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OBJECTIVE: To study and compare the preimplantation genetic testing for monogenic disorders (PGT-M) results, and to evaluate the treatment cycle outcomes of embryos derived from a single pronucleus (1PN) vs. two pronuclei (2PN). DESIGN: A retrospective cohort study from January 2018 to December 2022 involving in vitro fertilization (IVF)-PGT-M treatment cycles. SETTING: Single, academically affiliated fertility center. PATIENTS: A total of 244 patients underwent 351 IVF-PGT-M treatment cycles. INTERVENTION: Embryo biopsy with molecular testing for a monogenic disorder. MAIN OUTCOME MEASURES: The molecular diagnosis results and clinical outcomes after the transfer of embryos derived from 1PN and 2PN in IVF-PGT-M treatment cycles. RESULTS: Embryos derived from 1PN have a significantly low developmental potential with a lower rate of embryos that underwent biopsy compared with 2PN-derived embryos; 1PN-derived embryos demonstrated a significantly lower number of blastocysts (24% vs. 37.9%) and top-quality blastocysts (22.3% vs. 48.1%) compared with 2PN-derived embryos. Lower successfully completed and unaffected PGT-M results were achieved in 1PN compared with 2PN-derived embryos (47.1% vs. 65.5% and 18.7% vs. 31.6%, respectively), with significantly higher abnormal molecular results (39.6% vs. 22.7%). The embryo transfer of 24 1PN-derived embryos with no affected genetic disorder resulted in 5 (20.8%) clinical pregnancies and 4 (16.7%) live births (LBs). CONCLUSIONS: Within the limits of fewer embryos derived from 1PN that yielded unaffected embryos suitable for transfer, the clinical pregnancy and LB rate of 1PN embryos undergoing PGT-M are reassuring. We, therefore, suggest applying PGT-M to embryos derived from 1PN embryos to improve the cumulative clinical pregnancy and LB rates.

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OBJECTIVE: Most of the literature about postmortem sperm retrieval (PMSR) deals with the controversies surrounding ethical and legal aspects, while the optimal time interval between the death and viable sperm acquisition is indefinite. In an attempt to aid fertility specialists, while counseling whether to pursue and adopt PMSR, we aim to explore the maximal time frame from ejaculated sperm acquisition to sperm cryopreservation in different "culture" conditions, observations that might be extrapolated to PMSR requests. PATIENTS AND METHODS: Five healthy men with normal semen analysis were enrolled. The sperm specimen from each man was diluted to 6.5 mL. After extracting 0.5 mL for cryopreservation, the remaining 6 mL were divided into three tubes: one was maintained in room temperature (23-25 °C), the second in an incubator (37 °C), and the third in a refrigerator (4 °C). Thereafter, every day, a 0.5 mL of each sample was extracted, examined, and cryopreserved. A week later, all the cryopreserved samples were thawed and tested for sperm motility and viability. RESULTS: While at room temperature, frozen/thawed sperm were still motile (6.5%) and viable (9.9%) up to 96 h; those maintained in the refrigerator, following freezing/thawing were immotile already at 48 h in culture, but still viable (6.0%) up to 72 h in culture. Those maintained in the incubator demonstrated the worse results with negligible motility (1.5%) and viability (3.7%) following freezing/thawing, already after 48 h in culture. CONCLUSIONS: The timeframe cut-off between ejaculated sperm acquisition and cryopreservation should be 72 h, unless sperm was maintained at room temperature, where it might be longer. It would be prudent to check for sperm vitality prior to freezing in cases where only immotile sperms are present.

