Revascularization of human ovarian grafts is equally efficient from both sides of the cortex tissue.

RESEARCH QUESTION: Does revascularization of human ovarian grafts in a mouse model occur with equal efficiency from both sides of the cortex tissue? DESIGN: Twenty-four frozen-thawed ovarian cortex pieces from 12 women were transplanted to immunodeficient mice, for 8 days to analyse graft revascularization using immunohistochemical detection of murine CD31, or for 8 weeks to evaluate follicle density (follicles/mm3). The CD31-positive vessel area and density were quantified using a custom-designed application. Three regions of interest (ROI) were defined in each tissue section: the cortical side, the centre and the medullary side. Vessels were subdivided into three categories according to size: microvessels (<300 µm2), small vessels (300-1000 µm2) and large vessels (>1000-3000 µm2). RESULTS: No significant difference in the mean percentage of the CD31-positive vessel area was found between the three ROI (cortical side: 3.9% ± 0.2%; centre: 3.5% ± 0.2%; medullary side: 4.0% ± 0.3%; P = 0.17), but a significantly lower density of vessels was found in the centre of the human ovarian grafts compared with the cortical and medullary sides (cortical side: 323 ± 14 vessels/mm2; centre: 240 ± 12 vessels/mm2; medullary side: 301 ± 18 vessels/mm2; P < 0.001). Microvessels comprised 89-91% of all vessels in the three ROI. Follicle density in ungrafted cortex pieces was 51.8 ± 17.3 and 14.7 ± 3.7 follicles/mm3 after 8 weeks of xenografting, resulting in a follicle survival rate of 28%. CONCLUSIONS: Host revascularization was established equally efficiently from both sides of transplanted human ovarian cortex, suggesting that transplantation techniques ensuring revascularization from both sides of the ovarian graft could potentially facilitate faster graft revascularization.

Efficacy of assisted reproductive technology after ovarian tissue transplantation in a cohort of 11 patients with or without associated infertility factors.

PURPOSE: IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor reproductive outcomes. The aim of this study was to evaluate the efficacy of ovarian tissue transplantation (OTT) followed by assisted reproductive technology (ART) in women with or without associated infertility factors. METHODS: This is a prospective cohort study with retrospective data collection including eleven women, four of whom having an infertility factor (IF), who had undergone OTT in one university center between 2005 and 2017, followed by ART in six in vitro fertilization (IVF) centers. RESULTS: In total, 25 of the 85 cycles initiated (29%) were canceled, resulting in 60 oocyte retrievals. Ninety-five oocytes were retrieved: 36 were abnormal or immature, 29/39 fertilized (74%) after ICSI and 13/20 (65%) after IVF. Thirty-five embryos were transferred in seven patients (5/7 patients without IF and 2/4 patients with IF). After ART, one patient with IF experienced two pregnancies, one resulting in a live birth. For all patients, pregnancy rates and live birth rates were 7.4% and 3.7% per embryo transfer, respectively. Nine pregnancies and four live births occurred after spontaneous conception in five patients without IF, none in the infertility group. CONCLUSION: This study confirms that IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor outcomes. However, the chances of natural conception are high in women without IF. Patients with IF, without the possibility of spontaneous pregnancy, should be informed of poor reproductive outcomes after OTT followed by ART. TRIAL REGISTRATION NUMBER: NCT02184806.

Adipose tissue-derived stem cells protect the primordial follicle pool from both direct follicle death and abnormal activation after ovarian tissue transplantation.

