Hormonal response in patients transplanted with cryopreserved ovarian tissue is independent of whether freezing was performed in childhood or adulthood.

PURPOSE: This study evaluated the concentrations of hormones resulting from the transplantation of ovarian tissue (OTT) in relation to whether the tissue was frozen at a time close to puberty or above the age of 19 years. METHODS: Six girls and adolescents (aged 9-14 years) who underwent ovarian tissue cryopreservation (OTC) were followed after transplantation in adulthood. After OTT, the women were followed via regular blood samples to evaluate the concentrations of FSH, LH, oestradiol and AMH. Twenty-three women undergoing OTT (aged 19-36 years at the time of OTC) were included as a reference group. All of the women had postmenopausal levels of gonadotropins at the time of transplantation. RESULTS: The return of FSH and LH to normal premenopausal concentrations in adult women transplanted with ovarian tissue that was frozen at a time close to puberty was similar to the profiles in women from the reference group. Serum AMH levels were below the detection limit (via the Roche Elecsys assay) in many samples, but four out of six young girls showed measurable concentrations. Oestradiol similarly increased in the first 12 weeks following transplantation, after which it tended to be higher in women having frozen tissue in adulthood. CONCLUSIONS: Ovarian tissue that was excised from girls at a time close to puberty, after which it was frozen and transplanted in adulthood, interacts with pituitary tissue in a similar manner to ovarian tissue that is frozen from adult women. Follicles located in the ovarian tissue from young girls are equally sensitive to gonadotropin stimulation as follicles from adult women when exposed to postmenopausal levels of gonadotropins. This result indicates that it is not the ovaries that require maturation to sustain full reproductive potential but rather proper FSH and LH stimulation. Moreover, these results support the continued use of OTC in young women.

Quality and extent of implementation of a nurse-led care management intervention: care coordination for health promotion and activities in Parkinson's disease (CHAPS).

BACKGROUND: A recent nurse-led, telephone-administered 18-month intervention, Care Coordination for Health Promotion and Activities in Parkinson's Disease (CHAPS), was tested in a randomized controlled trial and improved care quality. Therefore, intervention details on nurse care manager activity (types and frequencies) and participant actions are needed to support potential dissemination. Activities include nurse care manager use of a holistic organizing framework, identification of Parkinson's disease (PD)-related problems/topics, communication with PD specialists and care coordination, participant coaching, and participant self-care actions including use of a notebook self-care tool. METHODS: This article reports descriptive data on the CHAPS intervention. The study setting was five sites in the Veterans Affairs Healthcare System. Sociodemographic data were gathered from surveys of study participants (community-dwelling veterans with PD). Nurse care manager intervention activities were abstracted from electronic medical records and logbooks. Statistical analysis software was used to provide summary statistics; closed card sorting was used to group some data. RESULTS: Intervention participants (n = 140) were primarily men, mean age 69.4 years (standard deviation 10.3) and community-dwelling. All received the CHAPS Initial Assessment, which had algorithms designed to identify 31 unique CHAPS standard problems/topics. These were frequently documented (n = 4938), and 98.6% were grouped by assigned domain from the Organizing Framework (Siebens Domain Management Model™). Nurse care managers performed 27 unique activity types to address identified problems, collaborating with participants and PD specialists. The two most frequent unique activities were counseling/emotional support (n = 387) and medication management (n = 349). Both were among 2749 total performed activities in the category Implementing Interventions (coaching). Participants reported unique self-care action types (n = 23) including use of a new notebook self-care tool. CONCLUSIONS: CHAPS nurse care managers implemented multiple activities including participant coaching and care coordination per the CHAPS protocol. Participants reported various self-care actions including use of a personalized notebook. These findings indicate good quality and extent of implementation, contribute to ensuring reproducibility, and support CHAPS dissemination as a real-world approach to improve care quality. TRIAL REGISTRATION: ClinicalTrials.gov as NCT01532986 , registered on January 13, 2012.

Distinct expression patterns of TLR transcripts in human oocytes and granulosa cells from primordial and primary follicles.

Ovulation has long been regarded as a process resembling an inflammatory response. Previously, luteinizing hormone (LH) was shown to induce Toll-like receptor 2 (TLR2) and TLR4 in granulosa cells from preovulatory hormone-dependent follicles. However, whether this could already initiate before the hormone-dependent phase is currently unknown. The aim of this study was to investigate TLR genes in human oocytes and granulosa cells from primordial and primary ovarian follicles during the hormone-independent phase. A class-comparison study of existing oocyte and granulosa cell RNA sequencing transcriptomes from primordial (n = 539 follicles) and primary (n = 261) follicles collected from three patients was examined. This revealed a distinct expression pattern of TLR3, TLR4 and TLR5 transcripts. Interestingly, the TLR3 protein was differentially detected in both the oocyte and the granulosa cells in primordial and primary follicles, suggesting that TLR3 is maternally contributed both as mRNA and protein. Intracellularly, the compartmentalized TLR3 dot-like staining in the intersection between the oocyte and the surrounding primordial granulosa cells. The TLR4 protein was detected in both primordial and primary follicles, with a notable staining in the granulosa cells. We functionally challenged ovaries in vitro, by polyinosinic:polycytidylic acid (poly I:C) and LPS, known to activate TLR3 and TLR4, respectively, and found a tendency for increased IL-6 production, which was particular evident in the LPS-treated group. Based on the expression of TLRs, it is notably that human primordial and primary follicles express genes that would allow them to respond to innate immune proteins and cytokines during follicle activation.

