Nucleolus Precursor Bodies Research Articles

Effects of unequal-sized pronuclei and their origin on embryo development and obstetric outcomes: a time-lapse observational study

Research QuestionDo maternal and paternal pronuclear (PN) sizes and their relative differences affect embryonic development, morphokinetics, and pregnancy outcomes in human embryos? DesignTotally, 2,516 fertilised oocytes with two PNs from 1,207 patients were assessed using a time-lapse culture system. The associations between the PN area immediately before PN breakdown and its relative ratio (PNR) and embryonic, pregnancy, and perinatal outcomes were retrospectively evaluated. Perinatal outcomes were obtained from a self-reported questionnaire. Zygotes were stratified by PNR and origin of the PNs; embryo development, morphokinetics, and morphological alterations were compared among the zygotes. ResultsAreas of maternal and paternal PN were not correlated with embryonic, pregnancy, and perinatal outcomes. Zygotes with a PNR lower than the median (<0.88, unequal-sized PNs) exhibited impaired embryo development (expanded blastocyst; P = 0.0100). Unequal-sized PNs resulted in a prolonged time interval between maternal and paternal PN appearance, decreased nucleolus precursor body (NPB) alignment, and increased incidence of asynchronous PN breakdown, asymmetric division, and multinucleation (P < 0.0001–0.0230). When the paternal PN was smaller than the maternal PN, the decreased NPB alignment, asynchronous PN breakdown, and abnormal cleavage were observed more frequently, resulting in significantly decreased blastocyst formation compared with the zygotes with equal-sized PNs (P < 0.0001–0.0030). ConclusionsZygotes with unequal-sized PNs exhibited impairments in pre-implantation development, particularly when the paternal PN was smaller than the maternal PN, without any adverse effects on maternal and obstetric outcomes. In addition to the number of PNs, evaluating PNR and PN origin would be beneficial when fertilisation is verified.

The kinetics of nucleolar precursor bodies clustering at the pronuclei interface: Positive correlations with the morphokinetic characteristics of cleaving embryos and euploidy in preimplantation genetic testing programs

Objective: This study investigated potential relationships between the kinetics of nucleolar precursor bodies (NPBs) in the pronucleus and developmental morphokinetics and euploidy in human preimplantation genetic testing for aneuploidy (PGT-A) cycles.Methods: The morphokinetic analysis of 200 blastocysts obtained from 53 PGT-A cycles was performed retrospectively in a time-lapse incubator. At the time of pronuclear breakdown (PNBD), we categorized the blastocysts into two groups based on the kinetic degree of clustering NPBs at the interface of the two pronuclei: clustered NPBs (CL) and non-clustered NPBs (NCL). We then compared morphokinetic parameters, abnormal behavioral events, and the rate of aneuploidy between the two groups.Results: Pronuclear fading and the first cleavage occurred earlier in the NCL group than in the CL group. However, the initiation of blastocyst formation and blastocyst expansion was delayed in the NCL group relative to the CL group. No differences were found in the rate of abnormal cleavage events, such as multinucleation at the 2-cell stage, direct cleavage from one to three cells, and from two to five cells between the CL and NCL groups. However, the fragmentation rate at the 8-cell stage was higher in the NCL group than in the CL group (10.3% vs. 1.9%, p&lt;0.05). Additionally, the euploid rate in the CL group was significantly higher than in the NCL group (37.9% vs. 12.4%, p&lt;0.05).Conclusion: These results demonstrate the effectiveness of combining NPB clustering at PNBD with morphokinetics as a parameter for selecting embryos with higher developmental potential in in vitro fertilization.

Essential roles of the nucleolus during early embryonic development: a regulatory hub for chromatin organization.

The nucleolus is the most prominent liquid droplet-like membrane-less organelle in mammalian cells. Unlike the nucleolus in terminally differentiated somatic cells, those in totipotent cells, such as murine zygotes or two-cell embryos, have a unique nucleolar structure known as nucleolus precursor bodies (NPBs). Previously, it was widely accepted that NPBs in zygotes are simply passive repositories of materials that will be gradually used to construct a fully functional nucleolus after zygotic genome activation (ZGA). However, recent research studies have challenged this simplistic view and demonstrated that functions of the NPBs go beyond ribosome biogenesis. In this review, we provide a snapshot of the functions of NPBs in zygotes and early two-cell embryos in mice. We propose that these membrane-less organelles function as a regulatory hub for chromatin organization. On the one hand, NPBs provide the structural platform for centric and pericentric chromatin remodelling. On the other hand, the dynamic changes in nucleolar structure control the release of the pioneer factors (i.e. double homeobox (Dux)). It appears that during transition from totipotency to pluripotency, decline of totipotency and initiation of fully functional nucleolus formation are not independent events but are interconnected. Consequently, it is reasonable to hypothesize that dissecting more unknown functions of NPBs may shed more light on the enigmas of early embryonic development and may ultimately provide novel approaches to improve reprogramming efficiency.

RRNA transcription is integral to phase separation and maintenance of nucleolar structure.

Transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus is necessary for ribosome biogenesis, which is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis results in tissue specific disorders termed ribosomopathies in association with alterations in nucleolar structure. However, how rRNA transcription and ribosome biogenesis regulate nucleolar structure during normal development and in the pathogenesis of disease remains poorly understood. Here we show that homozygous null mutations in Pol I subunits required for rRNA transcription and ribosome biogenesis lead to preimplantation lethality. Moreover, we discovered that Polr1a-/-, Polr1b-/-, Polr1c-/- and Polr1d-/- mutants exhibit defects in the structure of their nucleoli, as evidenced by a decrease in number of nucleolar precursor bodies and a concomitant increase in nucleolar volume, which results in a single condensed nucleolus. Pharmacological inhibition of Pol I in preimplantation and midgestation embryos, as well as in hiPSCs, similarly results in a single condensed nucleolus or fragmented nucleoli. We find that when Pol I function and rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus increases, which disrupts its phase separation properties, leading to a single condensed nucleolus. However, if a cell progresses through mitosis, the absence of rRNA transcription prevents reassembly of the nucleolus and manifests as fragmented nucleoli. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity during development and in the pathogenesis of disease.

O-052 Morphometric and morphokinetic differences in the sperm- and oocyte- originated pronuclei of male and female human zygotes: a time-lapse study

Abstract Study question Do morphometric and morphokinetic profiles of pronuclei (PN) following intracytoplasmic sperm injection (ICSI) vary between male and female human zygotes? Summary answer Male and female zygotes displayed different PN morphometrics and morphokinetics. Additionally, variations were identified between sperm-originated (SPN) and oocyte-originated (OPN) pronuclei. What is known already Previous studies have investigated the use of PN-associated parameters via static observations as indicators of zygote viability, including size equality or juxtaposition. However, recent clinical application of time-lapse videography (TLV) provides a novel opportunity to assess these pronuclear events with greater accuracy and precision of morphometric and morphokinetic measurement. A number of recent TLV studies have also investigated potential live birth prediction by such PN associated measures, however whether or not there are sex associated differences in such measures which could in turn confound live birth prediction is unknown. Study design, size, duration This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles (either fresh or frozen) performed between January 2019 and March 2020. Only ICSI cycles (maternal age &amp;lt;40 years) leading to a singleton live birth (43 males and 51 females) were included for analysis. All oocytes were placed in the EmbryoScope incubator for culture immediately post sperm injection with all annotation performed retrospectively by one embryologist (L-SO). Participants/materials, setting, methods Timings included 2nd polar body extrusion (tPb2), SPN(tSPNa)/OPN(tOPNa) appearance (differentiated by proximity to Pb2) and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2) and 0 hour before PNF (stage 3), measuring PN area (um2), PN juxtaposition, and nucleolus precursor body (NPB) arrangement. Means ± standard deviation were compared using student t test or logistic regression as odds ratio (OR) and 95% confidence interval (CI), and proportional data by chi-squared analysis. Main results and the role of chance Logistic regression indicated that male zygotes had longer time intervals of tPb2_tSPNa than female zygotes (4.8±1.5 vs 4.2±1.0 h, OR = 1.442, 95% CI 1.009-2.061, p = 0.044), but not tPb2_tOPNa (4.7±1.8 vs 4.5±1.3 h, OR = 1.224, 95% CI 0.868-1.728, p = 0.250) and tPb2_tPNF (19.9±2.8 vs 19.1±2.3 h, OR = 1.136, 95% CI 0.957-1.347, p = 0.144). SPN increased in size from stage 1 through 2 to 3 (435.3±70.2, 506.7±77.3, and 556.3±86.4 um2, p = 0.000) and OPN did similarly (399.0±59.4, 464.3±65.2, and 513.8±63.5 um2, p = 0.000), with SPN being significantly larger than OPN at each stage (p &amp;lt; 0.05 respectively). However, relative size difference between SPN and OPN was similar between male and female zygotes at 3 stages (33.6±61.7 vs 38.6±50.8 um2, p = 0.664; 38.5±53.1 vs 45.7±71.9 um2, p = 0.585; 38.4±77.4 vs 45.8±63.9 um2, p = 0.615; respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (77% vs 51%, p = 0.010), stage 2 (98% vs 86%, p = 0.048) and stage 3 (98% vs 86%, p = 0.048). Furthermore, more OPN showed aligned NPBs than in SPN at stage 1 (45% vs 29%, p = 0.023), but similar proportions at stage 2 (64% vs 50%, p = 0.056) and stage 3 (76% vs 72%, p = 0.618). There were no sex associated differences detected in NPB alignment in either SPN or OPN (p &amp;gt; 0.05 respectively). Limitations, reasons for caution The retrospective design does not allow for control of unknown confounders. Sample size is considered relatively small. PN area measurement may not truly represent volume as PN may not be perfectly spherical. Findings were based on women &amp;lt;40 years old so may not apply to older population. Wider implications of the finding These findings augment and extend previous studies investigating PN parameters via static observations. The reported variations between male and female embryos may confound live birth prediction when using pronuclei morphometrics and morphokinetics. Larger scaled studies are warranted to verify these findings. Study funding/competing interest(s) N/A. Trial registration number N/A.

