Good Quality Blastocysts Research Articles

The effect of male factors on embryo morphokinetics: a retrospective analysis of 2726 blastocysts.

Male infertility may influence fertilization rates, embryo morphology, and implantation rates in in vitro fertilization (IVF) cycles. Oocyte competence plays a major role in embryo development, but there is a limited understanding of the connection between sperm quality, embryo development, and morphokinetic parameters using donor oocytes. The study evaluated if sperm quality may influence the morphokinetic parameters in IVF cycles. A retrospective multicentric observational cohort study included 747 ICSI cycles using donor oocytes with fresh or frozen sperm. Embryos were cultured in time-lapse incubators until the blastocyst stage. The population was divided into three groups according to sperm concentration, as control group (> 16 mill/mL), severe oligospermia (0-5 mill/mL), and moderate oligospermia group (5-16 mill/mL). Morphokinetic analysis showed no difference in the time from the 2-cell to 6-cell stage of embryo development. A significant difference was observed on day 3 of embryo development, specifically at the 7-cell stage (t7), severe oligospermia 53.37 ± 9.81, moderate oligospermia 56.95 ± 9.78, and control 55.1 ± 8.85h post-insemination (hpi) (p = 0.024), and 8-cell stage (t8), severe oligospermia 55.41 ± 10.83, moderate oligospermia 61.86 ± 12.38 hpi (p < 0.001), and control 58.61 ± 11.33. Accordingly, the synchrony of the four cleavages going from 4 to 8 cells (s3) was found statistically different among the groups in the severe oligospermia 8.05 ± 9.99, moderate oligospermia 11.66 ± 11.04 hpi, and control 8.55 ± 8.58 (p = 0.009). Morphokinetic time ranges were obtained for t6, t7, t8, and s3 in order to identify the good-quality blastocysts. Poor sperm quality is associated with alterations in morphokinetic parameters on day 3 in IVF cycles with donor oocytes, underlining the important role of spermatozoa during embryo development.

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.

Oocyte Competence, Embryological Outcomes and miRNA Signature of Different Sized Follicles from Poor Responder Patients

Poor ovarian response (POR) patients often face the risk of not having enough competent oocytes. Then, aspirating small follicles could serve as a strategy to increase their number. Many efforts have been addressed to associate follicular size with oocyte competence, but results are controversial. Therefore, our study aimed to evaluate oocyte maturation and developmental competence, along with a non-invasive oocyte-maturation-related miRNA signature in oocytes retrieved from both large and small follicles. A total of 178 follicles, from 31 POR patients, were aspirated and measured on the day of ovarian puncture. Follicular diameters, oocyte collection, oocyte maturation, fertilization, blastocysts, and good-quality blastocyst rates were recorded. Simultaneously, follicular fluids were collected to quantify their miRNA expression. The efficacy of oocyte retrieval along with oocyte maturation, fertilization, and blastulation rates tended to increase with follicular size, but few significant differences were found. Despite there being significantly more collected oocytes from follicles &gt; 11.5 mm compared to follicles ≤ 11.5 mm (p &lt; 0.05), oocytes from the latter were also mature, with no significant differences in the miRNA signature, but only those &gt; 13.5 mm demonstrated developmental competence. In conclusion, 11.5 mm follicles can produce mature oocytes, but only those larger than 13.5 mm yielded transferable embryos.

Intraovarian PRP injection improves oocyte quality and early embryo development in mouse models of chemotherapy-induced diminished ovarian reserve.

Intraovarian injection of platelet-rich plasma (PRP) has been recently proposed, with encouraging results to provide an alternative option to patients diagnosed with POR or POI. However, the broad spectrum of PRP effects on the reproductive function and the mechanisms of action in follicular activation, response to stimulation, and embryo quality have not yet been studied. In this study, we first induced poor ovarian reserve (POR) and premature ovarian insufficiency (POI) ovarian phenotypes in CD1 mice undergoing PRP or sham intraovarian injection. PRP administration reduced those alterations induced by chemotherapy in ovarian stroma and follicle morphology in both the POR and POI conditions. After ovarian stimulation, we found that PRP did not modify the MII-oocyte yield. Nevertheless, the amount of obtained 2-cell embryos and fertilization rate were increased, being especially relevant for the POI model. Further in vitro embryo culture led to improved blastocyst formation rates and higher numbers of good quality blastocysts in PRP vs. sham females in both the POR and POI conditions. These positive results of PRP injection were also validated in the C57Bl/6 stain. Altogether, our findings suggest a possible effect on oocyte and embryo quality. This effect is likely due to the increase of local paracrine signaling through the released growth factors in PRP-treated ovaries.

A new decision-support tool in a multi-center randomized trial for personalized, optimized, and simplified fertility treatment in non-PCOS patients.

This study aimed to evaluate the effectiveness of a clinical decision support tool, Opt-IVF, in achieving the following outcomes: reducing the total cumulative dosage of Gonadotropins (Gns) used during controlled ovarian stimulation cycles and reducing the repeated ultrasonograms (USG) for monitoring follicular growth without compromising the number of good quality blastocysts obtained. The study design employed a multi-center randomized trial. The study enrolled 115 women aged 25-45 years undergoing IVF. Among the participants, 55 were randomly assigned to the intervention group (Opt-IVF), and 60 were randomly assigned to the control group. The intervention involved using a clinical decision support tool, Opt-IVF, to guide Gn dosing and trigger dates. The participants in the intervention group required significantly lower cumulative Gn dosage. The intervention group had higher numbers of oocytes retrieved and M2 retrieved than the control group. The number of good-quality blastocysts, the good-quality blastocyst rate, the ovarian sensitivity index (OSI), and the pregnancy rate in the intervention group were significantly higher than in the control group. The utilization of the clinical decision support tool led to several positive outcomes, including eliminating the need for ultrasound exams after day 5, reducing the dosage of Gn required, and yielding significantly higher numbers of high-quality blastocysts and higher pregnancy rates. Thus, Opt-IVF can successfully provide a personalized, optimized, and simplified approach to superovulation. Opt-IVF consistently outperformed the clinical teams in most of the outcomes. Clinical trials registration: ClinicalTrials.gov (ID - NCT05811065). Date of Registration: 15 March 2023. Date of enrollment of the first subject: 20 March 2023. The high cost of IVF is a result of costly drugs, fixed prices for infrastructure, extensive testing required, and labor costs for physicians and other healthcare personnel. Superovulation, which involves the drug-induced release of multiple eggs needed for IVF, accounts for a significant share of these costs. Current approaches to superovulation involve almost daily monitoring of follicle development using ultrasound and/or blood tests. The daily dosage of stimulatory hormones is then prescribed by physicians based on empirical data and clinical experience. However, the dose is not optimized for each patient, and overstimulation complications can occur. The cost of testing and drugs makes this stage very expensive. To overcome the shortcomings of this system, we have developed a decision support tool (Opt-IVF) that can provide a personalized model-optimized dosage profile for each patient. The clinical results show that Opt-IVF optimizes and personalizes dosage, reduces testing, and provides better outcomes for patients.