PURPOSE: To investigate whether adipose tissue-derived stem cells (ASCs) protect the primordial follicle pool, not only by decreasing direct follicle loss but also by modulating follicle activation pathways. METHODS: Twenty nude mice were grafted with frozen-thawed human ovarian tissue from 5 patients. Ten mice underwent standard ovarian tissue transplantation (OT group), while the remaining ten were transplanted with ASCs and ovarian tissue (2-step/ASCs+OT group). Ovarian grafts were retrieved on days 3 (n = 5) and 10 (n = 5). Analyses included histology (follicle count and classification), immunohistochemistry (c-caspase-3 for apoptosis and LC3B for autophagy), and immunofluorescence (FOXO1 for PI3K/Akt activation and YAP for Hippo pathway disruption). Subcellular localization was determined in primordial follicles on high-resolution images using structured illumination microscopy. RESULTS: The ASCs+OT group showed significantly higher follicle density than the OT group (p = 0.01). Significantly increased follicle atresia (p < 0.001) and apoptosis (p = 0.001) were observed only in the OT group. In primordial follicles, there was a significant shift in FOXO1 to a cytoplasmic localization in the OT group on days 3 (p = 0.01) and 10 (p = 0.03), indicating follicle activation, while the ASCs+OT group and non-grafted controls maintained nuclear localization, indicating quiescence. Hippo pathway disruption was encountered in primordial follicles irrespective of transplantation, with nuclear YAP localized in their granulosa cells. CONCLUSION: We demonstrate that ASCs exert positive effects on the ovarian reserve, not only by protecting primordial follicles from direct death but also by maintaining their quiescence through modulation of the PI3K/Akt pathway.

Disease-inducing potential of two leukemic cell lines in a xenografting model.

PURPOSE: Cryopreserved ovarian tissue transplant restores ovarian function in young cancer patients after gonadotoxic treatment. However, leukemia is associated with increased risk of malignant cell transmission. We aimed to assess the tumor-inducing potential of two different leukemic cell lines when xenografted to immunodeficient mice. METHODS: Fifty-four female immunodeficient mice were grafted with either 100, 200, 500, 1000, and 10,000 chronic myeloid leukemia in blast crisis (BV-173) cells or relapsed acute lymphoblastic leukemia (RCH-ACV) cells, embedded inside a fibrin scaffold along with 50,000 human ovarian stromal cells. Two mice per cell line received the fibrin matrix without leukemic cells as negative controls. Clinical signs of disease were monitored for 20 weeks. Grafts, liver tissue, and masses were collected for macroscopic analysis and gene expression of BCR-ABL1 and E2A-PBX fusion transcripts present in BV-173 and RCH-ACV respectively. RESULTS: BV-173 cells: Mice grafted with 100, 200, or 500 cells showed no sign of disease after and were negative for BCR-ABL1 expression. Three of the 5 animals grafted with 1000 cells and all mice with 10,000 cells developed disease and showed BCR-ABL1-positive expression. RCH-ACV cells: Two out of 4 mice grafted with 100 cells developed disease and were E2A-PBX1-positive. All the animals grafted with higher cell doses showed signs of disease and all but one were E2A-PBX1-positive. CONCLUSION: The present work proves that the disease-inducing potential of BV-173 and RCH-ACV leukemic cells xenografted to SCID mouse peritoneum differs between cell lines, depending on cell number, type, status, and cytogenetic disease profile when ovarian tissue is harvested.

Role of Stem Cells in the Ovarian Tissue Cryopreservation and Transplantation for Fertility Preservation.

Although the cancer survival rate has increased, cancer treatments, including chemotherapy and radiotherapy, can cause ovarian failure and infertility in women of reproductive age. Preserving fertility throughout cancer treatment is critical for maintaining quality of life. Fertility experts should propose individualized fertility preservation methods based on the patient's marital status, pubertal status, partner status, and the urgency of treatment. Widely practiced fertility preservation methods, including ovarian transposition and embryo and oocyte cryopreservation, are inappropriate for prepubertal girls or those needing urgent initiation of cancer treatment. Ovarian tissue cryopreservation and transplantation, an emerging new technology, may be a solution for these cancer patients. The use of stem cells in ovarian tissue cryopreservation and transplantation increases oxygenation, angiogenesis, and follicle survival rates. This review discusses the recent advances in ovarian tissue cryopreservation and transplantation with special focus on the use of stem cells to improve fertilization techniques.

A novel mathematical model of true ovarian reserve assessment based on predicted probability of poor ovarian response: a retrospective cohort study.

