Premature ovarian insufficiency: the need for evidence on the effectiveness of hormonal therapy.

Premature ovarian insufficiency (POI) - the loss of ovarian function before the age of 40 years, a decade before natural menopause - is a life-changing diagnosis for women. POI causes significant short-term and long-term morbidity related to estrogen deficiency. The condition is managed by providing exogenous estrogen replacement, usually as the oral contraceptive pill or hormone therapy. These preparations have different estrogen formulations and may have differing benefits and risks. At present, there are no robust data to inform clinical recommendations and women's decision-making about treatment that they may be taking for many years. The POISE study (Premature Ovarian Insufficiency Study of Effectiveness of hormonal therapy) has been designed to determine whether hormone therapy is superior to combined oral contraceptives on important clinical outcomes and patient-reported symptoms, based on the hypothesis that hormone therapy provides more physiological continuous hormone supplementation with natural estrogens. The study is an open and pragmatic, parallel, randomized controlled trial. The primary outcome is absolute bone mineral density assessed by dual-energy X-ray absorptiometry of the lumbar spine after 2 years of treatment. The study will also investigate cardiovascular markers, symptom relief and acceptability of treatment, and will continue to collect long-term data on fractures and cardiovascular events. Results will inform future guidance on management of POI.

Elective Egg Freezing for Non-Medical Reasons: Scientific Impact Paper No. 63.

Although a woman's fertility declines markedly in her late-30s and early-40s, gradually more and more women start a family at this stage of their lives, with the average age of childbirth progressively increasing. More women are storing their eggs (oocytes) to give them the potential opportunity to have a baby in the future. Nonetheless, the number of egg freezing cycles accounts for less than 2% of IVF cycles, and the number of cycles using stored eggs is even lower. The technology for freezing eggs changed dramatically about a decade ago with the development of a technique of rapid freezing called vitrification, which gives success rates almost as good as using fresh eggs. The growing use of this technique, and the publicity surrounding how this technique may have been promoted, has led to this paper. It is essential that women are very clearly informed about the likely success rates of egg freezing, particularly as it is entirely provided by the private sector, with the associated concerns of financial costs and inappropriate or inaccurate marketing. Its success is strongly dependent on the age of the woman at the time of freezing her eggs, with much higher success rates in those aged 35 years and under. Current legislation only allows women to store eggs for 10 years, which conflicts with the better success rates when women do so at a younger age. The reasons behind the increase in egg freezing are complex, but the most common reason given by women storing eggs is that they do not have a partner and are concerned that by the time they do find themselves in a relationship within which they wish to start a family, they may not be able to. We conclude that elective egg freezing provides women with an opportunity to take action about the drop in their fertility, but at present most women who are doing this are already in their later 30s when the success rates are limited. We strongly support the need for improved and continuing education of both women and men regarding the decline in female fertility with age.

Cancer risk in children born after donor ART.

