Post Insemination Research Articles

P-514 Unequal pronucleus size is indicative of higher rates of diploidy in 3PN blastocysts

Abstract Study question What proportion of blastocysts developed from 3PN embryos are diploid, and is this associated with the relative size of the pronuclei? Summary answer Most 3PN blastocysts with even-sized pronuclei are triploid, while most 3PN blastocysts with uneven-sized pronuclei are diploid. What is known already Fertilised oocytes with three pronuclei (3PN, or tripronuclear) are generally considered unsuitable for use due to the high likelihood of triploidy, which is not compatible with an ongoing healthy pregnancy. Recent data on 3PNs with uneven-sized pronuclei (micro-3PN, or 2.1PN) indicates that many may actually be diploid if they form a good quality blastocyst. Study design, size, duration Prospective multi-centre study from October 2020 to December 2023. 81 3PN blastocysts from 69 patients were biopsied. Participants/materials, setting, methods ICSI-inseminated embryos were assessed on Day 1 and pronuclear size at 18 h post insemination was measured using EmbryoViewer software. Biopsy was performed at the blastocyst stage and genetic testing was performed using the Human Karyomap-12 SNP array to ascertain ploidy status. Pronuclei were classified as “Equal”, “Unequal” (smallest pronucleus is 61%-75% of the largest), or “Micro” (smallest pronucleus is < 60% of the largest). Main results and the role of chance 94% of the 3PN blastocysts with equal size pronuclei were triploid, and the remaining 6% were complex aneuploid or had genome wide uniparental disomy (n = 36). Of the 3PN blastocysts with unequal pronuclei (n = 19), 42% were triploid, whereas none of the embryos with a micro pronucleus were triploid (n = 26). Limitations, reasons for caution Only 3PN embryos that formed a good quality expanded blastocyst were biopsied. Only ICSI-inseminated embryos were included, not IVF. No 3PN embryos were transferred in the study, so any additional pregnancy and birth risks from diploid 3PN embryos are unknown. Wider implications of the findings Blastocysts that develop from 3PN oocytes with uneven pronuclei have a greater than 80% chance of diploidy and could be considered for clinical use with PGT. More data is needed to inform clinical guidelines. Trial registration number N/A

P-196 Early blastocyst formation on day 4 is associated with improved clinical outcome and advocates for embryo quailty assessment on day 4

Abstract Study question Is early cavitation on day 4 associated with improved clinical outcome? Summary answer Early blastulation on day 4 is a positive predictor of implantation after single blastocyst transfer on day 5. What is known already In order to select the blastocyst with the highest implantation potential for transfer or freezing, several morphological and/or morphokinetic parameters have been proposed. For instance, early compaction on day 3 (D3) or blastocyst expansion on day 5 (D5) have been reported to be relevant when selecting embryos for transfer. However, day 4 has often been overlooked and very few studies focused on early blastulation on day 4 (D4). Indeed, data on early blastulation on day 4 is scarce. It was however recently demonstrated that embryos transferred on day 4 could provide acceptable pregnancy rates. Study design, size, duration This monocentric retrospective cohort study was conducted in all consecutive couples undergoing IVF cycles in 2017-2022 with single blastocyst transfer and observation on day 4. Participants/materials, setting, methods Morphological and morphokinetic assessment was performed by 2 operators at 93±2h, 116±2h (and 140±2h) post insemination, on day 4, 5 (and 6) respectively. A total of 26 507 D4 embryos were included in this study and grouped according to the observation of early cavitation or not : 23 244 were in the D4 control group (absence of early cavitation on D4) and 3263 were in the D4 blastocyst group (observation of early cavitation on D4). Main results and the role of chance The proportion of embryos used clinically for either transfer or freezing on day 5 or 6 was significantly higher in the D4 blastocyst group than in D4 control group (86.7% vs 50.1% respectively, p < 0.05). Concerning pregnancy rate, cinical pregnancy rate was significantly higher in the D4 blastocyst group than in D4 control group (p < 0.05). In the D4 control group, 5051 embryos were transferred and clinical pregnancy rate is 28.7%. In the D4 blastocyst group, 1286 embryos were transferred (fresh or frozen) and clinical pregnancy rate is 40.2%. Limitations, reasons for caution The retrospective and monocentric design calls for caution when considering the generalizability of our findings. Wider implications of the findings We postulate that D4 embryo quality assessment should be widely implemented in any blastocyst program in order to improve embryo quality assessment and subsequently clinical outcome. Trial registration number not applicable

Interferon-Tau in Maternal Peripheral Blood and Its Relationship with Progesterone and Pregnancy-Associated Glycoproteins in the Early Phases of Gestation in Water Buffalo.

