Early Cleavage Embryos Research Articles

The subcortical maternal complex modulates the cell cycle during early mammalian embryogenesis via 14-3-3

The subcortical maternal complex (SCMC) is essential for safeguarding female fertility in mammals. Assembled in oocytes, the SCMC maintains the cleavage of early embryos, but the underlying mechanism remains unclear. Here, we report that 14-3-3, a multifunctional protein, is a component of the SCMC. By resolving the structure of the 14-3-3-containing SCMC, we discover that phosphorylation of TLE6 contributes to the recruitment of 14-3-3. Mechanistically, during maternal-to-embryo transition, the SCMC stabilizes 14-3-3 protein and contributes to the proper control of CDC25B, thus ensuring the activation of the maturation-promoting factor and mitotic entry in mouse zygotes. Notably, the SCMC establishes a conserved molecular link with 14-3-3 and CDC25B in human oocytes/embryos. This study discloses the molecular mechanism through which the SCMC regulates the cell cycle in early embryos and elucidates the function of the SCMC in mammalian early embryogenesis.

Melatonin partially rescues defects induced by tranexamic acid exposure during oocyte maturation in mice.

Tranexamic acid (TXA) is widely used among young women because of its ability to whiten skin and treat menorrhagia. Nevertheless, its potential effects on oocyte maturation and quality have not yet been clearly clarified. Melatonin (MT) is an endogenous hormone released by the pineal gland and believed to protect cells from oxidative stress injury. In the present study, we used an in vitro maturation model to investigate the toxicity of TXA and the protective role of MT in mouse oocytes. Compared with the control group, the TXA-exposed group had significantly lower nuclear maturation (57.72% vs. 94.08%, P < 0.001) and early embryo cleavage rates (38.18% vs. 87.66%, P < 0.001). Further study showed that spindle organization (52.56% vs. 18.77%, P < 0.01) and chromosome alignment (33.23% vs. 16.66%, P < 0.01) were also disrupted after TXA treatment. Mechanistically, we have demonstrated that TXA induced early apoptosis of oocytes (P < 0.001) by raising the level of reactive oxygen species (P < 0.001), which was consistent with an increase in mitochondrial damage (P < 0.01). Fortunately, all these effects except the spindle defect were successfully rescued by an appropriate level of MT. Collectively, our findings indicate that MT could partially reverse TXA-induced oocyte quality deterioration in mice by effectively improving mitochondrial function and reducing oxidative stress-mediated apoptosis.NEW & NOTEWORTHY Tranexamic acid is increasingly used to whiten skin, reverse dermal damages, and treat heavy menstrual bleeding in young women. However, its potential toxicity in mammalian oocytes is still unclear. Our study revealed that tranexamic acid exposure impaired the mouse oocyte quality and subsequent embryo development. Meanwhile, melatonin has been found to exert beneficial effects in reducing tranexamic acid-induced mitochondrial dysfunction and oxidative stress.

P-173 An exploratory clinical study in patients with recurrent ART-failure due to severe fragmentation during early-cleavage: the effects of artificial removal of zona-pellucida at the pronuclear-stage

