This chapter describes the development of the science of cryopreservation of gametes and embryos of various species including human. It attempts to record in brief the main contributions of workers in their attempts to cryopreserve gametes and embryos. The initial difficulties faced and subsequent developments and triumphs leading to present-day state of the art are given in a concise manner. The main players and their contributions are mentioned and the authors' aim is to do justice to them. This work also attempts to ensure that credit is correctly attributed for significant advances in gamete and embryo cryopreservation. In general this chapter has tried to describe the historical development of the science of cryopreservation of gametes and embryos as accurately as possible without bias or partiality.
Early-life exposure to toxic chemicals causes irreversible morphological and physiological abnormalities that may last for a lifetime. The present study aimed to determine the toxicity effect of 3,4-Dichloroaniline (3,4-DCA) on Javanese medaka (Oryzias javanicus) embryos. Healthy embryos were exposed to various 3,4-DCA concentrations for acute toxicity (5, 10, 25, 50, and 100 mg.L-1) and sublethal toxicity (0.10, 0.50, 1.25, 2.50, and 5.00 mg.L-1) for 96 h and 20 days respectively. Acute toxicity test revealed that the median lethal concentration (96h-LC50) was 32.87 mg.L-1 (95 % CI = 27.90-38.74, R2 = 0.95). Sublethal exposure revealed that 1.25 mg.L-1 at 3 days post-exposure (3 dpe) has a significant lower heartrate (120 ± 12.3 beats/min., p embryos in 5.00 mg.L-1 and 2.50 mg.L-1 treatment groups survived at the end of the experiment. The results indicated a concentration-dependent response. The lowest observed effect concentration (LOEC) that exerted developmental deformities was 0.5 mg.L-1. Javanese medaka embryo have low sensitivity to acute toxicity of 3,4-DCA, but developmental abnormalities at sublethal concentrations were observed.
Techniques to evaluate gene expression profiling, including real-time quantitative PCR, TaqMan low-density arrays, and sufficiently sensitive cDNA microarrays, are efficient methods for monitoring human embryonic stem cell (hESC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a paucity of rapid, cost-effective, robust, yet sensitive methods for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all germ layers, including ecto-, meso-, and endoderm. To quantify the modulation of gene expression in hESCs during their propagation, expansion, and differentiation via embryoid body (EB) formation, the authors developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising set 1, and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2 to 4, respectively. The authors found that these 4 sets were not only effective in determining the relative differentiation in hESCs, but were easily reproducible. In this study, they used the HUES-7 cell line to standardize the technique, which was subsequently validated with HUES-9, NTERA-2, and mouse embryonic fibroblast cells. This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.
A new rat cytomegalovirus (RCMV) isolated from the placenta/uterus of a house rat (Rattus rattus diardii) was found to productively infect rat embryo fibroblast (REF) cells. The virus produced typical herpesvirus-like cytopathic effects characterized by a lytic infection. The well-known herpesvirus morphology was confirmed by electron microscopy. Its slow growth in cell culture indicated that the virus is belonging to subfamily Betaherpesvirinae. Electron microscopy techniques and immunohistochemistry confirmed the presence of herpesviral inclusion bodies and virus related particles in the cytoplasm and nucleus of infected cells. Hyperimmune serum against the Maastricht strain of RCMV revealed the virus identity in neutralization test, immunoperoxidase and immunofluorescence techniques. Despite typical characteristics of CMV, the viral genome is significantly different from that of Maastricht, English, UPM/Sg and UPM/Kn strains. The dissimilarities, which have not been reported before, had been confirmed by mean of restriction endonuclease analysis. The new RCMV strain, a virus that infects placenta and uterus of rats, has been named as ALL-03.
Tyrosinase inhibitors have become increasingly important targets for hyperpigmentation disease treatment. Kojic monooleate (KMO), synthesized from the esterification of kojic acid and oleic acid, has shown a better depigmenting effect than kojic acid. In this study, the process parameters include the speed of high shear, the time of high shear and the speed of the stirrer in the production of nanoemulsion containing KMO was optimized using Response Surface Methodology (RSM), as well as evaluated in terms of its physicochemical properties, safety and efficacy. The optimized condition for the formulation of KMO nanoemulsion was 8.04 min (time of high shear), 4905.42 rpm (speed of high shear), and 271.77 rpm (speed of stirrer), which resulted in a droplet size of 103.97 nm. An analysis of variance (ANOVA) showed that the fitness of the quadratic polynomial fit the experimental data with large F-values (148.79) and small p-values (p < 0.0001) and an insignificant lack of fit. The optimized nanoemulsion containing KMO with a pH value of 5.75, showed a high conductivity value (3.98 mS/cm), which indicated that the nanoemulsion containing KMO was identified as an oil-in-water type of nanoemulsion. The nanoemulsion remains stable (no phase separation) under a centrifugation test and displays accelerated stability during storage at 4, 25 and 45 °C over 90 days. The cytotoxicity assay showed that the optimized nanoemulsion was less toxic, with a 50% inhibition of cell viability (IC50) > 500 μg/mL, and that it can inhibit 67.12% of tyrosinase activity. This study reveals that KMO is a promising candidate for the development of a safe cosmetic agent to prevent hyperpigmentation.