Addition of GM-CSF during in vitro oocyte maturation improves embryo development and implantation and birth rate in mice.

The present study determined whether adding granulocyte macrophage colony stimulating factor (GM-CSF) during in vitro oocyte maturation (IVM) could improve oocyte developmental competence by examining embryo development and implantation and birth rates following embryo transfer in mice. In an initial dose response experiment, we demonstrated that the addition of 2 and 10 ng/mL of GM-CSF during IVM increased cumulus expansion (P<0.05) but did not affect fertilisation rate compared with the control group. The addition of 10 ng/mL increased blastocyst rate (17.0%; P<0.05) and tended to increase the number of good quality blastocysts present at 96 h of culture (+19.4%; P=0.06) and increased blastocyst inner cell mass (+25.2%; P<0.001), trophectoderm (+29.9%; P<0.01), and total cell numbers (+28.6%; P<0.05). GM-CSF also reduced the incidence of DNA damage in blastocysts in the 10 ng/mL group (-16.2%) compared with the control group. These improvements translated into increases in implantation rate (+21.0%; P<0.05) and birth rate (+17.0%; P<0.05) following the transfer of vitrified blastocysts.GM-CSF treatment did not alter any fetal and placental parameters. Together these results suggest that the addition of GM-CSF during IVM may improve livestock in vitro embryo production and human IVM.

P-212 Balancing success and safety in assisted reproductive technologies cycles: the outcomes of transferring poor-quality blastocyst or day 5 slow-growing embryos with good-quality blastocyst

Abstract Study question Does transferring two mixed-quality embryos improve the live birth rate (LBR) without increasing the multiple gestation rate compared to the single good-quality blastocyst (GQB) transfers? Summary answer The transfers of single GQB and two mixed-quality embryos show similar LBRs, however, the multiple gestation rate was significantly higher in double embryo transfer groups. What is known already Due to higher LBR after blastocyst transfer, the single embryo transfer (SET) strategy has gained popularity worldwide. Despite that double-embryos transfers (DET) demonstrate higher LBR than SET, multiple gestation rates (MGRs) are higher in DET. To reduce MGR but still accomplish high LBR, DETs could be performed using embryos of mixed-quality. It has been suggested that the transfer of GQB with poor-quality blastocyst (PQB) could increase LBR without increasing MGR. Alternatively, when the embryo transfer is scheduled in a fresh cycle, the DET could be performed with GQB and day 5 slow-growing embryo (SGE) – early blastocyst or morula. Study design, size, duration This study included assisted reproductive technologies (ART) cycles with fresh embryo transfer on day 5, between 2015 and 2021, for patients under 34 years. Selected ART cycles were assigned into five groups: SET with GQB (SET-GQB, n = 754); DET with two GQBs (DET-GQB, n = 306); DET with one GQB and one PQB (DET-GP, n = 75); DET with one GQB and one day 5 SGE (DET-GS, n = 386), DET with two day 5 SGE (DET-SGE, n = 209). Participants/materials, setting, methods According to Gardner and Schoolcraft’s grading system, blastocyst morphological assessment was performed on day 5. Blastocysts graded as AA, AB, BA, and BB with a degree of expansion ≥3 were classified as GQBs, whereas blastocysts graded ВС, СВ, СС with a degree of expansion ≥3 were classified as PQBs. Day 5 morulas and blastocysts with degree of expansion 1 were classified as slow-growing embryos. The cycles with donor gametes, preimplantation genetic testing were excluded. Main results and the role of chance The LBR was significantly higher in DET-GQB than in SET-GQB [53,59% (164/306) vs 40,58% (306/754); P &amp;lt; 0,001]. There were no significant differences in implantation rate (IR) and miscarriage rate (MR) between SET-GQB and DET-GQB [47,88% (361/754) vs. 44,12% (270/612) P = 0,165], [12,19% (44/361) vs. 10,00% (27/270) P = 0,389]. However, the MGR was significantly higher in DET-GQB compared to SET-GQB [36,59% (60/164) vs 1,31% (4/306) P &amp;lt; 0,001]. When SET-GQB was compared with DET-GS, DET-GP there were no significant differences in LBR: [40,58% (306/754) vs. 39,64% (153/386) P = 0,757; vs. 41,33% (31/75) P = 0,899]. The IR was significantly higher in SET-GQB than in DET-GS and DET-GP [47,88% (361/754) vs. 30,70% (237/772) P &amp;lt; 0,001; vs. 30,00% (45/150) P &amp;lt; 0,001]. There were no significant differences in MR between SET-GQB and DET-GS, DET-GP [12,19% (44/361) vs. 17,30% (41/237) P = 0,079; vs. 11,11% (5/45) P = 0,834]. However, the MGR was significantly lower in SET-GQB compared to DET-GS, DET-GP [1,31% (4/306) vs. 18,95% (29/153) P &amp;lt; 0,001; vs. 22,58% (7/31) P &amp;lt; 0,001]. The IR and LBR were significantly higher in SET-GQB compared to DET-SGE [47,88% (361/754) vs 16,99% (71/418) P &amp;lt; 0,001], [40,58% (306/754)vs 19,14% (40/209) P &amp;lt; 0,001]. The MR and MGR were significantly lower in SET-GQB than in DET-SGE [12,19% (44/361) vs 32,39% (23/71) P &amp;lt; 0,001], [1,31% (4/306) vs 15,00% (6/40) P &amp;lt; 0,001]. Limitations, reasons for caution The study is limited due to the uneven distribution and a low number of participants in group DET-GP. Blastocyst morphological assessment remains subjected to a human factor. It was impossible to form a group where two PQBs were transferred, as there were only a few cases of such transfers. Wider implications of the findings Transferring an additional poor-quality embryo (poor-quality blastocyst or day 5 slow-growing embryo) does not increase the live birth rates. However, it significantly raises the multiple gestation rate. Despite the low live birth rate when two day 5 slow-growing embryos are transferred, the multiple gestation rate significantly increases, compared to SET. Trial registration number Not applicable

P-264 Analysis of embryo development based on time of pronuclei appearance (tPNa) and aspects of overcoming delayed pronuclei appearance through morphokinetic patterns

