Autotransplantation Of Ovarian Tissue Research Articles

O-272 Oral administration of antioxidants can support follicle survival in xenografted human ovarian tissue

Abstract Study question Can oral administration of antioxidants improve follicle survival in human ovarian tissue through reduction of oxidative stress in a xenotransplantation model? Summary answer Daily oral administration of antioxidants for 7 days post-transplantation had a positive effect on follicle survival in grafted human ovarian tissue. What is known already Today cryopreservation and autotransplantation of ovarian tissue is an established fertility preservation method in many centres. Hundreds of live births have been achieved but the efficacy of this procedure is hampered by a critical loss of follicles following transplantation. The ovarian tissue is subjected to hypoxia and ischemia postoperative which leads to oxidative damage in the tissue. Postoperative supplementation with antioxidants has been shown to decrease oxidative stress in transplanted ovarian tissue and increase follicle survival, however, these results were mainly obtained through intraperitoneal injections to host animals. Oral administration of antioxidants could be a better alternative for clinical application. Study design, size, duration Pieces of frozen-thawed ovarian cortex tissue (total of 56) from 12 women were transplanted to immunodeficient mice in a short- and long-term xenograft study, for 7 and 28 days respectively. In each study the mice were administered daily doses of hazelnut cream (control group) for 7 days post-transplantation or hazelnut cream containing either 150 mg/kg N-acetylcysteine (NAC) or FERTILIX (containing Vitamin E, D and C, Mixed Tocopherols and Tocotrienols, Lycopene, and Coenzyme Q10). Participants/materials, setting, methods Cryopreserved ovarian tissue was donated by 12 women undergoing ovarian tissue cryopreservation at Copenhagen University Hospital. Grafts were retrieved after 7 and 28 days of xenografting and histologically processed for follicle counts, immunohistochemistry, and qPCR analysis. The number of surviving follicles and ovarian graft volume were evaluated histologically after 7 and 28 days xenografting. Gene expression analysis of antioxidant defence markers (Sod1, Hmox and Catalase) and angiogenic factor Vegfa were performed after 7 days xenografting. Main results and the role of chance After 7 days xenografting, the follicle density was similar for all groups (control group: 33.03±14.85 (mean±SEM) follicles/mm3; NAC-group: 26.34±10.54 follicles/mm3, FERTILIX-group: 35.12±10.88 follicles/mm3). However, the NAC- and FERTILIX-group had a 3- and 3.7-fold increase in follicle density respectively compared to the control group. However, after 28 days of xenografting there was a higher density of surviving follicles in both antioxidant groups, compared to control (control group: 9.51±2.9 follicles/mm3; NAC-group: 14.9±6.02 follicles/mm3, FERTILIX-group: 14.43±5.83 follicles/mm3), which resulted in a 3.2-fold increase in follicle density in the NAC-group and 2.4-fold increase in the FERTILIX-group compared to the control group. The findings were not statistically significantly different in either the 7 days or 28 days study (p = 0.7072; p = 0.5691). The relative gene expression of the antioxidant defence genes Hmox and Catalase was overall like the control for all treatment groups after 7 days xenografting, but for Sod1 the expression was significantly lower for the NAC- and FERTILIX-group compared to the control group (p = 0.0407; p = 0.0255). For Vegfa the expression was similar overall and not significantly different from the control group (p > 0.05). Limitations, reasons for caution The distribution of follicles in human ovarian cortex is highly heterogeneous and can vary tremendously between cortex pieces from the same woman and between patients. Statistically significant results were not obtained for the follicle data in the current study; thus, the conclusions should be interpreted with caution. Wider implications of the findings Oral administration of antioxidants had a positive effect on follicle survival in xenotransplanted human ovarian tissue. These findings provide a step toward potential clinical administration of antioxidants like NAC or FERTILIX postoperative of ovarian tissue transplantation, which could improve follicle outcomes in a feasible way for the patient. Trial registration number Not applicable

Effects of different subcutaneous sites on heterotopic autotransplantation of canine ovarian tissue.

Ovarian tissue transplantation makes it possible to restore fertility; however, the success of this technique depends on the transplant region used. Therefore, this study aimed to evaluate the effect of two subcutaneous regions on canine ovarian transplantation, pinna (Pi) and neck (Ne), for 7 and 15 days. Ovaries collected by ovariosalpingohysterectomy were fragmented using a punch device. Fresh fragments were fixed, and the others were immediately grafted onto the animal itself in the Pi and Ne regions for 7 and 15 days. Recovered fragments were evaluated for histology (morphology, development and stromal density), picrosirius (collagen fibers), and immunohistochemistry (fibrosis and cell proliferation). The results showed that follicular normality rates were lower in Pi-7 (78%) vs. control (90%) and Pi-15 (86%), similar in Ne-7 (92%) and superior in Ne-15 (97%) compared to the control, with the effect of the region Ne (94%) superior (P < 0.05) to Pi (82%). Stromal density reduced in both regions vs. control but was similar within 15 days. Fragments from both regions showed higher fibronectin labeling and deposition of type I and lower type III collagen fibers (P < 0.05) vs. control. Proliferation rates in Ne-7 were higher (P < 0.05) than in control, and Pi-15 was higher (P < 0.05) than Ne-15. In conclusion, the pinna may be a region with greater potential than the neck after a 15-day autotransplantation of canine ovarian tissue.

