Transplantation Of Frozen-thawed Ovarian Tissue Research Articles

UC-MSCs promote frozen-thawed ovaries angiogenesis via activation of the Wnt/β-catenin pathway in vitro ovarian culture system

BackgroundOvarian tissue cryopreservation and transplantation are novel therapeutic approaches for fertility preservation. However, follicle loss caused by ischemic and hypoxic damage is one of the issues after frozen-thawed ovarian tissue transplantation. Promoting angiogenesis in grafts is the key to restore cryopreserved ovarian function. Mesenchymal stem cells (MSCs) have been reported to facilitate angiogenesis in the cryopreserved ovarian tissue transplantation. However, the risk of embolization, immunogenic effect and tumorigenesis hinders the clinical application of MSCs to human organ transplantation. In this study, we established an in vitro ovarian culture system to restore frozen-thawed ovarian function before transplantation with the application of umbilical cord mesenchymal stem cells (UC-MSCs), and explored the effects of UC-MSCs on frozen-thawed ovaries in vitro ovarian culture system and the mechanisms of UC-MSCs on the angiogenesis of frozen-thawed ovaries.MethodsA simple in vitro three dimensional (3D) ovarian culture system using Matrigel was established to support to an ideal niche, and ovary was alone cultured in the 24-well plate as a control. We also evaluated the effects of UC-MSCs treatment on ovarian function with or without Matrigel support. All thawed ovaries were randomly divided into control group (Matrigel−/UC-MSCs−), Matrigel group (Matrigel+/UC-MSCs−), UC-MSCs group (Matrigel−/UC-MSCs+) and UC-MSCs + Matrigel group (Matrigel+/UC-MSCs+). HE staining was used to detect the histological structure of follicles and TUNEL staining was used to detect cell apoptosis. The number of microvessels was counted to evaluate neovascularization. The mRNA expression of VEGFA, IGF1 and ANGPT2 were detected by RT-PCR. Western blotting was used to measure the expression of GSK-3β, β-catenin and p-β-catenin.ResultsIn the absence of UC-MSCs, 3D culture system supported by Matrigel showed significantly improved follicular development and microvascular number. Additionally, UC-MSCs were also found to effectively improve follicular development and microvascular number regardless of the culture condition used. However, alleviated follicular apoptosis, increased mRNA expression of angiogenesis-related gene and activated Wnt/β-catenin pathway occurred only in the UC-MSCs + Matrigel group. Besides, with the application of IWP-2 in UC-MSCs + Matrigel group, Wnt//β-catenin pathway could be blocked by IWP-2 serving as one of Wnt/β-catenin pathway inhibitors.ConclusionsThis in vitro study showed the beneficial effects of UC-MSCs on thawed ovaries and explored a potential mechanism inducing angiogenesis. In particular, 3D ovarian culture system supported by Matrigel further improved UC-MSCs treatment. The in vitro culture system using Matrigel and UC-MSCs may provide a potential treatment strategy for improving the success rate of thawed ovaries transplantation.

Does it make sense to refreeze ovarian tissue after unexpected occurrence of endometriosis when transplanting the tissue?

BackgroundOvarian insufficiency is a major concern for long-term cancer survivors. Ovarian tissue cryopreservation for fertility preservation is an emerging technique that has proven successful over the past decade through transplantation of frozen-thawed ovarian tissue. Compared to other established techniques, such as oocyte freezing, ovarian tissue cryopreservation preserves actual organ function and thus the production of sex hormones. Endometriosis in perimenopausal women is rare, however it can be surprising diagnosis in the planned transplantation of cryopreserved ovarian tissue and the already thawed tissue may not be transplanted, so that it has to be refrozen.ResultsOvarian function returned in the patient two months after transplantation, as shown by estrogen production. Ten months after the ovarian tissue transplantation mild stimulation with FSH was initiated in accordance with a low-dose protocol. When ultrasonography revealed a follicle 17 mm in size in the ovarian graft, hCG was added and after follicular puncture one oocyte was obtained. The oocyte could be fertilized by IVF and transferred to the uterus. On day 14 after embryo-transfer, a positive hCG-Level was detected and after an uncomplicated pregnancy a healthy child was delivered. ConclusionsWe report the first pregnancy and live birth achieved using transplantation of thawed and refrozen ovarian tissue in a woman treated by chemotherapy and subsequent endometriosis surgery. Refreezing of cryopreserved ovarian tissue is not a hindrance to successful transplantation of ovarian tissue. Against the background of increasing numbers of candidates for transplantation of ovarian tissue is expected that the combination chemotherapy followed by endometriosis will increase.

