Poor Embryo Research Articles

Effect of frozen-thawed embryo transfer with a poor-quality embryo and a good-quality embryo on pregnancy and neonatal outcomes.

To evaluate the impact of embryo quality and quantity, specifically a poor quality embryo (PQE) in combination with a good quality embryo (GQE), by double embryo transfer (DET) on the live birth rate (LBR) and neonatal outcomes in patients undergoing frozen-thawed embryo transfer (FET) cycles. A study on a cohort of women who underwent a total of 1462 frozen-thawed cleavage or blastocyst embryo transfer cycles with autologous oocytes was conducted between January 2018 and December 2021. To compare the outcomes between single embryo transfer (SET) with a GQE and DET with a GQE and a PQE, propensity score matching (PSM) was applied to control for potential confounders, and a generalized estimating equation (GEE) model was used to determine the association between the effect of an additional PQE and the outcomes. Subgroup analysis was also performed for patients stratified by female age. After PS matching, DET-GQE + PQE did not significantly alter the LBR (adjusted odds ratio [OR] 1.421, 95% CI 0.907-2.228) compared with SET-GQE in cleavage-stage embryo transfer but did increase the multiple birth rate (MBR, [OR] 3.917, 95% CI 1.189-12.911). However, in patients who underwent blastocyst-stage embryo transfer, adding a second PQE increased the live birth rate by 7.8% ([OR] 1.477, 95% CI 1.046-2.086) and the multiple birth rate by 19.6% ([OR] 28.355, 95% CI 3.926-204.790), and resulted in adverse neonatal outcomes. For patients who underwent cleavage-stage embryo transfer, transferring a PQE with a GQE led to a significant increase in the MBR ([OR] 4.724, 95% CI 1.121-19.913) in women under 35 years old but not in the LBR ([OR] 1.227, 95% CI 0.719-2.092). The increases in LBR and MBR for DET-GQE + PQE compared with SET-GQE in women older than 35 years were nonsignificant toward. For patients who underwent blastocyst-stage embryo transfer, DET-GQE + PQE had a greater LBR ([OR] 1.803, 95% CI 1.165-2.789), MBR ([OR] 24.185, 95% CI 3.285-178.062) and preterm birth rate (PBR, [OR] 4.092, 95% CI 1.153-14.518) than did SET-GQE in women under 35 years old, while no significant impact on the LBR ([OR] 1.053, 95% CI 0.589-1.884) or MBR (0% vs. 8.3%) was observed in women older than 35 years. The addition of a PQE has no significant benefit on the LBR but significantly increases the MBR in patients who underwent frozen-thawed cleavage-stage embryo transfer. However, for patients who underwent blastocyst-stage embryo transfer, DET-GQE + PQE resulted in an increase in both the LBR and MBR, which may lead to adverse neonatal outcomes. Thus, the benefits and risks of double blastocyst-stage embryo transfer should be balanced. In patients younger than 35 years, SET-GQE achieved satisfactory LBR either in cleavage-stage embryo transfer or blastocyst-stage embryo transfer, while DET-GQE + PQE resulted in a dramatically increased MBR. Considering the low LBR in women older than 35 years who underwent single cleavage-stage embryo transfer, selective single blastocyst-stage embryo transfer appears to be a more promising approach for reducing the risk of multiple live births and adverse neonatal outcomes.

Evaluation of Oxidative and Nitrosative Stress Markers Related To Inflammation in The Cumulus Cells and Follicular Fluid of Women Undergoing Intracytoplasmic Sperm Injection: A Prospective Study.

Oxidative/nitrosative stress in the oocyte microenvironment could have an impact on intracytoplasmic sperm injection (ICSI) outcomes. The presence of reactive oxygen species (ROS) and reactive nitrogen species (RNS) can stimulate the secretion of pro-inflammatory cytokines, leading to chronic inflammation and potentially affecting embryo as well as oocyte quality. This study aimed to examine the relationship of lipid peroxidation [measured by the malondialdehyde (MDA) assay] with protein carbonyl [measured by the 2,4 dinitrophenylhydrazine (DNPH) assay] levels in cumulus cells (CCs), as well as nitric oxide (NO), peroxynitrite (ONOO-), and C-reactive protein (CRP) levels in follicular fluid (FF). The potential relationship of these levels with ICSI outcome was also evaluated. In this prospective study, 63 FF samples and their corresponding CCs were collected for ICSI procedures. Spectrophotometry was used to assess levels of DNPH, MDA, NO, and ONOO-. CRP levels were evaluated using an immunoturbidimetric assay. The patients under 37 years with normal ovarian reserve had significantly lower levels of MDA, DNPH, NO, ONOO-, and CRP compared to those over 37 years. Additionally, we observed higher levels of MDA, DNPH, NO, ONOO-, and CRP in the group with an oocyte maturity rate of less than 60%. No significant difference was observed between the DNPH levels and factors such as infertility duration, embryo quality, pregnancy, or the number of retrieved oocytes. A higher level of MDA, NO, ONOO-, and CRP was found to be significantly related to the lower number of retrieved oocytes, longer periods of infertility, poor embryo quality, and negative pregnancy outcomes. Oxidative/nitrosative stress, linking to inflammation in the oocyte microenvironment, can be considered as a potentially useful biomarker for assessing the development and competence of oocytes and embryos and predicting ICSI outcomes.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

Could the sperm epigenome become a diagnostic tool for evaluation of the infertile man?

Although male infertility is currently diagnosed when abnormal sperm parameters are found, the poor predictive ability of sperm parameters on natural fecundity and medically assisted reproduction outcome poses the need for improved diagnostic techniques for male infertility. The accumulating evidence about the role played by the sperm epigenome in modulation of the early phases of embryonic development has led researchers to focus on the epigenetic mechanisms within the sperm epigenome to find new molecular markers of male infertility. Indeed, sperm epigenome abnormalities could explain some cases of unexplained male infertility in men showing normal sperm parameters and were found to be associated with poor embryo development in IVF cycles. The present mini-review summarizes the current knowledge about this interesting topic, starting from a description of the epigenetic mechanisms of gene expression regulation (i.e. DNA methylation, histone modifications, and non-coding RNAs' activity). We also discuss possible mechanisms by which environmental factors might cause epigenetic changes in the human germline and affect embryonic development, as well as subsequent generations' phenotypes. Studies demonstrating sperm epigenome abnormalities in men with male infertility are reviewed, with particular emphasis on those with the more severe form of spermatogenic dysfunction. Observations demonstrate that the diagnostic and prognostic efficacy of sperm epigenome evaluation will help facilitate the management of men with male factor infertility.