Alpha-lipoic acid in ovarian vitrification solution for in vitro culture or autotransplantation as future strategies for the restoration of ovarian function in sheep

Vitrification is a promising method for ovarian cortex preservation, however, oxidative stress resulting from excessive free radical production can cause irreversible damage to the ovary and ovarian follicles. Therefore, this study aimed to evaluate the effect of 100 µM and 150 µM concentrations of alpha-lipoic acid (ALA) in the vitrification solution of sheep ovaries, after in vitro culture (IVC - 7 days) or autotransplantation (TRP - 15 days). For this, after IVC and TRP, the ovary was evaluated for follicular morphology and development; stromal cell density and proliferation; presence of mature and new vessels; tissue fibrosis, as well as, levels of reactive oxygen species (ROS), malonaldehyde (MDA), and nitrite in the culture medium after IVC. The results showed that ALA 100 µM or 150 µM maintained the morphology of primordial follicles and promoted follicular development after IVC. Although stromal cell density was not altered after IVC of the ovary vitrified with ALA 100 µM, a reduction was observed after TRP. The proliferation of these cells was also reduced after IVC or after TRP. The data also showed that after TRP, revascularization increased in the ovary vitrified with ALA 150 µM, but high levels of ROS and signs of fibrosis were observed. In conclusion, ALA is an excellent antioxidant agent that can be added to the vitrification solution to alleviate some of the negative effects observed after in vitro culture or autotransplantation of ovine ovarian tissue.

P-450 Mechanical injury improves follicle survival in xenotransplanted human ovarian cortex

Abstract Study question Can mechanical injury by needle puncturing of frozen thawed human ovarian cortex tissue prior to transplantation increase revascularization of xenografts and improve follicle survival? Summary answer Mechanical injury caused by needle puncture prior to transplantation improved follicle survival 2-fold in human ovarian cortex tissue after 4 weeks xenografting. What is known already Cryopreservation and autotransplantation of ovarian tissue is an established fertility preservation method which has resulted in numerous live births worldwide. However, the efficiency of the method is hampered by a critical follicle loss after transplantation. Faster revascularization of the transplanted graft would shorten the ischemic period and could potentially improve follicle survival. Controlled tissue damage by laser or needles can induce local angiogenesis by initiating the wound healing mechanisms and has been applied clinically in cardiovascular surgery for decades. Whether controlled mechanical tissue damage could be used to improve grafting of human ovarian cortex tissue has not yet been investigated. Study design, size, duration A total of 32 frozen-thawed human ovarian cortex pieces were cut in halves; one half serving as the untreated control and the other half was punctured approximately 200 times with a 29G needle immediately after thawing. The cortical pieces were transplanted subcutaneously to 10 immunodeficient mice. Ovarian grafts were retrieved after 3, 6 and 10 days (n = 8) to analyse graft revascularization and gene expression, and after 4 weeks to assess follicle density (n = 8). Participants/materials, setting, methods Ovarian cortex was donated by 16 women (aged 24-36 years) undergoing ovarian tissue cryopreservation for fertility preservation at a University Hospital. Gene expression analysis of proinflammatory cytokines (Tnf-α), apoptotic factors (Bcl2 and Bax), and angiogenic factors (Vegfa, Angptl4, Ang1, and Ang2) were performed by qPCR. Follicle density after 4 weeks xenografting were evaluated histologically. Graft-revascularization was assessed using immunohistochemical detection of murine Cd31. Main results and the role of chance Our results showed that the ovarian grafts which had been punctured with needles prior to transplantation contained a larger number of surviving follicles per mm3 compared to the untreated control (control group: 2.9 ± 0.6 (mean±SEM) follicles/mm3; needle group: 6.4 ± 2.4 follicles/mm3). Follicle densities between patients and groups varied from 0.8 to 26 follicles/mm3. Overall, a 2.3-fold increase in follicle density was found in the needle group compared to control, though this finding was not significantly different (p-value = 0.1182). The relative gene expression of Tnf-α and the Bax/Bcl2 ratio after 6 days grafting was similar in the needle and control group, indicating that the mechanical injury did not induce an additional inflammatory or apoptotic response in the grafts. The relative gene expression of Vegf, Angptl4, and Ang2 after 6 days grafting was highest in the needle group compared to the control group, though not significant (p&amp;gt;0.1). Results of the gene expression analysis from Day 3 and 10 are awaited, and so are the quantification of the Cd31 positive vessel area and vascular density from Day 3, 6, and 10, which will provide valuable insight into the initial graft revascularization. Limitations, reasons for caution The ovarian follicles are highly heterogeneously distributed in the ovarian cortex and can vary 100-fold between cortex pieces from the same woman and between patients. A high variability in follicle density within and between treatment groups and patients was found in the current study. Thus, solid conclusions cannot be made. Wider implications of the findings Our findings suggest a positive effect of mechanical injury on human ovarian grafts as needle puncturing prior to transplantation improved follicle survival after xenotransplantation. Clinically, this technique would be easy to implement since it is performed during the thawing process and does not involve any additional treatment of the patient. Trial registration number Not applicable

