Euploidy Rates Research Articles

Reproductive outcomes after preimplantation genetic testing in couples with sex chromosome abnormalities: a retrospective cohort study of 83 couples.

To explore the effects of parental sex chromosome abnormality on their preimplantation embryos. This is a retrospective cohort study including 83 couples with sex chromosome abnormalities undergoing preimplantation genetic testing (PGT) between 2013 to 2023. The preimplantation genetic testing results and pregnancy outcomes were compared to those of a control group consisting of 166 age-matched couples with normal karyotypes who underwent preimplantation genetic testing for monogenic disorders (PGT-M). Student's t-tests, chi-square or Fisher's exact tests were applied to compare clinical characteristics. The embryo euploidy rate was lower (58.94% vs 65.44%, P = 0.048, OR 0.76, 95%CI [0.58,0.99]) and the sex chromosomal aneuploidy rate was higher in the couples with sex chromosome abnormalities than control group (6.62% vs 2.63%, P = 0.004, OR 2.63, 95%CI [1.37,5.05]). The pregnancy outcomes including clinical pregnancy rates (48.57% vs 57.25%, P = 0.305) and live birth rates (47.14% vs 52.90%, P = 0.465) were similar between the two groups in their first embryo transfer cycles. To avoid high risk of embryo aneuploidy and sex chromosome abnormalities, preimplantation genetic testing should be recommended to couples with sex chromosome abnormalities.

Comparison of Recombinant Cumulase with Bovine Derived Hyaluronidase for Oocyte Denudation before ICSI in Sibling Oocytes

Assisted Reproductive Technology (ART) with Intra-cytoplasmic Sperm Injection (ICSI) commonly utilizes Bovine-Derived Hyaluronidase for oocyte denudation. The emergence of Recombinant Cumulase as an alternative merit an examination of its impact upon ICSI outcomes. We compared the effects of Recombinant Cumulase and Bovine-Derived Hyaluronidase on oocyte denudation during ICSI, investigating impact upon fertilization rates, blastulation rates, and euploidy rates, aiming to improve clinical practice and optimize ART for patient outcomes. This was a retrospective observational study involving 59 patients undergoing ICSI conducted at First IVF Fertility Centre, Abu Dhabi, United Arab Emirates. Oocytes from each patient were divided one group which employed Recombinant Cumulase, and the other group utilised Bovine-Derived hyaluronidase. Parameters such as age, number of oocytes retrieved, denudation outcomes, and euploidy rates were assessed to determine enzyme impact. Comparative analysis revealed no substantial difference in fertilisation rates or embryo development (blastocyst rate) between recombinant Cumulase and bovine-derived Hyaluronidase. However, the recombinant Cumulase group exhibited significantly higher (p<0.05) rates of normal euploid embryos (Recombinant Cumulase-48.5%; Bovine Derived Hyaluronidase- 38.0%), indicating a potential advantage. However, the results are from small cohort and need to be validated with large sample size. Selecting enzymes for ART procedures is challenging. Although no significant differences were observed in fertilisation rates, the Recombinant Cumulase group treated oocytes exhibited yielding higher normal euploid embryo rates. Not many studies have looked at the Euploidy rates with use of different enzymes for oocyte denudation. Further investigations are essential to refine best practices and advance patient outcomes in assisted reproduction.

Effects of ovarian stimulation on embryo euploidy: an analysis of 12874 oocytes and 3106 blastocysts in cycles with preimplantation genetic testing for monogenic disorders.

Does ovarian stimulation and the ovarian response affect embryo euploidy? Ovarian stimulation and the ovarian response in women undergoing preimplantation genetic testing for monogenic disorders (PGT-M) cycles did not affect the rates of blastocyst euploidy. Whether or not ovarian stimulation in IVF-embryo transfer has potential effects on embryo euploidy is controversial among studies for several reasons: (i) heterogeneity of the study populations, (ii) biopsies being performed at different stages of embryo development and (iii) evolution of the platforms utilized for ploidy assessment. Patients who undergo PGT-M cycles typically have no additional risks of aneuploidy, providing an ideal study population for exploring this issue. A retrospective cohort study including embryos undergoing PGT-M was conducted at a single academically affiliated fertility clinic between June 2014 and July 2021. A total of 617 women with 867 PGT-M cycles involving 12874 retrieved oocytes and 3106 trophectoderm biopsies of blastocysts were included. The primary outcome of the study was median euploidy rate, which was calculated by dividing the number of euploid blastocysts by the total number of biopsied blastocysts for each cycle. Secondary outcomes included the median normal fertilization rate (two-pronuclear (2PN) embryos/metaphase II oocytes) and median blastulation rate (blastocyst numbers/2PN embryos). Comparable euploidy rates and fertilization rates were observed across all age groups, regardless of variations in ovarian stimulation protocols, gonadotropin dosages (both the starting and total dosages), stimulation durations, the inclusion of human menopausal gonadotrophin supplementation, or the number of oocytes retrieved (all P > 0.05). Blastulation rates declined with increasing starting doses of gonadotropins in women aged 31-34 years old (P = 0.005) but increased with increasing gonadotrophin starting doses in women aged 35-37 years old (P = 0.017). In women aged 31-34, 35-37, and 38-40 years old, blastulation rates were significantly reduced with increases in the number of oocytes retrieved (P = 0.001, <0.001, and 0.012, respectively). Limitations include the study's retrospective nature and the relatively small number of patients of advanced age, especially patients older than 40 years old, leading to quite low statistical power. Second, as we considered euploidy rates as outcome measures, we did not analyze the effects of ovarian stimulation on uniform aneuploidy and mosaicism, respectively. Finally, we did not consider the effects of paternal characteristics on embryo euploidy status due to the fact that blastocyst aneuploidy primarily originates from maternal meiosis. However, sperm factors might have an effect on embryo development and the blastulation rate, and therefore also the number of blastocysts analyzed. The exclusion of patients with severe teratozoospermia and the fact that only ICSI was used as the insemination technique for women undergoing PGT-M contributed to minimize the effect of paternal factors. Ovarian stimulation and response to stimulation did not affect blastocyst euploidy rates in women undergoing PGT-M cycles. However, in women aged 31-40 years old, there was a significant decline in blastulation rates as the number of retrieved oocytes increased. This study was supported by the National Natural Science Foundation of China (Grant No. 81701407, 82301826); the National Key Research and Development Program of China (2022YFC2702901, 2022YFC2703004); China Postdoctoral Science Foundation (2022M710261), and China Postdoctoral Innovation Talent Support Program (BX20220020). There is no conflict of interest. N/A.

Cytoplasmic strings in human blastocysts: hypotheses of their role and implications for embryo selection.

What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts? Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality. Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial. Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers. ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics. About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10). Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes. Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment. None. N/A.