P-204 The pattern of the zygote cleavage to three cells must be considered before embryo transfer

Abstract Study question What is the clinical outcome of instant irregular cleavage (IDC) from zygote to three cells? Summary answer IDC embryos, did not develop to blastocysts and did not acieve pregnancy; FC embryos that reached blastocyst stage exerted similar pregnancy rates as control embryos What is known already Evaluation of the zygote morphology is one of the earliest embryonic stages investigated. Several zygote grading systems were proposed based on pronuclei size and NPB (nucleolus precursor bodies) distribution, thus is an effective indicator of the embryo’s potential to result in a life birth. The first cleavage of a zygote is a highly coordinated event. Disruptive timing of the first cleavage, named direct cleavage (DC, less than 5 hours), is associated with formation of poor embryo quality. Early identification of pathological embryos is challenging and impacts the timing of embryo fate decision in ART. Study design, size, duration A total of 1978 fresh IVF cycles from single unit were analyzed and a total of 4012 embryos were studied. Only 2pn embryos, cultured exclusively in time-lapse system for 5 days were included. The morphokinetics of embryos and their outcome were recorded, from z-score analysis through day 5. We discriminated between IDC, FC and normal cleavage pattern of embryos and compared clinical outcome of various morphokinetic parameters. We also evaluated possible early predictors for IDC. Participants/materials, setting, methods We generated three study groups according to embryo cleavage pattern: (I) Control, normal cleavage (n = 551); (II) FC, zygote to three cells within 5 hours (n = 1587); (III) IDC, instant cleavage from zygote to three cells (n = 922). The association between z-score of 138 IDC embryos and their sibling random controls was thoroughly investigated. All time lapse annotations were performed by senior embryologists. Statistical analysis was performed by SPSS software. Main results and the role of chance IDC embryos were mainly arrested at day 3 and the number of usable embryos (reached blastocyst stage and were suitable for embryo transfer or cryopreservation) was negligible; 4/922 (0.4%). In comparison, the amount of usable FC embryos was 108/1587 (6.6%) and control embryos usage reached 180/551 (32.7%). While the pregnancy rate of control and FC embryos, which reached embryo transfer were similar (40.35% and 42.55%, respectively); transfer of IDC embryos did not result in pregnancy. Additionally, the timetable as measured by time of PN fading and time from fading to first cleavage, differed significantly between the three groups. Therefore, we performed a thorough analysis of zygote morphology data of IDC embryos compared to control sibling embryos in search of early possible markers for these findings. We have not detected significant differences in z-score analysis, reflected by number and size of nucleoli, as well as pronuclear symmetry. Limitations, reasons for caution IDC embryos number used for z-score analysis was limited since many didn’t meet the inclusion criteria (lack of overlapping of the pronuclei, presence of random of control embryo and good quality image). Wider implications of the findings The decision regarding the fate of IDC and FC embryos should include the pattern of first cycle cleavage. Culture IDC and FC embryos for 5 days up to the blastocyst will spare transfer of embryos that are fated to arrest even when their morphological grade on day 3 is acceptable. Trial registration number 0043-22-MMC

Maternal age affects pronuclear and chromatin dynamics, morula compaction and cell polarity, and blastulation of human embryos.

Does maternal ageing impact early and late morphokinetic and cellular processes of human blastocyst formation? Maternal ageing significantly affects pronuclear size and intra- and extra-nuclear dynamics during fertilization, dysregulates cell polarity during compaction, and reduces blastocoel expansion. In ART, advanced maternal age (AMA) affects oocyte yield, fertilization, and overall developmental competence. However, with the exception of chromosome segregation errors occurring during oocyte meiosis, the molecular and biochemical mechanisms responsible for AMA-related subfertility and reduced embryo developmental competence remain unclear. In particular, studies reporting morphokinetics and cellular alterations during the fertilization and pre-implantation period in women of AMA remain limited. A total of 2058 fertilized oocytes were stratified by maternal age according to the Society for Assisted Reproductive Technology classification (<35, 35-37, 38-40, 41-42, and >42 years) and retrospectively analysed. AMA effects were assessed in relation to: embryo morphokinetics and morphological alterations; and the presence and distribution of cell polarity markers-Yes-associated protein (YAP) and protein kinase C-ζ (PKC-ζ)-involved in blastocyst morphogenesis. A total of 1050 cycles from 1050 patients met the inclusion criteria and were analysed. Microinjected oocytes were assessed using a time-lapse culture system. Immature oocytes at oocyte retrieval and mature oocytes not suitable for time-lapse monitoring, owing to an 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 patterns and embryo quality were annotated and compared among groups. Furthermore, 20 human embryos donated for research by consenting couples were used for immunofluorescence. Static microscopic observation revealed that blastocyst formation and expansion were impaired in the 41-42 and >42-year groups (P < 0.0001). The morphological grades of the inner cell mass and trophectoderm were poorer in the >42-year group than those in the <35-year group (P = 0.0022 and P < 0.0001, respectively). Time-lapse microscopic observation revealed a reduction in nucleolus precursor body alignment in female pronuclei in the 41-42 and >42-year groups (P = 0.0010). Female pronuclear area decreased and asynchronous pronuclear breakdown increased in the >42-year group (P = 0.0027 and P < 0.0122, respectively). Developmental speed at cleavage stage, incidence of irregularity of first cleavage, type and duration of blastomere movement, and number of multinucleated cells were comparable among age groups. Delayed embryonic compaction and an increased number of extruded blastomeres were observed in the >42-year group (P = 0.0002 and P = 0.0047, respectively). Blastulation and blastocyst expansion were also delayed in the 41-42 and >42-year groups (P < 0.0001 for both). YAP positivity rate in the outer cells of morulae and embryo PKC-ζ immunoflourescence decreased in the >42-year group (P < 0.0001 for both). At the cellular level, the investigation was limited to cell polarity markers. Cell components of other developmental pathways should be studied in relation to AMA. The study indicates that maternal ageing affects the key functions of embryo morphogenesis, irrespective of the well-established influence on the fidelity of oocyte meiosis. This study was supported by the participating institutions. The authors have no conflicts of interest to declare. N/A.