The aim of this study was to investigate the interferon tau (IFNt) concentration in the peripheral maternal blood during the early phase of pregnancy in buffalo cows and improve the knowledge on the physiological importance of circulating IFNt, evaluating the possible interaction with pregnancy-associated glycoproteins (PAGs) and progesterone (P4). Blood samples were taken from buffalo cows on day 0 (day of AI), 7, 14, 18, 28, and 40 post insemination for the IFNt, PAG, and P4 analysis and to determine the IFNt mRNA expression. The animals were categorized ex post into Pregnant, Non-pregnant and Embryo mortality groups. The interferon value was influenced by group (p = 0.003), being always higher in pregnant buffalo cows than in non-pregnant ones, while the embryo mortality group showed intermediate values between those for pregnant and non-pregnant animals. The mRNA expression of IFNt was not influenced by groups or any time points. The regression analysis that included IFNt as the independent variable showed that PAGs, from day 18 (p < 0.01), and P4, from day 28 (p < 0.05), were positively associated with IFNt values. The close associations among IFNt, PAGs and P4 demonstrate that all three molecules work together for fetal-placental well-being and pregnancy support. Unfortunately, the great individual variability in circulating IFNt makes this analysis unsuitable for early pregnancy diagnosis.

Dynamics of transcription is affected by oxygen tension and developmental speed during invitro production of bovine embryos.

This study examines the impact of oxygen tension and embryo kinetics on gene transcription dynamics in pathways crucial for embryonic preimplantation development, including lipid metabolism, carbohydrate transport and metabolism, mitochondrial function, stress response, apoptosis and transcription regulation. Bovine embryos were generated invitro and allocated into two groups based on oxygen tension (20% or 5%) at 18 h post insemination (hpi). At 40 hpi, embryos were categorized into Fast (≥4 cells) or Slow (2 cells) groups, resulting in four experimental groups: FCL20, FCL5, SCL20 and SCL5. Embryo collection also occurred at 72 hpi (16-cell stage; groups FMO20, FMO5, SMO20 and SMO5) and at 168 hpi (expanded blastocyst (BL) stage; groups FBL20, FBL5, SBL20 and SBL5). Pools of three embryos per group were analysed in four replicates using inventoried TaqMan assays specific for Bos taurus, targeting 93 genes. Gene expression patterns were analysed using the K-means algorithm, revealing three main clusters: genes with low relative abundance at the cleavage (CL) and 16-cell morula (MO) stages but increased at the BL stage (cluster 1); genes with higher abundances at CL but decreasing at MO and BL (cluster 2); and genes with low levels at CL, higher levels at MO and decreased levels at BL (cluster 3). Within each cluster, genes related to epigenetic mechanisms, cell differentiation events and glucose metabolism were particularly influenced by differences in developmental kinetics and oxygen tension. Fast-developing embryos, particularly those cultured under low oxygen tension, exhibited transcript dynamics more closely resembling that reported invivo-produced embryos.

Evaluation of the STAT1 mRNA expression in peripheral blood cells as a predictive marker of early pregnancy in Awassi ewes

The primary objective of this study was to measure the expression profile of the signal transducer activator transcription 1 (STAT1) in the circulatory leukocytes of non-pregnant/pregnant ewes and test the possibility that it could be useful for detecting early pregnancy. Whole blood was collected from 20 ewes (control) and another 5 samples were taken on days (15, 25, 35, and 50). Also, blood was collected from 30 ewes on day 15 post insemination (PI) and another 10 samples were taken on days (25, 35, and 50) PI. Total RNA was isolated from peripheral blood leukocytes (PBLs), and STAT1 expression levels were determined by quantitative real-time PCR (qRT-PCR). Gene expression for STAT1 mRNA was 5.8-fold and 5.1 times higher in gravid as opposed to non-gravid ewes. The reliable cut-off point &gt; 1.45 was established using the receiver operating characteristic (ROC) curve, and the accuracy (Acc) for detecting pregnancy was 82%, 93%, 70% and 60%, and the sensitivity (Se) was 83%, 80%, 60% and 33% and Specificity (Sp) 95% on days 15, 25, 35 and 50, respectively. The mRNA expression was lower in ewes that control non-pregnant than pregnant ewes on days 15 and 25. So The best period to detect pregnancy was 15-25 days PI.