STUDY QUESTION: Do children born after donor ART have an increased risk of developing childhood cancer in comparison to the general population? SUMMARY ANSWER: This study showed no overall increased risk of childhood cancer in individuals born after donor ART. WHAT IS KNOWN ALREADY: Most large population-based studies have shown no increase in overall childhood cancer incidence after non-donor ART; however, other studies have suggested small increased risks in specific cancer types, including haematological cancers. Cancer risk specifically in children born after donor ART has not been investigated to date. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study utilized record linkage to determine the outcome status of all children born in Great Britain (1992-2008) after donor ART. The cohort included 12 137 members who contributed 95 389 person-years of follow-up (average follow-up 7.86 years). PARTICIPANTS/MATERIALS, SETTING, METHODS: Records of all children born in Great Britain (England, Wales, Scotland) after all forms of donor ART (1992-2008) were linked to the UK National Registry of Childhood Tumours (NRCT) to determine the number who subsequently developed cancer by 15 years of age, by the end of 2008. Rates of overall and type specific cancer (selected a priori) were compared with age, sex and calendar year standardized population-based rates, stratifying for potential mediating/moderating factors including sex, age at diagnosis, birth weight, multiple births, maternal previous live births, assisted conception type and fresh/ cryopreserved cycles. MAIN RESULTS AND THE ROLE OF CHANCE: In our cohort of 12 137 children born after donor ART (52% male, 55% singleton births), no overall increased risk of cancer was identified. There were 12 cancers detected compared to 14.4 expected (standardized incidence ratio (SIR) 0.83; 95% CI 0.43-1.45; P = 0.50). A small, significant increased risk of hepatoblastoma was found, but the numbers and absolute risks were small (<5 cases observed; SIR 10.28; 95% CI 1.25-37.14; P < 0.05). This increased hepatoblastoma risk was associated with low birthweight. LIMITATIONS REASONS FOR CAUTION: Although this study includes a large number of children born after donor ART, the rarity of specific diagnostic subgroups of childhood cancer results in few cases and therefore wide CIs for such outcomes. As this is an observational study, it is not possible to adjust for all potential confounders; we have instead used stratification to explore potential moderating and mediating factors, where data were available. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to investigate cancer risk in children born after donor ART. Although based on small numbers, results are reassuring for families and clinicians. The small but significant increased risk of hepatoblastoma detected was associated with low birthweight, a known risk factor for this tumour type. It should be emphasized that the absolute risks are very small. However, on-going investigation with a longer follow-up is needed. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by Cancer Research UK (C36038/A12535) and the National Institute for Health Research (405526) and supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. The work of the Childhood Cancer Research Group (CCRG) was supported by the charity CHILDREN with CANCER UK, the National Cancer Intelligence Network, the Scottish Government and the Department of Health for England and Wales. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Engineering serendipity: High-throughput discovery of materials that resist bacterial attachment.

Controlling the colonisation of materials by microorganisms is important in a wide range of industries and clinical settings. To date, the underlying mechanisms that govern the interactions of bacteria with material surfaces remain poorly understood, limiting the ab initio design and engineering of biomaterials to control bacterial attachment. Combinatorial approaches involving high-throughput screening have emerged as key tools for identifying materials to control bacterial attachment. The hundreds of different materials assessed using these methods can be carried out with the aid of computational modelling. This approach can develop an understanding of the rules used to predict bacterial attachment to surfaces of non-toxic synthetic materials. Here we outline our view on the state of this field and the challenges and opportunities in this area for the coming years. STATEMENT OF SIGNIFICANCE: This opinion article on high throughput screening methods reflects one aspect of how the field of biomaterials research has developed and progressed. The piece takes the reader through key developments in biomaterials discovery, particularly focusing on need to reduce bacterial colonisation of surfaces. Such bacterial resistant surfaces are increasingly required in this age of antibiotic resistance. The influence and origin of high-throughput methods are discussed with insights into the future of biomaterials development where computational methods may drive materials development into new fertile areas of discovery. New biomaterials will exhibit responsiveness to adapt to the biological environment and promote better integration and reduced rejection or infection.

A biomechanical study of the effects of simulated ulnar deviation on silicone finger joint implant failure.

Silicone finger arthroplasties are used widely, especially for metacarpophalangeal joint replacement in patients with inflammatory arthritis. Implant failure is well recognized. The rates of failure in vivo differ substantially from experience in vivo. One cause of failure is felt to be post-operative ulnar deviation. The aim of our study was to test the effect of ulnar deviation testing on silicone finger implants. We tested 12 implants in three groups of four implants. The implants were submerged in a bath of Ringer's solution at 370 °C throughout the experiment and tested in a rig held in 0°, 10° and 20° deviation. The rig was cycled at 1.5 Hz from 0°-90°. The implants were inspected every 500,000 cycles until a total of 4 million cycles. There was consistently increased wear and supination plastic deformity in going from 0°-20° deviation. This study confirms the adverse effects of ulnar deviation on silicone finger implant wear. It is likely that this combines with lateral pinch forces and sharp bone edges to cause catastrophic silicone implant failure. LEVEL OF EVIDENCE: III.

Chemically diverse polymer microarrays and high throughput surface characterisation: a method for discovery of materials for stem cell culture†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4bm00054dClick here for additional data file.

