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.

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.

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.

Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease.

Mutations in mitochondrial DNA (mtDNA) are a common cause of genetic disease. Pathogenic mutations in mtDNA are detected in approximately 1 in 250 live births and at least 1 in 10,000 adults in the UK are affected by mtDNA disease. Treatment options for patients with mtDNA disease are extremely limited and are predominantly supportive in nature. Mitochondrial DNA is transmitted maternally and it has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease. Here we show that transfer of pronuclei between abnormally fertilized human zygotes results in minimal carry-over of donor zygote mtDNA and is compatible with onward development to the blastocyst stage in vitro. By optimizing the procedure we found the average level of carry-over after transfer of two pronuclei is less than 2.0%, with many of the embryos containing no detectable donor mtDNA. We believe that pronuclear transfer between zygotes, as well as the recently described metaphase II spindle transfer, has the potential to prevent the transmission of mtDNA disease in humans.

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.

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.

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.

Homologue disjunction in mouse oocytes requires proteolysis of securin and cyclin B1.

Disjunction of pairs of homologous chromosomes during the first meiotic division (MI) requires anaphase-promoting complex (APC)-mediated activation of separase in budding yeast and Caenorhabditis elegans, but not Xenopus laevis. It is not clear which model best fits the mammalian system. Here we show that homologue disjunction in mouse oocytes is dependent on proteolysis of the separase inhibitor securin and the Cdk1 regulatory sub-unit cyclin B1. Proteolysis of both proteins was entirely dependent on their conserved destruction box (D-box) motifs, through which they are targeted to the APC. These data indicate that the mechanisms regulating homologue disjunction in mammalian oocytes are similar to those of budding yeast and C.elegans.

Evaluation of epigenetic marks in human embryos derived from IVF and ICSI.

BACKGROUND: It has long been appreciated that environmental cues may trigger adaptive responses. Many of these responses are a result of changes in the epigenetic landscape influencing transcriptional states and consequently altering phenotypes. In the context of human reproductive health, the procedures necessary for assisted reproduction may result in altered phenotypes by primarily influencing DNA methylation. Among the well-documented effects of assisted reproduction technologies (ART), imprinted genes appear to be frequently altered, likely owing to their reliance on DNA methylation to impose parent-specific monoallelic expression. However, the generality of the potential deregulation of DNA methylation in ART-derived human embryos has not been evaluated. METHODS: In this study, we evaluate the genome-wide DNA methylation together with chromatin organisation in human embryos derived by either IVF (n = 89) or ICSI (n = 76). DNA methylation was assessed using an antibody against 5-methyl-cytidine, and chromatin organisation by DNA staining. RESULTS: Irrespective of the ART procedure, similar errors were observed in both groups and abnormal chromatin was positively correlated (P < 0.001) with inappropriate DNA methylation. Development up to the blastocyst stage was consistent with normal DNA methylation and chromatin organisation, reinforcing the importance of epigenetic regulation to form the early lineages of the blastocyst. Most importantly, we found no evidence that ICSI blastocysts were more severely affected than those derived by IVF. CONCLUSIONS: We conclude that ICSI does not lead to an increased incidence of epigenetic errors (DNA methylation and chromatin) compared with IVF.

Surgeons and scientists: working together in embryo research.

Most surgeons in academic hospitals will have had a request from an enthusiastic research scientist to take samples of tissue during an operation. It seems reasonable and most patients will respond positively. But of course it is not quite that simple. The regulation of donation of human tissue for basic research is clearly defined but usually less rigorous than that which covers translational research and clinical trials. An exception has been the donation of embryos for embryonic stem cell derivation. The specific issues related to obtaining cells from patients for this work has resulted in a different relationship between scientist and clinician. This will be considered.

Epidemiology and management of infertility: a population-based study in UK primary care.

BACKGROUND: Our current knowledge of the epidemiology of infertility is limited and outdated. Health care provision for infertility in the UK attracts public interest because of restrictions on access to services. OBJECTIVE: To describe the incidence, prevalence, referral patterns and outcomes of infertile couples, presenting in general practice in UK. METHODS: A population-based retrospective observational outcome study of infertile couples from general practices in Northumberland, Tyne and Wear, UK (population 1 043 513). Outcome data at 1 year were collected on all couples who presented to their GP between the 1st January 2005 and 30th June 2006 with a fertility problem. RESULTS: Thirty-four per cent of general practices in the study area contributed data (population 404 263). The incidence of infertility was 0.9 couples per 1000 general population. The average age of women was 31 years, and the average time attempting conception was 18 months. Treatment end points for half of all couples were in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Over half of the couples in the study were not eligible for National Health Service (NHS) fertility treatment on social criteria. At 12 months, 27% of all couples in the study achieved a pregnancy spontaneously and a further 9% with treatment. CONCLUSIONS: Infertile women present to their GP later in life compared with 20 years ago, and after a shorter period of infertility. Half of the couples required treatment with IVF or ICSI. Adopting the British Fertility Society recommendation of allowing couples, where one or both partners has a child in a previous relationship, will result in an additional 26% of infertile couples becoming eligible for NHS fertility treatment.

Patient experience of infertility management in primary care: an in-depth interview study.

