Abnormal Cleavage Research Articles

Sperm-borne proteins improve rabbit cloning efficiency via regulating embryonic cleavage and epigenetics.

Somatic cell nuclear transfer (SCNT) shows great application value in the generation of transgenic animals, protection of endangered species, and therapeutic cloning. However, the cloning efficiency is still very low, which greatly restricts its application. Compared to fertilized embryos, cloned embryos lack the sperm proteins, which are considered to play an important role in embryonic development. Here, we compared the sperm proteome, with that of donor fibroblasts and oocytes, and identified 342 proteins unique to sperm, with 42 being highly expressed. The 384 proteins were mainly enriched in the categories of post-translational modification and cytoskeletal arrangement. Extracts of soluble sperm or fibroblast proteins were injected into cloned embryos, and the result showed that injection of sperm protein significantly inhibited abnormal embryonic cleavage, significantly decreased the level of trimethylated histone H3 Lys9 (H3K9me3) and the apoptotic index, and increased the inner cell mass (ICM)-to-trophectoderm (TE) ratio. More importantly, the sperm proteins also significantly enhanced the birthrate. The results of in vitro and in vivo experiments demonstrate that sperm-derived proteins improve embryo cloning efficiency. Our findings not only provide new insights into ways to overcome low cloning efficiency, but also add to the understanding of sperm protein function.

O-288 Association of initial zygote cleavage, compaction pattern and euploidy rates: possible implications for embryo correction mechanisms

Abstract Study question Is the morula compaction pattern associated with early zygote cleavage anomalies and euploidy rates? Can it represent a possible mechanism of embryonic correction? Summary answer Abnormalities in the first divisions give rise to dysfunctional blastomeres, which were excluded from compaction process. This process represent a possible embryonic correction mechanism. What is known already IVF embryos have historically been analyzed based on morphological characteristics focusing on days 2/3 (cleavage stage) and 5/6 (blastocyst). The morula stage, usually reached on day 4, represent a transitional phase resulting from the embryonic genome activation and has received little attention in clinical embryology. However, during this crucial stage, cell differentiation begins, metabolism reaches higher levels, changes in cell shape and cell-destination mechanisms are enhanced, providing opportunities for “quality control”. At morula stage, embryos with severe genetic abnormalities are sorted out by natural selection or possibly self-correction events may occurs, reducing the load of aneuploid cells in mosaic embryos. Study design, size, duration We evaluated laboratory and clinical outcomes of 836 blastocysts (195 ICSI cycles) cultured in a time-lapse system and biopsied for preimplantation genetic testing for aneuploidy (PGT-A), from April-2018 to June-2020. Time-lapse videos were retrospectively analyzed to assess morphokinetic parameters of initial zygote cleavages and morula compaction. These parameters were evaluated and associated to genetic and embryonic outcomes. Blastocyst outcomes were also assessed according to maternal age <35 (n = 140), 35-37 (n = 175), 38-40 (n = 314) and >40 (n = 207). Participants/materials, setting, methods The zygote initial cleavages were classified as normal (1-2-4 cleavage) or anomalous: 1-3 cleavage (zygote splits directly into three blastomeres); 1-2-3 cleavage (2-3 in < 3h); and 1-2-5 cleavage (after the first normal cleavage, one blastomere splits directly into three blastomeres). Morula were classified as: Fully Compacted Morulae (FCM); Partially Compacted Morulae (PCM). The PCM could have cells excluded from the compaction process (Exc-PCM), extruded from an already compacted morula (Ext-PCM), or both situations (Exc/Ext-PCM). Main results and the role of chance Most blastocysts showed normal zygotic cleavage (83.3%). Anomalous cleavage such as 1-3 (0.8%), 1-2-3 (7.2%) and 2-5 (8.7%) were less prevalent and no differences were observed according to female age (p = 0.268). Overall, FCM (61.0%) was a more frequent pattern than PCM (39.0%). According to compaction pattern of PCM, Exc-PCM (25.1%) was more prevalent than Ext-PCM (11.5%) and Exc/Ext-PCM (2.4%). There was also no difference in the prevalence of the morulae compaction pattern according to female age (p = 0.124). FCM occurred in 511 embryos (73.4%) from zygotes with normal cleavage (n = 696), but no FCM was observed among anomalous cleaved zygotes (p < 0.001). Genetic and embryonic outcomes were displayed as follows: normal cleavage and FCM (NC-FCM, n = 511), normal cleavage and PCM (NC-PCM, n = 185), and anomalous cleavage and PCM (AC-PCM, n = 140). NC-FCM and AC-PCM groups showed statistically similar euploidy rates (39.3% and 38.6%, p = 0.870), that were both significantly higher than the NC-PCM group euploidy rate (23.8%, p < 0.001). However, morphokinetic scores (KidScore) and percentage of top-quality blastocysts at morphological evaluation were discordant: NC-FCM (6.4±1.7; 81.4%), NC-PCM (5.0±1.8; 47.6%) and AC-PCM (4.3±2.0; 37.9%). Euploidy rate decreased with female age for all groups: NC-FCM (<35:54.7%, 35-37:56.0%, 38-40:33.3%, >40:24.1%); NC-PCM (<35:37.5%, 35-37:19.0%, 38-40:25.0%, >40:19.0%); AC-PCM (<35:66.7%, 35-37:48.5%, 38-40:39.0%, >40:3.7%). Limitations, reasons for caution The retrospective design of the study represents a general limitation. In addition, this study evaluated only embryos that reached the blastocyst stage and showed enough quality to be biopsied. Therefore, the association between anomalies in the first and second mitotic cleavages and the embryonic development could not be evaluated. Wider implications of the findings Similar euploidy rates among NC-FCM and AC-PCM support the hypothesis that cell exclusion/extrusion in compaction stage of anomalous-cleaved zygotes can represent a potential correction mechanism, even though lower morphological/morphokinetic scores are seen on AC-PCM. That process is supposed to rescue embryos from the mosaic aneuploidy which occurs after abnormal cleavage. Trial registration number not applicable

