OBJECTIVE: To evaluate the embryological and pregnancy outcomes of women who failed in their first IVF treatment if they attempted a second cycle.
METHODS: For evaluating the embryological outcomes, the study cohort included 1,227 women who failed to obtain a live birth after the initial IVF cycle from September 2018 to August 2021 and returned for a second attempt. To evaluate reproductive outcomes including live birth rates (LBRs), 1227 women who returned for a second attempt were compared with 13,195 women undergoing their first oocyte retrieval with blastocyst culture attempted during the same study period.
RESULTS: In women who had a second cycle, the median number of oocyte retrieved (11 vs 9), fertilized oocytes (7 vs 5), usable embryos (6 vs 4) and blastocysts (3 vs 1) was higher in the second cycle compared to the first cycle (All p < 0.001). Blastocyst formation rates were significantly increased from 33% in the first cycle to 50% in the second cycle across the age group (p < 0.001). However, the primary transfer LBRs were significantly lower in the second cycle than that in the initial cycle (40.82% versus 51.79%, aOR: 0.74 [0.65, 0.84]). LBRs in the second cycle were 42.26%, 42.68%, 25.49% and 16.22% in women aged < 35, 35-37, 38-40, and > 40 years.
CONCLUSIONS: There was a notable enhancement in laboratory outcomes following the second attempt in women whose initial IVF cycles were unsuccessful. However, the uncertainty inherent in the successful implantation and the consequent progression to live birth remains a significant challenge.

UNASSIGNED: Polycystic ovary syndrome (PCOS) is main cause of anovulatory infertility in women with gestational age. There are currently four distinct phenotypes associated with individualized endocrinology and metabolism. Growth differentiation factor 9 (GDF9) is a candidate as potential biomarker for the assessment of oocyte competence. The effect on oocyte capacity has not been evaluated and analyzed in PCOS phenotypes.
UNASSIGNED: We aimed to screen the expression levels of GDF9 in mature follicles of women with controlled ovarian hyperstimulation (COS) with different PCOS phenotypes. To determine the correlation between the expression level of GDF9 and oocyte development ability.
UNASSIGNED: In Part 1, we conducted a retrospective study comparing the clinical outcomes and endocrine characteristics of patients with PCOS according to different subgroups (depending on the presence or absence of the main features of polycystic ovarian morphology (PCOM), hyperandrogenism (HA), and oligo-anovulation (OA)) and non-PCOS control group. We stratified PCOS as phenotype A (n = 29), phenotype B (n = 18) and phenotype D (n = 24). In Part 2, the expression of GDF9 in follicular fluid (FF) and cumulus cells (CCs) were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively.
UNASSIGNED: In Part 1, the baseline clinical, hormonal, and ultrasonographic characteristics of the study population were matched with the presence or absence of the cardinal features of each PCOS phenotypes showed a clear difference. Phenotypes A and D had statistically significant associations with blastocyst formation and clinical pregnancy compared with phenotypes B (p < 0.001). In Part 2, the levels of GDF9 in FF and CCs for phenotype A and B were significantly were higher than those of phenotype D (P = 0.019, P = 0.0015, respectively). Multivariate logistic regression analysis showed that GDF9 was an important independent predictor of blastocyst formation (P＜0.001). The blastocyst formation rate of phenotype A was higher than that of phenotype B and D (P＜0.001). Combining the results of the two parts, GDF9 appears to play a powerful role in the development of embryos into blastocysts.
UNASSIGNED: GDF9 expression varies with different PCOS phenotypes. Phenotype A had higher GDF9 levels and blastocyst formation ability.

