Search

Human embryonic stem cells (hESCs), produced from human embryos, are demonstrating: utility and promise in disease modeling; enhanced and unique understanding of early events in basic genetic or molecular or cellular or epigenetic development; novel human approaches to pharmaceutical screening; pathways toward the discoveries of disease treatments and cures; and foundational importance for regenerative medicine. The regulatory landscape is rigorous, and rightly so. Here, we discuss the current US federal and state regulatory environment. A unique approach of presenting anonymized embryo donor statements is provided to personalize the decision-making process of human embryo donation for hESC derivation. From the uses of preimplantation genetic-tested and affected human embryos to derived disease-specific hESCs, one can glean the much needed information on early human genetics and developmental biology, which are presented here. Finally, we discuss the future uses of hESCs, and other pluripotent stem cells, in general and reproductive medicine.

Oocyte quality is perhaps the most important limiting factor in female fertility; however, the current methods of determining oocyte competence are only marginally capable of predicting a successful pregnancy. We aim to review the predictive value of non-invasive techniques for the assessment of human oocytes and their related cells and biofluids that pertain to their developmental competence. Investigation of the proteome, transcriptome, and hormonal makeup of follicular fluid, as well as cumulus-oocyte complexes are currently underway; however, prospective randomized non-selection-controlled trials of the future are needed before determining their prognostic value. The biological significance of polar body morphology and genetics are still unknown and the subject of debate. The predictive utility of zygotic viscoelasticity for embryo development has been demonstrated, but similar studies performed on oocytes have yet to be conducted. Metabolic profiling of culture media using human oocytes are also limited and may require integration of automated, high-throughput targeted metabolomic assessments in real time with microfluidic platforms. Light exposure to oocytes can be detrimental to subsequent development and utilization of time-lapse imaging and morphometrics of oocytes is wanting. Polarized light, Raman microspectroscopy, and coherent anti-Stokes Raman scattering are a few novel imaging tools that may play a more important role in future oocyte assessment. Ultimately, the integration of chemistry, genomics, microfluidics, microscopy, physics, and other biomedical engineering technologies into the basic studies of oocyte biology, and in testing and perfecting practical solutions of oocyte evaluation, are the future for non-invasive assessment of oocytes.

Funder: Weston Havens Foundation; doi: https://doi.org/10.13039/100011223, Aneuploidy, the presence of an abnormal number of chromosomes, is a major cause of early pregnancy loss in humans. Yet, the developmental consequences of specific aneuploidies remain unexplored. Here, we determine the extent of post-implantation development of human embryos bearing common aneuploidies using a recently established culture platform. We show that while trisomy 15 and trisomy 21 embryos develop similarly to euploid embryos, monosomy 21 embryos exhibit high rates of developmental arrest, and trisomy 16 embryos display a hypo-proliferation of the trophoblast, the tissue that forms the placenta. Using human trophoblast stem cells, we show that this phenotype can be mechanistically ascribed to increased levels of the cell adhesion protein E-CADHERIN, which lead to premature differentiation and cell cycle arrest. We identify three cases of mosaicism in embryos diagnosed as full aneuploid by pre-implantation genetic testing. Our results present the first detailed analysis of post-implantation development of aneuploid human embryos.

We have previously demonstrated that cellulose nanocrystals modified with poly(ethylenimine) (PEI-f-CNC) are capable of capturing volatile organic compounds (VOCs) associated with malodors. In this manuscript, we describe our efforts to develop a scalable synthesis of these materials from bulk cotton. This work culminated in a reliable protocol for the synthesis of unmodified cellulose nanocrystals (CNCs) from bulk cotton on a 0.5 kg scale. Additionally, we developed a protocol for the modification of the CNCs by means of sequential 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) oxidation and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) coupling to modify their surface with poly(ethylenimine) on a 100 g scale. Subsequently, we evaluated the performance of the PEI-f-CNC materials that were prepared in a series of VOC capture experiments. First, we demonstrated their efficacy in capturing volatile fatty acids emitted at a rendering plant when formulated as packed-bed filter cartridges. Secondly, we evaluated the potential to use aqueous PEI-f-CNC suspensions as a spray-based delivery method for VOC remediation. In both cases, the PEI-f-CNC formulations reduced detectable malodor VOCs by greater than 90%. The facile scaled synthesis of these materials and their excellent performance at VOC remediation suggest that they may emerge as a useful strategy for the remediation of VOCs associated with odor.

