Role of the mTOR Signalling Pathway During Extracellular Acidosis in Tumour Cells.

The metabolic microenvironment of solid tumours is often dominated by extracellular acidosis which results from glycolytic metabolism. Acidosis can modulate gene expression and foster the malignant progression. The aim of the study was to analyse the effects of extracellular acidosis on the mTOR signalling pathway, an important regulator of anabolic and catabolic processes like cell proliferation and autophagy. The study was performed in two tumour cell lines, AT-1 prostate and Walker-256 mammary carcinoma cells. Cells were incubated at pH 7.4 or 6.6 for 3 h and 24 h. Then RNA and protein were extracted and analysed by qPCR and western blot. mTOR and P70-S6 kinase (P70-S6K), an important downstream target of mTOR, as well as the autophagic flux were studied. The effect of acidosis on P70S6K phosphorylation was compared to pharmacological mTOR inhibition with LY294002 and rapamycin. In both cell lines the total mTOR expression was not altered by acidosis, however, the mTOR phosphorylation was reduced after 3 h but not after 24 h. The P70S6K phosphorylation was reduced at both time points comparable to changes by pharmacological mTOR inhibitors. The autophagic flux, also a target of mTOR and measured by LC3-II expression, was increased in both cell lines after 24 h of acidosis. The results of this study indicate that mTOR signalling is inhibited by extracellular acidosis which then lead to a reduced activity of the P70-S6 kinase (modulating gene expression) and increased autophagy possibly mediated by ULK1/2 activity. These finding may offer new perspectives for therapeutic interventions in acidic tumours.

Determinants of transplantation success with cryopreserved ovarian tissue: data from 196 women of the FertiPROTEKT network.

STUDY QUESTION: What are the pregnancy and live birth rates for ovarian tissue transplantation and which factors are associated with the success rate? SUMMARY ANSWER: Pregnancy and live birth rates per transplanted woman are 32.7% and 26.5% and success rate is associated with female age and first versus repeated transplantation. WHAT IS KNOWN ALREADY: Live birth rates after ovarian tissue transplantations have been reported to be between around 24% and 41% per patient. Success rates seem to be negatively associated with increasing female age at the time of tissue cryopreservation and with pelvic radiation. Success rates are apparently not reduced after overnight transportation of ovarian tissue before freezing. STUDY DESIGN, SIZE, DURATION: Registry analysis of 244 transplantations in 196 women, performed by 26 FertiPROTEKT network centres from 2007 to 2019 with follow-up till December 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: Orthotopic ovarian tissue transplantations were performed in 196 women, 191 with previous malignant and 5 with previous non-malignant diseases. Size of transplanting centres varied between 1 and 100 transplantations per centre (median: 2). Factors possibly associated with success rate such as female age, first and repeated transplantation, experience of the transplanting centre and overnight transportation of the ovarian tissue before freezing were analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Average age of all 196 transplanted women was 31.3 years (SD 5.2; range 17-44) at the time of cryopreservation of tissue and 35.9 years (SD 4.8; range 23-47) at the time of transplantation. Pregnancy rate was 30.6% (95% CI, 24.2-37.6%) per first transplantation and 32.7% (95% CI, 26.1-39.7%) per patient. Pregnancy rate was higher after first transplantation (30.6% (95% CI, 24.2-37.6%)) compared to second and subsequent transplantations (11.8% (95% CI, 3.3-27.5%)). Live birth rate per first transplantation was 25.0% (95% CI, 19.1-31.7%) and per patient 26.5% (95% CI, 20.5-33.3%). Success rate decreased with increasing age at the time of ovarian tissue freezing. Live birth rate was 28.2% (95% CI, 20.9-36.3%) in women <35 years and 16.7% (95% CI, 7.9-29.3%) in women >35 years. Pregnancy rates after first transplantation were higher in centres who had performed ≥10 transplantations (35.1%) compared to centres with <10 transplantation (25.4%) (P = 0.12). Corresponding live birth rates were 27.0% and 18.6%. Success rates were not different in women with and without overnight transportation of tissue before cryopreservation. LIMITATIONS, REASONS FOR CAUTION: The data were drawn from a registry analysis. Data such as ovarian reserve and premature ovarian insufficiency were not available for all women. Data might be influenced by different follow-up policies of the centres. WIDER IMPLICATIONS OF THE FINDINGS: The study reveals the high potential of ovarian tissue freezing and transplantation, but only if freezing is performed in younger women. The study suggests focus should be placed on the first and not on repeated transplantations. It also opens the discussion of whether transplantation should rather be performed by experienced centres. STUDY FUNDING/COMPETING INTEREST(S): No funding. No competing interests. TRIAL REGISTRATION NUMBER: N/A.

