ABSTRACT
BACKGROUND: For in vitro fertilization (IVF), mitochondrial DNA (mtDNA) levels in the trophectodermal (TE) cells of biopsied blastocysts have been suggested to be associated with the cells' developmental potential. However, scholars have reached differing opinions regarding the use of mtDNA levels as a reliable biomarker for predicting IVF outcomes. Therefore, this study aims to assess the association of mitochondrial copy number measured by mitoscore associated with embryonic developmental characteristics and ploidy. METHODS: This retrospective study analyzed the developmental characteristics of embryos and mtDNA levels in biopsied trophectodermal cells. The analysis was carried out using time-lapse monitoring and next-generation sequencing from September 2021 to September 2022. Five hundred and fifteen blastocysts were biopsied from 88 patients undergoing IVF who met the inclusion criteria. Embryonic morphokinetics and morphology were evaluated at 118 h after insemination using all recorded images. Blastocysts with appropriate morphology on day 5 or 6 underwent TE biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Statistical analysis involved generalized estimating equations, Pearson's chi-squared test, Fisher's exact test, and Kruskal-Wallis test, with a significance level set at P < 0.05. RESULTS: To examine differences in embryonic characteristics between blastocysts with low versus high mitoscores, the blastocysts were divided into quartiles based on their mitoscore. Regarding morphokinetic characteristics, no significant differences in most developmental kinetics and observed cleavage dysmorphisms were discovered. However, blastocysts in mitoscore group 1 had a longer time for reaching 3-cell stage after tPNf (t3; median: 14.4 h) than did those in mitoscore group 2 (median: 13.8 h) and a longer second cell cycle (CC2; median: 11.7 h) than did blastocysts in mitoscore groups 2 (median: 11.3 h) and 4 (median: 11.4 h; P < 0.05). Moreover, blastocysts in mitoscore group 4 had a lower euploid rate (22.6%) and a higher aneuploid rate (59.1%) than did those in the other mitoscore groups (39.6-49.3% and 30.3-43.2%; P < 0.05). The rate of whole-chromosomal alterations in mitoscore group 4 (63.4%) was higher than that in mitoscore groups 1 (47.3%) and 2 (40.1%; P < 0.05). A multivariate logistic regression model was used to analyze associations between the mitoscore and euploidy of elective blastocysts. After accounting for factors that could potentially affect the outcome, the mitoscore still exhibited a negative association with the likelihood of euploidy (adjusted OR = 0.581, 95% CI: 0.396-0.854; P = 0.006). CONCLUSIONS: Blastocysts with varying levels of mitochondrial DNA, identified through biopsies, displayed similar characteristics in their early preimplantation development as observed through time-lapse imaging. However, the mitochondrial DNA level determined by the mitoscore can be used as a standalone predictor of euploidy.
Subject(s)
Blastocyst , Embryonic Development , Fertilization in Vitro , Time-Lapse Imaging , Humans , Blastocyst/cytology , Female , Retrospective Studies , Time-Lapse Imaging/methods , Adult , Embryonic Development/genetics , Embryonic Development/physiology , Fertilization in Vitro/methods , Pregnancy , DNA, Mitochondrial/genetics , Preimplantation Diagnosis/methods , Aneuploidy , Biopsy , Mitochondria/genetics , DNA Copy Number Variations , Embryo Culture TechniquesABSTRACT
PURPOSE: To study the effectiveness of federated learning in in vitro fertilization on embryo evaluation tasks. METHODS: This is a retrospective cohort analysis. Two datasets were used in this study. The ploidy status dataset consisted of 10,065 embryo records, 3760 treatments, and 2479 infertile couples from 5 hospitals. The clinical pregnancy dataset consisted of 4495 embryo records, 4495 treatments, and 3704 infertile couples from 4 hospitals. Federated learning and the gradient boosting decision tree algorithm were utilized for modeling. RESULTS: On the ploidy status dataset, the areas under the receiver operating characteristic curves of our model trained with federated learning were 71.78%, 73.10%, 69.39%, 69.72%, and 73.46% for 5 hospitals respectively, showing an average increase of 2.5% compared to those of our model trained without federated learning. On the clinical pregnancy dataset, the areas under the receiver operating characteristic curves of our model trained with federated learning were 72.03%, 56.77%, 61.63%, and 58.58% for 4 hospitals respectively, showing an average increase of 3.08%. CONCLUSIONS: Federated learning can improve data privacy and data security and meanwhile improve the performance of embryo selection tasks by leveraging data from multiple sources. This study demonstrates the effectiveness of federated learning in embryo evaluation, and the results show the promise for future application.