P-231 Embryo morphokinetics in male factor infertility

Abstract Study question Are embryo morphokinetics and division pattern susceptible to sperm origin? Summary answer Surgical retrieved sperm and Oligo-astheno-teratozoospermia have significant effect on morphokinetics and pattern of embryo development in IVF. What is known already Among couples seeking primary infertility treatment, approximately one third of them have male factor indication. Intracytoplasmic sperm injection (ICSI) has changed their prognosis to achieve successful pregnancy. Recently, there are many conflicting studies on the relation of sperm origin, embryo development and clinical outcome after fertilization burrier is passed by ICSI. Implementation of time-lapse monitoring system (TLM) allows us to analyze embryo development under stable conditions, and identify morphological, temporal, spatial parameters and cell cycle endpoints at which embryos with sperm of different origin exhibit distinct kinetics. The magnitude of variable morphokinetics in anticipating pregnancy outcome is not yet determined. Study design, size, duration This is a prospective cohort study that included 195 ICSI cycles stratified into three groups of different sperm origin (abnormal sperm (OAT), surgical sperm(SRS) and normosperm (CS)). Each group included around 400 zygotes for patients seeking IVF treatment in Fakih IVF/Dubai between December 2019 and December 2021 in Dubai / UAE. The mean age of patients is (22-40 years female/ 22-45 years male). Normospermia subset was limited to couples seeking gender selection and tubal factor. Participants/materials, setting, methods Embryo morphokinetics (time to polar body (tPB) until time to blastocyst (tB)) and cell cycle durations were analyzed. Embryos were examined for irregular cleavage phenotypes. The prevalence of each category of out of-range morphokinetics and abnormal cleavage among the different groups was compared. The portion of embryos displaying optimal ranges for the indicated morphokinetics was explored focusing on cell cycle duration. Finally, multi-variables influencing morphokinetics parameters were controlled. Chi2 test was performed in SPSS, P-value<0.05. Main results and the role of chance Embryos derived from SRS were later to reach tPNf and t2(26.66±9.16 and 29.03±8.52) than embryos derived from CS (25.15±4.20 and 27.84±4.98) and OAT (25.71±6.20 and 28.67±7.22). P < 0.05. Embryos derived from SRS reached t5(47.94±11.02) faster than both embryos derived from CS (49.61±8.89) and OAT (49.26±9.47). Embryos derived from SRS reached t6(51.64±10.44) faster than embryos from CS (53.10±8.96). P value=0.0. Embryos derived from SRS and OAT reached tSB (95.34±9.01 and 94.92 ±10.04) and tB(103.68±8.53 and 102.08±10.32) significantly later than embryos derived from CS (91.16±5.65 and 96.34±7.70). No correlation was found between sperm origin and irregular embryo phenotypes except for blastomere asymmetry and incomplete morulation. P = 0.0004. SRS and OAT had higher incidence of blastomere asymmetry at 2-, 3- and 4cell stage than CS. P = <0.05. For incomplete morulation, CS group had higher incidence of 1-3 fragments at morula stage(71.3%) than SRS and OAT (57.4% and 49%). However, OAT had higher incidence of 3-5 fragments at morula stage(35.9%) than SRS (224.1%) and CS (18.8%). Significantly, SRS displayed higher proportion of embryos out of range for most of the measured morphokinetics. P < 0.05. Accordingly, less proportion of embryos in SRS and OAT fall within optimal range for cell cycle duration P < 0.0. Antral follicle count, number of collected oocytes and male’s age had significantly affected morphokinetics and cleavage abnormality. Limitations, reasons for caution Selection of sperm by ICSI due to male- and severe male factor infertility may result in selecting live sperm with the best available normal morphology which may not reflect DNA fragmentation status of the overall sample where damage of DNA is more significant and lead to bias the results. Wider implications of the findings Embryo development potential with different sperm origin was compared based normal optimum morphokinetics or not. Further follow up study to investigate clinical outcome of implantation and clinical pregnancy rate, miscarriage rate and live birth rate is needed. Trial registration number DSREC-11/2021_07

O-208 Use of epididymal spermatozoa in in vitro fertilization cycles impacts the morphokinetics of embryos cultured in a time-lapse imaging incubator system

Abstract Study question Does sperm origin impact embryo morphokinetics (the moments at which an embryo reaches a developmental milestone) and clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles? Summary answer The use of epididymal sperm correlates with late cell cleavages, and higher multinucleation and abnormal cleavage rates compared to those from men without infertility factor. What is known already ICSI with sperm surgically retrieved from the epididymis (PESA) dramatically altered the pregnancy prognosis in couples with severe male factor infertility, including obstructive azoospermia. The integration of time-lapse imaging (TLI) into the in vitro fertilization (IVF) laboratory allowed for an in-depth analysis of embryonic development. However, the impact of the use of epididymal sperm on embryonic morphokinetics has been poorly studied. We hypothesized that factors related to sperm origin may interfere with the speed and pattern of cell divisions. Study design, size, duration Women undergoing ICSI with epididymal sperm between Jan/2019 and Dec/2020 in a private university-affiliated IVF center were included (PESA group, n = 32 cycles/276 embryos). A matching was performed to form two groups of women undergoing ICSI using ejaculated sperm from partners with idiopathic male factor infertility (IMF group, n = 32 cycles/284 embryos) or without male factor infertility (Control group, n = 32 cycles/246 embryos). Generalized linear models followed by Bonferroni post hoc were used to compare morphokinetics between groups. Participants/materials, setting, methods Embryos were cultured in the EmbryoScope incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), morulae (tM), start of blastulation (tSB) and blastulation (tB). Durations of second and third cell cycles (cc2 and cc3) and timing to complete synchronous divisions s1, s2, and s3 were calculated. The KIDScore ranking was recorded. Main results and the role of chance Embryos derived from epididymal sperm showed significantly slower divisions compared to those from ejaculated sperm (Table 1). The KIDScore rank was significantly lower for embryos deriving from epididymal sperm (3,1 ± 0.2) compared to those deriving from ejaculated sperm from IMF and control groups (5.4 ± 0.1 and 5.6 ± 0.2, p < 0.001, respectively). The incidence of multinucleation was significantly higher in embryos deriving from epididymal sperm (23.2%) compared to those deriving from ejaculated sperm from IMF and control groups (2.8% and 3.7%, p < 0.001, respectively). The incidence of abnormal cleavage patterns (direct or reverse) was significantly higher in embryos deriving from epididymal sperm (11.1%) compared to those deriving from ejaculated sperm from control group (4.3%, p = 0.001). Similar clinical outcomes were observed between the groups. Limitations, reasons for caution Retrospective nature of this study and the small sample size may be a reason for caution. Wider implications of the findings The use of epididymal sperm for ICSI correlates with delayed cell cleavage, and increased incidences of multinucleation and abnormal cleavage patterns. This finding highlights the importance of TLI for the identification and de-selection of slow-growing embryos for transfer, in ICSI cycles with surgical sperm retrieval. Trial registration number not applicable

O-100 Incidence, morphology, ploidy and developmental arrest in direct cleavage embryos: what are they teaching us? Morphokinetic analysis of 15.081 embryos