Abstract Study question Can the artificial removal of the zona pellucida (ZP) at the pronuclear (PN)-stage prevent severe fragmentation in early-cleavage embryos and improve good-quality blastocyst development rate? Summary answer The artificial removal of the ZP at the 2PN-stage could prevent significantly severe fragmentation in early-cleavage embryos and improved the good-quality blastocyst development rate. What is known already Recent analysis using time-lapse culture demonstrated that fragments during early-cleavage occur at the site of the perivitelline threads between the ZP and the embryo membrane (Reprod Biomed Online 2017;35:640-5,646-56). Based on these findings we conducted a pilot study on embryonic development after ZP-removal using abnormally-fertilized oocytes (J Assist Reprod Gene2020;37:1349-54). We showed that the inter-blastomere adhesion was maintained and fragmentation was significantly lower in the ZP-free embryos than in the ZP-intact embryos. Therefore, we attempted to apply this method to patients who have given full consent and had repeatedly failed to conceive due to severe fragmentation in early cleavage-stage embryos. Study design, size, duration This is a single-centre retrospective exploratory study of 34 patients that were scheduled to undergo ART-treatment in our clinic between February 2020 and January 2021. They had been previously treated in our facilities (January 2016 to January 2020) with less than 10% morphologically good-quality blastocysts due to severe fragmentation. None of these patients became pregnant during their initial treatments. Totally, 173 2PN-zygotes were obtained, with 101 allocated to ZP-free group and 72 to ZP-intact group. Participants/materials, setting, methods Normally-fertilized oocytes were placed in 0.125M sucrose-containing HEPES-medium (approximately 5min) and cultured under: 1) ZP-free, in which the ZP was completely removed from the ooplasm by laser-irradiation and a medium-blowing method with a biopsy-pipette, or 2) ZP-intact. ZP-free and ZP-intact zygotes were cultured in sequential medium. Embryos were either freshly transferred on Day-2(ZP-intact group only, according to patients’ wishes), or Day-5/6, or they were cryopreserved on Day-5/6/7 for future embryo transfer cycles for both groups. Main results and the role of chance Even allocation of the normally fertilised oocytes to the two groups was not possible, due to the patients’ request to include more zygotes in the ZP-free group. Therefore, no specific criteria were set other than the rule that all the normally fertilised oocytes of each patient must be allocated into both groups at the time of allocation. As a result, each patient had 2PN zygotes assigned to both ZP-free and ZP-intact groups. Rates of good-quality embryos, blastocyst development, morphologically good-quality blastocyst development and cryopreservation demonstrated significant improvement (all P &amp;lt; 0.01) in the ZP-free group compared to the ZP-intact group. Pregnancy rates in ZP-intact group were 5.9% per patient and 5.3% per embryo transferred, while in ZP-free group 37.5% per patient and 24.3% per embryo transferred. Our results demonstrate that in difficult-to-treat cases where embryos with good morphology cannot be obtained due to excessive fragmentation at the early-cleavage, embryonic development can be significantly improved by artificial removal of ZP at the 2PN-stage. We believe that the ZP-free method has opened a new door in assisted reproductive medicine and can provide the opportunity to obtain blastocysts with good-morphology from an embryo that would probably not have become a blastocyst otherwise. Limitations, reasons for caution A comparative analysis between the two groups was not possible due to the small number of patients, the inclusion of fresh embryos as well as frozen embryos, and the non-randomized data. The imbalance was attributed to the fact that patients wanted us to allocate more oocytes to the ZP-free group. Wider implications of the findings The development of treatment methods based on new ideas should always take into consideration the possibility of associated risks. In the future, we will examine the criteria for selecting patients for whom this method is useful, as well as verify its safety and effectiveness by conducting a prospective randomized analysis. Trial registration number not applicable

P-183 Cleavage patterns combined with fusion status at the compacting stage contribute to the selective single blastocyst transplantation