#243 : Comparison of IVF Outcome Following Follitropin Alfa and Follitropin Delta Utilization at Melati Clinic, Harapan Kita Women and Children Hospital

Background and Aims: Follitropin delta is the third recombinant FSH (rFSH) preparation developed with an individualized dosing regimen based on patient’s body weight and AMH level. According to the existing report, a daily dose of follitropin delta provides an equivalent ovarian response to that of follitropin alfa. However, the effect of follitropin delta on the resulting embryo and biochemical pregnancy remains to be elucidated. Hence, the present study aims to evaluate the effect of follitropin delta on embryo quality and biochemical pregnancy rates compared with that of follitropin alfa in an in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle. Methods: A retrospective cohort study was conducted between January and December 2022 at Melati Clinic, Harapan Kita Women and Children Hospital. A total of 59 IVF/ICSI cycles stimulated with follitropin alfa and 26 IVF/ICSI cycles treated with follitropin delta were subsequently analyzed. Results: After analysis, no significant difference was observed regarding the embryo quality of both treatments (P>0.05). The median of the good, moderate, and poor embryos in the follitropin alfa and follitropin delta group was 2 vs. 3, 1 vs. 2, and 2 vs. 2, respectively. Similarly, the biochemical pregnancy rate between the follitropin alfa and follitropin delta group was comparable (25% vs. 15%, P>0.05). Conclusions: In conclusion, our study suggests that stimulation with follitropin delta in IVF/ICSI cycles yields a similar embryo quality and biochemical pregnancy rates to those of follitropin alfa.

#328 : Fertilization and Embryo Quality of Human Mature Oocytes with Morphological Abnormalities After Intracytoplasmic Sperm Injection (ICSI)

Background and Aims: The dysmorphism of oocytes either extra or intracytoplasmic could decrease viability and developmental competence of oocytes. There are several reports describing that specific dysmorphisms are related with decrease in fertilization and embryo competence. The aim of this research is to investigate fertilization and embryo quality of specific dysmorphic mature oocytes obtained from ICSI. Method: A retrospective analysis was performed in 183 patients with 1700 mature oocytes. Mature oocytes were classified as normal morphology (control), oocytes with intra-cytoplasmic dysmorphism (fragile oocytes, inclusion bodies, vacuoles, mild granularity of cytoplasm, severe granularity of cytoplasm, and central granulation), and oocytes with extra-cytoplasmic dysmorphism (granulation in perivitelline space, polar body fragmentation, large perivitelline space, and abnormal zona pellucida). All oocytes were inseminated with ICSI. Oocytes were cultured and scored on day 3 then some of them were transferred. The rest of oocytes were extended culture until day 5. Day 3 embryos were classified based on grading by Kamardi et al., 2021. Grade 1 and 2 embryos are considered good quality embryos. Day 5 embryos were classified based on modified grading by Gardner and Schoolcraft., 1999. Results: Overall, fertilization rate was significantly lower in the dysmorphic oocytes compared to control (P<0.01). Good quality embryo development on day 3 was significantly lower in oocytes with severe granularity of cytoplasm compared to control (P<0.05). Looking at embryo development on day 5, oocytes with inclusion bodies and central granulation had significantly lower on good quality blastocyst (P<0.05). Conclusion: The fertilization potential of dysmorphic oocytes in our study was lower compared to normal morphology oocytes, however, only severe granularity of cytoplasm, inclusion bodies and central granulation related with poor embryo quality on day 3 and day 5. In this study, we were able to define several specific of oocyte dimorphism related with lower fertilization and embryo quality.

Analyzing the dynamic measurement of growth and development in children born after the transplantation of poor quality blastocyst embryos: a propensity matching study

Objective This study aimed to observe the dynamic measurement of growth and development in children (0–3 years) after transplantation of low-quality frozen-thawed single blastocysts. Methods This was a retrospective cohort study conducted from January 2016 to December 2019 at a single center. The follow-up data of the children were obtained from the Jiangsu Province Maternal and Child Database. A total of 350 singleton live births were included. Based on the blastocyst score, the live births were divided into good-quality blastocyst embryo (GQE) group (315 live births) and poor-quality blastocyst embryo (PQE) group (35 live births). To improve statistical efficiency and control for potential confounding factors, singletons conceived from PQEs were matched with a 3:1 ratio according to couple ages, BMI, occupation and women AMH levels. Ultimately, 32 children in the PQE group and 95 children (with one missing data) in the GQE group were included in the final analysis. Results After matching for parents’ age, BMI, occupation and maternal serum AMH level, there was no significant difference in growth and development of children between the PQE group and GQE group. However, the E2 level on trigger days, the rate of 2PN oocyte, and blastocyst formation rate in the PQE group were significantly lower than in the GQE group (p < .05). The number of embryo transfers (ETs) in the PQE group was higher than in the GQE group (p < .0001). The rate of cesarean section in the PQE group was significantly higher than in the GQE group (p < .05). The height (at 3 months) and head circumference (at 12 months) in the PQE group were lower than in the GQE group (p < .05). Conclusions Transplantation of PQEs did not affect the growth and development of offspring (0–3 years) compared to good-quality blastocysts. However, the oocyte and embryo development potential was lower in the PQE group than in the GQE group. These results provide clinical reference that the transfer of PQE could be acceptable for patients with only PQE embryos.