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OBJECTIVE: Nowadays, patients attempting social/elective egg freezing has spread globally. Ovarian stimulation (OS) with high daily gonatotropin doses, are commonly offered to this group of patients, aiming to achieve the maximal oocytes cohort with minimum IVF cycle attempts. We aim to assess the IVF-ET outcome, and specifically the oocyte yield, of patients undergoing two successive IVF cycle attempts for elective egg freezing (EEF), and whether changing the daily gonadotropin dose in the second IVF cycle attempt, affect the outcome. PATIENTS AND METHODS: All women admitted to our IVF unit for social/EEF, who underwent 2 consecutive IVF cycle attempts, with only those who used in the first attempt a starting daily gonadotropin dose of 300 IU were included. Ovarian stimulation characteristics, duration of OS, number of retrieved oocytes, number of mature oocytes were assessed and compared between the 1st and the 2nd IVF cycle attempts, and between the different daily gonadotropin doses and the oocyte yields in the 2nd cycle attempt (increase, decrease or no change). MAIN OUTCOME MEASURES: Oocytes and mature oocytes yield in the 2nd as compared to the 1st IVF cycle attempt. RESULTS: A reduced oocyte yield in the 2nd cycle attempt was observed in those who highly responded in the 1st attempt, regardless the daily dose in the 2nd cycle attempt (whether it was increased, no change and decreased). Moreover, the proportion of patients with same or more oocytes in the 2nd IVF cycle attempt was significantly lower in patients with high peak E2 levels, compared to those with peak E2 levels < 9175 pmol/L. Among patients with high peak E2 (> 9175 pmol/L), those who achieved a lower oocytes yield in the 2nd IVF cycle attempt had lower basal Day-3 FSH/LH ratio (1.5 + 0.5 vs 1.8 + 0.8, p < 0.03) and higher oocyte (range: 7-28, median:10; vs range: 2-15, median:7) and mature oocytes yields. With a cut-off of 9 oocytes, 78.8% of those with > 9 oocytes and 61.8% of those with < 9 oocytes will achieve lower/higher oocytes yield in the 2nd IVF cycle attempt, respectively. CONCLUSIONS: Ovarian stimulation with high daily gonatotropin doses (300 IU) should be offered to patients attempting social/EEF. Moreover, in their 2nd IVF cycle attempt, those with high peak E2 (> 9175 pmol/L) in the 1st attempt, and basal Day-3 FSH/LH ratio < 1.5 and/or more than 9 oocytes retrieved, should receive same OS protocol with no change in the daily gonadotropin dose.

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Oocytes eligible for intracytoplasmic sperm injection (ICSI) are those that have progressed through meiosis to metaphase 2 (MII). The remaining delayed mature oocytes can be injected, aiming to achieve more embryos and a better chance to conceive. We aimed to assess the outcome of delayed matured oocytes, derived from either germinal vesicles or metaphase 1 (MI), that reached maturity (MII) 24 h following retrieval. The study population consisted of 362 women who underwent 476 IVF cycles. While fertilization rates were comparable between the sibling delayed mature oocyte group compared with injection on day 0 group (58.4% vs 62%, respectively, P = 0.07), the top-quality embryo rate per injected MII day 0 oocyte was significantly higher compared with day 1 injected oocyte (57.5% vs 43.9% respectively, P < 0.001). Moreover, following fresh transfer of embryos derived from delayed mature oocytes, implantation rate and the clinical pregnancy (CPR) and live-birth rates (LBR) per transfer were 3.9%, 3.3% and 1.6% respectively. When considering the following thawed embryo transfer cycles, implantation, pregnancy and LBR were non-significantly higher (10%, 8.3% and 8.3%, respectively). Although clinical outcomes are significantly lower when using embryos derived from delayed mature oocyte to mature day 0 oocytes, the additional embryos derived from delayed mature oocytes might contribute to the embryo cohort and increase the cumulative live-birth rate per retrieval. Moreover, the embryos derived from delayed mature oocyte favour a transfer in a frozen-thawed cycle rather than in a fresh cycle.

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Embryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle.

