factors. Wider implications of the findings This is the first study that we are aware of to investigate the ploidy status of embryos created among non-male factor patients undergoing ICSI. Conservative use of ICSI can significantly decrease the cost of IVF. Larger studies are needed to further elucidate the role for ICSI among these patients. Trial registration number not applicable

P-398 Elective Oocyte Cryopreservation – a viable alternative as a means of fertility preservation

Abstract Study question What are the trends in women accessing elective oocyte cryopreservation (EOC) across a multi-site clinic in Sydney, Australia, thaw rates, utilisation and live birth rates Summary answer Over 9 years, there was a significant increase in women having EOC. 2.7% returned to use. Oocyte thaw survival rates were 89.5% with LBR 35.5%. What is known already Data from Australia/New Zealand and USA demonstrated a 3 to 8-fold increase in EOC cycles, with a reduction in mean age of women accessing treatment. <5% of women returned to use their frozen gametes and live birth rates were better in women aged < 35 years. HFEA data demonstrated the use of frozen oocytes did not appear to increase risk of adverse perinatal outcomes. Research has shown that almost half of the women who returned to use their frozen oocytes used donor sperm with an overall LBR of 20.9%. 91% of women had no regrets over their decision for EOC. Study design, size, duration This was a retrospective cohort study performed at a multi-site large private fertility clinic in Sydney, Australia. This clinic was the first in Sydney to offer EOC to women. All women accessing EOC from 2014 – September 2022 were included. Ethics approval was obtained. Participants/materials, setting, methods Data was collected from women undergoing EOC; including demographic information, controlled ovarian stimulation parameters, return to use, oocyte thaw survival rates, source of sperm used for fertilization, embryo biopsy rates, aneuploidy rates, as well as pregnancy rates. Main results and the role of chance There was a 17.5-fold increase in cycles of EOC over the 8-year study period; with a reduction in average age from 38.13 to 35.85 years. Overall the mean age was 36.6 of the 2278 women who underwent EOC, of which 30% were aged >38 years. The mean oocyte yield was 8.85. Of the 62 (2.7%) women who returned to use their frozen oocytes, 54.8% were < 38 years of age. Oocyte thaw survival rates were 89.5%. The majority of women who returned to use their frozen oocytes, used donor sperm for conception. Fertilization rates with intra-cytoplasmic sperm injection (ICSI) was 65.3%; with 8.3% of oocytes degenerating post ICSI. 41.6% of embryos developed into blastocysts with 27.8% utilization rate (good quality blastocyst formation rate). Overall LBR was 35.5% for all women. Clinical pregnancy rates (CPR) from warmed oocytes were 46.2% from women aged <38 years old and 22.2% from women >38 years old. Of women who returned for a frozen embryo transfer, LBR was 25%; all occurring in women who froze their oocytes prior to age 38. Limitations, reasons for caution In parallel to international studies, very few women returned to use their oocytes; results need to be interpreted with care when counselling women considering EOC. Lifestyle factors were unable to be accounted for. Most women have opted to continue storing and not dispose/donate their oocytes at the time of publication. Wider implications of the findings EOC proves to be a viable and useful option for fertility preservation. Due to the small proportion of women who return to use their oocytes; and paucity of data; it would be prudent to improve standardisation of data collection and outcome measure reporting. Trial registration number Not Applicable

O-051 Refreezing and rebiopsy – are these worth it?

Abstract Preimplantation genetic testing of embryos offers the opportunity to avoid the transmission of hereditary disorders to the offspring by selecting out embryos affected by a single gene mutation or mutations (PGT-M), structural rearrangements of chromosomes (PGT-SR), aneuploidy (PGT-A) as well as polygenic risk (PGT-P). Over the last two decades, laboratory techniques have seen tremendous developments including extended embryo culture to blastocyst stage, trophectoderm biopsy and vitrification. The latter technique is highly efficient and allows multiple freezing and warming cycles when performed in proficient laboratories. At the same time whole genome analysis techniques provide the opportunity to analyse embryonic DNA in great detail for multiple indications simultaneously. The PGT process is sensitive to both biological and technical issues which sometimes lead to a failed PGT result. Failure of PGT diagnosis is reported to occur in between 2-12% of PGT cycles depending on the methodology used. The main causes of inconclusive diagnosis are DNA amplification failure and non-concurrent results. Independent variables that influence inconclusive results are day of embryo biopsy, number of cells taken at biopsy and the quality of the IVF laboratory. In order to seize every opportunity in reaching a diagnosis and optimize reproductive outcome for the patient, rebiopsy and retesting (double biopsy and double cryopreservation) can be performed. In situations where embryos need to be biopsied for PGT for reasons of newly found genetic indications or the clinical need for PGT-A, and after an initial cryopreservation, single biopsy and double cryopreservation may be performed with potential effect on subsequent reproductive outcome. A limited number of studies have addressed both the feasibility as well as the success rate of rebiopsy and refreezing. Most studies conclude that the technique is efficient in terms of diagnostic efficiency and reproductive outcome, when performed in expert laboratories. A few studies have addressed the health of the children after multiple biopsy and multiple freezing and have not shown increased risks. Larger studies are needed to establish the true clinical and health-economic value of rebiopsy and refreezing. The speaker will give an overview of existing evidence in literature and assess whether it is safe and worthwile to go the extra mile and consider refreezing and rebiopsy for PGT. Trial registration number XXXX

P-727 New methods reveal the true incidence of DNA contamination in PGT-A samples for the first time and avoid errors that could result in serious misdiagnoses