PURPOSE: Oocytes and embryos can be vitrified with and without dimethyl sulfoxide (DMSO). Objectives were to compare no vitrification (No-Vitr), vitrification with DMSO (Vitr + DMSO), and vitrification without DMSO (Vitr - DMSO) on fresh/warmed oocyte survival, induced parthenogenetic activation, parthenogenetic embryo development, and embryonic maternal imprinted gene expression. METHODS: In this prospective controlled laboratory study, mature B6C3F1 female mouse metaphase II oocytes were treated as: i) No-Vitr, ii) Vitr + DMSO/warmed, and iii) Vitr - DMSO/warmed with subsequent parthenogenetic activation and culture to the blastocyst stage. Oocyte cryo-survival, parthenogenetic activation and embryo development, parthenogenetic embryo maternal imprinted gene expression were outcome measures. RESULTS: Oocyte cryo-survival was significantly improved in Vitr + DMSO versus Vitr - DMSO at initial warming and 2 h after warming. Induced parthenogenetic activation was similar between all three intervention groups. While early preimplantation parthenogenetic embryo development was similar between control, Vitr + DMSO, Vitr - DMSO oocytes, the development to blastocysts was significantly inferior in the Vitr - DMSO oocytes group compared to the control and Vitr + DMSO oocyte groups. Finally, maternal imprinted gene expression was similar between intervention groups at both the 2-cell and blastocyst parthenogenetic embryo stage. CONCLUSION(S): Inclusion of DMSO in oocyte vitrification solutions improved cryo-survival and developmental potential of parthenogenetic embryos to the blastocyst stage without significantly altering maternal imprinted gene expression.

The objective of the current study was to determine the fatty acid composition of sperm from Holstein bulls with different freezability (Good and Poor; n = 12). Fatty acids were extracted from frozen sperm in 1:2 (v/v) chloroform–methanol solvent, fractionated into neutral and polar fractions, and composition determined by gas chromatography–mass spectrometry. Thirty-four fatty acids were quantified and their concentrations and percentages within each lipid fraction were calculated. Overall, saturated fatty acids (SFA) were predominant, accounting for 71 to 80% of fatty acids in neutral and polar lipid factions. There were marked differences in fatty acid composition between the lipid fractions (P < 0.001). The branched chain fatty acid (BCFA) concentration (15 to 18 µg) was almost twice as much as polyunsaturated fatty acids (PUFA) concentration found in the polar lipid fraction (8 to 9 µg; P < 0.001). Sperm with different freezability phenotypes only had a few differences in 22:0, 18:1 cis 9, and 14:0 13-methyl fatty acids (P ≤ 0.011). These results are significant because they reveal key understandings of fatty acid composition of sperm membrane and lay a foundation for the manipulation of membrane integrity, fluidity, and stability to advance the assisted reproductive technologies.

INTRODUCTION AND OBJECTIVE:Molecular characterization studies suggest the neuronal subtype muscleinvasive bladder cancer (MIBC) is associated with a high response rate toimmune checkpoint inhibitor...

Intracytoplasmic sperm injection (ICSI) has allowed reproduction options through assisted reproductive technologies (ARTs) for men with no spermatozoa within the ejaculate (azoospermia). In men with non-obstructive azoospermia (NOA), the options for spermatozoa retrieval are testicular sperm extraction (TESE), testicular sperm aspiration (TESA), or micro-surgical sperm extraction (microTESE). At the initial time of spermatozoa removal from the testis, spermatozoa are immobile. Independent of the means of spermatozoa retrieval, the subsequent steps of removing spermatozoa from seminiferous tubules, determining spermatozoa viability, identifying enough spermatozoa for oocyte injections, and isolating viable spermatozoa for injection are currently performed manually by laboratory microscopic dissection and collection. These laboratory techniques are highly labor-intensive, with yield unknown, have an unpredictable efficiency and/or success rate, and are subject to inter-laboratory personnel and intra-laboratory variability. Here, we consider the potential utility, benefits, and shortcomings of developing technologies such as motility induction/stimulants, microfluidics, dielectrophoresis, and cell sorting as andrological laboratory add-ons to reduce the technical burdens and variabilities in viable spermatozoa isolation from testicular samples in men with NOA.