The impact of micronized progesterone on cardiovascular events - a systematic review.

Biologically identical menopausal hormone therapy (MHT) including micronized progesterone (MP) has gained much attention. We aimed to assess the impact of MP in combined MHT on venous and arterial thromboembolism (VTE/ATE) (e.g. deep venous thrombosis/pulmonary embolism, myocardial infarction [MI] and ischemic stroke). Articles were eligible if they provided endpoints regarding cardiovascular events and use of exogenous MP. Literature searches were designed and executed for the databases Medline, Embase, CINAHL, the Cochrane Library, ClinicalTrials.gov and interdisciplinary database Web of Science. Twelve studies consisting of randomized controlled trials (RCTs), case-control studies and prospective or retrospective cohort studies were included, and risk of bias was assessed. Only a minority assessed thromboembolic events as a primary endpoint, showing that in contrast to norpregnane derivatives, primary and recurrent VTE risk was not altered by combining estrogens with MP, which was also true for ischemic stroke risk. Similarly, in placebo-controlled RCTs assessing VTE/ATE as adverse events there were no significant intergroup differences. Studies on MI as a primary endpoint are missing. In conclusion, while available data suggest that MP as a component in combined MHT may have a neutral effect on the vascular system, more RCTs investigating the impact of MP alone or in combined MHT on vascular primary endpoints are needed.

Cerebral aspergillosis in the era of new antifungals: The CEREALS national cohort study Nationwide CEREbral Aspergillosis Lesional study (CEREALS).

BACKGROUND: Cerebral aspergillosis (CA) is a life-threatening disease for which diagnosis and management remain challenging. Detailed analyses from large cohorts are lacking. METHODS: We included 119 cases of proven (n = 54) or probable (n = 65) CA diagnosed between 2006 and 2018 at 20 French hospitals. Data were collected at baseline and during follow-up. Cerebral imaging was reviewed centrally by two neuroradiologists. RESULTS: The most frequent underlying conditions were hematological malignancy (40%) and solid organ transplantation (29%). Galactomannan was detected in the serum of 64% of patients. In 75% of cases, at least one of galactomannan, Aspergillus PCR, and ß-d-glucan was positive in the cerebrospinal fluid. Six-week mortality was 45%. Two distinct patterns of disease were identified according to presumed route of dissemination. Presumed haematogenous dissemination (n = 88) was associated with a higher frequency of impaired consciousness (64%), shorter time to diagnosis, the presence of multiple abscesses (70%), microangiopathy (52%), detection of serum galactomannan (69%) and Aspergillus PCR (68%), and higher six-week mortality (54%). By contrast, contiguous dissemination from the paranasal sinuses (n = 31) was associated with a higher frequency of cranial nerve palsy (65%), evidence of meningitis on cerebral imaging (83%), macrovascular lesions (61%), delayed diagnosis, and lower six-week mortality (30%). In multivariate analysis and in a risk prediction model, haematogenous dissemination, hematological malignancy and the detection of serum galactomannan were associated with higher six-week mortality. CONCLUSION: Distinguishing between hematogenous and contiguous dissemination patterns appears to be critical in the workup for CA, as they are associated with significant differences in clinical presentation and outcome.

Impact of progesterone on skin and hair in menopause - a comprehensive review.

In clinical practice, micronized progesterone (MP) is frequently recommended to treat signs and symptoms of skin and hair aging in menopausal women. The aim of this comprehensive review was to evaluate whether topically or systemically applied MP may effectively prevent or slow down signs of skin and hair aging. Three out of six identified studies reported an impact of MP on skin aging markers in menopausal women. Of these, two studies reported a benefit: one for topically applied MP, another for systemically applied combined menopausal hormone therapy (MHT) comprising MP as progestogen for endometrial protection. Tolerability and safety of MP were good. However, there was no study investigating the impact of MP on menopausal scalp hair. In conclusion, delay of skin aging comprises lifestyle adjustment, antioxidants, and several esthetic procedures. In menopausal women, MHT displays beneficial effects on skin aging. There is poor quality but promising scientific evidence for MP displaying anti-aging skin effects in menopausal women. However, good quality studies are needed.