The migration speed of nucleolar precursor bodies in pronuclei affects in vitro fertilization-derived human embryo ploidy status and live birth.

To study the relationship between clinical outcomes after assisted reproduction and the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN). NPB migration speed, embryo ploidy status, and live birth (LB) were retrospectively analyzed in IVF-derived zygotes. The central coordinates of the mPN, fPN, and NPBs were noted at multiple timepoints. The migration distance of NPBs between two sequential images was measured to calculate NPB migration speed. The NPB migration speeds in mPN and fPN were significantly faster in euploid zygotes than in aneuploid zygotes. In multivariate logistic analysis, NPB migration speed in mPN and the female age were associated with euploidy. The NPB migration speeds in mPN and fPN were also significantly faster in zygotes that led to LB than in zygotes that led to no pregnancy. In a receiver operating characteristic curve analysis of LB by NPB migration speed in mPN, the cut-off value was 3.74 μm/h (AUC: 0.825, 95%CI: 0.688-0.963). When the zygotes were categorized by this cut-off value, there were significantly more LBs in zygotes with migration speed ≥ the cut-off (78.9% vs. 21.1%). Zygotes with quickly migrating NPBs demonstrated the developmental potential to become a baby.

Heterogeneity of nucleolar morphology in four‐cell mouse embryos after IVF: association with developmental potential

AbstractIn mammals, around fertilization, the nucleolus of embryos transforms into the nucleolus precursor bodies (NPBs), which continue to mature until the blastocyst stage, leading to distinct morphological changes. In our study, we observed two types of nucleolar morphology in mouse in vitro fertilized embryos at the four‐cell stage, which we refer to single nucleolus (SN) and multiple nucleoli (MN). To visualize nucleolar morphology, four‐cell embryos were immunostained with anti‐NOPP140 antibody. These embryos were categorized into five types based on the number of blastomeres carrying SN: SN4/MN0, SN3/MN1, SN2/MN2, SN1/MN3, and SN0/MN4, with percentages of 13, 27, 21, 23 and 9, respectively. Next, using a light microscope, we divided the four‐cell in vitro fertilized embryos without fixation into two groups: those with at least two blastomeres displaying SN (SN embryos) and those without (MN embryos). Notably, significantly more SN embryos developed into blastocysts and offspring at 18.5 dpc compared with MN embryos. Furthermore, SN embryos displayed a higher NANOG‐positive cell number at the blastocyst stage, significantly lower body and placental weights, resulting in a higher fetal/placental ratio. These findings suggest a close association between nucleolar state at the four‐cell stage and subsequent developmental potential.

Embryo classification beyond pregnancy: early prediction of first trimester miscarriage using machine learning

PurposeFirst trimester miscarriage is a major concern in IVF-ET treatments, accounting for one out of nine clinical pregnancies and for up to one out of three recognized pregnancies. To develop a machine learning classifier for predicting the risk of cleavage-stage embryos to undergo first trimester miscarriage based on time-lapse images of preimplantation development.MethodsRetrospective study of a 4-year multi-center cohort of 391 women undergoing intra-cytoplasmatic sperm injection (ICSI) and fresh single or double embryo transfers. The study included embryos with positive indication of clinical implantation based on gestational sac visualization either with first trimester miscarriage or live-birth outcome. Miscarriage was determined based on negative fetal heartbeat indication during the first trimester. Data were recorded and obtained in hospital setting and research was performed in university setting.ResultsA minimal subset of six non-redundant morphodynamic features were screened that maintained high prediction capacity. Features that account for the distribution of the nucleolus precursor bodies within the small pronucleus and pronuclei dynamics were highly predictive of miscarriage outcome as evaluated using the SHapley Additive exPlanations (SHAP) methodology. Using this feature subset, XGBoost and random forest models were trained following a 100-fold Monte-Carlo cross validation scheme. Miscarriage was predicted with AUC 0.68 to 0.69.ConclusionWe report the development of a decision-support tool for identifying the embryos with high risk of miscarriage. Prioritizing embryos for transfer based on their predicted risk of miscarriage in combination with their predicted implantation potential is expected to improve live-birth rates and shorten time-to-pregnancy.