Abstract Study question What is the occurrence of contamination in embryo biopsy samples and could this lead to incorrect interpretation of preimplantation genetic testing for aneuploidy (PGT-A) results? Summary answer On average, contamination affects 0.4% of biopsy samples, but can be significantly more common in some clinics. Misdiagnosis can occur when contamination is not detected. What is known already Until recently, most commercially available platforms for PGT-A have utilised whole genome amplification followed by sequencing of a random selection of DNA fragments scattered across the genome using next generation sequencing (NGS). However, the simple quantitative measurements of DNA fragments derived from each chromosome, provided by such methods, cannot reveal when a biopsy sample is contaminated with non-embryonic DNA. Negative controls are seldom used during PGT-A and are inadequate as they do not evaluate contamination in the actual tube containing the biopsy specimen. The extent to which failure to detect DNA contamination is a problem for PGT-A is unknown. Study design, size, duration This was a retrospective study involving analysis of 49,287 trophectoderm biopsy samples that underwent PGT-A over a three-year period. Embryos found to have a contaminated biopsy specimen typically underwent a second biopsy. In such cases, results from the two samples were compared to ascertain whether the contaminated sample would have been misdiagnosed if the analysis had been restricted to examining only the relative chromosome copy number, as is the case for most NGS-based PGT-A methods. Participants/materials, setting, methods All trophectoderm biopsies underwent targeted DNA amplification and next generation sequencing using a highly validated PGT-A method that evaluates the relative chromosomal copy number, similar to traditional PGT-A methods, but combines this with analysis of variations in DNA sequence (single nucleotide polymorphisms - SNPs). The genotype of each SNP, and the relative quantity of DNA fragments containing each of the different alleles, allows detection of otherwise invisible states, such as triploidy, haploidy, and contamination. Main results and the role of chance From the 49,287 TE biopsies analysed, contamination with non-embryonic DNA was detected in 218 (0.44%). There was variation in the rates of contamination between the 25 clinics that provided samples, ranging between 0% and 1.5%. Additionally, one clinic had a contamination rate of 7.7%, but the number of biopsies derived from that site was considered too small for reliable evaluation (n = 26). 156 of the embryos with a contaminated biopsy specimen underwent secondary biopsy (71.5%), allowing the relative chromosome copy number result from the contaminated specimen to be compared to that obtained from an uncontaminated sample. The results were split into three categories: 1) no change in interpretation between the first (contaminated) and second biopsy specimens; 2) false positive – the contaminated sample was euploid but would have been wrongly interpreted as triploid and would have been erroneously discarded, potentially impacting the patients chances of achieving a pregnancy; 3) false negative – the contaminated sample was fully aneuploid but would have been incorrectly classified mosaic or euploid and could have been eligible for transfer, potentially leading to implantation failure or abnormal pregnancy. 19% of contaminated samples gave a false negative result, while 24% gave a false positive, appearing to be triploid. Limitations, reasons for caution It is not possible to determine the origin of contaminants with certainty without having DNA from the contamination source for comparison. Additionally, we were unable to conclude whether contamination is more likely to occur in IVF or ICSI cycles as only 3% of samples were fertilized using IVF. Wider implications of the findings Contamination detection during PGT-A is important to prevent misdiagnosis of embryos. Misclassification due to undetected contamination can lead to discard of potentially viable embryos. It can also lead to the transfer aneuploid embryos, wrongly classified as mosaic, which could lead to increased rates of implantation failure, miscarriage and aneuploid pregnancy. Trial registration number Not applicable

O-240 Does embryo categorisation by existing artificial intelligence, morphokinetic, or morphological embryo selection models correlate with blastocyst euploidy rates?

Abstract Study question Does embryo categorization by existing artificial intelligence, morphokinetic, or morphological embryo selection models correlate with blastocyst euploidy? Summary answer Our results show that existing blastocyst scoring models correlate with ploidy status. What is known already A previous study suggested that morphokinetic parameters should not be used yet as a surrogate for PGT-A to determine embryo ploidy in vitro. As for predicting pregnancy likelihood, AI models have been proposed for predicting ploidy status. Discrimination performance in terms of AUC has been reported of up to 0.80 when including metadata such as maternal age, and 0.63 when AI predictions were made based on time-lapse images alone. However, as the studies only looked at models trained for ploidy prediction, it is unclear how existing blastocyst scoring systems would perform at discriminating euploids and aneuploids. Study design, size, duration A total of 834 patients, 3,573 blastocysts were retrospectively analyzed. The cycles were stratified into five maternal age groups according to the Society for Assisted Reproductive Technology (SART) age groups (< 35, 35–37, 38–40, 41–42, and >42 years). The quality and scoring of embryos were assessed by iDAScore v1.0 (iDA, Vitrolife, Sweden), KIDScoreTM D5 v3 (KS; Vitrolife), and Gardner grading (GG). Participants/materials, setting, methods Embryos were cultured in the EmbryoScope+ and EmbryoScopeFlex (Vitrolife). iDA was automatically calculated using the iDAScore model running on the EmbryoViewer (Vitrolife). KS was calculated in EmbryoViewer after annotation of the required parameters. ICM and TE were annotated according to the Gardner grading. The degree of expansion in all blastocysts was Grade 4 due to our freezing policy. Furthermore, Gardner's scores were stratified into four grades (A: AA, B: AB BA, C: BB, D: others). Main results and the role of chance At first, the correlation between all assessment methods and euploid rates were analyzed using a trend-test. Euploidy rates significantly correlated with iDA in each age group (p = 0.035 for age <35 years, p < 0.001 for all other age groups) and decreased progressively with increasing maternal age in each score group. Furthermore, euploidy rates were significantly correlated with KS (P < 0.0001), except in the youngest age group (p = 0.07). They decreased progressively with increasing maternal age in each score group. Similarly, euploidy rates were significantly correlated with GG (P < 0.0001), except in the youngest age group (p = 0.06). They decreased progressively with increasing maternal age in each score group. Secondly, the performance of the euploidy prediction for each embryo scoring model was compared using the area under the curve (AUC) of the receiver operating characteristic curve. The AUCs for euploid prediction of iDA, KS, and GG for all ages were 0.666, 0.655, and 0.642, respectively. iDA and KS were significantly higher than Gardner grading (iDA vs. GG: p = 0.004; KS vs. GG: p = 0.02). Additionally, there was no significant difference between iDA and KS. Limitations, reasons for caution It was based on minimal stimulation and the use of natural cycle for IVF treatment, which involved only insemination by ICSI. Furthermore, the blastocyst assessment models that we tested were initially developed for pregnancy prediction and not for the prediction of the chromosomal status of an embryo. Wider implications of the findings Existing blastocyst assessment models that reflect blastocyst viability in view of implantation potential to some extent correspond with ploidy status. Therefore, existing blastocyst scoring models can be used to support the decision for embryo biopsy and/or to reduce the patient’s cost by deciding on which embryo(s) to biopsy. Trial registration number not applicable

P-731 The accuracy of truly non-invasive PGT using spent culture media is insufficient to justify routine clinical use