O-035 Ex vivo photodynamic therapy approach using a nanovesicular photosensitizer to allow safe autotransplantation of ovarian tissue from leukemia patients

Abstract Study question Is it possible to purge leukemia cells from ovarian tissue (OT) fragments before transplantation? Summary answer Our photodynamic therapy (PDT) approach showed to efficiently destroy leukemia cells from a tumor model, indicating the feasibility of this technique to purge OT samples. What is known already Autotransplantation of the cryopreserved OT is the most suitable option to preserve fertility for prepubertal girls and women that require immediate cancer treatment [1, 2]. Up to now, more than 200 live births have already been reported after OT cryopreservation and transplantation [3]. Leukemia is the 12th most common cancer in Europe among prepubertal girls and women of reproductive age [4]. Unfortunately, once their health has been restored, autotransplantation of cryopreserved OT for leukemia patients is not advised due to the high risk of transferring malignant cells back to the patient, leading to leukemia recurrence [3]. Study design, size, duration To safely transplant the OT from these patients and restore their fertility, our goal here was to develop a PDT strategy to eliminate leukemia ex vivo. To this end, we designed OR141-loaded niosomes (ORN) to create the most effective formulation for ex vivo purging OT fragments from myelogenous leukemia cells. Participants/materials, setting, methods After establishing the best ORN formulation, our PDT approach was used to eradicate HL60 from ex vivo tumor models prepared by microinjection of cancer cell suspension in ovarian fragments [5]. Additionally, we evaluated the effect of ORN-based PDT on follicle density, survival, and tissue quality was evaluated after 7-day xenotransplantation to SCID mice. Main results and the role of chance The ex vivo purging of tumor models demonstrated that the PDT strategy with ORN could selectively eradicate the malignant cells from tissue fragments without affecting ovarian tissue normal cells, as evidenced by PCR and immunohistochemical analysis. Regarding the effect of our PDT approach on follicle population and OT quality, our results after xenotransplantation revealed no significant difference between the follicle density of control (non-treated, grafted OT) and ORN-treated groups (2.38 ± 0.63 and 3.21 ± 1.94 morphologically normal follicles/mm2, respectively). In addition, the results showed that the control and ORN-treated ovarian tissue can be equally vascularized (7.65±1.45% and 9.89±2.21%, respectively). Moreover, the proportion of fibrotic area in control and ORN-treated groups were 15.96±5.94%, and 13.32±3.05%, which is not significantly different from the pre-graft fragments (14.22±6.65%). Limitations, reasons for caution This study did not use OT fragments from leukemia patients, but tumor models created after injection of HL60 cells in OT from healthy patients. Therefore, while the results are promising, it still remains to assess if our PDT approach will be equally successful to eliminate malignant cells from leukemia patients. Wider implications of the findings Our results showed that the purging procedure causes no significant impairing effect on follicles development and tissue quality, suggesting our novel PDT procedure could be a promising strategy to destroy leukemia cells in fragments of ovarian tissue allowing its safe transplantation in cancer survivors. Trial registration number not applicable

Autologous transplantation of fresh ovarian tissue in the ICR mice model

SUMMARY&#x0D; During the past 20 years, autologous transplantation of either frozen ovarian tissue for cancer patients or ovarian tissue pretreated with curative medications for premature ovarian insufficiency (POI) patients have been achieved worldwide. POI is characterized by amenorrhea before 40 years of age. As a result, juvenile cancer patients and POI patients have increased chances to give healthy birth based on this achievement. It also indicates that mastering the skills of ovarian auto-transplantation is required for researchers and doctors. In this study, we performed orthotopic auto-transplantation of fresh ovarian tissues by transplanting unilateral half ovarian tissue to the contralateral ovary in the ICR (Institute of Cancer Research) strain of outbred, heterogeneous mice to determine if the transplanted tissue could be functional. The transplantation surgery entailed taking partial ovarian tissue from the unilateral ovary of the adult ICR female mouse and then transplanting into the ovarian sac of the contralateral ovary, in which part of the ovarian tissue had been removed in advance. One week after surgery, we found that the freshly transplanted mouse ovarian tissue survived and functional, as histochemical and immunofluorescence assays have shown that not only both follicles at different developing stages and corpus luteum are available, but the morphology of them are properly maintained within the transplanted tissue. This study approved that for those who are interested in ovarian transplantation study, applying mouse model to practice ovarian tissue auto-transplantation is reliable and easy handled.