296 Comparison of Dna Methylation Patterns in Sperm Collected from Brahman Bulls with Differences in Embryo Development

Abstract The objective was to determine if methylation patterns in sperm differed between bulls with good vs poor embryo development. Semen collected from sexually mature Brahman bulls was purified from frozen-thawed straws using a gradient [50% (vol/vol) and 90% (vol/vol)] of Isolate (Irvine Scientific, Santa Ana, CA), and used to fertilize abattoir-derived cumulus oocyte complexes (n = 81-100/bull in 2 wells). Number of blastocysts were recorded on d 8, and bulls were classified as Good (GD; &amp;gt;35% blastocyst rate; n = 5) or Poor (PD; &amp;lt; 35% blastocyst rate; n = 4) embryo development. Methylation of sperm DNA was assessed by reduced representation sodium bisulfite sequencing. Differences in methylation were determined with the Dispersion Shrinkage for Sequencing method in conjunction with Wald test. Differentially methylated cytosines and regions located in promoters of genes and gene bodies were analyzed to reveal gene ontology terms associated with them. Proportion of sperm cells that were viable (before and after a three-hour stress test), with inceased reactive oxygen species (ROS), and with damaged chromatin was determined. Statistical analysis (PROC GLIMMIX; SAS 9.4) included the fixed effect of embryo development classification which was considered significant at P &amp;lt; 0.05. Mean separation was performed using LSmeans. The proportion of viable (P &amp;gt; 0.21; GD = 46 ± 5%; PD = 36 ± 5%) sperm cells did not differ before the stress test. Post-stress test, PD bulls had more viable sperm cells (P &amp;lt; 0.02; 37 ± 3%) than GD (25 ± 2%) bulls. There was no difference between GD and PD for ROS (H2O2; P &amp;gt; 0.16 and •O2; P &amp;gt; 0.59). Proportion of sperm cells with damaged chromatin was greater (P &amp;lt; 0.01) in PD (3 ± 0.3%) than GD bulls (1 ± 0.01%). There were 1,742 differentially methylated cytosines and 343 differentially methylated regions when GD bulls were compared with PD bulls. Genes whose promoters were differentially methylated included DDC, GTSF1L, PPP1R27, NTM, HCK, GPR173, and TNFRSF1A. Functionally enriched pathways for differentially methylated promoters included apoptosis and amyotrophic lateral sclerosis while some of the significant biological processes were regulation of apoptotic process involved in morphogenesis, negative regulation of neuron migration, pyrimidine nucleoside catabolic process, and regulation of cardiac muscle tissue development. Differentially methylated gene bodies were significantly related to cellular components such as cell surface, endomembrane system, synapse, and cell periphery while molecular functions included binding, protein binding, and transmembrane receptor protein tyrosine kinase activity. Biological processes impacted by differentially methylated gene bodies included system development, neurogenesis, animal organ development, anatomical structure development, generation of neurons, nervous system development, and multicellular organism development. In summary, differences in embryo development are likely due to differences in methylation patterns impacting developmental processes, as semen characteristics were mainly similar between bulls with good versus poor embryo development.

The following article for this Special Issue was published in a different issue. They can be found in their respective issue.

Increased sperm deoxyribonucleic acid damage leads to poor embryo quality and subfertility of male rats treated with methylphenidate hydrochloride in adolescence. ANDR. 2022; 10(8): 1632-1643. https://doi.org/10.1111/andr.13277.

Decreased AKAP4/PKA signaling pathway in high DFI sperm affects sperm capacitation.

The sperm DNA fragmentation index (DFI) is a metric used to assess DNA fragmentation within sperm. During in vitro fertilization-embryo transfer (IVF-ET), high sperm DFI can lead to a low fertilization rate, poor embryo development, early miscarriage, etc. A kinase anchoring protein (AKAP) is a scaffold protein that can bind protein kinase A (PKA) to subcellular sites of specific substrates and protects the biophosphorylation reaction. Sperm protein antigen 17 (SPA17) can also bind to AKAP. This study intends to explore the reason for the decreased fertilization rate observed in high sperm DFI (H-DFI) patients during IVF-ET. In addition, the study investigates the expression of AKAP, protein kinase A regulatory subunit (PKARII), and SPA17 between H-DFI and low sperm DFI (L-DFI) patients. SPA17 at the transcriptional level is abnormal, the translational level increases in H-DFI patients, and the expression of AKAP4/PKARII protein decreases. H 2 O 2 has been used to simulate oxidative stress damage to spermatozoa during the formation of sperm DFI. It indicates that H 2 O 2 increases the expression of sperm SPA17 protein and suppresses AKAP4/PKARII protein expression. These processes inhibit sperm capacitation and reduce acrosomal reactions. Embryo culture data and IVF outcomes have been documented. The H-DFI group has a lower fertilization rate. Therefore, the results indicate that the possible causes for the decreased fertilization rate in the H-DFI patients have included loss of sperm AKAP4/PKARII proteins, blocked sperm capacitation, and reduced occurrence of acrosome reaction.

Artificial oocyte activation with Ca2+ ionophore improves reproductive outcomes in patients with fertilization failure and poor embryo development in previous ICSI cycles.

Does artificial oocyte activation (AOA) by a calcium ionophore (ionomycin) improve the previous fertilization failure or poor embryo development of intracytoplasmic sperm injection (ICSI) account for male factor infertility or other infertility causes? This retrospective study involved 114 patients receiving ICSI-AOA in Shanghai First Maternity and Infant Hospital with previous ICSI fertilization failure or poor embryo development. The previous ICSI cycles of the same patients without AOA served as the control group. The fertilization rates, cleavage rates, transferable embryo rates and blastocyst formation rates of the two groups were compared. Additionally, the clinical pregnancy, implantation rate and live birth rates were also compared to assess the efficiency and safety of AOA. Furthermore, two subgroup analyses were performed in this study based on the cause of infertility and the reason for AOA. The fertilization rate, embryonic development potential and clinical outcome were compared among groups. Among 114 ICSI-AOA cycles, the fertilization rate, top-quality embryo rate, implantation rate, clinical pregnancy per patient and live birth rate per patient were improved significantly compared with previous ICSI cycles (p<0.05 to P< 0.001), and the miscarriage rate in the AOA group was significantly lower than that of the control group (p<0.001). In the AOA subgroups based on the cause of infertility, the fertilization rates of each subgroup were significantly improved compared with previous control cycles except for the mixed factor infertility subgroup (p<0.05 to p<0.001). In the AOA subgroups based on the reason for AOA, the fertilization rates of each subgroup were significantly increased compared with those in their previous ICSI cycle without AOA (p<0.001); however, there was no significant difference in the top-quality embryo rate. No significant improvement was found in the implantation rates and the clinical pregnancy rate in each subgroup except for the poor embryo development subgroup. In the 114 AOA cycles, 35 healthy infants (21 singletons and 7 twins) were delivered without major congenital birth defects or malformations. This study showed that AOA with the calcium ionophore ionomycin can improve the reproductive outcomes of patients with previous fertilization failure and poor embryo development after ICSI.