Characterization of the plasma proteome of nonhuman primates during Ebola virus disease or melioidosis: a host response comparison.

BACKGROUND: In-depth examination of the plasma proteomic response to infection with a wide variety of pathogens can assist in the development of new diagnostic paradigms, while providing insight into the interdependent pathogenic processes which encompass a host's immunological and physiological responses. Ebola virus (EBOV) causes a highly lethal infection termed Ebola virus disease (EVD) in primates and humans. The Gram negative non-spore forming bacillus Burkholderia pseudomallei (Bp) causes melioidosis in primates and humans, characterized by severe pneumonia with high mortality. We sought to examine the host response to infection with these two bio-threat pathogens using established animal models to provide information on the feasibility of pre-symptomatic diagnosis, since the induction of host molecular signaling networks can occur before clinical presentation and pathogen detection. METHODS: Herein we report the quantitative proteomic analysis of plasma collected at various times of disease progression from 10 EBOV-infected and 5 Bp-infected nonhuman primates (NHP). Our strategy employed high resolution LC-MS/MS and a peptide-tagging approach for relative protein quantitation. In each infection type, for all proteins with > 1.3 fold abundance change at any post-infection time point, a direct comparison was made with levels obtained from plasma collected daily from 5 naïve rhesus macaques, to determine the fold changes that were significant, and establish the natural variability of abundance for endogenous plasma proteins. RESULTS: A total of 41 plasma proteins displayed significant alterations in abundance during EBOV infection, and 28 proteins had altered levels during Bp infection, when compared to naïve NHPs. Many major acute phase proteins quantitated displayed similar fold-changes between the two infection types but exhibited different temporal dynamics. Proteins related to the clotting cascade, immune signaling and complement system exhibited significant differential abundance during infection with EBOV or Bp, indicating a specificity of the response. CONCLUSIONS: These results advance our understanding of the global plasma proteomic response to EBOV and Bp infection in relevant primate models for human disease and provide insight into potential innate immune response differences between viral and bacterial infections.

Mammary gland health of Santa Inês ewes at the drying and puerperium and evaluation of a dry-off terapy with gentamicin / Sanidade da glândula mamária de ovelhas Santa Inês na secagem e no puerpério e avaliação da terapia intramamária com gentamicina na secagem

Mastitis represents an important health problem for Santa Inês breed, causing losses to the producer, due to loss of ewes or the decrease in weight gain of lambs. The aim of this work was to assess the health of the mammary gland of Santa Inês ewes at the drying and puerperium and to investigate the efficacy of a dry-off therapy with gentamicin. In this study, 64 ewes were divided in a control group (GC) and treatment group (GT), and the health of the mammary gland was assessed at the drying and puerperium. The GT ewes received 250mg of gentamicin (Gentocin® DryCow/Schering-Plough, product indicated for use in dairy cows) in each mammary half. For diagnosis, clinical examination, California Mastitis Test, somatic cell count and milk culture was performed. In the GC, of the 45 (70.3%) healthy mammary halves at the drying, 12 developed subclinical mastitis and nine clinical mastitis at the puerperium. In the GT, among 51 (79.7%) healthy mammary halves at the drying, six developed subclinical mastitis and 11 clinical mastitis at the puerperium. No association was observed between treatment and the occurrence of mastitis at puerperium. Of the 19 (29.7%) mammary halves of the GC that presented subclinical mastitis at the drying, three remained with subclinical mastitis and five developed clinical mastitis at the puerperium. In the GT, of the 13 (20.3%) mammary halves that had subclinical mastitis at the drying, four remained with subclinical mastitis and four developed clinical mastitis. No association was observed between treatment and cure or persistence of mastitis at the puerperium. The main microorganisms isolated, at the drying and puerperium, from animals with subclinical or clinical mastitis were Staphylococcus spp., predominantly coagulase negative Staphylococcus (CSN). At the puerperium, 29 cases of clinical mastitis occurred, 19 with isolation, where 10 were CNS and six S. aureus. Mannheimia haemolytica was isolated in one case of subclinical mastitis and other of clinical mastitis. News protocols and different ways of handling at drying and at puerperium must be investigated.(AU)