Subject(s)
Fertilization in Vitro , Humans , Fertilization in Vitro/methods , Female , Pregnancy , Male , Retrospective Studies , Embryo Transfer/methods , Adult , ROC Curve , AlgorithmsABSTRACT
BACKGROUND: Several studies have demonstrated that iDAScore is more accurate in predicting pregnancy outcomes in cycles without preimplantation genetic testing for aneuploidy (PGT-A) compared to KIDScore and the Gardner criteria. However, the effectiveness of iDAScore in cycles with PGT-A has not been thoroughly investigated. Therefore, this study aims to assess the association between artificial intelligence (AI)-based iDAScore (version 1.0) and pregnancy outcomes in single-embryo transfer (SET) cycles with PGT-A. METHODS: This retrospective study was approved by the Institutional Review Board of Chung Sun Medical University, Taichung, Taiwan. Patients undergoing SET cycles (n = 482) following PGT-A at a single reproductive center between January 2017 and June 2021. The blastocyst morphology and morphokinetics of all embryos were evaluated using a time-lapse system. The blastocysts were ranked based on the scores generated by iDAScore, which were defined as AI scores, or by KIDScore D5 (version 3.2) following the manufacturer's protocols. A single blastocyst without aneuploidy was transferred after examining the embryonic ploidy status using a next-generation sequencing-based PGT-A platform. Logistic regression analysis with generalized estimating equations was conducted to assess whether AI scores are associated with the probability of live birth (LB) while considering confounding factors. RESULTS: Logistic regression analysis revealed that AI score was significantly associated with LB probability (adjusted odds ratio [OR] = 2.037, 95% confidence interval [CI]: 1.632-2.542) when pulsatility index (PI) level and types of chromosomal abnormalities were controlled. Blastocysts were divided into quartiles in accordance with their AI score (group 1: 3.0-7.8; group 2: 7.9-8.6; group 3: 8.7-8.9; and group 4: 9.0-9.5). Group 1 had a lower LB rate (34.6% vs. 59.8-72.3%) and a higher rate of pregnancy loss (26% vs. 4.7-8.9%) compared with the other groups (p < 0.05). The receiver operating characteristic curve analysis verified that the iDAScore had a significant but limited ability to predict LB (area under the curve [AUC] = 0.64); this ability was significantly weaker than that of the combination of iDAScore, type of chromosomal abnormalities, and PI level (AUC = 0.67). In the comparison of the LB groups with the non-LB groups, the AI scores were significantly lower in the non-LB groups, both for euploid (median: 8.6 vs. 8.8) and mosaic (median: 8.0 vs. 8.6) SETs. CONCLUSIONS: Although its predictive ability can be further enhanced, the AI score was significantly associated with LB probability in SET cycles. Euploid or mosaic blastocysts with low AI scores (≤ 7.8) were associated with a lower LB rate, indicating the potential of this annotation-free AI system as a decision-support tool for deselecting embryos with poor pregnancy outcomes following PGT-A.
Subject(s)
Preimplantation Diagnosis , Pregnancy , Female , Humans , Preimplantation Diagnosis/methods , Live Birth , Retrospective Studies , Artificial Intelligence , Genetic Testing/methods , Aneuploidy , BlastocystABSTRACT
Elective single-embryo transfers of euploid or low-level mosaic blastocysts were analyzed in this retrospective study to determine the correlations of live birth (LB) probability with embryonic developmental features of implanted day 5 (D5, n = 245) or day 6 (D6, n = 73) blastocysts using time-lapse (TL) monitoring. According to the logistic regression analyses (adjusted odds ratio [OR] = 0.341, 95% confidence interval [CI] = 0.169-0.685, P < 0.05), the LB probability was negatively associated with the D6 group. The LB rate of the D5 group was higher than the D6 group (88.2% vs. 75.3%; P < 0.05). Compared with the D5 blastocysts, the D6 blastocysts exhibited comparable dysmorphisms except for the multinucleation at the 4-cell stage (10.9% vs. 2.9%, P < 0.05). Moreover, D6 blastocysts had considerably slower developmental kinetics and poorer blastocyst morphologies. Further analysis confirmed that the LB rate was not associated with developmental kinetics or dysmorphisms but rather with blastocyst morphology (inner cell mass [ICM] grade ≤ C vs. ICM grade A, adjusted OR = 0.155, 95% CI = 0.04-0.596, P < 0.05; trophectoderm [TE] grade ≤ C vs. TE grade A, adjusted OR = 0.157, 95% CI = 0.032-0.760, P < 0.05). In conclusion, D6 implanted blastocysts have a considerably lower LB rate than D5 implanted blastocysts. As determined by TL monitoring, the diminished blastocyst morphology can be one of the primary reasons underlying the decreased likelihood of LB.