Changes on corpus luteum structure and progesterone synthesis pathway after hCG or GnRH treatment during the early luteal phase in sheep

This study investigated the effect of hCG or GnRH on structural changes of the corpora lutea (CL) and the regulation of the expression of steroidogenic enzymes involved in P4 secretion in post-ovulatory (po-CL) and accessory CL (acc-CL). Sixty-four ewes were assigned to three groups receiving: 300 IU of hCG (hCG) or 4 µg Buserelin (GnRH) or 1 mL of saline solution (Control) on Day (d) 4 post artificial insemination (FTAI). Laparoscopic ovarian were performed on d 4, 14 and, 21 post-FTAI to determine the numbers of CL. Blood samples were collected for serum LH and P4 analysis. On d 14 post-FTAI, both CL were removed from the ovary to determine large luteal cell (LLC) number and to evaluate the expression of steroidogenic enzymes (HSD3B1, STAR, CYP11A1). Only hCG and GnRH treated ewes generated acc-CL. The LLC in both po- and acc-CL were significantly greater in the hCG group compared to GnRH and Control groups (P<0.05). Overall, hCG group showed the greatest immunodetection of HSD3B1and STAR in both po- and acc-CL (P<0.05). rnRNA expression of HSD3B1, STAR and CYP11A1 in the acc-CL tended to be greater in hCG group than in GnRH group (P<0.1). The LH concentration was increased in GnRH group (P<0.05) and P4 concentration was greater in hCG group compared to the other groups (P<0.05). In conclusion, administration of hCG has a notably impact on acc-CL development and the expression of steroidogenic enzymes compared to GnRH treatment in ewes. This leads to elevated P4 concentration and improved luteal function.

Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes.

Somatic cell nuclear transfer or cytoplasm microinjection has widely been used to produce genome-edited farm animals; however, these methods have several drawbacks which reduce their efficiency. In the present study, we describe an easy adaptable approach for the introduction of mutations using CRISPR-Cas9 electroporation of zygote (CRISPR-EP) in buffalo. The goal of the study was to determine the optimal conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced buffalo zygotes by electroporation. Electroporation was performed using different combinations of voltage, pulse and time, and we observed that the electroporation in buffalo zygote at 20 V/mm, 5 pulses, 3 msec at 10 h post insemination (hpi) resulted in increased membrane permeability and higher knockout efficiency without altering embryonic developmental potential. Using the above parameters, we targeted buffalo POU5F1 gene as a proof of concept and found no variations in embryonic developmental competence at cleavage or blastocyst formation rate between control, POU5F1-KO, and electroporated control (EC) embryos. To elucidate the effect of POU5F1-KO on other pluripotent genes, we determined the relative expression of SOX2, NANOG, and GATA2 in the control (POU5F1 intact) and POU5F1-KO-confirmed blastocyst. POU5F1-KO significantly (p ≤ 0.05) altered the expression of SOX2, NANOG, and GATA2 in blastocyst stage embryos. In conclusion, we standardized an easy and straightforward protocol CRISPR-EP method that could be served as a useful method for studying the functional genomics of buffalo embryos.

Circulatory extracellular vesicle derived miR-195-5p promotes cellular apoptosis and suppresses cell proliferation in the buffalo endometrial primary cell culture

In pregnant animals, communication between the mother and conceptus occurs via extracellular vesicles (EVs) that carry several biomolecules such as nucleic acids (miRNAs, mRNAs), proteins, and lipids. At the time of implantation, the endometrium undergoes several morphological and physiological changes, such as angiogenesis, apoptosis, and cell proliferation regulation at the implantation site, to attain a receptive state. This study was conducted to detect pregnancy-specific miRNAs derived from extracellular vesicles in the systemic circulation of Bubalus bubalis (water buffalo) and to assess their functional significance in the modulation of endometrial primary cells. The extracellular vesicles were isolated from the blood plasma using a precipitation-based method and further characterized by various methods such as Differential light scattering, Nanoparticle tracking assay, Western blot, and transmission electron microscopy. The relative expression of the selected extracellular vesicles associated miRNAs (EV-miRNA) at different intervals (days 15, 19, 25, and 30) post artificial insemination (AI) was analyzed using RT-qPCR, and expression of miR-195-5p was found to be significantly higher (P < 0.01) in pregnant animals on day 19 post AI (implantation window) as compared to day 15 post AI. The elevated expression might indicate the involvement of this miRNA in the maternal-conceptus cross-talk occurring during the implantation period. The KEGG pathway enrichment and Gene Ontology analyses of the miR-195-5p target genes revealed that these were mostly involved in the PI3-Akt, MAPK, cell cycle, ubiquitin-mediated proteolysis, and mTOR signaling pathways, which are related to the regulation of cell proliferation. Transfecting the in vitro cultured cells with miR-195-5p mimic significantly suppressed (P < 0.05) the expression of its target genes such as YWHAQ, CDC27, AKT-3, FGF-7, MAPK8, SGK1, VEGFA, CACAND1, CUL2, MKNK1, and CACAN2D1. Furthermore, the downregulation of the miR-195-5p target genes was positively correlated with a significant increase in the apoptotic rate and a decrease in the proliferation. In conclusion, the current findings provide vital information on the presence of EV miR-195-5p in maternal circulation during the implantation window indicating its important role in the modulation of buffalo endometrium epithelial cells via promoting cell death. Altogether, the milieu of miR-195-5p may serve as a novel and potential molecular factor facilitating the implantation of the early embryo during the establishment of pregnancy in buffaloes. Thus, miR-195-5p may be identified as a unique circulatory EV biomarker related to establishing pregnancy in buffaloes as early as day 19 post-AI.

