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

Abstract Study question Can deep learning (DL) algorithms trained on time-lapse videos be used to detect and track the size and gender of pronuclei in developing human zygotes? Summary answer Our DL algorithm not only outperforms state-of-the-art models in detecting the pronuclei but can also accurately identify and track its gender and size over time. What is known already Recent researches have explored the use of DL to extract key morphological features of human embryos. Existing studies, however, focus either on blastocysts’ morphological measurements (Au et al. 2020) or on embryos’ general developmental stages classification (Gingold et al. 2018, Liu et al. 2019, Lau et al. 2019). So far, only one paper attempted to evaluate zygotes’ morphological components but stopped short of identifying the existence and location of their pronuclei (Leahy et al. 2020). We address this research gap by training a DL model that can detect, classify the gender, and quantify the size of zygotes’ pronuclei over time. Study design, size, duration A retrospective analysis using 91 fertilized oocytes from infertile patients undergoing IVF or ICSI treatment at Hanabusa Women’s Clinic between January 2011 and August 2019 was conducted. Each embryo was time-lapse monitored using Vitrolife which records an image every 15 minutes at 7 focal planes. For our study, we used videos of the first 1–2 days of the embryo from its 3 central focal planes, corresponding to 70–150 images per focal plane. Participants/materials, setting, methods All 273 timelapse videos were split into 30,387 grayscale still images at a 15-minute interval. Each image was checked and annotated by experienced embryologists where every pixel of the image was classified into 3 categories: male pronuclei, female pronuclei, and others. Images were converted into grayscale, resized into 500x500 pixels, and then fed into a neural network with the Mask R-CNN architecture and a ResNet101 backbone to produce a pronuclei instance segmentation model. Main results and the role of chance The 91 embryos were split into training (∼70% or 63 embryos) and validation (∼30% or 28 embryos). Our pronuclei model takes as input a single image and outputs a bounding box, mask, category, confidence score, and size measured in terms of pixel for each detected candidate. For prediction, we run the model on the 3 middle focal planes and merge candidates by using the one with the highest confidence score. We used the mean-average precision (mAP) score to evaluate our model’s ability to detect pronuclei and used the mean absolute percentage error (MAPE) between the actual size (as annotated by the embryologist) and the predicted one to check the model’s performance in tracking the pronuclei’s size. The mAP for detecting pronuclei, regardless of its gender, achieved by our model was 0.698, higher than the 0.680 value reported in the Leahy et al. paper (2020). Breakdown by gender, our model’s mAP for male and female pronuclei are 0.734 and 0.661 respectively. The overall MAPE for tracking pronuclei’s size is 21.8%. Breakdown by gender, our model’s MAPE for male and female pronuclei are 19.4% and 24.3% respectively. Limitations, reasons for caution Samples were collected from one clinic with videos recorded from one time-lapse system which can limit our results’ reproducibility. The accuracy of our DL model is also limited by the small number of embryos that we used. Wider implications of the findings: Even with a limited training dataset, our results indicate that we can accurately detect and track the gender and the size of zygotes’ pronuclei using time-lapse videos. In future models, we will increase our training dataset as well as include other time-lapse systems to improve our models’ accuracy and reproducibility. Trial registration number Not applicable

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

Human Reproduction ◽

10.1093/humrep/deab130.190 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

L S Orevich ◽

K Watson ◽

K Ong ◽

I Korman ◽

R Turner ◽

...

Keyword(s):

Logistic Regression ◽

Live Birth ◽

Polar Body ◽

Relative Size ◽

Time Lapse ◽

Area Measurement ◽

Size Difference ◽

Male And Female ◽

Stage 1 ◽

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 <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 < 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 > 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 <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

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P–561 Male and female blastocyst: any difference other than the sex?

Human Reproduction ◽

10.1093/humrep/deab130.560 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

B Carrasc. Canal ◽

M C Pons ◽

M Parriego ◽

M Boada ◽

S García ◽

...