Subject(s)
Embryo Implantation , Single Embryo Transfer , Pregnancy , Female , Humans , Live Birth/epidemiology , Retrospective Studies , Incidence , BlastocystABSTRACT
OBJECTIVE: To investigate whether the presence of vacuoles in biopsied blastocysts is associated with the likelihood of aneuploidy and clinical outcomes. DESIGN: Retrospective observational study. SETTING: A single reproductive center. INTERVENTION(S): None. PATIENT(S): This study retrospectively analyzed data obtained through preimplantation genetic testing for aneuploidy performed on 3351 blastocysts from 826 patients at a single reproductive center between August 2018 and July 2020. Ultimately, 167 single euploid blastocyst transfers were performed in these patients. Vacuoles existing in the trophectoderm or inner cell mass were observed using blastocyst biopsy. After the biopsy, all blastocysts were vitrified, and embryo transfer was performed in a subsequent treatment cycle. MAIN OUTCOME MEASURE(S): The associations between vacuoles and euploidy or live birth rates were assessed using logistic regression models and estimated adjusted odds ratios (ORs) and 95% confidence intervals (CIs). RESULT(S): Of the 3351 blastocysts from 826 patients, 903 (26.9%) were discovered to have vacuoles. The vacuole-positive group had a significantly lower percentage of euploid blastocysts after TE biopsy than the vacuole-negative group (28.8% vs. 35.5%). Embryos with vacuoles were significantly more likely to be poor quality (30.6% vs. 18.2%). Logistic regression analyses revealed that euploid blastocysts were positively associated with the absence of vacuoles, maternal age, and good embryo quality (vacuole-negative group: adjusted OR 1.291; 95% CI: 1.089-1.530; age <38 years: adjusted OR 1.989; 95% CI: 1.692-2.337; good embryo quality: adjusted OR 1.703; 95% CI: 1.405-2.064). The implantation and live birth rates were significantly lower for the transferred single euploid blastocysts with vacuoles than those without (35.5% vs. 56.6%; 29.0% vs. 52.2%, respectively). The live birth rate was positively associated with the absence of vacuoles (adjusted OR 2.792; 95% CI: 1.180-6.608). CONCLUSION(S): The formation of vacuoles in blastocysts is associated with lower rates of euploidy and live birth. Blastocysts without vacuoles should thus be prioritized for embryo transfer in vitro fertilization cycles.
Subject(s)
Birth Rate , Preimplantation Diagnosis , Pregnancy , Female , Humans , Adult , Vacuoles , Retrospective Studies , Embryo Implantation , Aneuploidy , Blastocyst , Live BirthABSTRACT
This study evaluated whether the concentration of biphasic O2 (5-2%) promotes the formation of qualified blastocysts (QBs) and euploid blastocysts and the probability of cycles with transferable blastocysts. The paired experimental design included a total 90 patients (180 cycles) without euploid blastocysts in previous monophasic O2 (5%) cycles were enrolled for an additional cycle of biphasic O2 (5-2%). In the biphasic O2 (5-2%) group, the QB rate (35.8%, 225/628) was significantly higher than that in the monophasic O2 (5%) group (23.5%, 137/582; p < 0.001). In addition, the euploid blastocyst number (0.5 ± 0.8) and the percentage of cycles with transferable blastocysts were significantly higher in the biphasic O2 (5-2%) group (57.8%, 52/90) than those in the monophasic O2 (5%) group (0 and 35.6%, 32/90, respectively; p < 0.01). Multivariable regression analysis also indicated that the QB rate and the probability of cycles with transferable blastocysts correlated with O2 tension (OR 1.535, 95% CI 1.325-1.777, and OR 3.191, 95% CI 1.638-5.679, respectively; p < 0.001). Biphasic O2 culture can be used as an alternative strategy to increase the euploid QBs and the probability of cycles with transferable blastocysts in patients with a poor prognosis.
