In chemical risk assessment, default uncertainty factors are used to account for interspecies and interindividual differences, and differences in toxicokinetics and toxicodynamics herein. However, ...these default factors come with little scientific support. Therefore, our aim was to develop an in vitro method, using acetylcholinesterase (AChE) inhibition as a proof of principle, to assess both interspecies and interindividual differences in toxicodynamics. Electric eel enzyme and human blood of 20 different donors (12 men/8 women) were exposed to eight different compounds (chlorpyrifos, chlorpyrifos-oxon, phosmet, phosmet-oxon, diazinon, diazinon-oxon, pirimicarb, rivastigmine) and inhibition of AChE was measured using the Ellman method. The organophosphate parent compounds, chlorpyrifos, phosmet and diazinon, did not show inhibition of AChE. All other compounds showed concentration-dependent inhibition of AChE, with IC
50
s in human blood ranging from 0.2–29 µM and IC
20
s ranging from 0.1–18 µM, indicating that AChE is inhibited at concentrations relevant to the in vivo human situation. The oxon analogues were more potent inhibitors of electric eel AChE compared to human AChE. The opposite was true for carbamates, pointing towards interspecies differences for AChE inhibition. Human interindividual variability was low and ranged from 5–25%, depending on the concentration. This study provides a reliable in vitro method for assessing human variability in AChE toxicodynamics. The data suggest that the default uncertainty factor of ~ 3.16 may overestimate human variability for this toxicity endpoint, implying that specific toxicodynamic-related adjustment factors can support quantitative in vitro to in vivo extrapolations that link kinetic and dynamic data to improve chemical risk assessment.
Preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm (TE) biopsy is widely applied in in vitro fertilization (IVF) to identify aneuploid embryos. However, potential safety ...concerns regarding biopsy and restrictions to only those embryos suitable for biopsy pose limitations. In addition, embryo mosaicism gives rise to false positives and false negatives in PGT-A because the inner cell mass (ICM) cells, which give rise to the fetus, are not tested. Here, we report a critical examination of the efficacy of noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) in the spent culture media of human blastocysts by analyzing the cell-free DNA, which reflects ploidy of both the TE and ICM. Fifty-two frozen donated blastocysts with TE biopsy results were thawed; each of their spent culture medium was collected after 24-h culture and analyzed by next-generation sequencing (NGS). niPGT-A and TE-biopsy PGT-A results were compared with the sequencing results of the corresponding embryos, which were taken as true results for aneuploidy reporting. With removal of all corona-cumulus cells, the false-negative rate (FNR) for niPGT-A was found to be zero. By applying an appropriate threshold for mosaicism, both the positive predictive value (PPV) and specificity for niPGT-A were much higher than TE-biopsy PGT-A. Furthermore, the concordance rates for both embryo ploidy and chromosome copy numbers were higher for niPGT-A than TE-biopsy PGT-A. These results suggest that niPGT-A is less prone to errors associated with embryo mosaicism and is more reliable than TE-biopsy PGT-A.
Single-cell genome analyses of human oocytes are important for meiosis research and preimplantation genomic screening. However, the nonuniformity of single-cell whole-genome amplification hindered ...its use. Here, we demonstrate genome analyses of single human oocytes using multiple annealing and looping-based amplification cycle (MALBAC)-based sequencing technology. By sequencing the triads of the first and second polar bodies (PB1 and PB2) and the oocyte pronuclei from same female egg donors, we phase the genomes of these donors with detected SNPs and determine the crossover maps of their oocytes. Our data exhibit an expected crossover interference and indicate a weak chromatid interference. Further, the genome of the oocyte pronucleus, including information regarding aneuploidy and SNPs in disease-associated alleles, can be accurately deduced from the genomes of PB1 and PB2. The MALBAC-based preimplantation genomic screening in in vitro fertilization (IVF) enables accurate and cost-effective selection of normal fertilized eggs for embryo transfer.
