Blastocyst Formation Rate Research Articles

P-771 Prospective comparison of different needle sizes (17G vs 18G) and aspiration pressures (110 vs 220 Hgmm) for egg collection in oocyte donors: randomized, pilot study

Abstract Study question How does higher aspiration pressure (220Hgmm) and thinner oocyte retrieval needle size (18G) affect egg retrieval rate, intervention duration, and post-operative pain in oocyte donors? Summary answer Egg retrieval rate and developmental potential did not vary according to needle thickness or aspiration pressure, whereas with higher aspiration pressure the intervention was shorter. What is known already Although transvaginal ultrasound guided egg collection has been successfully used for more than two decades, technical parameters of the egg collection are still not standardized and are often based on the manufacturer’s recommendations which have not been evaluated in a clinical setting. Only a handful of studies evaluated the use of thinner oocyte retrieval needle aiming at reducing ovarian trauma (Wikland 2011, Kushnir, 2013). Study design, size, duration After obtaining informed consent consecutive oocyte donors (n = 105) were prospectively assigned on the day of egg retrieval scheduling to three equal-sized groups (group 2: 17G needle, 220 Hgmm, group 1; 18G needle, 220 Hgmm, group 3: 17 G needle, 110 Hgmm) using two different oocyte retrieval needles (Kitazato, Japan) and two different aspiration pressures (Labotect, Germany). Oocyte donor age, BMI, baseline AMH, ≥14 mm and ≥10 mm follicular counts were not significantly different among groups. Participants/materials, setting, methods Oocyte donors were stimulated with GnRH antagonist protocol and oocyte maturation was triggered with GnRH agonist. Pre- and postoperative pain measured by visual analogue score (VAS:0-10) and duration of each egg collection performed under sedation was recorded. Retrieved mature eggs were assigned to previously matched recipients or electively vitrified for use in our centre’s egg bank. The developmental potential of retrieved eggs was evaluated by the fertilization and blastocyst formation rate of assigned, fresh oocytes. Main results and the role of chance As aspiration pressure and needle size decreased, the average duration of egg collection became longer (08:02, 09:36, 10:50 minutes, p = 0.002). Although postoperative pain scores were uniformly low, there was a marginally lower postoperative pain score in group 3, where a lower aspiration pressure was used (1.6±1.4, 1.9±1.9, 1±1.1, p < 0.0001). No oocyte donor experienced any significant postoperative complication. The number of retrieved eggs (27.3±12, 26.8±13.6, 26.1±12), oocyte retrieval rate per number of ≥ 14 mm follicles (104, 103, 104%), and the proportion of damaged eggs with ruptured zona pellucida (3.8, 2.7, 3.7%) were not significantly different among groups. Fertilization (72, 73, 73%) and blastocyst formation (51, 53, 46%) rates of injected fresh mature oocytes were not significantly different. Limitations, reasons for caution Due to the lack of similar studies, the size of each study group (n = 35) was determined by convenience instead of a previous sample size calculation. Wider implications of the findings Precise calibration of technical parameters of egg collection merits further attention and continuous monitoring, especially in hyper-stimulated oocyte donors where an excellent oocyte yield should be balanced against a shorter intervention, maximally reduced postoperative pain, and the lowest possible rate of complications. Trial registration number Not applicable

P-406 Metal-phenolic network cell-membrane modification-mediated assembly strategy for re-establishing oocyte-granulosa cell communications and improving oocyte developmental potential

Abstract Study question Re-establish the cell-cell communication of isolated oocyte and granulosa cells (GCs) to mimic the follicle microenvironment and promote the success rate of in-vitro maturation (IVM). Summary answer The Metal-phenolic network (MPN) nanoparticles-mediated cell membrane modification system was used to achieve self-assembly of GCs on the oocyte membrane and re-establishing the Oocyte-GCs communication. What is known already How to preserve the fertility of female patients of childbearing age is an important problem faced by reproductive medicine. IVM of oocytes is an important means for female fertility preservation, but the problem of oocyte maturation rate needs to be solved urgently. The Oocyte-GCs interaction determines the key events in the oocyte development and maturation. Biological materials are gradually used for IVM, but there are still significant disadvantages. MPN has been widely used for the functionalization of bacteria and cell surface due to its unique nano-network structure and good biocompatibility. Study design, size, duration The MPN nanoparticles are respectively modified on GCs and oocytes, and the GCs can be self-assembled on the surface of the oocytes to re-establish cell communication. Then explore the biosafety and cytotoxicity of MPN nanoparticles on GCs and oocytes to rule out potential impact on the secretory function of GCs, reveal the formation of gap junction and substance exchange of Oocyte-GCs, and evaluate the effects of this technology on oocyte maturation, fertilization and embryo development. Participants/materials, setting, methods The oocyte-GCs assembly could be achieved through the MPN(EGCG-Zn2+). Then MPN@GCs and MPN@Oocytes were characterized by fluorescent protein, Zeta, SEM and TEM; the cytotoxicity was assessed by Live/Dead staining, CCK-8 and flow cytometry assays. Gap junction was observed by TEM; the expression levels changes of signaling pathways for assembled oocyte and GCs were detected by single-cell RNA-seq, RT-qPCR and WB. The oocyte and embryo developmental potential were also analyzed. Main results and the role of chance We confirmed the formation of a nano-network surrounding GCs and oocytes and achieved the assembly of GCs on the surface of oocytes. Furthermore, we illustrated that MPN was non- cytotoxicity for GCs and oocytes and have no negative effect on the secretion function of GCs, indicating high biocompatibility of MPN and shows the potential application on the re-establishment of oocyte-GCs communication. Gap junctions formation and substance exchange establishment between GCs and oocytes were also proved in the study. The single-cell RNA-seq uncovered key pathways involved in oocyte-GCs interactions after the oocyte-GCs assembly. In detail, the SUMO1 ubiquitination modification of PTEN was inhibited after re-establishing cell-cell communication and decreased the localization of PTEN at the GCs membrane, which weakened its inhibition on PI3K. Higher PI3K level further up-regulated AKT level and stimulated its activation by increasing phosphorylation modification, finally improved the developmental potential and maturation rate of oocytes. The re-establishment of cell communication significantly down-regulated the P38/MAPK pathway in oocytes, including inhibited P38/MAPK expression, thereby reducing the phosphorylation of MSK1 and the activation of ATF-1, and effectively weakened the apoptosis controlled by P38/MAPK. Finally, a significantly higher fertilization and blastocyst formation rate were observed as compared to the control group. Limitations, reasons for caution Although this study has achieved promising results, there is still gap between basic research and applications, and more fundamental research should be conducted to make it a powerful tool for clinical application in the future. Wider implications of the findings This study not only provided a promising tool for establishing oocyte-GCs communication and improving oocyte maturation rate for IVM, but also showed a versatile and powerful cell-cell assembly platform for regenerative medicine including tissue engineering and organoid studies. Trial registration number not applicable

P-133 A study of the relationship between in vitro embryo development and maternal hypothyroidism