Comparative analysis of short versus long co-incubation of gametes on post-insemination outcomes and embryo morphokinetics: a sibling oocyte randomized study

Research questionDoes a short co-incubation of gametes in conventional IVF impact post-insemination outcomes and embryo morphokinetics? DesignSibling oocyte randomized pilot study performed between December 2020 and March 2023. Eligible couples (n=55) were female aged 18-43 years with body mass index (BMI) ≤35 and male normal semen parameters. Cumulus oocyte complexes (COCs) (min 6 - max 12) were randomized in a 1:1:2 proportion in long (16-18 hrs), or short (2 hrs) co-incubation IVF exposure, and ICSI, respectively. All oocytes inseminated via IVF were monitored by time-lapse (TL). Blastocysts of ≥BL3CC were subjected to trophectoderm (TE) biopsy on day (D)5, D6 or D7 by next generation sequencing (NGS). ResultsA total of 954 COCs were distributed to long co-incubation (n=235, 24.6%), short co-incubation (n=235, 24.6%), and ICSI (n=484, 50.3%). In total, 202 were MII after long co-incubation, 209 after short co-incubation and 394 at ICSI. Regression analyses showed both short co-incubation groups (OR: 0.72, 95% CI: 0.46–1.11, P=0.139) and ICSI (OR: 1.43, 05% CI: 0.95–2.15, P=0.090) did not significantly impact normal fertilization rates (2PN) when compared to long co-incubation. Usable blastocysts per MII, per 2PN, and euploid blastocysts per MII, per 2PN and per tested blastocysts were similar among the groups. The rate of embryo arrest (HR: HR: 0.79, 95% CI: 0.57-1.11, P=0.183) and timings to reach morphokinetic milestones among embryos reaching blastulation were similar between IVF groups (P>0.05, for all). ConclusionA short 2-hour co-incubation of gametes in IVF yields comparable fertilization, blastocyst, and euploidy rates without adverse embryo morphokinetic events, compared to standard overnight co-incubation.

Feasibility of preimplantation genetic testing for aneuploidy on frozen-thawed embryos following conventional IVF insemination.

Intracytoplasmic sperm injection (ICSI) is commonly employed in preimplantation genetic testing (PGT) to minimize the risk of foreign sperm DNA contamination. Cryopreserved embryos from patients with recurrent miscarriage or repeated implantation failure, who have undergone conventional in vitro fertilization (IVF), can be thawed and biopsied for PGT. Therefore, we aimed to assess the accuracy and effectiveness of preimplantation genetic testing for aneuploidy (PGT-A) on frozen embryos using conventional IVF (c-IVF) insemination methods. From January 2021 to November 2023, our center conducted 107 thawed cryopreserved embryo biopsy cycles to screen for PGT-A. Among them, 58 cycles used c-IVF insemination, and 49 used ICSI insemination. Basic patient clinical information, laboratory data, PGT test results, and clinical outcome data were collected. To minimize the confounding effects of patient characteristics and embryo quality on PGT-A outcomes, clinical outcomes, and contamination assessment, these variables were included in the analysis. We then evaluated the blastocyst euploidy rate, clinical outcomes, and accuracy of PGT-A results between the two groups and analyzed potential contamination in the c-IVF insemination group. A total of 320 blastocysts underwent PGT-A testing, with 179 blastocysts from c-IVF insemination and 141 from ICSI insemination. Considering participants' baseline characteristics and embryological outcomes, no significant differences were found between the two groups regarding infertility type, average age, body mass index, percentage of PGT-A indications, or quality of embryonic development. Regarding PGT-A results, all 320 biopsy samples were successfully analyzed, showing no statistical variance in chromosomal euploidy, abnormality, or mosaicism rates between the two insemination methods. No parental contamination was detected in the c-IVF insemination group. When assessing clinical outcomes, parameters such as biochemical pregnancy, clinical pregnancy, and miscarriage rates did not exhibit significant discrepancies between the two groups, and no misdiagnoses were reported during the study period. Embryo transfer and PGT-A results are not affected by potential parental contamination in frozen-thawed embryos conceived via c-IVF. PGT-A guided embryo transfer in thawed embryos conceived by c-IVF is a viable and clinically effective approach.

Progestin-primed ovarian stimulation

The use of progestins for the prevention of premature ovulation during ovarian stimulation is called progestin-primed ovarian stimulation (PPOS). Response to ovarian stimulation with PPOS and gonadotropin-releasing hormone (GnRH) analogs seem to be similar with the clinically insignificant differences being explained by endocrine characteristics as expected. PPOS has the advantage of oral administration and lower medication cost than GnRH analogs, but the mandatory cancellation of an otherwise possible fresh embryo transfer can render PPOS less cost-effective if a fresh transfer would have been possible. Oocytes collected in PPOS cycles have similar developmental potential, including blastocyst euploidy rates. Frozen embryo transfer outcomes of PPOS and GnRH analog cycles seem similar both in terms of ongoing pregnancy/live birth rates and obstetric and perinatal outcomes. While some studies reported lower cumulative live birth rates with PPOS, they suffer from methodological issues, including arbitrary definitions of cumulative live birth rates. PPOS has been used in all patient types with consistent results and seems a patient-friendly and cost-effective choice when a fresh embryo transfer is not intended.

Blastocysts originated from oocytes with smooth endoplasmic reticulum aggregates have a reduced euploidy rate: a retrospective cohort study.

Does the presence of smooth endoplasmic reticulum aggregates (SERa) in oocytes adversely impact the euploidy rate of subsequent blastocysts? We performed a retrospective cohort study with 671 young patients (< 38 years) undergoing their first preimplantation genetic testing for aneuploidy (PGT-A) between January 2019 and October 2022 at a reproductive medical center of university affiliated teaching hospitals in China. Cycles were categorized as either SERa(+) cycles (containing at least one SERa(+) oocyte) or SERa(-) cycles (all oocytes without SERa). In SERa(+) cycles, oocytes were further subdivided into the SERa(+) oocyte group and the sibling SERa(-) oocyte group, comprising oocytes with normal morphology. No significant differences were observed in the normal fertilization rate (72.9% vs. 75.4% vs. 72.6%, P=0.343), and cleavage rate (96.8% vs. 97.1% vs. 96.4%, P=0.839) among the SERa(-) cycle group, the SERa(-) oocyte group, and the SERa(+) oocyte group. Additionally, there were no statistically significant differences in the rates of good quality embryos (44.7% vs. 48.8% vs. 46.2%, P=0.177) or blastocyst formation (60.1% vs. 60.9% vs. 60.5%, P=0.893) among the groups. However, the euploidy rate of blastocysts derived from SERa(+) oocytes was significantly lower compared to those from SERa(-) oocytes in SERa(+) cycles and normal oocytes in SERa(-) cycles (39.3% vs. 51.2% vs. 54.5%, P=0.005). Despite this, there were no significant differences in pregnancy and neonatal outcomes after euploid embryo transfer among the three groups. Blastocysts derived from SERa(+) oocytes have a lower euploidy rate than those derived from SERa(-) oocytes. Nevertheless, comparable reproductive outcomes were achieved following euploid embryo transfer from both SERa(+) and SERa(-) oocytes.