A mastite é um problema sanitário importante em ovelhas da raça Santa Inês, ocasionando prejuízo ao produtor em virtude do descarte de matrizes e da queda no ganho de peso dos cordeiros. O objetivo deste trabalho foi avaliar a saúde da glândula mamária de ovelhas da raça Santa Inês na secagem e no puerpério e pesquisar a eficácia da terapia intramamária com gentamicina na secagem. Sessenta e quatro ovelhas foram divididas em grupos controle (GC) e tratamento (GT), cada um contendo 32 animais, e a saúde da glândula mamária avaliada na secagem e no puerpério. As ovelhas do GT receberam 250mg de gentamicina (Gentocin® Mastite Vaca Seca/ Schering-Plough Veterinária, produto indicado pela empresa para utilização em vacas de leite) em cada metade mamária. Para o diagnóstico, foram realizados exame físico da glândula mamária, California Mastitis Test, contagem de células somáticas e cultura do leite. No GC, das 45 (70,3%) metades mamárias sadias na secagem, 12 desenvolveram mastite subclínica e nove mastite clínica no puerpério. No GT, das 51 (79,7%) metades mamárias sadias na secagem, seis desenvolveram mastite subclínica e 11 mastite clínica no puerpério. Não houve associação entre o tratamento e a ocorrência de mastite no puerpério. Das 19 (29,7%) metades mamárias do GC que apresentaram mastite subclínica na secagem, três permaneceram com mastite subclínica e cinco desenvolveram mastite clínica no puerpério. No GT, das 13 (20,3%) metades mamárias com mastite subclínica na secagem, quatro permaneceram com mastite subclínica e quatro desenvolveram mastite clínica. Não houve associação entre o tratamento e a cura ou persistência da mastite no puerpério. Os principais micro-organismos isolados, na secagem e puerpério, de animais com mastite subclínica ou clínica foram Staphylococcus spp., com predominância de Staphylococcus Coagulase Negativa (SCN). No puerpério, ocorreram 29 casos de mastite clínica, sendo 19 com isolamento, 10 com SCN e seis com S. aureus. Mannheimia haemolytica foi isolado em um caso de mastite subclínica e um caso de mastite clínica. Novos protocolos e diferentes formas de manejo na secagem e no puerpério devem ser pesquisados.(AU)

A simple method to quantify follicle survival in cryopreserved human ovarian tissue.

STUDY QUESTION: Can follicle survival in frozen-thawed human ovarian tissue be quantified in situ using the dye Neutral Red (NR) to stain viable follicles specifically? SUMMARY ANSWER: A follicle survival rate within ovarian tissue can be calculated using NR followed by histological evaluation and evidence for a consistently high follicle survival in a series of ovarian tissue from 25 Danish girls and women undergoing ovarian tissue cryopreservation (OTC) was obtained. WHAT IS KNOWN ALREADY: Securing follicle survival in cryopreserved ovarian tissue is crucial for proper quality control when centers wish to implement OTC. The only established technique for validation of follicle survival is xenografting of thawed ovarian tissue to immunodeficient mice. However, this functional test is expensive, time consuming, requires animal facilities and only provides a qualitative-not quantitative-measure for follicle survival. STUDY DESIGN SIZE, DURATION: Quantification of follicle survival in human ovarian tissue donated from 30 girls and women having tissue cryopreserved for fertility preservation from 2000 to 2015 at the Laboratory of Reproductive Biology in Copenhagen, Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cryopreserved ovarian cortex was donated from 25 girls and young women aged 10-36 years (mean age: 25 years) and the average storage time in liquid nitrogen was 9.1 ± 5.6 years, ranging from 1.6 to 17.9 years. In 12 of the cases, the ovarian tissue was collected from the local hospital and in the other 13 cases the ovarian tissue was transported on ice up to 6 h prior to freezing. Donated fresh ovarian surplus tissue was obtained from five women aged 23-34 years (mean age: 27 years). Ovarian tissues were chopped into small fragments and incubated in culture medium containing 50 mg/ml NR for 3-4 h. Fragments of ovarian tissue containing clearly NR-stained follicles were selected for counting, encapsulated in 4% agar and were processed for histology to calculate a follicular survival rate. MAIN RESULTS AND THE ROLE OF CHANCE: The mean follicle survival rate in the 25 patients after freezing and thawing was 84% ± 11 (mean ±SD), ranging from 50% to 98%. The high follicle survival rate in this clinical series of patients reflects a constant high-quality service performed in our center and confirms the robustness of the slow freezing protocol. No significant association between follicle survival rates and storage time was found using linear regression analysis, suggesting that storage in liquid nitrogen does not affect viability of the tissue. No significant association in follicle survival rates was found between ovarian tissues collected at the local hospital compared to tissues transported on ice prior to freezing, supporting that prolonged cooling does not seem to greatly affect the follicle survival. For the fresh ovarian tissue, the average follicle survival rate was 91% ± 5 (mean ± SD) in five patients, ranging from 81% to 95%. LIMITATIONS, REASONS FOR CAUTION: Even though the NR staining requires active incorporation of the dye, the test is merely a short in situ test that cannot completely replace the functional value of xenografting studies in which the integrity and developmental potential of the ovarian follicles are assessed. WIDER IMPLICATIONS OF THE FINDINGS: OTC is now being employed around the world but to date it has been difficult for centers to evaluate the effectiveness of their program and perform proper quality control. NR staining combined with histological evaluation is the first quantitative method to provide a survival rate for follicles in frozen-thawed human ovarian tissue and offer a valuable and easily applicable tool to validate the cryopreservation procedure when implementing OTC or as routine quality control for the overall freezing performance within tissue banking facilities. STUDY FUNDING/COMPETING INTEREST(S): The Research Pools of Rigshospitalet, the Danish Cancer Foundation, Dagmar Marshalls Foundation, and the Novo Nordic Foundation are thanked for having funded this study. The authors have no conflicts of interest.

