Lack of involvement of known DNA methyltransferases in familial hydatidiform mole implies the involvement of other factors in establishment of imprinting in the human female germline

BACKGROUND: Differential methylation of the two alleles is a hallmark of imprinted genes. Correspondingly, loss of DNA methyltransferase function results in aberrant imprinting and abnormal post-fertilization development. In the mouse, mutations of the oocyte-specific isoform of the DNA methyltransferase Dnmt1 (Dnmt1o) and of the methyltransferase-like Dnmt3L gene result in specific failures of imprint establishment or maintenance, at multiple loci. We have previously shown in humans that an analogous inherited failure to establish imprinting at multiple loci in the female germline underlies a rare phenotype of recurrent hydatidiform mole. RESULTS: We have identified a human homologue of the murine Dnmt1o and assessed its pattern of expression. Human DNMT1o mRNA is detectable in mature oocytes and early fertilized embryos but not in any somatic tissues analysed. The somatic isoform of DNMT1 mRNA, in contrast, is not detectable in human oocytes. In the previously-described family with multi-locus imprinting failure, mutation of DNMT1o and of the other known members of this gene family has been excluded. CONCLUSIONS: Mutation of the known DNMT genes does not underlie familial hydatidiform mole, at least in the family under study. This suggests that trans-acting factors other than the known methyltransferases are required for imprint establishment in humans, a concept that has indirect support from recent biochemical studies of DNMT3L

Structural and biocompatibility challenges for 3D printed microfluidic devices for IVF

In Vitro Fertilization (IVF) is a widely used treatment for infertility, but success rates for UK women under 35 stand at approximately 32%. Culture conditions significantly affect in-vitro embryo development and treatment efficacy. Over 40 years, IVF procedures evolved, with microfluidic platforms emerging to enhance culture conditions. These platforms, designed to mimic a natural environment, show promise without negatively affecting development rates. However, potential impacts on embryo characteristics require further evaluation. The study introduces a microfluidic concept compatible with time-lapse microscopy. Two prototyping methods are compared, being soft lithography in PDMS and 3D printing in HTL resin. Results indicate successful prototype detection and loading efficiency, with the soft lithographic method showing a lower assembly yield. 3D printing facilitates rapid design, in particular for high aspect ratio microfluidic devices. However, viability assessments suggest additional steps are required to exclude material embryo toxicity of the resin

Intracellular oxygen metabolism during bovine oocyte and preimplantation embryo development

We report a novel method to profile intrcellular oxygen concentration (icO2) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO2 associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO2 and decreasing oxygen consumption to increase icO2. Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO2 measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO2 for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO2 of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO2 was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9–4 × 106 copies and did not correlate with icO2. These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos

Trophectoderm non-coding RNAs reflect the higher metabolic and more invasive properties of young maternal age blastocysts

Increasing female age is accompanied by a corresponding fall in her fertility. This decline is influenced by a variety of factors over an individual’s life course including background genetics, local environment and diet. Studying both coding and non-coding RNAs of the embryo could aid our understanding of the causes and/or effects of the physiological processes accompanying the decline including the differential expression of sub-cellular biomarkers indicative of various diseases. The current study is a post-hoc analysis of the expression of trophectoderm RNA data derived from a previous high throughput study. Its main aim is to determine the characteristics and potential functionalities that characterize long non-coding RNAs. As reported previously, a maternal age-related component is potentially implicated in implantation success. Trophectoderm samples representing the full range of maternal reproductive ages were considered in relation to embryonic implantation potential, trophectoderm transcriptome dynamics and reproductive maternal age. The long non-coding RNA (lncRNA) biomarkers identified here are consistent with the activities of embryo-endometrial crosstalk, developmental competency and implantation and share common characteristics with markers of neoplasia/cancer invasion. Corresponding genes for expressed lncRNAs were more active in the blastocysts of younger women are associated with metabolic pathways including cholesterol biosynthesis and steroidogenesis

O-275 Is there a case for ovarian tissue cryopreservation in young girls and Turner syndrome patients?