PURPOSE: To establish a mathematical model for assessing the true ovarian reserve based on the predicted probability of poor ovarian response (POR). METHODS: In this retrospective cohort study, a total of 1523 GnRH-antagonist cycles in 2017 were firstly analyzed. The ovarian responses were calculated based on the number of retrieved oocytes. The continuous variables were converted into categorical variables according to cutoff values generated by the decision tree method. The optimal model was identified using forward stepwise multiple logistic regression with 5-fold cross-validation and further verified its performances using outer validation data. RESULTS: The predictors in our model were anti-Müllerian hormone (AMH), antral follicle counts (AFC), basal follicle-stimulating hormone (FSH), and age, in order of their significance, named AAFA model. The AUC, sensitivity, specificity, positive predictive value, and negative predictive value of AAFA model in inner validation and outer validation data were 0.861 and 0.850, 0.603 and 0.519, 0.917 and 0.930, 0.655 and 0.570, and 0.899 and 0.915. Ovarian reserve of 16 subgroups was further ranked according to the predicted probability of POR and further divided into 4 groups of A-D using clustering analysis. The incidence of POR in the four groups was 0.038 (0.030-0.046), 0.139 (0.101-0.177), 0.362 (0.308-0.415), and 0.571 (0.525-0.616), respectively. The order of ovarian reserve from adequate to poor followed the order of A to D. CONCLUSION: We have established an easy applicable AAFA model for assessing true ovarian reserve and may have important implications in both infertile women and general reproductive women in Chinese or Asian population.

The growth and development conditions in mouse offspring derived from ovarian tissue cryopreservation and orthotopic transplantation.

PURPOSE: To investigate the potential development or metabolic risk in offspring derived from mice with transplanted frozen-thawed ovarian tissue. METHODS: Mice ovaries were intervened by vitrification (group V) and slow-freezing (group S) cryopreservation and orthotopic transplantation. Orthotopic transplantation of fresh ovarian (group F) and natural mating (group C) served as control groups. The fertility restoration and health conditions of generations were assessed by offspring counts, anti-fatigue and motor ability, and organ morphology. The methylation rate and expression level of imprinted genes (IGF2R, H19, SNRPN, and PLAGL1) were used to predict the potential risk of development in transplanted generations. RESULTS: Both the percentage of normal morphological follicles in different developmental periods and the litter size of receipt mice were comparable in all three transplanted groups. There was no significant difference in offspring mice's birth defects, body weight gain, anti-fatigue ability, or exercise capacity among the four groups. The methylation rate of IGF2R, H19, and PLAGL1 showed a significant variation in cryopreservation groups as compared with control groups, as well as a difference in gene expression. The SNRPN appeared to be stable in methylation status. There were no differences in mRNA expression in all groups. CONCLUSIONS: The different ovarian tissue cryopreservation methods did not influence either maternal fertility function or offspring growth. However, these technologies could affect the methylation rate and expression level of some development-related imprinting genes in the offspring, which may lead to some indeterminacy risk.

First pregnancy and live birth in Turkey following frozen-thawed ovarian tissue transplantation in a patient with acute lymphoblastic leukemia who underwent cord blood transplantation.

PURPOSE: To report the first live birth after frozen-thawed ovarian transplantation in Turkey and the second case for an acute lymphoblastic leukemia (ALL) survivor in the world. METHODS: A 19-year-old patient underwent ovarian tissue cryopreservation (OTC) before cord blood transplantation in 2010. She was diagnosed as ALL with a bone marrow biopsy revealing 90% blast ALL-L2 type, and karyotype analyses indicated reciprocal translocation at t(9;22)(q34;q11). The patient received the Berlin-Frankfurt-Munster (BFM) protocol, and complete remission was achieved before fertility preservation. Serum AMH level was measured as 1.5 ng/ml, and 12 antral follicles were counted on ultrasound. She was informed about fertility preservation options and decided to proceed with OTC, with her signed consent before cord blood transplantation in April 2011. Ovarian tissue transplantation (OTT) was performed in 2017 when the patient was menopausal with serum FSH levels > 100 IU/ml and estradiol < 20 pg/ml and hematologically in molecular remission. Detailed molecular analysis, standard histology, and immunohistochemistry demonstrated that the thawed tissue is free of malignant cells. RESULTS: Six months following OTT, she had spontaneous menstruation with serum FSH 11 IU/ml and estradiol 53 pg/ml. Two consecutive IVF cycles yielded three top-quality embryos. Following three embryo transfer cycles, one fresh and two frozen, a healthy term live birth was achieved. Frozen-thawed-transplanted tissues were extracted during caesarean delivery upon the patient's request after a total period of 25 months in vivo, and histopathological evaluation revealed that the tissue was free of leukemic infiltration. CONCLUSION: The authors report the first pregnancy and live birth in Turkey and the second live birth in the world following transplantation of frozen-thawed ovarian tissue in a leukemia survivor. As the transplanted tissues were removed during caesarean delivery, histological findings prove the functionality and the malignant-free status of the transplanted tissue during the grafted period.