Current Insights in the Repeat Breeder Cow Syndrome.

Cows can have difficulties becoming pregnant, and in certain cases, these reproductive failures do not have an evident cause. Furthermore, when these failures are repeated three or more times with estrous cycles of normal duration and in the absence of evident clinical signs, it is considered repeat breeder cow (RBC) syndrome. A substantive incidence of RBC syndrome has been reported all over the world, which severely affects the farm economy. This paper reviews those studies particularly focused on RBC syndrome from 2000 to 2023 but also includes consolidated information until this date. Hormonal imbalances, undetectable oviductal or uterine defects, or poor oocyte or embryo quality have been reported as causes of RBC syndrome, while subclinical endometritis has been considered a relevant causal agent. However, it is unresolved why this condition is recurrent in certain animals, despite the implementation of corrective management actions or treatments. Recent studies evaluate the putative role of certain genes, factors, hormones, or proteins in the pathogenesis of RBC syndrome. Numerous risk factors contribute to the appearance of this syndrome, and some of them could be mitigated to partially prevent this infertility, while others cannot be changed. Due to the complexity of this syndrome, it is important to increase knowledge about the mechanisms involved, develop new diagnostic tools to differentiate causal agents, and implement new treatments to restore fertility. There is consensus about the huge repercussions of this syndrome on farm profitability, but further studies are now needed to describe its economic impact.

O-252 Genetic characterization of human sperm centrosome defects in infertile couples with complex embryo aneuploidy

Abstract Study question Can we characterize the genetic aspects related to sperm centrosome dysfunction in couples with high embryo aneuploidy that cannot exclusively be attributed to the oocyte? Summary answer We characterized structural and genomic abnormalities responsible for sperm centrosome dysfunction in infertile couples with complex embryo aneuploidy not exclusively related to the female partner. What is known already Conventional semen analysis provides only limited information on the proper function and embryo developmental competence of the male gamete. Therefore, an ancillary test, such as sperm chromatin fragmentation (SCF) and particularly double-stranded DNA breaks, has been claimed responsible for structural chromosomal abnormalities. It remains puzzling the source of complex embryo aneuploidy and chaotic chromosomal mosaicism that cannot solely be attributed to the female partner. The availability of genomic analysis may shed light on the integrity and function of the sperm centrosome responsible for ordaining chromosomal distribution at the first mitotic division. Study design, size, duration In the past 4 years, we identified 13 couples with unexpected high poor embryo cleavage resulting in a higher number of embryos with complex aneuploidy not solely attributable to the female gamete, resulting in extremely poor embryo implantation. Therefore, ancillary testing on the spermatozoa of the male partners was carried out to determine if the couple’s reproductive failure was eventually due to a stealth male gamete deficiency. Participants/materials, setting, methods Thirteen couples with poor clinical outcomes, characterized by poor embryo cleavage, high and complex embryo aneuploidy not exclusively due to the oocyte, with poor embryo implantation in multiple IVF/ICSI cycles, were offered adjuvant male partner testing including centrosome assessment through immunofluorescence staining on centrin (normal ≥ 60%), sperm chromatin fragmentation testing through terminal deoxynucleotide dUTP transferase nick-end labeling (TUNEL, normal ≤ 15%), transmission electron microscopy (TEM), and whole exome sequencing (WES) on spermatozoal DNA. Main results and the role of chance In 13 couples with an average paternal age of 38.1±2 years, semen analysis revealed a volume of 22.2±1.4mL, concentration of 58.2±37x10^6/mL, motility of 35.2±18% and normal morphology of 2.6±1%. They underwent with female partners (maternal age of 37.2±2 years) a total of 16 ICSI cycles with aneuploidy testing. Only 11 cycles yielded embryos for transfer due to poor embryo development with high aneuploidy rate, and none of them achieved a pregnancy. Centrosome staining revealed that only 45.8±22% of the spermatozoa displayed this key organelle. An overall SCF was observed at 16.7±10%. TEM confirmed that approximately 70% of the spermatozoa had irregular proximal centrioles. Moreover, genomic analysis by WES revealed mutations on PLK4 gene responsible for centriole duplication, BRSK1 gene involved in centrosome duplication, TUBGCP6 gene responsible for centrosomal microtubule nucleation, MARK4 for centrosome localization, MAP1S for microtubule anchoring and DNA binding, RIC8A for chromosome distribution, and CALM3 for cytokinesis. Of this cohort, 3 couples that subsequently underwent 3 ICSI cycles had their spermatozoa processed by microfluidics, aiming at selecting male gametes with higher genomic integrity. A fertilization rate of 78.3% (18/23) was achieved generating euploid embryos that resulted in 3 successful pregnancies. Limitations, reasons for caution We identified sperm centrosome dysfunction responsible for the peculiar and recurrent embryo aneuploidy. Although we identified shared gene mutations in those couples, these data are still preliminary. With exception of improving spermatozoa selection, there is a lack of a single effective treatment option to address sperm centrosome abnormality. Wider implications of the findings Ancillary tests with semen analysis were able to assess the function and competence of the male gamete. Corroborative genomic study can help provide insights into the reason for poor embryo implantation particularly those with high complex aneuploidy. Trial registration number Not applicable

P-165 Artificial removal of the zona pellucida at the pronuclear stage in patients with poor quality embryos with severe fragmentation