Keyword(s):

Embryo Transfer ◽

Sperm Quality ◽

Time Lapse ◽

Blastocyst Stage ◽

Study Data ◽

Single Step ◽

Chromosomal Anomalies ◽

Low Oxygen ◽

Male And Female ◽

Normal Copy Number

Abstract Study question Is there any imbalance in the sex ratio (SR) and in the aneuploidy rate of male and female human blastocysts from a PGT-A programme? Summary answer Although SR in human blastocysts is significantly male-biased, more aneuploidies are observed among male blastocysts, resulting in comparable euploid male and female embryos available. What is known already More boys than girls are born worldwide, meaning that the SR at birth is biased towards males. Differences in the SR of children born after ART have been also reported. Factors such as the insemination technique or the day of embryo transfer have been shown to be related to the SR at birth, but whether the SR is shifted during the preimplantation and/or postimplantation development remains unknown. Study design, size, duration: Embryos from patients undergoing 921 PGT-A cycles from September 2017 to February 2020 were included in the study. Data from the chromosomal constitution of 2637 biopsied blastocysts was retrospectively analysed. Participants/materials, setting, methods Embryos were cultured in time-lapse incubators with low oxygen tension (5%) (Embryoscope®; Geri®) using single-step medium (Global®, LifeGlobal®; GTL™, Vitrolife). Blastocyst biopsy was performed between D5-D7 followed by immediate vitrification (Cryotop®, Kitazato). Trophectoderm samples were analysed by NGS. Embryos were categorized as euploid, aneuploid or mosaic. Embryos were called as mosaic when the deviation from the normal copy number was ≥30% and <70%. Main results and the role of chance Overall biopsies from 2637 blastocysts were analysed, 1320 on day 5 (50.1%), 1169 on day 6 (44.3%) and 148 on day 7 (5.6%). Sex distribution among the embryos analysed was skewed in favor of male sex with 1401 diagnosed as male (53.1%) and 1236 were female (46.9%), [OR (95%CI):1.13(1.05–1.22)]. As a consequence of this biased SR, more male embryos reached the blastocyst stage and were biopsied both on day 5/6 (708/1320, 53.6% on day 5 and 619/1169, 53% on day 6). Embryos biopsied on day 7 were balanced between sexes with 50% being male and 50% being female. Following biopsy and PGT-A, 1086 (41.2%) of the embryos were classified as euploid, 1349 (51.16%) as aneuploid, and 202 (7.7%) as mosaic embryos. More chromosomal anomalies were observed among male blastocysts when compared to the female ones, 738 (52.7%) vs 611 (49.4%). Similarly, mosaicism was more frequents in male as compared with female blastocysts, 123 (8.8%) vs 79 (6.4%). (P = 0.000). As more aneuploidies are observed among male blastocysts, the final number of available euploid blastocysts for embryo transfer was comparable between sexes (540 male/546 female), [OR (95%CI): 0.99 (0.87–1.11)]. Limitations, reasons for caution This is a retrospective study. Only embryos at the blastocyst stage have been analyzed. Potential confounding factors such as sperm quality or the female age have not been analyzed. No data regarding the SR at birth have been analyzed in these study. Wider implications of the findings: In our study, more male embryos develop to the blastocyst when compared to female ones. It can be hypothesized that female embryos can be more affected by an early arrest at cleavage stages. SR at birth would be expected to be similar as more aneuploidy is observed in male embryos. Trial registration number Not applicable

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Reported Cases of Sexual Assault in Civil Hospital Sanghar

10.53350/pjmhs211592251 ◽

2021 ◽

Vol 15 (9) ◽

pp. 2251-2252

Author(s):

Muhammad Qasim Ahmed Khan Memon ◽

Pardeep Kumar ◽

Nadia Aslam ◽

Ejaz Ahmed Awan ◽

Shabir Ahmed Cheena ◽

...

Keyword(s):

Retrospective Study ◽

Sexual Assault ◽

Study Design ◽

Associated Factors ◽

Time Lapse ◽

Sexual Assaults ◽

Male And Female ◽

Civil Hospital ◽

Assault Victims

Aim: To evaluate the sexual assault against male and female. Study design: Retrospective study Place and duration of study: Civil Hospital Sanghar from 1st January 2020 to 31st December, 2020 Methodology: Forty two cases of sexual assault and recognize the associated factors were enrolled. The age between 6-35 years and either gender were included. Result: There were 28.6% females and 71.4% males, highest victims were among age between 6-18 years was 17(40.5%), illiterate was 23(54.7%), assaulted by a neighbour was 30 (71.4%) and time lapse <1-2 days was 19(45.4%) sexual assaults. Conclusion: Majority of victims were males, assailant relationships of neighbour and illiterate. Keywords: Sexual assault, Victims, Assailants

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P–215 The degree of perivitelline space (PS) at the pronuclear stage affects subsequent embryonic development in human zygotes