Subject(s)
Oxygen , Preimplantation Diagnosis , Humans , Female , Pregnancy , Aneuploidy , Blastocyst , Embryo Culture Techniques , Retrospective StudiesABSTRACT
Continued efforts are made on the development of earth-abundant metal catalysts for dehydrogenation/hydrolysis of amine boranes. In this study, complex [K-18-crown-6-ether][(NO)2Fe(µ-MePyr)(µ-CO)Fe(NO)2] (3-K-crown, MePyr = 3-methylpyrazolate) was explored as a pre-catalyst for the dehydrogenation of dimethylamine borane (DMAB). Upon evolution of H2(g) from DMAB triggered by 3-K-crown, parallel conversion of 3-K-crown into [(NO)2Fe(N,N'-MePyrBH2NMe2)]- (5) and an iron-hydride intermediate [(NO)2(CO)Fe(µ-H)Fe(CO)(NO)2]- (A) was evidenced by X-ray diffraction/nuclear magnetic resonance/infrared/nuclear resonance vibrational spectroscopy experiments and supported by density functional theory calculations. Subsequent transformation of A into complex [(NO)2Fe(µ-CO)2Fe(NO)2]- (6) is synchronized with the deactivated generation of H2(g). Through reaction of complex [Na-18-crown-6-ether][(NO)2Fe(η2-BH4)] (4-Na-crown) with CO(g) as an alternative synthetic route, isolated intermediate [Na-18-crown-6-ether][(NO)2(CO)Fe(µ-H)Fe(CO)(NO)2] (A-Na-crown) featuring catalytic reactivity toward dehydrogenation of DMAB supports a substrate-gated transformation of a pre-catalyst [(NO)2Fe(µ-MePyr)(µ-CO)Fe(NO)2]- (3) into the iron-hydride species A as an intermediate during the generation of H2(g).
ABSTRACT
Background: To identify the correlation among female age, cellular aging markers, and aneuploidy rate in in vitro fertilization (IVF) and the preimplantation genetic test for aneuploidy (PGT-A) cycles. Methods: This is a prospective cohort study recruiting 110 infertile women between August 2017 and July 2018. They were divided into young-age (<38 years, n = 60) and advanced-age (≥38 years, n = 50) groups. Peripheral leukocytes were assessed, and the granulosa cells were pooled during oocyte pickup. Mitochondrial DNA (mtDNA) copy number and telomere length (TL) were measured using real-time polymerase chain reaction. PGT-A was performed on the NGS platform. Results: mtDNA copy number and TL were positively correlated in both leukocytes (rho = 0.477, p < 0.001) and granulosa cells (rho = 0.361, p < 0.001), but the two parameters in leukocytes were not correlated with those in granulosa cells. In the young-age group, TL in the granulosa cells was the only factor correlated with the aneuploidy rate (rho = −0.283, p = 0.044), whereas in the advanced-age group, age was the main factor (rho = 0.358, p = 0.018). Conclusions: TL in the granulosa cells was negatively correlated with the aneuploidy rate in the young-age group, supporting the application of PGT-A in younger women.
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BACKGROUND: For women undergoing in vitro fertilization (IVF), the clinical benefit of embryo transfer at the blastocyst stage (Day 5) versus cleavage stage (Day 3) remains controversial. The purpose of this study is to compare the implantation rate, clinical pregnancy rate and odds of live birth of Day 3 and Day 5 embryo transfer, and more importantly, to address the issue that patients were chosen to receive either transfer protocol due to their underlying clinical characteristics, i.e., confounding by indication. METHODS: We conducted a retrospective cohort study of 9,090 IVF cycles collected by Lee Women's Hospital in Taichung, Taiwan from 1998 to 2014. We utilized the method of propensity score matching to mimic a randomized controlled trial (RCT) where each patient with Day 5 transfer was matched by another patient with Day 3 transfer with respect to other clinical characteristics. Implantation rate, clinical pregnancy rate, and odds of live birth were compared for women underwent Day 5 transfer and Day 3 transfer to estimate the causal effects. We further investigated the causal effects in subgroups by stratifying age and anti-Mullerian hormone (AMH). RESULTS: Our analyses uncovered an evidence of a significant difference in implantation rate (p=0.04) favoring Day 5 transfer, and showed that Day 3 and Day 5 transfers made no difference in both odds of live birth (p=0.27) and clinical pregnancy rate (p=0.11). With the increase of gestational age, the trend toward non-significance of embryo transfer day in our result appeared to be consistent for subgroups stratified by age and AMH, while all analyses stratified by age and AMH were not statistically significant. CONCLUSIONS: We conclude that for women without strong indications for Day 3 or Day 5 transfer, there is a small significant difference in implantation rate in favor of Day 5 transfer. However, the two protocols have indistinguishable outcomes on odds of live birth and clinical pregnancy rate.
Subject(s)
Embryo Implantation , Embryo Transfer/statistics & numerical data , Adult , Embryo Implantation/physiology , Embryo Transfer/methods , Female , Fertilization in Vitro/methods , Fertilization in Vitro/statistics & numerical data , Humans , Live Birth , Pregnancy , Propensity Score , Retrospective Studies , TaiwanABSTRACT
PURPOSE: Our retrospective study is to investigate an end-to-end deep learning model in identifying ploidy status through raw time-lapse video. METHODS: By randomly dividing the dataset of time-lapse videos with known outcome of preimplantation genetic testing for aneuploidy (PGT-A), a deep learning model on raw videos was trained by the 80% dataset, and used to test the remaining 20%, by feeding time-lapse videos as input and the PGT-A prediction as output. The performance was measured by an average area under the curve (AUC) of the receiver operating characteristic curve. RESULT(S): With 690 sets of time-lapse video image, combined with PGT-A results, our deep learning model has achieved an AUC of 0.74 from the test dataset (138 videos), in discriminating between aneuploid embryos (group 1) and others (group 2, including euploid and mosaic embryos). CONCLUSION: Our model demonstrated a proof of concept and potential in recognizing the ploidy status of tested embryos. A larger scale and further optimization on the exclusion criteria would be included in our future investigation, as well as prospective approach.
