Alessandro Bartolacci

To assess if 2.1PN-derived embryos are diploid, euploid and suitable for embryo-transfer. This systematic review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed, Embase, and SCOPUS were systematically searched for peer-reviewed original papers using relevant keywords and Medical Subject Heading (MeSH) terms. Diploidy, confirmed by genotyping analysis, is defined as the primary outcome measure. Secondary outcomes included blastulation rates, good quality blastocysts, euploidy, clinical pregnancy, miscarriage and live birth (LB). A total of 7 studies were included in the systematic review. Six out of seven studies indicated that 2.1PN embryos have lower developmental competence than 2PN embryos, yet can still be euploid/diploid. Only one study advised against their use for embryo transfer. In our analysis of the evidence tables, 2.1PN-derived embryos showed a descriptive trend toward lower blastulation rates (- 22.50%), good-quality blastocyst formation (- 12.93%), diploidy (- 16.70%), and euploidy rates (- 28.37%) when compared with their 2PN counterparts. Four studies also analyzed LB in 2.1PN, reporting an overall LB of 30.4% (7/23). Finally, based on the crude aggregated blastulation (32.30%), euploidy (35.0%), and diploidy (82.0%) rates, the estimated probability for a single 2.1PN zygote to develop into a euploid and diploid blastocyst was 9.3%. Although 2.1PN-derived embryos exhibited lower developmental competence, they showed potential for healthy LB. Our findings support the clinical use of 2.1PN-derived embryos when diploidy is confirmed by a validated genotyping analysis, providing further empirical evidence to support more inclusive and flexible clinical recommendations, potentially improving cumulative success rates. CRD420251070366.

Purpose To assess the clinical applicability of shortened (SW) and ultra-shortened (USW) warming protocols for oocytes cryopreserved using standard vitrification (STD-V). Methods This prospective sibling study included 436 mature human oocytes from 47 patients, all vitrified using a single standard vitrification (STD-V) protocol. Experiment 1 compared the standard warming (STD-W) with the SW protocols (2 min), and Experiment 2 compared the SW protocol with USW protocol (1 min). Oocyte survival rates were assessed 1.5 h post-warming, and oocytes were immediately fixed. The analysis of meiotic spindle organization and chromosome alignment was assessed by detailed examination of individual optical sections and corresponding full Z-stack projections encompassing the entire spindle. Mitochondrial mass was quantified from Z-stack–based projections integrating fluorescence across all optical sections. We also performed a pooled analysis to directly compare STD-W, SW, and USW groups. Results Survival rates were comparable across protocols. Both SW and USW protocols were associated with significantly lower odds of normal chromosomal distribution, spindle organization, and normal MII apparatus compared with the STD-W protocol, with no significant differences between SW and USW. Spindle length and pole width were increased in SW and USW versus STD-W. Mitochondrial mass was significantly reduced in SW compared with STD-W. Conclusions Although survival rates were comparable, our data indicate that SW and USW protocols are associated with a higher incidence of spindle and chromosomal abnormalities than the traditional approach. This may affect oocyte developmental competence, highlighting the need for further investigation regarding the clinical use of SW/USW protocols for STD-V-cryopreserved oocytes.

Purpose To assess if 1PN-derived embryos are diploid/euploid and suitable for embryo transfer. Methods This systematic review and meta-analysis were conducted according to the Meta-Analysis of Observational Studies in Epidemiology (MOOSE) guidelines. PubMed, Embase, and SCOPUS were systematically searched for peer-reviewed original papers using relevant keywords and Medical Subject Heading (MeSH) terms: “1PN” OR “monopronucleated” OR “single pronucleus” AND “cleavage” OR “embryo quality” OR “blastocyst quality” OR “blastulation” OR “embryo development” OR “euploidy” OR “ploidy” OR “pregnancy” OR “live birth” OR “miscarriage” OR “clinical outcomes” OR “malformation.” Results A total of 21 studies met the inclusion criteria. Compared to 2PN-derived embryos, 1PN-derived embryos exhibited a lower blastulation rate and clinical outcomes when transferred without prior genetic testing. However, 1PN-derived embryos demonstrated similar euploidy and live birth rates following euploid blastocyst transfer, as well as comparable malformation rates to their 2PN counterparts. A non-significant trend toward a higher risk of abnormal ploidy constitution was observed in 1PN-derived embryos. Notably, pregnancy and live birth rates were significantly lower for 1PN-derived embryos from ICSI compared to those from conventional IVF. Conclusions 1PN-derived embryos, though less competent than 2PNs, can lead to healthy live births. They may be cautiously utilized in clinical practice, particularly when biparental diploidy has been confirmed. Clinical trial registration. CRD42024605305.