Transcripts encoding free radical scavengers in human granulosa cells from primordial and primary ovarian follicles.

PURPOSE: To study the presence and distribution of genes encoding free radical scavengers in human granulosa cells from primordial and primary ovarian follicles. METHODS: A class comparison study on existing granulosa cell transcriptome from primordial (n = 539 follicles) and primary (n = 261) follicles donated by three women having ovarian tissue cryopreserved before chemotherapy was performed and interrogated. RESULTS: In granulosa cells from primordial follicles, 30 genes were annotated 'mitochondrial dysfunction' including transcripts (PRDX5, TXN2) encoding enzymatic free radical scavengers peroxiredoxin 5 and thioredoxin 2. Several apoptosis regulation genes were noted (BCL2, CAS8, CAS9, AIFM1). In granulosa cells from primary follicles, mitochondrial dysfunction signalling pathway was annotated. High expression of transcripts encoding the free radical scavenger peroxiredoxin 3, as well as anti-apoptotic enzyme BCL2, was found. Interestingly, PARK7 encoding the deglycase (DJ-1) protein was expressed in granulosa cells from primary follicles. DJ-1 is implicated in oxidative defence and functions as a positive regulator of the androgen receptor and as a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/serine-threonine protein kinase (AKT) signalling pathway suppressor PTEN. CONCLUSIONS: The results indicate extensive energy production and free radical scavenging in the granulosa cells of primordial follicles with potential implications for ovarian ageing, cigarette smoking, premature ovarian failure and polycystic ovarian syndrome. Furthermore, DJ-1 may be involved in androgen responsiveness and the regulation of follicle growth via PI3K/PTEN/AKT signalling pathway regulation in the granulosa cells of primary follicles. The involvement of mitochondrial free radical production in the age-related decline of competent oocytes is becoming apparent.

Granulosa cells from human primordial and primary follicles show differential global gene expression profiles.

STUDY QUESTION: Can novel genetic candidates involved in follicle dormancy, activation and integrity be identified from transcriptomic profiles of isolated granulosa cells from human primordial and primary follicles? SUMMARY ANSWER: The granulosa cell compartment of the human primordial and primary follicle was extensively enriched in signal transducer and activator of transcription 3 (STAT3) and cAMP-response element binding protein (CREB) signalling, and several other putative signalling pathways that may also be mediators of follicle growth and development were identified. WHAT IS KNOWN ALREADY: Mechanistic target of rapamycin kinase (mTOR) signalling and the factors Forkhead Box L2 (FOXL2) and KIT proto-oncogene receptor tyrosine kinase (KITL) may be involved in defining the early steps of mammalian follicular recruitment through complex bidirectional signalling between the oocyte and granulosa cells. cAMP/protein kinase K (PKA)/CREB signalling is a feature of FSH-induced regulation of granulosa cell steroidogenesis that is essential to normal human fertility. STUDY DESIGN, SIZE, DURATION: A class comparison study was carried out on primordial follicles (n = 539 follicles) and primary follicles (n = 261) follicles) donated by three women having ovarian tissue cryopreserved before chemotherapy. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA samples from isolates of laser capture micro-dissected oocytes and follicles from the primordial and primary stage, respectively, were sequenced on the HiSeq Illumina platform. Data mapping, quality control, filtering, FPKM (fragments per kilobase of exon per million) normalization and comparisons were performed. The granulosa cell contribution in whole follicle isolates was extracted in silico. Modelling of complex biological systems was performed using Ingenuity Pathway Analysis (IPA). For validation of transcriptomic findings, we performed quantitative RT-PCR of selected candidate genes. Furthermore, we interrogated the in situ localization of selected corresponding proteins using immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: Our differentially expressed gene analysis revealed a number of transcripts in the granulosa cells to be significantly down- (736 genes) or up- (294 genes) regulated during the human primordial-to-primary follicle transition. The IPA analysis revealed enriched canonical signalling pathways not previously associated with granulosa cells from human primordial and primary follicles. Immunofluorescent staining of human ovarian tissue explored the intra-ovarian localization of FOG2, and FOXL2, which revealed the presence of forkhead box L2 (FOXL2) in both oocytes and granulosa cells in primary follicles, with a more enriched staining in the granulosa cells in primary follicles. Friend of GATA 2 (FOG2) stained strongly in oocytes in primordial follicles, with a shift towards granulosa cell as follicle stage advanced. LARGE SCALE DATA: http://users-birc.au.dk/biopv/published_data/ernst_et_al_GC_2017/. LIMITATIONS REASONS FOR CAUTION: This is a descriptive study, and no functional assays were employed. The study was based on a limited number of patients, and it is acknowledged that natural biological variance exists in human samples. Strict filters were applied to accommodate the in silico extraction of the granulosa cell contribution. In support of this, quantitative RT-PCR was used to confirm selected candidate genes, and immunofluorescent staining was employed to interrogate the intra-ovarian distribution of selected corresponding proteins. Moreover, it is unknown whether the primordial follicles analysed represent those still in the resting pool, or those from the cohort that have entered the growing pool. WIDER IMPLICATIONS OF THE FINDINGS: We present, for the first time, a detailed description of global gene activity in the human granulosa cell compartment of primordial and primary follicles. These results may be utilized in the development of novel clinical treatment strategies aimed at improving granulosa cell function. STUDY FUNDING/COMPETING INTEREST(S): E.H.E. was supported by the Health Faculty, Aarhus University and Kong Christian Den Tiendes Fond. K.L.H. was supported by a grant from Fondens til Lægevidenskabens Fremme and Kong Christian Den Tiendes Fond. No authors have competing interests to declare.