Subject(s)
Aneuploidy , Deep Learning , Preimplantation Diagnosis/methods , Time-Lapse Imaging/methods , Adult , Area Under Curve , Blastocyst , Calibration , Diploidy , Female , Fertilization in Vitro , Humans , Image Processing, Computer-Assisted/methods , Retrospective StudiesABSTRACT
Avoiding aneuploid embryo transfers has been shown to improve pregnancy outcomes in patients with implantation failure and pregnancy loss. This retrospective cohort study aims to analyze the correlation of time-lapse (TL)-based variables and numeric blastocyst morphological scores (TLBMSs) with different mosaic levels. In total, 918 biopsied blastocysts with time-lapse assessments at a uniform time-point were subjected to next-generation sequencing-based preimplantation genetic testing for aneuploidy. In consideration of patient- and cycle-related confounding factors, all redefined blastocyst morphology components of low-grade blastocysts, that is, expansion levels (odds ratio [OR] = 0.388, 95% confidence interval [CI] = 0.217-0.695; OR = 0.328, 95% CI = 0.181-0.596; OR = 0.343, 95% CI = 0.179-0.657), inner cell mass grades (OR = 0.563, 95% CI = 0.333-0.962; OR = 0.35, 95% CI = 0.211-0.58; OR = 0.497, 95% CI = 0.274-0.9), and trophectoderm grades (OR = 0.29, 95% CI = 0.178-0.473; OR = 0.242, 95% CI = 0.143-0.411; OR = 0.3, 95% CI = 0.162-0.554), were less correlated with mosaic levels ≤20%, <50%, and ≤80% as compared with those of top-grade blastocysts (p < 0.05). After converting blastocyst morphology grades into scores, high TLBMSs were associated with greater probabilities of mosaic levels ≤20% (OR = 1.326, 95% CI = 1.187-1.481), <50% (OR = 1.425, 95% CI = 1.262-1.608), and ≤80% (OR = 1.351, 95% CI = 1.186-1.539) (p < 0.001). The prediction abilities of TLBMSs were similar for mosaic levels ≤20% (AUC = 0.604, 95% CI = 0.565-0.642), <50% (AUC = 0.634, 95% CI = 0.598-0.671), and ≤80% (AUC = 0.617, 95% CI = 0.576-0.658). In conclusion, detailed evaluation with TL monitoring at the specific time window reveals that redefined blastocyst morphology components and converted numeric TLBMSs are significantly correlated with all of the threshold levels of mosaicism. However, the performance of TLBMSs to differentiate blastocysts with aberrant ploidy risk remains perfectible.
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PURPOSE: This study evaluated the potential viability of embryos with low mosaicism level (< 50%) by comparing the clinical outcomes of single mosaic versus euploid blastocyst transfer. In addition, the live birth outcomes for various types of mosaicism with respect to abnormalities in chromosome structure and content were analyzed. METHODS: This study included patients who underwent in vitro fertilization with preimplantation genetic testing for aneuploidy (PGT-A). The PGT-A cycles performed through next-generation sequencing with single euploid or mosaic embryo transfers were included. We collected 299 frozen single embryo transfer cycles-216 single euploid and 83 mosaic-between July 2016 and July 2018. This study analyzed clinical outcomes, including fetal karyotyping by using amniocentesis, gestational age at delivery, and live birth weight after single mosaic embryo transfer. RESULTS: The average birth weight of infants in the euploid and mosaic blastocyst transfer groups was 3146.2 and 2997.7 g, respectively. The karyotyping results of prenatal diagnosis in all pregnant women were normal. Our study indicated that mosaic embryos can develop into euploid healthy infants with various levels or types of mosaicism. No significant difference was observed between infants from euploid and mosaic blastocyst transfers. CONCLUSION: If patients have no euploid embryos, mosaic embryos can be transferred as they have potential for implantation and development into euploid healthy infants. This study is invaluable for counseling clinical results after single mosaic embryo transfers.