Offspring from oocytes grown in frozen-thawed ovarian tissues transplanted to male and female bodies

Abstract Objective: There are few reports of live births from heterotopic transplantation of frozen-thawed ovarian tissue. The purpose of this study is to assess the follicular development in the frozen-thawed ovarian tissues following heterotopic transplantation in both female and male bodies. Methods: Cluster of differentiation 1 (CD1) mice (6-8 weeks) were used in this study as ovarian tissue donors and foster mothers for embryo transfer. Sperm from CD1 male mice were used for insemination by intracytoplasmic sperm injection (ICSI). Nude severe combined immunodeficiency mice (8 weeks) were employed as recipients of ovarian tissue transplantation. The frozen-thawed ovarian tissues were transplanted to 4 sites on each mouse, female and male, subcutaneously. After 3 months, both female and male mice were injected with 5.0 IU gonadotropins intraperitoneally. Post 48 hours of injection, the mouse was killed for ovarian transplant collection. Only fully grown oocytes with contacted cumulus cells (cumulus-oocyte complexes) were then selected for maturation in vitro. In vitro matured oocytes were inseminated with fresh sperm by ICSI, and the developed blastocysts were frozen using the vitrification method and stored until embryo transfer. After thawing, the thawed blastocysts were incubated for at least 2 hours before the transfer. The foster mice mothers mated with vasectomised male 3 days previously. Live birth was monitored at 19 days after transfer, and the resulted offspring was raised for fertility test. Results: The relatively high recovery rates of the transplanted ovarian tissues were collected in both frozen-thawed and fresh ovarian tissue transplants from both female and male bodies. The fully grown immature oocytes became mature in vitro and the fertilized zygotes developed to blastocyst stage. There are no differences between frozen-thawed and fresh ovarian transplants in term of oocyte quality and embryo development to blastocyst rates. Nineteen-day post-transfer, 3 foster mothers from the frozen-thawed ovarian tissue transplant group delivered 13 pups and the 4 foster mothers of the fresh ovarian tissue transplant group delivered 12 live pups. The produced offspring were normal in appearance and grew healthy and fertile. Conclusions: Our results attest that the follicles can survive and develop in the frozen-thawed ovarian tissues following the subcutaneous transplant to adult male mouse's body regardless of basal endocrinal environment. Those fully grown oocytes can produce healthy and fertile offspring which will provide the possibility for further mechanistic understanding of endocrinology of folliculogenesis.

O-179 Safety of ovarian tissue cryopreservation and transplantation in patients with central nervous system cancers

Abstract Study question Is cryopreserved ovarian tissue transplantation safe in patients with central nervous system (CNS) tumors? Summary answer Cancer cell contamination was not detected in any ovarian samples from patients with CNS tumors by histological analysis, immunohistochemistry, molecular biology or long-term xenotransplantation. What is known already Frequently encountered CNS cancers in childhood include astrocytoma, medulloblastoma, ependymoma, glioblastoma and germinoma. CNS tumors have the capacity for extraneural metastases in 0.5-18% of cases. There are two publications reporting metastases to patients’ ovaries from medulloblastoma. Study design, size, duration Prospective experimental study conducted in an academic gynecology research laboratory using frozen-thawed ovarian tissue from 20 patients suffering from 6 types of CNS tumors, including the most common forms mentioned above and primitive neuroectodermal tumors (PNET). Five-month xenotransplantation was performed to severe combined immunodeficient (SCID) mice. Participants/materials, setting, methods Cryopreserved ovarian tissue from 20 patients with CNS cancers was thawed and analyzed for minimal disseminated disease and long-term xenografting to immunodeficient mice. The presence of malignant cells was assessed in both cryopreserved and xenografted ovarian tissue using histological analysis, immunohistochemistry for disease-­specific markers (neuron-specific enolase [NSE] and glial fibrillary acidic protein [GFAP]) and reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) for quantification of GFAP gene amplification. Main results and the role of chance No malignant cells were detected in frozen-thawed ovarian tissue from any of the patients by histology, immunolabeling for NSE and GFAP, RT-ddPCR for detection of GFAP gene amplification or xenotransplantation to SCID mice. One patient successfully underwent frozen-thawed ovarian tissue transplantation, resulting in the birth of 3 healthy children, but suffered a recurrence of her PNET 6 years after reimplantation and sadly died. Scrupulous analysis of her remaining frozen tissue showed no infiltration by malignant cells, neither after thawing nor long-term xenotransplantation. No relationship was ever established between the patient’s relapsed cancer and reintroduction of her cryopreserved ovarian tissue. The risk of reseeding cancer cells when transplanting ovarian tissue in patients with CNS cancers can therefore be considered low. Limitations, reasons for caution The risk of ovarian metastases cannot be completely ruled out for any type of tumor because we cannot analyze the actual fragments that will be reimplanted. Wider implications of the findings Our results indicate that the risk of disseminated disease in ovarian tissue from CNS patients is minimal. This is useful information for doctors when counseling women looking to undergo ovarian tissue transplantation. Trial registration number Not applicable

O-178 Reproductive and endocrine outcomes after fresh and frozen-thawed ovarian tissue transplantation based on age and anti-cancer therapy: A systematic review and individual patient data meta-analysis