Abstract Study question How much delay in the appearance of 2PN can be considered normal when predicting embryo transfer potential? Summary answer Embryos with tPNa &amp;lt;13hpi post-insemination develop into good-quality blastocysts; embryos with tPNa ≥13hpi develop blastocysts by shortening the time of tPNf-tPNa. What is known already Oocytes generally exhibit 2PNs at 17–20 hours, with pronuclei fading (time of pronuclei fading; tPNf) at 23–25 hours and developing into two cells (time of 2-cell division; t2) at 25–33 hours. Fertilization confirmation is typically performed 17–18 hours after insemination. However, a few oocytes with no visible PN (0PN) at the time of fertilization confirmation develop morphologically normal blastocysts, ultimately leading to pregnancy. The failure to identify PNs can be attributed to two scenarios: rapid fading or delayed appearance. Notably, there is still a lack of research on the normal range for delayed PN appearance. Study design, size, duration This study was conducted with 2153 embryos obtained from 390 intracytoplasmic sperm injection cycles (August 2021 to June 2023). All embryos were incubated for 5 days using a time-lapse system (EmbryoScopeTM+, Vitrolife). The blastocyst development rate and morphokinetic parameters according to the tPNa of embryos were analyzed using KIDScore D5 v3 and iDAScore v2.0 (VTH server+, Vitrolife). Clinical pregnancy was also analyzed. Participants/materials, setting, methods Morphokinetic parameters were analyzed from time of second polar body (tPB2) ∼ time of expanded blastocyst (tEB). The times taken for PN to appear after the second polar body release (tPNa–tPB2), for PN to fade (tPNf–tPNa) and for 2-cell division (t2–tPNf) were calculated and compared. Blastocysts were graded using the Gardner system, a grade of BB or higher divided into good quality blastocysts (GQ-BL). Main results and the role of chance tPNa was observed as 8.09±2.11hpi [1.93hpi∼32.46hpi; &amp;lt;5hpi (n = 49), 5∼6hpi (n = 474), 7∼8hpi (n = 1220), 9∼10hpi (n = 266), 11∼12hpi (n = 91), 13∼14hpi (n = 29), 15∼20hpi (n = 18) and &amp;gt;20hpi (n = 6)]. The rate of blastocysts was highest at 5∼6hpi (64.98%) and significantly lower at 9∼10hpi (54.14%), 11∼12hpi (42.86%) and 13∼14hpi (31.03%) (p &amp;lt; 0.005). Similarly, the rate of GQ-BL was also highest at 5∼6hpi (29.11%) and significantly lower at 9∼10hpi (19.17%), 11∼12hpi (10.99%) and 13∼14hpi (3.45%) (p &amp;lt; 0.005). No embryos developed into GQ-BL at 15∼20hpi, and no embryos developed into blastocysts at &amp;gt; 20hpi. The iDAScore was significantly different at &amp;lt; 13hpi and ≥13hpi (6.00±1.86 vs. 4.24±2.26, p &amp;lt; 0.005). Similarly, KIDScore D5 showed the same patterns (6.33±1.93 vs. 4.05±1.66, p &amp;lt; 0.005). At ≥ 13hpi, no blastocysts led to pregnancy. Morphokinetic parameters were analyzed to identify the factors influencing the development of blastocysts. The analysis revealed that tPNf-tPNa tended to gradually become shorter with delayed tPNa. Notably, there were significant differences in tPNf-tPNa between &amp;lt;13hpi and ≥13hpi (16.37±4.10 vs. 13.72±6.28, p &amp;lt; 0.005). At ≥ 13hpi, tPNf-tPNa was shorter in blastocysts than in cases of cleavage arrest (10.68±5.74 vs. 15.28±5.97, p &amp;lt; 0.05). It was particularly observed that tPNf-tPNa of GQ-BL in ≥ 13hpi was 7.90hpi, and t2 was not significantly different from blastocysts and GQ-BL in &amp;lt; 13hpi (25.06 vs. 25.66±3.18 vs. 25.19±2.97). Limitations, reasons for caution More data are needed for conclusive pregnancy results due to the small number of samples. Additionally, the maturation of oocytes had yet to be considered; further detailed studies related to oocyte maturity are needed. Wider implications of the findings Embryos with a tPNa of &amp;lt; 13hpi develop into GQ-BL. Embryos with delayed tPNa tend to overcome this by shortening the tPNf-tPNa, resulting in develop into blastocysts. Predicting blastocyst development can be achieved by considering factors such as tPNa∼t2. This can aid in improving the selection of embryos for cleavage-stage ET. Trial registration number N/A

P-594 Retrospective comparative study of follitropin delta vs follitropin alfa in progestin-primed ovarian stimulation (PPOS) protocol in IVF

Abstract Study question Could the individualized fixed-dose algorithm with follitropin delta show a similar pregnancy rate to conventional dosing with follitropin alfa in PPOS protocol? Summary answer Individualized fixed-dose algorithm with follitropin delta showed a similar pregnancy rate to follitropin alfa In PPOS protocol. What is known already Follitropin delta (REKOVELLE, Ferring Pharmaceuticals, Switzerland) is the first human cell line driven recombinant follicle-stimulating hormone (FSH) with an individualized fixed-dose algorithm based on serum AMH level and body weight. Follitropin alfa is a Chinese hamster ovarian cell driven recombinant FSH and its dosing is adjusted according to follicular response during the stimulation period. There was no significant difference between follitropin delta and follitropin alfa in terms of pregnancy rate under GnRH antagonist protocol. Study design, size, duration A retrospective analysis of 355 patients aged under 40 years who underwent ovarian stimulation in PPOS protocol using follitropin delta or follitropin alfa and on whom frozen-thawed single blastocyst transfer was conducted between August 2021 and November 2023 was performed. Participants/materials, setting, methods 193 cycles of women who used follitropin delta were compared to 162 cycles of women who used follitropin alfa. The following clinical outcomes on each treatment group were analyzed: chemical pregnancy rate (serum β hCG), clinical pregnancy rate (gestational sac), ongoing pregnancy rate(gestational sac and fetal heartbeat), serum E2 level at trigger day, cycle proportion of ≥ 10 oocytes retrieval, blastocyst formation rate, cycle proportion of ≥ 5 blastocysts and cycle proportion of ≥ 2 good quality blastocysts. Main results and the role of chance Baseline demographics in the follitropin delta group and the follitropin alfa group were not significantly different: age, 33.9 ± 3.5 years vs 34.5 ± 3.2 years; body weight, 53.5 ± 8.3 kg vs 53.8 ± 5.9 kg; number of previous IVF cycles, 0.2 ± 0.6 vs 0.3 ± 0.7; serum AMH level, 5.5 ± 2.9 ng/mL vs 5.3 ± 3.8 ng/mL, respectively. The chemical pregnancy rate (69.9% vs. 64.8%, P = 0.308), clinical pregnancy rate (58.0% vs. 56.2%, P = 0.748) and ongoing pregnancy rate (51.8% vs. 46.3%, P = 0.338) were comparable between the follitropin delta group and the follitropin alfa group. The individualized follitropin delta treatment resulted in a smaller cycle proportion of ≥ 10 oocytes retrieval (77.7% vs. 90.8%, P &amp;lt; 0.001) with lower serum E2 level at trigger day (3273 ± 1577 pg/mL vs. 3927 ± 1732 pg/mL, p &amp;lt; 0.001). However, the blastocyst formation rate was significantly higher in the follitropin delta group (75.5% vs. 68.7%, p &amp;lt; 0.001). Moreover, the cycle proportion of ≥ 5 blastocysts (71.0% vs. 76.4%, P = 0.286) and the cycle proportion of ≥ 2 good quality blastocysts (51.8% vs. 60.3%, P = 0.115) were comparable between the groups. Limitations, reasons for caution This is a non-controlled, retrospective study. Wider implications of the findings Follitropin delta in its individualized fixed-dose regimen could have the potential to decrease the risk of OHSS by minimizing excessive ovarian response, whereas a similar pregnancy rate to conventional dosing regimen could be expected due to the number of good quality blastocysts secured. Trial registration number not applicable