In reproductive biology, understanding the effects of novel techniques on early embryo development is of paramount importance. To date, the effects of electrical activation on oocytes prior to in vitro fertilization (IVF) are not well understood. The aim of this study was to investigate the effects of oocyte electroporation prior to IVF on embryo development and to differentiate between true embryos and parthenotes by using a TPCN2 knock-out (KO) male to evaluate the presence of the KO allele in the resulting blastocysts. The study consisted of three experiments. The first one examined oocyte electroporation with and without subsequent IVF and found that electroporated oocytes had higher activation rates, increased occurrence of a single pronucleus, and no effect on sperm penetration. Cleavage rates improved in electroporated oocytes, but blastocyst rates remained constant. Genotype analysis revealed a significant increase in the proportion of parthenotes in the electroporated groups compared to the IVF control (30.2 % vs. 6.8 %). The second experiment compared two electroporation media, Opti-MEM and Nuclease-Free Duplex Buffer (DB). DB induced higher oocyte degeneration rates, and lower cleavage and blastocyst rates than Opti-MEM, while parthenogenetic formation remained consistent (60.0 and 48.5 %). In the third experiment, the timing of electroporation relative to IVF was evaluated (1 h before IVF, immediately before IVF and 7 h after IVF). Electroporation immediately before IVF resulted in higher activation rates and different pronuclear proportions compared to the other timing groups. The penetration rate was higher in the immediate electroporation group, and cleavage rate improved in all electroporated groups compared to the control. Blastocyst rates remained constant. Genotyping revealed no significant differences in parthenote proportions among the timing groups, but these were higher than the control (56.25 %, 63.89 %, 51.61 %, 2.44 %, respectively), and showed higher mutation rates when electroporation was performed 7 h after IVF. Overall, this comprehensive study sheds light on the potential of electroporation for creating genetically modified embryos and the importance of media selection and timing in the process, the best media being the Opti-MEM and the more efficient timing regarding mutation rate, 7 h post-IVF, even when the parthenote formation did not differ among electroporated groups. Further studies are needed to reduce the parthenogenetic activation while maintaining high mutation rates to optimize the use of this procedure for the generation of gene-edited pig embryos by oocyte/zygote electroporation.

N-acetyltransferase 10 (NAT10)-mediated N4-acetylcytidine (ac4C) modification is crucial for mRNA stability and translation efficiency, yet the underlying function in mammalian preimplantation embryos remains unclear. Here, we characterized the ac4C modification landscape in mouse early embryos and found that the majority of embryos deficient in ac4C writer-NAT10 failed to develop into normal blastocysts. Through single-cell sequencing, RNA-seq, acetylated RNA immunoprecipitation combined with PCR (acRIP-PCR), and embryonic phenotype monitoring, Nop2 was screened as a target gene of Nat10. Mechanistically, Nat10 knockdown decreases the ac4C modification on Nop2 mRNA and reduces RNA and protein abundance by affecting the mRNA stability of Nop2. Then, depletion of NOP2 may inhibit the translation of transcription factor TEAD4, resulting in defective expression of the downstream lineage-specific gene Cdx2, and ultimately preventing blastomeres from undergoing the trophectoderm (TE) fate. However, exogenous Nop2 mRNA partially reverses this abnormal development. In conclusion, our findings demonstrate that defective ac4C modification of Nop2 mRNA hinders the morula-to-blastocyst transition by influencing the first cell fate decision in mice.

In the present study, we attempted to improve the developmental competence of vitrified immature porcine oocytes by the preservation of mitochondrial properties using Cyclosporin A (CsA, inhibitor of mitochondrial membrane permeability transition) and Docetaxel (stabilizer of microtubules, hence mitochondrial distribution). In Experiment 1, Mitotracker red staining revealed reduced mitochondrial activity (MA) in vitrified/warmed oocytes at 0 and 22 h of in vitro maturation (IVM) compared with fresh ones. However, by at 46 h of IVM, MA levels in vitrified oocytes were similar to those in fresh control. Treatment of oocytes with CsA or Docetaxel improved MA at 0 h and 22 h of IVM compared with non-treated vitrified oocytes. However, there were no significant differences among groups in percentages of survival, maturation and embryo development after subsequent IVM and parthenogenetic activation. Nevertheless, a pretreatment with a combination of 10 µg/mL CsA and 0.05 µM Docetaxel improved the blastocyst formation of vitrified oocytes compared with non-treatment counterparts (11.2 ± 1.6% vs 5.9 ± 1.6%, P < 0.05). In conclusion, vitrification reduced mitochondrial activity in GV-stage oocytes during 0-22 h of IVM; however, it was normalized by 46 h IVM. Docetaxel or CsA pretreatment alone did not improve development competence of vitrified oocytes. However, pretreatment with a combination of CsA and Docetaxel could improve blastocyst formation rates.