Abstract Study question Is artificial removal of the zona pellucida (ZP) at the pronuclear stage effective in patients with poor quality embryos with severe fragmentation? Summary answer Artificial removal of the ZP at the pronuclear stage significantly improved culture results in patients with poor quality embryos with severe fragmentation. What is known already In 2020, Yumoto et al. first reported that artificial removal of the ZP (ZP-free) at the pronuclear stage decreased the rate of fragmentation and improved the culture outcomes of 3 pronuclei (3PN) embryos reaching to the good blastocysts. Therefore, the ZP may not always be necessary for normal embryonic development after the pronuclear stage. The same group presented at the 37th ESHRE meeting in 2021, that the blastocyst transfer after ZP-free embryo of 2 pronuclei (2PN) resulted in normal pregnancy in two patients with poor-quality embryos resulting in live births. Study design, size, duration This study included 211 of 2PN embryos from 46 cycles for 30 couples obtained between July 2021 and December 2022, whose previous oocyte retrieval cycle showed massive cytoplasmic fragmentation at the first cleavage confirmed by a time-laps incubator (TLI). Average age of women was 38.8years. The embryos with artificial removal of the ZP at the pronuclear stage were cultured in a TLI up to 7 days. All blastocysts were cryopreserved for future embryo transfer cycles. Participants/materials, setting, methods All participants provided written informed consent, and Institutional Review Board approval was obtained. Artificial removal of the ZP at the pronuclear stage was performed by following procedure. 1) Embryos were placed in 0.125M sucrose-containing HEPES for cytoplasm shrinking. 2) The ZP without adhesion was removed with a laser system. 3) Ooplasms were completely separated from ZP by blowing the medium to adhesion point like a jet car wash. 4) ZP-free embryo was obtained. Main results and the role of chance Day 3 embryos were divided into 3 groups using the Veeck’s classification: 8 cell embryos with grade 2 or higher were defined as good, embryos with grade 4 or 5 regardless of the number of cells were defined as poor, and others were defined as fair. We compared the culture results of ZP-free cycle and previous oocyte retrieval cycle (ZP-intact). For day 3 embryos, good, fair and poor grade were 19.4% (41/211), 62.6% (132/211) and 18.0% (38/211) in ZP-free embryos compared with 2.9% (15/561), 37.4% (210/561), and 59.9% (336/561) in the past, respectively (p &amp;lt; 0.001). Good-quality blastocysts were defined as 3BB or more on day 5 by the Gardner’s classification. As to the blastocysts, blastocysts development and good-quality blastocysts rates were 45.0% (95/211) and 27.0% (57/211) in ZP-free embryos compared with 23.0% (112/488) and 6.8% (33/488) in the past, respectively (p &amp;lt; 0.001). Of these, 30 embryo transfers were performed resulting in 11 clinical pregnancies. We had 3 live births without any congenital anomalies and one miscarriage. One was delivered at 33 weeks of gestation with caesarian section, one mother had gestational diabetes. Limitations, reasons for caution This study was retrospective study without any control group and was conducted at only single fertility center. It is necessary to conduct sibling oocytes study to demonstrate the effectiveness of ZP-free culture, and to create criteria who are benefit from ZP-free culture. Moreover, safety of ZP-free culture should be evaluated. Wider implications of the findings ZP-free culture reduced fragments and improved culture outcomes in patients with poor quality embryos with severe fragmentation. ZP-free culture might bring light to the patients who have difficulty obtaining good-quality embryos with severe fragmentation. Trial registration number Not Applicable

O-288 Implantation, ultrastructure and metabolic profiling of embryos from PGT-M and PGT-A Cycles

Abstract Study question Can metabolic profiling predict embryos at risk of single gene defects and chromosomal abnormalities and how are these reflected in their ultrustructure? Summary answer Different metabolic profiles are observed between embryos with chromosomal abnormalities and monogenic disorders and normal embryos, linked to altered mitochondrial and other organelles’ structure/function What is known already Down’s syndrome embryos and Monosomy 21 embryos have previously been shown to have differential expression of metabolites compared to normal embryos, but limited studies, have investigated in detail the metabolic profiling of embryos with other abnormalities or those with monogenic disorders in comparison to chromosomally normal embryos or their reflection in their ultrustructure. Study design, size, duration 520 embryos were biopsied on day5 for PGT-A (n = 178-47 cycles) or on day3 for PGT-A (n = 178-33 cycles) and PGT-M (n = 164-24 cycles). Following transfer of normal embryos, spare embryos, rejected for transfer following day5 or day3 biopsy were processed for TEM (n = 60 day3 biopsied, n = 60 day5 biopsied/vitrified), 60 unbiopsied/vitrified embryos from egg donation cycles used as control. Culture media were collected from the embryos and analysed by hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS). Participants/materials, setting, methods PGT-A and PGT-M were performed in 2 IVF Units. Metabolic profiling was conducted in a Forensic Toxicology Laboratory by HILIC-MS/MS (100 metabolites). Ultrastructure analysis by TEM was carried out in an Academic Hospital and Histology/Embryology Laboratory following embryo fixation in 3% glutaraldehyde, 1% osmium tetroxide, washes in PBS and staining with 1% aqueous uranyl acetate. Main results and the role of chance The implantation rates (+ve hCG/ET) for the PGT-M cycles was 68.2% and the for the PGT-A (blastocyst and cleavage biopsy cycles) 64.5% and 65% respectively. Characteristic patient specific metabolic profiles after screening for &amp;gt;100 primary metabolites were observed which differed between normal embryos that had resulted in a viable pregnancy and aneuploid and chaotic embryos although it was more difficult to find clear patterns in embryos with monogenic disorders. Logistic regression analysis revealed a number of metabolites with high predictive value which in combination with embryo score could serve in the future as non-invasive markers for the detection of chromosomal abnormalities before embryo transfer. TEM analysis revealed differences in the quality of cells and organelle activity which were reflected in the embryo metabolic profiles. Abnormal but well developed hatching blastocysts had mainly cells with good mitochondrial morphology/ activity, nice Golgi apparatus and well developed rough and smooth endoplasmic reticulum but depending on the aneuploidy or gene mutation involved, inner cell mass cells with limited organelles and occasionally lipofuscin droplets in the trophectoderm were evident. Chaotic poor quality embryos showed a lower number of mitochondria, often with no cisternae, increased number of vacuoles, and frequently problems in junctions between cells. Limitations, reasons for caution Although metabolic profiles were compared between normal and abnormal embryos, all the normal embryos were transferred to the uterus or remain vitrified for clinical purposes. Therefore the ultrastructure analysis is based only on biopsied abnormal embryos and as control unbiopsied embryos from egg-donation cycles with high chance of being normal. Wider implications of the findings This study shows high implantation rates after PGT—M and PGT-A and identified distinct differences in the metabolic profiles of normal and abnormal embryos providing unique metabolites which in the future could serve as non-invasive biomarkers for the detection of abnormalities before embryo transfer. Trial registration number Not applicable