Pronuclear Differences Between Male and Female Human Zygotes: A Time-Lapse Study

Background: Recent time-lapse studies have investigated potential live birth prediction by pronuclei (PN) associated morphometric and morphokinetic measures. Whether there is gender associated difference in such measures is unknown. Aim: To determine if morphometric and morphokinetic profiles of PN vary between male and female zygotes. Method: This retrospective study included 94 consecutive autologous single day 5 transfers leading to a singleton live birth (43 males and 51 females) at Monash IVF Gold Coast between January 2019 and March 2020. Timing parameters included 2nd polar body extrusion (tPB2), sperm-originated PN (tSPNa) or oocyte-originated PN (tOPNa) appearance (differentiated by proximity to PB2) and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2) and 0 hour before PNF (stage 3), measuring PN area (um2), PN juxtaposition, and nucleolar precursor bodies (NPB) arrangement. Results: Male zygotes had longer time intervals of tPB2_tSPNa than female (4.8±0.2 vs 4.2±0.1 h, p=0.044), but not tPB2_tOPNa and tPB2_tPNF. SPN increased in size from stage 1 through 2 to 3 (435.3±7.2, 506.7±8.0, and 556.3±8.9 um2, p=0.000) and OPN did similarly (399.0±6.1, 464.3±6.7, and 513.8±6.5 um2, p=0.000), with SPN being significantly larger than OPN at each stage (p[Formula: see text]0.05 respectively). However, their relative size difference (SPN-OPN) was similar between male and female zygotes at all 3 stages. More male than female zygotes reached central PN juxtaposition at stage 1 (76.7% vs 51.0%, p=0.010), stage 2 (97.7% vs 86.3%, p=0.048) and stage 3 (97.7% vs 86.3%, p=0.048). Furthermore, more OPN showed aligned NPBs than in SPN at stage 1 (44.7% vs 28.7%, p=0.023). No gender associated differences were detected in NPB alignment in either SPN or OPN (p[Formula: see text]0.05 for each comparison). Conclusion: Zygotes with different genders display different morphokinetic and morphometric features. Embryo selection using such parameters may lead to unbalanced sex ratio in the resulting offspring.

Morphometric evaluation of two-pronucleus zygote images using image-processing techniques.

Identifying embryos with a high potential for implementation remains a challenge in in vitro fertilization (IVF) cycles. Despite progress in IVF treatment, only a minority of generated embryos has the ability to implant. Another drawback of this practice is the high frequency of multiple pregnancies. This problem leads to economic and health problems. Therefore, the transfer of a single embryo with high implantation potential is the ideal strategy. Morphometric evaluation of two-pronucleus zygote images is a helpful technique when aiming to transfer a single embryo with a high implantation potential. In this study, an automated zygote morphometric evaluation algorithm, called the zygote morphology evaluation (ZME) algorithm, was created to analyze the zygote and provide morphological measurements. The first and most crucial step of the ZME algorithm is the noise reduction step, which was first applied to zygote images. After that, the proposed algorithm detects different parts of the zygote that are indicators of embryo viability and normality, that is the oolemma, perivitelline space, zona pellucida, and nucleolar precursor bodies (NPBs). In addition, a novel dataset was prepared for this task. This dataset consisted of 703 human zygote images, and called the human zygote morphometric evaluation dataset (HZME-DS). Our experimental results in the HZME-DS showed that the ZME algorithm was able to achieve 79.58% average accuracy in identifying the oolemma region, 79.40% average accuracy in determining the perivitelline space, and 79.72% accuracy in identifying the zona pellucida. To calculate the accuracy of identifying NPBs, the proposed algorithm uses Recall and Precision measures, and their harmonic average (F1 measure) reached values of 81.14% and 79.53%, respectively. These encouraging results for our proposed method, which is an automatic and very fast method, showed that the ZME algorithm could help embryologists to evaluate the best zygotes in real time and the best embryos subsequently.

Human 1PN and 3PN zygotes recapitulate all morphokinetic events of normal fertilization but reveal novel developmental errors

Does mono- (1PN) and tri-pronuclear (3PN) fertilization recapitulate the morphokinetic changes of normal bi-pronuclear (2PN) fertilization? Abnormal fertilization retraces the overall choreography of normal fertilization but reveals novel morphokinetic phenomena and raises scientifically and clinically relevant questions. ART has allowed the extracorporeal observation of early human development. Time-lapse technology (TLT) has revealed the complexity of the morphokinetic changes underpinning fertilization and the importance of this process for the genetic and cellular integrity of the embryo. Abnormal fertilization has remained neglected, despite its relevance to the physiology and pathology of early human development. This retrospective study involved TLT observation of normally (2PN, N = 2517) and abnormally (1PN, N = 41; 3PN, N = 27) fertilized oocytes generated in ICSI cycles performed between October 2019 and December 2020. Oocyte retrieval was carried out after clomiphene citrate-based minimal ovarian stimulation. Oocytes of patients with different diagnoses of infertility were included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded. The study included 1231 couples treated for diverse infertility causes. The fraction of male factor cases was substantial (36.1%). Microinjected oocytes were assessed by a combined TLT-culture system. Oocytes not suitable for TLT assessment, owing to an 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 patterns and embryo quality were annotated and compared between groups. Extrusion of the second polar body (PBII) was observed in almost all 2PN/1PN (99.9% and 100.0%, respectively) and in a vast majority of 3PN zygotes (92.1%). Rates of PBII fusion with the ooplasm were much higher in 1PN and 3PN zygotes (P < 0.0001 versus 2PN). The cytoplasmic wave was observed not only in 2PN and 3PN but also in 1PN zygotes (positivity rates of 99.8% and 100% and 82.9%, respectively; P < 0.0001). More rarely, 2PN and 1PN zygotes emitted a third polar body (PBIII). The average times of this event were comparable. The presence and position of the cytoplasmic halo were comparable among the three classes of zygotes. In the 1PN group, the single PN was maternally or paternally derived in 17 and 24 zygotes, respectively, while in the vast majority of 3PN zygotes (121/127) the supernumerary PN was of maternal origin. Average times of maternal PN appearance were comparable, while average times of paternal PN appearance were delayed in 3PN zygotes (P = 0.0127). Compared with the control group, the area of the maternal PN was larger in 1PN zygotes, but smaller in 3PN zygotes (P < 0.0001). The paternal PNs displayed the same trend (P < 0.0001), although such values were consistently smaller than maternal PNs. The area of the third PN in the 3PN group was on average more than 50% smaller than those of maternal and paternal PNs. In maternal PNs of 3PN zygotes, nucleolus precursor bodies (NPBs) aligned along the area of PN juxtaposition at a lower rate compared with the 2PN group. The rate of NPB alignment was ∼50% smaller in 1PN zygotes (P = 0.0001). In paternal PNs, the rates of NPB alignment were not statistically different among the three groups. Asynchronous PN breakdown was increased in 3PN compared with 2PN zygotes (P = 0.0026). In 1PN zygotes, a developmental delay was observed starting from the disappearance of the cytoplasmic halo, reaching 9 h at the time of the first cleavage (P < 0.0001). Higher rates of abnormal cleavage patterns and blastomere fragmentation (P < 0.0001) were observed in 1PN compared to 2N and 3PN zygotes. Cleavage progression was increasingly affected after abnormal fertilization, especially 1PN, finally resulting in blastocyst formation rates of 70.2%, 12.2% and 53.5% in 2PN, 1PN and 3PN embryos, respectively (P < 0.0001). Both maternal and paternal ages were higher in cases involving 3PN fertilization. The study data were obtained from ICSI, but not standard IVF, treatments carried out in a single centre. The study findings therefore require independent verification. This study reports the first detailed morphokinetic map of human abnormal fertilization. Collectively, this evidence prompts new scientific hypotheses and raises clinical questions relevant to the aetiology and the treatment of abnormal fertilization. This study was supported by the participating institutions. The authors have no conflicts of interest to declare. N/A.

