Developing a novel device, Eggcell, to improve temperature stability during oocyte collection for IVF.

RESEARCH QUESTION: What temperature fluctuations are oocytes exposed to during oocyte retrieval? Can an alternative method of oocyte retrieval be designed to minimize these fluctuations? DESIGN: Mock oocyte retrieval procedures were performed to investigate the change in temperature when the follicular fluid is drained into collection tubes and when the fluid is subsequently poured into dishes to allow identification of the cumulus-oocyte complex (COC). A new device, the Eggcell, has been designed that addresses the problem of these temperature fluctuations. To confirm its safety and demonstrate the clinical applicability of Eggcell, laboratory validation was performed prior to use with human participants (n = 15). RESULTS: Eggcell meets its design specification to provide temperature stability within the physiological range for aspirated follicular fluid. The COC can be successfully retained within the chamber (n = 180) without evidence of loss or damage to the oocytes or compromise of fertilization rate, blastocyst development or clinical outcome. CONCLUSIONS: This study has demonstrated the successful first stages of development of a new medical device. Further studies are needed for comparative evaluation of clinical outcome with standard technology.

Erratum: Revisiting the Warnock rule.

This corrects the article DOI: 10.1038/nbt.4015.

Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease.

Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly, techniques used in proof-of-concept studies involving abnormally fertilized human zygotes were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. After optimization, mtDNA carryover was reduced to <2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease, but it may not guarantee prevention.

To freeze or not to freeze embryos: clarity, confusion and conflict.

Although embryo freezing is a routine clinical practice, there is little contemporary evidence on how couples make the decision to freeze their surplus embryos, or of their perceptions during that time. This article describes a qualitative study of 16 couples who have had in vitro fertilisation (IVF) treatment. The study question was 'What are the personal and social factors that patients consider when deciding whether to freeze embryos?' We show that while the desire for a baby is the dominant drive, couples' views revealed more nuanced and complex considerations in the decision-making process. It was clear that the desire to have a baby influenced couples' decision-making and that they saw freezing as 'part of the process'. However, there were confusions associated with the term 'freezing' related to concerns about the safety of the procedure. Despite being given written information, couples were confused about the practical aspects of embryo freezing, which suggests they were preoccupied with the immediate demands of IVF. Couples expressed ethical conflicts about freezing 'babies'. We hope the findings from this study will inform clinicians and assist them in providing support to couples confronted with this difficult decision-making.

Concise reviews: Assisted reproductive technologies to prevent transmission of mitochondrial DNA disease.

While the fertilized egg inherits its nuclear DNA from both parents, the mitochondrial DNA is strictly maternally inherited. Cells contain multiple copies of mtDNA, each of which encodes 37 genes, which are essential for energy production by oxidative phosphorylation. Mutations can be present in all, or only in some copies of mtDNA. If present above a certain threshold, pathogenic mtDNA mutations can cause a range of debilitating and fatal diseases. Here, we provide an update of currently available options and new techniques under development to reduce the risk of transmitting mtDNA disease from mother to child. Preimplantation genetic diagnosis (PGD), a commonly used technique to detect mutations in nuclear DNA, is currently being offered to determine the mutation load of embryos produced by women who carry mtDNA mutations. The available evidence indicates that cells removed from an eight-cell embryo are predictive of the mutation load in the entire embryo, indicating that PGD provides an effective risk reduction strategy for women who produce embryos with low mutation loads. For those who do not, research is now focused on meiotic nuclear transplantation techniques to uncouple the inheritance of nuclear and mtDNA. These approaches include transplantation of any one of the products or female meiosis (meiosis II spindle, or either of the polar bodies) between oocytes, or the transplantation of pronuclei between fertilized eggs. In all cases, the transferred genetic material arises from a normal meiosis and should therefore, not be confused with cloning. The scientific progress and associated regulatory issues are discussed.

Therapeutic potential of somatic cell nuclear transfer for degenerative disease caused by mitochondrial DNA mutations.