Effect of varying amounts of linseed oil or saturated fatty acids around insemination on reproductive and blood parameters of ewes.

The current study was designed to evaluate the effect of sequential low and high dietary linseed oil (LO; as omega-3 enriched fatty acid; FA) before and post insemination, respectively, on different plasma variables of ewes. Fat-tailed Qezel ewes were assigned randomly to be fed a diet enriched with 3% LO (n = 30) or the saturated FA (SFA; n = 30) three weeks before insemination (Day 0). The lipogenic diet supplemented with 6% LO or SFA was fed after insemination until Day +21. The control ewes were fed an isocaloric and isonitrogenous diet with no additional FA during the study. Estrus was synchronized by inserting a vaginal sponge (Spongavet®) for 12 days + 500 IU equine chorionic gonadotropin (eCG; Gonaser®), and ewes were inseminated via laparoscopic approach 56-59 h after eCG injection. The size of ovarian structures was assessed by transvaginal ultrasonography at -21, -14, -2, 0, and +10 days. Blood samples were collected weekly to measure the plasma's different biochemical variables and FA profile. Treatment did not affect the amounts of glucose, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, interleukin-10, interleukin-2, and non-esterified FA (p > 0.05). Conversely, concentrations of triglyceride, cholesterol, tumor necrosis factor-alpha, and insulin-like growth factor-1 were higher in SFA-fed ewes relative to control animals (p < 0.05). LO feeding resulted in greater amounts of n-3 FA isomers in plasma, while higher amounts of stearic acid were detected in SFA fed group 0 and +21 (p < 0.05). The number of ovarian follicles and corpora lutea also were not affected by treatment. Other reproductive variables were not affected by treatment except for the reproductive rate. It seems that LO or SFA feeding of fat-tailed ewes peri-insemination period was not superior to the isocaloric non-additional fat diet provided for the control group during the non-breeding season.

The effect of progesterone concentrations during superovulation of Holstein heifers in a randomized trial

The aim of this study was to evaluate the effect of different progesterone (P4) concentrations during the follicular growth on the intensity of estrous expression, ovarian response to the superovulatory treatment, and embryo production and quality in superovulated heifers. A total of 63 Holstein heifers were randomly assigned into 2 experimental groups: Low P4 (n = 31) and High P4 (n = 32). Animals received a pre-synchronization protocol followed by a protocol of superovulation that included the allocated P4 treatment. Activity was monitored continuously by an automated activity monitor, and estrus characteristics (maximum intensity and duration) were recorded. Embryo collection was performed 7 d post artificial insemination (AI). Embryos were counted and graded from good or excellent (1) to degenerated (4). The outcomes of interest were: number and diameter of follicles at the time of AI, ovulation success (confirmed 7 d post-AI), time to estrus event, maximum intensity and duration of estrus, number and quality of embryos. Data were analyzed according to the type of outcome variable using logistic, linear, or Poisson regression models. A total of 105 embryos (High P4: n = 42; Low P4: n = 63) were graded for quality. Different P4 levels did not affect the maximum intensity (High P4 = 497.8 ± 23.9%; Low P4 = 542.2 ± 23.5%) or the duration (High P4 = 13.5 ± 1.5 h; Low P4 = 14.3 ± 1.4 h) of estrus. Heifers in the High P4 treatment had greater number of follicles at time of AI (High P4 = 16.6 ± 1.6 follicles; Low P4 = 13.9 ± 1.2 follicles), but with smaller diameter (High P4 = 11.3 ± 0.1 mm; Low P4 = 12.0 ± 0.1 mm) compared with Low P4. High P4 heifers tended to have better embryo quality compared with Low P4 heifers (odds ratio = 1.98; 95% CI = 0.90-4.35). High P4 heifers had less embryos than Low P4 heifers, but this was modified by the CIDR (intravaginal implant of P4) removal to estrus interval (interval 0-21 h: mean ratio = 1.15, 95% CI = 0.42-1.87; interval 22-46 h: mean ratio = 0.58, 95% CI = 0.27-0.96). Although estrous expression was not associated with embryo quality, as the duration and the maximum intensity of estrous expression increased, the number of embryos recovered 7 d post-AI increased (duration: mean ratio = 1.04; 95% CI = 1.03-1.05; maximum intensity: mean ratio = 1.50; 95% CI = 1.42-1.58). In conclusion, P4 during the follicular growth, and intensity of estrus, are playing a role in regulating the quality and the number of embryos produced by superovulated heifers. This study was supported by contributions from Resilient Dairy Genome Project and the Natural Sciences and Engineering Research Council.