Transplantation of frozen-thawed ovarian tissue: an update on worldwide activity published in peer-reviewed papers and on the Danish cohort.

PURPOSE: The purpose of the study is to review all peer-reviewed published reports of women receiving ovarian tissue transplantation (OTT) with frozen/thawed tissue (OTC) with respect to age, diagnosis, transplantation site, fertility outcome, and potential side effects, including data from all women in the Danish program. METHODS: A systematic review of the literature was performed in PubMed combined with results from all patients who had received OTT in Denmark up to December 2017. RESULTS: OTT has been reported from 21 different countries comprising a total of 360 OTT procedures in 318 women. In nine women, malignancy was diagnosed after OTT; none were considered to be directly caused by the OTT. Despite a potential under reporting of cancer recurrence, there is currently no evidence to suggest that OTT causes reseeding of the original cancer. Renewed ovarian endocrine function was reported in 95% of the women. Half of all children born following OTT resulted from natural conception, and newborns were reported to be healthy except for one neonate with a chromosome anomaly with a family disposition. Women who conceived after OTT were significantly younger than those who failed. CONCLUSION: This study found no indications of sufficient numbers of malignant cells present in the ovarian tissue to cause recurrence of cancer after OTT. Further, it is unlikely that OTC affects the well-being of children born. OTC is now an established method of fertility preservation in Denmark with public reimbursement. The current data encourage that women who require gonadotoxic treatment should be offered an individual evaluation considering fertility preservation.

The polymorphic insertion of the luteinizing hormone receptor "insLQ" show a negative association to LHR gene expression and to the follicular fluid hormonal profile in human small antral follicles.

The luteinizing hormone receptor (LHCGR) has a little studied polymorphic 6 bp insertion (rs4539842/insLQ). This study has evaluated the insLQ polymorphism in relation to potential associations with hormonal characteristics of human small antral follicles (hSAFs). In total, 310 hSAFs were collected from 86 women undergoing fertility preservation. Analysis included hormonal profile of 297 follicular fluid (FF) samples and 148 corresponding granulosa cells samples were evaluated by qPCR for selected genes. Significantly reduced and non-detectable mRNA levels of anti-Müllerian hormone receptor II (AMHR2) and LHCGR, respectively, were observed for insLQ/insLQ compared to -/insLQ and the -/- genotypes. Moreover, LHCGR and CYP19a1 together with oestradiol and inhibin-B were significantly increased in -/insLQ compared to the -/- genotype. The homozygous insLQ genotype showed strong significant associations to GC specific genes LHCGR and CYP19a1, which may translate into significant changes in FF hormone profiles and an altered LH signaling.

Dormancy and activation of human oocytes from primordial and primary follicles: molecular clues to oocyte regulation.