Live Motile Sperm Sorting Device Improves Embryo Aneuploidy: A Retrospective Cohort Study

Background: Conventional sperm selection methods, involving centrifugation, exert a detrimental effect on sperm DNA integrity due to mechanical stress. The recent noninvasive sperm selection device, CA0, based on live sperm sorting technology, facilitates the retrieval of highly motile sperm and minimizes sperm DNA fragmentation (SDF). This study was to investigate the impact of various sperm separation methods, with and without centrifugation, on embryo ploidy status. Methods: The retrospective study comprised 82 intracytoplasmic sperm injection (ICSI) cycles involving preimplantation genetic testing for aneuploidy (PGT-A) cases, with a focus on recruiting egg donation cycles to thoroughly investigate the impact of male factors. Two populations are classified based on semen quality: normozoospermic ([Formula: see text] = 33) and non-normozoospermic ([Formula: see text] = 49). Subjects were allocated to either swim-up (SU) or CA0. Preimplantation genetic testing results were recorded. Results: When comparing male characteristics between subgroups, no significant differences were observed except for a lower normal morphology rate in the CA0 group compared to SU (SU: 3 [3–4] vs. CA0: 2 [2–2.8], [Formula: see text] &lt; 0.0001) in the non-normozoospermic cohort. There were no differences in female factors such as age and mature oocyte count (MII) number between subgroups, indicating that this model is ideal for assessing the impact of male factors on clinical outcomes. In the normozoospermic cohort, euploidy rates were similar between SU and CA0 (SU: 71.9% vs. CA0: 64.2%). However, in the non-normozoospermic cohort, CA0 showed a significantly higher euploidy rate compared to SU (SU: 53.6% vs. CA0: 74.2%) and a lower aneuploidy rate (SU: 37.1% vs. CA0: 25.8%). Additionally, CA0 minimized the incidence of mosaic embryos, whereas a mosaicism rate of 9.3% was observed with SU. This trend highlights CA0’s distinct advantage in optimizing outcomes for non-normozoospermic cases. Conclusions: CA0 is a reliable intervention to optimize paternal genetic quality before assisted insemination, thereafter effectively reducing the incidence of embryo aneuploidy associated with male factors.

The impact of low oocyte maturity ratio on blastocyst euploidy rate: a matched retrospective cohort study

ObjectiveTo investigate the association between a low oocyte maturity ratio from in vitro fertilization cycle and blastocyst euploidy.MethodsA total of 563 preimplantation genetic testing (PGT) cycles (PGT cycles with chromosomal structural rearrangements were excluded) were performed between January 2021 and November 2022 at our center (average oocyte maturity rate: 86.4% ± 14.6%). Among them, 93 PGT cycles were classified into the low oocyte maturity rate group (group A, < mean − 1 standard deviation [SD]), and 186 PGT cycles were grouped into the average oocyte maturity rate group (group B, mean ± 1 SD). Group B was 2:1 matched with group A. Embryological, blastocyst ploidy, and clinical outcomes were compared between the two groups.ResultsThe oocyte maturity (metaphase II [MII oocytes]), MII oocyte rate, and two pronuclei (2PN) rates were significantly lower in group A than in group B (5.2 ± 3.0 vs. 8.9 ± 5.0, P = 0.000; 61.6% vs. 93.0%, P = 0.000; 78.7% vs. 84.8%, P = 0.002, respectively). In group A, 106 of 236 blastocysts (44.9%) that underwent PGT for aneuploidy were euploid, which was not significantly different from the rate in group B (336/729, 46.1%, P = 0.753). However, euploid blastocysts were obtained only in 55 cycles in group A (55/93, 59.1%), which was lower than the rate in group B (145/186, 78.0%, P = 0.001). The clinical pregnancy rate in group B (73.9%) was higher than that in group A (58.0%) (P = 0.040).ConclusionOur results suggest that a low oocyte maturity ratio is not associated with blastocyst euploidy but is associated with fewer cycles with euploid blastocysts for transfer, lower 2PN rates, and lower clinical pregnancy rates.

P-074 The effect of sperm from male patients who use electronic cigarettes on the euploidy ratio of embryos during in vitro fertilization procedures

Abstract Study question What is the effect of electronic cigarettes as a substitute for regular cigarettes on sperm and subsequent embryo development in IVF patients? Summary answer E-cigarettes are just as harmful to male fertility as regular cigarettes, reduce the euploidy rate of the embryo, and have negative effects on pregnancy. What is known already Many harmful substances from smoking are known to negatively affect sperm development and function. It causes high elevations in sperm DNA fragmentation (SDF), which are associated with embryo euploidy rates. Recently, the use of e-cigarettes is increasing as an alternative to regular cigarettes because they are less harmful and safer. However, several animal studies have shown that e-cigarettes are just as harmful as regular cigarettes and can have negative effects on sperm and fertility. Preliminary data showed that SDF increased with e-cigarettes compared to regular cigarettes. Therefore, we would like to find out whether e-cigarettes affect embryo euploidy. Study design, size, duration In this study, from January 2022 to December 2023, 1,725 cycles of PGT-A (PGT for aneuploidy) were analyzed among the patients of IVF. The average ages of men and women were 40.0±4.6 and 38.7±4.0. A total of 4,796 embryos were analyzed (2.8 ± 2.0 per case) for euploidy rates. Participants/materials, setting, methods Blastocyst biopsy and PGT-A were performed, and embryo euploidy rates was confirmed. In male, the number of no smoking IVF cycles was 1061 and the number of embryo biopsies was 2.9 ± 2.1. The number of IVF cycles using regular cigarettes is 370, and the number of embryo biopsies is 2.8 ± 2.1. The number of IVF cycles using e-cigarettes was 294, and the number of embryo biopsies was 2.5 ± 1.6. Main results and the role of chance Based on the total female age, the embryo euploidy rate was nonsmoking (27.3%), regular cigarettes (23.6%), and e-cigarettes (18.8%). Among women under 38 years of age, e-cigarettes (29.9%) had a lower euploidy rate than regular cigarettes (37.4%), and nonsmoking (38.2%) had the highest rate. Among women over 38, the euploidy rate dropped significantly compared to those under 38, including nonsmoking (18.9%), but it was also lower for e-cigarettes (11.6%) than regular cigarettes (17.7%). Based on female age under 38 years old, the embryo euploidy rate among male age under 39 years old was nonsmoking (39.0%), regular cigarettes (38.3%), and e-cigarettes (31.7%). And among men over 40, the group with the lowest embryo euploidy rate was those who used e-cigarettes (no smoking (35.0%), regular cigarettes (32.6%), e-cigarettes (27.7%)). Based on the age of women over 38 years old, the euploidy rate among men under 39 years of age is nonsmoking (23.9%), regular cigarettes (21.6%), and e-cigarettes (12.7%). And the euploidy rate among men over 40 years of age is nonsmoking (16.8%), regular cigarettes (16.5%), and e-cigarettes (11.8%). nonsmoking is essential to maintain a high euploidy rate in embryos, and e-cigarettes may have a more adverse effect on pregnancy than regular cigarettes. Limitations, reasons for caution Because there are various types of e-cigarettes, it is expected that the degree of harmfulness will vary, but the number of patients was not sufficient to conduct research on each type of e-cigarette. Therefore, it is believed that the above problems should be solved through additional research. Wider implications of the findings This study is a study that links the harmful effects of cigarettes and the polyploidy of embryos. Electronic cigarettes, used as an alternative to regular cigarettes, may reduce embryo euploid production and cause recurrent pregnancy failure. Therefore, it is believed that electronic cigarettes should also be managed appropriately. Trial registration number not applicable