Abstract Study question To investigate the impact of various cleavage patterns combined with fusion status at the compacting stage on the chromosome ploidy of blastocysts and possible reasons. Summary answer It can be inferred that in single blastocyst transfer without PGT-A, the normal cleavage incomplete compacted embryo can be selected as a last resort. What is known already Previous research has shown that embryos with abnormal cleavage patterns possess lower developmental potential; however, once a blastocyst is formed, its chromosomal euploidy is not necessarily inferior to that of embryos with normal cleavage. It is possible that these embryos undergo self-repair through unfused cells during the compactification stage. The question remains: what is the chromosomal ploidy of embryos with normal cleavage that have unfused cells or fragments at the compacting stage? Is it similar to the chromosomal ploidy of embryos with abnormal cleavage that have unfused cells at the compacting stage? This study aims to provide clarity. Study design, size, duration We conducted a retrospective analysis on 600 PGT-A cycles at our reproductive center from January 2020 to August 2022. A total of 2,250 blastocysts that underwent biopsy and PGT-A testing were included in our analysis. Of these, 475 blastocysts underwent single blastocyst transfer. Participants/materials, setting, methods We classified the embryos into four groups based on their cleavage patterns and fusion status at the compacting stage: normal cleavage complete compacting group (n = 1128), normal cleavage incomplete compacting group (n = 735), abnormal cleavage incomplete compacting group (n = 371), and abnormal cleavage complete compacting group (n = 16). We compared the chromosomal ploidy of four groups, as well as the composition of aneuploidy. We also compared live birth outcomes among the four groups following euploidy thawed single blastocyst transfer. Main results and the role of chance The chromosomal euploidy rates were 55.59%, 50.61%, 59.57%, and 62.5% in the normal cleavage complete compacting group, normal cleavage incomplete compacting group, abnormal cleavage incomplete compacting group, and abnormal cleavage complete compacting group, respectively. There was a significant difference in euploidy rates between the normal cleavage incomplete dense group and the abnormal cleavage incomplete dense group (P = 0.026). Additionally, there was a significant difference in chromosome deletion rates in abnormal chromosomes between the normal cleavage incomplete compacting group (23.95%) and the abnormal cleavage incomplete compacting group (15.63%) (P = 0.005). When thawing and transferring euploid single blastocysts, there was no significant difference in live birth outcomes among the four groups (P = 0.984).In early normal cleavage embryos, if there are unfused cells or fragments at the compacting stage, the ratio of chromosomal abnormalities increases, possibly due to chromosomal loss during the compacting stage. In contrast, abnormal cleavage embryos had an increased euploidy rate once they formed usable blastocysts. The live birth outcomes of abnormal cleavage diploid blastocysts are comparable to those of normal cleavage diploid blastocysts. Limitations, reasons for caution The limitation of this study is a retrospective study with a small sample size. The conclusions of the studies need to be validated in larger-scale prospective studies. Wider implications of the findings Embryologists prefer normal cleavage embryos over abnormal cleavage embryos, and this study found that the low euploidy ratio of normal cleavage incomplete compacting embryos can help guide the screening of single blastocyst transfer without PGT-A. Trial registration number not applicable

Shape of the first mitotic spindles impacts multinucleation in human embryos

During human embryonic development, early cleavage-stage embryos are more susceptible to errors. Studies have shown that many problems occur during the first mitosis, such as direct cleavage, chromosome segregation errors, and multinucleation. However, the mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, in the present study, we image discarded living human two-pronuclear stage zygotes using fluorescent labeling and confocal microscopy without microinjection of DNA or mRNA and investigate the association between spindle shape and nuclear abnormality during the first mitosis. We observe that the first mitotic spindles vary, and low-aspect-ratio-shaped spindles tend to lead to the formation of multiple nuclei at the 2-cell stage. Moreover, we observe defocusing poles in many of the first mitotic spindles, which are strongly associated with multinucleation. Additionally, we show that differences in the positions of the centrosomes cause spindle abnormality in the first mitosis. Furthermore, many multinuclei are modified to form mononuclei after the second mitosis because the occurrence of pole defocusing is firmly reduced. Our study will contribute markedly to research on the occurrence of mitotic errors during the early cleavage of human embryos.