Abstract Study question Are there an association between the maternal thyroid condition and in vitro fertilization (IVF) outcomes? Summary answer Maternal hypothyroidism was found to be related to oocyte maturation and blastocyst development. What is known already Previous studies have shown that thyroid-stimulating hormone (TSH) levels affect ovarian stimulation in assisted reproductive technology (ART). In addition, changes in serum TSH levels have been linked to a sub-optimal environment for the implantation and development of the embryo. Thyroid-related receptors are present in granulosa cells, ovaries, and the endometrium. In addition, those receptors play a role in ovulation and folliculogenesis. This study aims to determine the association between maternal hypothyroidism and adverse implications for IVF outcomes in IVF/ICSI treated women. Study design, size, duration This retrospective study performed 333 cycles of 208 patients with TSH levels ranging from 0.043-16.627 µIU/mL, treated at the RMC National IVF Center between 2019 and 2022. The study population consisted of 1171 oocytes, which were divided into two groups based on their maternal basal TSH levels: normal TSH ≤ 2.5 µIU/mL (control group, n = 865, euthyroid) and higher TSH > 2.5 µIU/mL (case group, n = 306, hypothyroid). Participants/materials, setting, methods Maternal basal serum TSH levels were measured using Fluorescence Enzyme Immunoassay on the third day of the menstrual cycle. IVF outcome expressed oocyte maturation, fertilization, cleavage, and blastocyst formation rate. Oocytes were divided into mature (metaphase II) and immature (metaphase I and prophase I) groups. Blastocysts are graded by the Kato Ladies Clinic classification system and divided into high potential (A and B grades) and low potential (C, D, and E grades) groups. Main results and the role of chance In our study, there was no statistically significant difference in age and partner’s semen criteria between the two groups (p > 0.05). As regards oocyte maturation, MII oocytes retrieved significantly higher in the control group (82.2% vs. 75.8%). Logistic regression analysis found that TSH levels > 2.5 µIU/mL were related to statistically significantly increased risks of oocyte maturation stages like MI or GV (Age affect adjusted model: B = 0.456, p = 0.005; OR = 1.578, 95% CI = 1.146-2.175). The fertilization, cleavage, and blast formation rates have no differences between the groups. However, 91% (n = 324/356) of higher potential blastocysts in the control group were significantly higher than the case group (79.7%, n = 106/133). While, by the logistic regression analysis found that higher TSH levels ( > 2.5 µIU/mL) were related to statistically significantly increased risks of lower potential for implantation grades (B = 0.947, p = 0.001; OR = 2.579, 95%CI = 1.477-4.502). Limitations, reasons for caution The study relied on TSH as the primary indicator of thyroid function and excluded participants with abnormal prolactin levels. Other markers, such as FT4 and TSHR, should also be considered for a thorough evaluation of thyroid function. Wider implications of the findings Thyroid dysfunction can negatively impact IVF success, and screening and managing thyroid function in women undergoing IVF are essential. More research is needed to understand the mechanisms and develop strategies to improve oocyte and blastocyst quality. Trial registration number not applicable

P-633 Progesterone elevation on the day of trigger in the presence of low ovarian response signifies abnormal follicular environment and poorer oocyte health

Abstract Study question Is progesterone elevation (PE) in the presence of low number of follicles (unexpected PE) associated with oocyte or embryo quality? Summary answer PE on day of trigger in cycles with ≤4 follicles is associated with significantly reduced fertilisation rates and fewer top-quality blastocysts What is known already The negative effect of PE on pregnancy rates in fresh IVF cycles has been demonstrated by multiple studies and meta-analyses, and most studies support no negative effect on oocyte and embryo quality. However, as it is thought that PE is the product of cumulative secretion of low levels of progesterone from multiple developing follicles, PE in the presence of a low number of follicles (unexpected PE) is considered a different pathophysiological entity. It has been hypothesized, but never proven, that in these cases, PE is the result of follicular dysregulation and that it might indicate poorer follicular and oocyte health. Study design, size, duration This is a multi-centre retrospective analysis of a large cohort (n = 1,393) of IVF/ICSI cycles with a low response to ovarian stimulation (1-4 follicles ≥14mm) performed from January 2018 to April 2022. Only cycles of ovarian stimulation using gonadotrophins and GnRH analogues with a measurement of serum progesterone on the day of triggering final oocyte maturation were included. Natural cycles and cycles involving the administration of clomiphene were excluded. Participants/materials, setting, methods Unexpected PE was defined as serum progesterone levels greater than 4.77 nmol/L (1.5 ng/mL) on day of trigger. The primary outcome was the number of usable blastocysts. Secondary outcomes included the number of oocytes retrieved, two pronuclei (2PN) oocytes, number of top-quality embryos, fertilisation rate and blastocyst formation rate. Generalised estimating equations were used to account for the clustered nature of data. Multivariable regression analysis was performed to control for the effect of multiple confounders. Main results and the role of chance In the univariate regression analysis, the number of oocytes retrieved was not significantly different between cycles with and without PE (mean 2.3 vs. 2.1 respectively; p = 0.550). The number of 2PN oocytes was significantly (p = 0.021) lower in the PE group (mean 0.72, 95%CI 0.31-1.14) compared to the non-PE group (mean 1.21, 95%CI 1.12-1.31). The number of top-quality blastocysts was also significantly (p = 0.003) lower in the PE group (mean 0.03, 95%CI -0.03-0.10) compared to the non-PE group (mean 0.14, 95%CI 0.11-0.17). Fertilisation rates per oocyte retrieved, fertilisation rates per oocyte inseminated and the top-quality blastocyst rate per oocyte retrieved were consistently lower in the PE group at 33.3% (95% CI 19.0-47.6), 26.9% (95% CI 14.8-39.0) and 0.7% (95% CI -0.8-2.1) compared to 58.7% (95% CI 55.8-61.7), 52.3% (95% CI 49.3-55.2) and 5.5% (95% CI 4.3-6.8) in the non-PE group. The negative association of PE with the primary outcome measure of this study was confirmed in the multivariable regression analysis where the number of usable blastocysts was significantly lower in the PE group (adjusted mean 0.14, 95%CI 0.03-0.25) compared to the non-PE group (adjusted mean 0.27, 95%CI 0.23-0.31) after adjusting for confounders. Limitations, reasons for caution This is a retrospective observational study, thus not all confounders may be accounted for. Despite being one of the largest studies conducted in poor responders, the sample size of unexpected PE cycles is relatively small due to its rarity. Therefore, this study may be underpowered to detect smaller effect sizes. Wider implications of the findings This study demonstrates for the first time that unexpected PE on the day of trigger (i.e. in cycles with low ovarian response) is associated with poorer oocyte health as reflected in the reduced fertilisation rates and fewer blastocysts available for transfer. This information can aid in better understanding this phenomenon. Trial registration number Not applicable

O-006 Novel and scalable non-invasive metabolic imaging of early embryos using a lab-on-a-chip approach