Materials discovery provides the opportunity to identify novel materials that are tailored to complex biological environments by using combinatorial mixing of monomers to form large libraries of polymers as micro arrays. The materials discovery approach is predicated on the use of the largest chemical diversity possible, yet previous studies into human pluripotent stem cell (hPSC) response to polymer microarrays have been limited to 20 or so different monomer identities in each study. Here we show that it is possible to print and assess cell adhesion of 141 different monomers in a microarray format. This provides access to the largest chemical space to date, allowing us to meet the regenerative medicine challenge to provide scalable synthetic culture ware. This study identifies new materials suitable for hPSC expansion that could not have been predicted from previous knowledge of cell-material interactions.

Chemical and spatial analysis of protein loaded PLGA microspheres for drug delivery applications.

Polymer microspheres for controlled release of therapeutic protein from within an implantable scaffold were produced and analysed using complimentary techniques to probe the surface and bulk chemistry of the microspheres. Time of Flight - Secondary Ion Mass Spectrometry (ToF-SIMS) surface analysis revealed a thin discontinuous film of polyvinyl alcohol (PVA) surfactant (circa 4.5 nm thick) at the surface which was readily removed under sputtering with C(60). Atomic Force Microscopy (AFM) imaging of microspheres before and after sputtering confirmed that the PVA layer was removed after sputtering revealing poly(lactic-co-glycolic) acid(PLGA). Scanning electron microscopy showed the spheres to be smooth with some shallow and generally circular depressions, often with pores in their central region. The occurrence of the protein at the surface was limited to areas surrounding these surface pores. This surface protein distribution is believed to be related to a burst release of the protein on dissolution. Analysis of the bulk properties of the microspheres by confocal Raman mapping revealed the 3D distribution of the protein showing large voids within the pores. Protein was found to be adsorbed at the interface with the PLGA oil phase following deposition on evaporation of the solvent. Protein was also observed concentrated within pores measuring approximately 2 µm across. The presence of protein in large voids and concentrated pores was further scrutinised by ToF-SIMS of sectioned microspheres. This paper demonstrates that important information for optimisation of such complex bioformulations, including an understanding of the release profile can be revealed by complementary surface and bulk analysis allowing optimisation of the therapeutic effect of such formulations.

The application of ToF-SIMS to the analysis of herbicide formulation penetration into and through leaf cuticles.

Understanding the movement of the active ingredient in relation to the other formulation components following application is crucial to an overall understanding of herbicide performance. We describe the novel use of time-of-flight secondary ion mass spectrometry (ToF-SIMS) as a tool for following the movement of herbicide formulation components into and across plant cuticles. This technique provides new insights since it provides both high (sub-micron) spatial resolution combined with the chemical specificity associated with organic mass spectrometry. The components studied include the oligomeric ethoxylate surfactants Synperonic A7 and A20 and active ingredient Sulfosate (trimesium glyphosate). The movement of these molecules, both separately and when combined in a simple formulation, into the surface of Prunus laurocerasus leaves and across the isolated plant cuticle was investigated and clear differences in penetration/diffusion behaviour were identified. ToF-SIMS was uniquely able to (simultaneously) spatially resolve all the species involved, including the anion and cation components of the active ingredient. Also, using spectral reconstructions from the imaging raw data streams, the behaviour of individual oligomers within the surfactant distributions, could be assessed. The observations are discussed with reference to the action of surfactants identified in parallel micro-structural studies and the current understanding of herbicide uptake.

Predictors of ovarian reserve in young women with breast cancer.