Novel extra cellular-like matrices to improve human ovarian grafting.

PURPOSE: To investigate if human ovarian grafting with pure virgin human recombinant collagen type-1 from bioengineered plant lines (CollPlant™) or small intestine submucosa (SIS) yields better implantation results for human ovarian tissue and which method benefits more when combined with the host melatonin treatment and graft incubation with biological glue + vitamin E + vascular endothelial growth factor-A. METHODS: Human ovarian tissue wrapped in CollPlant or SIS was transplanted into immunodeficient mice with/without host/graft treatment. The tissue was assessed by follicle counts (including atretic), for apoptosis evaluation by terminal deoxynucleotidyl transferase assay and for immunohistochemical evaluation of neovascularization by platelet endothelial cell adhesion molecule (PECAM) expression, and for identification of proliferating granulosa cells by Ki67 expression. RESULTS: Human ovarian tissue transplanted with CollPlant or SIS fused with the surrounding tissue and promoted neovascularization. In general, implantation with CollPlant even without additives promoted better results than with SIS: significantly higher number of recovered follicles, significantly fewer atretic follicles, and significantly more granulosa cell proliferation. Moreover, results with CollPlant alone seemed to be at least as good as those after host and graft treatments. CONCLUSIONS: CollPlant is a biomaterial without any potential risks, and grafting ovarian tissue with CollPlant is easy and the procedure may be easily modified, with limited or no foreseeable risks, for auto-transplantation in cancer survivors. Further studies are needed using other novel methods capable of enhancing neovascularization and reducing apoptosis and follicle atresia.

Investigation on revascularization time and initial damage after transplantation of fresh and cryopreserved ovarian tissue in domestic cats.

The present study evaluated revascularization time of fresh and cryopreserved cat ovarian tissue after transplantation to subcutaneous tissue. Ovaries of five cats were used and eight pieces of ovarian tissue were taken from each pair of ovaries. Immediately after removal, three pieces were transplanted and one fixed for fresh control. The remaining four pieces were cryopreserved and, after thawing, one was fixed for cryopreservation control and three were transplanted. Grafts were recovered on days 2 (D2), 4 (D4) and 6 (D6) post-transplantation. Blood vessels were identified by immunohistochemistry and doppler ultrasound. Immunohistochemistry showed that the percentages of total tissue area occupied by blood vessels were similar (P > 0.05) in fresh and cryopreserved tissues. In both cases, blood vessel area was significantly higher (P < 0.05) on D4 and D6 compared to D0. Ultrasound analysis showed vascularization improvement on the periphery of grafts from D2 to D4 and from D4 to D6, both in fresh and cryopreserved tissue samples. Nonetheless, there was a significant decrease (P < 0.05) in the percentage of morphologically normal follicles (MNF) after transplantation compared to non-transplanted tissue (D0), both for fresh and cryopreserved samples. Moreover, the number of follicles found in samples was considerably smaller after grafting. In conclusion, revascularization of ovarian tissue autotransplanted to subcutaneous tissue in domestic cats occurs within 4 days after transplantation, both for fresh and cryopreserved tissue. However, large follicular loss has been observed in the first days post-transplantation, especially in cryopreserved tissues.

Peroxisome Proliferator-Activated Receptor Gamma Modulator Promotes Neonatal Mouse Primordial Follicle Activation In Vitro.