Indications for cryopreservation and autotransplantation of ovarian tissue.

Cryopreservation and autotransplantation of ovarian tissue as a fertility-preserving treatment are offered to prepubertal girls and women at high risk of developing premature ovarian insufficiency in more than 20 different countries. There is some controversy regarding the main criteria for offering patients this treatment. This article aims to describe the applied indications for cryopreservation and autotransplantation of ovarian tissue. This review article is based on literature searches in the Embase and Medline databases, restricted to articles published from 2010onwards. A total of twelve articles were included, with diagnoses for 1947patients in the age group 0-44years and 820patients aged less than 18years. The most frequent indications for cryopreservation of ovarian tissue were breast cancer (694of 1947, 36%) and lymphoma (416of 1947, 21%). Malignant diseases accounted for 86% of indications. In patients aged less than 18years, malignant neurological diseases (166of 820, 20%), leukaemia (156of 820, 19%), sarcomas (125of 820, 15%) and benign haematological disorders (124of 820, 15%) were the most common indications. In patients aged less than 18years, 26% of the indications were benign disorders. The most frequent indications for autotransplantation of ovarian tissue were lymphoma (74of 196, 38%) and breast cancer (53of 196, 27%). The frequency of the indications is consistent with the prevalence of the diseases, but further experience is necessary to improve the guidelines for the treatment. The risk of autotransplantation of malignant cells and radiation injury should be taken into account when assessing which patients should be offered treatment.

First Ovarian Tissue Cryopreservation and Autotransplantation in Patients with Malignancies in Hungary – Report of the First Three Cases

Fertility Preservation, Cryopreservation, Ovarian Tissue Autotransplantation and Malignancies. Abstract: Autotransplantation of cryopreserved ovarian tissue is one of the most advanced methods for fertility preservation of patients suffering from malignant diseases. Even though the method itself is still experimental, nearly a hundred live births have been documented worldwide, and its efficacy is comparable with the efficacy of any other assisted reproductive technology. Our prospective, nonrandomized study was the first in Hungary that aimed to examine the safety and efficacy of fertility preservation based on ovarian tissue cryopreservation and autotransplantation. Patients were included only with stage I-III malignancy confirmed by histological diagnosis with a high risk for post-treatment infertility. 13 patients met the inclusion criteria and were enrolled in the study. After successful treatment and recovery, cryopreserved ovarian tissue was thawed and autotransplanted in three cases. The ultrathin slices of ovarian cortex were transplanted on the remaining ovaries with laparoscopic or minilaparotomic intervention. Patients were discharged home after an uneventful postoperative period and are followed up currently. In summary, cryopreservation and autotransplantation of ovarian tissue is a safe technology for fertility preservation, which should be considered to offer and perform prior to gonadotoxic treatment, after individual evaluation of patients

Heterotopic autotransplantation of ovarian tissue in a large animal model: Effects of cooling and VEGF.

Heterotopic and orthotopic ovarian tissue autotransplantation techniques, currently used in humans, will become promising alternative methods for fertility preservation in domestic and wild animals. Thus, this study describes for the first time the efficiency of a heterotopic ovarian tissue autotransplantation technique in a large livestock species (i.e., horses) after ovarian fragments were exposed or not to a cooling process (4°C/24 h) and/or VEGF before grafting. Ovarian fragments were collected in vivo via an ultrasound-guided biopsy pick-up method and surgically autografted in a subcutaneous site in both sides of the neck in each mare. The blood flow perfusion at the transplantation site was monitored at days 2, 4, 6, and 7 post-grafting using color-Doppler ultrasonography. Ovarian grafts were recovered 7 days post-transplantation and subjected to histological analyses. The exposure of the ovarian fragments to VEGF before grafting was not beneficial to the quality of the tissue; however, the cooling process of the fragments reduced the acute hyperemia post-grafting. Cooled grafts compared with non-cooled grafts contained similar values for normal and developing preantral follicles, vessel density, and stromal cell apoptosis; lower collagen type III fibers and follicular density; and higher stromal cell density, AgNOR, and collagen type I fibers. In conclusion, VEGF exposure before autotransplantation did not improve the quality of grafted tissues. However, cooling ovarian tissue for at least 24 h before grafting can be beneficial because satisfactory rates of follicle survival and development, stromal cell survival and proliferation, as well as vessel density, were obtained.