Rendering is a global industry that recycles by-products resulting from butchering operations, which process billions of animals per year. About 50% of the weight of livestock is not consumed by humans and must be processed by rendering operations, which cook and separate the material into its protein and fat components. These products serve as a sustainable food source for livestock, feedstocks for oleochemicals, and raw material for biodiesel refineries. Due to the scale and nature of the raw materials and the cooking process, rendering operations emit a significant, but as yet poorly quantified, VOC load. Assessing this VOC load is important in order to calibrate the industry’s contribution to global VOC emissions, and to help address nuisance odor problems. We conducted VOC air sampling of two facilities in California, USA during the winter and summer seasons. VOC and reduced sulfur analyses were conducted using 8 h ambient air samples. Analyses for amines, ammonia, aldehydes/ketones, and volatile fatty acids were conducted using sampling pumps. These analyses detected 43 compounds at the facilities, and the number and concentration of detectable compounds were seasonally dependent. The compounds present at the highest concentrations included: ammonia (1600–2800 ppb, i.e., winter–summer levels), acetic acid (80–320 ppb, along with twelve other fatty acids ranging from ~0.5–140 ppb), acetone (55–241 ppb, along with nine other aldehyde/ketone products ranging from 0.4–60 ppb), and ethanol (15–81 ppb). These constituents have low odor thresholds and thus contribute to nuisance odor problems. Further, the overall VOC contribution arising from rendering facilities on a global scale is as yet very poorly characterized. This analysis will be useful to guide the development of new odor abatement strategies and strategies for the reduction of VOC emissions associated with this critical industry.

Spinocerebellar ataxia type 3 (SCA3) is a fatal, late-onset neurodegenerative disorder characterized by selective neuropathology in the brainstem, cerebellum, spinal cord, and substantia nigra. Here, we characterize the first NIH-approved human embryonic stem cell (hESC) line derived from an embryo harboring the SCA3 mutation. Referred here as SCA3-hESC, this line is heterozygous for the mutant polyglutamine-encoding CAG repeat expansion in the ATXN3 gene within the pathogenic repeat range for SCA3. We observed relevant molecular hallmarks of the human disease at all differentiation stages from stem cells to cortical neurons, including robust ATXN3 aggregation and altered expression of key components of the protein quality control machinery. Finally, antisense oligonucleotide-mediated reduction of ATXN3 prevented the formation of p62-positive aggresomes in SCA3-hESCs. The SCA3-hESC line offers a unique and highly relevant human disease model that holds strong potential to advance understanding of SCA3 disease mechanisms and facilitate the evaluation of possible SCA3 therapies.HighlightsGenerated first NIH-approved SCA3 human embryonic stem cell line (SCA3-hESC)SCA3–hESC exhibit robust ATXN3 aggregation pathology and form p62+ aggresomesAnti-ATXN3 antisense oligonucleotides rescue aggresome formation in SCA3-hESCDerived SCA3 neurons form aggregates and exhibit impaired protein quality control

INTRODUCTION AND OBJECTIVES:Previous studies demonstrated ERCC2 helicase domain mutations confer nucleotide excision repair (NER) deficiency and drive cisplatin sensitivity in muscle invasive bladd...

INTRODUCTION AND OBJECTIVES:The main dysregulated pathway of clear cell renal cell carcinoma (ccRCC), the von Hippel Lindau (VHL)/hypoxia inducible factor-α (HIF-α) axis, is a key regulator and reg...

Cytosolic lipids participate in the growth, development, and overall health of mammalian oocytes including many roles in cellular homeostasis. Significant emphasis has been placed on the study of lipids as a dynamic organelle, which in turn requires the development of tools and techniques to quantitate and compare how lipid content relates to cellular structure, function, and normalcy. Objectives of this study were to determine if nonlinear vibrational microscopy (e.g., coherent anti-Stokes Raman scattering or CARS microscopy) could be used for live-cell imaging to quantify and compare lipid content in mammalian oocytes during development and in relation to body composition; and compare its efficacy to methods involving cellular fixation and staining protocols. Results of this study demonstrate that CARS is able to identify lipids in live mammalian oocytes, and there exists quantifiable and consistent differences in percent lipid composition across ooctyes of different species, developmental stages, and in relation to body composition. Such a method of live-cell lipid quantification has (i) experimental power in basic cell biology, (ii) practical utility for identifying developmental predictive biomarkers while advancing biology-based oocyte/embryo selection, and (iii) ability to yield rationally supporting technology for decision-making in rodents, domestic species, and human assisted reproduction and/or fertility preservation.