Germline genome editing of IVF embryos is controversial because it is not directly health or lifesaving but is intended to prevent genetic diseases in yet-unborn future offspring. The following criteria are thus proposed for future clinical trials: (i) Due to medical risks, there should be cautious and judicious application while avoiding any non-essential usage, with rigorous patient counseling. (ii) Genome editing should only be performed on the entire batch of IVF embryos without initial PGT screening if all of them are expected to be affected by genetic disease. (iii) When there is a fair chance that some IVF embryos will not be affected by genetic diseases, initial PGT screening must be performed to identify unaffected embryos for transfer. (iv) IVF embryos with carrier status should not undergo germline genome editing. (v) If patients fail to conceive after the transfer of unaffected embryos, they should undergo another fresh IVF cycle rather than opt for genome editing of their remaining affected embryos. (vi) Only if the patient is unable to produce any more unaffected embryos in a fresh IVF cycle due to advanced maternal age or diminished ovarian reserves, can the genome editing of remaining affected embryos be permitted as a last resort.
To ascertain the embryotoxicity of peritoneal fluid from infertile women with endometriosis (PF-E), on mouse embryos in culture and to examine the effect of pyruvate in the culture medium on PF-E induced embryotoxicity.
This is the first report in South East Asia of a singleton frozen embryo donation pregnancy for hypergonadotrophic hypogonadism. The hormonal profile was compared with that of a control group of normal uncomplicated singleton pregnancies in Singapore. The plasma beta hCG levels were lower compared to those of our normal uncomplicated singleton pregnancies at 2 to 3 weeks after the embryo transfer but became comparable at 4 to 5 weeks after embryo transfer. The successful vaginal delivery and the obstetric complications developed in this case are discussed.
Verbena officinalis is widely used by women for maintaining general health and treating various gynaecological disorders during pregnancy. A case report has indicated that the consumption of V. officinalis induced an abortifacient effect. Hence, this study aimed to investigate the prenatal developmental toxicity of this plant according to OECD guideline (no. 414). A total of 50 pregnant female rats (dams) were distributed into five groups (n = 10); 500 mg/kg 1000 mg/2000 mg/kg and 3000 mg/kg of V. offcinalis extracts and the fifth group served as a normal control. All dams received their respective oral single daily treatment from the 6th to the 20th day of gestation. Maternal clinical toxicity signs, body weight and weight gain were recorded. Caesarean sections were performed on day 21 to evaluate embryo-foetal developmental toxicity. For dams, ovaries were harvested and weighed. The number of corpora lutea, implantation sites, and resorptions were recorded. No mortality was observed in dams, but their body weight gain was significantly reduced particularly in dams treated with 2000 and 3000 mg/kg V. officinalis. Asymmetrical distribution of implantation sites and embryos were observed. Embryo-fetotoxicity retardation was observed as evident by the decrease in foetal weight, head cranium, tail length, and higher incidence in the pre-and post-implantation loss. Some foetal skeleton abnormalities such as incomplete ossification of skull, sternebrae, and metatarsal bones were observed in foetuses of the 2000 and 3000 mg/kg V. officinalis-treated dams. LC/MS analysis identified the major constituents including geniposidic acid, tuberonic acid glucoside, luteolin 7, 3'-digalacturonide, iridotrial and apigenin. The glycosylated flavonoids such as apigenin and luteolin could be responsible for the reported prenatal developmental toxicity. In conclusion, the use of V. officinalis during pregnancy is not safe indicating evidence-based toxic effects on the reproductive performance of dams and dose-dependent risk potentials to the foetuses.
To investigate the effect of epigenetic modification on pattern, time and capacity of transcription activation of POU5F1, the key marker of pluripotency, in cloned bovine embryos.
Chromosomal abnormalities are implicated in a substantial number of human developmental syndromes, but for many such disorders little is known about the causative genes. The recently described 1q41q42 microdeletion syndrome is characterized by characteristic dysmorphic features, intellectual disability and brain morphological abnormalities, but the precise genetic basis for these abnormalities remains unknown. Here, our detailed analysis of the genetic abnormalities of 1q41q42 microdeletion cases identified TP53BP2, which encodes apoptosis-stimulating protein of p53 2 (ASPP2), as a candidate gene for brain abnormalities. Consistent with this, Trp53bp2-deficient mice show dilation of lateral ventricles resembling the phenotype of 1q41q42 microdeletion patients. Trp53bp2 deficiency causes 100% neonatal lethality in the C57BL/6 background associated with a high incidence of neural tube defects and a range of developmental abnormalities such as congenital heart defects, coloboma, microphthalmia, urogenital and craniofacial abnormalities. Interestingly, abnormalities show a high degree of overlap with 1q41q42 microdeletion-associated abnormalities. These findings identify TP53BP2 as a strong candidate causative gene for central nervous system (CNS) defects in 1q41q42 microdeletion syndrome, and open new avenues for investigation of the mechanisms underlying CNS abnormalities.