P-199 The development time of good quality blastocysts is dependent on their genetic status in patients over 36 years old

Abstract Study question Is there any difference in PGT-A results between good quality blastocysts depending on whether they reach this stage on the fifth or sixth day? Summary answer In patients over 36 years old, there is a significantly higher rate of aneuploidy in embryos that reach the blastocyst stage on Day 6. What is known already The level of chromosomal abnormalities in embryos increases with maternal age, as indicated by PGT-A data. Embryo quality also correlates with aneuploidy level. The normal timeframe for blastocyst formation is considered to be between the fifth and sixth day of development. In this study, the genetic status of good quality blastocysts was examined to determine a difference in the aneuploidy rate between blastocysts formed on the fifth day compared to those formed on the sixth day.This information could provide insights into the optimal timing for blastocyst formation and its relationship to genetic abnormalities. Embryo quality also correlates with aneuploidy level Study design, size, duration The retrospective study included 513 good quality blastocysts (3-4, A-B according to the Gardner classification) after trophectoderm biopsy and obtained from fresh oocytes of 141 patients aged 23-45 years. The PGT-A data were analyzed. The analysis considered the age of the patients, with one group of patients younger than 36 years old (35 patients, 174 blastocysts), and another group aged 36 years and older (106 patients, 339 blastocysts). Participants/materials, setting, methods Trophectoderm biopsy was performed on good quality embryos as they progressed in development, either on the fifth or sixth day. PGT-A was conducted using the NGS method, and the data was processed using the chi-square test. Main results and the role of chance Among patients younger than 36 years old, 58.5% of good quality blastocysts formed by the fifth day of development. In comparison, among patients 36 years old and older, 43.0% of good quality blastocysts formed by the fifth day. The difference in the growth rate of blastocysts of the same quality between the age groups was statistically significant (p &amp;gt; 0.01). In patients younger than 36 years old, 75.0% of blastocysts formed by the fifth day and 66.7% of blastocysts formed by the sixth day were euploid (with no significant difference). In patients 36 years old and older, 50.5% of blastocysts formed by the fifth day were euploid, while only 19% of blastocysts formed by the sixth day were euploid. This difference in euploidy rates between the fifth- and sixth-day blastocysts was statistically significant (p &amp;gt; 0.01). Limitations, reasons for caution The study was conducted with a relatively small sample size, which may limit the generalizability of the results. A larger and more diverse sample would be beneficial to confirm the findings. Wider implications of the findings In patients over 36 years of age, the majority of blastocysts develop by Day 6, and there is a significant increase in aneuploidy level. This suggests that there may be a higher risk of chromosomal abnormalities in embryos that take longer to develop into blastocysts in this group. Trial registration number Not applicable

P-164 The impact of oocyte morphological abnormalities on fertilization rate and blastocyst quality

Abstract Study question Do distinct morphological abnormalities in oocytes have an adverse effect on both fertilization rates and the quality of resulting blastocyst? Summary answer While most oocyte morphological abnormalities did not significantly affect outcomes, dark cytoplasmic color correlated with lower fertilization rates and the absence of good quality blastocysts What is known already Variability exists in the fertilization and developmental potential of mature (metaphase II - MII) human oocytes. A high-quality MII oocyte is characterized by a spherical shape, homogeneous cytoplasm, smooth appearance, a uniform zona pellucida (ZP), a normal-sized perivitelline space (PVS), and a single unfragmented polar body (PB). Dysmorphisms, such as variations in cytoplasmic features and anomalies in PVS size and PB regularity may occur. There are conflicting reports on the relationship between oocyte morphology and IVF outcomes. A recent meta-analysis highlighted associations between poor fertilization potential and specific dysmorphisms, including large/irregular PB, a large PVS, refractile bodies, and vacuoles. Study design, size, duration This study analyzed 367 MII oocytes obtained from 40 young patients. The fertilization rate (FR) and blastocyst quality (BQ) were compared among 132 dysmorphic and 235 normal MII oocytes (from October 2023 to January 2024). Dysmorphic oocytes were categorized as having a non-spherical shape, large PVS, PVS debris, abnormal ZP, fragmented PB, excess cytoplasmic granules, vacuoles, and dark cytoplasmic color. Participants/materials, setting, methods All MII oocyte denuded were examined under the microscope at x-400 magnification. Dysmorphic and normal oocytes were separated, injected, and cultured individually until day 5 under conditions of 37oC and 6% CO2. Fertilization was assessed 14 to 18 hours post-injection, and subsequent embryonic development was evaluated on day 2 and day 5. Blastocysts graded 4AA to 4BC are considered good quality, while those graded 4CC to 3CC are classified as fair quality. Main results and the role of chance The mean age of patients was 24.14 ± 3.77. In total, 36% of all oocytes exhibit at least one dysmorphism (n = 132). From the overall dataset analyzed, there was no significant difference (p &amp;gt; 0.9999) in FR between the normal 77.87% (183/235) and dysmorphic group of oocytes 78.03 (103/132). The odd ratio and 95% confidence interval are 0.9909 and 0.5957 to 1.633 respectively. However, this was not the same for oocytes with dark color cytoplasm. The presence of this dysmorphism significantly lowered FR (p = 0.0176) with an odd ratio of 5.544 (95% CI: 1.419 to 21.67). Similarly, the result showed no statistically significant difference (p = 0.1073) in the likelihood of obtaining a fair quality blastocyst between the normal oocytes 23.40% (55/235) and dysmorphic group of oocytes 15.79 (21/133). This was also the same for the likelihood of obtaining good quality blastocyst. There was no statistically significant difference (p = 0.1536) between the normal group 20.00% (47/235) and the dysmorphic group 26.52 (35/132). Oocyte with dark color cytoplasm gave no good quality blastocyst. Limitations, reasons for caution The exclusive use of oocytes from young patients may limit the generalizability of findings to a broader population of IVF patients. Also, the sample size may limit the study’s ability to detect subtle associations. A reason for caution when interpreting this study is its focus on morphological assessments alone. Wider implications of the findings The knowledge from our findings may contribute to optimizing oocyte selection strategies for intracytoplasmic sperm injection. Also, the knowledge could potentially be integrated into AI modules for oocyte morphological assessment. However, more studies would be required to develop a robust and scalable system 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-012 Euploid and good-quality blastocysts using testicular sperm from men with high sperm DNA fragmentation (SDF), oligozoospermia, and previous failed ICSI with ejaculated sperm