Evaluation of ovarian tissue transplantation: results from three clinical centers

To report ovarian tissue autotransplantation (AT) results and describe the relationship between technical and clinical factors and outcomes. Multicenter retrospective cohort study. Tertiary medical centers. Infertile patients who had stored ovarian tissue before sterilizing treatment and returned for AT with the aim of conceiving. Ovarian tissue cryopreservation (OTC) and AT, endocrine monitoring, invitro fertilization. Endocrine performance, pregnancy and live-birth rates. From 2004 to 2018, 70 patients underwent 87 ATs. Sixty patients undergoing 70 ATs met the inclusion criteria. After AT, menses returned in 94% of patients and median FSH dropped from 68 to 19 IU/mL. Fifty pregnancies and 44 deliveries were attained, with 50% of women achieving at least one pregnancy and 41.6% at least one delivery. Twelve patients underwent AT more than once and had their endocrine activity restored in case menses recurred after the first transplantation. Repeated transplantations yielded five live births in three patients, two of whom had already given birth after the first transplantation. Preharvesting chemotherapy was not associated with inferior outcomes. Of seven patients whose pelvis was exposed to radiation before AT, four conceived and delivered. Neither tissue dimensions nor surgical approach affected fertility outcomes. OTC is highly effective at restoring fertility in sterilized patients, and prior exposure to chemotherapy should not be considered a contraindication. Repeated AT should be contemplated in case of graft malfunction, especially if previous transplantation was successful. In selected cases, conception and delivery may be feasible after pelvic exposure to radiation.

History, Evolution and Current State of Ovarian Tissue Auto-Transplantation with Cryopreserved Tissue: a Successful Translational Research Journey from 1999 to 2020.

The loss of fertility and early menopause are common after gonadotoxic therapies and radical pelvic surgery. The strategy of ovarian tissue cryopreservation and auto-transplantation was introduced to prevent this significant quality of health issue. Ovarian transplantation with cryopreserved tissue has gone through remarkable evolution in the last 20years. In this review, we detail the history and evolution of ovarian transplantation with cryopreserved tissue from its origins to the present. Ovarian cryopreservation and transplantation approach was first tested with animal models. The approach was then validated in human ovarian xenografting models before being applied to patients in pioneering clinical studies. The first orthotopic and heterotopic approaches to ovarian transplantation was developed by Oktay et al. who reported the first successful restoration of ovarian function with these approaches beginning in 2000 with first embryo development in 2004. Controversy remains on when the first live birth occurred after orthotopic ovarian transplantation with cryopreserved tissue as the patient was ovulating with elevated progesterone levels in the case reported in 2004; first live birth is likely to be the one reported by Meirow et al. in 2005. Nevertheless, the technique has evolved to reach a level where most recent live birth rates are exceeding 35% and the procedure is no longer considered experimental by many.

Biopsying, fragmentation and autotransplantation of fresh ovarian cortical tissue in infertile women with diminished ovarian reserve.

Can ovarian biopsying per se and/or autotransplantation of fragmented ovarian cortical tissue activate dormant follicles and increase the number of recruitable follicles for IVF/ICSI in women with diminished ovarian reserve (DOR)? Ovarian biopsying followed by immediate autotransplantation of fragmented cortical tissue failed to increase the number of recruitable follicles for IVF/ICSI 10weeks after the procedure either at the graft site or in the biopsied ovary, but 12 of the 20 women subsequently had a clinical pregnancy during the 1-year follow-up. Infertile women with DOR constitute a group of patients with poor reproductive outcome mainly due to the low number of mature oocytes available for IVF/ICSI. Recent studies have shown that in vitro activation of residual dormant follicles by both chemical treatment and tissue fragmentation has resulted in return of menstrual cycles and pregnancies in a fraction of amenorrhoeic women with premature ovarian insufficiency. This is a prospective clinical cohort study including 20 women with DOR treated at the fertility clinic, Rigshospitalet, Denmark, during April 2016-December 2017. Non-pregnant patients were on average followed for 280days (range 118-408), while women who conceived were followed until delivery. Study follow-up of non-pregnant patients ended in September 2018. The study included infertile women aged 30-39years with preserved menstrual cycles, indication for IVF/ICSI and repeated serum measurements of anti-Müllerian hormone (AMH) ≤ 5pmol/L. Patients were randomized to have four biopsies taken from either the left or the right ovary by laparoscopy followed by fragmentation of the cortical tissue to an approximate size of 1mm3 and autotransplanted to a peritoneal pocket. The other ovary served as a control. Patients were followed weekly for 10weeks with recording of hormone profile, antral follicle count (AFC), ovarian volume and assessment for ectopic follicle growth. After 10weeks, an IVF/ICSI-cycle with maximal ovarian stimulation was initiated. No difference in the number of mature follicles after ovarian stimulation 10weeks after the procedure in the biopsied versus the control ovaries was observed (1.0 vs. 0.7 follicles, P= 0.35). In only three patients, growth of four follicles was detected at the graft site 24-268days after the procedure. From one of these follicles, a metaphase II (MII) oocyte was retrieved and fertilized, but embryonic development failed. Overall AMH levels did not change significantly after the procedure (P= 0.2). The AFC increased by 0.14 (95% CI: 0.06;0.21) per week (P< 0.005), and the biopsied ovary had on average 0.6 (95% CI: 0.3;-0.88) follicles fewer than the control ovary (P= 0.01). Serum levels of androstenedione and testosterone increased significantly by 0.63nmol/L (95% CI: 0.21;1.04) and 0.11nmol/L (95% CI: 0.01;0.21) 1week after the procedure, respectively, and testosterone increased consecutively over the 10weeks by 0.0095nmol/L (95% CI: 0.0002;0.0188) per week (P= 0.045). In 7 of the 20 patients, there was a serum AMH elevation 5 to 8weeks after the procedure. In this group, mean AMH increased from 2.08pmol/L (range 1.74-2.34) to 3.94pmol/L (range 3.66-4.29) from Weeks 1-4 to Weeks 5-8. A clinical pregnancy was obtained in 12 of the 20 (60%) patients with and without medically assisted reproduction (MAR) treatments. We report a cumulated live birth rate per started IVF/ICSI cycle of 18.4%. Limitations of the study were the number of patients included and the lack of a non-operated control group. Moreover, 9 of the 20 women had no male partner at inclusion and were treated with donor sperm, but each of these women had an average of 6.8 (range 4-9) unsuccessful MAR treatments with donor sperm prior to inclusion. Although 12 out of 20 patients became pregnant during the follow-up period, the current study does not indicate that biopsying, fragmenting and autotransplanting of ovarian cortical tissue increase the number of recruitable follicles for IVF/ICSI after 10weeks. However, a proportion of the patients may have a follicular response in Weeks 5-8 after the procedure. It could therefore be relevant to perform a future study on the possible effects of biopsying per se that includes stimulation for IVF/ICSI earlier than week 10. This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. The funders had no role in the study design, data collection and interpretation, or decision to submit the work for publication. None of the authors have a conflict of interest. NCT02792569.