Human Reproduction ◽

10.1093/humrep/deab130.214 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

K Yumoto ◽

T Shimura ◽

M Sugishima ◽

M Nakaoka ◽

Y Mio

Keyword(s):

Embryonic Development ◽

Time Lapse ◽

Perivitelline Space ◽

Blastocyst Development ◽

Intact Group ◽

Registration Number ◽

Group 3 ◽

Group 2 ◽

Human Zygotes ◽

Group 1

Abstract Study question Was embryonic development affected by the degree of perivitelline space (PS) at the pronuclear stage in human zygotes? Summary answer Zygotes with a fully surrounding PS showed less cytoplasmic fragmentation and a higher blastocyst development rate (BDR) than zygotes with a partially surrounding PS. What is known already We previously used abnormally-fertilized oocytes (zygotes with three pronuclei; 3PN), donated by ART patients in our clinic who gave written consent for the research. The zona pellucida (ZP) was artificially removed from these oocytes at the pronuclear stage, termed ZP-free culture. The resultant ZP-free 3PN embryos showed less cytoplasmic fragmentation and a higher rate of good-quality embryos (GQE) compared with ZP-intact embryos. Furthermore, in our clinical setting, the rate of GQE and BDR of normally-fertilized embryos were clearly improved by ZP-free culture in patients with recurrent failure of ART treatments due to severe cytoplasmic fragmentation at the early cleavage stage. Study design, size, duration This study included 49 patients who gave written informed consent for our study and were treated with ART in our clinic between March and December 2020. Embryonic development was compared between zygotes with a fully surrounding PS [PS(+)] with those with a partially surrounding PS [PS(-)] at the pronuclear stage. Furthermore, the ZP of PS(-) embryos were artificially removed at the pronuclear stage, and the rate of GQE and BDR were compared with ZP-intact embryos. Participants/materials, setting, methods The degree of PS in 128 zygotes was confirmed by hypertonic preparation using 0.125M sucrose-containing HEPES medium. PS(+) and PS(-) embryos were both cultured as ZP-intact, and the rate of GQE was compared. Furthermore, 223 zygotes were divided into three groups: 1) PS(-)/ZP-intact, 2) PS(-)/ZP-free, and 3) PS(+)/ZP-intact, and cultured in an incubator equipped with time-lapse monitoring up to Day 7, and the rate of GQE, BDR and useable embryos were compared between each groups. Main results and the role of chance The degree of PS was confirmed by a hypertonic preparation (shrinkage of the ooplasm) in 128 normally-fertilized zygotes obtained from 44 cases. There were 86 PS(-) (67.2%) and 42 PS(+) (32.8%) zygotes. The mean maternal age was 35.9 in PS(-) and 40.5 in PS(+) (P < 0.01), and the rate of GQE was significantly higher in PS(+) [64.3% (27/42)] than in PS(-)[38.4% (33/86)] (P < 0.01). In addition, of 223 normally-fertilized zygotes obtained from 41 cases, there were 51 PS(-)/ZP-intact (Group 1), 132 PS(-)/ZP-free (Group 2) and 40 PS(+)/ZP-intact (Group 3) zygotes. The rate of GQE was significantly lower in Group 1 [29.4% (15/51)] compared with Group 2 [59.8% (79/132)] and Group 3 [62.5% (25/40)] (P < 0.01). BDR was also significantly lower in Group 1 [51.3% (10/39)] compared with Group 2 [75.0% (99/132)] and Group 3 [65.0% (13/20) (P < 0.01). Limitations, reasons for caution Although the artificial removal of ZP at the pronuclear stage (ZP-free culture) clearly increased the rate of GQE, embryonic development was not improved in all cases. It seems that this procedure is only effective in embryos with a viable ooplasm. Wider implications of the findings: The degree of PS at the pronuclear stage affects subsequent embryonic development in human zygotes. The artificial removal of ZP at the pronuclear stage (ZP-free culture) helps to suppress fragmentation and leads to an increase in GQE and BDR, and eventually, improves pregnancy rate in cases with severe fragmentation. Trial registration number non

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O-219 Detailed morpho-kinetic analysis of the first cleavage can help in evaluating the viability of direct-cleaved human zygotes

Human Reproduction ◽

10.1093/humrep/deab128.043 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

T Shimura ◽

K Yumoto ◽

M Sugishima ◽

Y Mio

Keyword(s):