Abstract Study question Are euploid rate and blastocyst quality improved when testicular sperm is used in patients with elevated SDF and poor semen parameters or/and previous ICSI failure? Summary answer The use of testicular spermatozoa in non-azoospermic males does preserve blastocyst ploidy while favoring the proportion of good-quality embryos available for the couple. What is known already Sperm extracted from the testicle has been proposed as a strategy to improve reproductive outcomes in couples with elevated DNA fragmentation in the ejaculate and with severe male infertility or previous ICSI failure. Different studies and meta-analyses show to increase pregnancy and live birth rates, and one of the reasons could be the lower genetic damage of spermatozoa retrieved from the testicle. However, there is little information on the effect on the quality of the embryos generated and their ploidy since the use of testicular sperm tends to have a higher rate of aneuploidy. Study design, size, duration This is a prospective, blinded, randomized, and controlled trial conducted in a private university-affiliated IVF center between October 2020 and June 2023. In each patient (n = 6), oocytes were randomly microinjected by sperm from the testis (T-ICSI) or ejaculate (E-ICSI) assigned in a 1:1 ratio. All males had an SDF&amp;gt;30% (measured by Sperm Chromatin dispersion (SCD) test), severe oligozoospermia, and/or a previous ICSI failure. Participants/materials, setting, methods Males underwent testicular sperm extraction (TESE) to obtain spermatozoa, which were frozen along with an ejaculated semen sample obtained previously on the same day. Women were &amp;lt;38 years old with good ovarian reserve (antral follicle count&amp;gt;10). SDF level was compared between both sources. The primary outcome was the euploid rate (among a total number of biopsied embryos). Secondary outcomes were fertilization rate, blastocyst rate, good-quality blastocyst rate, and ongoing pregnancy rate per embryo transfer (ET). Main results and the role of chance Mean ejaculated SDF was 44.83±16.6 (IC95% 27.5 -62.2), while mean women age was 31.8±2.9 (28.76-34.9). An average of 14.5±5.8 metaphase II oocytes were microinjected per patient. A total of 87 oocytes were randomized to the T-ICSI group (n = 45) or E-ICSI group (n = 42). There was a 14% difference between the SDF of testicular (44.7% (31.3-58.0) and ejaculated sperm (58.5% (37.6-79.4) (p = 0.2). The fertilization rate was slightly lower in the T-ICSI group (53.3% (38.1-68.1) compared to the E-ICSI group (61.9 (61.8-76.0) (p = 0.6). A total of 21 embryos reached the blastocyst stage, being the blastocyst arrival rate per correctly fertilized oocyte 41.7% (10/24) (22.8-63.1) for T-ICSI and 42.3% (11/26) (24.0-62.8) for E-ICSI (p = 1.0). Of them, a higher proportion of good-quality blastocysts was found in the T-ICSI (29.2% (13.4-51.6)) group than in the E-ICSI (19.2% (7.3-40.0)) group, although the difference was not statistically significant. In addition, the euploidy rate was comparable between the T-ICSI (60% (27.4-86.3)) and E-ICSI (63.6% (31.6-87.6)) groups (p = 1.0). Seven embryos were transferred in 6 ET, three in each study group. Only one of the ET in the E-ICSI group resulted in pregnancy (33.3% (1.8-87.5), while the three transfers in the T-ICSI group resulted in an ongoing pregnancy (100% (31.0-100)) (p = 1.0). Limitations, reasons for caution Despite being a randomized controlled trial, the number of subjects recruited is small; it is necessary to increase the number of cases to obtain results with greater statistical power. TESE is a technique that is not risk-free, so its performance should be evaluated in terms of its cost-benefit for patients. Wider implications of the findings The use of less genome-damaged testicular spermatozoa in non-azoospermic males seems to be a reliable strategy for couples with high ejaculated SDF and severe oligospermia and/or previous ICSI failures. However, although we have found an improvement, remains to be statistically confirmed with more cases. Trial registration number NCT04795440

P-213 Effectiveness and safety of single poor quality blastocyst transfer compared with single good quality blastocyst transfer in the first transfer cycle

Abstract Study question Comparison of pregnancy and perinatal outcomes in blastocysts with poor inner cell mass quality or poor trophectoderm quality to optimize non-quality single blastocyst transfer strategies. Summary answer Poor trophoblast quality blastocysts have better pregnancy outcomes than poor inner cell mass quality blastocysts, with no statistically significant difference in perinatal outcomes What is known already There is no doubt that good-quality blastocysts have better pregnancy outcomes than poor-quality blastocysts, but the preference for trophectodermal poor-quality blastocysts or inner cell mass poor-quality blastocysts in non-good-quality blastocyst cycles is still uncertain. Study design, size, duration A total of 46,433 first single blastocyst transfer cycles from March 1, 2014, to December 31, 2022 were retrospectively analyzed, and blastocyst transfer cycles were divided into 3 groups according to the grade of trophoderm and inner cell mass: A/B A/B (Group 0, N = 30748), A/B C (Group 1, N = 15455), and C A/B (Group 2, N = 230). Participants/materials, setting, methods 46,433 patients who underwent their first single blastocyst transfer at Reproductive and Genetic Hospital of CITIC-Xiangya were included. Multivariate log-binomial regression model with COPY methods were performed to estimate relative risk of Group 2/1 versus Group 0 and Group 2 versus Group 1 for reproductive and neonatal outcomes. Five propensity score weighting methods, namely IPW, processing, overlap, matching, and entropy, were used for sensitivity analysis to explore the robustness of the results. Main results and the role of chance After adjusting for female age, BMI, duration of infertility, number of cycles, AFC, AMH, endometrial thickness, causes of infertility (tubal disorders, ovulatory disorders, endometriosis, unexplained, more than one causes, male factors), culture days (D5, D6), expansion of blastocyst (3, 4, 5, 6), and type of blastocyst (fresh blastocyst, thawed blastocyst, thawed cleavage cultured to blastocyst, PGT blastocyst), we found that clinical pregnancy rate (CPR) and live birth rate (LBR) of Group 0, Group 1 and Group 2 were successively significantly decreased (CPR: Group 1 vs Group 0, RR = 0.83 (0.81, 0.84), Group 2 vs Group 0, RR = 0.43 (0.35, 0.53), Group 2 vs Group 1, RR = 0.53 (0.42, 0.64); LBR: Group 1 vs Group 0, RR = 0.79 (0.77, 0.81), Group 2 vs Group 0, RR = 0.36 (0.27, 0.47), Group 2 vs Group 1, RR = 0.45 (0.34, 0.59); respectively), while early miscarriage rate was successively significantly increased (Group 1 vs Group 0, RR = 1.3 (1.22, 1.38), Group 2 vs Group 0, RR = 2.25 (1.46, 3.15), Group 2 vs Group 1, RR = 1.73 (1.12, 2.42)). Late miscarriage, preterm birth, singleton live birth, sex ratio of singleton, low birth-weight of singleton, and birth weight of singleton were not statistically different between three groups. Above results were robust in our two sensitivity analysis. Limitations, reasons for caution As a retrospective study, there are inevitably unmeasured confounding factors. This study only included a small number of Group 2. It’s therefore possible that our study was under powered. Risk of gestational hypertension, gestational diabetes, cesarean section and macrosomia should also be evaluated with a more comprehensive assessment of safety. Wider implications of the findings High-quality blastocysts have better pregnancy and live birth rates than poor-quality blastocysts, but there are no significant differences in preterm birth, sex ratio, or birth weight. In the absence of good quality blastocysts for transplantation, poor quality blastocysts of the trophectoderm are preferred over poor quality inner cell mass. Trial registration number not applicable