Pyroptosis is involved in cryopreservation and auto-transplantation of mouse ovarian tissues and pyroptosis inhibition improves ovarian graft function

The aim of this study was to investigate the role of pyroptosis in the cryopreservation and transplantation of mouse ovarian tissues; the effects of pyroptosis inhibitior on the ovarian graft function were also explored. ICR (institute of cancer research) mice were randomly divided into control group and experimental groups (n = 10 per group). The experimental groups included fresh graft group (autograft), cryopreserved graft group (cryopreservation + autograft), and pyroptosis inhibition group (cryopreservation + autograft + pyroptosis inhibitor). At the third day after auto-transplantation, caspase-1 and NLRP3 protein expression in grafts were assessed by Western blot; in the meantime, serum concentration of IL-1β was examined by ELISA. After 28 days of auto-transplantation, estradiol concentrations and follicular densities of grafts were evaluated. The caspase-1 and NLRP3 protein expression in grafts from all the experimental groups were significantly higher than that from control group respectively; moreover, there was a significant increase in serum concentrations of IL-1β in all experimental groups compared with control group. The concentration of estradiol and follicular densities of grafts in pyroptosis inhibition group were significantly higher than that in cryopreserved graft group. Pyroptosis is involved in cryopreservation and auto-transplantation of mouse ovarian tissues, and pyroptosis inhibition can improve the ovarian graft function.

Fertility and hormone preservation and restoration for female children and adolescents receiving gonadotoxic cancer treatments: A systematic review

Background/PurposeThe purpose of this systematic review by the American Pediatric Surgical Cancer Committee was to summarize evidence from the current medical literature regarding fertility restoration and hormone replacement for female children and adolescents treated with gonadotoxic treatments. MethodsUsing PRISMA guidelines, questions were addressed by searching Medline, Cochrane, Embase Central and National clearing house databases using relevant search terms. Eligible studies included those that addressed ovarian tissue cryopreservation (OTC), oocyte harvest, ovarian transposition, and ovarian tissue auto-transplantation for females under the age of 20. Four reviewers independently screened studies for eligibility, extracted data and assessed the risk of bias. Study outcomes were summarized in a narrative synthesis. ResultsTwo thousand two hundred seventy-six studies were identified by database search and manual review and 2185 were eliminated based on defined exclusion criteria. Ninety-one studies served as the basis for the systematic review. There were 1019 patients who underwent OTC with ages ranging from 0.4 to 20.4 years old, with 298 under the age of 13. Twenty patients aged 13–20 years old underwent successful oocyte harvest. Thirty-seven children underwent ovarian transposition as a means of fertility preservation. Eighteen patients underwent auto-transplantation of thawed ovarian cortical tissue that was harvested before the age of 21 years resulting in 10 live births. ConclusionsClinically accepted and experimental fertility preservation options such as OTC, oocyte cryopreservation, and ovarian transposition are available to females aged 20 years and younger who are at risk for premature ovarian insufficiency and infertility due to gonadotoxic treatments. There is a large cohort of pediatric-aged patients, with a wide variety of diagnoses and treatments, who have undergone fertility preservation. Currently, fertility and hormone restoration experience for patients who were 20- years of age or younger at the time of fertility preservation remains limited. Level of EvidenceIV.