Summary Self-renewing embryonic stem cells (ESCs) respond to environmental cues by exiting pluripotency or entering a quiescent state. The molecular basis underlying this fate choice remains unclear. Here, we show that histone acetyltransferase MOF plays a critical role in this process through directly activating fatty acid oxidation (FAO) in the ground-state ESCs. We further show that the ground-state ESCs particularly rely on elevated FAO for oxidative phosphorylation (OXPHOS) and energy production. Mof deletion or FAO inhibition induces bona fide quiescent ground-state ESCs with an intact core pluripotency network and transcriptome signatures akin to the diapaused epiblasts in vivo. Mechanistically, MOF/FAO inhibition acts through reducing mitochondrial respiration (i.e., OXPHOS), which in turn triggers reversible pluripotent quiescence specifically in the ground-state ESCs. The inhibition of FAO/OXPHOS also induces quiescence in naive human ESCs. Our study suggests a general function of the MOF/FAO/OXPHOS axis in regulating cell fate determination in stem cells.

Polyethylenimine (PEI) functionalized kaolinite clay was successfully prepared, characterized, and assessed for the remediation of volatile organic compounds (VOCs) comprising the aldehyde, carboxylic acid, and disulfide functional group classes. A gas chromatographic vapor capture assay evaluated the capability of unmodified and modified clay material to capture representative aldehyde, carboxylic acid, and disulfide VOCs in a laboratory setting. Unmodified kaolinite (Kao) clay was moderately or poorly effective at remediating these VOCs, while the poly(amine) functionalized Kao was capable of capturing VOCs in the vapor phase with reductions up to 100%. Sample cartridge tubes were packed with PEI-functionalized clay in order to assess their ability to reduce the detectable volatile fatty acid load at an open-air rendering plant in a relevant field test for applying these materials in a packed-bed scrubber application. The PEI-Kao packed cartridges were capable of significantly reducing the detectable concentration of volatile fatty acid effluent from the rendering operation. These volatile fatty acids are major contributors to nuisance odors associated with rendering.

Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5′ untranslated region (UTR) of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRP’s pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the Clustered Regularly Interspaced Palindromic Repeats/deficient CRISPR associated protein 9 (CRISPR/dCas9) system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCas9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 messenger RNA (mRNA) expression despite promoter and repeat methylation. However, these changes at the transcript level were not coupled with a significant elevation in FMRP protein expression in FXS cells. These studies demonstrate that directing a transcriptional activator to CGG repeats is sufficient to selectively reactivate FMR1 mRNA expression in Fragile X patient stem cells.

Approximately 15% of human couples of reproductive age have impaired fertility, and the male component accounts for about half of these cases. The etiology is usually unknown, but high correlation with the increase in obesity rates is documented. In this study, we show that diet-induced and genetically obese mice display copulatory behavior comparable to controls, but the number of females impregnated by obese males is remarkably low. Screening for changes in gene expression in the male reproductive tract showed decreased Crisp4 expression in testis and epididymis of obese mice. Lack of CRISP4 in the luminal membrane of epididymal cells indicated inadequate secretion. Consistent with CRISP4 action in acrosome reaction, sperm from mice fed a high-fat diet (HFD) had decreased fertilization capacity. CRISP4 treatment of sperm from HFD mice prior to in vitro fertilization improved fertilization rate. In leptin-deficient obese and infertile mice, leptin's effect to restore CRISP4 expression and function required gonadal hormones. Our findings indicate that the obesity-induced decline in sperm motility and fertilization capacity results in part from the disruption of epididymal CRISP4 expression and secretion.

Microfluidics can be considered both a science and a technology. It is defined as the study of fluid behavior at a sub-microliter level and the investigation into its application to cell biology, chemistry, genetics, molecular biology and medicine. There are at least two characteristics of microfluidics, mechanical and biochemical, which can be influential in the field of mammalian gamete and preimplantation embryo biology. These microfluidic characteristics can assist in basic biological studies on sperm, oocyte and preimplantation embryo structure, function and environment. The mechanical and biochemical characteristics of microfluidics may also have practical and/or technical application(s) to assisted reproductive technologies (ART) in rodents, domestic species, endangered species and humans. This review will consider data in mammals, and when available humans, addressing the potential application(s) of microfluidics to assisted reproduction. There are numerous sequential steps in the clinical assisted reproductive laboratory process that work, yet could be improved. Cause and effect relations of procedural inefficiencies can be difficult to identify and/or remedy. Data will be presented that consider microfluidic applications to sperm isolation, oocyte cumulus complex isolation, oocyte denuding, oocyte mechanical manipulation, conventional insemination, intracytoplasmic sperm injection, embryo culture, embryo analysis and oocyte and embryo cryopreservation. While these studies have progressed in animal models, data with human gametes and embryos are significantly lacking. These data from clinical trials are requisite for making future evidence-based decisions regarding the application of microfluidics in human ART.

Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage?The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods.The use of more 'steps' of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time.Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study.Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem-Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification.The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less osmotic stress resulting in better morphology, higher cell quality and improved developmental competence. This microfluidic procedure resulted in murine zygotes with a significantly smoother cell surface (P0.001), more spherical cellular morphology (P0.001), increased cytoplasmic lipid retention in vitrified and warmed bovine oocytes (P0.01), decreased membrane perforations and cytoplasmic leakage in CPA-exposed murine zygotes (P0.05) and improved developmental competence of vitrified and warmed murine zygotes (P0.05) than CPA exposure using the current clinically used manual pipetting method.It is necessary to design the microfluidic device to be more user-friendly for widespread use.The theory and approach of eliminating osmotic stress by decreasing shrinkage rate is complementary to the prevalent osmotic stress theory in cryobiology which focuses on a minimum cell volume at which the cells shrink. The auto-microfluidic protocol described here has immediate applications for improving animal and human oocyte, zygote and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sublethal damage should be broadly useful for cryobiology.This project was funded by the National Institutes of Health and the University of Michigan Reproductive Sciences Program. The authors declare no conflicts of interest.

Cryopreservation of gametes and embryos has played a critical role in successful assisted reproductive technologies in rodents, domestic farm species, endangered species and humans. With improved success, and changing needs, the utility of gamete or embryo cryopreservation has escalated. In this review we address some of the foundational history of mammalian cryobiology, species-specific utilities, fundamental understandings of cryoprotectant agents and their use in slow-rate freezing and vitrification, and expand on the recent success and uses of oocyte vitrification and warming. In the area of female gamete cryopreservation, emphasis will be placed on not just cell survival, but also perceived and measured affects of cryopreservation on intracellular structures and functions that affect subsequent completion of meiosis with chromatin segregation fidelity, normal fertilisation and embryonic developmental competence. We compare and contrast data from cow, mouse and humans with a focus on using species-comparative developmental biology to guide future studies for improving methodologies for all species. The application of the relatively new technology microfluidics is discussed in relation to moving gradually (i.e. changing the solution over cells in an automated fashion) compared with the stepwise manual movement of cells through changing solution currently used. This use of microfluidics to change the way cells are exposed to cryoprotectant agents can provide new insights into the effects of osmotic stress and cellular strain rates previously unappreciated, precise methods of computational and biological data acquisition and appreciation of morphometric changes to cellular structure in response to different osmotic stresses and strain rates achieved with varying cryoprotectant exposures. Collectively, these devices and methodologies provide a means of achieving incremental improvement of oocyte and zygote cryopreservation with normalised and improved developmental competence. Finally, we look to the past and the future to acknowledge the accomplishment of leaders in the field of mammalian gamete and embryo cryobiology, their inspirational works, their tireless dissemination of information and the potential of new technologies in bioengineering to improve the efficiency and safety of gamete and embryo cryopreservation.

The differentiation of mature sperm from male germ cells requires both chromatin remodeling and compaction as well as DNA double stranded break repair of sister chromatids. We examined the function of PTIP, a protein implicated in both DNA repair and histone methylation, during spermatogenesis by using a conditional, inducible mutation in adult male mice. Loss of PTIP led to the developmental arrest of spermatocytes, testicular atrophy, and infertility. By immunostaining with specific markers for different stages of spermatogenesis and for proteins involved in DNA damage and repair mechanisms, we conclude that the lack of PTIP results in genomic instability and DNA damage resulting in the cessation of spermatogenesis in meiosis I. These data underscore the importance of PTIP in the DNA repair process associated with the development of mature spermatozoa.