P-185 The migration speed of nucleolar precursor bodies in pronuclei affects in vitro fertilization-derived human embryo ploidy status

Abstract Study question Does the migration speed of nucleolar precursor bodies (NPBs) in male and female pronuclei (mPN and fPN) affect in vitro fertilization (IVF)-derived embryo ploidy status? Summary answer The NPB migration speed in mPN impacts the IVF-derived human embryo ploidy status and this indicator could be an attractive marker for noninvasive embryo selection. What is known already NPBs are not considered as simple nucleolar components transmitted from an oocyte to an embryo, and they could participate in genome remodeling during embryo development. NPBs are essential only shortly after fertilization, suggesting that they may actively participate in centromeric chromatin establishment. A previous study demonstrated that NPBs migrated faster in intracytoplasmic sperm injection-derived zygotes having the potential to develop into a blastocyst and eventually into a baby (Inoue et al., 2021). However, the relationship between NPB migration speed and IVF-derived embryo ploidy status is unclear. Study design, size, duration The relationship between the NPB migration speed and embryo ploidy status was retrospectively analyzed in patients with recurrent assisted reproductive technology failure (euploid n =18; aneuploid n =19; and total = 219 NPBs). Archived time-lapse videos (images were recorded every 5 min; Geri+) from incubation after IVF were retrieved after the patients were identified for the study, and the NPB migration speed was analyzed. The retrospective analyses were performed with the patient’s identities masked. Participants/materials, setting, methods mPN and fPN were identified by appearance location in a zygote (fPN appearance is just below the polar bodies). The mPN, fPN, and 2–3 NPBs/PN central coordinates were measured by Kinovea (motion capture software). Their central coordinates were confirmed/revised every image and were decided. The migration distance of NPBs between two sequential images was calculated as the standard of the central PN coordinates. Thereafter, the migration speed of NPBs was calculated. Main results and the role of chance Both NPB speeds were significantly faster in the euploid than in the aneuploid groups (mPN: 4.08±0.61 vs. 3.54±0.54 µm/h, P =0.003, power [1-β]: 0.999, fPN: 4.03±0.89 vs. 3.26±0.45 µm/h, P &amp;lt;0.003, 1-β: 0.987). The NPB speed in mPN was correlated with that in fPN (rs =0.523, P =0.001). The ploidy status was related to the NPB speeds in mPN and fPN (P &amp;lt;0.05) in univariate logistic analysis including male/female ages, ICM/TE grades, and 29 morphokinetic parameters. The factors associated with ploidy status were the NPB speed in mPN (odds ratio [OR], 10.2; 95% confidence interval [CI], 1.90–54.90; P =0.007) and female age (OR, 0.8; 95%CI, 0.64–0.98; P =0.03) in multivariate logistic analysis. The cutoff value for the NPB speeds in mPN and fPN were 3.65 μm/h (specificity, 73.7%; sensitivity, 77.8%; AUC, 0.78; 95%CI, 0.62–0.93) and 3.77 μm/h (specificity, 89.5%; sensitivity, 66.7%; AUC, 0.78; 95%CI, 0.62–0.94). When the zygotes were categorized by their cutoff values, the euploid rate in zygotes with NPB speeds greater than the cutoff value was significantly higher than that in zygotes with the speeds less than the cutoff value (mPN = 73.7% vs. 22.2% [P =0.003]; fPN = 85.7% vs. 26.1% [P &amp;lt;0.001]). Limitations, reasons for caution The NPB migration in the z-axis direction could not be analyzed. NPB tracking could not be performed when NPBs were large in number or drastically moved. Our findings should help in elucidating the relationship, although they did not completely explain the relationship between NPB migration and embryo development. Wider implications of the findings The migration speed of NPBs impacts human embryo ploidy status. NPB migration speed may add clinical value for embryo selection, which may be associated with live birth, and consequently, the time of the live birth could be shorter. The indicator could be an attractive marker for noninvasive embryo selection. Trial registration number Not applicable

Morphometric and morphokinetic differences in the sperm- and oocyte-originated pronuclei of male and female human zygotes: a time-lapse study.