Fertility Preservation in Women With Malignancy: Future Endeavors.

The area of fertility preservation is constantly developing. To date, the only noninvestigational and unequivocally accepted methods for fertility preservation are cryopreservation of embryos and unfertilized oocytes. This article is one of several in a monogram on fertility preservation. The debate, pros and cons, and equivocal data on the use of GnRH analogues for fertility preservation are elaborated by 3 other manuscripts, in this monogram. A repeat of the arguments, pros and cons of this debatable issue, would be a repetition and redundancy of what is already included in this monogram. The subject of ovarian cryopreservation for fertility preservation is also elaborated by several other authors in this monogram. It is possible that, in the not too far future, the technologies of in vitro maturation of primordial follicles to metaphase 2 oocytes, and the “artificial ovary,” will turn clinically available. These technologies may bypass the risk of resuming malignancy by autotransplantation of cryopreserved-thawed ovarian tissue in leukemia and diseases where malignant cells may persist in the cryopreserved ovarian tissue. We summarize here the suggested options for future endeavors in fertility preservation.

Immuno-Isolating Dual Poly(ethylene glycol) Capsule Prevents Cancer Cells from Spreading Following Mouse Ovarian Tissue Auto-Transplantation

For female cancer survivors, premature ovarian insufficiency (POI) is a common complication of anticancer treatments. Ovarian tissue cryopreservation before treatment, followed by auto-transplantation after remission is a promising option to restore fertility and ovarian endocrine function. However, auto-transplantation is associated with the risk of re-introducing malignant cells harbored in the stroma of the ovarian autograft. To mitigate this risk, we investigated in this pilot study whether an immuno-isolating dual-layered poly(ethylene glycol)(PEG) capsule can retain cancer cells, while supporting folliculogenesis. The dual PEG capsule loaded with 1000 4T1 cancer cells retained 100% of the encapsulated cells in vitro for 21 days of culture. However, a greater cell load of 10,000 cells/capsule led to capsule failure and cells’ release. To assess the ability of the capsule to retain cancer cells, prevent metastasis, and support folliculogenesis in vivo we co-encapsulated cancer cells with ovarian tissue in the dual PEG capsule and implanted subcutaneously in mice. Control mice implanted with 2000 non-encapsulated cancer cells had tumors formed within 14 days and metastasis to the lungs. In contrast, no tumor mass formation or metastasis to the lungs was observed in mice with the same number of cancer cells encapsulated in the capsule. Our findings suggest that the immuno-isolating capsule may prevent the escape of the malignant cells potentially harbored in ovarian allografts and, in the future, improve the safety of ovarian tissue auto-transplantation in female cancer survivors.

Autotransplantation of fragmented ovarian cortical tissue: a laparoscopic demonstration

To describe and demonstrate a simple and secure procedure for laparoscopic autotransplantation of fragmented ovarian cortical tissue in women with diminished ovarian reserve as part of invitro activation (IVA) of ovarian follicles. Step-by-step video explanation of the surgical procedure with still pictures and surgical video clips to demonstrate the detailed technique. Fertility clinic and obstetrics and gynecology department at a university hospital. Women with idiopathic diminished ovarian reserve and indication for invitro fertilization (IVF), aged 25 to 39years with an antral follicle count bilaterally of ≤5, antimüllerian hormone level of ≤5 pmol/L, and two ovaries. The laparoscopic autotransplantation consists of six steps: [1] obtaining ovarian cortical biopsy samples, [2] preparing the peritoneal pocket, [3] fragmenting the ovarian cortical tissue into pieces of approximately 1mm3, [4] installing the tissue fragments into a catheter, [5] transplanting the tissue fragments into the peritoneal pocket, and [6] closing the peritoneal pocket with a surgical clip. After the procedure, the patients are evaluated with blood samples and ultrasound scans followed by controlled ovarian stimulation. Ethics committee approval was obtained. Feasibility of a six-step laparoscopic autotransplantation procedure using fragmented ovarian cortical tissue. A simple, fast laparoscopic procedure for taking biopsy samples and autotransplanting cortical tissue fragments in an all-in-one procedure ensures the rapid handling and correct placement of the small tissue fragments. The procedure is performed in an outpatient setting with an operation time of 1hour. We have performed this procedure on 20 patients with no complications. Invitro activation is a new, developing option for women in fertility treatment who have diminished ovarian reserve. Fragmentation of murine ovarian tissue has shown to suppress the Hippo pathway, thereby initiating proliferation and growth. This surgical procedure resembles that used when transplanting pieces of frozen-thawed ovarian tissue for fertility restoration, but the fragmented ovarian tissue is only 1mm3, which makes it difficult to transplant. Until now no surgical procedures for transplanting small IVA fragments of cortical tissue has been published. With this video we report in detail a simple way of autotransplanting small fragments of IVA cortical tissue using what is already accessible in the operating theater. Among the many advantages of this procedure are its short duration (1hour) and outpatient setting, which enable fast recovery and minimal postoperative pain. The procedure also allows fast handling and minimal manipulation of the tissue (limited to the fragmentation). The effect of autotransplantation of fragmented tissue in women with diminished ovarian reserve is currently being studied in ongoing trials. If the technique is combined with chemical IVA, a better outcome may be seen.