Live Birth ◽

Birth Rate ◽

Live Birth Rate ◽

Time Lapse ◽

Cell Group ◽

Group 3 ◽

Group 2 ◽

Time Lapse Imaging ◽

Human Zygotes ◽

Group 1

Abstract Study question Why do some direct-cleaved human zygotes still lead to a live birth? Summary answer Direct-cleaved zygotes which have undergone the 2-cell stage can lead to a live birth, while zygotes cleaved from 1-cell to ≥ 3-cell do not. What is known already In recent years, zygotes that develop from 2-cell to 3-cell within 5 hours after the first cleavage have been evaluated as “direct-cleaved” zygotes, because normal cleavage takes approximately 12 hours to complete. It was reported that their implantation rate was significantly lower than zygotes with normal cleavage pattern, and eliminating direct-cleaved zygotes from transfer could improve the implantation rate. However, some direct-cleaved zygotes at the first cleavage could still lead to a live birth. Few reports have examined the difference between a cleavage from 1-cell to ≥ 3-cell and 2-cell to ≥ 3-cell within 5 hours after the first cleavage. Study design, size, duration A retrospective study involving 2,077 cycles of IVF/ICSI between July 2012 and July 2019. A total of 5,991 normally fertilized zygotes (2PN/2PB) were included. Of those, 3,508 were evaluated as usable good/fair quality embryos on Day2/3, and the rest (n = 2,483) were evaluated as poor quality and rejected from transfer or cryopreservation after 7 days of culture. Of 3,508 usable embryos, 884 were selected based on the availability of results of live birth for this study. Participants/materials, setting, methods Time-lapse imaging (5 slices along Z-axis every 10 minutes) was performed in EmbryoScopeTM. Zygotes were morphokinetically analyzed in detail and classified into four groups by their cleavage patterns: Group1 (1-cell→2-cell); Group 2 (1-cell→3-cell); Group 3 (1-cell→2-cell→≥3-cell within 5 hours after the first cleavage); and Group 4 (1-cell→2-cell→≥5-cell). The proportion, mean maternal age and live birth rate of each group were examined. Main results and the role of chance The proportion of Groups 1-4 was 83.6% (n = 739), 3.8% (n = 34), 5.9% (n = 52), and 6.7% (n = 59), respectively. 0f 884 zygotes examined in this study, the mean maternal age was significantly higher in Group 2 and 4 than in Group 1 (P < 0.05; 37.4±4.9 in Group1, 39.1±5.2 in Group 2, 38.6±6.0 in Group 3, and 38.7±5.1 in Group 4). The rate of confirmed gestational sac was significantly lower in Group 2 and 4 than in Group 1 [P < 0.01; 36.3% (n = 268/739), 0% (n = 0/34), 25.0% (n = 13/52), and 18.6% (n = 11/59) in Groups 1-4, respectively]. Furthermore, the live birth rate was significantly higher in Group 1 than in Groups 2, 3 and 4 [P < 0.01; 28.4% (n = 210/739), 0% (n = 0/34), 13.5% (n = 7/52), and 15.3% (n = 9/59) in Groups 1-4, respectively]. Above all, while zygotes in Group 2 showed no pregnancy and live birth at all, zygotes in Group 3 showed a live birth rate of 13.5%. However, they had a significantly higher miscarriage rate (42.9%, n = 6) compared to zygotes in Group 1 (19.5%, n = 55). Limitations, reasons for caution It is very difficult to capture cleavage patterns by routine observations because the timings of developmental events are different between embryos. A time-lapse imaging and culturing system is essential to solve this problem, however, it cannot visualize the distribution of chromosomes, and no chromosomal analysis was conducted in this study. Wider implications of the findings This study revealed that zygotes previously classified as “direct-cleaved” and eliminated from transfer included viable zygotes which could lead to a live birth. Therefore, it is crucial to optimize the use of time-lapse imaging of human zygotes in order to precisely evaluate the first cleavage. Trial registration number not applicable

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P–137 Male embryos take longer to develop to the blastocysts stage

Human Reproduction ◽

10.1093/humrep/deab130.136 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

J Fraire-Zamora ◽

M Martínez ◽

D García ◽

R Vassena ◽

A Rodríguez

Keyword(s):