Abstract Study question Is it feasible to safely determine metabolic imaging signatures to measure nicotinamide adenine dinucleotide (NADH) associated auto-fluorescence in early embryos using a scalable lab-on-a-chip approach? Summary answer We developed an optofluidic device capable of non-invasively obtain high-resolution 3D images of the metabolic activity of live mouse embryos using a lab-on-a-chip approach. What is known already Selecting the most suitable embryos for implantation and subsequent healthy live birth is crucial to the success rate of assisted reproduction and offspring health. Thus, developing non-invasive methods that are reliable to assess oocyte and embryo quality has been a significant aim for assisted reproduction. Besides morphological evaluation using optical microscopy, a promising alternative is the non-invasive imaging of live embryos to establish metabolic activity performance. However, metabolic imaging has been only achieved using highly complex advanced microscopy such as Fluorescence-lifetime imaging (FLIM) and hyperspectral microscopy, methods that are costly and challenging, limiting the potential for deployment in fertility clinics. Study design, size, duration The non-invasive nature of the system was investigated by assessing the development and viability of live embryos after embryo culture for 67hrs post metabolic imaging at the 2-cell embryo stage (n = 115), including a control for culture conditions and sham controls (system no-illuminated). Embryo quality of developed blastocysts was assessed by immunocytochemistry to quantify trophectoderm and inner mass cells (n = 75). Furthermore, inhibition of metabolic activity (FK866 inhibitor) during embryo culture was also assessed (n = 18). Participants/materials, setting, methods Optofluidic devices were manufactured by cast-moulding using a negative photoresist (SU8-2075; MicroChemicals GmbH, Ulm, Germany) by a standard UV-photolithography process. The microstructures fabricated of polydimethylsiloxane (PDSM) integrated Light Sheet Fluorescence Microscopy into a microfluidic system, including on-chip micro-lenses to generate a light sheet at the center of a microchannel. Super-ovulated F1 (CBA/C57Bl6) mice were used to produce 2-cell embryos and embryo culture experiments. Blastocyst formation rates and embryo quality (immunocytochemistry) were compared between study groups. Main results and the role of chance The optofluidic device was capable of non-invasively obtaining high-resolution 3D images of the metabolic activity in live mouse embryos. The system’s design allowed continuous tracking of the embryo location, including high control displacement through the light-sheet, fast imaging of the embryos (<2 second) and keeping a low dose of light exposure (16 J cm-2 and 8 J cm-2). Optimum settings for keeping sample viability showed that a modest light dosage was capable to obtain 30 times higher signal-noise-ratio images than images obtained with a confocal system (p < 0.00001; t-test). The results showed no significant differences between the control, illuminated and non-illuminated embryos (sham control) for embryo development as well as embryo quality at the blastocyst stage (p > 0.05; Yate’s chi squared test). Additionally, embryos with inhibited metabolic activity showed decreased blastocyst formation rates as well as 47% reduction in metabolic activity measured by non-invasive metabolic imaging (p < 0.0001; t-test). This study reports an optofluidic device capable of non-invasive metabolic imaging of live embryos using a similar concept as previously reported using FLIM technology and hyperspectral microscopy, but allowing a novel, scalable and affordable system with implementation potential in standard IVF laboratory equipment. Limitations, reasons for caution The study was conducted using a mouse model focused on early embryo development. Further safety studies are required to assess embryonic health by investigating the impact of the use of light during embryo development, live birth, embryo gene expression and epigenome stability. Wider implications of the findings This lab-on-a-chip is novel, scalable and has the potential to be used in fertility clinics as a technology platform to enable real-time monitoring of the metabolic function of embryos prior embryo transfer. Future applications include potential integration into existing IVF laboratory equipment such as bench and time-lapse incubators. Trial registration number N/A

P-227 ICSI outcomes after using in-situ microfluidics of fluidic walls versus DGC: a prospective non-inferiority comparative pilot study in sibling oocytes

Abstract Study question Does the novel strategy in-situ microfluidics (isM) yield comparable ICSI outcomes to the control sperm selection methodology density gradient centrifugation (DGC)? Summary answer ICSI outcomes show that handmade in-situ microfluidics of fluidic walls are as effective as DGC to achieve fertilization and usable day 5 blastocyst formation rate. What is known already Microfluidics technologies are proving their capability to select suitable sperm for ICSI. Increasingly, publications show novel and ingenious microfluidics strategies aimed to integrate sperm biomimicry during in vitro sperm selection while simplifying the IVF-workflow. We recently designed a microfluidics system which allows selecting sperm for ICSI in the same ICSI-dish, only using microfluidics and disregarding centrifugation, washing and plasticware. A previous proof-of-concept study showed that this methodology was efficient to select suitable sperm for ICSI as it could separate at least 20 progressive spermatozoa in less than 15 minutes in a clean microdroplet free of any remaining seminal plasma. Study design, size, duration The present pilot study included a total of 280 fresh MII-oocytes allocated in a 1:1 ratio to the study (isM-ICSI) and control (DGC-ICSI) groups. The statistical power was established at 80% at a CI 95%. For comparison we relied in the KPIs: ICSI normal fertilization rate (INFR) and Day 5 usable blastocyst rate (D5UBR). The non-inferiority margin (Δ) was established using 2-sigma warning limit and our historical control mean values (ΔINFR = 7% and ΔD5UBR = 10%). Participants/materials, setting, methods An informed consent was signed by all participants. The comparative study included 29 consecutive ICSI cases performed in fresh oocytes from women under 40 years old and with an oocyte yield ≥6 MII. All semen samples had ≥1 × 106/ml progressive spermatozoa and were split into three aliquots: 1-seminogram (100 µl), 2-isM (10 µl), 3-DGC (surplus). Embryos were cultured in bench-top Time-lapse incubators, using GTL-media under 6,4% CO2 and 5% O2 conditions up to blastocyst stage. Main results and the role of chance Sperm characteristics showed the following total mean values for total concentration (42,83 ± 31,1 × 106/ml), total motility (51,28 ± 14,16 %) and progressive sperm concentration (17 ± 12 × 106/ml). The mean number of MII per patient was 9,66. From the total number of oocytes (n = 280), 139 were allocated to isM (study group) and 141 to DGC (control group) groups. INFR was 72,0 % vs 76,6 %, respectively. Non-significant differences were observed (p = 0,54). We observed that the mean total number of usable blastocysts (day 5/6) per patient was 1,83 (53/29) in isM vs 1,66 (48/29) in DGC. Moreover, D5UBR was 53,0% vs 44,4 % (p = 0,26) with a difference between the study and the control of within the non-inferiority margin. We observed a lower rate of arrested embryos (up to day 6) in the study group (19,0% vs 30,0%), although results were not statistically significant (p = 0,08). Limitations, reasons for caution This pilot study includes a limited number of oocytes. The present isM protocol depends on a free-hand preparation. Although the use of templates and the training reduces inter and intra-operator variability, a ready-to-use surface would benefit the operability. Further investigation is required to evaluate clinical pregnancy and live birth outcomes. Wider implications of the findings Sperm DNA fragmentation is cited among the causes of embryo arrest. As the use of isM resulted in a lower proportion of arrested embryos, exploring its capacity to select non-fragmented sperm warrants further interest. The isM protocol streamlines sperm selection for ICSI, reduces costs and risks along the procedure. Trial registration number na

O-301 Can advanced sperm selection techniques improve blastocyst euploidy rate of abnormal sperm DNA fragmentation cases to the level of normal ones; prospective randomized controlled trial