Abstract Study question Can non-invasive PGT (niPGT), using a protocol that avoids manipulation of the embryo before spent culture medium is collected, accurately determine embryo chromosomal status? Summary answer Accuracy using a truly non-invasive PGT method was low, suggesting this strategy is not appropriate for clinical use and should be restricted to research studies. What is known already PGT requires embryo biopsy to obtain small numbers of cells for genetic testing. The procedure requires skilled personnel and specialist equipment, significantly adding to the cost of PGT while also creating training and logistical bottlenecks that reduce patient access to PGT. Additionally, there have been concerns that biopsy could damage embryos. These considerations have stimulated interest in potential non-invasive PGT strategies. Thus far, niPGT methods have mostly focused on cell-free embryonic DNA found in spent culture media, but studies reporting highest accuracy have tended to involve embryo manipulations likely to promote DNA release and therefore cannot be considered truly non-invasive. Study design, size, duration Samples of spent culture medium (SCM) associated with 128 embryos were collected. Oocytes had been denuded of cumulus cells, fertilised using ICSI and the resulting embryos thoroughly washed on day-4 before transfer to a fresh drop of medium. SCM samples were collected on day-5 or day-6 of culture. Embryos had not previously been cryopreserved and underwent minimal manipulation prior to SCM collection. Results of SCM analysis were compared to those obtained following conventional ‘invasive’ PGT. Participants/materials, setting, methods Media samples were subjected to whole genome amplification using the PG-Seq Rapid Non-Invasive PGT kit. Concentrations of sequencing libraries were measured, and next-generation sequencing (NGS) was undertaken. The resulting NGS data was analysed with PG-Find Software to predict chromosomal status. In parallel, trophectoderm biopsies from the corresponding embryos were analysed using a well-established and highly validated PGT method, based upon amplification of thousands of sites across the genome, followed by NGS. Main results and the role of chance Despite the implementation of rigorous measures to prevent contamination of SCM samples, extraneous female DNA, likely of cumulus cell origin, was frequently observed. Depending on the clinic, 16.7% to 38.1% of media samples associated with male embryos gave a discordant (female) result. This highlights the difficulty of avoiding inadvertent sampling of maternal DNA. There were no instances of male DNA contamination affecting SCM samples from female embryos. We assessed whether clinic of origin, ploidy status of the embryo, and day of SCM collection influenced the quality of the sequencing libraries (ANOVA). Higher concentrations of libraries and superior sequencing quality scores were observed in SCM collected on day-6 compared to day-5 (p = 0.011 and p = 0.002, respectively). Chromosomal assessments obtained from SCM were compared to those from standard, trophectoderm-based PGT in a blinded fashion. 50% of SCM samples gave an identical predicted karyotype to their corresponding trophectoderm biopsy (concordant copy number for all chromosomes). When applying a simple aneuploid/euploid classification, 80% of embryos classified chromosomally abnormal using the validated PGT method were also aneuploid according to analysis of the associated SCM sample, but without necessarily having identical karyotypes, while 62% of embryos classified euploid by PGT received the same designation after SCM analysis. Limitations, reasons for caution SCM samples were designated ‘contaminated’ when embryos of one sex (determined using invasive PGT) were incorrectly classified as the opposite sex using niPGT. However, our study was unable to determine when SCM samples were contaminated with DNA of the same sex as the embryo. Consequently, contamination rates could be higher. Wider implications of the findings Most niPGT methods are not truly non-invasive, as they include embryo manipulations that increase likelihood of cell death and DNA release. Our results show that obtaining reliable niPGT results is difficult without deviating significantly from optimal embryological protocols. Currently, the accuracy of niPGT seems insufficient to justify routine clinical use. Trial registration number Not applicable

P-723 Pre-fertilization genetic testing to select healthy gametes in a mammalian Marfan syndrome model

Abstract Study question Is it possible to identify disease-free gametes before insemination to prevent vertical transmission of an autosomal dominant disease? Summary answer In a mammalian Marfan syndrome model, we successfully generated and identified healthy androgenotes to be utilized as male gametes to produce unaffected conceptuses. What is known already In patients with autosomal dominant genetic disorders such as Marfan syndrome, germline heterozygosity poses a risk to offspring health. For these patients, preimplantation genetic testing for monogenic disorders (PGT-M) allows us to select unaffected conceptuses for embryo replacement. However, PGT-M is inherently inefficient and can lead to embryo wastage. This has sparked interest in replicating the genome of individual gametes for (1) pre-fertilization genetic testing and (2) subsequent use as genotyped gamete substitutes during IVF. Study design, size, duration For this study, we selected 4 male B6 mice that were heterozygous for the Fbn1tm1Hcd mutation and displayed classic manifestations of Marfan syndrome. Spermatozoa from these mice were used to inseminate enucleated wildtype mouse oocytes and generate haploid androgenetic embryos for (1) Fbn1tm1Hcd genotyping and (2) use as unaffected male gamete substitutes. Participants/materials, setting, methods Using tail clippings and embryo biopsy specimens, a Fbn1tm1Hcd allele-specific PCR primer was developed and validated. Haploid androgenetic embryos were allowed to progress to the 8-cell stage. Subsequently, a single-haploid pseudo-blastomere was isolated for genotyping, while the remainder were individually grafted into activated recipient wildtype oocytes and fused using inactivated Sendai virus to generate biparental conceptuses. Resulting diploid conceptuses were cultured in a time-lapse incubator and then genotyped to confirm the healthy paternal haplotype. Main results and the role of chance A total of 175 wildtype B6 oocytes were enucleated yielding 169 ooplasts (96.6% survival rate). The ooplasts were inseminated with spermatozoa from mutant Fbn1tm1Hcd mice, yielding 144 (85.2%) monopronucleated haploid androgenetic embryos. Of those, 100 (69.4%) progressed to the 8-cell stage. In 47 (47.0%) of them, genetic analysis by PGT-M on an individual pseudo-blastomere characterized the embryo as unaffected because it harbored the wildtype Fbn1 allele. The remaining 7 haploid pseudo-blastomeres from 5 Fbn1 wildtype androgenetic embryos were individually isolated and grafted into 35 recipient oocytes to generate 31 (88.6%) biparental conceptuses with the desired paternal haplotype. These achieved a 61.3% (19/31) blastocyst development rate after 96 hours, displaying normal embryo morphokinesis. A blastocyst biopsy was carried out (n = 19), confirming the unaffected paternal haplotype void of the Fbn1tm1Hcd mutation. Limitations, reasons for caution Although this technique is feasible for pre-fertilization genetic testing, it needs to be reproduced with the same efficiency in humans. A major concern is related to the dynamics and roles of the sperm centrosome in the first mitotic division of human zygotes, which may be missing in the pseudo-gametes. Wider implications of the findings This approach to pre-fertilization genetic testing will benefit patients with heritable genetic conditions. In addition, the inherited decondensation of the male genome by the ooplasm may enable genome editing experiments to be performed. Trial registration number Not applicable

P-707 Evaluating DualStim/PGTA in poor prognosis patients: Comparison of double ICSI-PGTA vs. oocyte cryopreservation plus single PGTA in luteal phase through laboratory, genetic and clinical results.