Serum anti-Müllerian hormone concentration and follicle density throughout reproductive life and in different diseases-implications in fertility preservation.

STUDY QUESTION: How do anti-Müllerian hormone (AMH) serum concentrations and follicle densities (FDs) change with age and disease and what are the implications for fertility preservation? SUMMARY ANSWER: AMH concentrations and FD do not correlate in young women, and AMH but not FD is reduced in some diseases, limiting the value of AMH as a predictive parameter of ovarian tissue transplantation. WHAT IS KNOWN ALREADY: AMH is widely used as a parameter to estimate the ovarian reserve. However, the reliability of AMH to predict total number of follicles and the FD is questionable. Women with lymphoma and leukaemia have been shown to have reduced AMH concentrations, but it is unknown if the FD is also reduced. In fertility preservation it is essential to estimate the correct total number of follicles and the FD, as ovarian tissue should only be cryopreserved if ovarian reserve is high. Furthermore, the amount of tissue to be transplanted should be based on the estimation of the real FD. STUDY DESIGN, SIZE, DURATION: This retrospective observational study included 830 women (mean ± SD age, 28.2 ± 6.81 years; range, 4-43 years) with malignant (n = 806) and benign (n = 24) diseases who cryopreserved tissue in a single centre as part of a national fertility preservation programme. Females with ovarian surgery or known predispositions for a reduced ovarian reserve were excluded. AMH concentrations and FD were evaluated from March 2011 to September 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: AMH concentrations were analysed before gonadotoxic therapies. Standardized biopsies, obtained from different areas of ovarian cortex, were collected. FD was analysed after tissue digestion and calcein staining and was expressed as average number of primordial and primary follicles count per 3 mm biopsy and per cubic millimeter tissue. AMH concentrations and FD were analysed in relation to age and diagnosis group. Both parameters were age adjusted, and associations between the different diagnosis groups and AMH versus FD were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Mean ± SD AMH concentration was 3.1 ± 2.81 g/ml, mean FD per 3 mm biopsy was 137 ± 173.9 and 19.4 ± 24.60 per mm3. Maximum AMH concentrations were found in children and teenagers at the age of 6-10 years (5.71 ng/ml) and in adults at the age of 21-25 years (3.33 ng/ml). FD was highest in young children up to an age of 15 years and decreased with increasing age. AMH and FD were not correlated in women ≤20 years and weakly to moderately correlated in women 21-40 years (r = 0.24-0.39). Age-adjusted correlations between AMH and FD were demonstrated in several diagnosis groups such as breast cancer, leukaemia, sarcoma, gastrointestinal cancer and gynaecological cancer but not in the groups exhibiting Hodgkin's and non-Hodgkin's lymphoma, cerebral cancer, other types of malignancies and other types of benign diseases. Further statistical analysis supported the finding that, in some diagnosis groups such as Hodgkin's lymphoma and in gynaecological cancer, AMH concentrations but not FDs are reduced, questioning the prognostic accuracy of AMH for the FD in these diseases. LIMITATIONS, REASONS FOR CAUTION: Even though biopsies were taken from different sites, heterogenous distribution of follicles might have had some effect on the accuracy of the analysis. WIDER IMPLICATION OF THE FINDINGS: AMH should be used with care to estimate the total ovarian reserve and FD of cancer patients in young women in some diseases. Therefore, calculating the amount of ovarian tissue to be transplanted based solely on AMH might be inaccurate whereas FD might be a better parameter. STUDY FUNDING/COMPETING INTEREST(S): The study did not receive any exterior funding.

Evaluation of a Chitosan Hemostat in a Porcine Laparoscopic Partial Nephrectomy Model: A Pilot Study.