Association of early cleavage, morula compaction and blastocysts ploidy of IVF embryos cultured in a time-lapse system and biopsied for genetic test for aneuploidy

IVF embryos have historically been evaluated by morphological characteristics. The time-lapse system (TLS) has become a promising tool, providing an uninterrupted evaluation of morphological and dynamic parameters of embryo development. Furthermore, TLS sheds light on unknown phenomena such as direct cleavage and incomplete morula compaction. We retrospectively analyzed the morphology (Gardner Score) and morphokinetics (KIDScore) of 835 blastocysts grown in a TLS incubator (Embryoscope+), which were biopsied for preimplantation genetic testing for aneuploidy (PGT-A). Only the embryos that reached the blastocyst stage were included in this study and time-lapse videos were retrospectively reanalysed. According to the pattern of initial cleavages and morula compaction, the embryos were classified as: normal (NC) or abnormal (AC) cleavage, and fully (FCM) or partially compacted (PCM) morulae. No difference was found in early cleavage types or morula compaction patterns between female age groups (< 38, 38–40 and > 40 yo). Most of NC embryos resulted in FCM (≅ 60%), while no embryos with AC resulted in FCM. Aneuploidy rate of AC-PCM group did not differ from that of NC-FCM group in women < 38 yo, but aneuploidy was significantly higher in AC-PCM compared to NC-FCM of women > 40 yo. However, the quality of embryos was lower in AC-PCM blastocysts in women of all age ranges. Morphological and morphokinetic scores declined with increasing age, in the NC-PCM and AC-PCM groups, compared to the NC-FCM. Similar aneuploidy rates among NC-FCM and AC-PCM groups support the hypothesis that PCM in anomalous-cleaved embryos can represent a potential correction mechanism, even though lower morphological/morphokinetic scores are seen on AC-PCM. Therefore, both morphological and morphokinetic assessment should consider these embryonic development phenomena.

A comparative study of preimplantation embryos development of young and aged mice treated with L-carnitine

Female aging is one of the main factors influencing reproductive fertility. The existing experimental study was planned to investigate the L-carnitine (LC) treatment effect on the body weight of adult young 8–10 weeks old and aged 26–28 weeks old female mice and the preimplantation embryonic development of their embryos. This study involved 40 mature young (n=20) and aged (n=20) old female mice. The animals were weighed and divided into 4 groups according to their age (n = 10). The control groups of young and aged mice groups were orally administered distilled water, while the young and aged mice that were treated orally with 10 mg/kg LC daily for 2-3 estrous cycles were considered as treated groups. Then, all female mice were mated with adult males. The weight of pregnant mice on 1-day post coitum was recorded and then euthanized to harvest early cleavage embryos. The embryo development was examined and evaluated their grading according to A, B, C and D. The results showed that the body weight of mice in both young LC and aged LC groups reduced significantly (P≤0.01). The grade A embryo in 1-day post coitum in young and aged LC groups improved significantly (P≤0.01). However, the development of embryos grade A in the young LC group was higher than that of the aged LC group. It was concluded from these findings that the oral supplementation of LC can reduce body weight and improve the preimplantation embryonic development proportions.

Melatonin improves the quality of rotenone-exposed mouse oocytes through association with histone modifications

Rotenone, an insecticide that inhibits mitochondrial complex I and generates oxidative stress, is responsible for neurological disorders and affects the female reproductive system. However, the underlying mechanism is not fully understood. Melatonin, a potential free-radical scavenger, has been shown to protect the reproductive system from oxidative damage. In this study, we investigated the impact of rotenone on mouse oocyte quality and evaluated the protective effect of melatonin on oocytes exposed to rotenone. Our results showed that rotenone impaired mouse oocyte maturation and early embryo cleavage. However, melatonin prevented these negative effects by ameliorating rotenone-induced mitochondrial dysfunction and dynamic imbalance, intracellular Ca2+ homeostasis damage, ER stress, early apoptosis, meiotic spindle formation disruption, and aneuploidy in oocytes. Additionally, RNA sequencing analysis showed that rotenone exposure changed the expression of multiple genes involved in histone methylation and acetylation modifications that result in mouse meiotic defects. However, melatonin partially rescued these defects. These findings suggest that melatonin has protective effects against rotenone-induced mouse oocyte defects.