BACKGROUND: GPs do not have a full range of diagnostic resources to help manage infertile couples. Little is known about the patient experience of infertility management in primary care. OBJECTIVE: To explore the patient experience of infertility management from a primary care perspective. METHODS: This was a nested qualitative study with infertile couples in North-East England. In-depth interviews of infertile couples identified in the course of an observational study describing the incidence, prevalence, referral patterns and pregnancy outcomes for infertile couples. A grounded approach was used, with the interviews and analysis proceeding together using the method of constant comparison. Emergent themes and their links gave an overall explanation to the interview data. RESULTS: We interviewed 22 patients in 13 interviews. Factors that influenced a couple's experience of infertility management were personal and professional relationships, patient autonomy in decision making and access to services. CONCLUSIONS: This study provides insights into the experiences of infertile couples seeking assisted reproduction from their GP. A good experience was linked to a strong personal relationship, a patient-centred professional relationship fostering informed decision making by the couple, GPs using diagnostic resources, interpreting restrictive clinical and social criteria and referring appropriately.

Obesity and female fertility: a primary care perspective.

Infertility affects approximately one in six couples during their lifetime. Obesity affects approximately half of the general population and is thus a common problem among the fertile population. Obese women have a higher prevalence of infertility compared with their lean counterparts. The majority of women with an ovulatory disorder contributing to their infertility have polycystic ovary syndrome (PCOS) and a significant proportion of women with PCOS are obese. Ovulation disorders and obesity-associated infertility represent a group of infertile couples that are relatively simple to treat. Maternal morbidity, mortality and fetal anomalies are increased with obesity and the success of assisted reproductive technology (ART) treatments is significantly reduced for obese women. Body mass index (BMI) treatment limits for ART throughout the UK vary. The mainstay for treatment is weight loss, which improves both natural fertility and conception rates with ART. The most cost-effective treatment strategy for obese infertile women is weight reduction with a hypo-caloric diet. Assisted reproduction is preferable in women with a BMI of 30 kg/m(2) or less and weight loss strategies should be employed within primary care to achieve that goal prior to referral.

Putative role of hyaluronan and its related genes, HAS2 and RHAMM, in human early preimplantation embryogenesis and embryonic stem cell characterization.

Human embryonic stem cells (hESC) promise tremendous potential as a developmental and cell therapeutic tool. The combined effort of stimulatory and inhibitory signals regulating gene expression, which drives the tissue differentiation and morphogenetic processes during early embryogenesis, is still very poorly understood. With the scarcity of availability of human embryos for research, hESC can be used as an alternative source to study the early human embryogenesis. Hyaluronan (HA), a simple hydrating sugar, is present abundantly in the female reproductive tract during fertilization, embryo growth, and implantation and plays an important role in early development of the mammalian embryo. HA and its binding protein RHAMM regulate various cellular and hydrodynamic processes from cell migration, proliferation, and signaling to regulation of gene expression, cell differentiation, morphogenesis, and metastasis via both extracellular and intracellular pathways. In this study, we show for the first time that HA synthase gene HAS2 and its binding receptor RHAMM are differentially expressed during all stages of preimplantation human embryos and hESC. RHAMM expression is significantly downregulated during differentiation of hESC, in contrast to HAS2, which is significantly upregulated. Most importantly, RHAMM knockdown results in downregulation of several pluripotency markers in hESC, induction of early extraembryonic lineages, loss of cell viability, and changes in hESC cycle. These data therefore highlight an important role for RHAMM in maintenance of hESC pluripotency, viability, and cell cycle control. Interestingly, HAS2 knockdown results in suppression of hESC differentiation without affecting hESC pluripotency. This suggests an intrinsic role for HAS2 in hESC differentiation process. In accordance with this, addition of exogenous HA to the differentiation medium enhances hESC differentiation to mesodermal and cardiac lineages. Disclosure of potential conflicts of interest is found at the end of this article.

General practitioners' perceptions and attitudes to infertility management in primary care: focus group study.

BACKGROUND: Infertility management in primary care is variable. National Institute of Clinical Excellence have recommended hysterosalpingography (HSG) as a first-line investigation for tubal assessment. Aim To explore general practitioners' (GPs) perceptions of, and attitudes to, the initial management of the infertile couple and their views on open access to HSG. DESIGN: Qualitative study using three focus groups. SETTING: Seven general practices in Newcastle upon Tyne and Northumberland. SUBJECTS: We purposively selected the three focus groups to provide a range of GPs' views. In total 13 practitioners participated: 11 GPs, one GP registrar and one nurse practitioner. RESULTS: The key themes to emerge were: (1) perceived professional responsibilities, (2) uncertainty and lack of knowledge, (3) consistency of approach to the initial management of infertility, and (4) access to infertility services. Some GPs felt that they should do all they possibly could, while others felt it was the responsibility of the infertility specialist. Uncertainty and lack of knowledge was linked to the relative infrequency of primary care infertility consultations and the difficulty 'keeping up to date' with rapidly advancing reproductive technologies in tertiary care. Some GPs subscribed to the notion of one suitably trained clinician delivering the service on behalf of a group of GPs. Some were unsure where HSG fitted into the overall management plan, but they were comfortable with following recommended guidelines. CONCLUSIONS: GPs recognize an advocacy role and many take on a significant degree of clinical responsibility welcoming the introduction of a new technology in primary care. Nevertheless, GPs feel that they lack proficiency and have little opportunity to rehearse the necessary skills. These findings contribute to an understanding of the management of infertility, an infrequently presenting problem in primary care.

Erratum: Revisiting the Warnock rule.

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