Background and Objective: The ideal hemostatic agent for laparoscopic partial nephrectomy (LPN) would provide complete hemostasis and sealing of the collecting system at a low cost. Chitosan (CS) is an established topical hemostatic agent, but standard sterilization techniques affect its functional and biologic properties, thereby preventing parenteral uses. This study sought to characterize the safety and efficacy of an implanted CS hemostat sterilized with either a standard technique, electron beam (e-beam) irradiation, or a novel technique, nonthermal nitrogen plasma, in a porcine LPN model. Methods: Laparoscopic partial nephrectomies were performed on six farm pigs and hemostasis achieved using only a CS hemostatic agent (Clo-Sur P.A.D.) that was e-beam (n = 3) or plasma sterilized (PS) (n = 3). Number of pads needed to achieve hemostasis, estimated blood loss, operative time, mass of kidney resection, and warm ischemia time were measured. Animals were monitored for 14 weeks and at harvest, retrograde ureteropyelography and histologic analysis were performed. Results: Complete hemostasis and collection system sealing were achieved in both groups. There was a trend toward less pads required for hemostasis (p = 0.056) and reduced blood loss (p = 0.096) with PS pads, although this did not achieve statistical significance. No complications were observed for 14 weeks and gross examination showed the implanted CS was encapsulated in a fibrous capsule. Histologic analysis revealed a healed nephrectomy site with residual CS and associated chronic inflammation, reactive fibrosis, and foreign body giant cell formation. Importantly, the adjacent renal tissue was intact and viable with no residual parenchymal inflammation or cytologic damage. Conclusion: CS pads alone provided safe and effective hemostasis in a porcine LPN model. PS may enhance hemostatic efficacy and resorption compared with e-beam.

Fertility preservation: ovarian response to freeze oocytes is not affected by different malignant diseases-an analysis of 992 stimulations.

PURPOSE: To study if ovarian response is affected by the type of disease if fertility preservation is required. METHODS: A registry of the trinational fertility preservation network FertiPROTEKT including 992 patients aged 18-40 years undergoing ovarian stimulation and follicle aspiration for fertility preservation from 1/2007 until 3/2016 was analysed. The number of collected oocytes, days of stimulation, total gonadotropin dosage and gonadotropin dosage per day were evaluated. RESULTS: Total oocyte number was negatively correlated with increasing age (r = 0.237, p < 0.0001). Oocyte numbers were in women < 26 years 15.4 ± 8.8, 26-30 years 13.1 ± 8.5, 31-35 years 12.2 ± 7.7 and 36-40 years 9.9 ± 8.0. Age-adjusted oocyte numbers were not different in women with Hodgkin's lymphoma (12.6 ± 8.8), non-Hodgkin's lymphoma (12.4 ± 8.2), leukaemia (11.7 ± 8.2), sarcoma (11.8 ± 8.2), cerebral cancer (16.5 ± 8.1), gastrointestinal cancer (13.2 ± 8.1) gynaecological cancer (10.8 ± 8.2) and other types of malignancies (15.8 ± 8.1) apart from ovarian cancer with lower oocyte yield (7.3 ± 8.3, p < 0.001) compared to women with breast cancer (13.3 ± 8.8). The total gonadotropin dose used for stimulation was only elevated in Hodgkin's and non-Hodgkin's lymphoma compared to women with breast cancer (p < 0.05). Oocyte yield was lower in women with versus without ovarian cancer (p < 0.0001). CONCLUSIONS: As ovarian response is not affected by the type of cancer, ovarian stimulation can be performed with the same oocyte yield in different malignant diseases. However, oocyte yield is reduced if ovarian surgery is required and in older women.

Optimizing the impact of alcohol and drug screening and early intervention in a high-risk population receiving services in New York City sexual health clinics: A process and outcome evaluation of Project Renew.

Unhealthy substance use is associated with increased rates of STDs, including HIV. Within three high-risk New York City (NYC) sexual health clinics between 2008 and 2012 (n = 146,657), 17% of patients screened positive for a current SUD but only 5.3% ever received prior treatment. The goal of Project Renew was to expand the reach of substance use early intervention services within and across sexual health clinics citywide and decrease substance use, poor mental health, and risky sexual behavior. To accomplish this goal, Screening, Brief Intervention, and Referral to Treatment (SBIRT), an evidence-based substance use early intervention model, was implemented in all eight NYC sexual health clinics February 2012-January 2015. Clinic patients were screened for substance misuse using the AUDIT/DAST-10, and those who screened positive were eligible for on-site brief intervention. Overall, 130,597 substance misuse screenings were conducted (66,989, or 51%, positive), and 17,474 on-site brief interventions and 1238 referrals were provided (not unique to individual patients). A 10% sample of 14,709 unique patients who screened positive were interviewed using a federal data collection tool at baseline and six months later to assess changes in substance use, sexual risk behaviors, mental health, and health status (n = 1328). At six-month follow-up, patients reported reduced substance use, less sexual activity, improved overall health, and fewer days of depression and anxiety compared to measures at baseline (p < 0.05). Based on positive results, Project Renew SBIRT services have been sustained, ensuring essential care which may help prevent acquisition of HIV/STDs among a large population of high-risk New Yorkers.