Abstract The development of new cancer drugs and drug combinations has led to a dramatic increase in the survival rates of prepubertal and postpubertal girls undergoing myeloablative treatment for malignant or non-malignant diseases. This includes girls who will receive high doses of alkylating agents, total body irradiation, or high radiation doses to the craniospinal, abdominal or pelvic area. Chemotherapy is also used as a conditioning regimen before hematopoietic stem cell transplantation for treatment of, for example, beta thalassaemia or homozygous sickle-cell anaemia. The corollary of improved treatment outcomes of childhood diseases is that many survivors are now at risk of multi-faceted, chronic morbidity, impaired quality-of-life and psychosocial problems. Depending on the nature and duration of their treatment some, but not all, young patients facing fertility threatening diagnosis and treatments will have an increased risk of adverse pregnancy outcomes and premature ovarian insufficiency (POI) as their future fertile potential will be defined by the number of follicles remaining in their ovaries following completion of their chemotherapy/radiotherapy. While a fully functioning hypothalamic-pituitary-ovarian axis and uterus are vital to the reproductive potential of adult females, Ovarian tissue cryopreservation (OTC) is now being used to safeguard the future fertility of prepubertal and adolescent girls and young women undergoing treatments that have a high risk of causing POI. OTC is increasingly advocated for young girls receiving an early diagnosis of the most common sex chromosome abnormality- Turner Syndrome (TS) who have a high risk of POI. In theory the ovaries of girls, including TS patients with an adequate ovarian reserve, are well suited to OTC as their ovaries contain high numbers primordial follicles which can be used to preserve future fertility. Ovarian tissue can be removed laparoscopically and early staged follicles cryopreserved in situ within slices of cortex. The operative approaches used for tissue removal from paediatric and adolescent patients are variable and invasive and are dependent on patient age, health and ovarian reserve at the time of tissue recovery. Extensive research has shown that ovarian cryopreservation techniques are robust and that experienced practitioners can achieve high levels of survival of both the ovarian stroma and follicle compartments post thaw. Cryobanked ovarian cortex collected from prepubertal and postpubertal girls has been shown to function normally and support fertility, pregnancy to term and the birth of healthy babies following orthotopic autografting in adulthood. Regardless of patient age at tissue preservation, autografting of frozen-thawed ovarian cortex at either the site of the retained ovary or into the peritoneal cavity results in an immediate and significant follicle loss due to ischaemia and accelerated apoptosis. Those primordial follicles that survive graft revascularization have been shown to have the capacity to grow to preovulatory stages, to reinstate hormonal function and to undergo ovulation and restore natural fertility. Alternatively, follicle growth can be induced in autografted tissue by controlled ovarian stimulation before oocyte harvest for insemination in vitro using assisted reproductive technologies. Graft functional longevity remains variable between patients and may extend from months to years. Alternative methods for fertility restoration such as the complete in vitro growth and maturation of oocytes and the construction of artificial ovaries remain unproven in humans. While OTC can be recommended for young girls at high risk of developing ovarian insufficiency for genetic reasons or following high dose chemotherapy and/or irradiation, to date very few young patients with stored ovarian tissue have used their tissue for fertility restoration. OTC therefore remains experimental and further research is needed to establish the risks, benefits, efficacy, safety and ethical and clinical frameworks for offering fertility preservation to girls during childhood and adolescence and to girls with TS.</jats:p

Therapeutic Potential of In Vitro–Derived Oocytes for the Restoration and Treatment of Female Fertility

Considerable progress has been made with the development of culture systems for the in vitro growth and maturation (IVGM) of oocytes from the earliest-staged primordial follicles and from the more advanced secondary follicles in rodents, ruminants, nonhuman primates, and humans. Successful oocyte production in vitro depends on the development of a dynamic culture strategy that replicates the follicular microenvironment required for oocyte activation and to support oocyte growth and maturation in vivo while enabling the coordinated and timely acquisition of oocyte developmental competence. Significant heterogeneity exists between the culture protocols used for different stages of follicle development and for different species. To date, the fertile potential of IVGM oocytes derived from primordial follicles has been realized only in mice. Although many technical challenges remain, significant advances have been made, and there is an increasing consensus that complete IVGM will require a dynamic, multiphase culture approach. The production of healthy offspring from in vitro–produced oocytes in a secondary large animal species is a vital next step before IVGM can be tested for therapeutic use in humans