Ovarian reserve can be diminished following treatment for breast cancer. This study evaluated biochemical and biophysical parameters of ovarian reserve in these patients. Biochemical and biophysical tests of ovarian reserve were performed simultaneously in young (age 22-42 years), regularly menstruating women with breast cancer (n=22) and age-matched controls (n=24). All tests were performed before (baseline) and after transient ovarian stimulation in the early follicular phase. Patients were recruited both before and after completion of chemotherapy, with some patients being followed up prospectively. Serum samples were analysed for follicle-stimulating hormone (FSH), luteinising hormone (LH), oestradiol (E(2)), inhibins A and B, and antimullerian hormone (AMH). Biophysical (ultrasound) tests included ovarian volume, antral follicle count (AFC), ovarian stromal blood flow and uterine dimensions. Significant differences were revealed (when compared with the controls) for basal FSH (11.32+/-1.48 vs 6.62+/-0.42 mIU ml(-1), P<0.001), basal AMH (0.95+/-0.34 vs 7.89+/-1.62 ng ml(-1), P<0.001) and basal inhibin B (19.24+/-4.56 vs 83.61+/-13.45 pg ml(-1), P<0.001). Following transient ovarian stimulation, there were significant differences in the increment change (Delta) for inhibin B (3.02+/-2.3 vs 96.82+/-16.38 pg ml(-1), P<0.001) and E(2) (107.8+/-23.95 vs 283.2+/-40.34 pg ml(-1), P<0.01). AFC was the only biophysical parameter that was significantly different between patients and the controls (7.80+/-0.85 vs 16.77+/-1.11, P<0.001). Basal and stimulated biochemical (serum AMH, FSH, inhibin B and E(2)) and biophysical (AFC) tests may be potential markers of ovarian reserve in young women with breast cancer.

Fertility outcomes in women with hypopituitarism.

BACKGROUND: Women with hypopituitarism are known to have a poor outcome once pregnancy has been achieved by ovulation induction. There are no data, however, recording the efficacy of ovulation induction and pregnancy rates in this group of subfertile women. METHODS: The outcome of fertility treatments in all 19 women with hypopituitarism attending the fertility clinics of University College London Hospitals over the past 20 years was audited. RESULTS: Ovulation was achieved in almost all women (95%) but occurred in only 60% of treatment cycles. Pregnancy was achieved in 47% of women or 11% of cycles resulting in a live birth rate of 6.7% per cycle. Seven of the 18 pregnancies (39%) miscarried. Only 42% of women treated achieved a live birth. CONCLUSION: Ovulation induction in women with hypopituitarism yields relatively low pregnancy rates in comparison to other causes of anovulation and a high miscarriage rate. Pituitary hormone deficiency beyond gonadotrophins has a major adverse effect on achieving pregnancy.

Structural study of DNA condensation induced by novel phosphorylcholine-based copolymers for gene delivery and relevance to DNA protection.

Poly[2-(dimethylamino)ethyl methacrylate-b-2-methacryloyloxyethyl phosphorylcholine] (DMA-MPC) is currently under investigation as a new vector candidate for gene therapy. The DMA block has been previously demonstrated to condense DNA effectively. The MPC block contains a phosphorylcholine (PC) headgroup, which can be found naturally in the outside of the cell membrane. This PC-based polymer is extremely hydrophilic and acts as a biocompatible steric stabilizer. In this study, we assess in detail the morphologies of DNA complexes obtained using the diblock copolymer series DMA(x)MPC30 (where the mean degree of polymerization of the MPC block was fixed at 30 and the DMA block length was systematically varied) using transmission electron microscopy (TEM) and liquid atomic force microscopy (AFM). Both techniques indicate more compact complex morphologies (more efficient condensation) as the length of the cationic DMA block increases. However, the detailed morphologies of the DMA(x)MPC30-DNA complexes observed by TEM in vacuo and by AFM in aqueous medium are different. This phenomena is believed to be related to the highly hydrophilic nature of the MPC block. TEM studies revealed that the morphology of the complexes changes from loosely condensed structures to highly condensed rods, toroids, and oval-shaped particles as the DMA moiety increases. In contrast, morphological changes from plectonemic loops to flower-like and rectangular block-like structures, with an increase in highly condensed central regions, are observed by in situ AFM studies. The relative population of each structure is clearly dependent on the polymer molecular composition. Enzymatic degradation assays revealed that only the DMA homopolymer provided effective DNA protection against DNase I degradation, while other highly condensed copolymer complexes, as judged from TEM and gel electrophoresis, only partially protected the DNA. However, AFM images indicated that the same highly condensed complexes have less condensed regions, which we believe to be the initiation sites for enzymatic attack. This indicates that the open structures observed by AFM of the DNA complexation by the DMA(x)MPC30 copolymer series are closer to in vivo morphology when compared to TEM.