Practical recommendations for fertility preservation in women by the FertiPROTEKT network. Part I: Indications for fertility preservation.

PURPOSE: Most guidelines about fertility preservation are predominantly focused on scientific evidence, but are less practically orientated. Therefore, practically oriented recommendations are needed to support the clinician in daily practice. METHODS: A selective literature search was performed based on the clinical and scientific experience of the authors, focussing on the most relevant diseases and gynaecological cancers. This article (Part I) provides information on topics that are essential for the fertility preservation indication, such as disease prognosis, disease therapy and its associated risks to fertility, recommending disease-specific fertility preservation measures. Part II specifically focusses on fertility preservation techniques. RESULTS: In breast cancer patients, fertility preservation such as ovarian tissue and oocyte cryopreservation is especially recommended in low-stage cancer and in women < 35 years of age. In Hodgkin's lymphoma, the indication is mainly based on the chemotherapy regime as some therapies have very low, others very high gonadotoxicity. In borderline ovarian tumours, preservation of fertility usually is achieved through fertility sparing surgery, ovarian stimulation may also be considered. In cervical cancer, endometrial cancer, rheumatic diseases and other malignancies such as Ewing sarcoma, colorectal carcinoma, non-Hodgkin lymphoma, leukaemia etc., several other factors must be considered to enable an individual, stage-dependent decision. CONCLUSION: The decision for or against fertility preservation depends on the prognosis, the risks to fertility and individual factors such as prospective family planning.

Markers of oxidative damage to lipids, nucleic acids and proteins and antioxidant enzymes activities in Alzheimer's disease brain: A meta-analysis in human pathological specimens.

Oxidative stress and decreased cellular responsiveness to oxidative stress are thought to influence brain aging and Alzheimer's disease, but the specific patterns of oxidative damage and the underlying mechanism leading to this damage are not definitively known. The objective of this study was to define the pattern of changes in oxidative-stress related markers by brain region in human Alzheimer's disease and mild cognitive impairment brain tissue. Observational case-control studies were identified from systematic queries of PubMed, ISI Web of Science and Scopus databases and studies were evaluated with appropriate quality measures. The data was used to construct a region-by-region meta-analysis of malondialdehyde, 4-hydroxynonenal, protein carbonylation, 8-hydroxyguanine levels and superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase activities. We also evaluated ascorbic acid, tocopherol, uric acid and glutathione levels. The analysis was complicated in several cases by publication bias and/or outlier data. We found that malondialdehyde levels were slightly increased in the temporal and occipital lobes and hippocampus, but this analysis was significantly impacted by publication bias. 4-hydroxynonenal levels were unchanged in every brain region. There was no change in 8-hydroxyguanine level in any brain region and protein carbonylation levels were unchanged except for a slight increase in the occipital lobe. Superoxide dismutase, glutathione peroxidase and reductase and catalase activities were not decreased in any brain region. There was limited data reporting non-enzymatic antioxidant levels in Alzheimer's disease brain, although glutathione and tocopherol levels appear to be unchanged. Minimal quantitative data is available from brain tissue from patients with mild cognitive impairment. While there is modest evidence supporting minor regional changes in markers of oxidative damage, this analysis fails to identify a consistent pattern of pro-oxidative changes and accumulation of oxidative damage in bulk tissue analysis in the setting of Alzheimer's disease, as has been widely reported.

Cellular hormetic response to 27-hydroxycholesterol promotes neuroprotection through AICD induction of MAST4 abundance and kinase activity.

The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer's Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driven, hormetic pathway providing insight for therapeutic approaches against cholesterol associated neuronal disorders.

Fertility preservation issues in pediatric hematopoietic stem cell transplantation: practical approaches from the consensus of the Pediatric Diseases Working Party of the EBMT and the International BFM Study Group.