Abstract Study question To compare PGT-A outcomes of Physiological ICSI (PICSI) and magnetic activated cell sorting (MACS) sperm selection techniques to a control of normal sperm DNA fragmentation. Summary answer PGT-A outcomes did not differ between using normal sperm DNA fragmentation semen to the semen processed by PICSI or MACS. What is known already Sperm DNA fragmentation can negatively affects pre-implantation embryo development in all stages after ICSI. Advanced sperm selection techniques like PICSI and MACS are thought to be efficient to improve fertilization, cleavage, and blastocyst formation rates in cases with abnormal SDF. It has become evident that a blastocyst's aneuploidy is not only contributed by the oocyte,but can also be caused by the sperm.The relation between sperm selection techniques such as PICSI or MACS and blastocyst euploidy is not investigated yet. Study design, size, duration A prospective randomized controlled trial of 351 ICSI cycles in a private fertility center, from October 2019 to December 2022. Cases were included in the study if: female had ≥5 MII oocytes, male had abnormal sperm DNA fragmentation (SDF) index (>20% by TUNEL assay), all blastocysts (≥4BB) had trophoectoderm biopsy. Participants/materials, setting, methods Included cases were randomized on the day of ICSI to PICSI or MACS group, along with a control arm of cases with normal SDF needed for comparison. PGT-A was done using Illumina platform (Miseq), and the results were reviewed by artificial intelligence (Cooper Surgical). Cases were categorized according to the female age into 2 subgroups(<35, and > 35 years). Statistical analysis of all pre-implantation embryo development parameters and PGT-A results were done using SPSS. Main results and the role of chance We found no significant differences between the female age, male age, count, motility, SDF, no. mature oocytes between the studied groups. Limitations, reasons for caution The results included all pre-implantation embryo parameters but, subsequent post-implantation events like implantation, pregnancy and miscarriage rates will add more data about the competency of the resulted embryos. Wider implications of the findings The usage of advanced sperm selection techniques like PICSI or MACS has showed comparable results to that of normal SDF cases, through eliminating the adverse effects of SDF on ICSI and PGT-A outcomes. Trial registration number NCT05494216

P-130 Progesterone-primed cycles result in slower embryos without compromising the implantation potential, with the advantage of oral administration and potential cost reduction: A time-lapse imaging study

Abstract Study question Could exogenous progestin replace the use of an antagonist of GnRH (GNRH-antagonist) without any effects on embryo morphokinetics and implantation rate in freeze-all cycles? Summary answer Exogenous progestin leads to slower embryo development and increased cyclés cancellation rate, with increased implantation rate when compared to cycles using an GnRH-antagonist. What is known already Improvements in cryopreservation techniques associated with the expansion of elective-single embryo transfer have steadily increased the use of deferred embryo transfers. This gives the opportunity to break away from the standard sequence of stimulation–retrieval–transfer, and to consider new strategies for pharmacological control of follicle growth. Usual ovarian stimulation regimens use an analog of the GnRH to prevent the LH surge and premature ovulation. Since progesterone is able to block the LH surge question remains on whether exogenous progesterone may replace the use of an GnRH analogue without compromising embryo development, in cycles followed by embryo cryopreservation. Study design, size, duration This cohort study, performed in a private university-affiliated IVF center, from Mar/2019 - Mar/2021, included 288 freeze-all ICSI cycles and its 2,768 respective embryos. Patients were age matched, according to the age, into groups depending on the protocol used to prevent the LH surge: Progestin-Primed-Group (n = 144 cycles and 1,360 embryos) and GnRH-Antagonist-Group (n = 144 cycles and 1,408 embryos). Embryos were cultured in a time-lapse imaging incubation system (TLI) and embryo morphokinetics were compared among the groups Participants/materials, setting, methods Evaluated kinetic markers were timing to pronuclei appearance (tPNa) and fading (tPNf), to two, three, four, five, six, seven, and eight cells (t2 - t8), to morulation (tM), start of blastulation (tSB) and blastulation (tB). Durations of the second (t3-t2) and third (t5-t3) cell cycles (cc2 and cc3) and timing to complete synchronous divisions s1 (t2-tPNf), s2 (t4-t3), and s3 (t8-t5) were also calculated. The KID-Score ranking, laboratorial and clinical outcomes were also evaluated. Main results and the role of chance Mean maternal and paternal ages were 37.0 ± 3.8 and 39.0 ± 6.4 years old. Slower tPNa (6.2 ± 0.2 vs. 7.0 ± 0.2, p = 0.008), t2 (27.2 ± 0.3 vs. 26.2 ± 0.3, p = 0.045), t7 (56.4 ± vs. 54.7 ± 0.5, p = 0.046), tM (89.3 ± 0.8 vs. 87.1 ± 0.6, p = 0.045), tSB (101.5 ± 0.8 vs. 110.8 ± 0.1, p = 0.012), tB (111.0 ± 0.8 vs. 108.5 ± 0.7, p = 0.034), were observed among embryos derived from Progestin-Primed cycles. Similar tPNf, t3, t4, t5, t6, t8, timing to cc2, cc3, s1, s2, and s3 were observed among the groups. No significant differences were noted in the Kid-score D5, number of aspirated follicles, retrieved oocytes, oocyte yield, mature oocytes, and rates of mature oocytes, fertilization, and blastocyst formation. The cyclés cancelation (15.7% vs. 2.0%, p = 0.004) and implantation rates (63.1% ± 6.1 vs. 44.4% ± 6.3, p = 0.004) were increased in the Progestin-Primed-Group, however no significant differences were noted in the cumulative pregnancy (64.4% vs. 49.0%, p = 0.104) and miscarriage rate (2.6% vs. 8.6%, p = 0.554) for Progestin-Primed and GnRH-Antagonist groups, respectively. The expense for the ovulation prevention using the GnRH-Antagonist was U$318.18, while by using progestins U$ 11.05 was sufficient to inhibit the premature LH surge. Limitations, reasons for caution This study has limitations, such as the retrospective design and small sample size. Wider implications of the findings Despite the increased implantation rate and potential cost reduction, the high cancellation rate and slower embryo development in the progestin-protocol cannot be ignored. Delayed embryo transfer due to the freeze-only approach may also be inconvenient. Before considering any protocols for LH surge prevention, pros and cons must be carefully evaluated. Trial registration number N/A

P-236 IL-6 levels in follicular-fluid and day5/6 spent culture drops indicate its role as a biomarker of oocyte maturation and an embryokine influencing embryo developmental competence