This study evaluates the effects of double cryopreservation and re-biopsy on embryo viability and clinical outcomes. Studies of interest were selected from an initial cohort of 1027 potentially relevant records retrieved. PubMed was systematically searched for peer-reviewed original papers identified by keywords and medical subject heading terms. Moreover, we elaborated the evidence tables for double cryopreservation and re-biopsy separately. Data were systematically extracted, focusing on live birth, survival, clinical pregnancy, and miscarriage rates. For each study, we identified absolute numbers (numerator and denominator) related to clinical outcomes. Finally, for each outcome, we calculated the percentage change between the control and study groups. Among studies on double cryopreservation, 13 out of 22 reported no effect on clinical outcomes, suggesting contradictory results. Similarly, findings on re-biopsy were controversial, with seven out of 12 studies showing negative effects on survival and clinical outcomes, while five reported no impact. In our analysis of the evidence tables, we observed a reduction in live birth rates of 22.2% and 39.3% in blastocysts undergoing double vitrification and re-biopsy, respectively. These findings suggest that repeated micromanipulations can impair embryo competence. Therefore, double cryopreservation and re-biopsy should be limited in the selected cases without other options by consulting patients about the possible harmful effects.

Purpose Recent evidence showed that the phase between pronuclear fading and the first cleavage is a perilous bridge connecting the zygote and the embryo. Indeed, delay in the short interval between pronuclear breakdown (PNBD) and the first cytokinesis may result in chromosome segregation errors. We tested the hypothesis that delays in this final phase of fertilization are associated with a detrimental impact on embryo development. Methods This is a retrospective study of 1315 zygotes cultured using time lapse technologies generated in 205 first ICSI-cycles. Results We observed an association between increasing times of the pronuclear fading-first cleavage interval (t2-tPNf) and the rates of trichotomous/direct unequal cleavage at the first (DUC-1) and second (DUC-2) mitotic cycle. Moreover, we observed a reduced blastulation rate. No significant associations were observed between rates of direct unequal cleavage at the third mitotic cycle (DUC-3) and top-quality blastocysts, euploidy, and live births. To evaluate whether the interval t2–tPNf could have a predictive value for the onset of DUC-1 and DUC-2, ROC curve analyses were performed. The area under the curve values obtained for DUC-1 showed a significant prediction accuracy. The best cut-offs to identify zygotes with a high risk of DUC-1 and DUC-2 occurrence were t2–tPNf > 2.78 (hours) and t2–tPNf > 2.50 (hours), respectively. Conclusion Delay in the short interval between PNBD and the first cytokinesis result in trichotomous cleavage and early developmental arrest. However, if the embryos reach the blastocyst stage, rates of euploidy and live birth do not appear to be compromised.

Oocyte cryopreservation has transformed assisted reproductive technology, providing enhanced flexibility and efficacy in assisted reproductive technology treatments. The shift from traditional slow freezing to vitrification has markedly improved survival rates by preventing ice crystal formation, which can damage cellular structures. Despite these advances, rapid warming of vitrified oocytes remains a challenge due to the unique sensitivity of oocytes to thermal and osmotic stress, which can compromise spindle integrity and developmental competence. Recent studies indicate that using both rapid vitrification and rapid warming protocols may reduce processing time and enhance workflow efficiency, yet the impact of rapid warming on oocytes previously cryopreserved using standard methods is not fully understood. Although promising results have emerged from preclinical and clinical studies using rapid warming on embryos, the applicability to human oocytes requires further research. For oocytes, establishing a universal rapid warming protocol that balances efficiency and safety is essential, especially considering the millions of oocytes already preserved with conventional techniques.