Purpose: Our center is known as a pioneer center initiating oncofertility service since 2010 in Japan. We demonstrate our transition of this service in regional university hospitals ingenuously. Methods: We compared two phases of service: initial phase (2011 and 2012) and current phase (2019). The comparison included the number of women attending the oncofertility unit, diversity of breast cancer cases, the acceptability of preservation service, and the type of fertility preservation (FP) option offered in between these phases. Results: A total of 58 women were seen during the initial phase as compared with 41 women in the later phase. The mean age at diagnosis was not significantly different between the two periods. The majority of them were married and diagnosed with stage II luminar type. The current phase had a tendency to have a higher anti-Müllerian hormone level although not reaching significance. At least 50% of them declined FP and 84.5% never received ovarian control stimulation in the initial phase. Otherwise, 61% used aromatase inhibitor in the current phase. Only 15.5% in the initial phase received control ovarian stimulation whereas 63.4% in the current phase received it. The ovarian tissue cryopreservation was highly chosen during the initial phase (25.9%), whereas embryo cryopreservation (39%) was highly opted for during the current phase. All of our parameters are comparable between these two phases (p > 0.05). Conclusion: The significant changes of oncofertility practice were observed mainly due to the understanding of the oncofertility concept among reproductive physicians and the acceptance environment, including standard guidelines, supportive society, as well as advancements in cryobiology technique.
In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.
BACKGROUND: Ooplasmic transfer (OT) technique or cytoplasmic transfer is an emerging technique with relative success, having a significant status in assisted reproduction. This technique had effectively paved the way to about 30 healthy births worldwide. Though OT has long been invented, proper evaluation of the efficacy and risks associated with this critical technique has not been explored properly until today. This review thereby put emphasis upon the applications, efficacy and adverse effects of OT techniques in human.
MAIN BODY: Available reports published between January 1982 and August 2017 has been reviewed and the impact of OT on assisted reproduction was evaluated. The results consisted of an update on the efficacy and concerns of OT, the debate on mitochondrial heteroplasmy, apoptosis, and risk of genetic and epigenetic alteration.
SHORT CONCLUSION: The application of OT technique in humans demands more clarity and further development of this technique may successfully prove its utility as an effective treatment for oocyte incompetence.
MicroRNAs are small non-coding RNAs that play crucial roles in the regulation of gene expression and protein synthesis during brain development. MiR-3099 is highly expressed throughout embryogenesis, especially in the developing central nervous system. Moreover, miR-3099 is also expressed at a higher level in differentiating neurons in vitro, suggesting that it is a potential regulator during neuronal cell development. This study aimed to predict the target genes of miR-3099 via in-silico analysis using four independent prediction algorithms (miRDB, miRanda, TargetScan, and DIANA-micro-T-CDS) with emphasis on target genes related to brain development and function. Based on the analysis, a total of 3,174 miR-3099 target genes were predicted. Those predicted by at least three algorithms (324 genes) were subjected to DAVID bioinformatics analysis to understand their overall functional themes and representation. The analysis revealed that nearly 70% of the target genes were expressed in the nervous system and a significant proportion were associated with transcriptional regulation and protein ubiquitination mechanisms. Comparison of in situ hybridization (ISH) expression patterns of miR-3099 in both published and in-house-generated ISH sections with the ISH sections of target genes from the Allen Brain Atlas identified 7 target genes (Dnmt3a, Gabpa, Gfap, Itga4, Lxn, Smad7, and Tbx18) having expression patterns complementary to miR-3099 in the developing and adult mouse brain samples. Of these, we validated Gfap as a direct downstream target of miR-3099 using the luciferase reporter gene system. In conclusion, we report the successful prediction and validation of Gfap as an miR-3099 target gene using a combination of bioinformatics resources with enrichment of annotations based on functional ontologies and a spatio-temporal expression dataset.
Ultra-slow cortical oscillatory activity of 1-100 mHz has been recorded in human by electroencephalography and in dissociated cultures of cortical rat neurons, but the underlying mechanisms remain to be elucidated. This study presents a computational model of ultra-slow oscillatory activity based on the interaction between neurons and astrocytes. We predict that the frequency of these oscillations closely depends on activation of astrocytes in the network, which is reflected by oscillations of their intracellular calcium concentrations with periods between tens of seconds and minutes. An increase of intracellular calcium in astrocytes triggers the release of adenosine triphosphate from these cells which may alter transmission at nearby synapses by increasing or decreasing neurotransmitter release. These results provide theoretical support for the emerging awareness of astrocytes as active players in the regulation of neural activity and identify neuron-astrocyte interactions as a potential primary mechanism for the emergence of ultra-slow cortical oscillations.