Statistical Difference ◽

Small Sample Size ◽

Time Lapse ◽

Study Groups ◽

Blastocyst Stage ◽

Small Sample ◽

Preimplantation Embryos ◽

Specific Difference ◽

Male And Female ◽

Morphokinetic Parameters

Abstract Study question Are there any differences in developmental timings between male and female preimplantation embryos? Summary answer There is a tendency for statistical difference in the time to reach blastocyst stage for male embryos compared to female embryos What is known already Differences in gene expression and metabolic uptake between male and female preimplantation embryos have been found in animal models and humans. These differences could affect the developmental timings of embryos resulting in differences in either sex. Morphokinetic parameters can precisely assess developmental timings. Only a few studies have analyzed morphokinetic parameters between male and female preimplantation embryos and no consensus has been reached on whether there is any sex-specific difference. The objective of this study is to compare morphokinetic parameters between male and female preimplantation embryos to determine any sex-specific developmental differences. Study design, size, duration This is a retrospective study including 102 preimplantation embryos from February 2018 to February 2020. The morphokinetic parameters obtained from time-lapse records of each embryo were: time to pronuclear fading (tPNf), times to 2–8 cells (t2, t3, t4, t5, t6, t7, t8), time to start of blastulation (tSB) and time to full blastocyst stage (tB). A two-tailed Student’s t-test was used to compare morphokinetic parameters between embryo sexes. A p < 0.05 was considered statistically significant. Participants/materials, setting, methods The study included retrospective time-lapse data from preimplantation embryos giving rise to 51 baby boys and 51 baby girls, as seen at birth. This is a single-center study with standardized culture conditions. Embryos in both study groups issued from cycles with donated oocytes. Only elective blastocyst stage single-embryo transfers (SET) on day 5 were assessed. Main results and the role of chance A tendency to statistical difference (p = [0.1–0.05]) was observed for blastocyst-related morphokinetic parameters: tSB (mean time was 89.6±6.3 hours in male embryos vs. 86.9±8.1 hours in female embryos, p = 0.06) and tB (100.2±5.9 hours versus 97.9±6.5 hours, p = 0.07). Male embryos showed an increased average time of 2.7 hours to tSB and 2.3 hours to tB, while no differences were found in the mean times of all the other morphokinetic paraments measured (p > 0.50): tPNf (∼21.8±3.0 hours) t2 (∼24.4±3.2 hours); t3 (∼35.6±3.9 hours); t4 (∼36.6±4.6 hours); t5 (∼46.9±6.0 hours); t6 (∼53.5±7.0 hours); t7 (∼54.1±7.3 hours) and t8 (∼54.1±7.3 hours). This finding suggests a sex-specific difference in reaching blastocyst stages. Limitations, reasons for caution The main limitation of the study is its retrospective nature and the small sample size. We analyzed the data of embryos leading to a live birth (high-quality embryos), therefore, caution should be made when generalizing results to non-implanting embryos (of potentially lower quality). Wider implications of the findings: Sex-specific differences in developmental timings of preimplantation embryos at blastocyst stage, as evidenced by time-lapse data, should be considered to avoid selection biases during embryo transfers in ART clinic. Trial registration number Not applicable

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P–193 First cleavage division perpendicular to the pronuclear axis adversely affects the clinical outcome in human embryos

Human Reproduction ◽

10.1093/humrep/deab130.192 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

M Nakaoka ◽

K Yumoto ◽

T Shimura ◽

Y Mio

Keyword(s):