Temporal analysis of circulating nucleic acid in early days of pregnancy in buffalo.

Improving reproductive efficiency in livestock relies mainly on the ability to detect pregnancy quickly and accurately. Recently, circulating nucleic acids (CNAs) have been exploited for prenatal diagnosis in humans and animals. In the current investigation, serum samples were collected from pregnant animals (n = 30) and non-pregnant animals (n = 20) on 0th, 6th, 12th, and 18th day post artificial insemination. Total DNA was isolated from these serum samples. Two CNA tags (Bov-B and ART2A) derived from repetitive sections of the bovine genome were amplified using DNA extracted from serum samples. The expression analysis of these CNAs was done using real-time polymerase chain reaction assay, and copy number of each tag was calculated in pregnant and non-pregnant animals. The average number of copies of Art2A increased approximately threefold (P < 0.01) from day zero of pregnancy (7,000 copies) to the day 18 of pregnancy (> 21,000). Similarly, BovB levels in the pregnant group increased significantly (approximately 2.9-fold) from day zero (93,900 copies) till day 18 (> 2, 72,310 copies) (P < 0.01). There was no significant change observed on the 6th and 12th day of pregnancy and on the 18th day in the non-pregnant animals. In conclusion, based on these findings, the defined cut-off value can distinguish between pregnant and non-pregnant animals with a sensitivity of nearly80% and specificity of nearly70%. It is possible to employ these two CNA tags as biomarkers for early detection of pregnancy in buffaloes.

P-135 No difference in morphokinetic patterns between male and female preimplantation embryos

Abstract Study question Do morphokinetic parameters vary between male and female preimplantation embryos? Summary answer We observed no significant differences in morphokinetic characteristics between male and female preimplantation embryos What is known already Studies across various mammalian species, including humans, suggest that male and female embryos differ in their gene expression and metabolic phenotypes. These variations may inherently affect the timing and duration of key preimplantation events. In the context of ART, such developmental differences may ultimately bias embryo selection, leading to an unbalanced sex ratio in the resulting offspring. To date, only a few studies have explored the relationship between morphokinetic patterns and human embryo sex. While some suggest subtle sex-specific variations in human embryo growth dynamics, results have been conflicting and currently no clear consensus has been reached. Study design, size, duration Retrospective study of morphokinetic data obtained from 175 preimplantation embryos. The study included 175 cycles performed at two IVF centers, between March 2018 and June 2021. Only fresh oocyte donation cycles using intracytoplasmic sperm injection (ICSI) and fresh single embryo transfer (SET) were assessed. Sex of the embryos was obtained from live birth data. Overall, our analysis included 92 female and 83 male embryos. Participants/materials, setting, methods All embryos were cultured in a timelapse incubator in standardized conditions. The morphokinetic parameters assessed included: time to pronuclear fading (tPNf), times to 2–5 cells (t2, t3, t4, t5), time to 8 cells (t8), time to start of blastulation (tSB) and time to full blastocyst stage (tB). All parameters were measured in hours post insemination (hpi). A two-tailed Student’s t-test was used to compare morphokinetics between embryo sexes. A p&amp;lt;0.05 was considered statistically significant. Main results and the role of chance We observed no significant differences in morphokinetic parameters when comparing cycles resulting in female vs. male live births: tPNf (21.8 ± 3.3 vs. 22.3 ± 3.4 hpi; p &amp;gt; 0.39); t2 (24.6 ± 2.5 vs. 25.0 ± 2.5 hpi; p &amp;gt; 0.34); t3 (35.3 ± 3.3 vs. 35.8 ± 3.1 hpi; p &amp;gt; 0.28); t4 (36.3 ± 3.4 vs. 36.9 ± 3.7 hpi; p &amp;gt; 0.20); t5 (47.9 ± 4.7 vs. 48.0 ± 4.8 hpi; p &amp;gt; 0.88); t8 (54.0 ± 6.5 vs. 54.1 ± 6.5 hpi; p &amp;gt; 0.91); tSB (86.3 ± 14.6 vs 85.7 ± 15.5 hpi; p &amp;gt; 0.78) and tB (93.0 ± 16.9 vs. 93.2 ± 17.2 hpi; p &amp;gt; 0.94). These findings suggest that the timing of key developmental events does not vary between male and female ICSI-derived preimplantation embryos. Limitations, reasons for caution The main limitation of this study is its retrospective nature and sample size. We analyzed the data of embryos leading to a live birth; therefore, caution is warranted when generalizing results to non-implanting embryos. Wider implications of the findings Male and female human embryos display similar morphokinetic patterns throughout preimplantation development in our study. Accordingly, embryo selection in a timelapse setting based on developmental characteristics does not bias sex ratios in resulting live births. Trial registration number not applicable