Aims Mutations of cardiac sarcomere genes have been identified to cause HCM, but the molecular mechanisms that lead to cardiomyocyte hypertrophy and risk for sudden death are uncertain. The aim of this study was to examine HCM disease mechanisms at play during cardiac differentiation of human HCM specific pluripotent stem cells. Methods and results We generated a human embryonic stem cell (hESC) line carrying a naturally occurring mutation of MYPBC3 ( c . 2905 + 1 G > A ) to study HCM pathogenesis during cardiac differentiation. HCM-specific hESC-derived cardiomyocytes (hESC-CMs) displayed hallmark aspects of HCM including sarcomere disarray, hypertrophy and impaired calcium impulse propagation. HCM hESC-CMs presented a transient haploinsufficiency of cMyBP-C during cardiomyocyte differentiation, but by day 30 post-differentiation cMyBP-C levels were similar to control hESC-CMs. Gene transfer of full-length MYBPC3 during differentiation prevented hypertrophy, sarcomere disarray and improved calcium impulse propagation in HCM hESC-CMs. Conclusion(s) These findings point to the critical role of MYBPC3 during sarcomere assembly in cardiac myocyte differentiation and suggest developmental influences of MYBPC3 truncating mutations on the mature hypertrophic phenotype.

The growth of mammalian embryos in the laboratory has progressed nicely over the last 60 to 70 years. This has been important for basic studies of preimplantation embryo regulation of growth, development, and differentiation. It has also been critical for the establishment of assisted reproductive technologies in the clinical setting to treat infertility. During the first 40 to 50 years, the primary focus was on defining soluble media components, which fortunately has yielded improved success in the culture of embryos. More recently, attention has shifted to other aspects of culture beyond media composition. In this review we discuss recent advances in preimplantation embryo culture that deal with modifying the insoluble culture environment, producing culture environments that are dynamic and not static, systems that allow more precise gas phase and/or media pH regulation/monitoring, and platforms that integrate with culture systems to allow analysis of embryos and thus assist in selection of the embryos with the greatest implantation potential.

In the last decades significant advances have been made in successful cryopreservation of mammalian oocytes. Human oocyte cryopreservation has practical application in preserving fertility for individuals at risk of compromised egg quality due to cancer treatments or advanced maternal age. While oocyte cryopreservation success has increased over time, there is still room for improvement. Oocytes are susceptible to cryodamage; which collectively entails cellular damage caused by mechanical, chemical or thermal forces during the vitrification and warming process. This review will delineate many of the oocyte intracellular and extracellular structures that are/may be stressed and/or compromised during cryopreservation. This will be followed by a discussion of the theoretical basis of oocyte vitrification and warming, and a non-exhaustive review of current experimental data and clinical expectations of oocyte vitrification will be presented. Finally, a forward-thinking vision of a potential means of modifying and improving vitrification and warming procedures and success will be proposed. This review addresses theoretical and experimental evidence accumulated over the last two decades supporting the application of vitrification and warming to oocyte cryopreservation. Issues ranging from clinical needs for oocyte cryopreservation, cryopreservation-induced stresses and normal oocyte function, practical application of vitrification–warming of oocytes, and potential future directions will be discussed. In addition, we debate commonly discussed technical methods of oocyte vitrification–warming that may not necessarily be grounded in scientific knowledge. Instead these methodologies are many times theoretical, potentially empirical and commonly lack significant testing and scientific rigor. Questions include: (i) what is the best cryoprotectant? (ii) are some cryoprotectants more toxic compared with others? (iii) how should cryosolutions be mixed with cells? (iv) is there a best container for vitrification? (v) is there a threshold cooling–warming rate or is a faster rate always better? and finally (vi) should oocytes be vitrified with or without adjacent cells? With this said, it is recognized that important advancements have been made in the past decade in oocyte cryopreservation, many times through empirical findings. Finally, we propose some new areas of research that may influence future success of oocyte vitrification and warming, fully recognizing that these theories require mechanical and biological experimental testing.

Objective To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. Design Controlled clinical study. Setting An assisted reproductive technology laboratory. Patient(s) Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. Intervention(s) One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24 hours or maintained at room temperature for 4 and 24 hours. Aliquots used to assess separation techniques were processed by the following methods: washed and centrifuged in media, swim-up from washed sperm pellet, density gradient separation, density gradient followed by swim-up. DNA integrity was then measured by SCD. Main Outcome Measure(s) DNA fragmentation as measured by SCD. Result(s) There was no significant difference in fragmentation among the snap frozen, slow frozen, and wet-ice groups. Compared to other storage methods short-term storage at room temperature did not impact DNA fragmentation yet 24 hours storage significantly increased fragmentation. Swim-up, density gradient and density gradient/swim-up had significantly reduced DNA fragmentation levels compared with washed semen. Postincubation, density gradient/swim-up showed the lowest fragmentation levels. Conclusion(s) The effect of sperm processing methods on DNA fragmentation should be considered when selecting storage or separation techniques for clinical use.