Pregnancy Rate ◽

Pregnancy Outcome ◽

Mitotic Spindle ◽

Cleavage Plane ◽

Time Lapse ◽

Human Embryos ◽

Cleavage Division ◽

Male And Female ◽

Spindle Poles ◽

Parallel Group

Abstract Study question Does the direction of formation for the first cleavage plane relative to the pronuclear axis affect clinical outcome? Summary answer A first cleavage division perpendicular to the pronuclear axis adversely affects the rate of embryo utilization for transfer or cryopreservation and the pregnancy outcome. What is known already It remains unclear how the first cleavage plane is determined in human embryos. By using time-lapse monitoring, our previous study (presented in ESHRE 2019) suggested that both the axis and locations of male and female pronuclei are involved in determining the first embryonic cleavage plane. Furthermore, by using immunofluorescence analysis, it was also revealed that most analyzed zygotes showed two pericentrin signals aligned around the interface between the male and female pronuclei. Our findings suggest that the pronuclear axis strongly influences the positions of the centrosomes, which become mitotic spindle poles and define the first cleavage plane. Study design, size, duration: From January 2015 to December 2017, time-lapse imaging (EmbryoScope®) of 3397 intracytoplasmic sperm injection (ICSI) oocytes was conducted. Of those, the relationship between the pronuclear axis and the first cleavage plane was analyzed in 607 normally fertilized embryos that cleaved to two cells and were obtained in 2015. Furthermore, of 3397 ICSI oocytes, 749 transferred embryos were classified based on the first cleavage patterns relative to the pronuclear axis, and the pregnancy rate was examined. Participants/materials, setting, methods A straight line connecting the centers of the pronuclei was defined as the 2PN axis. Based on the direction of the first cleavage relative to the 2PN axis, embryos were classified into three groups: parallel, perpendicular and intermediate. Fresh embryos were transferred on Day 2/3 (fresh-ET). Frozen and thawed embryos were transferred on Day 2/3 or Day 5 (F/T-ET). Clinical pregnancy was defined as confirmed gestational sac in the uterine cavity. Main results and the role of chance Of 607 analyzed embryos, 506 produced suitable images and were assigned to one of three groups: parallel (84.4%, n = 427), perpendicular (9.7%, n = 49) and intermediate (5.9%, n = 30). Embryos that formed a cleavage furrow parallel to the 2PN axis were significantly more frequent than others (perpendicular, intermediate) (P < 0.001). The embryo utilization rate for transfer or cryopreservation was significantly lower in the perpendicular group than in the parallel group (30.7% vs. 69.3%, P < 0.01). Furthermore, of 749 transferred embryos, 504 assigned to the parallel and perpendicular groups were selected (n = 470 and n = 34, respectively), and the pregnancy outcome was analyzed. The mean maternal age was not significantly different between groups. The pregnancy rate of embryos was 24.2% (n = 45/186) from fresh-ET and 39.4% (n = 112/284) from F/T-ET in the parallel group, and 0% (n = 0/14) from fresh-ET and 15.0% (n = 3/20) from F/T-ET in the perpendicular group. Regardless of the types of embryo transfer (fresh or F/T), the pregnancy rate was significantly lower in the perpendicular group than in the parallel group (P < 0.01). In addition, one of three patients who became pregnant from the transfer of an embryo in the perpendicular group had a miscarriage. Limitations, reasons for caution Since only ICSI embryos were analyzed in this study, the influence of fertilization methods on subsequent development could not be investigated. Further studies including preimplantation genetic testing for aneuploidy may help determine the reasons why pregnancy rates differ between groups. Wider implications of the findings: We suggest that the 2PN axis is essential for determining the first cleavage plane because it seems to be involved in positioning the mitotic spindle poles. The direction of the first cleavage plane relative to the 2PN axis can be an important indicator for predicting embryo development and pregnancy outcome Trial registration number none

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Sharp Differences in the Timing of Male and Female Spring Arrival in the European Stonechat, Saxicola Rubicola, and the Whinchat, S. Rubetra (Passeriformes, Muscicapidae), in North-Eastern Ukraine

Vestnik Zoologii ◽

10.2478/vzoo-2019-0043 ◽

2019 ◽

Vol 53 (6) ◽

pp. 483-490

Author(s):

M. V. Banik

Keyword(s):

Migratory Birds ◽

Time Lapse ◽

Male And Female ◽

Proximate Cause ◽

North Eastern ◽

Breeding Grounds ◽

Arrival Dates ◽

Spring Arrival ◽

Widespread Phenomenon ◽

The Difference

Abstract Protandry, or the arrival of males prior to females to the breeding grounds is a widespread phenomenon in migratory birds though rarely examined in related species in which its manifestation can vary. European Stonechat and Whinchat are such a pair studied with use of individual marking in North-Eastern Ukraine in 1993–2008. An apparent protandry was found in Whinchat but not in European Stonechat. The difference between the arrival dates of male and female Whinchats (6 days) was significant. The mean time span between territory establishment by a male and subsequent pair formation was 10.6 days. By contrast, 38% of the first records of European Stonechats in spring were those of already paired birds and the difference between arrival dates of both sexes was non-significant. The proximate cause of protandry in Whinchat and its’ absence in European Stonechat seems to be the differences (or the lack thereof) in the onset of spring migration. The time lapse between the start of migration of male and female Whinchats originates at African wintering grounds and is maintained en route. The absence of the protandry in European Stonechat is probably a relict behaviour from the residency. The protandry in migratory populations of this species is yet to be developed.

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