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OBJECTIVE: To study the effect of patients' immunization after coronavirus disease 2019 (COVID-19) infection or messenger ribonucleic acid (mRNA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine on frozen-thawed embryo transfer (FET). DESIGN: Cohort retrospective study. SETTING: Tertiary university affiliated medical center. PATIENT(S): All consecutive patients undergoing FET cycles in our center. The study group (immune group) consisted of patients treated during the COVID-19 pandemic (between January 2021 and August 2021) who either recovered from COVID-19 infection or received the mRNA SARS-CoV-2 vaccine. The control groups consisted of patients treated during the COVID-19 pandemic (between January 2021 and August 2021) but were not infected or did not receive the mRNA SARS-CoV-2 vaccine (not-immune2021 group) and those treated between January 2019 and August 2019 (before the pandemic) (not-immune2019 group). INTERVENTION(S): Frozen-thawed embryo transfer cycles. MAIN OUTCOME MEASURE(S): Ongoing pregnancy rates and FET cycles' characteristics. Data on patient age and variables related to infertility treatment were collected from the patient records. RESULT(S): During the study periods, 428 patients underwent 672 FET cycles. The immune group consisted of 141 patients who underwent 264 FET cycles (44 in postinfection and 220 in postvaccination), whereas the not-immune2021 and not-immune2019 groups consisted of 93 and 194 patients undergoing 125 and 283 FET cycles, respectively. Patients' characteristics and the types of endometrial preparations were comparable between the study groups. The implantation rate and clinical and ongoing pregnancy rates per transfer were similar between the study groups (immune group, postinfection and postvaccination; not-immune2021 group; not-immune2019 group). CONCLUSION(S): Coronavirus disease 2019 infection or vaccination did not affect patients' performance or implantation in their subsequent FET cycle.

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RESEARCH QUESTION: What are the cumulative clinical pregnancy rates (CPR) and live births rates (LBR) in intracytoplasmic sperm injection (ICSI) cycles using testicular motile compared with immotile spermatozoa, obtained from testicular sperm aspiration (TESA) or extraction (TESE)? DESIGN: A retrospective analysis of ICSI cycles using TESA or TESE over a period of 7 years. Cycles were divided into two groups according to the motility of the retrieved spermatozoa: Group A consisted of couples with motile spermatozoa; Group B of couples with immotile spermatozoa. Group B was subdivided into two groups: B1 consisted of couples with motile spermatozoa and B2 with immotile spermatozoa after the addition of pentoxifylline. RESULTS: No differences in CPR and LBR per transfer was found between the study groups after fresh embryo transfer. No pregnancies were achieved by vitrified-warmed embryo transfer in group B2. Fertilization rates decreased when using immotile spermatozoa (64.4%, 56%, 37.9%, for groups A, B1 and B2, respectively, P < 0.001). Top-quality embryo rates were higher in groups A and B1 compared with B2 (40.7% and 40.1% versus 19.1%, respectively, P = 0.015). Cumulative CPR (53%, 41.7%, 13.6% for groups A, B1 and B2, respectively, P = 0.005) and LBR (42.4%, 30%, 13.6% for groups A, B1 and B2, respectively P = 0.03) per oocyte retrieval was significantly higher when using motile spermatozoa compared with motile or immotile spermatozoa after adding pentoxifylline. CONCLUSIONS: Although fertilization, top-quality embryo rates, cumulative CPR and LBR decreased when using immotile spermatozoa, ICSI is still valid; therefore, it should be considered and offered to couples before embarking on a donor sperm insemination cycle, or cryopreserving oocytes for future additional testicular sperm retrieval.

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A balanced progression through mitosis and cell division is largely dependent on orderly phosphorylation and ubiquitin-mediated proteolysis of regulatory and structural proteins. These series of events ultimately secure genome stability and time-invariant cellular properties during cell proliferation. Two of the core enzymes regulating mitotic milestones in all eukaryotes are cyclin dependent kinase 1 (CDK1) with its coactivator cyclin B, and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Discovering mechanisms and substrates for these enzymes is vital to understanding how cells move through mitosis and segregate chromosomes with high fidelity. However, the study of these enzymes has significant challenges. Purely in vitro studies discount the contributions of yet to be described regulators and misses the physiological context of cellular environment. In vivo studies are complicated by the fact that each of these enzymes, as well as many of their regulators and downstream targets, are essential. Moreover, long-term in vivo manipulations can result in cascading, indirect effects that can distort data analysis and interpretation. Many of these challenges can be circumvented using cell-free systems, which have historically played a critical role in identifying these enzymes and their contributions under quasicellular environments. Here, we describe the preparation of a newly developed human cell-free system that recapitulates an anaphase-like state of human cells. This new toolkit complements traditional cell-free systems from human cells and frog eggs and can be easily implemented in cell biology labs for direct and quantitative studies of mitotic signaling regulated by phosphorylation, APC/C-mediated proteolysis, and beyond.