Abstract Study question Which strategy is best for poor-prognosis patients: Two ICSI-PGTA cycles fresh oocytes (follicular plus luteal phase) or one ICSI-PGTA cycle with vitrified and fresh oocytes?" Summary answer Laboratory outcomes, ploidy rates and clinical results after euploid blastocyst transfer are similar between the two DualStim/PGTA strategies. What is known already Luteal phase ovarian stimulation (LPOS) after follicular phase ovarian stimulation (FPOS) is an effective strategy to obtain more oocytes in a single cycle and reduce time-to-pregnancy. The main goal of controlled ovarian stimulation in PGTA patients is to obtain a sufficient number of oocytes to have at least one euploid blastocyst. The standard treatment for achieving this goal is DuoStim plus oocyte cryopreservation. However, this method may decrease oocyte competence in older patients. To address this, we propose comparing the outcomes of two consecutive fresh ICSI-PGTA cycles to our standard protocol in older patients, as an alternative approach. Study design, size, duration This multicentric retrospective study evaluated 91 poor-prognosis PGTA cycles (≤ 5 antral follicle count) using the DualStim approach (182 oocyte pick-ups) in the same menstrual cycle, from January to October 2022. Fifty-three patients (group 1; 746 oocytes retrieved) underwent two consecutive egg retrievals, ICSI, and blastocyst biopsy, after FPOS and LPOS, while 38 patients (group 2; 743 oocytes retrieved) underwent oocyte vitrification after FPOS, followed by LPOS, egg retrieval, oocyte warming, ICSI, and blastocyst biopsy. Participants/materials, setting, methods We compared various parameters including oocyte number, maturity, fertilization rates, blastocyst quality, biopsy rates, and genetic results between two groups after both egg retrievals. Additionally, clinical outcomes after euploid embryo cryotransfer were also compared. Oocytes were microinjected, cultured in Geri® time-lapse incubators, and underwent embryo biopsy on day 5/6. Statistical analysis was conducted using R software (v.4.2.0), including maternal age as a confounding factor. Results were considered significant with a p-value less than 0.05. Main results and the role of chance Patient age was found to be significantly different between groups (40.5±2.2 in group-1 vs 38.5±2.7 in group-2; p < 0.001) and was used as a confounding factor in statistical comparisons. No significant differences were found in terms of oocytes retrieved per patient (14±7.1 vs 13.6±5.8), maturity rate (79.4 % vs 77.9%), fertilization rate (75.3 % vs 74.6%), or day-5 blastocyst rate (54.8% vs 51.7%). However, a significant difference was observed in the percentage of blastocysts biopsied per group (50.2% group-1 vs 43.0% group-2; p = 0.023). After genetic analysis, euploidy rate was found to be lower in group-1 compared to group-2 although not statistically significant (19.8% and 32.6%, respectively, p = 0.007/p=0.309 after adjusting for maternal age), and this led to similar average number of euploid embryos per DuoStim cycle (1.1±1.3 group-1 vs 1.3±1.4 group-2). Aneuploidy (73.9% vs 62%) and mosaicism rates (6.3% vs 5.4%) were also comparable between the two groups. No statistical differences were observed between the follicular and luteal phases in each group in terms of laboratory and genetic results. Preliminary clinical outcomes showed similar pregnancy rates with 12 pregnancies achieved in group 1 after 23 euploid embryo transfers (52.2%), and 14/20 in group 2 (70.0%) with no significant difference between groups. Limitations, reasons for caution This study is a retrospective analysis and further validation through prospective study is recommended. Additionally, while the average age of the study groups is statistically different due to bias in the clinical prescription of treatment, this difference was statistically corrected. Wider implications of the findings Cryopreserving oocytes obtained after FPOS and warming them after the subsequent LPOS does not negatively impact the quality or final results of a DuoStim PGTA cycle. This approach should be considered as it can reduce workload for the IVF laboratory and minimize costs for patients. Trial registration number none

P-795 Identification of a specific paternal haplotype in the germline and perpetuating progenies by male gamete replication