To study the morphometric and morphokinetic profiles of pronuclei (PN) between male and female human zygotes. This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles leading to a singleton live birth. All oocytes were placed in the EmbryoScope + incubator post-sperm injection with all annotations performed retrospectively by one embryologist (L-SO). Timing parameters included 2nd polar body extrusion (tPB2), sperm-originated PN (tSPNa) or oocyte-originated PN (tOPNa) appearance, and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2), and 0h before PNF (stage 3), measuring PN area (um2), PN juxtaposition, and nucleolar precursor bodies (NPB) arrangement. Male zygotes had longer time intervals of tPB2_tSPNa than female zygotes (4.8 ± 0.2 vs 4.2 ± 0.1h, OR = 1.442, 95% CI 1.009-2.061, p = 0.044). SPN increased in size from stage 1 through 2 to 3 (435.3 ± 7.2, 506.7 ± 8.0, and 556.3 ± 8.9 um2, p = 0.000) and OPN did similarly (399.0 ± 6.1, 464.3 ± 6.7, and 513.8 ± 6.5 um2, p = 0.000), with SPN being significantly larger than OPN at each stage (p < 0.05 respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (76.7% vs 51.0%, p = 0.010), stage 2 (97.7% vs 86.3%, p = 0.048), and stage 3 (97.7% vs 86.3%, p = 0.048). More OPN showed aligned NPBs than in SPN at stage 1 only (44.7% vs 28.7%, p = 0.023). Embryos with different sexes display different morphokinetic and morphometric features at the zygotic stage. Embryo selection using such parameters may lead to unbalanced sex ratio in resulting offspring.

EXOSC10/Rrp6 is essential for the eight-cell embryo/morula transition

The conserved 3′-5′ exoribonuclease EXOSC10/Rrp6 is required for gametogenesis, brain development, erythropoiesis and blood cell enhancer function. The human ortholog is essential for mitosis in cultured cancer cells. Little is known, however, about the role of Exosc10 during embryo development and organogenesis. We generated an Exosc10 knockout model and find that Exosc10−/− mice show an embryonic lethal phenotype. We demonstrate that Exosc10 maternal wild type mRNA is present in mutant oocytes and that the gene is expressed during all stages of early embryogenesis. Furthermore, we observe that EXOSC10 early on localizes to the periphery of nucleolus precursor bodies in blastomeres, which is in keeping with the protein's role in rRNA processing and may indicate a function in the establishment of chromatin domains during initial stages of embryogenesis. Finally, we infer from genotyping data for embryonic days e7.5, e6.5 and e4.5 and embryos cultured in vitro that Exosc10−/− mutants arrest at the eight-cell embryo/morula transition. Our results demonstrate a novel essential role for Exosc10 during early embryogenesis, and they are consistent with earlier work showing that impaired ribosome biogenesis causes a developmental arrest at the morula stage.

P–191 Time-lapse videography reveals morphometric and morphokinetic differences in the pronuclei of male and female human zygotes

