Mouse embryo assay to evaluate polydimethylsiloxane (PDMS) embryo-toxicity

In vitro embryo culture to support In Vitro Fertilization (IVF) procedures is a well-established but still critical technique. In the last decade first attempts to use microfluidic devices in IVF have shown positive results, enabling to control the culture conditions and to preserve the quality of the embryos during their development. In this study we completed an industry standard mouse embryo assay (MEA) to exclude potential toxic effects of PDMS

Survival from cancer in young people: An overview of late effects focusing on reproductive health

This paper provides a summary of the areas of survival from childhood, teenage and young adult cancers and the significant late effects that can arise from treatment; with particular focus on the area of reproductive health and the impact on both fertility and pregnancy. To complete this review, Web of Science and MEDLINE were used. Search terms included: “"survival AND childhood OR teenage OR young adult cancer", "late effects", "childhood cancer", "teenage AND/OR young adult cancer", AND "fertility after cancer" OR "pregnancy AND cancer" OR "fertility preservation”. Additionally, the clinical expertise of the authors was drawn upon. Childhood cancer is a thankfully rare occurrence; however, the incidence is increasing. Survival rates remain high and this means that a growing population of childhood and young adult cancer survivors are reaching adulthood. For some of these adults, although cured of their cancer, they are now facing a future with lasting effects on their health from their treatments. These effects, commonly referred to as late effects, are defined as health problems related either directly to the underlying cancer or to its treatment and which occur months or years after treatment has finished. Reproductive health is an important consideration for these patients, and although many will be able to conceive naturally, some will exhibit impaired fertility after their treatments. This can include difficulties at all points along the path from conception to delivery of a live, healthy offspring. High‐quality, large‐population evidence is sparse in many areas relating to fertility risk from treatment and the maternal and fetal health of childhood cancer survivors. Yet given the potential for complications, the authors advocate consideration of fertility at the time of diagnosis and before potentially gonadotoxic treatment

The use of a novel microfluidic culture device and predictive metabolic profiling as a means to improve murine embryo developmental competence in vitro

Successful embryo development in vitro is directly dependent on the provision of an optimal culture environment that supports coordinated embryonic cell division, metabolism, and genetic and epigenetic development. A number of attempts have been made over recent years to use microfluidic devices in IVF as a means to control the culture environment and so improve embryo developmental competence (quality) in vitro. In this study we have designed, engineered and tested a novel microfluidic device for the in vitro production of murine embryos from the 1 cell zygote stage to the blastocyst stage. Soft lithography was used to prepare microfluidic devices in polydimethylsiloxane (PDMS). The microfluidic device consists of a 400 nL circular chamber (radius 750 mm) where 10 embryos can be loaded, kept in static culture for the full period of culture and visualized by optical and fluorescent microscopy. A series of 2 experiments were conducted to evaluate the efficacy of our microfluidic device for mouse embryo culture. Cryopreserved, IVF-derived, mouse embryos of strain C57BL/6N, provided by MRC Harwell, UK were cultured in KSOM media. Microfluidic culture was used in conjunction with non-invasive analysis of glucose (G), pyruvate (P) and lactate (L) metabolism in spent zygote culture media as a means to improve embryo quality. In both experimental series, data from microfluidic cultures were compared to equivalent end point analyses of control embryos grown in conventional microdrop cultures under oil. Experiment 1: 2 cell embryos were thawed and cultured in groups of 8-10 in microfluidic devices (n=46) or 10µl control (n=32) drops for 3 days at 37oC under 5%CO2/5%O2/N2 balance. Embryos were removed to individual culture drops for 24h for analysis of energy substrate turnover using the method of Guerif et al. (PLOS ONE, 2013) followed by transfer to fibronectin-coated dishes for assessment of attachment and outgrowth according to the method of Hannan et al. (Endocrinology, 152 (12), p4948-4956, 2011). Blastocyst grade, hatching, attachment, outgrowth rates, and pyruvate and glucose consumption were assessed and were similar between device and control groups (P>0.05). However, lactate output was significantly reduced following device culture vs controls (4.1±0.8 vs 1.4±0.3 pmol/embryo/hr, P=<0.0001). GPL metabolism did not predict embryo attachment or outgrowth in either culture environment. Experiment 2: 1 cell zygotes were cultured individually overnight for analysis of GPL metabolism and assigned to culture groups based on pyruvate consumption, with 1 device and 1 microdrop group per tertile per culture with 10 embryos per group (total n=60 device and n=60 control). Following group culture, individual blastocyst pyruvate consumption was reduced (5.4±2.2 vs 12±1.5 pmol/embryo/hr, P=<0.0001). Pyruvate consumption tertile was unaffected by device culture. Device culture was non-toxic and did not affect embryo development. However, blastocyst pyruvate consumption and lactate output were reduced compared to controls. This may suggest microfluidic culture can be utilised to achieve a controlled, moderate metabolic phenotype, reducing variation between embryo metabolism

Isolation and expression of the human gametocyte-specific factor 1 gene (GTSF1) in fetal ovary, oocytes, and preimplantation embryos