Abstract Study question Can we identify and replicate a male germ cell genome to generate progenies carrying a specific genotype? Summary answer We were able to replicate male genome and identify the androgenote with a specific genotype to generate conceptuses with identical paternal haplotype. What is known already For patients with heritable genetic disorders, current treatment relies on preimplantation genetic testing for monogenic disorders (PGT-M) to select unaffected embryos for replacement; however, this can lead to embryo wastage, causing emotional distress. Although genomic editing of gametes is being attempted, CRISPR-based technologies are inaccurate and can lead to loss of heterozygosity in cases where the embryo uses the maternal genome as a template to edit the paternal genome. Therefore, the use of haploid androgenotes may offer a useful tool to select male gametes with genes of interest or a successfully edited genome before the creation of an embryo. Study design, size, duration To generate haploid androgenotes, spermatozoa from hemizygous transgenic mice with GFP expression (B6-EGFP x B6D2F1) were used to inseminate enucleated wildtype oocytes. Constructs were cultured to the 8-cell stage. Two pseudo-blastomeres were isolated for genotyping by DNAseq. A blastomere with GFP expression was used as a male gamete. Experiments were repeated using mice with the Fbn1tm1Hcd mutation as a model for Marfan syndrome. Biparental embryos were created to preserve the mutation as a disease model. Participants/materials, setting, methods To generate haploid androgenotes, metaphase II oocytes from wildtype B6 mice were enucleated and inseminated using spermatozoa from hemizygous GFP transgenic mice. Haploid androgenetic embryos were cultured in a time-lapse incubator up to the 8-cell stage. Biparental embryos were created by fusing a selected androgenote with an activated oocyte from another cohort. Resulting embryos were genotyped to confirm the paternal haplotype. Experiments were reproduced using spermatozoa from heterozygous Fbn1tm1Hcd-mutant mice. Main results and the role of chance In the first set of experiments using spermatozoa from transgenic mice hemizygous of GFP, 200 wildtype oocytes were enucleated and generated 195 (97.5%) ooplasts. Insemination of those ooplasts yielded 168 (86.2%) monopronucleated androgenetic embryos, and subsequently generated 116 (69.0%) 8-cell haploid androgenetic embryos, of which 59 (50.8%) expressed GFP. Up to 2 sibling blastomeres were genotyped individually by DNAseq and confirmed to have an identical paternal haplotype. The remaining blastomeres were used to create biparental embryos and yielded a 97.2% fertilization rate after grafting into 145 oocytes, comparable with the fertilization rate of ICSI controls at 95.6%. Although control zygotes yielded an 88.0% blastulation rate, the experimental embryos yielded a lower blastulation rate of 58.2% (P<0.0001). Albeit with a lower developmental rate, all experimental blastocysts expressed GFP and displayed normal embryo morphokinesis. For the experiment using spermatozoa from Fbn1tm1Hcd mice, 50 8-cell stage haploid androgenetic embryos were generated. Two individual pseudo-blastomeres from 35 haploid embryos were used for genotyping. The remaining 210 haploid blastomeres were used to construct biparental embryos and yielded 119 (56.7%) blastocysts. PCR on embryo biopsies confirmed that the resulting blastocysts inherited the mutant (n = 63) or wildtype (n = 56) paternal haplotype from the original gamete. Limitations, reasons for caution This technique allowed the selection of male gametes with the desired genotype to generate conceptuses with a known haplotype in a reproducible manner. A more prospective experiment is ongoing to simulate clinical application by cryopreserving haploid androgenetic embryos and using only androgenotes with a confirmed genotype to create biparental embryos. Wider implications of the findings This approach to utilize gamete substitutes before fertilization ensures the maintenance of rare and unique genetic materials to generate conceptuses with the desired genotype in transgenic mice in disease models. Indeed, once this technique is implemented in humans, genotyped androgenotes can be used to characterize germline heterozygosity. Trial registration number N/A

P-724 The choice of genetic service provider can have significant implications for the outcome of IVF cycles using preimplantation genetic testing for aneuploidy (PGT-A)

Abstract Study question Does the choice of PGT-A provider have any impact on clinical results, such as rates of aneuploidy, pregnancy and miscarriage? Summary answer The laboratory providing PGT can have a significant impact on various important clinical outcomes. PGT-A providers are not equivalent and should be chosen with care. What is known already A growing number of IVF cycles include PGT-A to assist in the identification of euploid embryos for transfer to the uterus. All modern PGT laboratories utilise a method called next generation sequencing (NGS) to predict the copy number of each chromosome in embryo biopsy samples. Given this technical convergence, it is often supposed that genetic results will be similar regardless which PGT laboratory is used, and consequently a clinic choosing a genetics provider need only consider convenience, quality of support and price. Here we consider whether the choice of PGT-A provider might have more profound affects, potentially impacting clinical results. Study design, size, duration A large network of IVF clinics switched from PGT-A provider ‘A’ to provider ‘B’. The final 6 months of clinical data using provider A was compared to 6 months of data after the switch to B. There were no changes in any aspect of patient population, treatment protocols, or embryological practice occurred between the two time periods evaluated. Genetic results (aneuploidy/euploidy rates) and clinical outcomes (implantation, miscarriage, ongoing pregnancy/birth) were compared to identify any differences. Participants/materials, setting, methods Within the two time periods considered, 9,091 embryos underwent PGT-A with provider A and 10,281 using B. The average female age was 39.0 and 39.3, respectively. Embryos were biopsied at the blastocyst stage and the cells were shipped to the genetics laboratories. Provider A was in the same country as the IVF clinics, while provider B was located overseas. Both PGT-A laboratories used NGS, but employed different methods for DNA amplification, sequencing and data analysis. Main results and the role of chance A higher proportion of embryos were classified euploid by provider B in comparison to A (49.3% versus 39.8%; p < 0.0001). Differences in reported aneuploidy were particularly striking for younger patients (<38 years; 67.5% euploid according to B versus 52.5% reported by A; p < 0.0001). For patients over 37 years, the lower aneuploidy frequency reported by B resulted in fewer cycles with all embryos classified ‘abnormal’ and more cycles with a transfer (52.2% of 2115 cycles in this age group had a transfer, compared to 48.6% of 1915 cycles for A; p = 0.02). Having more embryos classified euploid also benefitted younger patients, providing opportunities to combine morphological selection with genetic evaluation. Improved selection may explain a higher ongoing pregnancy rate observed for patients <38 years (63.2% ongoing after the first embryo transfer for B versus 55.3% for A). Differing miscarriage rates were also observed (18.1% when using A versus 15.1% for B; p = 0.047). We speculate that higher rates of euploidy and pregnancy when using provider B might be a consequence of viable embryos being excluded due to incorrect classification as ‘abnormal’ by A. Additionally, failure to detect some aneuploidies, leading to transfer of abnormal embryos, might explain the higher miscarriage rate associated with A. Limitations, reasons for caution There were no detectable differences in the composition of patient populations receiving PGT-A from the two providers, and no apparent changes to protocols used during the two time periods assessed. Nevertheless, it must be acknowledged that the conclusions are based on a retrospective review of data, not a controlled study. Wider implications of the findings PGT-A and NGS are umbrella terms encompassing multiple methods with widely varying levels of validation and accuracy. Our results suggest that the choice of PGT-A provider has important implications for clinical results, affecting the number of transferrable embryos, pregnancy, and miscarriage rates. The observed differences likely reflect differing PGT-A accuracies. Trial registration number Not applicable

P-006 In men with Raised Sperm DNA Fragmentation Index, would advance Sperm Preparation techniques alter Embryo Euploidy?