Checkpoint Kinase 1 Is a Key Signal Transducer of DNA Damage in the Early Mammalian Cleavage Embryo

After fertilization, remodeling of the oocyte and sperm genome is essential for the successful initiation of mitotic activity in the fertilized oocyte and subsequent proliferative activity of the early embryo. Despite the fact that the molecular mechanisms of cell cycle control in early mammalian embryos are in principle comparable to those in somatic cells, there are differences resulting from the specific nature of the gene totipotency of the blastomeres of early cleavage embryos. In this review, we focus on the Chk1 kinase as a key transduction factor in monitoring the integrity of DNA molecules during early embryogenesis.

Toxicity of the Mycotoxin Deoxynivalenol on Early Cleavage of Mouse Embryos by Fluorescence Intensity Analysis.

Deoxynivalenol is a mycotoxin, produced by Fusarium from contaminated corn, wheat, and other grains, that induces multiple effects in humans and animals, including cytotoxic, genotoxic, immunotoxic, and carcinogenic effects. Recent studies show that deoxynivalenol also affects the reproductive system of mammals, including oocyte quality. However, the effects of deoxynivalenol on early embryonic development have not been reported. In this study, fluorescence intensity analysis was used to show that deoxynivalenol disrupted the first cleavage of the zygote. The high deoxynivalenol dose disturbed the movement of the pronucleus after fertilization, while the low deoxynivalenol dose caused aberrant spindle morphology during the metaphase of the first cleavage. Further analysis showed that the reactive oxygen species level increased in the deoxynivalenol-exposed two-cell embryos, indicating oxidative stress. Moreover, deoxynivalenol caused DNA damage in the embryos, as positive γH2A.X signals were detected in the nucleus. These events led to the early apoptosis of mouse embryos, which was confirmed by autophagy. Taken together, our study provides evidence for the toxicity of deoxynivalenol during early embryonic development in the mouse model.

Drosophila melanogaster Oocytes after Space Flight: The Early Period of Adaptation to the Force of Gravity

The effect of space flight factors and the subsequent adaptation to the Earth's gravity on oocytes is still poorly understood. Studies of mammalian oocytes in space present significant technical difficulties; therefore, the fruit fly Drosophila melanogaster is a convenient test subject. In this study, we analyzed the structure of the oocytes of the fruit fly Drosophila melanogaster, the maturation of which took place under space flight conditions (the "Cytomehanarium" experiment on the Russian Segment of the ISS during the ISS-67 expedition). The collection of the oocytes began immediately after landing and continued for 12 h. The flies were then transferred onto fresh agar plates and oocyte collection continued for the subsequent 12 h. The stiffness of oocytes was determined by atomic force microscopy and the content of the cytoskeletal proteins by Western blotting. The results demonstrated a significant decrease in the stiffness of oocytes in the flight group compared to the control (26.5 ± 1.1 pN/nm vs. 31.0 ± 1.8 pN/nm) against the background of a decrease in the content of some cytoskeletal proteins involved in the formation of microtubules and microfilaments. This pattern of oocyte structure leads to the disruption of cytokinesis during the cleavage of early embryos.

Are Early Embryo Cleavage Kinetics Affected by Energy Substrates in Different Culture Media?

This study aimed to investigate the influence of different culture media on early embryonic cleavage kinetics using time-lapse analysis and to determine the possible relationships between energy substrates in culture media and the cleavage kinetics. A total of 10 021 embryos from 1310 couples were cultured in time-lapse incubators. Embryos cultured in Vitrolife media were allocated to group I, and those in COOK media to group II. Embryo cleavage time points up to the 8-cell stage (t2-t8) were observed after pronuclei fading. The baseline demographic features, in vitro fertilization indications, ovarian stimulation protocol, oocyte-cumulus complexes, fertilization rate, together with pregnancy and perinatal outcomes were similar (P>0.05) between groups I and II. According to the time-lapse analysis, all embryos in group I showed significantly faster cleavage speed than those in group II (P<0.05). Furthermore, there was better synchrony in division (s3) and a longer length of the third cell cycle duration (cc3) in group II. Interestingly, implanted embryos in group II showed faster cleavage speed than those in group I, especially at t4 and t7. The glucose contents and multiple major amino acids were similar between the two groups. Lactic and pyruvic acid contents were generally higher in group I than those in group II. Because different commercial culture media may influence cleavage kinetics of embryos, it is essential for embryologists to take culture media into consideration in selecting a potential embryo when using a time-lapse system before implantation.