P-612 Euploidy rates between cycles triggered with gonadotrophin-releasing hormone agonist (GnRHa), human chorionic gonadotrophin (hCG) and dual trigger

Abstract Study question Is there any difference in euploidy rates between in vitro fertilization (IVF) cycles triggered with GnRHa, hCG or dual trigger? Summary answer There is no difference in the euploidy rate in preimplantation genetic testing for aneuploidy (PGT-A) cycles according to the trigger method used. What is known already The use of PGT-A has shown that the success of meiosis in the IVF laboratory is inversely correlated with maternal age. However, other uninvestigated factors involved in the IVF process may interfere with normal oocyte physiology. Since the final maturation obtained after ovarian stimulation depends on the use of drugs to promote the resumption of meiosis, it is worth investigating whether the triggering method could impact on oocyte and embryo euploidy. This study aims is to evaluate differences in embryo euploidy rates between PGT-A cycles triggered with GnRHa, hCG or dual trigger (GnRHa + hCG). Study design, size, duration This is a single-center retrospective study of 934 patients who underwent 1085 PGT-A cycles by Next-Generation Sequencing (NGS) from January 2017 to December 2023, using one of three possible trigger methods to induce final oocyte maturation: GnRHa, hCG, or dual trigger. The couples included in the study had normal karyotypes and no severe male factor. Participants/materials, setting, methods Data on the number of oocytes retrieved, ratio of metaphase II (MII) oocytes and fertilization rate, blastocyst development and embryo euploidy were assessed and compared between the GnRHa, hCG and dual trigger treatments. A secondary analysis was performed to evaluate data stratified by patient age. Main results and the role of chance A total of 1085 PGT-A cycles involving 934 patients (38.07 ± 3.01 years) and 2803 biopsied blastocysts were included in the analysis. Dual trigger was used in 726 (77.73%) cycles, GnRHa in 287 (26,4%) cycles and hCG in 72 (7.71%) cycles. The number of oocytes retrieved was significantly higher in the GnRHa group (GnRHa: 18.72 ± 8.59; hCG: 9.88 ± 5.64; dual trigger: 10.58 ± 5.65; p &amp;lt; 0.001) with similar maturation rate (GnRHa: 77.46%; hCG: 74.58%; dual trigger: 76.03%; p = 0.789), fertilization rate (GnRHa: 78.12%; hCG: 76.37%; dual trigger: 79.12%; p = 0.168), blastulation rate (GnRHa: 61.03%; hCG: 58.32%; dual trigger: 58.73%; p = 0,629), high-quality blastocyst rate (GnRHa: 27,88%; hCG: 33,67%; dual trigger: 27,88%; p = 0.543) and embryo euploidy per biopsied embryo (GnRHa: 42.48%; hCG:45.49%; dual trigger:46.98%; p = 0.112). Analysis of the data stratified by age showed similar rates of euploidy rates with the three trigger methods in women younger than 41 years (&amp;lt;35 years: GnRHa 66.50%; hCG 66.67%; dual trigger 56.97% p = 0.290; 35-37 years: GnRHa 49.45% hCG 51.04%; dual trigger 50.23%; p = 0.988; 38-40 years: GnRHa 40.93%; hCG 41.27%; dual trigger 39.13% p = 0.257). However, in patients ≥41 years old the euploidy rate was statistically higher in the GnRHa group (GnRH 31.51%; hCG 18.75%; dual trigger 16.89%; p = 0.032). Limitations, reasons for caution The main limitations of the study are: its retrospective design, the possibility that the trigger method was chosen based on the ovarian response and the limited number of patients in both the hCG trigger and women ≥ 41 years groups. Wider implications of the findings Our results suggest that triggering with GnRHa, hCG or dual trigger leads to comparable euploidy rates. Triggering with GnRHa should be recommended in older patients since it has been associated with a higher rate of embryonic euploidy. Trial registration number Not Applicable

P-009 Impact of sperm preparation methods on embryo development rate and embryo ploidy status: ICSI egg donation as a model