Abstract Study question To elucidate the role of IL-6 in folliculogenesis and embryo development during human in-vitro culture Summary answer High IL-6 levels in follicular-fluid positively correlate with oocyte maturity status whereas higher IL-6 secretome in day5/day6 spent culture media inversely correlates with blastocyst/ICM grades What is known already Although some studies have attempted to explore the correlation of FF IL-6 with fertilization and embryo development rates; the results have been inconclusive. Similarly, studies comparing FF IL-6 levels with endometrial receptivity and pregnancy outcomes have been ambiguous and contradictory. Recent research has reported improvement in bovine blastocyst ICM grades in culture media supplemented with IL-6. No study has yet evaluated the role of IL-6 as a biomarker of oocyte maturity nor investigated IL-6 as an embryokine that could regulate embryo developmental competence in humans. We have employed the FF metabolomic and Secretomic approach for IL-6 in human IVF cycles. Study design, size, duration Prospective study in women (25-35 years) undergoing IVF cycles (n = 256) for unexplained/tubal-factor infertility from 2018-2020. Only those women whose FF and day5/6 spent culture media sample could be obtained, were included. Women with endometriosis, PID, Genital Kochs were excluded. IL-6 levels were measured by diagnostic kit in spent micro-drops and FF (pooled per patient per cycle). Cycles were divided into Low and High IL-6 groups (FF: ≤ 47.5 and >47.5pg/ml; Spent Medium: <0.5 and ≥0.5pg/ml). Participants/materials, setting, methods Antagonist cycles involving COH with r-FSH and ICSI for all. Noted oocyte Nuclear maturity (GV/MI/MII). Oocyte Cytoplasmic maturity graded as Top and non-Top, based on presence of granulation, refractile bodies, sER, visco-elasticity. Post ICSI, oocytes cultured individually in 50µl single-step media micro-drops till day3 and shifted to fresh 50µl micro-drops for extended culture till day5/6. Blastocyst gradation was done using Gardner’s classification for expansion, ICM and trophectodermal cells. Statistical analysis done using graph-pad prism VI Main results and the role of chance High FF/IL-6 group (n = 103) had significantly higher proportion of MII (92.08 vs. 76.33%; p < 0.001) and top-quality (88.10 vs. 70.0%; p < 0.001) oocytes and fertilization rates (89.45 vs. 77.30%; p = 0.009) compared to Low FF/IL-6 group (n = 153) although total number of days of stimulation/gonadotropin dose, mean oocytes retrieved did not differ significantly between the two groups. Cleavage (84.64 vs. 75.92%; p = 0.061) and blastocyst formation (53.86 vs. 42.37%; p = 0.053) rate, although higher, did not differ significantly between the two FF/IL-6 groups. FF/IL-6 level correlated strongly with overall oocyte maturity status (Pearson r = 0.61, 95% CI 0.52-0.8). Threshold FF/IL-6 level for obtaining top-quality MII oocytes was >39.2pg/ml (ROCAUC = 86%, Sensitivity = 78%, Specificity = 83%). In D5/6 spent culture drops, likelihood of top-quality blastocyst formation (expansion grade ≥3 and ICM/TE grades AA/AB/BA) increased by 41% (odds ratio: 0.44, 95%CI: 0.4-0.5; χ2 p < 0.0001) if IL-6 concentration was <0.5 pg/ml. Lower culture-drop IL-6 levels correlated strongly with top ICM grades (Pearson r = −0.66, 95% CI 0.6-0.7). Highest IL-6 levels were found in spent culture-drops containing grade-C blastocysts whether on day5 or day6. IL-6 levels were comparatively higher in day6 than in day5 micro-drops, correlating with higher percentage of top-quality blastocysts on day5 than on day6. Threshold micro-drop IL-6 level for top-grade blastocyst <0.42 pg/ml. Limitations, reasons for caution We estimated IL-6 levels in pooled FF (per patient, per cycle). Although cumbersome & time-consuming, it would be worthwhile to evaluate individual FF (per follicle, per patient, per cycle). Also, measuring IL-6 in spent media involving group embryo culture, may facilitate study of the probable paracrine effects of this ‘embryokine’. Wider implications of the findings IL-6 is a pleiotropic cytokine and its role/s in folliculogenesis, embryo development and endometrial receptivity is not yet clearly etched out. This study attempts to elucidate the functions of IL-6 in a systematic manner by deriving threshold values for obtaining top quality oocytes and blastocysts in human in-vitro culture conditions. Trial registration number NOT APPLICABLE

P-218 Does Artificial Oocyte Activation (AOA) improve the outcome beyond fertilization rate in ICSI cycles?

Abstract Study question Does CultActive© improve reproductive outcomes of ICSI cycles in cases of low fertilization rate and normal fertilization rate? Summary answer The application of CultActive© after ICSI improves fertilization rates only in clinical indication of fertilization failure. What is known already Oocyte activation deficiency is attributed in the majority of cases to fertilization failure in ICSI cycles, and these can be corrected by increasing initial levels of calcium (Ca2+) using assisted oocyte activation techniques (AOA), such as the use of Ca2+ ionophores. These agents enhance intracellular calcium release and increase the membrane permeability facilitating the influx of extracellular Ca2+, which initiates the activation cascade. Previous results suggest that Ca2+ ionophore treatment can increase the live birth rate after failed ICSI cycles in couples with poor fertilization rates, embryo developmental arrest, and also increase the number of good quality embryos. Study design, size, duration This was a retrospective observational study. There were 30 patients included and the first group consisted of 30 ICSI cycles, 231 oocytes and 56 embryos (without AOA). The second group consisted of the same patients (n = 30) with 30 ICSI cycles using AOA, 219 oocytes and 47 embryos. Subgroups were defined according to clinical indications (normal fertilization rate group: ≥ 65 % and low fertilization rate group: <65% ). All data was collected from January-December 2022. Participants/materials, setting, methods 450 oocytes were assessed in the study. 231 oocytes belonged to the pre-AOA and 219 belonged to the AOA. The oocytes were exposed for 15 minutes after ICSI in a solution containing the Ca2+ ionophore A23187, CultActive© (Gynemed, Germany). Fertilization, good quality blastocyst formation and pregnancy rate were analyzed.Good quality blastocyst were defined as ≥ 3BB. Statistical significance was analyzed using the Paired Student T test (p < 0.05 was considered statistically significant). Main results and the role of chance The cohort of patients with normal fertilization rate (without clinical indications),was not significantly different. The AOA treatment gave a fertilization rate of 61% compared to 71% of the control cycles (p = 0.1398). There was no increase in the number of good quality blastocysts formation when the study group was compared to the control (p = 0.1660). In the cohort of patients with an indication of low fertilization, a significantly higher fertilization rate was recorded compared to the control (51% and 37%, respectively p = 0.0238). There were no significant difference in the quantity of good quality blastocysts compared with the control (p = 0.3328). Regardless of normal or low fertilization rates there was no significant difference in pregnancy rate when CultActive© was used in treatment (p > 0.05). Limitations, reasons for caution There are limitations in our study. First, the retrospective design and limited study population are major limitations. The data should be interpreted cautiously in the subgroup analyses due to potential bias, specifically the small population in the group without clinical indication. Wider implications of the findings AOA was only beneficial for couples with poor fertilization. Embryo quality and pregnancy rate did not improve with CultActiveive©. External variation was minimized by using the same patients in each cohort. This suggests AOA should not be used without clinical indication or for poor embryo quality/blastulation rate. Trial registration number not applicable

P-137 pronuclear characteristics and chromosomal constitutions of mono-pronuclear zygotes