Perfluorooctanoic acid (PFOA) exposure affects early embryonic development and offspring oocyte quality via inducing mitochondrial dysfunction.

Perfluorooctanoic acid (PFOA) is a synthetic perfluorinated compound that is extensively used as an integral surfactant in commercial production. Owing to its hydrophilicity and persistence, PFOA can accumulate in living organisms and induce severe disease in animals and humans. It has been reported that PFOA exposure can affect ovarian function and induce reproductive toxicity; however, the effects and potential mechanism of PFOA exposure during gestation on early embryonic development and offspring remain unclear. This study found that PFOA exposure in vitro disrupted spindle assembly and chromosome alignment during the first cleavage of early mouse embryos, which impacted early embryonic cleavage and blastocyst formation. Moreover, PFOA exposure caused mitochondrial dysfunction and oxidative stress by inducing aberrant Ca2+ levels, liquid drops(LDs), and mitochondrial membrane potential in the 2-cell stage. Furthermore, we found that PFOA exposure resulted in DNA damage, autophagy, and apoptosis in 2-cell stage by inhibiting SOD2 function. Gestational exposure to PFOA significantly increased ovarian apoptosis and disrupted follicle development in F1 offspring. In addition, oocyte maturation competence was decreased in F1 offspring. Finally, single-cell transcriptome analysis revealed that PFOA-induced oocyte deterioration was caused by mitochondrial dysfunction and apoptosis in the F1 offspring. In summary, our results indicated that gestational exposure to PFOA had potential toxic effects on ovarian function and led to a higher incidence of meiotic defects in F1 female offspring.

PLCζ can stably regulate Ca2+ fluctuations in early embryo

Phospholipase C zeta (PLCζ) is an important inducer of Ca2+ oscillations in mammalian sperm. To explore the influence of PLCζ on early embryonic Ca2+ fluctuations during sperm-egg binding, this study used PLCζ from sheep sperm to construct an early embryonic Ca2+ fluctuation model. First, sheep MII oocytes were cultivated and screened using microinjection technology. Then, a pEGFP-N1-PLCζ plasmid was constructed to activate oocytes in the test group. Ionomycin combined with 6-Dimethylaminopurine (6-DMAP) was used for the control group to explore the effects on early embryonic development and regulation of Ca2+ fluctuations during development. The results demonstrated that both the PLCζ and ionomycin combined with 6-DMAP activation methods induced sheep oocyte parthenogenetic activation and development in early embryos. In comparisons, the cleavage rate of ionomycin combined with 6-DMAP activation was significantly higher than that of PLCζ (60.9% ± 19.4% vs 76.1% ± 0.7%, respectively; p < 0.001), and the blastocyst rates were 16.2% ± 0.62% and 21.1% ± 0.92%, respectively (p < 0.05). Additionally, when comparing the distribution of Ca2+ in early embryos at different stages, Ca2+ in both treatment groups was mainly distributed in the cytoplasm, but the temporal pattern of Ca2+ fluctuations differed. PLCζ resulted in Ca2+ peaks that appeared at the cleavage and morula stages of early embryos, and Ca2+ returned to normal levels at the morula stage. However, the Ca2+ concentration after ionomycin combined with 6-DMAP activation was always much higher than that with PLCζ, and its single peak appeared later than in the PLCζ group. In summary, the PLCζ gene promoted stable regulatory effects on Ca2+ fluctuations at different stages during early embryonic development.

Effects of Trichostatin A on the Timing of the First Cleavage and In Vitro Developmental Potential of Bovine Somatic Cell Nuclear Transfer Embryos.