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OBJECTIVE: No information exists in the literature regarding the effect of mRNA SARS-CoV-2 vaccine on subsequent IVF cycle attempt. We therefore aim to assess the influence of mRNA SARS-CoV-2 vaccine on IVF treatments. DESIGN: An observational study. SETTING: A tertiary, university-affiliated medical center. PATIENTS AND METHODS: All couples undergoing consecutive ovarian stimulation cycles for IVF before and after receiving mRNA SARS-CoV-2 vaccine, and reached the ovum pick-up (OPU) stage. The stimulation characteristics and embryological variables of couples undergoing IVF treatments after receiving mRNA SARS-CoV-2 vaccine were assessed and compared to their IVF cycles prior to vaccination. MAIN OUTCOME MEASURES: Stimulation characteristics and embryological variables. RESULTS: Thirty-six couples resumed IVF treatment 7-85 days after receiving mRNA SARS-CoV-2 vaccine. No in-between cycles differences were observed in ovarian stimulation and embryological variables before and after receiving mRNA SARS-CoV-2 vaccination. CONCLUSIONS: mRNA SARS-CoV-2 vaccine did not affect patients' performance or ovarian reserve in their immediate subsequent IVF cycle. Future larger studies with longer follow-up will be needed to validate our observations.

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BACKGROUND: We aimed to assess whether the survival rate of embryos is influenced by the number of embryos/oocytes loaded on a single cryo-carrier during vitrification. METHODS: This was a retrospective study that included 974 patients who underwent thawing of 1896 embryo-warming cycles between September 2016 and January 2020. A distinct analysis was made for cleavage stage embryos (2-10-cell stage) and blastocysts. For vitrification, embryos were placed in a Cryotop™ open device using a SAGE vitrification kit following the manufacturer's instructions. Warming was carried using a SAGE warming vitrification kit according the manufacturer's instructions. RESULTS: Total post-vitrification survival rates of embryos at the cleavage stage or blastocyst stage was 94.8%. At the cleavage stage, cryo-preserving three embryos per single cryo-carrier gave the highest full intact embryo survival rate (91.5%) compared with one or two embryo(s) per single cryo-carrier (85.7%, P < 0.0002 and 87.3%, P < 0.004). Conversely, post warmed full intact blastocyst survival rate for two blastocysts was significantly lower compared with one blastocyst (76.7% vs. 87.9%, P < 0.0193) per single cryo-carrier. CONCLUSION: Post-thawing survival rate following vitrification is affected by the number of embryos per single cryo-carrier undergoing the vitrification equilibration phase, with the optimum number of three cleaved embryos or one blastocyst per single cryo-carrier. Further studies are required to determine the optimum number of cleaved embryos or blastocysts that should be loaded onto a single cryo-carrier vitrification device.

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We sought to compare ICSI outcomes of cycle using fresh versus thawed TESE spermatozoa obtained during the previous fresh TESE. All consecutive couples undergoing ICSI cycles using fresh TESE spermatozoa, followed by ICSI cycle using cryopreserved sperm remaining from the previous fresh TESE procedure were included. Ovarian stimulation (OS)/laboratory variables and cycle outcome were assessed and compared between those utilising fresh versus thawed TESE spermatozoa. Seventy-five couples were evaluated, with no in-between groups differences in OS nor embryological variables. While implantation and LBR per embryo transfer were nonsignificantly higher in the frozen as compared to the fresh TESE, there was a trend towards higher LBRs per patient in the frozen TESE group. The cumulative miscarriage rate (4% versus 14.7%, p < .022 respectively) was significantly lower and the cumulative LBR (34.7% versus 16%, p < .007 respectively) was significantly higher using frozen TESE spermatozoa. Moreover, significantly higher proportion of frozen TESE sperm samples used pentoxifylline to enhance sperm motility. In conclusion, the results of ICSI cycles using frozen TESE spermatozoa are as good, or even better than using fresh TESE spermatozoa. Further studies are required to explore the factors responsible for the improved ICSI outcome, while using frozen versus fresh TESE sperm samples.

