Purpose The huge loss of ovarian follicles after transplantation of frozen/thawed ovarian tissue is considered a major drawback on the efficacy of the procedure. Here we investigate whether Er:YAG laser treatment prior to xenotransplantation can improve re-vascularization and subsequently follicle survival in human ovarian tissue. Methods A total of 99 frozen/thawed human ovarian cortex pieces were included of which 72 pieces from 12 woman were transplanted to immunodeficient mice. Tissues from each woman were included in both an 8-day and an 8-week duration study and treated with either full-beam laser (L1) or fractionated laser (L2), or served as untreated controls. Vascularization of the ovarian xenografts were evaluated after 8 days by qPCR and murine Cd31 immunohistochemical analysis. Follicle densities were evaluated histologically 8 weeks after xenografting. Results Gene expression of Vegf/VEGF was upregulated after L1 treatment (p=0.002, p=0.07, respectively), whereas Angpt1, Angpt2, Tnf-α, and Il1-β were significantly downregulated. No change in gene expression was found in Cd31/CD31, ANGPT1, ANGPT2, ANGTPL4, XBP1, or LRG1 after any of the laser treatments. The fraction of Cd31 positive cells were significantly reduced after L1 and L2 treatment (p<0.0001; p=0.0003, respectively), compared to controls. An overall negative effect of laser treatment was detected on follicle density (p=0.03). Conclusions Er:YAG laser treatment did not improve re-vascularization or follicle survival in human ovarian xenografts after 8 days and 8 weeks grafting, respectively. However, further studies are needed to fully explore the potential angiogenic effects of controlled tissue damage using different intensities or lasers.

Background: Salamander limb regeneration is a complex biological process that entails the orchestration of multiple cellular and molecular mechanisms in a three-dimensional space. Hence, a comprehensive understanding of this process requires whole-structure level explorations. Recent advances in imaging and optical clearing methods have transformed the study of regenerative phenomena, allowing the three-dimensional visualization of structures and entire organisms.Results: Here we introduce Salamander-Eci, a rapid and robust optical clearing protocol optimized for the widely used axolotl model, which allows simultaneous immunohistochemistry and Click-chemistry detection with minimal volume disruption. We provide examples of its application, from whole larva to adult limbs and organs, and complement it with an image analysis pipeline for volumetric cell quantification. Further, we offer a detailed 3D quantitation of cell proliferation throughout axolotl limb regeneration.Conclusions: Salamander-Eci enables the comprehensive volumetric analysis of regenerative phenomena at both local and systemic levels.

