Follicular growth after xenotransplantation of cryopreserved/thawed human ovarian tissue in SCID mice: dynamics and molecular aspects.

PURPOSE: To study the influence of xenotransplantation on follicular recruitment and growth in cryopreserved/thawed human ovarian tissue. METHOD: Two 3-mm pieces of cryopreserved/thawed human ovarian tissue obtained from female cancer patients (n = 11) were xenotransplanted into a subcutaneous neck pouch of 6-week-old ovarectomized SCID mice (n = 33) for 4 (n = 18) and 12 (n = 15) weeks. RESULT: Thirty-two out of 33 mice survived the entire observation periods. Graft recovery rate was 95.58 % (65 of 68 grafts). The percentages of primordial follicles after 4 weeks (P < 0.001) and 12 weeks (P = 0.009) of grafting were significantly lower in comparison to pregraft controls. The percentage of secondary follicle was significantly higher after 4 weeks of grafting (P = 0.018) and after 12 weeks (P = 0.001) of grafting in comparison to pregraft controls. Ki67 immunohistochemistry showed that proliferative follicles were significantly higher after 4 and 12 weeks of grafting compared to pregraft controls (P < 0.001). All follicles analyzed by TUNEL staining appeared healthy after xenotransplantation. The expression level of PTEN was reduced by 2.47-fold after 4 weeks of xenotransplantation, and this result was significant when 2-ΔCt were analyzed (P = 0.042). CONCLUSION: The higher proportion of growing follicles compared to resting follicles observed after xenotransplantation is most likely due to downregulation of PTEN gene expression followed by acceleration of follicular recruitment.

Novel dynamic culture system to support initiation of primordial follicle growth in prepubertal mouse ovaries.

OBJECTIVE: To evaluate the impact of dynamic in vitro culture on initiation of early follicular growth in prepubertal mouse ovaries. DESIGN: Ovaries from 8-day-old BALB/c mice were cultured either in a dynamic system (n=28) or in a static system (n=20) for 4 days. Uncultured 8-day-old (n=9) or 12-day-old (n=17) ovaries served as baseline or in vivo controls, respectively. SETTING: Academic research center. ANIMAL(S): Newborn female BALB/c mice (n=37). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Histologic follicle classification and counting and assessment of follicular viability via immunofluorescent staining. RESULT(S): The percentage of secondary follicles after dynamic culture was identical to the 12-day-old in vivo control. In contrast, after static culture ovaries showed a significantly higher percentage of secondary follicles. For immunofluorescent viability assessment 6.78 follicles per ovary could be isolated after dynamic culture, whereas only 3.8 follicles per ovary could be isolated after static culture. CONCLUSION(S): Dynamic in vitro culture supports physiologic follicular growth initiation, comparable to that observed in vivo. In contrast, accelerated follicular growth was observed after static culture. These findings add additional evidence to the idea that dynamic culture might be a beneficial first step to initiate follicle growth in vitro within the context of fertility preservation.

Quantitative levonorgestrel plasma level measurements in patients with regular and prolonged use of the levonorgestrel-releasing intrauterine system.

BACKGROUND: The levonorgestrel-releasing intrauterine system (LNG-IUS) is well accepted as an easy-to-use contraceptive with an excellent side-effect profile. It contains a reservoir of 52 mg of levonorgestrel (LNG) with continuous release of the steroid. Its contraceptive use is approved for 5 years. The aim of this study was to determine the plasma concentration of LNG and its variation with time in patients with in-dwelling LNG-IUS Mirena®. STUDY DESIGN: In this study, we determined LNG plasma concentrations in 110 women with LNG-IUS at different time points of use. Time from insertion of the system in the study population ranged from 20 days to 11.1 years. Quantitative LNG levels were determined using a validated liquid chromatography-tandem mass spectrometry assay. RESULTS: The mean±SD LNG plasma level in all women was 147±59 pg/mL. A highly significant negative correlation between LNG plasma level and LNG-IUS time of use could be demonstrated. In the first year of use, LNG plasma level was as high as 191±71 pg/mL, decreasing to 157±68 pg/mL in the second year and 134±41 pg/mL in the third year. Even after exceeding the recommended period of LNG-IUS use, systemic LNG concentrations were detectable: 133±38 pg/mL in the sixth year, 133±48 pg/mL in the seventh year and 117±45 pg/mL in the eighth year. Furthermore, a significant negative correlation between LNG plasma level and body mass index could be shown. CONCLUSION: Systemic LNG concentrations can be found in all patients with LNG-IUS IUS. However, concentrations are much lower than in other forms of LNG application. Moreover, this study demonstrates that a systemic effect of LNG-IUS can also be found after the recommended contraceptive lifespan of 5 years.