O-307 Aneuploidy in oocytes of women of advanced maternal age: impact on embryo development and evidence for a novel mechanism of meiotic error

Abstract Study question In oocytes of advanced maternal age (AMA) women, what is the association of aneuploidy with embryo development and what are the mechanisms leading to aneuploidy? Summary answer We confirmed precocious separation of sister chromatids as the main cause of aneuploidy. Known chromosome segregation errors explained 90.4% of the observed abnormal chromosome copy numbers in polar bodies. What is known already Meiotic chromosomal aneuploidies in oocytes correlate with AMA (&amp;gt;35 years), and can affect over half of oocytes in this age group. This underlies the rationale for polar body (PB) biopsy as a form of early PGT-A, as performed in the ‘ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis’ (ESTEEM) randomised controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), meiosis II (MII) nondisjunction (ND) and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. Study design, size, duration Data was sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridisation (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs, and on embryo morphology in a subset of embryos, up to day 6 post insemination (dpi), from both the intervention (PB biopsy for PGT-A) and from control (no biopsy) arms. Participants/materials, setting, methods ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (2 pronuclei) in the PB biopsy group and 1082 in the control group were compared using age-adjusted multilevel mixed-effect ordinal logistic regression for each day of embryo scoring and summarized with adjusted odds ratios (aOR) and 95% confidence intervals (CI). Main results and the role of chance Overall, 461/693 PB pairs showed abnormal segregation in 1162/10810 chromosomes. The main observed abnormal segregations were compatible with PSSC in meiosis I (MI) (n = 568/1162; 48,9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%) and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9,6%), we observed a chromosome copy number in PB1 and PB2 that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 dpi compared to the control group, indicating that polar body biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day (using the before-mentioned embryo scoring subcategories), aneuploidy was associated with a decrease in embryo quality on day 3 (aOR 0.62, 95% CI 0.43-0.90), day 4 (aOR 0.15, 95% CI 0.06-0.39) and day 5 (aOR 0.28, 95% CI 0.14-0.58). Limitations, reason for caution RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. Wider implications of the findings Our finding of frequent unexplained chromosome copy numbers in polar bodies indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by missegregation of chromosomes during meiosis I and II. Study funding/competing interest(s) Data was mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. None of the authors have competing interests. This work is dedicated to the memory of Professor Joep Geraedts. Trial registration number NCT01532284 Study funding Yes Funding source Funding by national/international organization(s)

Predicting Cell Cleavage Timings from Time-Lapse Videos of Human Embryos

Assisted reproductive technology is used for treating infertility, and its success relies on the quality and viability of embryos chosen for uterine transfer. Currently, embryologists manually assess embryo development, including the time duration between the cell cleavages. This paper introduces a machine learning methodology for automating the computations for the start of cell cleavage stages, in hours post insemination, in time-lapse videos. The methodology detects embryo cells in video frames and predicts the frame with the onset of the cell cleavage stage. Next, the methodology reads hours post insemination from the frame using optical character recognition. Unlike traditional embryo cell detection techniques, our suggested approach eliminates the need for extra image processing tasks such as locating embryos or removing extracellular material (fragmentation). The methodology accurately predicts cell cleavage stages up to five cells. The methodology was also able to detect the morphological structures of later cell cleavage stages, such as morula and blastocyst. It takes about one minute for the methodology to annotate the times of all the cell cleavages in a time-lapse video.

Effects of fetal bovine serum on trophectoderm and primitive endoderm cell allocation of in vitro-produced bovine embryos.