Abstract Study question What is the impact of direct cleavage (DC) occurrence during embryo/blastocyst development regarding morphology grade, embryo arrest and ploidy status? Summary answer Blastocyst formation and morphology grade are significantly reduced in DC when compared to non-DC embryos; however, there is no difference in blastocyst ploidy status. What is known already Chromosome instability leading to aneuploidy during early cleavage is well known in humans. DC is defined as the abrupt cleavage of one into three or more daughter blastomeres as the result from an irregular mitosis. Three or more mitosis poles can impair early embryo development; however, how some embryos with these irregular divisions might be able to develop into expanded blastocysts is still unknown. Time-lapse system provides an uninterrupted evaluation of embryo morphological and dynamic parameters, allowing our understanding in such events and if those embryos may be associated with a self-correction mechanism to reach blastocyst stage. Study design, size, duration Retrospective cohort study from 15.081 embryos, 3.962 cycles and 3.106 patients that were undergoing in vitro fertilization (IVF) treatment according to medical referral in a single private ART center from December 2018 to November 2021. Only two-pronuclear (2PN) embryos cultured in time-lapse incubators (EmbryoScope, Vitrolife, Sweden) were included in the study. Direct cleavage occurance was annotated when a single blastomere directly cleaved into three or more daughter blastomeres. Participants/materials, setting, methods DC embryos were also classified in DC 1-3: abnormal cleavage occurred in zygote (1-cell) resulting in 3–4 blastomeres and in DC 2+: abnormal cleavage occurred at the 2-cell stage resulting in 5 or 6 blastomeres. Embryo arrest, blastocyst formation rate and morphology grade (Gardner and Schoolcraft, 1999) were annotated. Blastocyst biopsy reports for ploidy status were also considered for analysis. Mann-Whitney, chi-squared and Fisher tests were applied for statistical analysis. p < 0,05 was considered significant. Main results and the role of chance There were 1168 DC embryos (7,7%) from 896 cycles/841 patients; 502 DC1-3 (3,3%) from 391 cycles/366 patients and 666 DC2+ embryos (4,4%) from 475 patients/505 cycles. In DC group, around 21% of patients have the incidence of more than 1 DC embryo per cycle. Maternal age was lower in DC groups: 38,0±3,61 (DC1-3) and 38,60±3,21 (DC2+) years old versus not-DC (39,02±3,01, p < 0,0001). Embryo arrest was higher (DC1-3:85,5% and DC2+:56,8% versus not-DC:35,4%, p < 0,0001) and blastocyst formation rate was lower (14,5% and 43,2% versus 64,6%, p < 0,0001) in both DC groups and also between DC groups (p < 0,0001). There were 4124 biopsied not-DC blastocysts, 24 in DC1-3 and 154 in DC2+. There was no difference in ploidy between not-DC (57,9%, 40,2%, 1,9%, aneuploid, euploid and mosaic respectively) and DC groups (54,2%, 41,7%, 4,2% and 61%, 37% and 1,9% respectively, p = 0.86) and neither between DC-groups (p = 0.69). Inner cell mass morphology grades were distinct in all groups (not-DC A:45,8%, B:45,2% C:9,0%, DC1-3 A:35,6%, B:33,9%, C:30,5% and DC2+ A:23,1%, B:57,0% C:19,9%, p < 0,0001) and between DC-groups (p < 0,0001). Trophoectoderm morphology grades were also higher for not-DC group (not-DC A:30,5%, B:48,9% C:20,6%, DC1-3 A:15,3%, B:47,5%, C:37,3% and DC2+ A:13,6%, B:44,4% C:42,0%, p < 0,0001), however there was no difference between DC-groups (p = 0.80). Limitations, reasons for caution Limited data are available on the implantation potential of embryos with DC, as they are not prioritize for transfer, although our study did not demonstrated a higher aneyploidy rate. Patient parameters, with the exception of maternal age, were not considered in this study. Wider implications of the findings Although the morphology grades are lower and embryo arrest is higher in DC embryos, probably due to unequal cleavage, if they reach blastocyst stage, the ploidy status of these embryos are similar to not-DC embryos, speculative for a self-correction mechanism. Future steps should focus on their performance in pregnancy/live-birth. Trial registration number Not Applicable

P-152 The first morphokinetic map of human abnormal fertilisation

Abstract Study question What are the similarities and differences between the morphokinetics of abnormal, one- (1PN) and three-pronuclear (3PN) and normal bi-pronuclear (2PN) fertilisation? Summary answer The morphokinetic analysis of 1PN/3PN fertilisation reveals novel aspects of abnormal early development. What is known already Assisted reproduction technology has allowed the observation of early human development. Initially assessed statically at a single time point, fertilization has revealed its complexity once observed by Time-Lapse Microscopy (TLM). Detailed morphokinetic analysis of fertilisation has been reported in the last few years, unveiling previously unknown cytoplasmic phenomena (e.g. the cytoplasmic wave and halo) and the importance of cell symmetry for embryo development. At present, abnormal fertilization remains neglected, despite potential for understanding the physiology and pathology of early human development. Study design, size, duration This retrospective study involved TLM observation of normally (2PN, n = 2,685) and abnormally (1PN, n = 41; 3PN, n = 127) fertilised oocytes generated in ICSI cycles. Oocyte retrievals were carried out after the clomiphene citrate-based minimal ovarian stimulation, between October 2019 and December 2020. Oocytes of patients with different diagnoses of infertility were included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded. Participants/materials, setting, methods Microinjected oocytes were assessed by a combined TLM-culture system (Embryoscope). Oocytes not suitable for TLM assessment, due to excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena, relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage pattern, and embryo quality, were annotated and compared between groups. Main results and the role of chance Second polar body (PBII) extrusion was observed in all 1PN- and in a majority of 3PN-zygotes (92.1%). A 0.3-hour delay in PBII extrusion was confirmed in 3PN-zygotes (P = 0.0439). In a significant proportion of 3PN-zygotes, a third (female) PN formed from reabsorption of the PBII. The cytoplasmic wave was observed not only in 2PN- and 3PN-, but also in 1PN-zygotes. The presence and position of cytoplasmic halo were comparable among the three classes of zygotes. However, the duration of the cytoplasmic halo was prolonged in 1PN-zygotes (P < 0.0001). PN juxtaposition immediately before PN breakdown was less frequent in 3PN- compared with 2PN-zygotes (P = 0.0159). Furthermore, asynchronous PN breakdown was increased in 3PN- compared with 2PN-zygotes (P = 0.0026). The PN area of 1PN- was larger than that of 2PN-zygotes; however, the PN area of 3PN-zygotes was smaller than that of 2PN-zygotes. In 1PN-zygotes, a developmental delay was observed starting from the disappearance of the cytoplasmic halo, reaching 9 hours at the time of cleavage (P < 0.0001). A higher incidence of abnormal cleavage (P = 0.0019) and blastomere fragmentation (P < 0.0001) was observed in 1PN-zygotes. Cleavage progression was increasingly affected especially in 1PN-zygotes, resulting in blastocyst formation rates of 70.2%, 12.2% and 53.5% in 2PN-, 1PN- and 3PN-zygotes, respectively (P < 0.0001). Limitations, reasons for caution The study data derive from treatments carried out in a single centre. The study findings therefore require independent verification from other research groups. Wider implications of the findings These observations suggest that 1PN and 3PN fertilisation follow the general pattern of normal fertilization. Crucially, they also shed light on diverse and previously undescribed phenomena - e.g. reabsorption of the PBII in 3PN zygotes - underpinning the origins of abnormal fertilization and potentially clinically relevant. Trial registration number not applicable

Morphological and morphokinetic associations with aneuploidy: a systematic review and meta-analysis.