Abstract Study question Limited studies have explored the correlation between the pronuclear characteristics, developmental potential, and genetic constitution of mono-pronuclear (1PN) zygotes. Summary answer Those 1PN zygotes developed into blastocysts have unique pronuclear characteristics and significantly higher rate of diploidy euploidy in comparison with the arrested embryos. What is known already It has been reported that 1PN blastocysts have similar morphokinetic features to two-pronuclear (2PN) embryos. Cytogenetic analyses reveal that some of the 1PN embryos have a normal chromosomal constitution. Study design, size, duration This retrospective cohort study included the 1PN zygotes from December 2021 to September 2022 (n = 388). Participants/materials, setting, methods Patients who underwent IVF or intracytoplasmic sperm injection (ICSI) in the Women and Children’s Hospital of Chongqing Medical University were recruited. Only 1PN zygotes cultured in the time-lapse incubators from the oocyte retrieval day were included. The pronuclear characteristics, including the area and diameter of the pronuclear, the number of nuclei, and the distance between the pronuclear and the near-polar body (DPNP), and the genetic constitutions were investigated. Main results and the role of chance The overall blastocyst formation and good-quality blastocyst rates in 1PN zygotes were 22.94% and 16.24%, significantly lower than that of 2PN zygotes (63.25% and 50.23%, respectively, P = 0.000). Compared to arrested embryos, 1PN zygotes that developed into blastocysts showed significantly larger area (752.57 ± 131.53 vs. 653.65 ± 116.58, P = 0.000), longer diameter of pronuclear (30.10 ± 2.90 vs. 27.28 ± 2.83, P = 0.000), a greater number of nuclei (11.92 ± 4.06 vs. 7.72 ± 3.07, P = 0.000), and shorter DPNP (13.73 ± 8.65 vs. 24.20 ± 13.51, P = 0.000). Of the tested embryos, the diploidy euploidy rate was significantly higher in good-quality blastocysts in comparison with the arrested embryos (66.67% vs. 11.76%, P = 0.000), which was also significantly higher in IVF-1PN blastocysts than in ICSI-1PN blastocysts (75.44% vs. 25.00%, P = 0.001). Limitations, reasons for caution The correlation between pronuclear characteristics and genetic constitution was investigated only in limited embryos. The small sample size might weaken the conclusions. In addition, part of the 1PN blastocysts were tested by NGS, which has inherent limitations because it could not find the uniparental diploidy. Wider implications of the findings The 1PN blastocysts, especially from IVF-1PN zygotes, have high diploidy euploidy rates. The utility of 1PN blastocysts is potentially beneficial for patients without other viable embryos. Trial registration number Chongqing Science and Technology Committee (grant number: CSTB2022NSCQ-MSX0253), Chongqing Health Committee (grant number: 2021MSXM108), Yuzhong District Science and Technology Committee (grant number: 20190143), and Women and Children’s Hospital of Chongqing Medical University (grant number: 2021YJQN07).

P-307 Is multinucleation as bad as we ever thought?

Abstract Study question Could the presence of multicleation at early stages influence further embryo development? Summary answer The presence of multinucleation at 2 cell stages is independent on the success, but not at 4 cell stage. What is known already The presence of multinucleated blastomeres (MNB) in the cleaving embryo has been linked to poor embryo development and adverse in vitro fertilization (IVF) outcomes, when detecte on days 2 and 3 of embryo culture under inverted microscope inspections once a day. Multinucleation has been associated with a lower rate of blastocyst formation, increased rates of aneuploidy and chromosomal abnormalities, leading to chromosomal chaos and a decreased rate of implantation and arrested embryo development. With the introduction of Time-Lapse (TL) technology, it is been demostrated that multinucleation at the 2 cell stage is a very frequent issue. Study design, size, duration It is a retrospective observational study. 195 embryos from 28 patients that underwent and IVF treatment at the Unit of Medicine Reproductive of the Hospital General Universitario de Valencia were included in this study from 2017 to 2021 to study embryo development. Those patients whose embryos could be visualized with quality in a Time-lapse incubator were selected. Participants/materials, setting, methods The study of the 195 embryos from the 28 patients was carried out in the MIRI® TL6 Time-Lapse incubator. These embryos were checked at the 2-cell, 4-cell and blastocyst stages. The Shapiro-Wilk test was used to determine whether the sample had a normal distribution. and the Pearson test was used to determine if there were significant differences between the samples. Besides, the Chi-square test was used to determine the correlation between 2 qualitative variables. Main results and the role of chance 108 fertilized embryos were studied, 78.7% of them did develop blastocyst formation; 50% did not show multinucleation and 28.7% did, the analysis indicated that there was no correlation between blastocyst formation and the presence of multinucleation at 2 cell-stage (p = 0.364). The analysis indicated that multinucleated cells showed a prolongation of the division time, the analysis indicated that it had a normal distribution (p < 0.001) and it exist significant differences (p < 0.001). Pregnancy was obtained in 5% and 9% of the embryos with multinucleation at 2 cell-stages. Finally, analysis of autocorrections on day 2 showed that these occurred in 60% and 40% of 2 cell-stage. Limitations, reasons for caution As these data correspond to patient profiles from our unicenter study, the data could be different in other assisted reproduction centers. In addition, a larger study sample size is necessary in order to confirm our results. Wider implications of the findings These results can help us to decide the fate of the embryos and not to penalize those embryos that present multinucleations at the 2-cell stage. Trial registration number not applicable

P-466 The effect of SARS-CoV-2 infection or vaccination on controlled ovarian stimulation and in vitro fertilization: A multicenter retrospective cohort study

Abstract Study question Are the ovarian response to controlled ovarian stimulation (COS) and embryo development after in vitro fertilization (IVF) affected by SARS-CoV-2 infection and immunization? Summary answer Both SARS-CoV-2 infection and SARS-CoV-2 mRNA or inactivated vaccinations do not affect ovarian responsiveness to COS or embryo development following IVF. What is known already SARS-CoV-2 infects the target cell by binding to angiotensin-converting enzyme 2 (ACE2) via its surface spike protein. ACE2 receptors are present in the vagina, uterus, fallopian tubes, and ovaries of the female reproductive system. They regulate ovarian steroidogenesis, follicular development, oocyte maturation, and follicle atresia. The virus downregulates ACE2 levels, resulting in decreased conversion of Angiotensin II (ANGII) to Ang (1-7) and increased accumulation of (ANGII). This may impair reproductive fertility. However, initial studies conclude that both SARS-CoV-2 infection and vaccination would not affect reproductive outcomes, but it is necessary to develop new studies that confirm their applicability. Study design, size, duration A retrospective multicentric cohort study of 428 patients who underwent COS for oocyte donation between January 1st, 2018 and July 31th, 2022 were screened for eligibility and followed up to September 18th, 2022. Oocyte donation cycles were utilized because this model is an excellent choice for controlling known female characteristics Participants/materials, setting, methods Eligibility criteria were donors between 18 to 32 years. Patients were categorized into the vaccinated group if they had received SARS-CoV-2 vaccines (BNT162b2 mRNA Pfizer-Biontech, mRNA-1273 Moderna or inactivated SARS-CoV-2 vaccine Sinovac). The control group was chosen from medical records before March 2020 ensure they were neither infected or vaccinated. Demographic, cycle characteristics, and laboratory outcomes were compared. Normally, distributed data were compared across groups by univariate ANOVA. All P-values were considered significant at < 0.05. Main results and the role of chance In total, 428 patients were included in the study, with 12 recovering from SARS–CoV-2 infection (Group 1), 92 reporting vaccination (Group 2), and 324 patients who underwent COS before March 2020 assigned to the non-exposed group (Group 3). The mean time from recovery to oocyte aspiration was 122 days (range 39-315 days) and 213 days from the first vaccine dose to oocyte aspiration (range 17–249 days). The study's participants had an average age of 24.8 years. Age was found to be normally distributed and did not differ significantly between the three groups (P-value 0,953). Similarly, ovarian reserve markers, as measured by AFC and AMH, did not differ between groups (P-values of 0,787 and 0,423 respectively). No significant differences were observed regarding stimulation duration and total gonadotropin dose. The number of retrieved oocytes, MII oocytes, fertilized oocytes, and good-quality day 5 embryos were also similar between the three groups. Comparing oocyte retrieval rate, mature oocyte rate, normal fertilization rate, and blastocyst formation rate consistently revealed no statistically significant differences. Limitations, reasons for caution The main limitation is its retrospective design. Other limitations include the small sample size of recovered patients, the absence of sperm analyses, and data of live birth rates and neonatal outcomes. Wider implications of the findings SARS-CoV-2 infection and vaccination seem unrelated to detrimental effects on ovarian response or embryo development. These findings contribute to the growing body evidence that supports vaccination in women trying to conceive. Further prospective studies with larger cohort sizes and longer follow-ups are warranted to validate our conclusion. Trial registration number NCT05562479