Fertility preservation is an urgent challenge in the transplant setting. A panel of transplanters and fertility specialists within the Pediatric Diseases Working Party of the European Society for Blood and Marrow Transplantation (EBMT) and the International BFM Study Group provides specific guidelines. Patients and families should be informed of possible gender- and age-specific cryopreservation strategies that should be tailored according to the underlying disease, clinical condition and previous exposure to chemotherapy. Semen collection should be routinely offered to all postpubertal boys at the diagnosis of any disease requiring therapy that could potentially impair fertility. Testicular tissue collection might be offered to postpubertal boys; nevertheless, its use has been unsuccessful to date. Oocyte collection after hormonal hyperstimulation should be offered to postpubertal girls facing gonadotoxic therapies that could be delayed for the 2 weeks required for the procedure. Ovarian tissue collection could be offered to pre-/post-pubertal girls. Pregnancies have been reported after postpubertal ovarian tissue reimplantation; however, to date, no pregnancy has been reported after the reimplantation of prepubertal ovarian tissue or in vitro maturation of pre-/post-pubertal ovarian tissue. Possible future advances in reproductive medicine could change this scenario. Health authorities should prioritize fertility preservation projects in pediatric transplantation to improve patient care and quality of life.

Follicular flushing in natural cycle IVF does not affect the luteal phase - a prospective controlled study.

In contrast to multifollicular IVF, follicular flushing seems to increase the efficacy of monofollicular IVF treatments such as natural cycle IVF (NC-IVF). However, because follicular flushing causes loss of granulosa cells, it might negatively affect luteal phase length and endocrine function of the luteal body. A prospective cohort Phase II study was performed in 24 women undergoing NC-IVF. Women underwent a reference cycle with human chorionic gonadotrophin-induced ovulation without follicle aspiration and analysis of the length of the luteal phase and luteal concentrations of progesterone and oestradiol. In addition, they underwent a NC-IVF cycle which was performed identically but follicles were aspirated and flushed three times. The luteal phase was shorter in 29.2%, equal in 16.7% and longer in 50.0% of cases following flushing of the follicles. Overall, neither difference in luteal phase length was significant [median duration (interquartile range) in reference cycle: 13 (12; 14.5), IVF (flushing) cycle: 14 (12.5; 14.5), median difference (95% CI): 0.5 (-0.5 to 1.5)] nor median progesterone and oestradiol concentrations. In conclusion, follicular flushing in NC-IVF affects neither the length of the luteal phase nor the luteal phase concentrations of progesterone and oestradiol, questioning the need for luteal phase supplementation.

State-of-the-art fertility preservation in children and adolescents undergoing haematopoietic stem cell transplantation: a report on the expert meeting of the Paediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT) in Baden, Austria, 29-30 September 2015.

Nowadays, allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a well-established treatment procedure and often the only cure for many patients with malignant and non-malignant diseases. Decrease in short-term complications has substantially contributed to increased survival. Therefore long-term sequelae are reaching the focus of patient care. One of the most important risks of stem cell transplant survivors is infertility. As well as in the field of allo-HSCT also the field of reproductive medicine has achieved substantial advances to offer potential options for fertility preservation in both boys and girls. Access to these procedures as well as their financing differs significantly throughout Europe. As all European children and adolescents should have the same possibility, the Paediatric Diseases Working Party of the European Society for Blood and Marrow Transplantation organised an expert meeting in September 2015. This manuscript describes the recommendations for the diagnosis and pre-emptive procedures that should be offered to all children and adolescents in Europe who have to undergo an allo-HSCT.

Gonadotrophin stimulation in IVF alters the immune cell profile in follicular fluid and the cytokine concentrations in follicular fluid and serum.