A time lapse system (TLS) is utilized in some fertility clinics with the aim of predicting embryo viability and chance of live birth during IVF. It has been hypothesized that aneuploid embryos display altered morphokinetics as a consequence of their abnormal chromosome complement. Since aneuploidy is one of the fundamental reasons for IVF failure and miscarriage, attention has focused on utilizing morphokinetics to develop models to non-invasively risk stratify embryos for ploidy status. This could avoid or reduce the costs associated with pre-implantation genetic testing for aneuploidy (PGT-A). Furthermore, TLS have provided an understanding of the true prevalence of other dysmorphisms. Hypothetically, the incorporation of morphological features into a model could act synergistically, improving a model's discriminative ability to predict ploidy status. The aim of this systematic review and meta-analysis was to investigate associations between ploidy status and morphokinetic or morphological features commonly denoted on a TLS. This will determine the feasibility of a prediction model for euploidy and summarize the most useful prognostic markers to be included in model development. Five separate searches were conducted in Medline, Embase, PubMed and Cinahl from inception to 1 July 2021. Search terms and word variants included, among others, PGT-A, ploidy, morphokinetics and time lapse, and the latter were successively substituted for the following morphological parameters: fragmentation, multinucleation, abnormal cleavage and contraction. Studies were limited to human studies. Overall, 58 studies were included incorporating over 40000 embryos. All except one study had a moderate risk of bias in at least one domain when assessed by the quality in prognostic studies tool. Ten morphokinetic variables were significantly delayed in aneuploid embryos. When excluding studies using less reliable genetic technologies, the most notable variables were: time to eight cells (t8, 1.13 h, 95% CI: 0.21-2.05; three studies; n = 742; I2 = 0%), t9 (2.27 h, 95% CI: 0.5-4.03; two studies; n = 671; I2 = 33%), time to formation of a full blastocyst (tB, 1.99 h, 95% CI 0.15-3.81; four studies; n = 1640; I2 = 76%) and time to expanded blastocyst (tEB, 2.35 h, 95% CI: 0.06-4.63; four studies; n = 1640; I2 = 83%). There is potentially some prognostic potential in the degree of fragmentation, multinucleation persisting to the four-cell stage and frequency of embryo contractions. Reverse cleavage was associated with euploidy in this meta-analysis; however, this article argues that these are likely spurious results requiring further investigation. There was no association with direct unequal cleavage in an embryo that progressed to a blastocyst, or with multinucleation assessed on Day 2 or at the two-cell stage. However, owing to heterogeneous results and poor-quality evidence, associations between these morphological components needs to be investigated further before conclusions can be reliably drawn. This first systematic review and meta-analysis of morphological and morphokinetic associations with ploidy status demonstrates the most useful morphokinetic variables, namely t8, t9 and tEB to be included in future model development. There is considerable variability within aneuploid and euploid embryos making definitively classifying them impossible; however, it is feasible that embryos could be prioritized for biopsy. Furthermore, these results support the mechanism by which algorithms for live birth may have predictive ability, suggesting aneuploidy causes delayed cytokinesis. We highlight significant heterogeneity in our results secondary to local conditions and diverse patient populations, therefore calling for future models to be robustly developed and tested in-house. If successful, such a model would constitute a meaningful breakthrough when accessing PGT-A is unsuitable for couples.

Case Analysis of Accessory Abductor Pollicis Longus Tendons and Brevis Muscles with Implications for Occupational Therapy

INTRODUCTIONVariations in muscles to the pollex are relatively common. Abnormal cleavage of the thenar muscles during development can form accessory muscle bellies, and forearm muscles sending long tendons to the pollex can develop extra tendons. While these abnormalities are typically asymptomatic and often go undetected, serious sequelae can result that affect pollex function. Pollex abduction, generated primarily by the abductor pollicis longus (APL) and brevis (APB) muscles, is an essential movement for hand function. APL has been reported with accessory tendons, split muscle bellies, and abnormal attachments, but variations in APB are less common. The addition of an accessory APL (aAPL) tendon in the 1st extensor compartment may cause crowding of APL and extensor pollicis brevis (EPB) tendons and result in inflammation. This condition is known as de Quervain’s tenosynovitis and often generates pain during abduction and extension of the pollex – implications that are pertinent to Occupational Therapy (OT). The objective of this study is to analyze a unique case of bilateral aAPL tendons which distally attach to accessory APB (aAPB) muscles and discuss their implications for OT practice.METHODSBilateral aAPL tendons distally attaching to aAPB muscles were discovered during routine cadaver dissection at the University of Nebraska Medical Center. The aAPL tendons, aAPB muscles, and surrounding anatomy were cleaned of extraneous tissue to better view the course, structure, and attachments of the anomalies. The bilateral anomalies were comparable, so only the left aAPL tendon and aAPB muscle were photographed in situand post‐evisceration. Bilateral tendon measurements and maximal isometric forces (Fmax) for each muscle were used to determine comparability and thereafter discussed as means. The normal APL and APB were also analyzed bilaterally to gauge accessory muscle impact. A literature review was conducted to discuss rarity and clinical implications of these muscles for OT practice.RESULTSThe regular APL tendon displayed a normal insertion on the base of the 1st metacarpal. The aAPL tendon originated from the APL muscle belly and exhibited two distal attachments: the main tendon (137.5 mm length) to the aAPB muscle belly and a short distal branch of the main tendon to the trapezium. Compression of the aAPL tendon in the 1st extensor compartment was grossly visible. The literature consistently references de Quervain’s tenosynovitis as an expected result from similar cases, but no studies describe a distal bifurcation of the aAPL tendon that distinctly attaches to both aAPB and trapezium. Mean Fmax for APB, aAPB, and APL were 12.1 N, 8.2 N, and 40.8 N, respectfully.CONCLUSIONS & SIGNIFICANCEThe present study reports a unique case of an aAPL tendon with a distinct bifurcation to an aAPB and the trapezium. The short tendon to trapezium allows aAPB to use the aAPL tendon as an anchor (origin) but limits APL’s ability to abduct the pollex via pull on aAPB. This report may help OT providers better determine the presence of accessory abductor pollicis tendons and/or muscles when treating patients with de Quervain’s tenosynovitis, adapt treatment modalities, and note this in their medical record for possible future use in tendon transfer surgeries.