Induced pluripotent stem cells (iPSCs) hold much promise in the quest for personalised cell therapies. However, the persistence of founder cell mitochondrial DNA (mtDNA) mutations limits the potential of iPSCs in the development of treatments for mtDNA disease. This problem may be overcome by using oocytes containing healthy mtDNA, to induce somatic cell nuclear reprogramming. However, the extent to which somatic cell mtDNA persists following fusion with human oocytes is unknown. Here we show that human nuclear transfer (NT) embryos contain very low levels of somatic cell mtDNA. In light of a recent report that embryonic stem cells can be derived from human NT embryos, our results highlight the therapeutic potential of NT for mtDNA disease, and underscore the importance of using human oocytes to pursue this goal.

Comparing the burden: what can we learn by comparing regulatory frameworks in abortion and fertility services?

In the UK, regulation of clinical services is being restructured. We consider two clinical procedures, abortion and IVF treatment, which have similar ethical and political sensitivities. We consider factors including the law, licensing, inspection, amount of paperwork and reporting requirements, the reception by practitioners and costs, to establish which field has the greater 'regulatory burden'. We test them based on scientific, ethical, social, political factors that might explain differences. We find that regulatory burden borne by IVF services is greater than in abortion, but none of the explanatory theses can provide a justification of this phenomenon. We offer an alternative explanation based on regulatory 'overspill' from research regulation and policy making, conceptualisation of risk regulation and a high public profile that locks a regulator into self-preservation.

Production and validation of a good manufacturing practice grade human fibroblast line for supporting human embryonic stem cell derivation and culture.

INTRODUCTION: The development of reproducible methods for deriving human embryonic stem cell (hESC) lines in compliance with good manufacturing practice (GMP) is essential for the development of hESC-based therapies. Although significant progress has been made toward the development of chemically defined conditions for the maintenance and differentiation of hESCs, efficient derivation of new hESCs requires the use of fibroblast feeder cells. However, GMP-grade feeder cell lines validated for hESC derivation are not readily available. METHODS: We derived a fibroblast cell line (NclFed1A) from human foreskin in compliance with GMP standards. Consent was obtained to use the cells for the production of hESCs and to generate induced pluripotent stem cells (iPSCs). We compared the line with a variety of other cell lines for its ability to support derivation and self-renewal of hESCs. RESULTS: NclFed1A supports efficient rates (33%) of hESC colony formation after explantation of the inner cell mass (ICM) of human blastocysts. This compared favorably with two mouse embryonic fibroblast (MEF) cell lines. NclFed1A also compared favorably with commercially available foreskin fibroblasts and MEFs in promoting proliferation and pluripotency of a number of existing and widely used hESCs. The ability of NclFed1A to maintain self-renewal remained undiminished for up to 28 population doublings from the master cell bank. CONCLUSIONS: The human fibroblast line Ncl1Fed1A, produced in compliance with GMP standards and qualified for derivation and maintenance of hESCs, is a useful resource for the advancement of progress toward hESC-based therapies in regenerative medicine.

A novel isolator-based system promotes viability of human embryos during laboratory processing.

In vitro fertilisation (IVF) and related technologies are arguably the most challenging of all cell culture applications. The starting material is a single cell from which one aims to produce an embryo capable of establishing a pregnancy eventually leading to a live birth. Laboratory processing during IVF treatment requires open manipulations of gametes and embryos, which typically involves exposure to ambient conditions. To reduce the risk of cellular stress, we have developed a totally enclosed system of interlinked isolator-based workstations designed to maintain oocytes and embryos in a physiological environment throughout the IVF process. Comparison of clinical and laboratory data before and after the introduction of the new system revealed that significantly more embryos developed to the blastocyst stage in the enclosed isolator-based system compared with conventional open-fronted laminar flow hoods. Moreover, blastocysts produced in the isolator-based system contained significantly more cells and their development was accelerated. Consistent with this, the introduction of the enclosed system was accompanied by a significant increase in the clinical pregnancy rate and in the proportion of embryos implanting following transfer to the uterus. The data indicate that protection from ambient conditions promotes improved development of human embryos. Importantly, we found that it was entirely feasible to conduct all IVF-related procedures in the isolator-based workstations.