Is early embryo development in mice influenced by RNA binding protein with multiple splicing 2 (RBPMS2), a maternal factor that accumulates and is stored in the cytoplasm of mature oocytes?
The expression patterns of RBPMS2 in mouse were analysed using quantitative real-time PCR (qRT PCR) and immunofluorescence staining. The effect of knockdown of RBPMS2 on embryo development was evaluated through a microinjection of specific morpholino or small interfering RNA. RNA sequencing was performed for mechanistic analysis. The interaction between RBPMS2 and the bone morphogenetic protein (BMP) pathway was studied using BMP inhibitor and activator. The effect on the localization of E-cadherin was determined by immunofluorescence staining.
Maternal protein RBPMS2 is highly expressed in mouse oocytes, and knockdown of RBPMS2 inhibits embryo development from the morula to the blastocyst stage. Mechanistically, RNA sequencing showed that the differentially expressed genes were enriched in the transforming growth factor-β (TGF-β) signalling pathway. BMPs are members of the TGF-β superfamily of growth factors. It was found that the addition of BMP inhibitor to the culture medium led to a morula-stage arrest, similar to that seen in RBPMS2 knockdown embryos. This morula-stage arrest defect caused by RBPMS2 knockdown was partially rescued by BMP activator. Furthermore, the localization of E-cadherin to the membrane was impaired in response to a knockdown of RBPMS2 or inhibition of the BMP pathway.
This study suggests that RBPMS2 activates the BMP pathway and thus influences the localization of E-cadherin, which is important for early mouse embryo development during blastocyst formation.

Follicular fluid has been found as a possible source of metabolic predictors for oocyte competence, and it is conveniently accessible during ovum pick-up (OPU). We used the OPU procedure to recover oocytes from 41 Holstein heifers for in vitro embryo production in this study. Follicular fluid was collected during OPU in order to establish a link between follicular amino acids and blastocyst formation. Each heifer\'s oocytes were collected, matured in vitro for 24 h and fertilized separately. The heifers were then divided into two groups based on blastocyst formation: those that produced at least one blastocyst (the blastocyst group, n = 29) and those that did not (the failed group, n = 12). The blastocyst group had higher follicular glutamine concentrations and lower aspartate levels than the failed group. Furthermore, network and Spearman correlation analyses revealed a link between blastocyst formation and aspartate (r = -0.37, p = 0.02) or glutamine (r = 0.38, p = 0.02). The receiver operator characteristic curve revealed that glutamine (AUC = 0.75) was the greatest predictor of blastocyst formation. These findings revealed that follicular amino acid levels in bovines can be used to predict blastocyst development.

OBJECTIVE: How does nucleus status at the two-cell stage predict blastocysts formation and clinical outcome after single blastocyst transfer?
CONCLUSIONS: Binucleated embryos at the two-cell stage (2BI) show higher rates of good quality blastocyst formation, pregnancy and live birth compared to those with one nucleus in each blastomere (2MONO), whereas true multinucleated embryos at the two-cell stage (2MULTI) show lower rates of good quality blastocyst formation and pregnancy compared to 2MONO embryos.
BACKGROUND: The introduction of time-lapse culture has made it possible to study nucleus status at the two-cell stage more consistently and it shows that multinucleation at the two-cell stage (2MN) is a common event. The effect of 2MN is still unclear. High numbers of 2MN with the potential to develop to blastocysts that become clinical pregnancies and result in birth of healthy babies with no impaired perinatal outcome have been reported. However, some studies have found 2MN to be associated with impaired implantation and live birth. Furthermore, knowledge on how the different subgroups of multinucleation affects the IVF outcome is limited.
UNASSIGNED: A non-interventional retrospective study was performed in a public fertility clinic. Blastocyst formation data from 223 women attending their first IVF cycle between May 2016 and December 2018, and clinical outcome data from 1314 single blastocyst transfers between May 2014 and December 2018 were used for the study. Fresh and frozen-thawed embryo transfers were included.
METHODS: Embryos were cultured until the blastocyst stage in a time-lapse incubator and nucleus status at the two-cell stage, the Gardner score and other morphokinetic parameters were annotated. We compared blastocyst development and clinical outcome, including positive hCG, ongoing pregnancy and live birth, of embryos with 2BI and/or 2MULTI blastomeres to 2MONO embryos.
RESULTS: Embryos with 2BI in one blastomere (2BI1) were twice as likely to develop to good quality blastocysts (odds ratio (OR) 2.54, 95% CI 1.30-4.95, P = 0.006) compared to 2MONO embryos. Embryos with 2MULTI in both blastomeres (2MULTI2) were significantly less able to develop to good quality blastocysts (OR 0.38, 95% CI 0.23-0.63, P < 0.001) compared to 2MONO embryos. Embryos with 2BI in both blastomeres (2BI2) had a significantly better chance of resulting in a positive hCG (OR 2.40, 95% CI 1.11-5.20, P = 0.027), ongoing pregnancy (OR 2.79, 95% CI 1.29-6.04, P = 0.009) and live birth (OR 3.16, 95% CI 1.43-6.95, P = 0.004) compared to 2MONO blastocysts after single blastocyst transfer. In contrast, 2MULTI2 embryos were significantly less likely to result in a positive hCG (OR 0.58, 95% CI 0.35-0.97, P = 0.036) and ongoing pregnancy (OR 0.51, 95% CI 0.28-0.94, P = 0.030) compared to 2MONO blastocysts.
CONCLUSIONS: Discrepancies among the existing studies regarding the definition of multinucleation may lead to different conclusions. Even though the distinction between binucleation and true multinucleation was a strength in our study design, a further distinction between true multinucleated and micronucleated embryos could be interesting to investigate in future studies. Also, we included any anucleated embryos in the 2MONO group. For the study of clinical outcomes, the patients were allowed to be included with more than one transfer cycle. Both fresh and thawed transfers were included.
CONCLUSIONS: We find it important to discriminate between binucleation and true multinucleation when evaluating embryo nucleus status at the two-cell stage. Embryos displaying 2BI1 and 2BI2 have significantly better good quality blastocyst formation rates and clinical outcome after single blastocyst transfers, respectively. 2MULTI2 embryos have impaired blastocyst development potential and poorer clinical outcomes.
BACKGROUND: H.S.N. received an unrestricted grant from Merck for 3 months\' normal salary for a medical Doctor (A.L.T.) to write the manuscript. Merck was not involved in the study design, analysis, interpretation of data, writing the paper or the decision to submit the manuscript for publication. H.S.N. has received speaker\'s fees from Ferring Pharmaceuticals, Merck Denmark A/S, Astra Zeneca, Cook Medical and Ibsa Nordic (outside the submitted work). N.l.C.F. has received a grant from Gedeon Richter (outside the submitted work). The other authors did not report any potential conflicts of interest. All authors declared no conflicts of interest regarding this work.
BACKGROUND: N/A.