Angiopoietin-1 and -2 and vascular endothelial growth factor expression in ovarian grafts after cryopreservation using two methods.

ObjectiveThe favored method of preserving fertility in young female cancer survivors is cryopreservation and autotransplantation of ovarian tissue. Reducing hypoxia until angiogenesis takes place is essential for the survival of transplanted ovarian tissue. The aim of this study was to investigate the role of angiopoietin-1 (Angpt-1), angiopoietin-2 (Angpt-2), and vascular endothelial growth factor (VEGF) in ovarian tissue grafts that were cryopreserved using two methods.MethodsOvarian tissues harvested from ICR mice were divided into three groups: group I (control), no cryopreservation; group II, vitrification in EFS (ethylene-glycol, ficoll, and sucrose solution)-40; and group III, slow freezing in dimethyl sulfoxide. We extracted mRNA for VEGF, Angpt-1, and Angpt-2 from ovarian tissue 1 week following cryopreservation and again 2 weeks after autotransplantation. We used reverse transcriptase-polymerase chain reaction to quantify the levels of VEGF, Angpt-1, and Angpt-2 in the tissue.ResultsAngpt-1 and Angpt-2 expression decreased after cryopreservation in groups II and III. After autotransplantation, Angpt-1 and Angpt-2 expression in ovarian tissue showed different trends. Angpt-1 expression in groups II and III was lower than in group I, but Angpt-2 in groups II and III showed no significant difference from group I. The vitrified ovarian tissues had higher expression of VEGF and Angpt-2 than the slowfrozen ovarian tissues, but the difference was not statistically significant.ConclusionOur results indicate that Angpt-2 may play an important role in ovarian tissue transplantation after cryopreservation although further studies are needed to understand its exact function.

Ovarian endometriosis and fertility preservation: a challenge in 2018.

Endometriosis is a benign, estrogen-dependent gynecological disorder. It is now well established that three different forms of endometriosis can occur in the pelvic cavity, namely peritoneal endometriosis, ovarian endometriosis and deep endometriotic nodules of the rectovaginal septum. Treatment of endometriosis-associated infertility has been investigated using both medical and surgical modalities. Surgery for ovarian endometriosis can lead to premature ovarian insufficiency (POI) and further infertility, so one of the most important goals of therapy should be preserving fertility. Procedures combining stripping and ablation should be considered the first-line approach for ovarian endometrioma-related infertility, in order to protect the ovarian reserve, which may already be depleted even before surgery. Autotransplantation of ovarian tissue should be considered in case of mandatory radical treatment (oophorectomy), or if conservative treatment poses a risk. For patients at risk of POI, particularly those undergoing gonadotoxic treatment, two main fertility preservation options exist: oocyte vitrification or reimplantation of fresh or frozen ovarian tissue. Both have advantages and disadvantages.

Expression of inhibitor proteins that control primordial follicle reserve decreases in cryopreserved ovaries after autotransplantation

Even with 86 live births reported globally so far, the mechanism of primordial follicle loss following autotransplantation of the frozen-thawed ovarian tissue needs further evaluation. Pten, Tsc1, p27, and Amh are the inhibitor proteins that play crucial roles in suppressing the transition from the primordial follicle to primary state, maintaining the primordial follicle reserve. In this study, we aimed to evaluate whether the expression patterns of these proteins change and it may be related to the global primordial follicle loss after autotransplantation of the frozen-thawed ovarian tissue. Four groups were established in rats: fresh-control, frozen/thawed, fresh-transplanted, and frozen/thawed and transplanted. After slow freezing and thawing process, two ovarian pieces were transplanted into the back muscle of the same rat. After 2weeks, grafts were harvested, fixed, and embedded into the paraffin block. Normal and atretic primordial/growing follicle count was performed in all groups. Ovarian tissues were evaluated for the dynamic expressions of the Pten, Tsc1, p27, and Amh proteins using immunohistochemistry, and H-score analyses were done. Ovarian tissue cryopreservation does not change the expression patterns of inhibitory proteins that control ovarian reserve. Both in fresh and frozen/thawed autotransplanted groups, the expression of inhibitory proteins and Amh decreased significantly in primordial follicles and in growing follicles, respectively. In control group and in frozen/thawed group, primordial follicle counts were similar but decreased by almost half in both fresh-transplanted and frozen/thawed and transplanted groups. One of the causes of primordial follicle loss after transplantation of ovarian graft may be decreased expression of the inhibitory proteins that guard the ovarian reserve and transplantation itself seems to be the major cause for disruption of inhibitory molecular signaling. Our findings highlight important molecular aspects for future clinical applications for fertility preservation in humans.