No abstract

Study question Can human Platelet Lysate (hPL) and umbilical cord plasma (UCP) be used as alternative serum sources in the culture of isolated human pre-antral follicles? Summary answer hPL significantly increased the growth and survival of human follicles cultured for 8 days compared to fetal bovine serum (FBS) and human serum albumin (HSA). What is known already Culture of human ovarian follicles is a potential new source of mature oocytes for fertility preservation and an important model system to study basic biology. However, only a few studies have produced mature human oocytes after culturing pre-antral stage follicles. Moreover, optimising culture systems is challenging due to the scarcity of human material. Platelet-rich plasma solutions have been used extensively in regenerative medicine due to their high platelet content, which upon activation, release a multitude of growth factors, hormones, and cytokines. Clinically cell-based therapies have used platelet-rich solutions, such as hPL or UCP, successfully as an animal-free serum. Study design, size, duration Human pre-antral follicles (n = 378; mean diameter: 78 µm; range: 46-237 µm) were isolated from ovarian medulla tissue. The follicles were encapsulated in 0.5% alginate and cultured for 8 days in media supplemented with one of four sources of serum; 5% FBS (n = 74), 2.5% HSA (n = 74), 5% hPL (n = 140), and 5% UCP (n = 90). The primary endpoints were follicular growth and survival. Secondary endpoints included follicular gene expression analysis and media hormone concentrations. Participants/materials, setting, methods Ovarian surplus tissue was donated by 7 women (aged 19-32 years) undergoing unilateral oophorectomy and ovarian tissue cryopreservation for fertility preservation. Pre-antral follicles were isolated enzymatically and growth and survival were assessed every second day during culture by microscopy. AMH and Estradiol concentrations were measured by ELISA in media collected at day 4 and 8. At day 8, surviving follicles were snap-frozen and the expression of AMH, FSHR, GDF9, and BMP15 was analysed by qPCR. Main results and the role of chance After 8 days in culture, the follicle survival rate in the hPL group (86%; n = 120/140) was significantly higher compared to the FBS group (60%; n = 45/74; p &lt; 0.0003), but comparable to the HSA group (76%; n = 56/74; p = 0.292). On the contrary, the follicle survival rate in the UCP group (33%; n = 30/90) was significantly lower compared to any of the other groups (p &lt; 0.0001 for all three groups). The average diameter of surviving follicles was statistically significantly larger in the hPL and UCP group compared to the FBS and HSA groups (hPL: 149 ±4.6 µm; UCP: 138 ±7.6 μm; FBS: 110 ±3.8 μm; HSA: 115 ±4.2 μm; p &lt; 0.001 for hPL compared to FBS and HSA; p = 0.0129 for UCP compared to HSA). The relative growth of the follicles compared to their initial diameter was 45% and 33% higher in the hPL group compared to the FBS and HSA groups, respectively, which was statically significant in both groups (p &lt; 0.001). In the UCP group, follicle growth was also significantly higher by 25% compared to the FBS group (p &lt; 0.001) and by 14% compared to the HSA group (p = 0.0376). Hormone measurements from the culture media and follicle gene expression analysis are awaited. Limitations, reasons for caution One limitation is the variability in the initial follicle sizes. Extrapolating results according to primordial, primary, and secondary follicles would require more follicles but could provide valuable insight. Moreover, long term culture studies are needed to evaluate the effects of hPL and UCP on antral follicle development and oocyte maturation. Wider implications of the findings Our findings show that hPL can be used as an alternative serum source in the culture of human pre-antral follicles as it increased follicle growth and survival compared to FBS and HSA. UCP also increased follicle growth but had detrimental effects on follicle survival which needs further studies. Trial registration number Not applicable

Background Ovarian tissue transplantation can restore fertility in young cancer survivors, however the detrimental loss of follicles following transplantation of cryopreserved ovarian tissue is hampering the efficiency of the procedure. This study investigates whether needle puncturing prior to transplantation can enhance revascularization and improve follicle survival in xenotransplanted human ovarian cortex. Methods Cryopreserved human ovarian cortex pieces (N = 36) from 20 women aged 24–36 years were included. During the thawing process, each piece of tissue was cut in halves; one half serving as the untreated control and the other half was punctured approximately 150–200 times with a 29-gauge needle. The cortex pieces were transplanted subcutaneously to immunodeficient mice for 3, 6 and 10 days (N = 8 patients) and for 4 weeks (N = 12 patients). After 3, 6 and 10 days, revascularization of the ovarian xenografts were assessed using immunohistochemical detection of CD31 and gene expression of angiogenic factors (Vegfα, Angptl4, Ang1, and Ang2), and apoptotic factors (BCL2 and BAX) were performed by qPCR. Follicle density and morphology were evaluated in ovarian xenografts after 4 weeks. Results A significant increase in the CD31 positive area in human ovarian xenografts was evident from day 3 to 10, but no significant differences were observed between the needle and control group. The gene expression of Vegfα was consistently higher in the needle group compared to control at all three time points, but not statistically significant. The expression of Ang1 and Ang2 increased significantly from day 3 to day 10 in the control group (p < 0.001, p = 0.0023), however, in the needle group this increase was not observed from day 6 to 10 (Ang2 p = 0.027). The BAX/BCL2 ratio was similar in the needle and control groups. After 4-weeks xenografting, follicle density (follicles/mm3, mean ± SEM) was higher in the needle group (5.18 ± 2.24) compared to control (2.36 ± 0.67) (p = 0.208), and a significant lower percentage of necrotic follicles was found in the needle group (19%) compared to control (36%) (p = 0.045). Conclusions Needle puncturing of human ovarian cortex prior to transplantation had no effect on revascularization of ovarian grafts after 3, 6 and 10 days xenotransplantation. However, needle puncturing did affect angiogenic genes and improved follicle morphology.