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We aim to evaluate our experience, comparing intracytoplasmic sperm injection (ICSI) outcomes of cycle using fresh versus thawed electroejaculated spermatozoa. All consecutive couples undergoing ICSI cycles using electroejaculated spermatozoa, during a 16-year period, were evaluated. Embryological/laboratory variables of the ICSI cycles were assessed and compared between those utilising fresh (fresh group) versus thawed (thawed group) electroejaculated spermatozoa. Fifty-seven couples were evaluated, 30 used a fresh electroejaculated spermatozoa in 55 ICSI cycles, while 27 used a thawed sperm sample in 41 ICSI cycles. There were no in-between group differences in the mean numbers of oocytes retrieved per oocyte retrieval nor the percentage of MII oocytes. The fresh group demonstrated significantly higher fertilisation (71.5% vs. 64.1%, respectively, p < .05), top-quality embryos (66.5% vs. 54.9%, respectively, p < .02), clinical pregnancy per transfer (41.3% and 21.2%, respectively, p < .05) and cumulative clinical pregnancy (58.2% vs. 26.8%, respectively, p < .001) rates, as compared to the thawed group. Independent of the source of spermatozoa used, no pregnancy was achieved following ICSI utilising immotile spermatozoa. In conclusion, ICSI cycles using ejaculated spermatozoa of patients suffering from neurologic or psychogenic anejaculation are reassuring. The use of fresh ejaculated spermatozoa retrieved on the day of the female spouse oocyte retrieval might improve outcome. Whenever a thawed electroejaculated spermatozoa yield no motile spermatozoa, emergency electroejaculation is mandatory.

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E2F8 is a transcriptional repressor that antagonizes E2F1 at the crossroads of the cell cycle, apoptosis, and cancer. Previously, we discovered that E2F8 is a direct target of the APC/C ubiquitin ligase. Nevertheless, it remains unknown how E2F8 is dynamically controlled throughout the entirety of the cell cycle. Here, using newly developed human cell-free systems that recapitulate distinct inter-mitotic and G1 phases and a continuous transition from prometaphase to G1, we reveal an interlocking dephosphorylation switch coordinating E2F8 degradation with mitotic exit and the activation of APC/CCdh1. Further, we uncover differential proteolysis rates for E2F8 at different points within G1 phase, accounting for its accumulation in late G1 while APC/CCdh1 is still active. Finally, we demonstrate that the F-box protein Cyclin F regulates E2F8 in G2-phase. Altogether, our data define E2F8 regulation throughout the cell cycle, illuminating an extensive coordination between phosphorylation, ubiquitination and transcription in mammalian cell cycle.

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Autophosphorylation of receptor and non-receptor tyrosine kinases is a common molecular switch with broad implications for pathogeneses and therapy of cancer and other human diseases. Technologies for large-scale discovery and analysis of autophosphorylation are limited by the inherent difficulty to distinguish between phosphorylation and autophosphorylation in vivo and by the complexity associated with functional assays of receptors kinases in vitro. Here, we report a method for the direct detection and analysis of tyrosine autophosphorylation using integrated microfluidics and freshly synthesized protein arrays. We demonstrate the efficacy of our platform in detecting autophosphorylation activity of soluble and transmembrane tyrosine kinases, and the dependency of in vitro autophosphorylation assays on membranes. Our method, Integrated Microfluidics for Autophosphorylation Discovery (IMAD), is high-throughput, requires low reaction volumes and can be applied in basic and translational research settings. To our knowledge, it is the first demonstration of posttranslational modification analysis of membrane protein arrays.

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Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system's potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.

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Animal cell division ends with the cutting of the microtubule and membrane intercellular bridge connecting the 2 daughter cells. This process, known as cytokinetic abscission (abscission), is widely regarded as the last step of cytokinesis, i.e., the last step of the cell cycle. Major breakthroughs have been recently achieved, illuminating mechanistic aspects of abscission; however, the timing of abscission with respect to the mammalian cell cycle remains unclear. In this study, we carefully measured the onset and progression of abscission in dividing cells expressing a G1 reporter. We conclude that abscission commences long after cells enter the G1 phase. Affiliating abscission with G1 is beyond semantics since it essentially postulates that the last step of the cell cycle is regulated in, and probably by, the following cycle.

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