O-018 Impact of atypical bacterial semen infections on spermocytogram parameters and Spermatozoa DNA fragmentation and denaturation

Abstract Study question Does Spermocytogram parameters and Spermatozoa-DNA-Fragmentation (SDF) / Denaturation (SDD) levels are affected by atypical-bacterial semen infections in North-African men during the period between 2013-2022? Summary answer SDD presented positive-correlations with the presence of 3/5 atypical bacteria in the semen. Various Spermocytogram-parameters were also found to be negatively associated with atypical-bacterial-semen-infection. What is known already Atypical-Bacterial semen infections such as infections by Chlamydia-trachomatis (CT), Mycoplasma-hominis (MH) and genitalium (MG), Ureaplasma urealyticum (UU) and parvum (UP), were shown to be associated with poor sperm function (Prabha.et.al.,2009; Agarwal.et.al.,2012). In infections caused by CT, SDF was observed to be increased significantly and associated with male infertility (Karinen.et.al.,2004; Gallegos.et.al.,2008). In addition, MH and UU appeared to cause induction of nuclear decondensation and SDD, which damages sperm and has effects on sperm parameters. However, other studies failed to show any effect of MH, UU and CT on sperm parameters and SDD (Lee.et.al.,2013; Gdoura.et.al.,2008; Huang.et.al.,2015; Farsimadan and Motamedifar,2020). Study design, size, duration Retrospective study, including Sperm-culture-analyzes, Spermocytograms and SDF/SDD tests performed between January,2013 and December,2022 of 10.386 patients from North-Africa aged between 19 and 73 years-old (y.o) (average-age: 43.2 y.o ± 7.16). Patients were classified in 2 main groups according to the presence (Group-1: n = 613) or not (Group-2: n = 9773) of atypical-bacteria in the semen and 5 subgroups of monobacterial-infected-semen which were extracted from Group-1 as: CT (n = 14), MH (n = 61), MG (n = 10), UU (n = 147) and UP (n = 73) subgroups. Participants/materials, setting, methods 8201-Tunisian-patients(79%), 1142-Algerians(11%) and 1038-Libyans(10%) presented for semen-analyzes. Samples were analyzed according to World-Health-Organization (WHO) guidelines. Bacterial-semen-infection was detected by real-time-polymerase-chain-reaction(RT-PCR). Spermocytogram analyzes were performed according to WHO2010-recommendations using Sperm-class-analyzer-software (SCA5/6(CASA-system)) and SCA-scope (Microptic®). SDF analyzed with terminal-uridine-nucleotide-end-labeling(TUNEL)/sperm chromatin dispersion(SCD) techniques. SDD analyzed with the Aniline-Blue-staining-method. Statistical-analyzes were performed using SPSS22.0 for Windows-software. Kolmogorov–Smirnov-test for normality-analysis and comparisons by Student-t-test/Mann–Whitney U-test, as appropriate. Pearson/Spearman’ tests for correlations were used as appropriate, P-value&amp;lt;0.05 was considered as significant. Main results and the role of chance Sperm concentration was significantly lower in atypical-bacterial-semen-infected-group (Group-1) compared to controls (Group-2) with 11.39[0-311] vs 20.06[0-694]x106spz/mL, (p = 0.014); respectively, as well as progressive motility (14.6%[0%-64.47%] vs 21.89%[0%-81.35%], (p = 0.002); respectively) and typical morphology (5.34%[0%-18%] vs 9.29[0%-21%] (p &amp;lt; 0.001); respectively). However, Leucocytes concentration and SDD presented significant higher levels in Group-1 compared to Group-2 with 1.8[0.1-8.5] vs 0.5[0.1-2.45]x106 leucocytes/mL, (p &amp;lt; 0.001) and 21%[4%-60%] vs 15%[2%-75%], (p = 0.04); respectively. Similar significant differences were observed with 3/5 of the monobacterial-semen-infected-subgroups in comparison with Group-2 (MH, UU and UP-subgroups). No significant differences were observed in MG and CT-subgroups in except of SDD which presented significant higher levels in CT-subgroup in comparison with Group-2 (52%[4%-85%] vs 15%[2%-75%], (p = 0.002);respectively). Correlation-analyzes showed that SDD was positively correlated to MH (r = 0.069; p = 0.022) and UU (r = 0.084; p = 0.005) which is in accordance with the study of .Lee.et.al.(2013). Leucocytes concentration was also correlated positively to MH, CT, UU and UP (r = 0.047; p&amp;lt;0.001; r = 0.035; p=0.005; r = 0.074; p&amp;lt;0.001; r = 0.045; p=0.05; respectively). Sperm concentration, Progressive motility and Typical morphology presented Negative correlations with MH (r=-0.038; p=0.034; r=-0.048; p=0.05; r=-0.047; p=0.05; respectively), UU (r=-0.034;p=0.003; r=-0.04; p&amp;lt;0.001; r=-0.067; p=0.007; respectively) and UP (r=-0.072; p&amp;lt;0.001; r=-0.078; p=0.018; r=-0.057; p&amp;lt;0.05; respectively). Our results are in accordance with other studies such as of Prabha.et.al.(2009) and Agarwal. et al. (2012). However, SDF did not show any significant variation between our studied groups. Limitations, reasons for caution Our study is a retrospective-statistical survey that included patients presenting to the laboratory for fertility diagnosis or for a diagnosis due to a pathology and/or inflammation of the genital tract. Meta-analysis studies in addition to more prospective-randomized-controlled-trials in collaboration with IVF or other medical-analyzes-laboratories are necessary to confirm/refute our results. Wider implications of the findings These results should interest urologists, microbiologists, gynecologists, reproductive science fundamentalists, epidemiologists and embryologists who want to improve the investigations on the relationship between bacteria and semen parameters to the reasons of infertility, recurrent miscarriages and poor quality embryos in IVF. Trial registration number Not applicable

P-277 Can a validated, livebirth predictive, in-house morphokinetic algorithm, score embryos according to maternal age; providing more information, for patients, regarding chance of success?