P-130 Comparing head-first and tail-first injection techniques in guiding new embryologists for human oocyte Piezo-ICSI

Abstract Study question Which technique is more effective for new embryologists in human oocyte Piezo-ICSI: Head-first or tail-first injection? Summary answer Tail-first injection is superior as it significantly increases fertilization rates without adversely affecting embryo development compared to head-first injection in Piezo-ICSI. What is known already ICSI is an essential technique in assisted reproductive technology. Recently, some researchers have reported that Piezo-ICSI is clinically more useful than Standard-ICSI. In most papers, Piezo-ICSI has shown significantly higher fertilization rates than Standard-ICSI. This is because, in Piezo-ICSI, it eliminates the need to aspirate the cytoplasm into the ICSI needle upon membrane breakage. After membrane breakage, the sperm is injected into the cytoplasm. A few papers evaluated the effect of sperm injection direction (head-first or tail-first) on ICSI results for human oocytes. However, little information is available to guide new embryologists on whether to use head-first or tail-first injection. Study design, size, duration During their Piezo-ICSI training, new embryologists were taught either head-first or tail-first injection techniques before commencing clinical procedures. Only the head part of the sperm was injected into the cytoplasm in the head-first group, and half of the whole sperm was injected in the tail-first group. This study divided the new embryologists into two groups (head-first or tail-first) and collected and retrospectively analyzed their first 200 oocyte ICSI results between January 2017 and November 2023. Participants/materials, setting, methods This study involved five new embryologists and a total of 1,000 oocytes. Two embryologists performed head-first injection for 400 oocytes, and three performed tail-first injection for 600 oocytes. The rates of degeneration, 2PN, 0PN, 1PN, ≥3PN, good quality day-3 embryo (≥7cell, ≤15%fragmemtation) per 2PN, blastocyst, and good quality blastocyst (better than BB with Gardner criteria) were compared. Statistical analysis was performed using either a t-test, Welch’s t-test, or Fisher’s exact test as appropriate. Main results and the role of chance The average ages of women and their husbands in the head-first and tail-first injection groups were 37.8 vs. 37.2 and 40.1 vs. 39.2, respectively, with average total motile sperm counts of 52.2 x106 vs. 52.6 x106. No significant difference was observed in these comparison items between the groups. Theoretical injected medium volume into the cytoplasm with sperm of the head-first and tail-first injection groups was 104μm3 vs. 520μm3. The 2PN rate of the tail-first injection group (88.3%; 530/600) was significantly higher (P = 0.0151) than head-first (82.8%; 331/400). The 0PN rate of the tail-first group (4.0%; 24/600) was significantly lower (P = 0.0007) than the head-first (9.5%; 38/400). The degeneration rates, 1PN rates, and ≥3PN rates of head-first and tail-first injection groups were 1.5% (6/400) vs. 1.3% (8/600), 3.5% (14/400) vs. 2.3% (14/600), and 2.8% (11/400) vs. 4.0% (24/600). No significant difference was observed in these comparison items between the groups. The good quality day-3 embryo per 2PN rates, blastocyst rates, and good quality blastocyst rates of head-first and tail-first injection groups were 64.7% (214/331) vs. 64.3% (341/530), 62.1% (192/309) vs. 62.7% (289/461), 35.3% (109/309) vs. 35.6% (164/461). No significant difference was observed in these comparison items between the groups. Limitations, reasons for caution The estimated volumes of medium injected into the cytoplasm for both head-first and tail-first injections were theoretical. We calculated the length of the sperm head and whole sperm as 6μm and 60μm. Some day-3 embryos were transferred or cryopreserved, so the blastocyst and good-quality blastocyst rates were reference ranges. Wider implications of the findings Despite the larger volume of medium injected with sperm, tail-first injection increases fertilization rates without adversely impacting embryo development compared to head-first injection. Therefore, the tail-first injection is better to guide the new embryologists for human oocytes Piezo-ICSI than the head-first injection. Trial registration number 17-0450221102

P-503 Euploidy rate of day 4 fully developed blastocysts

Abstract Study question Do embryos that fully developed to blastocyst at day 4 have high aneuploidy rates as assessed by preimplantation genetic analysis? Summary answer Fully developed blastocysts at day 4 had a high euploidy rate. What is known already In ART, it is considered important to use good quality blastocysts to increase the pregnancy rate after embryo transfer. Some reports have demonstrated a high pregnancy rate for embryos that fully developed to blastocysts on day 4 after IVF (Ozgur et al., 2021). Furthermore, it has been reported that the speed of embryo development is related to the rate of chromosome aneuploidy (Taylor et al., 2014). There are though, limited reports on the euploidy rate of embryos developed to blastocysts at day 4. Study design, size, duration This retrospective study analyzed 763 embryos (from 184 PGT-A cases) that underwent TE biopsy on day 5 between January to December 2022 to determine their ploidy status. Participants/materials, setting, methods Embryos cultured in a time-lapse incubator were divided into two groups: those that developed to blastocyst (Gardner’s classification: ≥ expansion grade 3) at day 4 (96-100 h) (D4(+) group) and those that did not develop to full blastocyst at day 4 (D4(-) group) and the euploidy rates were compared. Statistical analysis was performed using Fisher’s exact test to determine significant differences (P &amp;lt; 0.05). Main results and the role of chance The euploidy rate of the D4(+) group was 36.5% (80/219), which was significantly higher than the D4(-) group, 23.3% (127/544) (p &amp;lt; 0.01). Moreover, when considering the age of the female, for age ≤39 years, the rates were significantly higher in the D4(+) group (52.1% (61/117) and 35.4% (97/274) p &amp;lt; 0.01 respectively ). For age 40≥ years, it tended to be higher but was not significantly different in the D4(+) group vs D4(-) group (18.6% 19/102 and 11.1% 30/270 respectively). Even when limited to embryos that were graded as good quality blastocysts at day 5, the D4(+) group was significantly higher (p &amp;lt; 0.01) than the D4(-) group (55.0% (60/109) vs 36.1% (90/249)). Limitations, reasons for caution This was a retrospective study limited to day 5 embryos with biopsies taken after vitrification-warming. Wider implications of the findings These results demonstrate that embryos that had already developed to blastocyst at day 4 had a higher rate of euploidy compared to day 5 blastocysts. Thus, when performing PGT-A, biopsy of embryos that have already developed to blastocyst at day 4 are expected to be prioritised for transfer. Trial registration number not applicable

P-265 Should we divide two types of direct cleavage at the first division in human embryos observed by time-lapse imaging?