O-234 Constant antioxidant supplementation increases blastocyst formation from oocyte donors

Abstract Study question Does constant supplementation of antioxidants to the culture media increase blastocyst formation from oocyte donors? Summary answer The constant supplementation of antioxidants to the culture media increases blastocyst formation from oocyte donors What is known already The use of a combination of various antioxidants in embryo culture media has recently been investigated to explore the potential benefit for embryo development and clinical outcomes. However, the concentration of antioxidants added to the culture has only been determined by its effect. In this study, we performed a repeated antioxidants supplementation to resemble a physiological oxidation reduction environment. Therefore, we investigated the effect of antioxidants added every 12 hours to the culture media on blastocyst formation and expansion in oocyte donors. Study design, size, duration This prospective study was conducted at CITMER, Mexico from April 2020 to November 2022.We included a total of 258 recipients from oocyte donors undergoing IVF/ICSI. Participants/materials, setting, methods A total of 2403 zygotes were divided into 4 groups and cultured in the following conditions until blastocyst stage: Group 1A: 563 zygotes 20% O2 with antioxidants every 12 hours, Group 1B: 1109 zygotes 20% O2 with antioxidants at the beginning, Group 2A: 339 zygotes 5% O2 with antioxidants every 12 hours, Group 2B: 392 zygotes 5% O2 with antioxidants at the beginning. Embryo development was assessed. Odds ratio and Fisher test were performed. p < 0.05=significant Main results and the role of chance For both oxygen tensions, we found a significant increase in the total blastocyst formation rate (day 5+day 6) when antioxidants were added repeatedly (1A: 54.7% vs 1B: 45.9%, p = 0.0007*; 2A: 58.7% vs 2A: 46.9%, p = 0.001*). In addition, the rate of expansion at days 5 + 6 was also significantly higher than in the groups where the antioxidants were added only at the beginning of the culture (1A: 35.9% vs 1B: 29.9%, p = 0.01*; 2A: 41.3% vs 2A: 31.6%, p = 0.006*). Limitations, reasons for caution Given this is a sibling zygotes study, patients are their own controls. Wider implications of the findings Constant supplementation of antioxidants to the culture media increases blastocyst formation from oocyte donors as well as the expansion rate, which may significantly improve clinical outcomes. Trial registration number 06162020-aox04

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

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

P-716 Genetic mutations in women with recurrent assisted reproductive technology failure

Abstract Study question Are there genetic mutations which may lead to recurrent failures of assisted reproductive technology? Summary answer In this study, some genes were found to be candidates for causing repeated ART failures. What is known already Although reproductive aging is one of the greatest hindrances to successful pregnancy, many patients remain infertile, enduring recurrent ART failures, and approximately 20% of infertility is idiopathic. At present, the mutation of 8 genes, PADI6, NLRP2, TLE6, NLRP5, BTG4, KHDC3L, REC114 and OOEP has been reported to cause early embryonic arrest, while the mutation of PATL2 with GV stage arrest; TUBB8 and TRIP13 with failure of polar body extrusion; PANX1 with oocyte death; ZP1, ZP2, ZP3 with empty follicle syndrome; WEE2, TLE6 and CDC20 with fertilization failure have been found. Study design, size, duration Between January and October 2021, at Hanabusa Women’s Clinic, 25 infertile women, who experienced IVF / ICSI failure more than 5 times, with blastocyst formation rates of less than 10%, were recruited, after informed consent was obtained. 10 staff members of Hanabusa Women’s Clinic who conceived and delivered naturally were compared as normal controls. Participants/materials, setting, methods Genomic DNA was extracted from whole blood and genomic DNA samples were subjected to whole exome sequencing. High impact variants with allele frequencies of ≦0.20 in the 1000 Genome EAS, which were not found in the control subjects, were selected. Data from the Human Genetic Variation Database (HGVD) was used for the Hardy-Weinberg equation (HWE) to evaluate possible candidates for recurrent ART failures caused by genetic mutations. Main results and the role of chance High impact homozygous variants were detected in 57 genes among the study subjects of infertile women, but not among the control subjects. Among these 57 genes, significant differences in the allele frequencies between 8.3KJPN and the patients were detected in ADAM33 (p = 0.009), CEP89 (p = 0.012), CRIPAK (p < 0.001), LGALS9B (p < 0.001), PDZRN3 (p = 0.001), RAET1E (p = 0.007) and SPATA31A3 (p = 0.045). The HWE of each gene was calculated based on the data from HGVD. Among the 57 genes mentioned above, significantly lower HWE was detected in patients as compared to the HGVD in ADAM33 (p < 0.001), CEP89 (p = 0.033), MICA (p < 0.001), OR2T29 (p = 0.003), OR52J3 (p = 0.0497), RABL2A (p < 0.001), RNF17 (p = 0.0499), SPATA31C1 (p = 0.030) and WWTR1 (p < 0.001). Having identified one of the piRNA pathway genes, RNF17, which had significantly reduced HWE, the localization of RNF17 in the primordial follicle was examined. Immunofluorescent staining showed that RNF17 was sporadically localized in the cytoplasm of primordial follicles. Limitations, reasons for caution The sample size of this study was relatively small, and precise information such as age, gender in the database used in this study did not match that of the study patients, thus, the interpretation of the results is limited and further research is required to confirm our findings. Wider implications of the findings Among abovementioned genes, one of the piRNA pathway genes, RNF17, and one of the Hippo signal pathway genes, WWER1, were the most likely causes of repeated ART failures. The findings may provide potential diagnostic markers for patients with recurrent ART failures, helping us understand the genetic basis of female infertility. Trial registration number Not applicable

P-196 PIEZO-intracytoplasmic sperm injection (P-ICSI) can improve the clinical outcomes of ICSI