Purpose: Gametocyte-specific factor 1 has been shown in other species to be required for the silencing of retrotransposons via the Piwi-interacting RNA (piRNA) pathway. In this study, we aimed to isolate and assess expression of transcripts of the gametocyte-specific factor 1 (GTSF1) gene in the human female germline and in preimplantation embryos. Methods: Complementary DNA (cDNA) libraries from human fetal ovaries and testes, human oocytes and preimplantation embryos and ovarian follicles isolated from an adult ovarian cortex biopsy were used to as templates for PCR, cloning and sequencing, and real time PCR experiments of GTSF1 expression. Results: GTSF1 cDNA clones that covered the entire coding region were isolated from human oocytes and preimplantation embryos. GTSF1 mRNA expression was detected in archived cDNAs from staged human ovarian follicles, germinal vesicle (GV) stage oocytes, metaphase II oocytes, and morula and blastocyst stage preimplantation embryos. Within the adult female germline, expression was highest in GV oocytes. GTSF1 mRNA expression was also assessed in human fetal ovary and was observed to increase during gestation, from 8 to 21 weeks, during which time oogonia enter meiosis and primordial follicle formation first occurs. In human fetal testis, GTSF1 expression also increased from 8 to 19 weeks. Conclusions: To our knowledge, this report is the first to describe the expression of the human GTSF1 gene in human gametes and preimplantation embryos

On-chip mouse embryo culture: Evaluation of effects of uterine cells-conditioned media on embryo development and gene expression

Microfluidics has recently been proposed as a method to overcome the limitations of traditional oocyte and embryo culture methods. In this work, we report the use of a microfluidic polydimethylsiloxane device as promising alternative for in vitro embryo culture, and we have evaluated the effects of cells- conditioned media (CM) on embryo development. The microfluidic device was fabricated using traditional soft-lithographic technique. To produce CM, mouse uterine epithelial cells (Creative Bioarray, USA) were cultured in KSOM (Merck Millipore, UK) for 24 h. The CM was used to culture groups of 12, 1 cell murine embryos (B6C3F1xB6D2F1 strain, EmbryoTech, USA) into a microfluidic device. Control embryos were cultured in the device using KSOM. We compared blastocyst rates of embryos cultured in CM with those obtained using KSOM. The effect of treatment on embryo gene expression was assessed in cDNAs generated from individual stage matched, blastocysts using a custom, real time PCR array. Developmental ability of mouse embryos in the presence of CM was significantly higher (p<0.05) in comparison with control media. Blastocyst rates for the CM (n=15 devices, 180 embryos) and control media (n=15 devices, 180 embryos) groups were 68.9% and 45.1%, respectively. qPCR results showed that expression of Makorin Ring Finger Protein (MKRN, p=0.036), DNA Methyltransferase 3β (DNMT3β, p=0.012), DNA (Cytosine -5-)-Methyltransferase 3-Like (DNMT3L, p=0.043), Histone Acetyltransferase 1 (HAT1, p=0.006), Keratin 18 (KRT18, p=0.028), and Ubiquitin Like With PHD And Ring Finger Domains 1 (UHRF1, p=0.043) was significantly different between the treatment groups. Specifically, we observed in the CM group increased expression of DNMT3β and DNMT3L, which play an important role in early embryo development. Those finding revealed that the new microfluidic device supports mouse preimplantation embryo development in vitro. Uterine epithelial cells-conditioned medium has the potential to enhance blastocyst development. Further investigations are required to identify the mechanism of this effect

The impact of maternal age on gene expression during the GV to MII transition in euploid human oocytes