The relationship between amyloid-beta and brain capillary endothelial cells in Alzheimer's disease.

Neurovascular dysfunction, as an integral part of Alzheimer’s disease, may have an important influence on the onset and progression of chronic neurodegenerative processes. The blood-brain barrier (BBB) pathway is one of the main pathways that mediates the clearance of amyloid-beta (Aβ) in the brain parenchyma. A large number of studies have shown that receptors and ATP-binding cassette transporters expressed on endothelial cells play an important role in Aβ transport across the BBB, but the specific mechanism is not clear. In this review, we summarize the possible mechanisms of Aβ production and clearance, and in particular the relationship between Aβ and brain capillary endothelial cells. Aβ is produced by abnormal cleavage of the amyloid precursor protein via amyloidogenic processing under pathological conditions. Dysregulation of Aβ clearance is considered to be the main reason for the massive accumulation of Aβ in the brain parenchyma. Several pathways mediating Aβ clearance from the brain into the periphery have been identified, including the BBB pathway, the blood-cerebrospinal fluid barrier and arachnoid granule pathway, and the lymphoid-related pathway. Brain capillary endothelial cells are the key components of Aβ clearance mediated by BBB. Receptors (such as LRP1, RAGE, and FcRn) and ATP-binding cassette transporters (such as P-gp, ABCA1, and ABCC1) expressed on endothelial cells play a critical role in Aβ transcytosis across the BBB. The toxic effects of Aβ can induce dysregulation of receptor and transporter expression on endothelial cells. Excessive Aβ exerts potent detrimental cerebrovascular effects by promoting oxidative stress, inducing chronic inflammation, and impairing endothelial structure and functions. All of these are main causes for the reduction in Aβ clearance across the BBB and the accumulation of Aβ in the brain parenchyma. Therefore, studies on the interactions between Aβ and brain capillary endothelial cells, including their receptors and transporters, studies on inhibition of the toxic effects of Aβ on endothelial cells, and studies on promoting the ability of endothelial cells to mediate Aβ clearance may provide new therapeutic strategies for Aβ clearance in Alzheimer’s disease.

Targeted proteolytic products of τ and α-synuclein in neurodegeneration.

CNS pathological inclusions comprising τ or α-synuclein (αSyn) define a spectrum of neurodegenerative diseases, and these can often present concurrently in the same individuals. The aggregation of both proteins is clearly associated with neurodegeneration and the deleterious properties of each protein is further supported by mutations in each gene (MAPT and SNCA, respectively) resulting in disease. The initiating events in most sporadic neurodegenerative diseases are still unclear but growing evidence suggests that the aberrant proteolytic cleavage of τ and αSyn results in products that can be toxic and/or initiate aggregation that can further spread by a prion-like mechanism. The accumulation of some of these cleavage products can further potentiate the progression of protein aggregation transmission and lead to their accumulation in peripheral biofluids such as cerebrospinal fluid (CSF) and blood. The future development of new tools to detect specific τ and αSyn abnormal cleavage products in peripheral biofluids could be useful biomarkers and better understand of the role of unique proteolytic activities could yield therapeutic interventions.

A novel variant of NPPC causes abnormal post-translational cleavage: A candidate gene for premature ovarian insufficiency

ObjectivePremature ovarian insufficiency (POI) is a clinical disease that is diagnosed by the loss of ovarian function before the age of 40. Despite recent progress in molecular diagnosis, the genetic etiology of POI is not well established. The aim of this study is to reveal pathogenic genetic variants involved in POI. Study design and main outcome measuresTo reveal pathogenic genetic variants involved in POI, whole exome sequencing was performed in nonconsanguineous family members with POI. Constitutional variants were filtered against population databases and a missense mutation of natriuretic peptide C (NPPC) (c.131A>G, p.Q44R) was selected as a convincing candidate mutation among 14 heterozygous mutant alleles in 13 genes. ResultsThe wild-type NPPC and mutant NPPC (NPPC131A>G) were expressed in HeLa cells, and cells expressing NPPC131A>G secreted unique peptides. The ProP 1.0 Server, a neural network prediction tool, predicted the presence of a cleavage site at the substituted arginine residue (p.Q44R) of NPPC. The molecular weight of predicted cleaved peptides processed from mutant NPPC precursor corresponded to that of the actual mutant peptide. The cGMP synthetic activity of NPR2-expressing cells was significantly decreased by interaction with the mutant NPPC peptide compared with wild-type NPPC. ConclusionsThe peptide generated by a rare mutation of NPPC might influence paracrine C-type natriuretic peptide (CNP)-mediated preantral follicle development and/or sustain meiotic arrest in oocytes. We therefore suggest that a mutation of the NPPC gene is involved in the pathogenesis of POI.

Morphokinetic parameter comparison between embryos from couples with high or low sperm DNA fragmentation index

To study whether time-lapse imaging can identify morphokinetic events impacted by a high sperm DNA fragmentation index (DFI). Historical cohort study. Private university-affiliated invitro fertilization center. A total of 978 zygotes cultured until day 5 in a time-lapse imaging incubator between March 2019 and August 2020, derived from 118 patients undergoing intracytoplasmic sperm injection as a result of idiopathic male factor infertility. Kinetic markers from the point of insemination were recorded. Generalized linear mixed models adjusted for potential confounders followed by the Bonferroni post hoc test were used to compare the timing of specific events in patients with a low (<30%) or high (≥30%) sperm DFI. The recorded kinetic markers were the following: timing to pronuclei appearance and fading; timing to 2, 3, 4, 5, 6, 7, and 8 cells; and timing to start blastulation and blastulation. Timing to blastulation. Embryos derived from sperm samples with ≥30% DFI showed significantly slower divisions compared with those with <30% DFI (mean differences of 0.7 hours in timing to pronuclei appearance, 1.2 hours in timing to pronuclei fading, 1.5 hours in timing to 2 cells, 2.5 hours in timing to 3 cells, 1.8 hours in timing to 4 cells, 3.3 hours in timing to 5 cells, 3.1 hours in timing to 6 cells, 3.2 hours in timing to 7 cells, 2.7 hours in timing to 8 cells, 8.4 hours in timing to start blastulation, and 3.8 hours in timing to blastulation). The incidences of reverse or direct cleavages (9.3% vs. 4.4%; odds ratio [OR], 2.24; 95% confidence interval [CI], 1.32-3.77) and multinucleation at 2-cell (18.9% vs. 12.0%; OR, 1.70; 95% CI, 1.12-2.58) and 4-cell (14.2% vs. 6.4%; OR, 2.42; 95% CI, 1.57-3.74) stages were significantly higher in embryos deriving from ≥30% DFI than from <30% DFI. The KIDScore ranked significantly different between embryos derived from samples with <30% and ≥30% DFI. Continuous DFI was positively correlated with all timings of specific events and with the incidences of abnormal cleavage patterns (OR, 1.042; 95% CI, 1.025-1.059) and multinucleation at 2-cell stage (OR, 1.053; 95% CI, 1.030-1.076) and inversely correlated with the KIDScore rank (B, -0.218; 95% CI, -0.044 to -0.007). No significant differences were observed in clinical outcomes between the groups. Embryo morphokinetic parameters are negatively impacted by high sperm DFI, resulting in delayed cell cleavage and blastulation.