Abstract Study question Are there any differences on clinical outcomes between two types of direct cleavage at the first division (DC1) in human embryos observed by time-lapse imaging? Summary answer Blastocyst development of DC1 without any two-cell stage was lower than DC1 with momentary two-cell stage. Once they became blastocysts, they didn’t affect pregnancy outcomes. What is known already In recent years, time-lapse imaging (TLI) has been used to analyze dynamics on embryo development. It clarified that some embryo showed direct cleavage, where one cell divided into three or more cells directly (DC1). It has been reported that DC1 embryos showed chromosomal abnormalities and negative effect on the embryonic development compared to normal cleavage without DC1. Some reports revealed that once embryos with DC1 became blastocysts, they didn’t affect pregnancy outcomes compared to normal cleavage without DC1. No study has been published to date that categorizes DC1 into two types: DC1 embryos with or without momentary two-cell stage. Study design, size, duration This study included patients whose oocytes had been retrieved between January 2020 to December 2022. A retrospective study was conducted on 11,366 cleavage embryos derived from 2PN2PB in 2,735 cycles using time-lapse imaging (Geri®, Genea BIOMEDX, Australia, 5-minute intervals, 9 slices) after intracytoplasmic sperm injection (ICSI) with ejaculated spermatozoa. We focused on DC1 only and analyzed pregnancy outcomes on 248 cycles with single blastocyst transfer obtained from this study. Participants/materials, setting, methods Among 1,817 cycles where DC1 was observed by TLI, DC1 were classified into two types: DC1 embryos without any two-cell stage (DC1A) and with momentary two-cell stage (DC1B). We comparatively analyzed for blastocyst developmental rates, good-quality blastocyst (Grade 3BB or more by the Gardner’s classification) rates and pregnancy outcomes with single blastocyst transfer (clinical pregnancy rate, CPR; live birth rate, LBR). Statistical analysis was performed using student t-test and chi-square test. Main results and the role of chance Among 2,735 cycles where ICSI was performed, female age was 39.3 ± 4.8 years (mean ± SD), previous ART cycles were 4.6 ± 5.5 times, and number of retrieved oocytes was 10.0 ± 8.7. DC1 embryos were found in 32.2% (3,663/11,366). Blastocyst development and good-quality blastocyst rates were 28.3% (826/2,921) and 14.6% (427/2,921) in DC1 embryos, whereas 67.5% (3,512/5,204) and 41.4% (2,157/5,204) in embryos without direct cleavage (non-DC), respectively. The former was significantly lower than the latter (P &amp;lt; 0.001). Among DC1 embryos, we confirmed DC1A was relatively fewer (21.2%) than DC1B (78.8%). Blastocyst development and good blastocyst rates were 11.7% and 2.1% in DC1A embryos, whereas 32.7% and 11.0% in DC1B embryos, respectively. The former was significantly lower than the latter (P &amp;lt; 0.001). CPRs, miscarriage rates and LBRs per single blastocyst transfer of DC1A were 44.4% (4/9), 11.1% (1/9), and 33.3% (3/9), while those of DC1B were 33.8% (47/139), 12.2% (17/139), and 21.6% (30/139), respectively. On the other hand, those of non-DC were 44.7% (232/519), 13.1% (68/519), and 30.6% (159/519). Both embryos with DC1A and DC1B produced healthy babies, except for one case with congenital hydronephrosis from DC1B. Limitations, reasons for caution We excluded embryos with direct cleavage in the second division (DC2) in this study, because we focused on DC1 only. DC2 embryos may have complex effects besides DC1. The number of blastocysts from DC1A embryos may not be sufficient for comparison of pregnancy outcomes. Wider implications of the findings Since blastocyst development and good-quality blastocyst rates were of DC1A was significantly lower than those of DC1B, it may be necessary to categorizes DC1 into two types: DC1 embryos with or without momentary two-cell stage. However, once they became blastocysts, they didn’t affect pregnancy outcomes. Trial registration number not applicable

P-173 An exploratory clinical study in patients with recurrent ART-failure due to severe fragmentation during early-cleavage: the effects of artificial removal of zona-pellucida at the pronuclear-stage

Abstract Study question Can the artificial removal of the zona pellucida (ZP) at the pronuclear (PN)-stage prevent severe fragmentation in early-cleavage embryos and improve good-quality blastocyst development rate? Summary answer The artificial removal of the ZP at the 2PN-stage could prevent significantly severe fragmentation in early-cleavage embryos and improved the good-quality blastocyst development rate. What is known already Recent analysis using time-lapse culture demonstrated that fragments during early-cleavage occur at the site of the perivitelline threads between the ZP and the embryo membrane (Reprod Biomed Online 2017;35:640-5,646-56). Based on these findings we conducted a pilot study on embryonic development after ZP-removal using abnormally-fertilized oocytes (J Assist Reprod Gene2020;37:1349-54). We showed that the inter-blastomere adhesion was maintained and fragmentation was significantly lower in the ZP-free embryos than in the ZP-intact embryos. Therefore, we attempted to apply this method to patients who have given full consent and had repeatedly failed to conceive due to severe fragmentation in early cleavage-stage embryos. Study design, size, duration This is a single-centre retrospective exploratory study of 34 patients that were scheduled to undergo ART-treatment in our clinic between February 2020 and January 2021. They had been previously treated in our facilities (January 2016 to January 2020) with less than 10% morphologically good-quality blastocysts due to severe fragmentation. None of these patients became pregnant during their initial treatments. Totally, 173 2PN-zygotes were obtained, with 101 allocated to ZP-free group and 72 to ZP-intact group. Participants/materials, setting, methods Normally-fertilized oocytes were placed in 0.125M sucrose-containing HEPES-medium (approximately 5min) and cultured under: 1) ZP-free, in which the ZP was completely removed from the ooplasm by laser-irradiation and a medium-blowing method with a biopsy-pipette, or 2) ZP-intact. ZP-free and ZP-intact zygotes were cultured in sequential medium. Embryos were either freshly transferred on Day-2(ZP-intact group only, according to patients’ wishes), or Day-5/6, or they were cryopreserved on Day-5/6/7 for future embryo transfer cycles for both groups. Main results and the role of chance Even allocation of the normally fertilised oocytes to the two groups was not possible, due to the patients’ request to include more zygotes in the ZP-free group. Therefore, no specific criteria were set other than the rule that all the normally fertilised oocytes of each patient must be allocated into both groups at the time of allocation. As a result, each patient had 2PN zygotes assigned to both ZP-free and ZP-intact groups. Rates of good-quality embryos, blastocyst development, morphologically good-quality blastocyst development and cryopreservation demonstrated significant improvement (all P &amp;lt; 0.01) in the ZP-free group compared to the ZP-intact group. Pregnancy rates in ZP-intact group were 5.9% per patient and 5.3% per embryo transferred, while in ZP-free group 37.5% per patient and 24.3% per embryo transferred. Our results demonstrate that in difficult-to-treat cases where embryos with good morphology cannot be obtained due to excessive fragmentation at the early-cleavage, embryonic development can be significantly improved by artificial removal of ZP at the 2PN-stage. We believe that the ZP-free method has opened a new door in assisted reproductive medicine and can provide the opportunity to obtain blastocysts with good-morphology from an embryo that would probably not have become a blastocyst otherwise. Limitations, reasons for caution A comparative analysis between the two groups was not possible due to the small number of patients, the inclusion of fresh embryos as well as frozen embryos, and the non-randomized data. The imbalance was attributed to the fact that patients wanted us to allocate more oocytes to the ZP-free group. Wider implications of the findings The development of treatment methods based on new ideas should always take into consideration the possibility of associated risks. In the future, we will examine the criteria for selecting patients for whom this method is useful, as well as verify its safety and effectiveness by conducting a prospective randomized analysis. Trial registration number not applicable