Abstract Study question Do different sperm separation techniques result in distinguishable contributions to embryo development rates and ploidy status? Summary answer CA0 sperm separation device, devoid of centrifugation, yielded a higher euploidy rate than conventional swim-up (50% vs. 74%, p = 0.02), notably observed in abnormozoospermic cases. What is known already Sperm DNA fragmentation (SDF) emerges as a key paternal risk factor associated with embryo aneuploidy, increasing the risk of ART failures. Factors like advanced male age, prolonged ejaculatory abstinence, and the detrimental effects of centrifugation during sperm separation jeopardize sperm DNA integrity. A recent centrifugation-free sperm separation device (CA0), employing live sperm sorting technology, exhibits efficiency in producing highly motile sperm with minimized SDF. This retrospective cohort study solely focusing on ICSI egg donation cycles to assess the influence of sperm separation methods (conventional swim-up vs. CA0) on early embryo development, and embryo ploidy. Study design, size, duration This retrospective study included 83 couples who underwent ICSI egg donation cycles at Chachava Clinic, Tbilisi, Georgia. Thirty-three semen specimens were processed using conventional swim-up (SU) and 427 Mii were inseminated. Forty-four semen samples were separated by CA0 and 519 Mii were inseminated. Clinical outcome measures included fertilization rate (number of 2PN over number of inseminated oocyte), good embryo rate (embryo number with more than 8 cells over inseminated oocyte), and euploidy rate using PGT-A. Participants/materials, setting, methods Male age and basic semen parameters via CASA (CEROS II, Hamilton-Thorne) were recorded. SU involved layering 1 mL neat semen to 1.2 mL HTF medium, inclining the tube at 45° for 60 min at 37 °C, collecting 1 mL uppermost, adding 2 mL HTF for centrifugation at 500×g for 5 min, and resuspending sperm pellet with 0.5 mL HTF. Semen samples were sorted using LensHooke® CA0 device following the manufacturer’s instructions. Main results and the role of chance In comparing demographic characteristics between SU and CA0 arms, CA0 exhibited higher median total motility (SU: 40% vs. CA0: 54%, p = 0.02), with no significant differences in female age and Mii oocyte count. This study assessed the impact of sperm separation methods on early embryo development, finding no significant differences in fertilization rate (SU: 79% vs. CA0: 77%) and good embryo rate (SU: 64% vs. CA0: 62%). However, CA0 demonstrated a significantly higher euploidy rate than SU (SU: 59% vs. CA0: 69%, p = 0.03). Further analysis within subpopulations, including normozoospermic and asthenozoospermic (total motility &amp;lt;40%), revealed equivalent euploidy rates between SU and CA0 for normospermic specimens (SU: 66% vs. CA0: 68%). However, a significant differentiation in euploidy rate emerged for asthenozoospermic cases (SU: 59% vs. CA0: 74%, p = 0.02). Specifically, comparing outcomes in normozoospermic, a significant reduction was observed in asthenozoospermic cases using SU (66% vs. 50%, p = 0.02), whereas CA0 provided improved euploidy rates regardless of semen quality (68% for normozoospermic and 74% for asthenozoospermic). In conclusion, this study suggests that the non-invasive live sperm sorting device enhances embryo euploidy rates, serving as a reliable sperm preparation method, ensuring selection consistency despite sample variations. Limitations, reasons for caution This study’s limitations include its retrospective nature and single-center trial design. To more effectively assess CA0 efficiency, a randomized controlled trial targeting diverse patient eligibility criteria is warranted. Wider implications of the findings Attributed to the improved embryo euploidy with CA0, we anticipate enhanced ART outcomes, such as increased embryo transfer rate and prevention of ART failures related to embryo aneuploidy. Additionally, CA0 offers practical benefits by streamlining and standardizing the sperm separation process with only three steps involved. Trial registration number not applicable

P-090 Is preimplantation genetic testing for aneuploidy (PGT-A) necessary in the treatment strategy for ICSI with testicular sperm?

Abstract Study question Does the combination of testicular sperm extraction (TESE) and ICSI increase the risk of embryo aneuploidy? Summary answer Embryo aneuploidy occurrence is not increased when using testicular sperm and is not affected by the etiology of azoospermia. What is known already TESE-ICSI, especially in patients with non-obstructive azoospermia (NOA), has been reported to result in poor outcomes and difficulty in obtaining good blastocysts, which may be due to immature sperm retrieved by TESE. In addition, patients who require TESE have been shown to be at particularly high risk of chromosomally abnormal sperm. These factors may increase the risk of embryo aneuploidy in TESE-ICSI and contribute to poor outcomes. However, few reports have investigated the results of PGT-A following TESE-ICSI. This study was therefore designed to investigate whether using testicular sperm is more likely to result in embryo aneuploidy. Study design, size, duration This retrospective study included 81 couples and 197 TESE-ICSI cycles undergoing PGT-A (average maternal age: 38.1±5.4 years) between September 2016 and November 2023. Of the total cycles, 94 cycles were case of obstructive azoospermia (OA) and 53 cycles were cases of NOA. The etiologies of NOA included were unexplained (22 cycles), Klinefelter’s syndrome (KS) (10 cycles), post-chemotherapy (3 cycles), post-orchiopexy (11 cycles), and microdeletion of azoospermia factor (AZF) c on the Y chromosome (7 cycles). Participants/materials, setting, methods ICSI outcomes were compared between different etiologies (OA vs. NOA). A total of 356 blastocysts acquired were analyzed using PGT-A with next-generation sequencing (NGS). The PGT-A results were classified according to maternal age (&amp;lt;38, 38-41, &amp;gt;41 years) and paternal age (&amp;lt;40, ≥40 years). In addition, TESE-ICSI cycles were limited to the maternal age of &amp;lt; 38 years to remove the effect of maternal factors, the euploidy rates were compared between different etiologies or morphological grade. Main results and the role of chance The rates of fertilization and blastocyst formation for TESE-ICSI were significantly lower in NOA than OA (47.4% vs. 61.6%, P &amp;lt; 0.001 and 37.3% vs. 50.7%, P &amp;lt; 0.01, respectively). The rate of good-quality blastocyst (16.6% vs. 21.2%) was also lower, but not statistically significant. The euploidy rate of all evaluated TESE-ICSI embryos decreased with increasing maternal age (52.9% in &amp;lt; 38, 29.3% in 38-41, 10.0% in &amp;gt; 41 years), but was not affected by the paternal age (40.6% in &amp;lt; 40, 42.5% in ≥ 40 years). For cycles with the maternal age of &amp;lt; 38 years, the euploidy rate was not significantly different between OA and NOA patients (55.3% vs. 49.3%). In addition, the association between embryo euploidy and morphological grade (using Gardner’s classification, ≥BB for good-quality vs. &amp;lt;BB for poor-quality embryos) was evaluated, and the euploidy rate was significantly higher in good-quality embryos compared to poor-quality embryos (60.3% vs. 33.3%, P &amp;lt; 0.01). These results indicate the frequency of embryo aneuploidy in TESE-ICSI is comparable to commonly used ejaculated sperm. Furthermore, it suggests that embryo aneuploidy is dependent on maternal age or morphological grade of embryo, but not on paternal age or the etiology of azoospermia. Limitations, reasons for caution Patients with chromosomal abnormalities, i.e. KS, could not be evaluated due to the small number of cases and patients with chromosomal translocation were not included in this study. Therefore, the effects of male factors in NOA on embryo aneuploidy require extended investigation. Wider implications of the findings In TESE-ICSI, especially in the case of NOA, it is difficult to obtain good quality embryos due to poor ICSI outcomes. However, if good quality embryos are obtained, the euploidy rate is high and there is no need to perform PGT-A on the sole basis of using testicular sperm. Trial registration number not applicable

P-547 Impact of SARS-COV-2 infection on embryo euploidy rate: a retrospective cohort study of 498 PGT-A cycles in China