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•Whole-genome amplification and sequencing of single human oocytes using MALBAC method•First comprehensive study of crossovers and genetic interference in human oocytes•Phasing the genome of a female pronucleus by sequencing its polar bodies•Selection of a viable egg without aneuploidy or point mutations by sequencing its polar bodies
MALBAC genome amplification and high-throughput sequencing of the two polar bodies allowed inference of the health status of the oocyte, both in terms of aneuploidy and single-nucleotide variants associated with Mendelian diseases, demonstrating proof of principle for MALBAC-based preimplantation genomic screening in IVF.
In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal ...cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre‐incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre‐incubation with progesterone. In experiments 1 and 2, IVF was performed in the presence of porcine oviduct epithelial cells. The removal of cumulus cells from equine oocytes after in vitro maturation tended to increase the percentage of fertilization when fresh sperm was used (1/33 vs. 4/31, p > 0.05) but had no effect when frozen sperm was used (1/32 vs. 1/32). Equine sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/14 vs. 2/18 for fresh, 1/29 vs. 1/25 for frozen). In experiments 3 and 4, IVF was performed after pre‐incubation of sperm with porcine oviductal fluid. The removal of cumulus cells tended to increase the percentage of fertilization when fresh sperm was used (1/24 vs. 3/26, p > 0.05). Sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/39 vs. 2/36 for fresh, 2/37 vs. 1/46 for frozen), but two 3–4 cell stage zygotes were obtained with fresh sperm pre‐incubated with progesterone. This is an encouraging result for the setting up of an efficient IVF procedure in equine.
Cartilage tissue engineering has primarily focused on the generation of grafts to repair cartilage defects due to traumatic injury and disease. However engineered cartilage tissues have also a strong ...scientific value as advanced 3D culture models. Here we first describe key aspects of embryonic chondrogenesis and possible cell sources/culture systems for in vitro cartilage generation. We then review how a tissue engineering approach has been and could be further exploited to investigate different aspects of cartilage development and degeneration. The generated knowledge is expected to inform new cartilage regeneration strategies, beyond a classical tissue engineering paradigm.
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Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and ...reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins-1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.
The objective of this study was to determine the effects of insulin-like growth factor-I (IGF-I) (0, 60, 120, 180, and 240 ng/mL) and follicular fluid (FF) derived from 2 to 5 and 6 to 10 mm diameter ...follicles (SpFFs and LpFFs, respectively) added during in vitro maturation (IVM) of porcine oocytes on nuclear maturation and IVF. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium supplemented with SpFFs or LpFFs and various IGF-I concentrations. The COCs were cultured for 44 hours, and then fertilized in vitro. Maturation and IVF results were recorded 18 hours after insemination. The IVM (%) was higher (P < 0.05) in the COCs matured in LpFFs than with SpFFs when 0 (90.0 ± 6.9 vs. 76.3 ± 10.7) or 60 ng/mL IGF-I (92.0 ± 8.1 vs. 81.8 ± 10.2) was added. In SpFFs media, there was a quadratic relationship (P < 0.01) between IGF-I concentration and IVM (peak results at IGF-I = 129 ng/mL). However, when the COCs were matured with LpFFs, there was a decreasing linear effect between IGF-I concentration and IVM. At all concentrations of IGF-I, the percentage of degenerated oocytes was higher in COCs matured in SpFFs than in LpFFs. Penetration (%) did not differ (P > 0.05) between COCs matured with SpFFs or LpFFs when 60 (66.8 ± 9.4 vs. 72.7 ± 11.3) or 180 ng/mL of IGF-I (75.7 ± 10.4 vs. 73.8 ± 13.2) were used. Monospermy (%) was similar between SpFFs and LpFFs only with addition of 120 ng/mL IGF-I. The IVF performance (%) did not differ between COCs matured with SpFFs or LpFFs when IGF-I concentrations of 120 (28.5 ± 8.8 vs. 38.5 ± 8.3) and 180 ng/mL (24.3 ± 10.2 vs. 30.12 ± 8.2) were used. There was no effect of IGF-I concentration or of FF type on the number of penetrated sperm per oocyte and on male pronuclear formation. For COCs matured with SpFFs, there was a quadratic relationship between IGF-I concentration and penetration, monospermy, and IVF performance (peak results at IGF-I = 179, 122, and 135 ng/mL, respectively). Thus, on the basis of the observed quadratic relationships, we inferred that when using SpFFs, the addition of IGF-I (122–179 ng/mL) to the IVM medium produced results similar to those obtained with LpFFs without adding IGF-I. In conclusion, the addition of IGF-I to the IVM medium supplemented with SpFFs increased maturation and improved IVF results. Alternatively, IGF-I had no effect on IVM or IVF when used with LpFFs.