background: Despite advances in in vitro manipulation of preimplantation embryos, there is still a reduction in the quality of embryos produced leading to lower pregnancy rates compared with embryos produced in vivo. We hypothesized that a dynamic microfunnel embryo culture system would enhance outcomes by better mimicking the fluid-mechanical and biochemical stimulation embryos experience in vivo from ciliary currents and oviductal contractions. methods and results: Mouse embryos were cultured in microdrop-static control, microfunnel-static control or microfunneldynamic conditions with microfluidics. All groups tested had greater than 90% total blastocyst development from zygotes after 96 h culture. Blastocyst developmental stage was significantly enhanced (P , 0.01) under dynamic microfunnel culture conditions as evidenced by an increased percentage of hatching or hatched blastocysts (Microdrop-control 31%; Microfunnel-control 23%; Microfunnel-pulsatile 71%) and significantly higher (P , 0.01) average number of cells per blastocyst (Microdrop-control 67+ 3; Microfunnel-control 60+ 3; Microfunnel-pulsatile 109+ 5). Blastocyst cell numbers in dynamic microfunnel cultures (109+ 5) more closely matched numbers obtained from in vivo grown blastocysts (144+ 9). Importantly, dynamic microfunnel culture significantly improved embryo implantation and ongoing pregnancy rates over static culture to levels approaching that of in utero derived preimplantation embryos. conclusions: The improved pregnancy outcomes along with the simple and user-friendly design of the microfluidic/microfunnel system has potential to alleviate many inefficiencies in embryo production for biomedical research, genetic gain in domestic species and assisted reproductive technologies in humans.

Male factor infertility is the sole cause of infertility in approximately 20% of infertile couples, with an additional 30% to 40% secondary to both male and female factors. Current means of evaluation of male factor infertility remains routine semen analysis including seminal volume, pH, sperm concentration, motility, and morphology. However, approximately 15% of patients with male factor infertility have a normal semen analysis and a definitive diagnosis of male infertility often cannot be made as a result of routine semen analysis. Attention has focused on the role of sperm nuclear DNA integrity in male factor infertility. Here we review the structure of human sperm chromatin, the etiology and mechanisms of sperm DNA damage, current tests available to assess sperm DNA integrity, and effect of sperm DNA integrity on reproductive outcomes.

Human embryonic stem cell (hESC) culture is routinely performed using inactivated mouse embryonic fibroblasts (MEFs) as a feeder cell layer (FL). Although these cells maintain pluripotency of hESCs, the molecular basis for this is unknown. Objectives of this study were to determine whether timing between MEF inactivation and their use as a FL influenced hESC growth and differentiation, and to begin defining the mechanism(s) involved. hESCs were plated on MEFs prepared 1 (MEF-1), 4 (MEF-4), and 7 (MEF-7) days earlier. hESC colony morphology and Oct3/4 expression levels were evaluated to determine the influence of different FLs. Significant enhancement of hESC growth (self-renewal) was observed on MEF-1 compared with MEF-4 and/or MEF-7. Conditioned media (CM) collected from MEF-1 supported significantly better hESC growth in a FL-free system compared to MEF-7 CM. Effects of MEFs on hESC growth were not caused by differences in cell density or viability, although indications of apoptosis were observed in MEF-7. Scanning electron microscopy demonstrated that MEF-7 were morphologically distinct from MEF-1 and MEF-4. Microarray analysis identified 19 genes related to apoptosis with significantly different levels of expression between MEF-1 and MEF-7. Several differentially expressed RNAs had gene ontology classifications associated with extracellular matrix (ECM) structural constituents and growth factors. Because members of Wnt signaling pathway were identified in the array analysis, we examined the ability of the Wnt1 CM and secreted frizzled-related proteins to affect hESC growth and differentiation. The addition of Wnt1 CM to both MEF-1 and MEF-7 significantly increased the number of undifferentiated colonies, while the addition of Sfrps promoted differentiation. Together, these results suggest that microenvironment, ECM, and soluble factors expressed by MEF-1 are significantly better at maintaining self-renewal and pluripotency of hESCs. Our findings have important implications in the optimization of hESC culture when MEFs are used as FL or CM is used in FL-free culture.