Subject(s)
Abortion, Spontaneous/genetics , Fertilization in Vitro , Live Birth/epidemiology , Preimplantation Diagnosis , Abortion, Spontaneous/pathology , Adult , Aneuploidy , Blastocyst/metabolism , Embryo Implantation/genetics , Embryo Transfer/methods , Female , High-Throughput Nucleotide Sequencing , Humans , Karyotyping , Live Birth/genetics , Mosaicism , Pregnancy , Pregnancy Rate , Single Embryo Transfer/methodsABSTRACT
This retrospective study attempts to elucidate the relevance of the interval between human chorionic gonadotropin priming and oocyte pick-up (hCG-OPU) to the euploidy probability of biopsied blastocysts in preimplantation genetic tests for aneuploidy (PGT-A) cycles. A total of 1889 blastocysts from 511 patients undergoing PGT- A cycles were used. An analysis of generalized estimating equations (GEE) was used to identify whether the hCG-OPU interval is associated with euploidy probabilities of blastocysts. Accordingly, maternal age (OR: 0.925, 95% CI: 0.903-0.948, p < 0.001) and the hCG-OPU interval (OR: 1.138, 95% CI: 1.028-1.260, p = 0.013) were the two significant factors associated with the euploidy probabilities. The Cochran-Armitage trend test demonstrated that the blastocyst euploidy percentage increased progressively with the increasing hCG-OPU interval in normal responders (p = 0.006) and advanced maternal age (age ≥38 years; p = 0.020) groups. In normal responders, the euploidy rate was highest in the 38-39 h interval (43.1%, 47/109). In contrast, the euploidy rate was lowest in the 34-35 h interval (28.7%, 29/105). In conclusion, the present study demonstrated that at an hCG-OPU interval between 34-39 h, the longer the hCG-OPU interval, the higher the probability of euploidy for blastocysts.
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We previously developed pluripotent rabbit embryonic stem cells (rbES) using a culture system supplemented with basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF), noggin and Y-27632 (referred to as iFLY). In present work, we explored multiple approaches to enhance the chance of deriving domed pluripotent rbES cells by inhibition of MEK, GSK, and PKC signaling pathways. Domed stated rbES were derived in defined medium supplemented with 15% KOSR, 103 IU/mL mouse LIF, 10 ng/mL bFGF and three inhibitors to the MEK (PD0325901, 1 µM), GSK3 (CHIR99021, 3 µM) and PKC (Gö6983, 5 µM) (3i). Domed rbES were passaged every 3-4 days till passage 3-4 for the designated experiments. We showed that bFGF and LIF are indispensable for the derivation and maintenance of rbES; whereas the 3i medium containing inhibitors to the MEK (PD0325901), GSK3 (CHIR99021) and PKC (Gö6983) were necessary for deriving domed rbES. Domed rbES possessed naïve ES markers as Rex1 and ERAS in addition to Oct4, Klf4, Sox 2 and c-myc by RT-PCR. Domed rbES showed positive staining for Rex1, Fgf4, Klf4, Nanog and Oct4 by immunofluorescence chemistry. Further deleting either one factor in 3i medium as CHIR99021, PD0325901, Gö6983 or bFGF resulted in disappearing of domed rbES colonies. The optimal concentrations of 3i contained 0.75 µM PD0325901, 2.25 µM CHIR99021, and 4.5 µM Gö6983. Our work, in combination of different inhibitors for deriving rabbit ES, supports that the network of signal pathways plays an important role in ES self-renew, propagation and maintenance, and sheds light on deriving authentic properties of rbES in an important yet understudied model animal species.
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OBJECTIVE: Clinical genetic testing to diagnose germline mutations often requires blood sample or saliva smear from a cancer-affected individual. This rules out testing in families when cancer-affected individuals are deceased. We explored the use of a next-generation sequencing (NGS) platform to diagnose germline pathogenic mutations from tumors. METHODS: Archival tumors (ovarianâ¯=â¯26, breastâ¯=â¯25, othersâ¯=â¯9) were retrieved from 60 cancer patients who have undergone multi-gene panel blood testing. Genomic DNA was extracted and sequenced for BRCA1/2 using a NGS platform. 41/60 specimens were sequenced for 5 other genes (APC, ATM, PALB2, PTEN, TP53). Tumor testing and results interpretation were performed blinded to the blood test result. RESULTS: All 38 patients with no BRCA1/2 mutations on blood testing were correctly tested negative on tumor. Tumor testing correctly diagnosed BRCA1/2 pathogenic mutations in 15/22 (68%) patients while in 7/22 (32%) patients, the mutation was either detected but incorrectly classified as VUS (nâ¯=â¯3) or not detected at all (nâ¯=â¯4). Overall concordance rate for tumor and blood testing for BRCA1/2 mutations was 88%, with 0% false positive and 32% false negative rate for pathogenic mutations. Tumor testing correctly diagnosed 1/2 pathogenic germline ATM mutation, 1/1 pathogenic germline PALB2 mutation and 2/2 pathogenic germline TP53 mutations. False positive germline mutations were diagnosed in 4 genes at a rate of 2.4%-10.3% (APCâ¯=â¯2.4%, PALB2â¯=â¯2.4%, PTENâ¯=â¯4.9%, TP53â¯=â¯10.3%). CONCLUSION: Tumor testing for BRCA1/2 germline mutations using an NGS platform is fairly reliable with no false positive findings, and correctly diagnosed more than two-thirds of pathogenic germline BRCA1/2 mutations. However, it is not reliable to diagnose pathogenic germline mutations in genes frequently mutated in sporadic cancers, such as PTEN and TP53.
