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PURPOSE: Extensive follicle loss has been demonstrated in ovarian grafts post transplantation, reducing their productivity and lifespan. Several mechanisms for this loss have been proposed, and this study aims to clarify when and how the massive follicle loss associated with transplantation of ovarian tissue graft occurs. An understanding of the mechanisms of follicle loss will pinpoint potential new targets for optimization and improvement of this important fertility preservation technique. METHODS: Frozen-thawed marmoset (n = 15), bovine (n = 37), and human (n = 46) ovarian cortical tissue strips were transplanted subcutaneously into immunodeficient castrated male mice for 3 or 7 days. Histological (H&E, Masson's trichrome) analysis and immunostaining (Ki-67, GDF9, cleaved caspase-3) were conducted to assess transplantation-associated follicle dynamics, with untransplanted frozen-thawed tissue serving as a negative control. RESULTS: Evidence of extensive primordial follicle (PMF) activation and loss was observed already 3 days post transplantation in marmoset, bovine, and human tissue grafts, compared to frozen-thawed untransplanted controls (p < 0.001). No significant additional PMF loss was observed 7 days post transplantation. Recovered grafts of all species showed markedly higher rates of proliferative activity and progression from dormant to growing follicles (Ki-67 and GDF9 staining) as well as higher growing/primordial (GF/PMF) ratio (p < 0.02) and higher collagen levels compared with untransplanted controls. CONCLUSIONS: This multi-species study demonstrates that follicle activation plays an important role in transplantation-induced follicle loss, and that it occurs within a very short time frame after grafting. These results underline the need to prevent this activation at the time of transplantation in order to retain the maximal possible follicle reserve and extend graft lifespan.

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PURPOSE: The present study evaluated the association between oxidative parameters in embryo cryopreservation medium and laboratory and clinical outcomes. METHODS: This prospective laboratory study was conducted in an IVF unit in a university-affiliated hospital with 91 IVF patients undergoing a frozen-thawed embryo transfer cycle. Following thawing, 50 µL of embryo cryopreservation medium was retrieved from each cryotube and tested by the thermochemiluminescence (TCL) assay. TCL amplitudes after 50 (H1), 150 (H2), and 280 s (H3) were recorded in counts per second (CPS) and the TCL ratio determined for comparison with implantation and pregnancy rates. RESULTS: A total of 194 embryos were transferred in 85 frozen-thaw cycles. Twenty-one pregnancies (24.7 %) occurred. Implantation and overall and clinical pregnancy rates were higher when the median TCL H1 amplitude was <32 CPS compared to ≥32 CPS (14.6 vs. 5.3 %, 37.5 vs. 17 %, 28.1 vs. 9.4 %, respectively). No pregnancies occurred when the H1 amplitude was ≥40 CPS. Logistic regression multivariate analysis found that only the median TCL H1 amplitude was associated with the occurrence of pregnancy (OR = 2.93, 95 % CI 1.065-8.08). The TCL ratio inversely correlated with the duration of embryo cryopreservation (r = -0.37). CONCLUSIONS: The results indicate that thawed embryos may express oxidative processes in the cryopreservation medium, and higher oxidative levels are associated with lower implantation rates. These findings may aid in the improved selection of frozen-thawed embryos for IVF.

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STUDY QUESTION: What are the effects of thin ovarian grafts compared with grafts of the standard thickness on follicle loss post-transplantation? SUMMARY ANSWER: Transplantation of reduced-thickness ovarian grafts led to intense activation and 'burn-out' a short time after transplantation resulting in significant folllicle loss. WHAT IS KNOWN ALREADY: Transplantation of fresh and frozen-thawed ovarian tissue has been proved successful, but techniques vary and are not optimised, often resulting in significant follicular loss. Follicle loss is mostly related to the freezing-thawing process and to post-transplantation hypoxia. STUDY DESIGN, SIZE, DURATION: Bovine ovarian tissue strips (n = 55) were prepared in two groups of conventional-thickness strips (1-2 mm) or thin strips (0.5-0.9 mm). Fresh or frozen-thawed samples were xenotransplanted into sterilized immune-deficient mice (n = 49). Non-transplanted conventional size fresh samples were used as controls (n = 6). Grafts from all study groups were recovered after 7 days for analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Morphometric differential counting of follicle classes was performed by two observers. Immunohistochemistry was conducted for proliferation (Ki67), cortical fibrosis (Masson tri-chrome) and blood-vessel density (CD31). Results were expressed as the mean number of dormant or growing follicle (GF) type per section or total follicle counts per graft. Blood-vessel density was calculated per mm(2). P-values <0.05 were considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: The loss of all follicle types, and most noteably of primordial follicles (PMFs), was observed 7 days post-transplantation (P < 0.05). The relatively high number of GFs and the positive Ki67 staining in all recovered grafts indicated that follicle activation was depleting the resting follicle pool. The reduced graft thickness had an adverse effect on the number of recovered follicles, especially on the resting non-GFs in the fresh, and more so in the frozen-thawed, samples (P < 0.05). Extensive stromal fibrosis and high blood-vessel density were observed in all grafts with no advantage in the thin prepared grafts. LIMITATIONS, REASONS FOR CAUTION: This study used only one species of ovaries (bovine) for xenotransplantation. The immediate post-transplantation events were not visualized directly nor were the molecules involved in follicle activation studied. WIDER IMPLICATIONS OF THE FINDINGS: Follicle activation and 'burn-out' appear to be important in follicle loss after transplantation. Reducing graft thickness in an attempt to improve freezing conditions and reduce post-transplantation ischemia has adverse effects on the graft follicle pool due to increased activation and loss. Agents which prevent 'burn-out' will potentially improve follicle pool survival. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by research grants from the Israeli Science Foundation (No. 1675/10), the Israeli Jack Craps foundation and the Israel Cancer Research Fund (ICRF No. 12-3081). The authors have no competing interest to declare.

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OBJECTIVE: To compare contemporary and historical clinical presentation of complete moles (CMs) and the rates of gestational trophoblastic neoplasia (GTN). STUDY DESIGN: A study was conducted of a current cohort of 108 consecutive cases of CM from 3 medical centers in Israel accrued during a 5-year period and 87 CM cases reported by the New England Trophoblastic Disease Center (NETDC) from the years 1988 to 1993. Clinical presentation and the rate of GTN of our cohort of CM and cases from the NETDC were compared. RESULTS: Fewer current CMs presented with vaginal bleeding than historic NETDC cases (52% vs. 84%, p <0.001, respectively), and a greater proportion of current patients with CM were referred to termination of the pregnancy due solely to ultrasonographic findings (38% vs. 9%, p < 0.001, respectively). GTN rates were significantly lower in the current patients with CM compared to NETDC controls (14% vs. 23%, p<0.05, respectively). CONCLUSION: First-trimester ultrasound examination leads to early diagnosis of molar and of nonviable pregnancies subsequently histologically diagnosed as CM. The early evacuation of the molar pregnancy is associated with a reduction in the rate of GTN.

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