Abstract Study question In men with Raised Sperm DNA Fragmentation Index (DFI), Would advance sperm preparation techniques at Intra-Cytoplasmic Sperm Injection (ICSI) alter embryo euploidy status? Summary answer In men with raised Sperm DFI, use of advance sperm selection techniques are safe and do not appear to alter ploidy status of the embryos What is known already MACS (Magnetic Activated Cell Sorting), Microfluidic Sperm Sorting (MF), surgically retrieved Testicular Sperm (TESA) and Double Density Gradient sperm processing (DDG) have been shown to optimize sperm selection at ICSI for men with raised sperm DFI. Would any of these sperm selection methods alter embryo ploidy status is not proven Study design, size, duration This was a retrospective observational study conducted at a private fertility centre in-between years 2015 & 2022. Couples where women (<37yrs) had idiopathic Recurrent Implantation Failures (RIF) and men had raised sperm DFI (>25% DFI) and underwent Pre-Implantation Genetic Testing (PGT) were evaluated (n = 473). Based on sperm selection method the study population was divided into four groups. TESA (n = 84), MACS (n = 105), MF(n = 135), DDG (n = 148).A total of 1802 embryos were evaluated in this study. Participants/materials, setting, methods All women underwent Controlled Ovarian Stimulation (COS) and oocyte retrieval and male partners underwent sperm processing as per the clinics SOP. Appropriate counselling and written consents were obtained from all couples, where applicable. All oocytes subjected to ICSI, extended blastocyst culture and trophoectoderm (TE) biopsy. Post biopsy embryos were vitrified and TE tissue subjected to Next-Generation Sequencing (NGS) to assess the ploidy status. Ploidy status between various sperm selection methods was compared. Main results and the role of chance Euploidy rates between the study groups were as follows: TESA (n = 311 embryos screened)- 48% MACS (n = 445 embryos screened) – 51% MF (n = 489 embryos screened) – 61% DDG (n = 557 embryos screened)- 54% Though numerically the ploidy status was highest in MF group for euploid embryos, there seems to be no statistical significance between the groups (p-value = 0.973). Different sperm selection techniques do not seem to alter embryo ploidy status. Ejaculated sperm or Testicular sperms had similar euploidy status. Use of Testicular sperm, which is considered immature doesn’t seem to increase embryo aneuploidy. MACS, MF and DDG all seemed to have similar euploid embryos. This further warrants research to validate the role of MACS and MF as a routine intervention to optimize sperm selection and reproductive outcomes. Limitations, reasons for caution Retrospective design, un-equal group size and small sample size, a well-designed study is needed to further validate the findings of this study. Wider implications of the findings Role of advance sperm selection methods to optimize embryo implantation is yet to be proven. Data from this study showed no alarming increase in embryo aneuploidy in younger women and was comparable with published literature. Use of advance sperm selection techniques seems safe and doesn’t alter embryo ploidy status. Trial registration number not applicable

Electronic witnessing in the medically assisted reproduction laboratory: insights and considerations after 10 years of use.

What have we learnt after 10 years of electronic witnessing? When applied correctly, an electronic witnessing system can replace manual witnessing in the medically assisted reproduction lab to prevent sample mix-up. Electronic witnessing systems have been implemented to improve the correct identification, processing, and traceability of biological materials. When non-matching samples are simultaneously present in a single workstation, a mismatch event is generated to prevent sample mix-up. This evaluation investigates the mismatch and administrator assign rate over a 10-year period (March 2011-December 2021) with the use of an electronic witnessing system. Radiofrequency identification tags and barcodes were used for patient and sample identification. Since 2011, IVF and ICSI cycles and frozen embryo transfer cycles (FET) were included; IUIs cycles were included since 2013. The total number of tags and witnessing points were recorded. Witnessing points in a particular electronic witnessing system represent all the actions that have been performed from gamete collection through embryo production, to cryopreservation and transfer. Mismatches and administrator assigns were collected and stratified per procedure (sperm preparation, oocyte retrieval, IVF/ICSI, cleavage stage embryo or blastocyst embryo biopsy, vitrification and warming, embryo transfer, medium changeover, and IUI). Critical mismatches (such as mislabelling or non-matching samples within one work area) and critical administrator assigns (such as samples not identified by the electronic witnessing system and unconfirmed witnessing points) were selected. 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 witnessing points. The overall mismatch rate was 0.251% (2132/849 650) per witnessing point and 1.944% per cycle. In total, 144 critical mismatches occurred over the different procedures. The yearly mean critical mismatch rate was 0.017 ± 0.007% per witnessing point and 0.129 ± 0.052% per cycle. The overall administrator assign rate was 0.111% (940/849 650) per witnessing point and 0.857% per cycle, including 320 critical administrator assigns. The yearly mean critical administrator assign rate was 0.039 ± 0.010% per witnessing point and 0.301 ± 0.069% per cycle. Overall mismatch and administrator assign rates remained fairly stable during the evaluated time period. Sperm preparation and IVF/ICSI were the procedures most prone to critical mismatch and administrator assigns. The procedures and methods of integration of an electronic witnessing system may vary from one laboratory to another and result in differences in the potential risks related to sample identification. Individual embryos cannot (yet) be identified by such a system; this makes extra manual witnessing indispensable at certain critical steps where potential errors are not recorded. The electronic witnessing system still needs to be used in combination with manual labelling of both the bottom and lid of dishes and tubes to guarantee correct assignment in case of malfunction or incorrect use of radiofrequency identification tags. 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 proper training and attention of the staff is required. It may also induce new risks, i.e. blind witnessing of samples by the operator. No funding was either sought or obtained for this study. J.S. presents webinars on RIW for CooperSurgical. The remaining authors have nothing to declare. N/A.

