Abstract Study question Does enhancing oxygen availability during dynamic in vitro culture of bovine ovarian cortical tissue (BOCT) improve follicle growth and health? Summary answer Enhancing oxygen availability during dynamic in vitro culture of BOCT in perifusion bioreactors (PB) does improve follicle health and yield to secondary follicles What is known already Oxygen availability has been demonstrated to represent a key factor in follicle health and growth during in vitro culture of bovine and human ovarian cortical tissue (HOCT) under static culture conditions. Disruption of solutes gradients and application of physiological fluid mechanical stress, through in vitro dynamic culture of HOCT in a newly designed perifusion bioreactor have been shown to further enhance follicle growth and health. As it shows striking similarities with human, bovine folliculogenesis is considered a valuable model to study follicle growth in vitro Study design, size, duration Bovine ovaries from animals aged 8-24 months were collected at slaughterhouse. In each experiment (n = 3), BOCT strips from the same ovary were cultured for 6 days in perifusion bioreactors (PB, dynamic culture) and conventional dishes (CD, static culture). Culture outcome in static culture was analysed and compared to two bioreactor configurations in which medium oxygenation was kept low by using a standard tube reservoir (StPB) or was enhanced by using a gas-permeable dish reservoir (PB+O2). Participants/materials, setting, methods Slices of BOCT 0.5mm thick were cut with a tissue slicer and chopped into 1x1mm strips. In each experiment, fresh (D0) and cultured tissue (groups of ten strips) were analyzed. Follicle stages and health were assessed by histology (hematoxylin-eosin staining). Follicle viability was estimated by labelling with live-dead far-red and propidium iodide followed by clearing before analysis at the confocal laser scanning microscope. Main results and the role of chance Overall, 2417 follicles were analyzed (histology, 1476; viability, 941). At day 0 most follicles were primordial (primordial, 88.7%; primary, 10.6%; secondary, 0.7%), and had good quality (grade 1-2, 92.2%; grade 3, 7.8%), and high viability (91.8%). At day 6, follicle growth and health in StPB was superior than in CD (StPB vs CD - staging: primordial, 6.8 vs 16.3, P < 0.01; primary, 70.7 vs 74.1, NS; secondary, 22.5 vs 9.6%, P < 0.01; grading: grade 1 + 2, 71.4 vs 44.8, P < 0.01; grade 3, 28.6 vs 55.2%, P < 0.01). Dynamic culture in StPB better-preserved follicle viability compared to static culture in CD (StPB vs CD: 77.75 vs 64.9%, P < 0.01). Enhancing oxygen availability during dynamic culture increased follicle progression and viability (PB+O2 vs StPB - staging: primordial, 5.1 vs 6.8, NS; primary, 65.4 vs 70.7, NS; secondary, 29.5 vs 22.5%, P < 0.05; viability - 92 vs 77.75, P < 0.01). Overall, the obtained results demonstrate that i) disruption of stagnant layers of medium and application of shear stress to BOCT through dynamic culture improves follicle activation, growth and health; ii) enhancing oxygen availability by means of a gas-permeable medium reservoir further increases follicle progression and viability. Limitations, reasons for caution Although the bovine is considered a reliable model for human folliculogenesis, the study should be validated on human ovarian tissue. Wider implications of the findings A limiting step in the in vitro production of mature oocytes starting from primordial follicles is the low yield of secondary follicles after organ culture. The adoption of a newly designed dynamic bioreactor and modulation of oxygen availability could represent a valuable tool for multistep in vitro folliculogenesis. Trial registration number none

To calculate annual costs of endometriosis-associated symptoms, to identify the most important cost drivers, and to estimate the annual economic burden of endometriosis-associated symptoms at country level in 2009. A retrospective, international, multi-centre questionnaire-based survey to measure endometriosis costs in 12 tertiary care centres in 10 countries. Women with laparoscopically confirmed endometriosis and with at least one centre-specific contact related to endometriosis-associated symptoms in 2008 were eligible for participation. Women reported costs relating to August-September 2009, which were then multiplied by six to an annual basis. Cost analysis included direct health care costs (physician visits, medication, monitoring, informal care, hospitalization, other treatments), direct non-health care costs (transportation and support for household activities) and indirect costs of productivity loss (work absence/reduced productivity at work). Of the 1,450 women who provided informed consent, 931 (64%) women (mean age 36 years; 56% diagnosed with moderate-severe endometriosis; 6 year time interval between first symptoms and diagnosis) returned the questionnaires. The average annual total societal cost per woman was $16,116 (95% CI: $15,130-$17,101). Per woman, costs of productivity loss ($13,048) were nearly three times higher than direct costs ($3,066). Healthcare costs were mainly related to hospitalization (32%), monitoring tests (26%), physician visits (24%), and medication (7%). Total annual burden of endometriosis-associated symptoms varied from $2 billion in Denmark to $119 billion in the United States. Endometriosis imposes a substantial economic burden on society, mainly related to productivity loss. Costs are likely to decrease if time to diagnosis and treatment can be shortened.

The aim of this experiment was to design a suitable mouse model for male subfertility in which to study the effect of decreased sperm quality on embryo quality in vivo and in vitro. To achieve male subfertility, testes of adult male mice were immersed in water at either 42 degrees C (heated) or 33 degrees C (controls) during 20 min. Twenty-eight days after treatment, all heat stressed males showed a significant decrease in relative testis weight [384.7 mg in controls (286.7-460.6) versus 323 mg in stress heated groups (117.9-405.6); P < 0.001], sperm concentration [3.75 x 10(6)/ml (2.75-7.25) versus 1.00 x 10(6)/ml (0-4.00); P < 0.001] and progressive sperm motility [57.5% (48.0-79.0) versus 42.5% (14.0-66.0); P < 0.001]. Moreover, after mating to heat exposed males, not only the number of pregnant females (20/22 versus 18/30) but also the weight of their embryos [275.4 mg (78.7-339.4) versus 261.8 mg (68.1-339.0); P < 0.001] was significantly lower at 14.5 days post coitum when compared to controls. Neither the number of resorption sites nor the number of viable embryos per pregnant female was significantly different between groups. Also, the in-vitro fertilization rate of oocytes, fertilized by spermatozoa collected from heat stressed males, was significantly lower (44.9%; P < 0.0001) when compared to controls (65.1%; P < 0.0001). In conclusion, the results of this study suggest that male subfertility induced by acute scrotal heating may result in impaired sperm quality, reduced embryo weight in vivo and decreased fertilization rate in vitro.