Supplementing embryonic culture medium with fetal bovine serum (FBS) renders this medium undefined. Glucose and growth factors present in FBS may affect the results of cell differentiation studies. This study tested the hypothesis that FBS supplementation during in vitro culture (IVC) alters cell differentiation in early bovine embryo development. Bovine embryos were produced in vitro and randomly distributed into three experimental groups at 90 h post insemination (90 hpi): the KSOM-FBS group, which consisted of a 5% (v/v) FBS supplementation; the KSOM33 group, with the renewal of 33% of medium volume; and the KSOM-Zero group, without FBS supplementation nor renewal of the culture medium. The results showed that the blastocyst rate (blastocyst/oocytes) at 210 hpi in the KSOM-FBS group was higher than in the KSOM-Zero group but not different from the KSOM33 group. There were no significant changes in metabolism-related aspects, such as fluorescence intensities of CellROX Green and MitoTracker Red or reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+). Immunofluorescence analysis of CDX2 revealed that the lack of FBS or medium supplementation reduced the number of trophectoderm (TE) cells and total cells. Immunofluorescence analysis revealed a reduction of SOX17-positive cell numbers after FBS supplementation compared with the KSOM33 group. Therefore, we concluded that FBS absence reduced blastocyst rates; however, no reduction occurred when there was a 33% volume renewal of the medium at 90 hpi. We also concluded that FBS supplementation altered TE and primitive endoderm cell allocation during early bovine embryo development.

Warming Oocytes with a Universal Warming Medium and Protocol is as Good as the Company Media and Protocols

Background: A universal warming media and protocol would simplify exchange and use of oocytes and embryos between different IVF centres. Aim: The aim of this study was to compare a universal warming medium and protocol (Parmegiani et al, 2014 RBMonline 28, 614-23) with 3 other warming media and protocols which were used with their corresponding vitrification media (2 commercial, one in house) measuring survival (pre and post insemination combined), fertilization and blastocyst rate. Method: This was a prospective study on 300 mature cumulus-free F1 mouse oocytes divided between three vitrification protocols, SAGE™ vitrification (S), RapidVit™ Omni by Vitrolife (V), and the in house ‘MCE-Trehalose’ (M) designed for mouse embryos. Oocytes for each vitrification group (S,V &amp; M) were divided equally to be warmed either by the corresponding warming protocols (S/S, V/V &amp; M/M and or the universal warming protocol (S/U, V/U and M/U). Surviving oocytes were inseminated in vitro with 250,000 motile sperm/ml 2h after warming (18 oocytes were not retrieved post-warm). Results: V/V (25%), V/U (42%) had significantly lower survival than all other groups S/S (72%), S/U (76%), M/M (91%), M/U (82%; P&lt;0.01, Fishers Exact), but no other differences were observed. Fertilization rates [S/S (43%), S/U (40%), V/V (62%), V/U (57%), M/M (59%), M/U (45%)] and blastocyst rates [S/S (61%), S/U (41%), V/V (60%), V/U (75%), M/M (54%), M/U (60%)] did not differ. Conclusion: In conclusion, the universal warming protocol had similar outcomes to the other 3 warming protocols, thus shows promise for warming oocytes vitrified by any vitrification protocol within and between species. Also, demonstrated is that IVF can be used for vitrified oocytes, and thus can provide an option tother than using ICSI clinically, but more studies finding the most suitable clinical media and protocol are needed.

The Role of Transrectal Sonography in Pregnancy Diagnosis in Cattle

Objective: A reproduction management strategy is an essential component of any cattle production operation. Options include palpation per rectum, transrectal sonography, or biochemical blood tests. To determine the best method, veterinary costs must be weighed against operational costs of maintaining open cattle. Materials and Methods: An online literature search using PubMed, Medline, CINAHL, EBSCO, and Google Scholar was completed to find relevant articles regarding pregnancy detection in cattle, costs of reproductive management in cattle, and pregnancy loss in cattle. Priority was given to original research articles pertaining to palpation per rectum, transrectal ultrasound, fetal gender, and pregnancy loss. Results: Transrectal sonography can detect a pregnancy up to 15 days earlier than palpation per rectum and has sensitivities and specificities as high as 97% when performed between days 21 and 35 post artificial insemination. Conclusion: Cattle producers can be confident that transrectal sonography is a useful tool in the beef and dairy cattle industry. Its use facilitates reproductive management decisions, such as re-breeding or culling, thus reducing expenses, and increasing profitability.