Abstract Study question Does SARS-CoV-2 infection before oocyte retrieval have any impacts on embryo euploidy rate? Summary answer SARS-CoV-2 infection before oocyte retrieval had no detrimental impact on embryo euploidy rate, indicating that cycle cancellation due to SARS-CoV-2 infection may not be necessary. What is known already SARS-CoV-2 infection has significant effects on the health of people worldwide and poses a serious threat to reproductive health. According to the previous studies, SARS-CoV-2 infection had negative impacts on the blastocyst formation rate, top-quality embryos rate. However, the association between the infection and the embryo euploidy rate is indeterminate. It is still worth exploring the impact of SARS-CoV-2 infection on embryo euploidy rate. Especially this is the key question to be addressed for providing appropriate counseling to couples seeking for preimplantation genetic testing for aneuploidy (PGT-A) treatments during the ongoing pandemic. Study design, size, duration This was a retrospective cohort study of 498 couples who underwent PGT-A cycles with next-generation sequencing technology from November 1st, 2022 to June 30th, 2023 at a tertiary-care medical center in China. Participants/materials, setting, methods Women tested positive for SARS-CoV-2 before oocyte retrieval were included in the COVID-19 group, while patients who were asymptomatic and tested negative during the same period were included in the non-COVID-19 group. The primary outcome was the euploidy rate. Subgroup analysis was conducted according to the time interval between infection and oocyte retrieval. Univariate and multivariate logistic regression models were applied to adjust for potential confounders. Main results and the role of chance A total of 278 and 220 women were included in the COVID-19 and non-COVID-19 group, respectively. In the COVID-19 group, the maturated oocyte rate (79.5% vs. 83.7%, p＜0.001), number of total viable embryos (2 vs. 3, p=0.038), and number of euploid blastocyst (1 vs. 1, p=0.028) were significantly decreased compared with non-COVID-19 group. The euploidy rate (38.5% vs.40.3%, p = 0.451), aneuploidy rate (45.0% vs. 43.8%, p=0.616), and mosaicism rate (16.5% vs. 15.9%, p=0.746) were all comparable between the two groups. Results of multivariate logistic regressions revealed no association between SARS-CoV-2 infection and euploidy rate, aneuploidy rate, or mosaicism rate. Similarly, no association was detected between the time interval of infection and oocyte retrieval (≤30 days, 31-60days, 61-90 days, 91-120 days, or ≥ 120 days prior to oocyte retrieval) and euploidy rate. Limitations, reasons for caution The retrospective nature of the study and limited sample size for subgroup analysis limits the generalizability of the findings. Other potential confounders, such as the level of SARS-CoV-2 antibody, symptoms of infection, are needed to be taken into consideration as well. Large-scale prospective studies are warranted to confirm the conclusion. Wider implications of the findings Our study demonstrated that SARS-CoV-2 infection before oocyte retrieval had no detrimental impact on embryo euploidy rate, indicating that cycle cancellation due to SARS-CoV-2 infection may not be necessary. Trial registration number Not Applicable

P-050 Microfluidic chip-sorted spermatozoa selection yields embryo euploidy rate and reproductive outcomes similar to density-gradient centrifugation

Abstract Study question Does microfluidic chip-sorted spermatozoa selection (MSS) improve good quality blastocyst (GQB) and embryo euploidy rates compared to density gradient centrifugation (DGC)? Summary answer The use of MSS before intracytoplasmatic sperm injection (ICSI) does not improve good quality blastocist and euploidy rates comparing to DGC. What is known already The integrity of sperm DNA plays a crucial role in embryonic development. Conventional sperm selection methods, such as DGC, may potentially damage sperm DNA due to oxidative stress generated during centrifugation. One of the recent advances in sperm selection techniques is the microfluidic chamber, which, requiring less processing time and eliminating the need for centrifugation, allows for the selection of sperm with better mobility, morphology, and reduced DNA damage. Consequently, improved GQB and euploid embryo rates, as well as enhanced reproductive outcomes, are expected. Study design, size, duration Retrospective study of 1739 blastocysts from 404 couples undergoing 544 autologous cycles of ICSI/PGT-A (preimplantation genetic testing for aneuploidies) from January 2020 to December 2022. Those patients with sperm concentration &amp;lt;5 × 106/mL or motility &amp;lt;10%, altered karyotype, women aged &amp;gt; 43 years, uterine anomalies and thrombophilia were excluded. Besides comparing euploidy rates and blastocyst formation, reproductive outcomes were analyzed and compared. Participants/materials, setting, methods According to the sperm selection technique, two groups were established. One group underwent MSS (ZyMōt ICSI® or ZyMōt Multi®), resulting in 295 blastocysts from 96 cycles. It was compared with a control group (DGC) including 1444 blastocysts from 448 ICSI/PGT-A cycles. PGT-A was performed in day 5/6 using Next Generation Sequencing (NGS) technology. For a 95% confidence interval, statistical significance was considered at p &amp;lt; 0.05. Chi-square analysis was used for the statistical evaluation. Main results and the role of chance Both groups were comparable in terms of demographic data, including age, sperm analysis parameters, and cycle characteristics. There were no significant differences between the DGC and MSS groups in the women’s age (mean ± SD) (37.9 ± 3.9 vs. 38 ± 3.9 years, p = 0.99) or men’s age (38.4 ± 7.1 vs. 39.8 ± 5.9 years, p = 0.11). No significant differences were found between the DGC and MSS groups in terms of GQB rate (49.3% vs. 50.7%, p = 0.55), euploidy rate (35.8% vs. 32.2%, p = 0.23), fertilization rate (69.5% vs. 69.5%, p = 0.99), clinical pregnancy rate per transfer (63% vs. 76%, p = 0.13), clinical miscarriage (9% vs. 21%, p = 0.33), and live birth rate per transfer (44% vs. 37%, p = 0.42). The results also showed a higher number of euploid embryos per initiated cycle in the DGC group (1.154) compared to the MSS group (0.989). Despite this difference, the results were not statistically significant (p &amp;gt; 0.05). Limitations, reasons for caution The limitations of this study include the potential impact of selection bias, as it is a retrospective study in which groups were not randomly assigned. Additionally, the low number of blastocysts in the MSS group and the potential bias of DNA fragmentation, which was not evaluated in the study. Wider implications of the findings Both methods demonstrated similar effectiveness. While MSS could potentially be more effective in samples with high DNA fragmentation and they feature a user-friendly application, its major limitation lies in its challenging application with samples having a low concentration of motile sperm. Specific semen samples may need different sperm selection methods. Trial registration number Not Applicable

P-218 Should embryos with direct unequal cleavage be given a chance or directly deprioritized? A follow-up on blastocyst formation and ploidy