STUDY QUESTION: Do specific transcriptome dynamics in human oocytes from primordial and primary follicles identify novel pathways in oocyte activation? SUMMARY ANSWER: The transcriptomic profiles in oocytes from primordial and primary follicles, respectively, revealed several new canonical pathways as putative mediators of oocyte dormancy and activation. WHAT IS KNOWN ALREADY: Cellular signaling pathways including PI3K/AKT and AKT/mTOR as well as TGF-ß and IGF signaling are known to regulate the primordial-to-primary transition in mammalian follicle development. STUDY DESIGN, SIZE, DURATION: We performed a class comparison study on human oocytes from primordial (n = 436) and primary (n = 182) follicles donated by three women having ovarian tissue cryopreserved before chemotherapy. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA was extracted from oocytes from primordial and primary follicles isolated by Laser Capture Microdissection, and submitted to the HiSeq Illumina platform. Data mapping, quality control, filtering and expression analysis were performed using Tophat (2.0.4), Cufflinks (2.0.2), BWA (0.6.2) and software R. Modeling of complex biological systems was performed using the IPA® software. Finally, qPCR and immunohistochemistry were employed to explore expression and localization of selected genes and products in human ovarian tissue. MAIN RESULTS AND THE ROLE OF CHANCE: We found 223 and 268 genes down-regulated and up-regulated, respectively, in the oocytes during the human primordial-to-primary follicle transition (P < 0.05 and/or FPKM fold-change >2). IPA® enrichment analysis revealed known pathways ('mTOR Signaling', 'PI3K/AKT Signaling' and 'PTEN Signaling') as well as enriched canonical pathways not previously associated with human ovarian follicle development such as 'ErB Signaling' and 'NGF Signaling' in the down-regulated category and 'Regulation of eIF4 and P70S6K Signaling' and 'HER-2 Signaling in Breast Cancer' in the up-regulated group. Additionally, immunohistochemistry on human ovarian tissue explored the intraovarian localization of VASA, FOXO1 and eIF4E. LARGE SCALE DATA: http://users-birc.au.dk/biopv/published_data/ernst_2017/. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive analysis and no functional studies were performed. The study was based on a limited number of patients and the experimental design could not take into account the natural biological variance in human samples. Therefore, qPCR was used to confirm selected genes alongside immunohistochemical stainings. WIDER IMPLICATIONS OF THE FINDINGS: This study shows, for the first time, a detailed molecular description of global gene transcription activities in oocytes from primordial and primary follicles, respectively. Knowing the global transcription profiles of human oocyte dormancy and activation are important in developing new clinical applications. STUDY FUNDING/COMPETING INTEREST(S): E.H.E. was supported by Health Faculty, Aarhus University and Kong Christian Den Tiendes Fond. K.H. and S.F. were supported by an MRC (UK) project grant MR/M012638/1. K.L.H. was supported by grants from Fonden til Lægevidenskabens Fremme, Kong Christian Den Tiendes Fond. K.L.H. and L.S. were supported by the IDEAS grant from Aarhus University Research Foundation (AUFF). There are no conflicts of interest.

Inactivation of West Nile virus in serum with heat, ionic detergent, and reducing agent for proteomic applications.

Research involving biosafety level 3 pathogens such as West Nile virus (WNV) is often limited by the limited space and technical constraints of these environments. To conduct complex analytical studies outside of high containment, robust and reliable inactivation methods are needed that maintain compatibility with downstream assays. Here we report the inactivation of WNV in spiked serum samples using a commercially available SDS-PAGE sample buffer for proteomic studies. Using this method, we demonstrate its utility by identification proteins differentially expressed in the serum of mice experimentally infected with WNV.

Antibiotic-dependent perturbations of extended spectrum beta-lactamase producing Klebsiella pneumoniae proteome.

Extended spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-KP) causes life-threatening infections in susceptible and immuno-compromised individuals. Because of the emergence of multidrug resistance and tolerance, it is crucial to better understand the mechanisms by which ESBL-KP can adapt to antibiotic stress. The aim of this study was to provide an overview of the global proteome changes occurring in ESBL-KP in response to sub-lethal concentrations of the antibiotics doxycycline (DC, bacteriostatic) and streptomycin (SM, bactericidal), which both impair ribosomal synthesis of bacterial proteins. These results represent the greatest experimental coverage of the ESBL-KP proteome yet described. The 1538 proteins, representing 30% of the 5126 predicted KP gene products were identified from the combined experimental groups. Antibiotic stress resulted in significantly elevated levels of 42 proteins for DC and 55 for SM treatments, whereas 53 proteins were reduced for DC- and six for SM-treated bacteria. Specifically, the ESBL-KP response to DC was accompanied by the reduced levels of the porins LamB, CirA, FepA, and OmpC. In contrast to DC, the stress response to SM demonstrated a dramatic increase in the peroxidase detoxification pathway proteins PutA, KatG, KatE, and Dps, which prevent harmful hydroxyl radical formation. The results from this proteomic study are important for understanding adaptive responses to antibiotics, and may provide novel targets for the development of new therapeutic strategies.

Direct Observation of Enhanced Nitric Oxide in a Murine Model of Diabetic Nephropathy.