Embryo biopsy and vitrification applied to a commercial embryo transfer program in Argentina

Use of embryo vitrification (EV) would have multiple advantages in a commercial embryo transfer (ET) program. EV would allow embryos to be transferred so that foals were born in early spring, which is preferred in Argentina due to health problems in foals that are born in late spring-summer. Currently, many high-level Polo mares compete in the spring, and the potential for embryo production from these mares is thus lost. EV would allow embryo storage against future loss or sale of a mare and allow owners to control which embryos were transferred in any given season. EV would also help the ET Center, by spreading work out through the breeding season and simplifying recipient synchronization. To explore the use of EV in our program, we compared pregnancy rates after transfer of vitrified-thawed vs. fresh embryos. Embryos were recovered on Day 7 to 8 post-ovulation from donor mares in our clinical program at Doña Pilar in September 2021 to May 2022 (Season 1) and then vitrified, and in September 2022 to January 2023 (Season 2) and transferred fresh. All embryos were transferred during Season 2. Fresh embryos were either biopsied via micromanipulation for sexing by PCR (BF, n=136) or were not biopsied (client did not request sexing orthe embryo was considered too small to biopsy, <∼300 µm diameter; F, n=62). Vitrified embryos were vitrified using a commercial human kit (CryoTech) after collapse of the blastocoele by micromanipulation, with biopsy (BV, n = 69) or without biopsy (MV, n = 9), or were vitrified without micromanipulation (<∼300 µm diameter; V). To date, the pregnancy rates per transferred embryo were BF 77/109 (70.6%); F 47/59 (79.6%); BV 24/36 (66.7%); MV 4/4 (100%) and V 9/14 (64.2%). There were no significant differences between treatments (Fishers exact test, p > 0.2). We conclude that embryo biopsy did not affect viability of fresh embryos, and that notably, the pregnancy rate was equivalent between fresh biopsied embryos (71%) and vitrified biopsied embryos (67%). The numerically lowest pregnancy rate resulted from embryos vitrified without manipulation. To the best of our knowledge, this is the first report on pregnancy rates of equine embryos vitrified after biopsy. These results indicate that a biopsy and vitrification program is feasible in a commercial embryo transfer center in Argentina. We foresee that in the near future most embryos collected after December 31st in our program will be vitrified and transferred between September and October of the following year.

Application of preimplantation genetic diagnosis in 91 equine ICSI embryos

Preimplantation genetic diagnosis (PGD) of in vitro-produced (IVP) equine embryos has been reported in research and clinical settings. In the previous reports, 4-16% biopsied samples failed to yield PCR results of disease-causing genes. There is little information on the feasibility of a second biopsy and refreezing of those PGD-failed frozen IVP equine embryos. In this report, we evaluated biopsy results from 91 IVP equine embryos produced from 2018 to 2022 and tested for hereditary equine regional dermal asthenia (HERDA), glycogen branching enzyme deficiency (GBED) and hyperkalemic periodic paralysis (HYPP) genes. We also examined the pregnancy and foaling rates of twice biopsied-frozen (2BF) embryos by comparing them with fresh or once biopsied-frozen (1BF) embryos. IVP embryos were produced by intracytoplasmic sperm injection and in vitro culture of shipped immature oocytes. Biopsy of embryos was performed as previously reported with minor modifications (Choi et al., Reprod Fertil Dev 2016;28:1382-1389). Biopsies were kept frozen for a maximum of 7 days and shipped to the University of California, Davis for genetic analysis. A total of 15 fresh, 39 1BF and 5 2BF IVP embryos were transferred to recipients at embryo transfer facilities. The non-transferred vitrified embryos, 12 1BF and 1 2BF IVP embryos, were kept frozen accordingto owners’ instructions. Among the 91 IVP biopsies, 7 (8%) failed to yield PCR results and 84 samples were genetically analyzed; 14 (24%) were homozygous for HERDA in 58 samples, 4 (21%) were homozygous for GBED in 19 samples and 0 were homozygous for HYPP in 7 samples. The molecular sexes of 84 samples were 44 males and 40 females. Among the 7 2BF IVP biopsies, 1 failed to yield PCR results and 6 were genetically analyzed; 1 was homozygous for GBED. The molecular sexes of 6 2BF samples were 1 male and 5 females. The pregnancy rates of fresh, 1BF and 2BF IVP embryos were not significantly different among groups (60% (9/15), 64% (25/39) and 80% (4/5), respectively, Fisher's exact test). Of these, 3 pregnancies from 1BF were removed from the foaling rate calculation because of unknown and ongoing pregnancies. The foaling rates of fresh, 1BF and 2BF IVP embryos were not significantly different among groups (47% (7/15), 33% (12/36) and 60% (3/5), respectively, Fisher's exact test). Out of 22 foals analyzed, all matched with molecular genders. In summary, PGD for disease-causing genes can be further improved by a second biopsy and refreezing of frozen IVP equine embryos. In addition, two biopsy-freezing cycles of IVP equine embryos were not detrimental to in vivo embryo development.

A study on methods for preimplantation genetic diagnosis on equine embryos

Determination of embryonic sex or the presence of alleles associated with genetic conditions is commonly applied in equine reproduction. To date, two methods for preimplantation genetic diagnosis (PGD) have been described for equine embryos: trophoblast cell biopsy (TCB) or blastocele fluid aspiration (BFA). While TCB has been described in both in vivo- (IVV) and in vitro-produced (IVP) embryos, BFA has been reported only for IVV embryos. Alternative methods for PGD, including analysis of cell-free DNA (CFD) in the medium where IVP has been cultured, are commonly used in humans, but not for equine embryos. In the current study, three experiments were performed to determine the agreement among methods for PGD on both IVV and IVP equine embryos. In Experiment 1, IVV (n = 10) and IVP (n = 13) embryos were subjected to BFA, cultured for 24 hours, subjected to TCB, and then cultured for additional 24 hours. No detrimental effect on embryonic diameter was observed for either embryo group (IVV: before BFA: 740 ± 618 µm vs. after TCB: 718 ± 526 µm; IVP: before BFA: 174 ± 8 µm vs. after TCB: 176 ± 10 µm; P > 0.05). In Experiment 2, the agreement among BFA, TCB, and the whole embryo (Whole) was studied by detecting the genes SRY (Y chromosome) and AR (X chromosome) using PCR. For IVV embryos (n = 14),there was a 100% agreement (14/14 embryos) between Whole and TCB for both SRY and AR, while a 64% agreement (9/14 embryos) between Whole and BFA, or between BFA and TCB. On the other hand, for IVP embryos (n = 13), there was a 75% agreement (10/13 embryos) between Whole and TCB for both SRY and AR, while a 31% agreement (4/13 embryos) between Whole and BFA, and a 46% agreement (6/13 embryos) between BFA and TCB. In Experiment 3, CFD was performed in IVP embryos (n = 10), to determine embryonic sex by means of whole genome amplification (WGA) followed by detection of the genes SRY (Y chromosome) and AMEL (X and Y chromosome) using PCR. For this, WGA was performed in both Whole, CFD samples, and Control medium (negative control). Overall, CFD samples yielded results for SRY and AMEL in 6/10 embryos (60%); the sex determined in CFD samples corresponded to the same results for the Whole group. None of the Control samples amplified any DNA. In conclusion, equine embryos can be subjected to 2 biopsy procedures without apparent detrimental effects on embryonic size. For IVV, but not IVP embryos, BFA can be considered a potential alternative to TCB for PGD. Finally, CFD can be further explored as a non-invasive method for PGD in IVP equine embryos.