Peroxisome proliferator-activated receptor gamma (PPARγ) is known as a regulator of cellular functions, including adipogenesis and immune cell activation. The objectives of this study were to investigate the expression of PPARγ and identify the mechanism of primordial follicle activation via PPARγ modulators in mouse ovaries. We first measured the gene expression of PPARγ and determined its relationship with phosphatase and tensin homolog (PTEN), protein kinase B (AKT1), and forkhead box O3a (FOXO3a) expression in neonatal mouse ovaries. We then incubated neonatal mouse ovaries with PPARγ modulators, including rosiglitazone (a synthetic agonist of PPARγ), GW9662 (a synthetic antagonist of PPARγ), and cyclic phosphatidic acid (cPA, a physiological inhibitor of PPARγ), followed by transplantation into adult ovariectomized mice. After the maturation of the transplanted ovaries, primordial follicle growth activation, follicle growth, and embryonic development were evaluated. Finally, the delivery of live pups after embryo transfer into recipient mice was assessed. While PPARγ was expressed in ovaries from mice of all ages, its levels were significantly increased in ovaries from 20-day-old mice. In GW9662-treated ovaries in vitro, PTEN levels were decreased, AKT was activated, and FOXO3a was excluded from the nuclei of primordial follicles. After 1 month, cPA-pretreated, transplanted ovaries produced the highest numbers of oocytes and polar bodies, exhibited the most advanced embryonic development, and had the greatest blastocyst formation rate compared to the rosiglitazone- and GW9662-pretreated groups. Additionally, the successful delivery of live pups after embryo transfer into the recipient mice transplanted with cPA-pretreated ovaries was confirmed. Our study demonstrates that PPARγ participates in primordial follicle activation and development, possibly mediated in part by the PI3K/AKT signaling pathway. Although more studies are required, adapting these findings for the activation of human primordial follicles may lead to treatments for infertility that originates from poor ovarian reserves.

Enhancing the safety of ovarian cortex autotransplantation: cancer cells are purged completely from human ovarian tissue fragments by pharmacological inhibition of YAP/TAZ oncoproteins.

STUDY QUESTION: Is it possible to eliminate metastasized cancer cells from ovarian cortex fragments prior to autotransplantation without compromising the ovarian tissue or follicles? SUMMARY ANSWER: Ex vivo pharmacological inhibition of YAP/TAZ by Verteporfin enabled us to efficiently eradicate experimentally induced small tumours, derived from leukaemia and rhabdomyosarcoma, from human ovarian tissue fragments. WHAT IS KNOWN ALREADY: Autotransplantation of ovarian tissue fragments that contain metastasized tumour cells may reintroduce the malignancy to the recipient. In order to enhance safety for the patient there is a strong need for protocols that effectively purges the ovarian tissue from malignant cells ex vivo prior to transplantation, without compromising ovarian tissue integrity. STUDY DESIGN, SIZE, DURATION: Tumour foci were experimentally induced in human ovarian cortex tissue fragments derived from at least three patients by micro-injection of cancer cell lines. Next, the tissue fragments were cultured to allow formation of metastasis-like structures followed by a 24 h ex vivo treatment with the YAP/TAZ inhibitor Verteporfin to eradicate the cancer cells. A control treatment was included in all experiments. The purged ovarian cortex fragments were cultured for an additional 6 days to allow any possibly surviving cancer cells to establish new metastatic foci. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human ovarian tissue was obtained after female-to-male sex reassignment surgery. Human rhabdomyosarcoma, leukeamia, breast cancer and Ewing's sarcoma cell lines were utilized for the induction of tumour foci. Tumour specific (immuno)histochemistry and RT-PCR were used for the detection of residual cancer cells after ex vivo treatment. Ovarian tissue and follicle integrity after exposure to Verteporfin was evaluated by histology, a follicular viability assay and a glucose uptake assay. MAIN RESULTS AND THE ROLE OF CHANCE: Metastasized rhabdomyosarcoma and leukaemia cells could be effectively purged from ovarian cortex tissue by a 24 h ex vivo treatment with Verteporfin, while breast cancer and Ewing's sarcoma did not respond to this treatment. Ovarian tissue integrity was not affected by purging, as no statistically significant difference (P > 0.05) was observed in the percentage of morphologically normal follicles, percentage of follicles with apoptotic cells, follicular viability or glucose uptake between the control treated ovarian cortex and Verteporfin treated ovarian cortex. LIMITATIONS, REASONS FOR CAUTION: Our tumour model is based on growth of human cancer cell lines. It is unclear whether these cells reflect the behaviour of malignant cells that have metastasized to the ovary during natural disease progression. Furthermore, the functionality of the ovarian tissue after ex vivo treatment requires further investigation in vivo. WIDER IMPLICATIONS OF THE FINDINGS: The results indicate that ex-vivo tumour cell purging of human ovarian cortex fragments intended for fertility preservation purposes is feasible by short-term pharmacological treatment. Effective purging of the ovarian cortex tissue enhances safety of ovarian cortex autotransplantation for the patient. This increases the likelihood that this form of fertility restoration may become an option for patients with malignancies for which ovarian cortex transplantation is currently considered unsafe. STUDY FUNDING/COMPETING INTEREST(S): Unconditional funding was received from Merck B.V. The Netherlands (Number 2016-FERT-1) and the foundation 'Radboud Oncologie Fonds' (Number KUN 00007682). The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: NA.