Modified polyethylenimines as non-viral gene delivery systems for aerosol gene therapy: investigations of the complex structure and stability during air-jet and ultrasonic nebulization.

Polyelectrolyte complexes between DNA and polyethylenimine (PEI) are promising non-viral delivery systems for pulmonary inhalation gene therapy and thus require sufficient stability during nebulization. The structure and stability of four different PEI-DNA polyplexes, namely branched (bPEI), linear (linPEI), poly(ethylene glycol)-grafted PEI (PEGPEI), biodegradable (bioPEI) PEI with DNA, were investigated. Using atomic force microscopy, the morphology of DNA and polyplexes before and after both air-jet and ultrasonic nebulization was characterized. The influence of nebulization on physico-chemical properties, particle size and zeta potential, was studied. Efficient DNA condensation to spherical particles was achieved with bPEI (90 nm) and PEGPEI (110 nm). By contrast, incomplete DNA condensations, seen as flower structures, were observed with linPEI (110 nm) and bioPEI (105 nm). Air-jet nebulization altered the polyplex structure to a greater extent than ultrasonic nebulization and resulted mainly in smaller and non-spherical particles (30-200 nm). Ultrasonic nebulization did not change the spherical structure or particle size of the polyplexes. In particular, the shape and size of the PEGPEI polyplexes did not change. We conclude that ultrasonic nebulization is a milder aerosolization method for gene delivery systems based on PEI. Additionally, PEGPEI-DNA polyplexes seem to be more stable than their counterparts, which may be advantageous in pulmonary inhalation gene therapy.

Direct atomic force microscopy observations of monovalent ion induced binding of DNA to mica.

Multivalent ions in solution are known to mediate attraction between two like-charged molecules. Such attraction has proved useful in atomic force microscopy (AFM) where DNA may be immobilized to a mica surface facilitating direct imaging in liquid. Theories of DNA immobilization suggest that either 'salt bridging' or fluctuation in the positions of counter ions about both the mica surface and DNA backbone secure DNA to the mica substrate. Whilst both theoretical and experimental evidence suggest that immobilization is possible in the presence of divalent ions, very few studies identify that such immobilization is possible with monovalent ions. Here we present direct AFM evidence of DNA immobilized to mica in the presence of only monovalent ions. Our data depict E. coli plasmid pBR322 adsorbed onto the negatively charged mica both after short (10 min) and long (24 h) incubation periods. These data suggest the need to re-explore current theories of like-charge attraction to include the possibility of monovalent interactions. We suggest that this DNA immobilization strategy may offer the potential to image natural processes with limited immobilization forces and hence enable maximum conformational freedom of the immobilized biomolecule.

AFM studies of the crystallization and habit modification of an excipient material, adipic acid.

Atomic force microscopy (AFM) has been used to investigate the (1 0 0) face of crystalline adipic acid, both in air and liquid environments. In air, surface reorganization occurred during scanning of the AFM probe, which has been investigated using single point force-distance analysis under a controlled relative humidity (RH) environment. We suggest such reorganization can be attributed to the influence of a network of water molecules bound to the hydrophilic (1 0 0) surface permitting local AFM tip-enhanced dissolution and reorganization of the solute. In situ imaging was also carried out on the crystals, revealing etch-pit formation during dissolution, and rapid growth at higher levels of supersaturation (sigma), both of which are direct consequences of the hydrophilic nature of the (1 0 0) face. Also presented here are nanoscale observations of the effect of octanoic acid, a structurally-related habit modifier, on crystalline adipic acid. Using AFM, we have been able to show that the presence of octanoic acid at low concentration has little observable affect on the development of the (1 0 0) face; however, as this concentration is increased, there are clear changes in step morphology and growth mode on the (1 0 0) face of the crystal. At a concentration of 1.26 mmol dm(-3) (a concentration corresponding to a molar ratio of approximately 1:175 octanoic acid:adipic acid), growth on the (1 0 0) face is inhibited, with in situ AFM imaging indicating this is a direct consequence of octanoic acid binding to the surface, and pinning the monomolecular growth steps.