Study question: Are the immune cell profiles and the cytokine concentrations in follicular fluid (FF) and serum at the preovulatory stage different in conventional exogenous gonadotrophin stimulated IVF (c-IVF) compared with natural cycle IVF (NC-IVF)? Summary answer: The cell counts of CD45+ leucocytes and T cell subpopulations and the cytokine concentrations in FF and serum are different in c-IVF compared to NC-IVF. What is known already: FF-derived cells are heterogeneous. Immune cells are involved in intra-ovarian processes and cytokines are required for normal follicular development. Gonadotrophins stimulate the regulatory intrafollicular system and influence the local distribution of immune cells and the intrafollicular release of cytokines. Administration of exogenous gonadotrophins may have a significant effect on this local regulatory system, which then in turn could influence oocyte quality. Study design, size, duration: The study included 105 patients, 69 undergoing c-IVF and 36 undergoing NC-IVF. c-IVF was performed by exogenous ovarian stimulation with hMG and GnRH antagonists. Participants/materials, setting, methods: FF samples were collected from the first dominant follicle in c-IVF without pooling and from single leading preovulatory follicles in NC-IVF. Three different approaches were used to analyze FF samples: (i) microscopic investigation of CD45+ leucocytes, (ii) fluorescence-activated cell sorting to determine CD19+ B cells and CD3+ T cells including T cell subpopulations (CD4+, CD8+), and (iii) evaluation of tumour necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ), interleukins (IL)-2, -6, -8, -10 and vascular endothelial growth factor (VEGF) levels in matched FF and serum samples using the Bio-Plex® platform. Main results and the role of chance: FF obtained from c-IVF contained proportionally more CD45+ leucocytes (P = 0.0384), but fewer CD8+ cytotoxic T cells than FF from NC-IVF. CD3+ T lymphocytes were the most common type of lymphocytes, and the number thereof was comparable in the two study groups. In c-IVF, serum VEGF levels were higher (P = 0.007) than in NC-IVF while FF contained marginally decreased concentrations of IL-8 in c-IVF in comparison to NC-IVF. The cytokine concentration gradient between FF and serum in c-IVF was 10-fold for IL-8 and 8-fold for VEGF and thereby markedly lower than in NC-IVF, where the differences were 32-fold and 30-fold, respectively. Strong positive correlations were determined between FF- IL-10 and FF- VEGF in c-IVF (r = 0.85, P < 0.0001) and in NC-IVF (r = 0.81, P < 0.0001). Large scale data: N/A. Limitations, reasons for caution: The ovulation of NC-IVF follicles was induced by the exogenous administration of hCG, which means that the environment did not fully correspond to the physiological situation. Wider implications of the findings: The differences in the immune profile and the cytokine concentrations in c-IVF and NC-IVF follicles support the hypothesis that conventional ovarian stimulation affects indirectly and heterogeneously the intrafollicular milieu, and thereby possibly affects the oocyte quality and the IVF outcome. However, further studies are needed to confirm our findings and to refine stimulation protocols in the context of optimizing the intrafollicular environment during oocyte maturation. Study funding/competing interest(s): The study was supported by a research grant from IBSA Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH. The authors are clinically involved in low dose mono-follicular stimulation and IVF-therapies, using gonadotrophins from all gonadotrophins distributors on the Swiss market, including Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH.

Intrafollicular inflammatory cytokines but not steroid hormone concentrations are increased in naturally matured follicles of women with proven endometriosis.

PURPOSE: The aim of this study was to assess whether the intrafollicular cytokine profile in naturally developed follicles is different in women with endometriosis, possibly explaining the lower reproductive outcome in endometriosis patients. METHODS: A matched case-control study was conducted at a university-based infertility and endometriosis centre. The study population included 17 patients with laparoscopically and histologically confirmed endometriosis (rAFS stages II-IV), each undergoing one natural cycle IVF (NC-IVF) treatment cycle between 2013 and 2015, and 17 age-matched NC-IVF women without diagnosed endometriosis (control group). Follicular fluid and serum was collected at the time of follicle aspiration. The concentrations of inflammatory cytokines (IL-1ß, IL-6, IL-8, IL-15, IL-18, TNF-α) and hormones (testosterone, estradiol, AMH) were determined in follicular fluid and serum by single or multiplexed immunoassay and compared between both groups. RESULTS: In the follicular fluid, IL-1ß and IL-6 showed significantly (P < 0.001 and 0.01, respectively) higher median concentrations in the endometriosis group than in the control group and a tendency towards endometriosis severity (rAFS stage) dependence. The levels of the interleukins detectable in follicular fluid were significantly higher than those in the serum (P < 0.01). Follicular estradiol concentration was lower in severe endometriosis patients than in the control group (P = 0.036). Follicular fluid IL-1ß and IL-6 levels were not correlated with estradiol in the same compartment in neither patient group. CONCLUSIONS: In women with moderate and severe endometrioses, some intrafollicular inflammatory cytokines are upregulated and not correlated with intrafollicular hormone concentrations. This might be due to the inflammatory microenvironment in endometriosis women, affecting follicular function and thereby possibly contributing to the reproductive dysfunction in endometriosis.