FERTILITY PRESERVATION: Follicle reserve loss in ovarian tissue transplantation.

Ovarian tissue cryopreservation and transplantation (OTCP-TP) has progressed over the past decade from a revolutionary experimental procedure to a well-accepted treatment in many centers for young patients with a high risk of ovarian failure after cancer treatment. The procedure is remarkably successful, with studies reporting return of ovarian function in up to 95% of graft recipients and pregnancy rates of between 30 and 50%. The most significant limitation of OTCP-TP is the massive loss of follicles that occurs following transplantation, which is primarily attributed to ischemic damage and follicle activation. We review the current approaches to reducing follicle loss and maximizing graft lifespan via pharmacological agents which reduce ischemic damage and follicle activation. We further discuss the value and disadvantage of inducing follicle activation in the graft as a means of generating mature follicles in the immediate short term.

FERTILITY PRESERVATION: Construction and use of artificial ovaries.

Increasing numbers of patients are now surviving previously fatal malignant diseases, so for women of childbearing age, fertility concerns are paramount once they are cured. However, the treatments themselves, namely chemo- and radiotherapy, can cause considerable damage to endocrine and reproductive functions, often leaving these women unable to conceive. When such gonadotoxic therapy cannot be postponed due to the severity of the disease or for prepubertal girls, the only way to preserve fertility is cryobanking their ovarian tissue for future use. Unfortunately, with some types of cancer, there is a risk of reimplanting malignant cells together with the frozen-thawed tissue, so it is not recommended. A safer approach involves grafting isolated preantral follicles back to their native environment inside a specially created transplantable artificial ovary for their protection. This bioengineered ovary must mimic the natural organ and therefore requires an appropriate scaffold to encapsulate not only isolated follicles, but also autologous ovarian cells, which are needed for follicles to survive and develop. Here we review the indications for use of this artificial ovary and advances in the field that are bringing us ever closer to clinical implementation.

FERTILITY PRESERVATION: Progress and prospects for developing human immature oocytes in vitro.

Ovarian cryopreservation rapidly developed from basic science to clinical application and can now be used to preserve the fertility of girls and young women at high risk of sterility. Primordial follicles can be cryopreserved in ovarian cortex for long-term storage and subsequently autografted back at an orthotopic or heterotopic site to restore fertility. However, autografting carries a risk of re-introducing cancer cells in patients with blood-born leukaemias or cancers with a high risk of ovarian metastasis. For these women fertility restoration could only be safely achieved in the laboratory by the complete in vitro growth (IVG) and maturation (IVM) of cryopreserved primordial follicles to fertile metaphase II (MII) oocytes. Culture systems to support the development of human oocytes have provided greater insight into the process of human oocyte development as well as having potential applications within the field of fertility preservation. The technology required to culture human follicles is extremely challenging, but significant advances have been made using animal models and translation to human. This review will detail the progress that has been made in developing human in vitro growth systems and consider the steps required to progress this technology towards clinical application.