Subject(s)
Breast Neoplasms/genetics , Genes, BRCA1/physiology , Genes, BRCA2/physiology , Germ-Line Mutation/genetics , Ovarian Neoplasms/genetics , Adult , Aged , Breast Neoplasms/diagnosis , DNA, Neoplasm/genetics , False Positive Reactions , Female , Humans , Middle Aged , Ovarian Neoplasms/diagnosis , Prospective Studies , Sequence Analysis, DNA , Tumor Suppressor Protein p53/metabolismABSTRACT
RESEARCH QUESTION: Are the morphokinetics of euploid blastocysts evaluated by a generally applicable algorithm associated with the clinical outcomes of single-embryo transfer (SET)? DESIGN: Time-lapse microscopy was used to compare morphokinetic variables between expanded blastocysts derived from preimplantation genetic testing for aneuploidy cycles using high-resolution next-generation sequencing (hr-NGS). The clinical efficacy of the morphokinetic algorithm KIDScore D5 was evaluated after euploid SET. RESULTS: Compared with euploid blastocysts, low-level mosaic blastocysts presented comparable morphokinetic and morphological features. However, high-level mosaic blastocysts exhibited significant delays in t5 (median 51.9 h post insemination (hpi), Pâ¯=â¯0.034) (where t is the time for the embryo to reach the specific stage in hours after ICSI or conventional IVF) and t8 (median 58.6 hpi, Pâ¯=â¯0.032) accompanied by a prolonged time period for the third cell cycle (median 14.7 h, Pâ¯=â¯0.012). A significantly higher incidence (Pâ¯=â¯0.011) of multinucleation indicated a susceptibility of high-level mosaic blastocysts to mitotic errors. Only a delay in the time for the embryo to reach the full blastocyst stage (median 106.0 hpi, Pâ¯=â¯0.039) was revealed in aneuploid blastocysts, reflecting the reduced formation of good-quality blastocysts (42.6% versus 65.7%, P < 0.001). Euploid blastocysts with specific morphokinetic characteristics were graded using the KIDScore D5 algorithm. Grade C embryos achieved significantly lower rates of clinical pregnancy, implantation and ongoing pregnancy (25%, 25% and 10%, respectively) compared with the grade A (76.2%, 79.4% and 68.3%, respectively) or grade B (62.5%, 66.7% and 62.5%, respectively) embryos (Pâ¯=â¯0.0171 to <0.0001). CONCLUSIONS: Although morphokinetic features appear dissimilar in embryos with different diploid-aneuploid mosaic levels, predicting chromosomal abnormalities using morphokinetics alone is still insufficient. When combined with hr-NGS, use of the generally applicable KIDScore D5 algorithm has the potential to discriminate euploid blastocysts with different developmental competence.