Measuring and visualizing single molecular interactions in biology.

In recent years, considerable attention has focused upon the biological applications of the atomic force microscope (AFM), and in particular in its ability to explore biomolecular interaction events at the single molecule level. Such measurements can provide considerable advantages, as they remove the data averaging inherent in other biophysical/biochemical approaches that record measurements over large ensembles of molecules. To this end AFM has been used for both the high-resolution imaging of a range of individual biological molecules and their complexes, and to record interaction forces between single interacting molecules. In a recently initiated project we have begun to utilize these approaches to explore the interactions of a range of biologically important peptides with model and cell membrane surfaces. In this review, the potential value of AFM for the investigation of a range of biomolecular interaction events will be discussed, but highlighting in particular its potential for the study of interactions of peptides/proteins with biological membranes.

Controlled biological response on blends of a phosphorylcholine-based copolymer with poly(butyl methacrylate).

Phosphorylcholine (PC)-based polymers have been used in a variety of medical device applications to improve biocompatibility. Here, solutions containing poly(butyl methacrylate) (PBMA) and 2-methacryloyloxyethyl phosphorylcholine-co-lauryl methacrylate (MPC-co-LMA(2)) copolymer were spin-coated onto glass coverslips at various ratios ranging from 5:1 to 1:0 for each of the two components, respectively. The resulting blend coatings were shown to be phase-separated on the nanometre-scale by atomic force microscopy, the PC copolymer within the blend being preferentially expressed at the surface. The adsorption of two key blood proteins, fibrinogen and albumin were investigated using surface plasmon resonance as an indicator of biocompatibility. The adsorption of protein to a biomaterials' surface can then stimulate further biological responses. This study therefore, also investigates the materials ability to elicit an inflammatory response by studying the adhesion of human mononuclear cells to the material surface. The materials ability to support the adhesion and growth of other tissue cells was also evaluated, looking specifically at the adhesion and proliferation of rabbit corneal epithelial cells. Results suggest that the adsorption of proteins and the adhesion of both corneal epithelial cells and mononuclear cells are dependent on the composition of the PBMA:MPC-co-LMA(2) copolymer.

Electrostatic interactions observed when imaging proteins with the atomic force microscope.

The atomic force microscope (AFM) is now an established and valuable tool for the study of biological macromolecules in aqueous environments. In this paper we form a patterned boundary via the microcontact printing of individually isolated proteins, covalently attached to a solid support. We use this boundary to investigate electrostatic interactions that can occur between an AFM tip and a protein surface during imaging in solution. The observed height variations of the protein film are found to be a combination of not only structural considerations and thickness of the protein film, but also the repulsive contribution from electrostatic interactions between the AFM tip and the sample. These variations in measured heights of the protein surface can be described by Derjaguin, Landau, Verway, Overbeek (DLVO) theory. Our experimental results show that height measurements can be manipulated either negatively or positively by adjusting the pH and concentration of the electrolyte buffer that is utilised.

Interactions of 3T3 fibroblasts and endothelial cells with defined pore features.

The colonization of biodegradable polymer scaffolds with cell populations has been established as the foundation for the engineering of a number of tissues, including cartilage, liver, and bone. Within these scaffolds, the cells encounter a porous environment in which they must migrate across the convoluted polymer surface to generate a homogenous cell distribution. Predicting the interactions between cells and pores is important if scaffold characteristics are to be optimized. Therefore, we investigated the behavior of two model cell types over a range of defined pore features. These pore features range from 5 to 90 microm in diameter and have been fabricated by photolithographic techniques. Quantitatively, the behavior of the cells is dependent on three factors: 1) percentage cell coverage of the surface; 2) pore size; and 3) cell type. Fibroblast cells displayed a co-operative pattern of cell spreading in which pores with diameters greater than the cell dimensions were bridged by groups of cells using their neighbors as supports. Endothelial cells were unable to use neighbors as support structures and failed to bridge pores greater than the cell diameter.