Abstract Study question How does a livebirth (LB) prediction algorithm, built in-house on data from 6228 known outcomes from all ages, perform when categorised by female age? Summary answer The algorithm scored and ranked embryos accurately for clinical outcome (FH) regardless of female age; with proportions of poor prognosis embryos highest in older patients. What is known already Time-lapse incubation enables development of predictive algorithms using key morphokinetic variables to score embryos according to potential for clinical outcomes. Maternal age is associated with embryo quality and implantation. Understanding whether kinetic events are influenced by increasing age may benefit embryo selection and patient information provision. There is little evidence of a correlation between advancing maternal age and morphokinetics, although incidence of abnormal cleavage is linked to aneuploidy and reduced blastulation. Predictive algorithms are generally applied to embryos irrespective of oocyte age but are they effective in selecting the optimum embryo for transfer comparatively across differing maternal age groups? Study design, size, duration A retrospective, comparative analysis of 10034 transferred embryos from 2011 to 2022 in 11 sister fertility clinics. Embryos were cultured for 4 - 6 days in time-lapse incubation before transfer (Embryoscope, Vitrolife). A score was derived using a prospectively validated in-house statistical algorithm, assessing 6 manually annotated morphokinetic variables (t3,t4,t5,t8,tSB,and tB) and trophectoderm grade. Embryos were scored 1 to10 for increasing live birth (LB) potential. Participants/materials, setting, methods All transferred embryos were assessed for age of the patient or donor at treatment, traceable to an algorithm score (1-10) with known implantation, defined by presence of fetal heart (KID-FH). Embryos were assigned to two age groups, &amp;lt; 38 years (n6948) or &amp;gt; = 38 years (n3086) and compared according to the distribution of algorithm scores. Comparative significance of the two groups, against each other and the whole cohort, was assessed for significance using chi-squared. Main results and the role of chance Comparison of the KID-FH per score, for age groups revealed increasing implantation rates and effective ranking per score in each age group, &amp;lt;38 KID-FH scores 1 to10; 9.6%,17.8%, 25.7%, 24.3%, 28.3%, 38.2%, 41.4%, 48.4%, 54.6% and 57.8% and for &amp;gt; = 38 score 1-10; 2.0%, 3.8%, 5.9%, 9.9%, 10.8%, 17.9%, 22.2%, 22.0% 31.6% and 36.8%. KID-FH increased incrementally with increasing score in both maternal age groups, demonstrating that the algorithm is predictive irrespective of maternal age. The percentage incidence of scores were compared, &amp;lt;38 years, score 1 to 10; 3.8%, 4.6%, 4.5%, 5.0%, 6.2%, 7.5%, 9.3%, 12.4%, 16.8%, 29.8% versus &amp;gt; = 38 years 1 to 10; 8.0%, 9.4%, 7.1%, 9.2%, 8.7%, 9.4%,10.5%,11.0%, 11.2%,15.4%. There were significantly higher numbers of embryos with lower scores (p = 0.01) in the &amp;gt; = 38 age group (scores 1-6) than the &amp;lt;38 age group. The younger ages conversely, had significantly greater high scoring embryos 9-10, (p = 0.01), score 7 to 8 were comparable across the age groups. The algorithm score is effective in determining implantation potential irrespective of maternal age. Embryos with poorer implantation potential are present in significantly greater numbers in the older age group and this age group attains significantly fewer high potential embryos when compare to younger patient embryos. Limitations, reasons for caution The data for this analysis was determined from multiple clinics within a fertility group operating under uniform practices. The derived algorithm variables and outcomes may not be transferable to another clinic setting. Follow up is required for known live birth outcomes to confirm comparative prediction to KID-FH per age group. Wider implications of the findings The algorithm score infers embryo competence irrespective of age with differences existing only in the prevalence of low and high scores. This algorithm differentiation promises to inform clinicians and patients regarding their chance of success, when utilised to assess embryos. Morphology alone does not have this breadth or predictive capability Trial registration number N/A

P-405 Nuclear transfer overcomes poor embryo development of in vitro grown mouse oocytes