This study examined the relationship between the timing of the first cleavage and in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos treated with trichostatin A (TSA). SCNT embryos were visually assessed at 22, 26, and 48 hours after activation. Each embryo with two or more distinct blastomeres was transferred into a microwell and cultured until day 7. Irrespective of TSA treatment, approximately half of the cleaved embryos were observed at 22 hours, and a significantly higher blastocyst formation rate was shown in the SCNT embryos cleaved at 22 hours than those cleaved at ≥26 hours. The blastocyst formation rate of TSA-treated embryos cleaved at 22 hours (80%) was slightly higher than that of the control embryos (70%). In addition, interferon-τ (IFN-τ) expression was significantly lower in control SCNT embryos and late-cleaving (>26 hours) TSA-treated embryos than in in vitro fertilized (IVF) embryos. However, a significant difference was not observed between TSA-treated SCNT embryos cleaved at 22 and 26 hours, and IVF embryos. These results suggest that TSA treatment has no influence on the timing of the first cleavage of SCNT embryos; however, it slightly improves the blastocyst formation rate and the expression level of IFN-τ in early-cleaving embryos.

Spatial and Temporal Scaling of Microtubules and Mitotic Spindles.

During cell division, the mitotic spindle, a macromolecular structure primarily comprised of microtubules, drives chromosome alignment and partitioning between daughter cells. Mitotic spindles can sense cellular dimensions in order to adapt their length and mass to cell size. This scaling capacity is particularly remarkable during early embryo cleavage when cells divide rapidly in the absence of cell growth, thus leading to a reduction of cell volume at each division. Although mitotic spindle size scaling can occur over an order of magnitude in early embryos, in many species the duration of mitosis is relatively short, constant throughout early development and independent of cell size. Therefore, a key challenge for cells during embryo cleavage is not only to assemble a spindle of proper size, but also to do it in an appropriate time window which is compatible with embryo development. How spatial and temporal scaling of the mitotic spindle is achieved and coordinated with the duration of mitosis remains elusive. In this review, we will focus on the mechanisms that support mitotic spindle spatial and temporal scaling over a wide range of cell sizes and cellular contexts. We will present current models and propose alternative mechanisms allowing cells to spatially and temporally coordinate microtubule and mitotic spindle assembly.

153&#x2003;Effects of ewe age on oocyte viability and timing of early embryo cleavage.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Impact of oxygen tension according to embryo stage of development: a prospective randomized study

Human embryo culture under 2–8% O2 is recommended by ESHRE revised guidelines for good practices in IVF labs. Nevertheless, notably due to the higher costs of embryo culture under hypoxia, some laboratories perform embryo culture under atmospheric O2 tension (around 20%). Furthermore, recent meta-analyses concluded with low evidence to a superiority of hypoxia on IVF/ICSI outcomes. Interestingly, a study on mice embryos suggested that oxidative stress (OS) might only have an adverse impact on embryos at cleavage stage. Hence, we aimed to demonstrate for the first time in human embryos that OS has a negative impact only at cleavage stage and that sequential culture conditions (5% O2 from Day 0 to Day 2/3, then «conventional» conditions at 20% O2 until blastocyst stage) might be a valuable option for human embryo culture. 773 IVF/ICSI cycles were included in this randomized clinical trial from January 2016 to April 2018. At Day 0 (D0), patients were randomized using a 1:2 allocation ratio between group A (20% O2; n = 265) and group B (5% O2; n = 508). Extended culture (EC) was performed when ≥ 5 Day 2-good-quality-embryos were available (n = 88 in group A (20% O2)). In subgroup B, 195 EC cycles were randomized again at Day 2 (using 1:1 ratio) into groups B’ (5% O2 until Day 6 (n = 101)) or C (switch to 20% O2 from Day 2 to Day 6 (n = 94). Fertilization rate, cleavage-stage quality Day 2-top-quality-embryo (D2-TQE), blastocyst quality (Day 5-top-quality-blastocyst (D5-TQB) and implantation rate (IR) were compared between groups A and B (= cleavage-stage analysis), or A(20% O2), B’(5% O2) and C(5%-to-20% O2). Overall, characteristics were similar between groups A and B. Significantly higher rates of early-cleaved embryos, top-quality and good-quality embryos on Day 2 were obtained in group B compared to group A (P < 0.05). This association between oxygen tension and embryo quality at D2 was confirmed using an adjusted model (P < 0.05). Regarding blastocyst quality, culture under 20% O2 from Day 0 to Day 6 (group A) resulted in significantly lower Day 5-TQB number and rates (P < 0.05) compared to both groups B’ and C. Furthermore, blastocyst quality was statistically equivalent between groups B’ and C (P = 0.45). At Day 6, TQB numbers and rates were also significantly higher in groups B’ and C compared to group A (P < 0.05). These results were confirmed analyzing adjusted mean differences for number of Day 5 and Day 6 top quality embryos obtained in group A when compared to those respectively in groups B’ and C (P < 0.05). No difference in clinical outcomes following blastocyst transfers was observed. These results would encourage to systematically culture embryos under hypoxia at least during early development stages, since OS might be detrimental exclusively before embryonic genome activation.