Abstract Study question Should embryos with direct unequal cleavage (DUC) be given a chance to develop to blastocyst for a quality and ploidy assessment or directly deprioritized? Summary answer Although DUC embryos are clearly associated with poorer outcomes, if DUC embryos develop to good quality blastocysts the euploidy rate is comparable to non-DUC embryos. What is known already DUCs were first defined by time-lapse imaging and their implantation rate was found to be significantly lower than embryos with a normal cleavage pattern (1.2% vs. 20.2%, respectively) leading to the suggestion that rejection of these embryos for transfer could improve implantation rates (Rubio et al., 2012). Another study concluded that blastocyst formation, implantation potential and euploidy rate are significantly reduced in DUC embryos and that they should be deselected for day 3 transfers but should be cultured to blastocyst stage for possible embryo transfer (Zhan et al., 2016). However, in practice, DUCs are deselected and are therefore rarely transferred. Study design, size, duration This retrospective study included 1191 DUC embryos and 5058 non-DUC embryos incubated in the Embryoscope in 2022. Twenty-eight DUC and 185 non-DUC embryos were transferred in a fresh cycle. Preimplantation genetic testing for aneuploidy (PGT-A) was performed on 128 DUC and 1477 non-DUC blastocysts. Five DUC embryos were transferred in a frozen-thawed cycle. Participants/materials, setting, methods A total of 2430 IVF patients treated in a single private clinic were included in this retrospective study. The mean female age of the cohort was 36.5. Some of the blastocysts were subjected to PGT-A mainly for advanced maternal age. CHLOE (Fairtility) software analyzed time-lapse videos and identified pronuclei, DUCs and blastulation. DUC embryo outcomes (ploidy, blastulation and blastocyst quality) were assessed. Direct cleavages from 2 to 5 cells were not included in the study. Main results and the role of chance The outcomes of DUC embryos (n = 1191) were compared to non-DUC embryos (n = 5058). The blastulation rate was 23.8% for DUC embryos and 50.2% for non-DUC embryos. The good-quality blastocyst rate was 15.1% and 42.5%, respectively. The top-quality blastocyst rate was 2.4% and 18.5%, respectively. PGT-A was performed for 128 DUC blastocysts; 52 were euploid (40%) and 14 were mosaic (11%). The euploidy rate for non-DUC blastocysts was 43% and the mosaicism rate was 9.4%. Thirty-three DUC embryos were transferred giving 6 live births (18%). Limitations, reasons for caution The discrimination between fragments and blastomeres within the 5 hours from the first division is challenging for AI algorithms. Therefore, some DUC embryos may have been misclassified. Wider implications of the findings In conclusion, although DUC embryos are not prioritized for transfer, our results show that, once achieving the blastocyst stage with a good quality, their euploidy rate is comparable to non-DUC blastocysts. Morphokinetic analysis may propose additional markers to identify higher implantation potential DUC blastocysts. Trial registration number N/A

P-583 The ploidy status in blastocysts generated from progestin primed ovarian stimulation and GnRH antagonist protocols: A comparison of 14.000 trophectoderm biopsies

Abstract Study question Is there any difference in the euploidy rate in a large cohort of blastocysts when produced after a progestin-primed or GnRH antagonist protocol? Summary answer Euploidy rates in trophectoderm biopsies when tested with next-generation sequencing (NGS) were similar after progestin-primed or GnRH antagonist protocol. What is known already Preimplantation genetic testing (PGT) might be employed in patients with advanced female age to improve efficiency of embryo transfer. Given the fact that there is no fresh embryo transfer in that policy, progestin primed ovarian stimulation (PPOS) protocol might be preferred over GnRH analogs due to its lower cost and advantage of oral administration instead of s.c injections. However, due to small sample sizes within the limited number of studies, there is lack of consensus whether ploidy status might be affected when an embryo is generated after a PPOS or GnRH analogs protocol. Study design, size, duration A retrospective cohort study was designed among patients that has undergone in-vitro fertilization (IVF) treatment between January-2018 and June-2023 in Bahceci Health Group. For the aim of PGT, only patients treated with PPOS or GnRH antagonists were included. PPOS protocol referred to utilization of either medroxyprogesterone acetate (10 mg/day) or dydrogesterone (20-30 mg/day) to avoid premature ovulation during ovarian stimulation. In GnRH antagonist protocol, flexible or fixed approach was preferred for pituitary suppression. Participants/materials, setting, methods During the study period, 11.616 and 2.480 trophectoderm biopsies had been performed from blastocysts (D5/6) generated after GnRH antagonist or PPOS protocol, respectively. Of them, 9.390 and 1.801 of them were analyzed with NGS. A total of 303 (3.2%) and 17 (0.9%) biopsies were excluded due to failure or non-informative results after testing. For the final analysis, 9.087 and 1.784 biopsies were included in the antagonist and PPOS arms for the current study. Main results and the role of chance The mean age of women treated with GnRH antagonist and PPOS was comparable (36.5±5.3 vs. 36.7±5.4 years, p = 0.17). The respective numbers of euploid embryos were 3.912 and 1.784. Per biopsied embryos, the rate of euploidy was 33.7% and 34.8% (p = 0.30). The rate of euploidy per metaphase II oocyte and 2-PN were 10.6% and 13.5% in the GnRH antagonist arm and they were 11.3% and 14.4% in the PPOS arm (p = 0.08 and p = 0.07) without any statistical significance. When only patients included in whom all available blastocysts were biopsied, the linear regression model revealed only female age (RR: -0.32 CI%95: -0.38 to – 0.26, p &amp;lt; 0.001) as the independent factor for the prediction of ploidy rate (euploid/euploid + aneuploid embryos) when body mass index, duration of infertility, AMH, number of oocytes, number of metaphase-II oocytes, and the type of ovarian stimulation protocol were entered into the model. This model represented female age as the sole independent factor (RR: -0.14 CI%95: -0.18 to – 0.11, p &amp;lt; 0.001) when euploidy rate per 2PN was tested with the same variables. Limitations, reasons for caution The inherent risks cannot be excluded due to the retrospective nature of the current study. Wider implications of the findings The current study includes the largest number of trophectoderm biopsies generated from PPOS and GnRH antagonist protocol, giving the opportunity to compare them with each other. Due to lack of any superiority of one protocol regarding ploidy status, financial cost and easiness might favor PPOS when PGT is planned. Trial registration number None

P-221 The efficiency of a PGT-A program is negatively impacted by the utilization of frozen-thawed oocytes in patients of all ages