FERTILITY PRESERVATION: Freezing of ovarian tissue and clinical opportunities.

Ovarian tissue cryopreservation (OTC) is mainly used for fertility preservation in girls and women facing a gonadotoxic treatment. If the woman subsequently becomes menopausal, the ovarian tissue may be transplanted to regain ovarian function, including fertility. The method was developed more than two decades ago and today thousands of women worldwide have undergone OTC. Fewer than 500 patients have had tissue transplanted and close to 100% of those regain ovarian function. Several technical aspects of OTC are now becoming more established, including high quantitative follicle survival, defining the size of the tissue resulting in optimal tissue revascularisation and follicle loss resulting from transport of ovarian tissue prior to freezing. We have used OTC to safeguard fertility in patients with genetic diseases, which for some diagnoses is purely experimental, as no transplantations is yet been performed. Usage of OTC beyond fertility is now also being considered; here, the endocrine function of follicles is the focus. It has been suggested that ovarian tissue stored in the reproductive years may be used to avoid premature ovarian insufficiency (POI) when there is a familial disposition or to postpone menopause in patients with an increased risk of osteoporosis or cardiovascular diseases. The benefit of OTC beyond fertility requires, however, actual clinical studies. The current review includes several recent technical aspects with contributions from Denmark building on some of the early work by Roger Gosden.

Translational research aiming to improve survival of ovarian tissue transplants using adipose tissue-derived stem cells.

There is now sufficient evidence to support the feasibility and efficacy of ovarian tissue (OT) cryopreservation and transplantation for both fertility preservation and restoration purposes. However, there are still issues to address regarding the grafting procedure itself, since transplanted tissue suffers massive follicle loss in the early post-grafting period. To improve follicle survival after transplantation, our group recently developed a two-step transplantation technique for OT transplantation in a xenografting model using adipose tissue-derived stem cells (ASCs). The aim of this narrative review is to describe and discuss the previously reported findings. ASCs were initially characterized by flow cytometry as positive for CD29, CD44, CD73, CD90, CD105 and CD166 (>95%) and negative for CD34, CD14, CD31, CD45 and Lin1. ASCs were used in a model of xenotransplantation, were they were embedded in a fibrin scaffold and transplanted to the peritoneum of immunodeficient mice. The goal of the first step was to increase levels of partial pressure of oxygen (pO2 ) and revascularization in the peritoneal transplantation site for further OT transplantation. As ASCs showed the ability to differentiate into endothelial-like cells and vessels in our model, OT transplantation was then performed with ASC grafts in a controlled experiment. At 7 days post-transplantation, the ASC group showed: (1) significantly higher pO2 levels; (2) significantly greater human and murine CD34-positive endothelial areas; (3) significantly higher primordial follicle survival rates; (4) and significantly lower numbers of apoptotic follicles compared with the control group. Our research model demonstrates that by adding ASCs to a fibrin scaffold before OT transplantation, faster and better graft reoxygenation and revascularization may be obtained, resulting in increased follicle survival and reduced follicle apoptosis.

Evaluation of Z-VAD-FMK as an anti-apoptotic drug to prevent granulosa cell apoptosis and follicular death after human ovarian tissue transplantation.

PURPOSE: To evaluate the efficiency of ovarian tissue treatment with Z-VAD-FMK, a broad-spectrum caspase inhibitor, to prevent follicle loss induced by ischemia/reperfusion injury after transplantation. METHODS: In vitro, granulosa cells were exposed to hypoxic conditions, reproducing early ischemia after ovarian tissue transplantation, and treated with Z-VAD-FMK (50 µM). In vivo, cryopreserved human ovarian fragments (n = 39) were embedded in a collagen matrix containing or not Z-VAD-FMK (50 µM) and xenotransplanted on SCID mice ovaries for 3 days or 3 weeks. RESULTS: In vitro, Z-VAD-FMK maintained the metabolic activity of granulosa cells, reduced HGL5 cell death, and decreased PARP cleavage. In vivo, no improvement of follicular pool and global tissue preservation was observed with Z-VAD-FMK in ovarian tissue recovered 3-days post-grafting. Conversely, after 3 weeks of transplantation, the primary follicular density was higher in fragments treated with Z-VAD-FMK. This improvement was associated with a decreased percentage of apoptosis in the tissue. CONCLUSIONS: In situ administration of Z-VAD-FMK slightly improves primary follicular preservation and reduces global apoptosis after 3 weeks of transplantation. Data presented herein will help to guide further researches towards a combined approach targeting multiple cell death pathways, angiogenesis stimulation, and follicular recruitment inhibition.