P–282 A time lapse analysis of 36,671 embryos to compare the incidence of early stage abnormal cleavage events in ICSI and IVF derived embryos

Abstract Study question Is this incidence of early stage abnormal cleavage events different between embryos created following ICSI compared with IVF? Summary answer Embryos derived from ICSI are more likely to exhibit abnormal cleavage compared with those from IVF. This difference is most marked in women ≥35 years. What is known already Time lapse imaging (TLI) has been instrumental in allowing detailed annotation of early embryo development to provide an objective aid for embryo selection in ART cycles. Amongst several abnormal cleavage events reported, rapid cleavage and multichotomous mitosis/direct cleavage, during the first days after fertilisation have been demonstrated to be associated with lower blastulation rates, reduced implantation potential, increased aneuploidy and poor pregnancy outcomes. With ICSI being utilised commonly, and being the insemination method of choice in some clinics, the incidence of abnormal cleavage was investigated in association with insemination method, ICSI or IVF. Study design, size, duration The incidence of abnormal cleavage events was evaluated in a large multicentre retrospective analysis of 36,671 embryos from 6689 patients treated in 8 IVF clinics enabled with time lapse imaging, between 2011 - 2019. This constituted 10931 IVF embryos and 25740 ICSI embryos. Participants/materials, setting, methods Following ICSI or after IVF fertilisation check, embryos were time-lapse imaged every 10 minutes and annotated using the EmbryoScope. Second cell cycle durations were calculated as follows: time to reach 3-cell (t3) from 2-cell (t2) (t3-t2 = cc2). These were analysed using a welch t-test as three groups of abnormal cleavage: direct cleavage/trichotomous mitosis (DC) - where cc2=0 hours(h), rapid cleavage within 2h (R2) - where 0&amp;lt;cc2&amp;lt;2h and rapid cleavage between 2–5h (R5) where 2h&amp;lt;cc2&amp;lt;5h. Main results and the role of chance The incidence of DC, R2 and R5 in the whole cohort of embryos was 5%, 8% and 9% respectively. In the subpopulation of IVF embryos the incidence of DC, R2 and R5 was 4%, 8% and 9% respectively. In the subpopulation of ICSI embryos the incidence of DC, R2 and R5 was 6%, 8% and 9% respectively. The incidence of DC was significantly higher in ICSI embryos compared with IVF (p &amp;lt; 0.001) whilst R2 and R5 were the same. ICSI derived embryos had a mean (± SE) cc2 value of 9.39 ± 0.03h, compared with 9.56 ± 0.05h for IVF embryos (p &amp;lt; 0.0038). Examination of data split by maternal age demonstrated that ICSI oocytes from women of advanced maternal age (≥35) also had significantly more embryos exhibiting rapid cleavages R2 and R5 than IVF oocytes (p &amp;lt; 0.007). There were no significant differences however, in rates of abnormal cleavages between ICSI and IVF in embryos from women aged &amp;lt;30 (p = 0.06). Male-factor diagnoses showed no significant differences in abnormal cleavage values between ICSI or IVF in all three abnormal cleavage categories. Limitations, reasons for caution This analysis could not control for all potential confounders therefore it is possible that the increased abnormal cleavages observed in this investigation are a result of another, or combination of factors. Despite quality assurance programs being in place across all clinics, there is a risk of annotation bias. Wider implications of the findings: There is a higher incidence of early abnormal cleavage in embryos derived from ICSI, particularly in those from women of increased age and this research may help elucidate the reasons for this and add to the debate regarding the appropriateness of the increasing use of ICSI. Trial registration number Not applicable

O-003 The puzzling unknowns of abnormal fertilization and first cleavage

Abstract text Fertilization is a critical event in development in that it provides the connection between the gametes and the earliest stages of embryogenesis. Yet, despite the central importance of this process in contributing to embryo developmental fate, clinical embryologists have historically assessed fertilization merely by the number of pronuclei and, if two are present, perhaps, by the presence of two polar bodies. Even though over 20 years ago, time lapse imaging was applied for defining early events of fertilization (Payne et al., 1997), it is only with contemporary time-lapse imaging systems in the last few years that detailed evaluation of spatial and temporal events of fertilization have been described (Iwata &amp; Yasuyuki, 2016; Cottichio et al., 2018). These careful analyses allow us to describe typical and atypical events of fertilization and how they are each associated with timing of the first cleavage division and subsequent embryo development. In this lecture, we will first describe the fundamental underpinnings of fertilization and highlight the normal events associated with this process. We will then discuss gross morphological abnormalities as visualized by light microscopy and highlight the unknowns associated with these events. Finally, we will focus on time-lapse imaging studies, which have revealed the remarkable spatial and temporal coordination of meiotic resumption, pronuclear dynamics, chromatin organization and cytoplasmic/cortical modifications that occur during fertilization and the implications of aberrations for the first cleavage division. At the conclusion of this presentation, attendees should be able to: Review the normal events associated with fertilization and the first cleavage division. 1 Describe gross morphological aberrations of these two fundamental processes. 2 Discuss temporal and spatial abnormalities in the coordinated sequence of events that underly these processes. 3 State the potential application of these abnormalities as predictors of abnormal embryo development. 4 Summarize the puzzling unknowns that underly these abnormalities.