Uncoupling of nitric oxide synthase (NOS) secondary to redox signaling is a central mechanism in endothelial and macrophage activation. To date studies on the production of nitric oxide (NO) during the development of diabetic complications show paradoxical results. We previously showed that recoupling eNOS by increasing the eNOS cofactor tetrahydrobiopterin (BH4) could restore endothelial function and prevent kidney injury in experimental kidney transplantation. Here, we employed a diabetic mouse model to investigate the effects of diabetes on renal tissue NO bioavailability. For this, we used in vivo NO trapping, followed by electron paramagnetic resonance spectroscopy. In addition, we investigated whether coupling of NOS by supplying the cofactor BH4 could restore glomerular endothelial barrier function. Our data show that overall NO availability at the tissue level is not reduced sixteen weeks after the induction of diabetes in apoE knockout mice, despite the presence of factors that cause endothelial dysfunction, and the presence of the endogenous NOS inhibitor ADMA. Targeting uncoupled NOS with the BH4 precursor sepiapterin further increases NO availability, but did not modify renal glomerular injury. Notably, glomerular heparanase activity as a driver for loss of glomerular barrier function was not reduced, pointing towards NOS-independent mechanisms. This was confirmed by unaltered increased glomerular presence of cathepsin L, the protease that activates heparanase.

Cryopreservation of ovarian tissue for fertility preservation in a large cohort of young girls: focus on pubertal development.

STUDY QUESTION: Is there an association between the need for medical puberty induction and the diagnosis or treatment received in girls who have undergone cryopreservation of ovarian tissue for fertility preservation? SUMMARY ANSWER: There was a clear association between the intensity of treatment received and requirement for medical puberty induction but no association with the diagnosis. WHAT IS KNOWN ALREADY: Although it cannot be predicted which girls will become infertile or develop premature ovarian insufficiency (POI) following intensive chemotherapy or irradiation, patients who are at high risk of POI should be offered ovarian tissue cryopreservation (OTC). This includes girls who are planned to receive either high doses of alkylating agents, conditioning regimen before stem cell transplantation (SCT), total body irradiation (TBI) or high radiation doses to the craniospinal, abdominal or pelvic area. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study. In total, 176 Danish girls under 18 years of age have had OTC performed over a period of 15 years. An overview of the girls' diagnoses and mean age at OTC as well as the number of deceased is presented. Of the 176 girls, 38 had died and 46 girls were still younger than 12 years so their pubertal development cannot be evaluated yet. For the 60 girls who had OTC performed after 12 years of age, the incidence of POI was evaluated and in the group of 32 girls who were younger than 12 years at OTC, the association between the diagnosis and received treatment and the requirement for medical puberty induction was examined. PARTICIPANTS/MATERIALS, SETTING, METHODS: The need for medical puberty induction was assessed in 32 girls who were prepubertal at the time of OTC. MAIN RESULTS AND THE ROLE OF CHANCE: Indications for OTC were allogeneic SCT for leukaemia, myelodysplastic syndrome or benign haematological disorders, autologous SCT for lymphoma or sarcoma, and irradiation to the pelvis or to the spinal axis. The mean age at OTC of the 176 girls were 11.3 years. The two most prevalent diagnoses of the 176 girls were malignant tumours and malignant haematological diseases. Among the 32 prepubertal girls, 12 received high dose chemotherapy and either TBI prior to SCT or irradiation to the pelvis, abdomen or the spinal axis, 13 received high dose alkylating agents but no irradiation prior to SCT, six received alkylating agents as part of conventional chemotherapy and one patient had a genetic metabolic disorder and did not receive gonadotoxic treatment. Among these 32 girls, 23 did not undergo puberty spontaneously and thus received medical puberty induction. Among the nine girls, who went through spontaneous puberty, four had received high dose alkylating agents and five had received conventional chemotherapy. LIMITATIONS REASONS FOR CAUTION: All information was retrieved retrospectively from patient records, and thus some information was not available. WIDER IMPLICATIONS OF THE FINDINGS: OTC should be recommended to all young girls, who present a high risk of developing ovarian insufficiency and/or infertility following high dose chemotherapy and/or irradiation. STUDY FUNDING/COMPETING INTERESTS: The Childhood Cancer Foundation (2012-2016) and the EU interregional project ReproHigh are thanked for having funded this study. They had no role in the study design, collection and analysis of the data or writing of the report. The authors have no conflict of interest to disclose.

Development of a liquid chromatography high resolution mass spectrometry method for the quantitation of viral envelope glycoprotein in Ebola virus-like particle vaccine preparations.