Abstract Study question Do morphometric and morphokinetic profiles of pronuclei (PN) following intracytoplasmic sperm injection (ICSI) vary between male and female human zygotes? Summary answer Male and female zygotes displayed different PN morphometrics and morphokinetics. Additionally, variations were identified between sperm-originated (SPN) and oocyte-originated (OPN) pronuclei. What is known already Previous studies have investigated the use of PN-associated parameters via static observations as indicators of zygote viability, including size equality or juxtaposition. However, recent clinical application of time-lapse videography (TLV) provides a novel opportunity to assess these pronuclear events with greater accuracy and precision of morphometric and morphokinetic measurement. A number of recent TLV studies have also investigated potential live birth prediction by such PN associated measures, however whether or not there are gender associated differences in such measures which could in turn confound live birth prediction is unknown. Study design, size, duration: This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles (either fresh or frozen) performed between January 2019 and March 2020. Only ICSI cycles (maternal age &amp;lt;40 years) leading to a singleton live birth (43 males and 51 females) were included for analysis. All oocytes were placed in the EmbryoScope incubator for culture immediately post sperm injection with all annotation performed retrospectively by one embryologist (L-SO). Participants/materials, setting, methods Timings included 2nd polar body extrusion (tPb2), SPN(tSPNa)/OPN(tOPNa) appearance (differentiated by proximity to Pb2) and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2) and 0 hour before PNF (stage 3), measuring PN area (um2), PN juxtaposition, and nucleolus precursor body (NPB) arrangement. Means ± standard deviation were compared using student t test or logistic regression as odds ratio (OR) and 95% confidence interval (CI), and proportional data by chi-squared analysis. Main results and the role of chance Logistic regression indicated that male zygotes had longer time intervals of tPb2_tSPNa than female zygotes (4.8±1.5 vs 4.2±1.0 h, OR = 1.442, 95% CI 1.009–2.061, p = 0.044), but not tPb2_tOPNa (4.7±1.8 vs 4.5±1.3 h, OR = 1.224, 95% CI 0.868–1.728, p = 0.250) and tPb2_tPNF (19.9±2.8 vs 19.1±2.3 h, OR = 1.136, 95% CI 0.957–1.347, p = 0.144). SPN increased in size from stage 1 through 2 to 3 (435.3±70.2, 506.7±77.3, and 556.3±86.4 um2, p = 0.000) and OPN did similarly (399.0±59.4, 464.3±65.2, and 513.8±63.5 um2, p = 0.000), with SPN being significantly larger than OPN at each stage (p &amp;lt; 0.05 respectively). However, relative size difference between SPN and OPN was similar between male and female zygotes at 3 stages (33.6±61.7 vs 38.6±50.8 um2, p = 0.664; 38.5±53.1 vs 45.7±71.9 um2, p = 0.585; 38.4±77.4 vs 45.8±63.9 um2, p = 0.615; respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (77% vs 51%, p = 0.010), stage 2 (98% vs 86%, p = 0.048) and stage 3 (98% vs 86%, p = 0.048). Furthermore, more OPN showed aligned NPBs than in SPN at stage 1 (45% vs 29%, p = 0.023), but similar proportions at stage 2 (64% vs 50%, p = 0.056) and stage 3 (76% vs 72%, p = 0.618). There were no gender associated differences detected in NPB alignment in either SPN or OPN (p &amp;gt; 0.05 respectively). Limitations, reasons for caution The retrospective design does not allow for control of unknown confounders. Sample size is considered relatively small. PN area measurement may not truly represent volume as PN may not be perfectly spherical. Findings were based on women &amp;lt;40 years old so may not apply to older population. Wider implications of the findings: These findings augment and extend previous studies investigating PN parameters via static observations. The reported variations between male and female embryos may confound live birth prediction when using pronuclei morphometrics and morphokinetics. Larger scaled studies are warranted to verify these findings. Trial registration number Not applicable

Asymmetrical deposition and modification of histone H3 variants are essential for zygote development.

The pericentromeric heterochromatin of one-cell embryos forms a unique, ring-like structure around the nucleolar precursor body, which is absent in somatic cells. Here, we found that the histone H3 variants H3.1 and/or H3.2 (H3.1/H3.2) were localized asymmetrically between the male and female perinucleolar regions of the one-cell embryos; moreover, asymmetrical histone localization influenced DNA replication timing. The nuclear deposition of H3.1/3.2 in one-cell embryos was low relative to other preimplantation stages because of reduced H3.1/3.2 mRNA expression and incorporation efficiency. The forced incorporation of H3.1/3.2 into the pronuclei of one-cell embryos triggered a delay in DNA replication, leading to developmental failure. Methylation of lysine residue 27 (H3K27me3) of the deposited H3.1/3.2 in the paternal perinucleolar region caused this delay in DNA replication. These results suggest that reduced H3.1/3.2 in the paternal perinucleolar region is essential for controlled DNA replication and preimplantation development. The nuclear deposition of H3.1/3.2 is presumably maintained at a low level to avoid the detrimental effect of K27me3 methylation on DNA replication in the paternal perinucleolar region.

The slippery slope antedating syngamy: pronuclear activity in preparation for the first cleavage.

Nucleolus precursor bodies (NPB) are aggregations of intrapronuclear material observed in the pronuclei of fertilized human eggs. They derive from and evolve into nucleoli of the growing oocyte and the early embryo, respectively. Decades-old observations suggest that the patterns of their distribution represent a morphological marker of embryo developmental competence. Recent time-lapse microscopy (TLM) data now indicate that the vectorial characteristics of NPB movement can predict blastocyst formation, euploidy, and implantation. Since distributions of NPB and chromatin coincide, chromatin rearrangement in preparation for the first mitotic cleavage is emerging as a crucial process of early development.

Migration speed of nucleolus precursor bodies in human male pronuclei: a novel parameter for predicting live birth.

To study the relationship between the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN) and human embryo development during assisted reproduction. The migration speed of 263 NPBs from 47 zygotes was quantitated, and embryonic development was observed until the blastocyst stage. The central coordinates of mPN, fPN, and NPBs were noted at multiple timepoints. Then, the distance traveled by the NPBs between two sequential images was measured, and migration speed was calculated. Additionally, we investigated the relationship between NPB migration speed and ploidy status (N = 33) or live birth/ongoing pregnancy (LB/OP) (N = 60) after assisted reproduction. The NPB migration speed in both mPN and fPN was significantly faster in the zygotes that developed into blastocysts (N = 25) than that in the zygotes that arrested (N = 22). The timing of blastulation was negatively correlated with NPB migration speed in the mPN. Faster NPB migration was significantly correlated with LB/OP. In multivariate logistic analysis, NPB migration speed in the mPN was the only morphokinetic parameter associated with LB/OP. In a receiver-operating characteristic curve analysis of LB/OP by the NPB migration speed in the mPN, the cut-off value was 4.56 μm/h. When this cut-off value was applied to blastocysts with preimplantation genetic testing for aneuploidy, 100% of the blastocysts faster than or equal to the cut-off value were euploid. The NPBs migrated faster in zygotes having the potential to develop into a blastocyst, and eventually into a baby. This predictor could be an attractive marker for non-invasive embryo selection.