O-085 Oocyte cryopreservation: a comprehensive analysis of live birth rate by age and indication of treatment provides reassurance for young cancer patients

Abstract Study question Does the outcome of oocyte cryopreservation vary across different age groups and treatment indications, i.e. cancer diagnosis, other medical conditions or non medical fertility preservation? Summary answer Live birth rate (LBR) is significantly influenced by a woman's age, although not by indication of treatment. What is known already OC is increasingly applied in assisted reproduction, although little is about the outcomes, including livebirth according to indication. Study design, size, duration Retrospective cohort study analyzing all OC and subsequent warmed oocyte cycles in two fertility centers - Melbourne IVF and the Royal Women’s Hospital, Melbourne, Australia, between the years 2014 to 2022. Participants/materials, setting, methods Women undergoing autologous IVF treatment using vitrified and warmed oocytes. Outcome of stimulation cycles and warmed oocyte cycles were assessed according to women’s age at OCP (≤ 35 years, 35-40 years and ≥ 40 years) and treatment indication- cancer diagnosis, other medical conditions and non medical. Main results and the role of chance 2689 women underwent a total of 3801 cycles of OC, comprising 702 cycles for cancer diagnosis (18.6%), 1146 cycles for other medical (30.5%), and 1953 cycles for non-medical (50.9%). Regardless of indication, younger age groups exhibited a higher median number of mature oocytes: &amp;lt;35 (11, 8-17), 35-40 (9, 6-11), and &amp;gt;40 (6, 3-9) (H(2)=15.1, p &amp;lt; 0.05). Among 582 warmed oocytes cycles, the &amp;gt;40 group demonstrated lower rate cycles with at least one good-quality blastocyst (76.7%) compared to 35-40 (87.1%) and &amp;lt;35 (87.2%), p &amp;lt; 0.05. Cumulative live birth rates (cLBR) were also lower in the &amp;gt;40 age group (14.9%) compared to 35-40 (34.9%) and &amp;lt;35 (46.5%), p &amp;lt; 0.01. When analyzing cycles by indication, patients with cancer had lower oocyte survival rate (80.8% vs 84.8% in other medical and 82.1% in non medical, p &amp;lt; 0.01). Nonetheless, no differences in rate of cycles with at least one good quality embryo rates (cancer diagnosis 87.2%, other medical 82.1% and non medical 89.3%, p = 0.06). Patients with cancer diagnosis had higher clinical miscarriage rate (25%) compared with other medical (7%) and non medical (10.5%), p &amp;lt; 0.01. However, cLBR did not differ between the indication groups (29.7% in cancer diagnosis, 35% in other medical and 38.1% in non medical, p = 0.5. Limitations, reasons for caution Retrospective data collection. Wider implications of the findings Our study offers comprehensive data that may assist patients desiring OC, and provides reassurance that even with a cancer diagnosis, outcomes are as favorable as those having non-medical OC. Given the substantial impact of age, health care providers should, when possible, advise to complete this procedure at a young age. Trial registration number Not applicable

P-022 Klinefelter syndrome couples are expected higher live birth rates than unexplained NOA couples after ICSI with testicular spermatozoa

Abstract Study question What are sperm retrieval rates (SRR) by microdissection TESE (micro TESE), fertilization rates, and pre- and post- implantation development in Klinefelter syndrome (KS) couples? Summary answer In KS couples, SRR, clinical pregnancy rates (CPR) per embryo transfer (ET), and live birth rates (LBR) per patient were higher than unexplained NOA couples. What is known already Micro TESE, in combination with ICSI, is presently used to treat infertility in cases of NOA including KS, which is the most common sex-chromosome disorder among infertile males, with a prevalence of 1 in 660 men and is a frequent cause of hypogonadism and infertility. There were few reports regarding ICSI outcomes in the couples of KS. The aim of this study is to assess the prevalence and the significance including SRR by micro TESE of ICSI outcomes with embryonic development in KS couples. Study design, size, duration A retrospective study was conducted in 212 patients with non-mosaic KS and 963 unexplained NOA patients with 46, XY without past history (unexplained NOA; not including after orchidopexy, KS, cryptozoospermia, post chemotherapy, mumps orchitis, etc) who underwent micro TESE and ICSI with fresh or cryopreserved testicular spermatozoa for their wives between September 2013 to November 2023. Participants/materials, setting, methods A total of 2204 azoospermic patients were examined for chromosomal analysis. We evaluated SRR of microdissection TESE, two pronuclei (2PN) rates, blastocyst development rates, good-quality blastocyst (Grade 3BB and above on day 5 by the Gardner scoring) rates, CPR per ET, and LBR per couple patient who underwent micro TESE. We did not undergo preoperative hormonal therapy for KS patients. Statistical analysis was performed using unpaired t-tests and chi-squared tests. Main results and the role of chance We identified 212 KS out of 2204 azoospermic patients (9.6%). SRR of the first attempt micro TESE in KS (91/175=52.0%) was significantly higher than unexplained NOA (166/768=21.6%) (p &amp;lt; 0.001). 2PN, blastocysts development, and good-quality blastocysts rates were 49.2%, 42.9%, 16.9% in KS and 50.8, 45.8%, 21.0% in unexplained NOA respectively. Only good-quality blastocysts rate of KS was significantly lower than that of unexplained NOA (p &amp;lt; 0.01). Taking into account deflection of wives age, each result was calculated at their age 38 or under, but they were the same. CPR and LBR per ET in KS (42.6%, 30.4%) were significantly higher than unexplained NOA (33.1%, 23.7%) (p &amp;lt; 0.01, p &amp;lt; 0.05). The couples were 26.4% (56/212) in KS and 12.0% (116/963) in unexplained NOA (p &amp;lt; 0.001) who have given birth or ongoing pregnancy up to now after underwent micro TESE. That is, KS couples were significantly easier to give birth than unexplained NOA. Rates of congenital disorders had no significant difference between KS and unexplained NOA. Limitations, reasons for caution We did not show the data using ejaculated sperm with KS. The natal outcomes and development of these children has not been fully investigated, long-term follow-up of babies of the KS couples is needed. Wider implications of the findings Micro TESE is particularly helpful for KS cases, but embryonic development depend on the quality of spermatozoa. However, if a blastocyst can be obtained, the expectation of having a child is considered to be high. These results were shown to be useful in informing azoospermic couples undergoing micro TESE. Trial registration number not applicable