First report of DNA detection and sex determination in leaked fluid after needle collapse of large equine blastocysts

The current method for sex determination of equine blastocysts involves aspiration of blastocoel fluid followed by PCR, which can be challenging due to the need for specialized equipment and often requires the embryo to be shipped to a separate lab for analysis. In this study, we investigated a new method for sex determination using leakage from manual collapse prior to vitrification, a technique which previously resulted in a pregnancy in our laboratory (1 of 3, unpublished results). We hypothesized that leaked blastocele fluid into the surrounding medium could provide sufficient DNA for amplification and sex determination. From a total of 14 embryos (900 to 1200 μm diameter), 4 were collapsed by micromanipulator-assisted aspiration (Micro group), and 10 were collapsed by manual puncture (Needle group) using a 32-G, penta-point needle (BD Nano, Canada). Additional blastocoel leakage (up to 80% of original volume) was obtained by forced pipetting using a 275 μm flexipet. All manipulations were conducted in TALP without serum. Samples were snap-frozen (30 μl) and stored at -80°C. DNA amplification was performed by multiplex PCR targeted to sex chromosome-linked zinc finger protein genes (ZFx/ZFy) for both sex DNA detection and the Y-encoded testis-specific protein (TSPY) for Y chromosome detection. The primer sequences were ZFx/y-F 5’-ATAATCACATGGAGAGCCACAAGCT-3’, ZFx/y-R 5’-GCACTTCTTTGGTATCTGAGAAAGT–3’, and TSPY-F 5′-GAAGTCAGGCACACCAGTGA-3′, TSPY-R 5′-TAAGGCTGCAGTTGTCATGC-3′, with 445bp and 280bp products, respectively. The reaction was carried out in a 25 μl final volume with 4 μl of PROMEGA Green GOTaq 5X Buffer, 0.2 μM primers, 1 mM MgCl2, 5 μl DNA sample, and 1.25 IU Taq DNA polymerase. The cycle conditions were 4 min 94°C, 40 cycles of 30 sec 94°C, 30 sec 58°C, and 20 sec 72°C followed by final elongation of 1 min 72°C. Equine male and female DNA was purified from whole blood (PROMEGA WizardTM Genomic DNA purification kit) and used as positive controls; female and male band patterns were used as reference. Our results showed that sex was confirmed in 4/4 (100%; 3 male and 1 female) of embryos biopsied by micromanipulation (Micro group) and in 5/10 (50%; 1 male and 4 female) of embryos collapsed by manual needle puncture (Needle group). Although the proportion of sex determination in the manually collapsed group is still low, it could be attributed to sample dilution effects. This report provides proof of concept of DNA detection and sex determination in the medium surrounding manually collapsed embryos without blastocoel aspiration. Our findings suggest that this approach to embryo biopsy for sex determination is promising, but more studies are needed to confirm its effectiveness.

Effect of breed and embryo sexing on the efficiency of a commercial OPU-ICSI program in mares

Breeders of Polo pony mares favour females over males as their sport performance is shown to be superior. This sex preference in breeding Polo horses is a challenge for stud veterinarians as sex sorted semen in horses is not available commercially due to technical difficulties. An alternative is the sexing of embryos. Ovum pick-up (OPU) and ICSI have become popular in show jumping (SJ) horses over the last decade, with the overall efficiency of the process improving steadily, with reported embryo per OPU session of > 1.5 embryos. The in vitro production of embryos not only allows a higher yield of blastocysts compared with in vivo derived embryos, but also their cryopreservation with similar pregnancy results similar to those achieved with fresh embryos. In addition, the possibility of sexing embryos before freezing would be an attractive option to the Polo breeding industry. Although there are several reports on the efficiency of an OPU-ICSI commercial program in SJ, information in Polo mares OPU-ICSI programs is scarce. The objective of this study was to describe the OPU-ICSI outcomes of a commercial program of Polo mares (n=77 cycles) compared to show jumping mares (n=73 cycles), including the likelihood of pregnancy after transferring sexed cryopreserved embryos. A general linear model of variance was created to test the effect of breed (Polo vs. SJ)on antral follicles available for aspiration, recovered oocytes, and number of embryos per OPU-ICSI session. Furthermore, the effect of embryo sexing prior to cryopreservation on pregnancy rate was determined by chi-square test. All OPUs were performed in the UK and oocytes were shipped to an external laboratory in Europe. Pre-implantation gender diagnosis was performed by extended embryo culture and sexing of extruded cells in 53 embryos of Polo mares. Polo mares had a lower (P<0.001) number of punctured follicles and recovered oocytes (14.1±0.6 and 8.2±0.5) compared to SJ mares (19.2±0.7 and 11.0±0.5), respectively. The mean number of embryos per OPU-ICSI session was similar (P>0.1) for SJ and Polo mares (1.38 and 1.41, respectively). Embryos derived from SJ mares (15/22, 68%) were more likely (P<0.05) to develop into a pregnancy than embryos from Polo mares (22/51, 43%). The ongoing pregnancy rate of sexed embryos (6/26, 23.1%) was lower (P=0.016) than that of unsexed embryos (14/25, 56%) from Polo mares, which explains the overall lower pregnancy outcome of Polo compared to SJ mares (unsexed embryos). In conclusion, Polo mares had a lower antral follicle pool than SJ mares, but their oocytes showed similar developmental potential to those of SJ mares. Embryo sexing decreased the chances of having a viable pregnancy after an OPU-ICSI session.

WS09.06 Genetic testing donors of gametes for cystic fibrosis should be mandatory

In vitro fertilization (IVF) has made it possible for many cystic fibrosis (CF) patients to become parents. It is a routine practice worldwide to test the partner, whether he/she is a genetic carrier, and if proven positive preimplantation diagnosis (PGD) is offered. However, PGD due to its high cost is not often performed when donor gametes are used, presuming the donors are healthy.