Abstract Study question Can P-ICSI improve the clinical outcomes of ICSI, such as degeneration, fertilization and development to blastocyst, compared to conventional ICSI (C-ICSI)? Summary answer P-ICSI can improve the clinical outcomes of ICSI by increasing the number of fertilized oocytes and decreasing the number of degenerated oocytes after ICSI. What is known already In human IVF-ET, some of oocytes are damaged after ICSI when retrieved oocytes are inseminated by ICSI. The application of P-ICSI has been limited although it was developed over 30 years ago. Recently, it has been reported that P-ICSI can decrease damage after ICSI and improve fertilization and embryonic development compared to C-ICSI. Study design, size, duration This study included 50 subjects undergoing IVF and was performed between April and October 2022. Sibling oocytes were randomly assigned to C-ICSI and P-ICSI. Participants/materials, setting, methods A total of 1,207 oocytes were retrieved from 50 patients. Among them, 852 oocytes with visible spindle were inseminated by C-ICSI or P-ICSI in sibling oocytes. Four hundred twenty-five oocytes were inseminated by C-ICSI and 427 oocytes were by P-ICSI. Fertilization, degeneration after ICSI and blastocyst development on day 5 or day 6 were compared between the P-ICSI and C-ICSI. The differences between C-ICSI and P-ICSI were statistically analyzed using Mann-Whitney U test. Main results and the role of chance Normal fertilization rate of P-ICSI (78.2 ± 16.5%) were higher than that of C-ICSI (71.2 ± 18.9%). The difference was statistically significant (P = 0.0447). The oocyte degeneration rates after ICSI were 9.3 ± 11.8% (C-ICSI) and 4.8 ± 9.4% (P-ICSI). The degeneration rate of C-ICSI was significantly higher than that of P-ICSI (P = 0.04338). Blastocyst formation rate of C-ICSI was 58.6 ± 25.0% and that of P-ICSI was 64.3 ± 26.3%. The blastocyst formation rate of P-ICSI was higher than that of C-ICSI but the difference was not statistically significant (P = 0.2113). The rates of blastocysts that were available for embryo replacement or cryopreservation were higher in P-ICSI (56.0 ± 25.4%) than in C-ICSI (50.4 ± 24.7%) although the difference was not significant (P = 0.26272). Limitations, reasons for caution Pregnancy outcomes data were not available. The data of this study were obtained from single fertility center. More large-scale multicenter studies will be needed to confirm the effectiveness of P-ICSI. Wider implications of the findings Consistent with other studies, these results show that P-ICSI reduces the damage rate of oocytes after ICSI. The implementation of P-ICSI has the potential to improve clinical outcomes of human IVF although it is more complicated and takes longer than that of C-ICSI. Trial registration number not applicable

Melatonin promotes parthenogenetic development of vitrified-warmed mouse MII oocytes, potentially by reducing oxidative stress through SIRT1

Previous studies have demonstrated that melatonin could ameliorate oxidative stress during the cryopreservation of mouse MII oocytes and their in vitro culture after parthenogenetic activation. However, the underlying molecular mechanism remained poorly understood. This study was conducted to investigate whether melatonin could modulate the oxidative stress in the parthenogenetic 2-cell embryos derived from vitrified-warmed oocytes through SIRT1. The results showed that the reactive oxygen species levels increased, the glutathione levels and SIRT1 expression decreased significantly in parthenogenetic 2-cell embryos derived from cryopreserved oocyte, and the parthenogenetic blastocyst formation rates significantly decreased when compared to those derived from control oocytes. These unfavorable phenomena were prevented by the addition of either 10−9 mol/L melatonin or 10−6 mol/L SRT-1720 (SIRT1 agonist), and it was restored by the supplementation of 10−9 mol/L melatonin in combination with 2 × 10−5 mol/L EX527 (SIRT1 inhibitor). Therefore, the findings from the present study concluded that melatonin may reduce oxidative stress via regulating SIRT1, and potentially promote the parthenogenetic development of vitrified-warmed mouse MII oocytes.

Acute exposure to organophosphorus pesticide metabolites compromises buffalo sperm function and impairs fertility

Agrichemicals such as organophosphorus pesticides’ metabolites (OPPMs) are more hazardous and pervasive than their parent pesticides. Parental germline exposure to such xenobiotics leads to an elevated susceptibility towards reproductive failures e.g. sub- or in-fertility. This study sought to examine the effects of low-dose, acute OPPM exposure on mammalian sperm function using buffalo as the model organism. The buffalo spermatozoa were briefly (2 h) exposed to metabolites of the three most prevalent organophosphorus pesticides (OPPs) viz. Omethoate (from Dimethoate), paraoxon-methyl (from methyl/ethyl parathion) and 3, 5, 6-trichloro-2-pyridinol (from chlorpyrifos). Exposure to OPPMs resulted in compromised structural and functional integrity (dose-dependent) of the buffalo spermatozoa typified by elevated membrane damage, increased lipid peroxidation, precocious capacitation and tyrosine phosphorylation, perturbed mitochondrial activity and function and (P < 0.05). This led to a decline in the in vitro fertilizing ability (P < 0.01) of the exposed spermatozoa, as indicated by reduced cleavage and blastocyst formation rates. Preliminary data indicate that acute exposure to OPPMs, akin to their parent pesticides, induces biomolecular and physiological changes in spermatozoa that compromise their health and function ultimately affecting their fertility. This is the first study demonstrating the in vitro spermatotoxic effects of multiple OPPMs on male gamete functional integrity.

Oxidative stress in OPU mares can delay embryo development in vitro

The application of ovum pickup-intracytoplasmic sperm injection (OPU-ICSI) is well established for the commercial equine in vitro production (IVP). There is a marked individual mare effect on the OPU-ICSI outcome, but little is known about the influence of the mare's health conditions. This study aimed to determine a potential association between the concentrations of interleukin-6 (IL-6) and reactive oxygen metabolites (d-ROMs) in the serum of the donor mare on the efficiency of equine IVP. Just before OPU, a blood sample was collected from 28 Warmblood donor mares, that were subjected to a routine OPU-ICSI program. The serum concentrations of IL-6 and d-ROMs were photometrically assayed using the equine Nori® (Genorise Scientific, USA) and Diacron® (Diacron International, Italy) kits, respectively. The kinetics of blastocyst development and the blastocyst rate (blastocysts/injected oocytes) were recorded. For the blastocyst forming mares, Pearson's and Spearman's correlation coefficients were calculated between the blastocyst rate and the serum concentration of IL-6 and d-ROMs. The serum concentrations were also compared between mares yielding at least one and mares yielding no blastocyst or between mares with fast (at day 7-8 post ICSI) and slow (≥ day 9 post ICSI)growing blastocysts using the independent t-test or Mann-Whitney U-test. The average blastocyst formation rate was 24.68±5.16 % and the average concentrations of IL-6 and d-ROMs were 519.59±157.08 pg/mL and 171.30±4.55 carratelli units (UCARR), respectively. Serum concentrations of IL-6 and d-ROMs, respectively, were not significantly correlated with blastocyst formation rates and not markedly different between the blastocyst forming (552.68±235.18 pg/mL and 168.36±5.56 UCARR) and non-forming (468.47±179.99 pg/mL and 175.85±7.89 UCARR) mares. Serum concentrations of d-ROMs were significantly lower in mares with fast growing (148.10±8.13 UCARR) compared to those with slow growing blastocysts (179.41±4.89 UCARR; P= 0.003). Taken together, the serum concentration of IL-6 and d-ROMs are not associated with the mares’ ability to produce blastocysts after OPU-ICSI. Although it may be questioned whether a single sample is representative of the mare's health status, changes in serum metabolites related to oxidative stress at the time of oocyte retrieval may diminish the OPU-ICSI outcome via interfering with the embryonic development in mares. Funded by the Ministry of Higher Education of the Arab Republic of Egypt.