Background: Despite recent advances that have improved the pregnancy success rates that can be achieved via in vitro fertilization (IVF) therapy, it is not yet clear which blastocyst morphological ...parameters best predict the outcomes of single blastocyst transfer. In addition, most of the previous studies did not exclude the effect of embryo aneuploidy on blastocysts transfer. Thus, the present study investigated the predictive value of various parameters on the pregnancy outcomes achieved via the transfer of frozen euploid blastocysts.
Methods: The study retrospectively analyzed 914 single euploid blastocyst transfer cycles that were performed at the Peking University Third Hospital Reproductive Medical Center between June 2011 and May 2016. The expansion, trophectoderm (TE), and inner cell mass (ICM) quality of the blastocysts were assessed based on blastocyst parameters, and used to differentiate between "excellent", "good", "average", and "poor"-quality embryos. The relationship between these embryo grades and the achieved pregnancy outcomes was then analyzed via the Chi-square and logistic regression tests.
Results: For embryo grades of excellent, good, average and poor, the clinical pregnancy rates were 65.0%, 59.3%, 50.3% and 33.3%, respectively; and the live-birth rates were 50.0%, 49.7%, 42.3% and 25.0%, respectively. Both the clinical pregnancy rate (χ2 = 21.28, P = 0.001) and live-birth rate (χ2 = 13.50, P < 0.001) increased with the overall blastocyst grade. Both rates were significantly higher after the transfer of a blastocyst that exhibited either an A-grade or B-grade TE, and similarly, an A-grade ICM, than after the transfer of a blastocyst that exhibited a C-grade TE and/or ICM. The degree of blastocyst expansion had no apparent effect on the clinical pregnancy or live-birth rate. All odds ratio were adjusted for patient age, body mass index, length (years) of infertility history, and infertility type.
Conclusions: A higher overall euploid blastocyst quality is shown to correlate most strongly with optimal pregnancy outcomes. The study thus supports the use of the described TE and ICM morphological grades to augment current embryo selection criteria.
In vitro follicle development (IVFD) is an adequate model to obtain basic knowledge of folliculogenesis and provides a tool for ovarian toxicity screening. IVFD yielding competent oocytes may also ...offer an option for fertility and species preservation. To promote follicle growth and oocyte maturation in vitro, various culture systems are utilized for IVFD in rodents, domestic animals, wild animals, nonhuman primates, and humans. Follicle culture conditions have been improved by optimizing gonadotropin levels, regulatory factors, nutrient supplements, oxygen concentration, and culture matrices. This review summarizes quality assessment of oocytes generated from in vitro-developed antral follicles from the preantral stage, including oocyte epigenetic and genetic profile, cytoplasmic and nuclear maturation, preimplantation embryonic development following in vitro fertilization, as well as pregnancy and live offspring after embryo transfer. The limitations of oocyte quality evaluation following IVFD and the gaps in our knowledge of IVFD to support proper oocyte development are also discussed. The information may advance our understanding of the requirements for IVFD, with a goal of producing competent oocytes with genetic integrity to sustain embryonic development resulting in healthy offspring. Summary Sentence Although advances in current IVFD systems have greatly enhanced oocyte growth and maturation, further optimization is required to improve oocyte competence with genetic integrity for proper embryonic development.
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