Maximizing the reproductive performances of anestrus dairy buffalo cows using GnRH analogue-loaded chitosan nanoparticles during the low breeding season

The present study was designed to evaluate the efficiency of GnRH-loaded chitosan-TPP nanoparticles (GnRH-CsNPs) to improve the reproductive performance of anestrus buffaloes during the summer. Egyptian buffaloes (n = 50) were synchronized for estrus either with the ovsynch protocol (OVSs, n = 20) or with the modified ovsynch protocol (OVSm, n = 30) using GnRH-CsNPs. Sera samples were collected on days 0, 7, and 10 (insemination day); and days15 and 30 post insemination for estradiol (E2) and progesterone (P4) determination. The follicles were monitored on day 0 and day 10. CLs diameter and pregnancy diagnosis were scanned on day 30 post insemination. Using a half dose of GnRH as GnRH-CsNPs in the OVSm improved (P < 0.05) the number of total and large follicles, and the total surface area of subordinate and large follicles compared to the OVSs protocol. The OVSm increased (P < 0.05) the largest follicles diameter on day 10 and CLs diameter on day 30 post insemination compared to the OVSs protocol. E2 concentration was greater (P < 0.05) on insemination day, and P4 concentrations were higher (P < 0.05) on days 15 and 30 post insemination in the OVSm than in the OVSs. The estrus induction rate was 80 % higher (P < 0.05) in the OVSm and cows exhibited estrus within 17.54 h earlier than the OVSs (50 % within 42 h), respectively. The conception rate increased in OVSm compared to OVSs (75 vs. 40 %, respectively). In conclusion, nanofabrication of GnRH allowed to reduce hormonal dose to 50 % without any adverse effects on fertility, and improved ovarian activity and reproductive performance of anestrus buffaloes during the low breeding season.

Performing Early Pregnancy Tests in Milk and Their Effect on Cow Welfare and Reproductive Performance Compared to Rectal Pregnancy Tests 40 to 45 Days Post Insemination

The main pregnancy test method for dairy cows is rectal palpation performed around 40–45 days from last insemination. This is an invasive examination that disrupts the cow’s routine, potentially affecting its well-being. We examined the effectiveness of a commercial kit for performing pregnancy tests on milk samples compared to rectal palpation at 28- and 42-days post insemination. Accordingly, the purpose of the current study was to check if performing early pregnancy test instead of rectal palpation would result in better welfare and improvement of the reproductive performance, at least in part, of the cows. At 28 days, we examined the effect of early pregnancy testing on reproductive performance, especially number of days open. At 42 days, we determined the pregnancy test methods’ effects on cow welfare measures and milk production. For the day 28 experiment, cows on one farm were divided into two groups: one milk-tested for pregnancy on 28 to 30 days after insemination, and the other tested by routine veterinary examination (42 to 45 days after insemination). The milk test resulted in a 16-day reduction in days open for cows identified as non-pregnant on day 28 compared to day 42 (rectal examination), potentially improving reproductive performance on the dairy farm. For the day 42 experiment, test effects on cow welfare as seen in pedometer data and milk production were examined on two large dairy farms. Test-day milk production did not differ significantly between milk-tested and rectally palpated groups, but well-being indices of milk-tested cows improved, especially in winter months. The reason for this can be because, in the summer, cows are interrupted due to colling activities. In addition to fewer days open and economic improvement, early milk tests for pregnancy can free up the practitioner for other tasks on the dairy farm.

LincRNA#1 knockout alone does not affect polled phenotype in cattle heterozygous for the celtic POLLED allele

A long intergenic non-coding RNA (lincRNA#1) is overexpressed in the horn bud region of polled (hornless) bovine fetuses, suggesting a potential role in horn bud suppression. Genome editing was used to test whether the absence of this sequence was associated with the horned phenotype. Two gRNAs with high mutation efficiencies targeting the 5′ and the 3′ regions flanking the lincRNA#1 sequence were co-injected with Cas9 as ribonucleoprotein complexes into bovine zygotes (n = 121) 6 h post insemination. Of the resulting blastocysts (n = 31), 84% had the expected 3.7 kb deletion; of these embryos with the 3.7 kb deletions, 88% were biallelic knockouts. Thirty-nine presumptive edited 7-day blastocysts were transferred to 13 synchronized recipient cows resulting in ten pregnancies, five with embryos heterozygous for the dominant PCPOLLED allele at the POLLED locus, and five with the recessive pp genotype. Eight (80%) of the resulting fetuses were biallelic lincRNA#1 knockouts, with the remaining two being mosaic. RT-qPCR analysis was used to confirm the absence of lincRNA#1 expression in knockout fetuses. Phenotypic and histological analysis of the genotypically (PCp) POLLED, lincRNA#1 knockout fetuses revealed similar morphology to non-edited, control polled fetuses, indicating the absence of lincRNA#1 alone does not result in a horned phenotype.