To investigate whether blastocysts that divide irregularly reduce subsequent blastocyst euploidy.
Retrospective study.
Private clinic.
A total of 122 blastocysts for which consent for disposal and research use was obtained.
None.
Results of next-generation sequencing analysis of the blastocysts and whether blastomeres by normal or irregular divisions subsequently participated in blastocyst formation or not.
The embryos were classified according to their dynamics until the second cleavage. The blastocyst euploidy rates were 33.3% (19/57) in the normal cleavage (NC) group, 38.3% (18/47) in the direct cleavage (embryos with one cell dividing into 3 cells) (DC) group, and 72.2% (13/18) in the reverse cleavage (RC) (embryos with fused cells once divided) group. The rate of the RC group was significantly higher than that of the NC group. The blastocyst participation rate of the blastomeres were 95.6% in the NC group and 56.5% in that derived from DC of the first cleavage, and 91.7% in that of blastomeres derived from normal division of the second cleavage and 53.6% in that derived from DC of the second cleavage, both of which were significantly lower in the latter. In the RC group, the rates of fused and nonfused blastomeres were 62.1% and 87.5%, respectively, with no significant difference.
The blastomeres generated by DC were often excluded from blastocyst formation, and we speculate that this is one reason why their division does not reduce blastocyst euploidy. The association between RC and euploidy of blastocysts merits further study.

Metadata analysis of public microarray datasets using bioinformatics tools has been successfully used in several biomedical fields in the search for biomarkers. In reproductive science, there is an urgent need for the establishment of oocyte quality biomarkers that could be used in the clinical environment to increase the chances of successful outcomes in treatment cycles. Adaptive cellular processes observed in cumulus oophorus cells reflect the conditions of the follicular microenvironment and may thus bring relevant information of oocyte\'s conditions. Here we analyzed human cumulus cells gene expression datasets in search of predictors of oocyte quality, a strategy which uncovered several cellular processes positively and negatively associated with embryo development and pregnancy potential. Secondly, the expression levels of genes that were present in the majority of processes observed were validated in house with clinical samples. Our data confirmed the association of the selected biomarkers with blastocyst formation and pregnancy potential rates, independently of patients\' clinical characteristics such as diagnosis, age, BMI, and stimulation protocol applied. This study shows that bioinformatic analysis of cellular processes can be successfully used to elucidate possible oocyte quality biomarkers. Our data reinforces the need to consider clinical characteristics of patients when selecting relevant biomarkers to be used in the clinical environment and suggests a combination of positive (PTGS2) and negative (CYPB1) quality biomarkers as a robust strategy for a complementary oocyte selection tool, potentially increasing assisted reproduction success rates. Also, GPX4 expression as pregnancy potential biomarker is indicated here as a possibility for further investigations.