Ovulation induction with clomifene: a primary care perspective.

Infertility affects one in seven couples during their lifetime. Approximately one-quarter of these will have an ovulatory disorder contributing to their inability to conceive. Ovulatory disorders represent the simplest form of infertility to treat, and where this is not a result of ovarian failure or poor ovarian reserve most women require ovulation induction with clomifene citrate (CC). This review aims to examine the role of CC in a general practice setting. CC is a simple, relatively safe, easily administered and well-tolerated efficacious drug. There is, however, a 10% risk of multiple births associated with its use. CC has been used in general practice for many years and continues to be used. Currently, guidelines do not describe its use in the general practice setting and the evidence for monitoring its use with mid-luteal progesterone estimation or ultrasound scanning is conflicting.

The procurement of cells for the derivation of human embryonic stem cell lines for therapeutic use: recommendations for good practice.

The donation of human embryos for the derivation of embryonic stem cell lines that may be used in the development of therapeutic products raises more complex ethical, practical and regulatory problems than the donation of embryos for non-clinical research. This review considers these issues and offers recommendations for good practice.

Mitochondrial DNA disease: new options for prevention.

Very recently, two papers have presented intriguing data suggesting that prevention of transmission of human mitochondrial DNA (mtDNA) disease is possible. [Craven, L., Tuppen, H.A., Greggains, G.D., Harbottle, S.J., Murphy, J.L., Cree, L.M., Murdoch, A.P., Chinnery, P.F., Taylor, R.W., Lightowlers, R.N. et al. (2010) Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature, 465, 82-85. Tachibana, M., Sparman, M., Sritanaudomchai, H., Ma, H., Clepper, L., Woodward, J., Li, Y., Ramsey, C., Kolotushkina, O. and Mitalipov, S. (2009) Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature, 461, 367-372.] These recent advances raise hopes for families with mtDNA disease; however, the successful translational of these techniques to clinical practice will require further research to test for safety and to maximize efficacy. Furthermore, in the UK, amendment to the current legislation will be required. Here, we discuss the clinical and scientific background, studies we believe are important to establish safety and efficacy of the techniques and some of the potential concerns about the use of these approaches.

Human epidermal neural crest stem cells (hEPI-NCSC)--characterization and directed differentiation into osteocytes and melanocytes.

Here we describe the isolation, characterisation and ex-vivo expansion of human epidermal neural crest stem cells (hEPI-NCSC) and we provide protocols for their directed differentiation into osteocytes and melanocytes. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. Multipotency and self-renewal were determined by in vitro clonal analyses. hEPI-NCSC generate all major neural crest derivatives, including bone/cartilage cells, neurons, Schwann cells, myofibroblasts and melanocytes. Furthermore, hEPI-NCSC express additional neural crest stem cell markers and global stem cell genes. To variable degrees and in a donor-dependent manner, hEPI-NCSC express the six essential pluripotency genes C-MYC, KLF4, SOX2, LIN28, OCT-4/POU5F1 and NANOG. hEPI-NCSC can be expanded ex vivo into millions of stem cells that remain mulitpotent and continue to express stem cell genes. The novelty of hEPI-NCSC lies in the combination of their highly desirable traits. hEPI-NCSC are embryonic remnants in a postnatal location, the bulge of hair follicles. Therefore they are readily accessible in the hairy skin by minimal invasive procedure. hEPI-NCSC are multipotent somatic stem cells that can be isolated reproducibly and with high yield. By taking advantage of their migratory ability, hEPI-NCSC can be isolated as a highly pure population of stem cells. hEPI-NCSC can undergo robust ex vivo expansion and directed differentiation. As somatic stem cells, hEPI-NCSC are conducive to autologous transplantation, which avoids graft rejection. Together, these traits make hEPI-NCSC novel and attractive candidates for future cell-based therapies and regenerative medicine.