Time-lapse monitoring of fertilized human oocytes focused on the incidence of 0PN embryos in conventional in vitro fertilization cycles

We aimed to investigate why the incidence of embryos derived from oocytes with no pronuclei (0PN) decreases using time-lapse monitoring (TLM) versus fixed-point assessment in conventional IVF cycles. We analyzed 514 embryos monitored with TLM 6–9 h after insemination and 144 embryos monitored using microscopic assessment 18–21 h after insemination. The primary endpoint of this study was the incidence of 0PN-derived embryos in short insemination followed by TLM. The secondary endpoint was the duration of insemination. As exploratory endpoints, we analyzed the blastulation rate and cryo-warmed blastocyst transfer outcome of embryos with early PN fading, whereby PN disappeared within < 20 h following the initiation of insemination. The incidence of 0PN-derived embryo reduced more significantly through TLM than through fixed-point observation. The microscopic assessment time was more significantly delayed in the 0PN-derived embryo than that in the 2PN-derived embryo. The embryo with early PN fading formed good-quality blastocysts, and their pregnancy outcomes were similar to those of other embryos. Most 0PN-derived embryos in the fixed-point assessment might have resulted from missed observation of PN appearance in the early-cleaved embryos. TLM or strict laboratory schedule management may reduce 0PN-derived embryos by reducing missed PN observations.

Embryonic Environmental Niche Reprograms Somatic Cells to Express Pluripotency Markers and Participate in Adult Chimaeras.

The phenomenon of the reprogramming of terminally differentiated cells can be achieved by various means, like somatic cell nuclear transfer, cell fusion with a pluripotent cell, or the introduction of pluripotency genes. Here, we present the evidence that somatic cells can attain the expression of pluripotency markers after their introduction into early embryos. Mouse embryonic fibroblasts introduced between blastomeres of cleaving embryos, within two days of in vitro culture, express transcription factors specific to blastocyst lineages, including pluripotency factors. Analysis of donor tissue marker DNA has revealed that the progeny of introduced cells are found in somatic tissues of foetuses and adult chimaeras, providing evidence for cell reprogramming. Analysis of ploidy has shown that in the chimaeras, the progeny of introduced cells are either diploid or tetraploid, the latter indicating cell fusion. The presence of donor DNA in diploid cells from chimaeric embryos proved that the non-fused progeny of introduced fibroblasts persisted in chimaeras, which is evidence of reprogramming by embryonic niche. When adult somatic (cumulus) cells were introduced into early cleavage embryos, the extent of integration was limited and only cell fusion-mediated reprogramming was observed. These results show that both cell fusion and cell interactions with the embryonic niche reprogrammed somatic cells towards pluripotency.