Xenotransplantation of goat ovary as an alternative to analyse follicles after vitrification.

The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.

Two-step transplantation with adipose tissue-derived stem cells increases follicle survival by enhancing vascularization in xenografted frozen-thawed human ovarian tissue.

STUDY QUESTION: Do adipose tissue-derived stem cells (ASCs) enhance vascularization and follicle survival in xenografted ovarian tissue using a two-step transplantation approach? SUMMARY ANSWER: Higher rates of oxygenation and vascularization of ovarian tissue, as well as increased follicle survival rates, were detected in the early post-grafting period. WHAT IS KNOWN ALREADY: ASCs have multilineage differentiation potential, proangiogenic properties and enhance vascularization in a peritoneal grafting site. Some studies suggest that using ASCs may improve ovarian tissue quality by enhancing graft angiogenesis. STUDY DESIGN, SIZE, DURATION: A total of 15 severe combined immunodeficient (SCID) mice were intraperitoneally grafted with frozen-thawed human ovarian tissue (OT) from five different patients. A peritoneal transplantation site had been previously prepared in a first step using either empty fibrin (Fi+OT group [n = 5]) or ASC-loaded fibrin (Fi/ASCs+OT group [n = 5]) for 14 days prior to grafting. Five mice underwent the standard one-step transplantation procedure and served as controls (OT group). Lithium phthalocyanine (LiPc) crystals were inserted into all grafted human ovarian tissue before transplantation. Levels of partial pressure of oxygen (pO2) in grafts were monitored in vivo by electron paramagnetic resonance (EPR) oximetry on Days 3 and 7. Samples for histology and immunohistochemistry (IHC) were collected after euthanizing the mice on Day 7 following EPR. One piece of ovarian tissue per patient was fixed for analysis to serve as non-grafted controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prospective experimental study conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to perform pO2 measurements (EPR oximetry), histological (haematoxylin and eosin staining), immunohistochemistry (anti-mouse and human double CD34 and anti-human Ki-67) and TUNEL analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant increase in pO2 was observed in all groups between Days 3 and 7 (P < 0.001). A significantly higher pO2 level was observed in the Fi/ASCs+OT group compared to the OT group on Day 7 (P = 0.028). Total CD34-positive vessel area on Day 7 was greater in the Fi/ASCs+OT group than in any other group (vs non-grafted group: P = 0.0014; vs OT group: P = 0.013; vs Fi+OT group: P = 0.018). Primordial follicle survival rates after grafting were higher in the Fi/ASCs+OT group than in the OT (P = 0.0059) or Fi+OT groups (P = 0.0307). TUNEL-positive follicle percentages after grafting were significantly lower in the Fi/ASCs+OT group than in any other grafted tissue (vs OT group: P = 0.045; vs Fi+OT group: P = 0.0268). Percentages of Ki-67-positive primordial follicles were significantly higher in all grafted groups compared to non-grafted tissue controls (P < 0.01). LIMITATIONS REASONS FOR CAUTION: As demonstrated by our results, the proposed two-step ovarian tissue transplantation procedure using ASCs enhances vascularization in the early post-grafting period, leading to increased follicle survival rates and decreased apoptosis. However, mechanisms involved in the proangiogenic behavior of ASCs remain to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that the proposed transplantation procedure with ASCs is a promising step towards potentially solving the problem of massive follicle loss after ovarian tissue grafting. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, grant Télévie No. 7.6515.16 F to DDM and grant 5/4/150/5 awarded to MMD and CAA is research associate, FRS-FNRS), Fonds Spéciaux de Recherche, Fondation St Luc, and Foundation Against Cancer, and donations from the Ferrero family.