Abstract Study question Are there any differences in the use of fresh or frozen-thawed oocytes in a PGT-A program? Summary answer While euploidy rates are comparable, the efficiency of a PGT program is adversely impacted when utilizing frozen-thawed oocytes. What is known already Oocyte vitrification has become one of the key assisted reproduction procedures. Some studies have reported rates of embryonic development, clinical pregnancy, and embryo implantation comparable between fresh and cryopreserved oocytes in egg donor patients. Vitrified oocyte accumulation before the blastocyst biopsy is a commonly used strategy in PGT programs to enhance the chances of obtaining a euploid blastocyst with the patient’s own oocytes. Further research is needed to assess the impact of frozen-thawed oocytes on aneuploidy and the efficiency of PGT programs. Study design, size, duration We conducted a retrospective comparison of 777 PGT-A cycles, using either fresh or frozen-thawed oocytes, regarding embryo development (fertilization rates, blastocyst formation, and rates of good-quality blastocysts), PGT outcomes (euploidy and mosaicism rates), and the overall efficiency of the PGT cycle, measured as the number of euploid blastocyst per available mature oocytes, between April 2018 and November 2023. The study groups were stratified based on the age of the patients (≤35 and &amp;gt;35 years old). Participants/materials, setting, methods PGT-A cycles were categorized into two groups: patients who utilized fresh oocytes (n = 641) and those who utilized frozen-thawed oocytes (n = 136). A total of 6,370 fresh oocytes and surviving oocytes from the initially vitrified ones (972 out of 1,153) were fertilized and cultured to the blastocyst stage, at which point biopsies were performed. The biopsied cells were subjected to NGS screening, and statistical data analysis was conducted using the Chi-square test, with significance set at P &amp;lt; 0.05. Main results and the role of chance In the group of patients aged ≤35 years old, significant differences were observed when comparing PGT cycles using fresh or frozen-thawed oocytes in terms of the fertilization rate (78.4% vs. 72.0%), blastocyst formation rate (47.4% vs. 37.7%), and the efficiency of the PGT cycle (24.0% vs. 16.0%), corresponding to a 33.3% reduction in efficiency. No differences were observed between fresh and frozen-thawed oocytes regarding euploidy rate (64.6% vs. 68.4%), mosaicism rate (4.9% vs. 5.1%), and the percentage of good-quality embryos (60.0% vs. 56.1%). Similarly, in patients aged &amp;gt;35 years old, fertilization (81.5% vs. 73.5%), blastocyst formation (40.5% vs. 33.4%), and the efficiency of the PGT cycle (11.7% vs. 7.2%), which correspond to a 38.5% reduction in efficiency, were improved when using fresh oocytes. No differences were observed regarding euploidy (35.6% vs. 35.3%) or mosaicism rate (3.8% vs. 5.3%), and the percentage of good-quality embryos (55.2% vs. 54.7%). The oocyte survival rate was 84.3%. Limitations, reasons for caution Our study is limited by its sample size and retrospective design, as well as the inclusion of a non-selected and general infertile population, which could amplify the impact of confounding variables. Wider implications of the findings We found that the utilization of frozen-thawed oocytes reduces the PGT program efficiency by decreasing fertilization and blastocyst formation rates. This suggests that the oocyte accumulation strategy should be approached with caution. However, vitrification did not lead to an increase in the rates of aneuploidy, mosaicism, or poor-quality blastocysts. Trial registration number NOT APPLICABLE

P-568 Correlation of ploidy analysis in two types of zygotes with 3 pronuclei (3PN) observed: same size 3PNs and one micronucleus 3PNs

Abstract Study question Do all zygotes with three observed pronuclei have an actual triploid chromosomal complement? Summary answer Not all zygotes with 3 pronuclei observed are triploid, most of embryos with two even pronuclei and one smaller micronucleus are diploid. What is known already In the in vitro fertilization (IVF) setting, normal oocyte fertilisation is commonly confirmed 16–19 hours after conventional insemination or intracytoplasmic sperm injection (ICSI) with the appearance of two pronuclei (2PN). The presence three pronuclei (3PN) shows an abnormal oocyte fertilization. The incidence of 3PN zygotes in IVF is between 5.0%- 8.1% in conventional insemination and between 2.0%- 6.2% in ICSI. 3PN fertilized embryos are commonly discarded due to the suspected increased risk of a triploid chromosomal complement. However, no definitive genetic evidence has been obtained to show that all 3PN zygotes are chromosomally abnormal. Study design, size, duration An Ethics Committee for Investigation accredited this prospective observational study performed in a private centre between June 2023 and January 2024. 120 3PN zygotes from 113 cycles were cultivated until blastocyst stage. 46 were biopsied to clarify ploidy and euploidy status. 3PNs were divided into 2 groups based on the size of their pronuclei: Micro Triploid (MT) (smallest pronucleus &amp;lt;70% of the diameter of the largest pronucleus) and Pure Triploid (PT) (smallest &amp;gt;70% of the largest). Participants/materials, setting, methods Patients with 1 or more 3PN embryo were offered to participate, culturing and assessing their 3PNs. If 3PNs reached blastocyst stage, they were biopsied and analysed using Next-Generation Sequencing strategy with Single Nucleotide Polymorphisms technology without the need for parental samples, to assess ploidy and aneuploidy status. Measures of the pronuclei size were taken to sort the 3PNs in both groups. Comparisons between these groups regarding blastocyst rates, ploidy status and aneuploidy rates were assessed. Main results and the role of chance 120 3PNs zygotes were identified in 1054 cycles, representing 1.9% of all fertilized embryos. The blastocyst rate (BR) for overall 3PNs was 38.3%, while the BR for 2PNs on those 113 cycles was 58.3%, showing significant differences (p &amp;lt; 0.001). 3PN zygotes were classified into the MT and PT groups described above. The MT group had 40.4% BR, and the PT group had 37.0% BR. No significant differences were found. Of the 46 biopsied embryos, 37 were informative (80.4%), 17 for the MT group and 20 for the PT group. The diploidy rate for the overall 3PNs was 37.8% (n = 14). The MT group had 82.4% (n = 14) of diploidy rate, while the PT group had 0% diploid rate (n = 0), showing significant differences between them. (p &amp;lt; 0.001). The overall euploidy rate for 3PN zygotes was 28.6% (n = 12) (36.2 mean age), which was similar to the euploidy rate (30.3%) for our overall 2PNs biopsied during 2023 (38.2 mean age). No differences found. Out of the 14 diploid 3PNs, 28.6% (n = 4) were euploid, showing no differences with our 2023 euploidy rate. Two diploid 3PNs were mosaic, resulting in a total of 6 embryos that could potentially be transferred. Limitations, reasons for caution This study is limited by the number of triploid embryos recruited. As blastocyst rate for 3PNs population is lower than observed in the 2PN population the number of biopsied embryos is reduced. Euploidy rates for 3PNs and 2PNs are very similar, but the mean age differs by 2 years. Wider implications of the findings It is very important to distinguish between both types of 3PNs observed: PT are not worthy to keep and analyse, while MT embryos are very likely to be diploid, and it is worthwhile to culture and analyse them. Patients with few embryos available in particular could benefit from this study. Trial registration number not applicable