P–210 Abnormal cleavage patterns during embryo preimplantation development and their effect on blastulation: an overview from IVF patients with multiple IVF cycles in a time-lapse incubator

Abstract Study question How common abnormal cleavage patterns (ACP) are in IVF and what are their consequences on embryo developmental competence? Summary answer ACP might affect up to 25% of the 2PN-zygotes, independently from patients’/cycles’ characteristics, and mostly cause embryo developmental arrest around the 4-to–8-cell transition. What is known already Since its implementation in IVF, time-lapse-microscopy (TLM) allowed the standardization of embryo culture within undisturbed incubators, but it has not improved embryo selection especially if blastocyst transfer is performed. Nevertheless, TLM holds the potential for boosting our knowledge of embryo preimplantation development. In particular, a continuous observation of embryo morpho-dynamics unveiled peculiar blastomere cleavage patterns previously unidentifiable with a static morphological assessment. These events are possibly associated with massive mitotic errors, affecting both chromosomes and cytoskeletal components, as well as downstream metabolic imbalances. Still, the causes of ACP and their consequences on embryo developmental/reproductive competence require further investigation. Study design, size, duration Observational study including 75 patients (age:38.6±3.7yr, FSH:8.8±3.6IU/l, AMH:1.7±1.3ng/ml; BMI:21.4±2.4) who conducted multiple IVF cycles (N = 160; 8.7±5.0 cumulus-oocyte-complexes and 6.3±3.6 metaphase-II collected; 201±245 days between first and second cycles) in a time-lapse incubator between 2014–2020. All annotations were performed blindly by two operators and confirmed by a third in case of discordance. The outcomes were the blastulation rate after any ACP, their association between each other and with patients’/cycles’ characteristics. Participants/materials, setting, methods We included only ICSI-cycles after ovarian-stimulation with blastocyst culture conducted in the Embryoscope. Overall, 981 metaphase-II were inseminated and 677 2PN-zygotes annotated. The ACP investigated were: (i)cytokinesis-failure, formation of cytoplasmic septa without cell division; (ii)Chaotic-cleavage, disordered and uneven cleavages; (iii)Direct-unequal-cleavage (DUC), cleavage of zygotes or single blastomeres directly into 3; (iv)Rapid-cleavage, t3-t2&amp;lt;5hr; (v)Reverse-cleavage, fusion of 2 blastomeres into 1; (vi)Fragmentation, presence of numerous non-nucleated fragments; (vii)Blastomeres’ exclusion/extrusion, nucleated cells excluded/extruded from the morula. Main results and the role of chance Among the 2PN-zygotes, the prevalence of cytokinesis-failure was 5.9% (N = 40/677), 15.7% for chaotic-cleavage (N = 106/677), 18.6% for DUC (N = 126/677), 4.1% for rapid-cleavage (N = 28/677), 3.5% for reverse-cleavage (N = 24/677) and 24.1% for fragmentation (N = 163/677). Among the morulae, the prevalence of blastomere exclusion/extrusion was 27% (N = 109/410;1.5±1.2 excluded/extruded cells,range:1–7). The risk for reverse-cleavage was higher among 2PN-zygotes facing failed-cytokinesis (N = 8/40,20% versus N = 16/637,2.5%, OR:9.7,95%CI:3.9–24.3,p&amp;lt;0.01). Fragmentation was instead higher among 2PN-zygotes undergoing chaotic cleavage (N = 47/106,44.3% versus N = 116/571,20.3%, OR:3.1,95%CI:2–4.8,p&amp;lt;0.01) or DUC (N = 46/126,36.5% versus N = 117/551,21.2%, OR:2.1,95%CI:1.4–3.2,p&amp;lt;0.01). Lastly, higher prevalence of blastomeres’ exclusion/extrusion were reported among morulae obtained after chaotic-cleavage (N = 17/29,58.6% versus N = 92/381,24.1%, OR:4.4,95%CI:2–9.7,p&amp;lt;0.01), DUC (N = 26/37,70.3% versus N = 83/373,22.3%, OR:8.3,95%CI:3.9–17.4,p&amp;lt;0.01) and in presence of fragmentation (N = 79/195,75.2% versus N = 30/305,9.8%, OR:27.8,95%CI:15.6–49.8,p&amp;lt;0.01); only a higher trend after rapid-/reverse-cleavage. No predictive factor of ACP was identified among patients’ and cycles’ characteristics, except for higher risks of fragmentation (OR:2.6,95%CI:1.1–6.3,p= 0.04) and blastomeres’ exclusion/extrusion (OR:2.7,95%CI:1.1–7.2,p=0.04) among patients with previous experience with these events. The viable-blastocyst rate per 2PN-zygote was 45.1% (N = 305/677). It was lower in case of failed-cytokinesis (N = 12/40,30% versus N = 293/637,46%, OR:0.5,95%CI:0.25–0.99,p=0.05), chaotic cleavage (N = 20/106,18.9% versus N = 285/571,49.9%, OR:0.23,95%CI:0.14–0.39,p&amp;lt;0.01), DUC (N = 27/126,21.4% versus N = 278/551,50.5%, OR:0.27,95%CI:0.17–0.42,p&amp;lt;0.01), rapid-cleavage (N = 6/22,21.4% versus N = 299/649,46.1%, OR:0.32,95%CI:0.13–0.8,p=0.02), and reverse-cleavage (N = 5/19,20.8% versus N = 300/653,45.9%, OR:0.31, 95%CI:0.11–0.84,p=0.02). No difference was instead shown in case of fragmentation and/or blastomeres’ exclusion/extrusion. Limitations, reasons for caution The patients included were poor-prognosis women undergoing ≥2 cycles. We are expanding the sample size to account for all cycles conducted in time-lapse incubators. Larger sample size will provide also statistical-power to investigate the effect of ACP on blastocysts’ chromosomal and implantation competence, and more visualizations of rapid-/reverse-cleavage events. Wider implications of the findings: After ACP,developmental-arrest mostly occurs around the 4-to–8-cell transition (50–70% versus ∼30%), when embryonic-genome-activation takes place. Surviving embryos often fragment and/or exclude/extrude blastomeres at morulation, without further impact on blastulation-rates. Moreover, ACP seem independent from patients’/cycles’ characteristics. These evidence incite future Research on the biological/genetic mechanisms triggering ACP and their consequences. Trial registration number None

Early embryo development anomalies identified by time-lapse system: prevalence and impacting factors