To investigate the luteal phase endometrial expression of leukemia inhibitor factor (LIF), insulin-like growth factor 1 (IGF-1), progesterone receptor (PR), claudin 4 (CLDN4), vascular-endothelial growth factor receptor 3 (VEGFR-3), bone morphogenetic protein 4 (BMP-4) and citokeratin 7 (CK-7), we obtained luteal phase endometrial samples from 52 women. Samples were dated and integrated using a tissue microarray (TMA). Samples were immunostained for LIF, IGF-1, PR, CLDN4, VEGFR-3, BMP-4 and CK-7. Frequencies of positive expressions at the early, mid and late luteal phases were compared by two proportions test. Concomitant expression of these proteins was assessed with Chi-square or Fischer's test. The frequency of LIF was positively correlated to the frequency of IGF-1 (r = 0.99; p0.05) and PR (r = 0.99; p0.05), and the correlation between IGF-1 and PR tended to be significant (r = 0.98; p0.1). The expression of PR was associated with the absence of CLDN4 (p0.001). Thus, expression of LIF, IGF-1 and PR are correlated during the luteal phase, and immunohistochemistry for these proteins might be used to assist in the assessment of endometrial maturation. In addition, the expression of CLDN4 and PR was not concomitant, warranting further investigation on the relationship of their endometrial expression.

Objective To evaluate influences of vitrification and warming of metaphase II (MII) mouse oocytes on survival, spindle dynamics, spindle morphology, and chromatin alignment on metaphase plates. Design Experimental animal study. Setting University animal laboratory. Animal(s) Eight-week-old B6D2F1 mice. Intervention(s) Denuded MII oocytes were used fresh (control), exposed to vitrification/warming solutions (Sol Expos), or vitrified and warmed (Vitr). Main Outcome Measure(s) Oocyte recovery and survival after warming and the influence of solution exposure and cryopreservation on spindle dynamics and chromatin alignment. Result(s) Cryopreservation of two or 10 oocytes per straw resulted in recovery (100% ± 0% and 95% ± 4%, respectively; mean ± SE) and survival (95% ± 2% and 98% ± 2%, respectively). Immediately after warming (Vitr), significantly fewer oocytes assessed with immunocytochemistry contained spindles, compared with control and Sol Expos. When oocytes were placed into a 37°C environment for 2 hours after exposure or warming, the ability to recognize spindles by immunocytochemistry was not significantly different between groups. Using live-cell time-lapse imaging with LC-Polscope, similar time-dependent spindle formation dynamics were observed. At 2 hours after collection or treatment, spindle morphology and length were not significantly different between the groups, nor was the incidence of aberrant alignment of chromatin on metaphase plates. Conclusion(s) Immediately after warming of vitrified MII oocytes, β-tubulin is depolymerized and chromatin remains condensed on the metaphase plate. Within a 2-hour period, β-tubulin repolymerizes, forming morphologically normal metaphase spindles with properly aligned chromatin.

Examination of factors regulating oocyte chromatin remodeling is crucial to circumvent embryonic aneuploidy and resulting defects. Aurora kinases (AURK) are involved in regulation of chromatin remodeling, however, little attention has been paid to AURKs in regard to oocyte maturation. Meiotically incompetent mouse oocytes contain transcripts for all three Aurk isoforms: A, B and C. Upon achieving meiotic competence, oocytes showed significant increases in transcript levels of all three Aurk isoforms and transcript levels remained unchanged as oocytes progressed through meiosis, with AurkA being the predominant isoform. Inhibition of oocyte AURKs during the prophase–metaphase I (MI) transition via inhibitor ZM447439 (ZM) had no effect on germinal vesicle breakdown. However, meiotic spindles were malformed, and microtubule organizing centers and chromatin were scattered. Chromosomal spreads of MI oocytes indicated AURK inhibition resulted in abnormal chromosome condensation. Furthermore, inhibition of AURK during prophase I–MII prevented completion of MII and extrusion of the polar body. Inhibition of AURKs during the MI–MII transition resulted in significantly fewer cells progressing to MII and induced aberrant chromatin remodeling. Further analysis indicated that inhibition of AURKs resulted in absence of histone-H3 phosphorylation at serine 10 and 28. These data suggest a ZM-sensitive AURK may be an oocyte histone-H3 kinase capable of regulating chromatin remodeling throughout oocyte meiosis, both pre- and post-MI.