BACKGROUND: Ebola virus like particles (EBOV VLPs, eVLPs), are produced by expressing the viral transmembrane glycoprotein (GP) and structural matrix protein VP40 in mammalian cells. When expressed, these proteins self-assemble and bud from 'host' cells displaying morphology similar to infectious virions. Several studies have shown that rodents and non-human primates vaccinated with eVLPs are protected from lethal EBOV challenge. The mucin-like domain of envelope glycoprotein GP1 serves as the major target for a productive humoral immune response. Therefore GP1 concentration is a critical quality attribute of EBOV vaccines and accurate measurement of the amount of GP1 present in eVLP lots is crucial to understanding variability in vaccine efficacy. METHODS: After production, eVLPs are characterized by determining total protein concentration and by western blotting, which only provides semi-quantitative information for GP1. Therefore, a liquid chromatography high resolution mass spectrometry (LC-HRMS) approach for accurately measuring GP1 concentration in eVLPs was developed. The method employs an isotope dilution strategy using four target peptides from two regions of the GP1 protein. Purified recombinant GP1 was generated to serve as an assay standard. GP1 quantitation in 5 eVLP lots was performed on an LTQ-Orbitrap Elite and the final quantitation was derived by comparing the relative response of 200 fmol AQUA peptide standards to the analyte response at 4 ppm. RESULTS: Conditions were optimized to ensure complete tryptic digestion of eVLP, however, persistent missed cleavages were observed in target peptides. Additionally, N-terminal truncated forms of the GP1 protein were observed in all eVLP lots, making peptide selection crucial. The LC-HRMS strategy resulted in quantitation of GP1 with a lower limit of quantitation of 1 fmol and an average percent coefficient of variation (CV) of 7.6 %. Unlike western blot values, the LC-HRMS quantitation of GP1 in 5 eVLP vaccine lots exhibited a strong linear relationship (positive correlation) with survival (after EBOV challenge) in mice. CONCLUSIONS: This method provides a means to rapidly determine eVLP batch quality based upon quantitation of antigenic GP1. By monitoring variability in GP1 content, the eVLP production process can be optimized, and the total amount of GP1 needed to confer protection accurately determined.

A Multicomponent Animal Virus Isolated from Mosquitoes.

RNA viruses exhibit a variety of genome organization strategies, including multicomponent genomes in which each segment is packaged separately. Although multicomponent genomes are common among viruses infecting plants and fungi, their prevalence among those infecting animals remains unclear. We characterize a multicomponent RNA virus isolated from mosquitoes, designated Guaico Culex virus (GCXV). GCXV belongs to a diverse clade of segmented viruses (Jingmenvirus) related to the prototypically unsegmented Flaviviridae. The GCXV genome comprises five segments, each of which appears to be separately packaged. The smallest segment is not required for replication, and its presence is variable in natural infections. We also describe a variant of Jingmen tick virus, another Jingmenvirus, sequenced from a Ugandan red colobus monkey, thus expanding the host range of this segmented and likely multicomponent virus group. Collectively, this study provides evidence for the existence of multicomponent animal viruses and their potential relevance for animal and human health.

Outcomes of transplantations of cryopreserved ovarian tissue to 41 women in Denmark.

STUDY QUESTION: What are the results of transplanting cryopreserved ovarian tissue? SUMMARY ANSWER: The transplanted ovarian tissue can last up to 10 years, with no relapses following the 53 transplantations, and the chance of a successful pregnancy is currently around one in three for those with a pregnancy-wish. WHAT IS KNOWN ALREADY: Cryopreservation of ovarian tissue is now gaining ground as a valid method for fertility preservation. More than 36 children worldwide have now been born following this procedure. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study of 41 women who had thawed ovarian tissue transplanted 53 times over a period of 10 years, including 1 patient who was lost to follow-up. PARTICIPANTS/MATERIALS, SETTING, METHODS: The 41 Danish women, who had in total 53 transplantations, were followed for ovarian function and fertility outcome. Safety was assessed by monitoring relapse in cancer survivors. MAIN RESULTS, AND THE ROLE OF CHANCE: Among 32 women with a pregnancy-wish, 10 (31%) had a child/children (14 children in total); this included 1 woman with a third trimester on-going pregnancy. In addition, two legal abortions and one second trimester miscarriage occurred. A total of 24 clinical pregnancies were established in the 32 women with a pregnancy-wish. The tissue remained functional for close to 10 years in some cases and lasted only a short period in others. Three relapses occurred but were unlikely to be due to the transplanted tissue. LIMITATIONS, REASONS FOR CAUTION: Self-report through questionnaires with only in-one hospital formalised follow-up of transplanted patients could result in unreported miscarriages. The longevity of the tissue may vary by few months compared with those reported because some patients simply could not remember the date when the tissue became non-functional. WIDER IMPLICATIONS OF THE FINDINGS: Cryopreservation of ovarian tissue is likely to become integrated into the treatment of young women, with cancer, who run a risk of losing their fertility. The full functional lifespan of grafts is still being evaluated, because many of the transplanted women have continued to maintain ovarian activity. Some of our first cases have had tissue functioning for ∼ 10 years.