Research questionWhat is the prevalence of embryo abnormal early cleavage (ACL) identified by time lapse and factors related to patients and treatment that explain ACL occurrence? DesignA single-centre, retrospective cohort study. Data were collected on all IVF cycles for which embryos were observed in the EmbryoScope® between December 2015 and August 2017. Only diploid zygotes cleaved on day 2 were included. The study included 318 cycles (250 couples and 1343 embryos). Embryo videos were retrospectively analysed for ACL. The prevalence of each type of ACL was recorded. The influence of clinical factors (whether they were intrinsic to patients or specific to IVF treatment) on ACL occurrence was analysed in multivariate multilevel mixed-effect logistic regression analysis. ResultsA high prevalence of ACL was observed: 37.6% (505/1343) of embryos presented at least one ACL, 22.8% (306/1343) a trichotomous mitosis, 25.8% (347/1343) a rapid cleavage, 6.7% (90/1343) a cell fusion and two or more ACL (16.1%). Part of the variation (12–25%) in ACL occurrence could be explained by embryo origin. Trichotomous mitosis and two or more ACL phenotypes were less likely to occur in women with endometriosis or tubal pathology and tubal pathology alone, respectively. No factor related to IVF cycles was found to be statistically associated with ACL occurrence. ConclusionsOur findings emphasize the importance of considering embryo origin when interpreting studies focusing on embryo characteristics and factors that could affect their quality. The present study is limited by a small sample size of known embryo implantations and monocentric criterion.

Role of Monoamine Oxidase Activity in Alzheimer's Disease: An Insight into the Therapeutic Potential of Inhibitors.

Despite not being utilized as considerably as other antidepressants in the therapy of depression, the monoamine oxidase inhibitors (MAOIs) proceed to hold a place in neurodegeneration and to have a somewhat broad spectrum in respect of the treatment of neurological and psychiatric conditions. Preclinical and clinical studies on MAOIs have been developing in recent times, especially on account of rousing discoveries manifesting that these drugs possess neuroprotective activities. The altered brain levels of monoamine neurotransmitters due to monoamine oxidase (MAO) are directly associated with various neuropsychiatric conditions like Alzheimer’s disease (AD). Activated MAO induces the amyloid-beta (Aβ) deposition via abnormal cleavage of the amyloid precursor protein (APP). Additionally, activated MAO contributes to the generation of neurofibrillary tangles and cognitive impairment due to neuronal loss. No matter the attention of researchers on the participation of MAOIs in neuroprotection has been on monoamine oxidase-B (MAO-B) inhibitors, there is a developing frame of proof indicating that monoamine oxidase-A (MAO-A) inhibitors may also play a role in neuroprotection. The therapeutic potential of MAOIs alongside the complete understanding of the enzyme’s physiology may lead to the future advancement of these drugs.

Sperm DNA fragmentation measured by sperm chromatin dispersion impacts morphokinetic parameters, fertilization rate and blastocyst quality in ICSI treatments.

To determine the effects of sperm DNA fragmentation (SDF) on embryo morphokinetic parameters, cleavage patterns and embryo quality, this retrospective study analyzed 151 intracytoplasmic sperm injection (ICSI) cycles (1152 embryos collected) between November 2016 and June 2019. SDF was assessed using sperm chromatin dispersion. The cycles were divided into two groups based on the SDF rate: SDF < 15% (n = 114) and SDF ≥ 15% (n = 37). The embryo morphokinetic parameters, cleavage patterns, and embryo quality were compared between the two groups. The morphokinetic parameters tPNf, t2, t3, t4, t5, t6, and t8 were achieved significantly earlier in the SDF < 15% group compared with in the SDF ≥ 15% group. The fertilization and 2PN rates seemed to be significantly higher in the SDF < 15% group compared with in the SDF ≥ 15% group, while the abnormal cleavage rates were similar. However, a significantly higher rate of chaotic cleavage (CC) was observed in the SDF ≥ 15% group. The D3 high-quality embryo and available embryo rates were similar between the two groups. The blastocyst formation, high-quality blastocyst, and available blastocyst rates in the SDF < 15% group were significantly higher than those in the SDF ≥ 15% group. With an increase in SDF level, the chemical pregnancy, clinical pregnancy and implantation rates tended to decrease, while the miscarriage rate increased. This study demonstrated that SDF ≥ 15% reduces the fertilization rate of ICSI cycles and affects certain morphokinetic parameters. A higher SDF level can also induce a higher rate of CC, with subsequent decreases in the blastocyst formation rate and blastocyst quality.

Abnormal cleavage is involved in the self-correction of bovine preimplantation embryos

Chromosome instability leading to aneuploidy during early cleavage is well known in humans and cattle. Partial compaction (PC), which occurs only in some blastomeres, is suggested as a self-correction mechanism through which human embryos avoid aneuploid mosaicism. Partially compacted embryos show abnormal cleavages more frequently during early development; however, the mechanism by which blastomeres are excluded has not been elucidated. Here, we confirmed PC in approximately half of the tested bovine embryos, similar to that in human embryos. DNA sequencing of single-cell and intact embryos revealed that the morulae that excluded some blastomeres had euploidy, but many of the excluded blastomeres had aneuploidy. Time-lapse imaging of zygotes without the zona pellucida revealed that the excluded blastomeres underwent reverse and direct cleavages, which are abnormal cleavages, more frequently than the blastomeres involved in compaction. These results suggest the potential role of abnormal cleavage in the self-correction mechanism during the development of mammalian preimplantation embryos.

Morphokinetic evaluation of embryos generated from vitrified oocytes maintaining the meiotic spindle

Vitrification is a technique for preservation of human oocytes. There is still a lack of basic research about the possible effects of vitrification on subsequent embryos following oocyte vitrification. The purpose of this study was to evaluate the embryo morphokinetic parameters formed after fertilization of vitrified-warmed oocytes, where an intact meiotic spindle (MS) was observed pre- and post-cryopreservation. Matured oocytes after in vitro maturation were collected and MS evaluation was performed. The oocytes with MS were divided into two groups: fresh and post vitrification. After intra-cytoplasmic sperm injection, the oocytes were cultured in time lapse monitoring (TLM) and time of second polar body extrusion (SPBE), pronuclei appearance (tPNA), pronuclei fading (tPNF), formation of two to eight cells (t2 to t8), and irregular cleavage events [direct cleavage (DC), reverse cleavage (RC)] and vacuolation were assessed. The fertilization rate was not significantly different between the groups, although the rate of abnormal fertilization was higher in vitrification group compared with fresh group (23.5% VS 7.7%). Analysis of the TLM showed a significant delay in time points, including SPBE, tPNA, tPNF, t 2-cells cleavage in vitrification group (p = 0.02, p = 0.00, p = 0.002, P = 0.00, P = 0.01, respectively). In addition, t3 and t4 time points tended to be delayed in vitrification group (p = 0.05). Moreover, the higher level of DC, RC and vacuolation were noticed in the vitrification group (P˂0.05). In conclusion, despite MS maintenance after warming, TLM evaluation showed both a delay and abnormal cleavage patterns in generated embryos.