Abstract Study question Is the nuclear transfer technology able to overcome the compromised embryonic development of in vitro cultured secondary follicles from young B6D2 mice? Summary answer Nuclear transfer technology was able to restore embryonic development to levels similar of in vivo grown oocytes. What is known already In vitro follicle culture has been proposed as a strategy for fertility preservation in cancer patients. Studies in human follicle culture remain scarce, due to the low availability of human tissue. Mouse models have been extensively studied to improve follicle maturation and investigate the potential of in vitro grown (IVG) gametes. Despite significant improvements reported over the years, including increased maturation rates, advanced embryonic development and generation of fertile offspring, the quality of IVG oocytes remains inferior compared to their in vivo grown counterparts. Study design, size, duration The experimental study was conducted between October 2020 and January 2023, after approval by the Animal Ethics Committee of Ghent University Hospital (ECD no 19/60). In total, 108x 16-days-old B6D2 females were used for secondary follicle isolation and in vitro culture, 81x 8-12-week-old females for collection of in vivo grown oocytes, 4x 9-14-week-old B6D2 males for sperm freezing, 5x 10-24-week-old CD1 vasectomised males and 10x 8-12-weeks-old CD1 females as surrogate mothers for embryo transfer. Participants/materials, setting, methods Follicles isolated from 16-days-old B6D2 mice were cultured for 9 days, followed by maturation for 16-18hrs. Mature oocytes were assessed on diameter, spindle morphology, calcium releasing ability, mitochondrial membrane and embryonic developmental potential. In vivo grown oocytes from stimulated mice were used as controls and cytoplasmic donors for nuclear transfer. Spindle (ST) and Pronuclear transfer (PNT) were applied to overcome poor development of IVG embryos, by transferring the spindle/pronuclei of IVG oocytes/zygotes to enucleated controls. Main results and the role of chance In total, 1509 secondary follicles were cultured and 73.6% survived to Day 9. From the 719 cumulus oocyte complexes, 364 (50.6%) oocytes matured. Oocyte diameters were significantly smaller between IVG (67.4μm) and controls (73.1μm) (p &amp;lt; 0.001). Spindle staining revealed a similar number of normal spindles between the two groups (71.4% and 82.6% respectively, p = 0.138). Calcium release was significantly lower in IVG oocytes (1.6) vs controls (5.7) (p &amp;lt; 0.001), as well as mitochondrial membrane potential ((0.9) vs (2.2), p &amp;lt; 0.001). These data implicate a cytoplasmic inferiority in IVG oocytes. Following parthenogenetic activation, 2-cell (59.4%) and blastocyst rate (36.3%) in IVG group was significantly lower than control (89.4% and 88.2% respectively, p &amp;lt; 0.001). For this reason, we performed ST, using controls as cytoplasmic recipients for IVG spindles. The 2-cell rate significantly increased to 100% (p &amp;lt; 0.001) in ST oocytes, reaching 100% blastocyst formation. Next, to create bi-paternal embryos, we performed IVF. From the IVG group, 2-cell (40%) and blastocyst (0%) rates were significantly lower than control (94.4% and 83.3%, p &amp;lt; 0.001). Embryo development was restored following PNT (98% 2-cell and 86% blastocyst rate, p &amp;lt; 0.001). Genetic analysis of PNT embryos revealed that 5/6 were chromosomally normal, similar to controls (8/10). Embryo transfer of PNT and control embryos is ongoing. Limitations, reasons for caution Both ST and PNT are promising technologies to overcome poor embryonic development of IVG oocytes, but the benefit for human IVG oocytes remains to be investigated, along with the safety of the technique. Wider implications of the findings Our results demonstrate that ST and PNT have the potential to restore embryonic developmental competence of IVG oocytes, without increasing chromosomal abnormalities. This technology could be investigated in the future, as a mean to overcome poor quality of IVG oocytes in human. Trial registration number Not applicable

O-149 Endometrial thickness as a predictor of live birth and perinatal outcomes following oocyte donation

Abstract Study question Does endometrial thickness (EMT) predict the occurrence of live birth following oocyte donation? Summary answer Our study found that EMT is predictive of live birth in oocyte donation. What is known already EMT is a widely used surrogate marker of endometrial receptivity and is believed to be associated with in vitro fertilization pregnancy outcomes. However, the evidence thus far is mixed, with many studies (and even meta-analyses) suggesting that EMT may have little/no predictive value. One potential reason for the heterogeneity in results may be that most studies assessed mostly fresh autologous embryo transfers, without accounting for embryo quality nor the effect exogenous ovarian stimulation may have on endometrial receptivity and subsequent pregnancy/perinatal outcomes. Moreover, most subcategorized EMT to facilitate interpretation, which may have inadvertently introduced bias to the analyses. Study design, size, duration This retrospective, multicentre, cohort study analyzed all single blastocyst embryo transfers following oocyte donation from 2010-2019 subdivided into regular intervals of EMT (&amp;lt;7.0 mm, 7.0-8.9 mm, 9.0-10.9 mm, ≥11.0 mm). EMT was also evaluated as a continuous variable, comparing the best-fitting fractional polynomial against the linear function, to minimize bias due to residual confounding that may occur following the categorization of continuous variables. Confounder adjustment was performed using multivariable generalized estimating equations regression analysis. Participants/materials, setting, methods The oocyte donation model was chosen to minimize the influence of potential confounding factors such as poor embryo quality and the effect of ovarian stimulation on endometrial receptivity. The main objective of the study was to compare live birth rates (LBR). Our secondary outcomes included other surrogate pregnancy (hCG-positive pregnancy, clinical pregnancy, and miscarriage rates) and perinatal (gestational age at birth, preterm birth under 37 weeks, birthweight z-score, small and large for gestational age) outcomes. Main results and the role of chance In total, 33915 embryo transfers were analyzed. Confounder variables included were female donor and recipient age, recipient BMI, female factor infertility, male factor infertility, number of mature oocytes donated, oocyte status (fresh versus vitrified), sperm source (partner or donor), embryo status (fresh versus vitrified), embryo quality, endometrial preparation (natural or artificial cycle) and year of transfer. When compared to the reference EMT of 9.0-11.0 mm, EMT &amp;lt;7.0 mm was associated with lower hCG positive pregnancy rates (51.3% vs 57.6%; aOR 0.82 CI 0.75-0.89), lower clinical pregnancy (41.9% vs 49.1%; aOR 0.79 CI 0.73-0.86), higher miscarriage rates per hCG positive pregnancy (37.5% vs 31.4%; aOR 1.31 CI 1.16-1.47) and lower LBRs per transfer (32.1% vs 39.5%; aOR 0.76 CI 0.69-0.83). Regarding perinatal outcomes, we found significantly higher preterm delivery rates following an EMT &amp;lt;7.0 mm (14.1% vs 9.9%; aOR 1.54 CI 1.21-1.96) and higher large for gestational age rates associated with EMT ≥11.0 mm (16.4% vs 12.2%; aOR 1.40 CI 1.07-1.83). The fractional polynomial analysis revealed that the subcategorization of EMT caused clinically-relevant residual confounding. Specifically, LBRs assumed an approximate bell-shaped relationship with EMT, with LBR rates decreasing for both thinner EMTs (&amp;lt;7 mm subgroup) and thicker EMTs (≥11.0 mm subgroup). Limitations, reasons for caution The retrospective nature of the study and the inherent risk of bias related to unmeasured confounding may have impacted the results. Another potential limitation is the lack of perinatal outcome data due to loss of follow-up, which should be considered when interpreting these results. Wider implications of the findings Our results support the use of EMT as a predictor of live birth and as a marker of endometrial receptivity and competence. Moreover, it stresses the potential danger of subcategorizing all EMT &amp;lt;7.0 mm and &amp;gt;11.0 mm as equivalent, as the study found that these subgroups have varying LBR. Trial registration number not applicable

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 &amp;lt; 0.001) and it exist significant differences (p &amp;lt; 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