Subject(s)
Aneuploidy , Embryo, Mammalian/cytology , Embryo, Mammalian/diagnostic imaging , Pregnancy Outcome , Preimplantation Diagnosis , Single Embryo Transfer , Adult , Blastocyst/cytology , Blastocyst/physiology , Cell Shape , Cells, Cultured , Cleavage Stage, Ovum/cytology , Cleavage Stage, Ovum/physiology , Embryo Culture Techniques , Female , Fertilization in Vitro , Genetic Testing/methods , High-Throughput Nucleotide Sequencing , Humans , Pregnancy , Preimplantation Diagnosis/methods , Retrospective Studies , Single Embryo Transfer/methods , Single Embryo Transfer/standards , Time-Lapse ImagingABSTRACT
The porcine epidemic diarrhoea virus (PEDV) devastates the health of piglets but may not infect piglets whose CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene is mutated (knockouts, KO) by using CRISPR/Cas9 gene editing techniques. This hypothesis was tested by using KO piglets that were challenged with PEDV. Two single-guide RNAs targeting the CMAH gene and Cas9 mRNA were microinjected into the cytoplasm of newly fertilized eggs. Four live founders generated and proven to be biallelic KO, lacking detectable N-glycolylneuraminic acid (NGNA). The founders were bred, and homozygous offspring were obtained. Two-day-old (in exps. I, n = 6, and III, n = 15) and 3-day-old (in exp. II, n = 9) KO and wild-type (WT, same ages in respective exps.) piglets were inoculated with TCID50 1x103 PEDV and then fed 20 mL of infant formula (in exps. I and II) or sow's colostrum (in exp. III) every 4 hours. In exp. III, the colostrum was offered 6 times and was then replaced with Ringer/5% glucose solution. At 72 hours post-PEDV inoculation (hpi), the animals either deceased or euthanized were necropsied and intestines were sampled. In all 3 experiments, the piglets showed apparent outward clinical manifestations suggesting that infection occurred despite the CMAH KO. In exp. I, all 6 WT piglets and only 1 of 6 KO piglets died at 72 hpi. Histopathology and immunofluorescence staining showed that the villus epithelial cells of WT piglets were severely exfoliated, but only moderate exfoliation and enterocyte vacuolization was observed in KO piglets. In exp. II, delayed clinical symptoms appeared, yet the immunofluorescence staining/histopathologic inspection (I/H) scores of the two groups differed little. In exp. III, the animals exhibited clinical and pathological signs after inoculation similar to those in exp. II. These results suggest that porcine CMAH KO with nullified NGNA expression are not immune to PEDV but that this KO may lessen the severity of the infection and delay its occurrence.
Subject(s)
Cytidine Monophosphate/analogs & derivatives , Genetic Predisposition to Disease/genetics , Porcine epidemic diarrhea virus/genetics , Animals , CRISPR-Cas Systems , Coronavirus Infections/virology , Cytidine Monophosphate/genetics , Diarrhea/virology , Disease Susceptibility/metabolism , Enterocytes/pathology , Female , Gene Expression Regulation/genetics , Neuraminic Acids , Porcine epidemic diarrhea virus/pathogenicity , Pregnancy , Swine , Swine Diseases/virologyABSTRACT
Reaction of compounds [(NO)2Fe(TMEDA)] and [BH4]- affords the first hydride-containing dinitrosyl iron complex (DNIC) [(NO)2Fe(η2-BH4)]- (2). Hydride-insertion reactivity of DNIC 2 promotes the reductive transformation of CS2 into DNIC [(NO)2Fe(η3-HCS2)]-, the first DNIC featuring both linear/bent NO ligands and Fe 3dz2-to-HCS2 π* backbonding interaction.
ABSTRACT
In artificial photosynthesis, water splitting plays an important role for the conversion and storage of renewable energy sources. Here, we report a study on the electrocatalytic properties of the electrodeposited-film electrodes derived from irreversible electro-reduction/-oxidation of a molecular dinitrosyl iron complex (DNIC) {Fe(NO)2}9 [(Me6tren)Fe(NO)2]+ (Me6tren = tris[2-(dimethylamino)ethyl]amine) for the hydrogen evolution reaction (HER) and the oxygen evolution reaction (OER) in alkaline solution, individually. For HER, the overpotential and Tafel slope for the electrodeposited-film cathode are lower than those of the equiv.-weight Pt/C electrode. The electrodeposited-film anode for the OER is stable for 139 h. Integration of the electrodeposited-film cathode and anode into a single electrode-pair device for electrocatalytic water splitting exhibits an onset voltage of 1.77 V, achieving a geometrical current density of 10 mA cm-2.
ABSTRACT
This study was conducted to confirm that 1-site and 4-site ppU6-GGTA1-gRNA CRISPR vectors together with the pCX-Flag2-NLS1-Cas9-NLS2 plasmid can both generate KO pigs by direct pronuclear microinjection. In total, 41 and 53 fertilized eggs were microinjected on 1-site and 4-site strategies, respectively. The 1-site construction generated a litter of 8 piglets, and 2 were mono-allelic mutant (mMt). The injection of 4-site constructions resulted in one biallelic mutant (bMt) and one mMt piglet in a litter of 7. Those 3 mMt pigs had a 4 bp deletion, 5 bp insertion, or 7 bp insertion at site I, and the bMt pig had 5 types of mutations at cleavage sites I and III. The expression of alpha-Gal on the bMt peripheral blood mononuclear cells (PBMCs) was reduced, and survival rate of bMt PBMCs was maintained as indicated by results of cultivation with sera of humans or Formosan Macaques. We concluded that mutant pigs could be generated by direct pronuclear microinjection of ppU6-GGTA1-gRNA CRISPR vectors with the pCX-Flag2-NLS1-Cas9-NLS2 plasmid and that the 4-site strategy has a better mutant efficiency. Porcine U6 promoter was firstly used to express KO vectors and effectively generate mutant pigs, worthily to adopt for future KO studies.