Ninety-five orthotopic transplantations in 74 women of ovarian tissue after cytotoxic treatment in a fertility preservation network: tissue activity, pregnancy and delivery rates.

STUDY QUESTION: What is the success rate in terms of ovarian activity (menstrual cycles) as well as pregnancy and delivery rates 1 year after orthotopic ovarian transplantations conducted in a three-country network? SUMMARY ANSWER: In 49 women with a follow-up >1 year after transplantation, the ovaries were active in 67% of cases and the pregnancy and delivery rates were 33 and 25%, respectively. WHAT IS KNOWN ALREADY: Cryopreservation of ovarian tissue in advance of cytotoxic therapies and later transplantation of the tissue is being performed increasingly often, and the total success rates in terms of pregnancy and delivery have been described in case series. However, published case series have not allowed either a more detailed analysis of patients with premature ovarian insufficiency (POI) or calculation of success rates based on the parameter 'tissue activity'. STUDY DESIGN, SIZE, DURATION: Retrospective analysis of 95 orthotopic transplantations in 74 patients who had been treated for cancer, performed in the FertiPROTEKT network from 2008 to June 2015. Of those 95 transplantations, a first subgroup (Subgroup 1) was defined for further analysis, including 49 women with a follow-up period >1 year after transplantation. Of those 49 women, a second subgroup (Subgroup 5) was further analysed, including 40 women who were transplanted for the first time and who were diagnosed with POI before transplantation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Transplantation was performed in 16 centres and data were transferred to the FertiPROTEKT registry. The transplantations were carried out after oncological treatment had been completed and after a remission period of at least 2 years. Tissue was transplanted orthotopically, either into or onto the residual ovaries or into a pelvic peritoneal pocket. The success rates were defined as tissue activity (menstrual cycles) after 1 year (primary outcome) and as pregnancies and deliveries achieved. MAIN RESULTS AND THE ROLE OF CHANCE: The average age of all transplanted 74 women was 31 ± 5.9 years at the time of cryopreservation and 35 ± 5.2 at the time of transplantation. Twenty-one pregnancies and 17 deliveries were recorded. In Subgroup 1, tissue was cryopreserved at the age of 30 ± 5.6 and transplanted at 34 ± 4.9 years. Ovaries remained active 1 year after transplantation in 67% of cases (n = 33/49), the pregnancy rate was 33% (n = 16/49) and the delivery rate was 25% (n = 12/49). In Subgroup 5, tissue was cryopreserved at the age 30 ± 5.9 years and transplanted at 34 ± 5.2 years. Ovaries remained active 1 year after transplantation in 63% of cases (n = 25/40), the pregnancy rate was 28% (n = 11/40) and the delivery rate was 23% (n = 9/40). The success rates were age dependant with higher success in women who cryopreserved at a younger age. In Subgroup 5, tissue was exclusively transplanted into the ovary in 10% (n = 4/40) of women and into a peritoneal pocket in 75% (n = 30/40), resulting in spontaneous conceptions in 91% of patients (n = 10/11). LIMITATIONS, REASONS FOR CAUTION: The data were drawn from a retrospective analysis. The cryopreservation and transplantation techniques used have changed during the study period. The tissue was stored in many tissue banks and many surgeons were involved, leading to heterogeneity of the procedures. However, this does reflect the realistic situation in many countries. Although patients with POI were evaluated before transplantation to allow specific analysis of the transplanted tissue itself, the possibility cannot be excluded that residual ovarian tissue was also reactivated. WIDER IMPLICATIONS OF THE FINDINGS: This is the largest case series worldwide to date and it confirms that cryopreservation and transplantation of ovarian tissue can be a successful option for preserving fertility. Persistent tissue activity 12 months after transplantation suggests that the pregnancy and delivery rates may increase further in the future. As transplantation into the peritoneum results in a high success rate, this approach may be an alternative to transplantation into the ovary. However, in order to establish the best transplantation site, a randomized study is required. STUDY FUNDING/COMPETING INTEREST: This study was in part funded from the Deutsche Forschungsgemeinschaft (# DI 1525) and the Wilhelm Sander Foundation (2012.127.1) and did not receive any funding from a commercial company. No competing interests. TRIAL REGISTRATION NUMBER: None.