STUDY QUESTION Are there age-related differences in gene expression during the germinal vesicle (GV) to metaphase II (MII) stage transition in euploid human oocytes? SUMMARY ANSWER A decrease in mitochondrial-related transcripts from GV to MII oocytes was observed, with a much greater reduction in MII oocytes with advanced age. WHAT IS KNOWN ALREADY Early embryonic development is dependent on maternal transcripts accumulated and stored within the oocyte during oogenesis. Transcriptional activity of the oocyte, which dictates its ultimate developmental potential, may be influenced by age and explain the reduced competence of advanced maternal age (AMA) oocytes compared with the young maternal age (YMA). Gene expression has been studied in human and animal oocytes; however, RNA sequencing could provide further insights into the transcriptome profiling of GV and in vivo matured MII euploid oocytes of YMA and AMA patients. STUDY DESIGN, SIZE, DURATION Fifteen women treated for infertility in a single IVF unit agreed to participate in this study. Five GV and 5 MII oocytes from 6, 21–26 years old women (YMA cohort) and 5 GV and 6 MII oocytes from 6, 41–44 years old women (AMA cohort) undergoing IVF treatment were donated. The samples were collected within a time frame of 4 months. RNA was isolated and deep sequenced at the single-cell level. All donors provided either GV or MII oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS Cumulus dissection from donated oocytes was performed 38 h after hCG injection, denuded oocytes were inserted into lysis buffer supplemented with RNase inhibitor. The samples were stored at −80°C until further use. Isolated RNA from GV and MII oocytes underwent library preparation using an oligo deoxy-thymidine (dT) priming approach (SMART-Seq v4 Ultra Low Input RNA assay; Takara Bio, Japan) and Nextera XT DNA library preparation assay (Illumina, USA) followed by deep sequencing. Data processing, quality assessment and bioinformatics analysis were performed using source-software, mainly including FastQC, HISAT2, StringTie and edgeR, along with functional annotation analysis, while scploid R package was employed to determine the ploidy status. MAIN RESULTS AND THE ROLE OF CHANCE Following deep sequencing of single GV and MII oocytes in both YMA and AMA cohorts, several hundred transcripts were found to be expressed at significantly different levels. When YMA and AMA MII oocyte transcriptomes were compared, the most significant of these were related to mitochondrial structure and function, including biological processes, mitochondrial respiratory chain complex I assembly and mitochondrial translational termination (false discovery rate (FDR) 6.0E−10 to 1.2E−7). These results indicate a higher energy potential of the YMA MII cohort that is reduced with ageing. Other biological processes that were significantly higher in the YMA MII cohort included transcripts involved in the translation process (FDR 1.9E−2). Lack of these transcripts could lead to inappropriate protein synthesis prior to or upon fertilisation of the AMA MII oocytes. LARGE SCALE DATA The RNA sequencing data were deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo), under the accession number: GSE164371. LIMITATIONS, REASONS FOR CAUTION The relatively small sample size could be a reason for caution. However, the RNA sequencing results showed homogeneous clustering with low intra-group variation and five to six biological replicates derived from at least three different women per group minimised the potential impact of the sample size. WIDER IMPLICATIONS OF THE FINDINGS Understanding the effects of ageing on the oocyte transcriptome could highlight the mechanisms involved in GV to MII transition and identify biomarkers that characterise good MII oocyte quality. This knowledge has the potential to guide IVF regimes for AMA patients. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Medical Research Council (MRC Grant number MR/K020501/1)

Endometrial stromal cells of women with recurrent miscarriage fail to discriminate between high- and low-quality human embryos

Background The aetiology of recurrent miscarriage (RM) remains largely unexplained. Women with RM have a shorter time to pregnancy interval than normally fertile women, which may be due to more frequent implantation of non-viable embryos. We hypothesized that human endometrial stromal cells (H-EnSCs) of women with RM discriminate less effectively between high-and low-quality human embryos and migrate more readily towards trophoblast spheroids than H-EnSCs of normally fertile women. Methodology/Principal Findings Monolayers of decidualized H-EnSCs were generated from endometrial biopsies of 6 women with RM and 6 fertile controls. Cell-free migration zones were created and the effect of the presence of a high-quality (day 5 blastocyst, n = 13), a low-quality (day 5 blastocyst with three pronuclei or underdeveloped embryo, n = 12) or AC-1M88 trophoblast cell line spheroid on H-ESC migratory activity was analyzed after 18 hours. In the absence of a spheroid or embryo, migration of H-EnSCs from fertile or RM women was similar. In the presence of a low-quality embryo in the zone, the migration of H-EnSCs of control women was inhibited compared to the basal migration in the absence of an embryo (P<0.05) and compared to the migration in the presence of high-quality embryo (p<0.01). Interestingly, the migratory response H-EnSCs of women with RM did not differ between high- and low-quality embryos. Furthermore, in the presence of a spheroid their migration was enhanced compared to the H-EnSCs of controls (p<0.001). Conclusions H-EnSCs of fertile women discriminate between high- and low-quality embryos whereas H-EnSCs of women with RM fail to do so. H-EnSCs of RM women have a higher migratory response to trophoblast spheroids. Future studies will focus on the mechanisms by which low-quality embryos inhibit the migration of H-EnSCs and how this is deregulated in women with RM

A Fast and Reliable Method for Simultaneous Waveform, Amplitude and Latency Estimation of Single-Trial EEG/MEG Data

The amplitude and latency of single-trial EEG/MEG signals may provide valuable information concerning human brain functioning. In this article we propose a new method to reliably estimate single-trial amplitude and latency of EEG/MEG signals. The advantages of the method are fourfold. First, no a-priori specified template function is required. Second, the method allows for multiple signals that may vary independently in amplitude and/or latency. Third, the method is less sensitive to noise as it models data with a parsimonious set of basis functions. Finally, the method is very fast since it is based on an iterative linear least squares algorithm. A simulation study shows that the method yields reliable estimates under different levels of latency variation and signal-to-noise ratioÕs. Furthermore, it shows that the existence of multiple signals can be correctly determined. An application to empirical data from a choice reaction time study indicates that the method describes these data accurately

Preservation of Auditory P300-Like Potentials in Cortical Deafness

The phenomenon of blindsight has been largely studied and refers to residual abilities of blind patients without an acknowledged visual awareness. Similarly, “deaf hearing” might represent a further example of dissociation between detection and perception of sounds