Abstract Study question Do reproductive and endocrine outcomes from fresh and frozen-thawed ovarian transplants differ based on age and anti-cancer therapy before cryopreservation? Summary answer There was a significant difference in reproductive outcomes of women who have their tissue cryopreserved before or at the age of 35 years. What is known already Ovarian tissue cryopreservation (OTC) and transplantation is emerging as a new fertility preservation method. Despite being available for two decades, there is a marked variation in the delivery of this procedure worldwide. Most of the data are based on case reports from specialised centres with expertise in providing this procedure, but there are many unreported cases. Through this review, we aim to collate reproductive and endocrine outcomes from ovarian tissue transplantation. In particular the outcomes in women based on age at cryopreservation and whether they had anti-cancer therapy before cryopreservation were explored. Study design, size, duration This study was a systematic review and individual participant level meta-analysis to synthesize the existing evidence on the use of fresh and cryopreserved ovarian tissue transplantation. The review protocol was registered with PROSPERO (CRD42018115233) in November 2018 and the review was concluded in December 2020, including 87 studies (768 women). Participants/materials, setting, methods Literature search was performed using MEDLINE, EMBASE, CINAHL and Cochrane Central Register of Controlled Trials from inception to October 2020. After screening 20,566 abstracts, 87 studies (768 women) were included in the review. Patient-level data was extracted for 388 women and study-level data for 380 women. Authors were also contacted for data if relevant outcomes were not reported in published manuscripts. Meta-analysis was performed using inverse-variance weighting to calculate summary estimates using a fixed-effects model. Main results and the role of chance Age at cryopreservation was provided for 319 out of 388 (82%) women at participant level data. Of these, 283 (88.7%) had ovarian tissue retrieved at ≤ 35 years of age. A subgroup of four studies that reported data on participants age at cryopreservation and transplantation were included in meta-analysis. Pregnancy rates were higher in participants at ≤ 35 years of age at cryopreservation, with results being statistically significant (OR, 0.35; 95% CI: 0.13 to 0.92; z = 2.13; P 0.03, I2= 0%). Return of hormonal function shown as a decrease in FSH (IU/L) was also lower in this group (MD, 4.38; 95% CI: -4.29 to 13.05; z = 0.99; P 0.32, I2= 0%. Whether a participant had received chemotherapy before cryopreservation was explicitly reported in 122 out of 388 (31%) participants and 56 of them (46%) had received anti-cancer treatment before OTC. Thirty-five pregnancies and twenty-four live births were reported in these women. A further meta-analysis from 5 studies showed that although the results were not statistically significant for return of endocrine function, a decrease in FSH, an increase in oestrogen and increased pregnancy rates were noted in participants who did not receive anti-cancer therapy before cryopreservation. Limitations, reasons for caution Although we gathered 768 cases of ovarian transplants published in the literature, most were case reports and therefore not included in the meta-analysis. Of the studies included in the meta-analysis, information such as age and anti-cancer therapy were not always provided for individual participants but as an aggregate. Wider implications of the findings There was no difference in reproductive and endocrine outcomes for anti-cancer therapy before OTC. Previous chemotherapy alone should therefore not be a deterrent in offering young girls and women OTC. Furthermore, the ideal age to achieve higher pregnancy and live birth rates from OTC is less than 35 years. Trial registration number PROSPERO (CRD42018115233)

Cryopreservation Options to Preserve Fertility in Female Cancer Patients: Available Clinical Practice and Investigational Strategies from the Oncology Guidelines Point of View

In recent years, advances in cancer treatment have improved the survival rate of cancer patients significantly. However, destructive damage to ovaries due to the therapies or cancer itself can cause different degrees of infertility in women of reproductive age that can affect their quality of life seriously. In this study, fertility cryopreservation options for female cancer patients in oncology guidelines were reviewed. Cryopreservation methods have a long history in reproductive biology and oncology. However, embryo and oocyte cryopreservation were the eligible restoration strategies in clinical oncology practice. Ovarian tissue cryopreservation (OTC) is the latest option recommended for fertility preservation in pre-pubertal and adult patients who cannot delay their treatment or in whom taking IVF hormones may have adverse effects on their cancer. Reports show that frozen-thawed ovarian tissue transplantation has led to more than 130 live births so far in patients, most of whom were cancer patients. Although OTC is indeed generally recognized as an investigational method, it is recommended in some important guidelines, such as ASCO 2018. Therefore, based on many clinical pieces of evidence , it is predicted that the investigational label will soon be removed, and OTC might be considered as one of the main fertility preservation options for female cancer patients in clinical oncology practice.

Transplantation of frozen-thawed ovarian tissue: an update on worldwide activity published in peer-reviewed papers and on the Danish cohort.

The purpose of the study is to review all peer-reviewed published reports of women receiving ovarian tissue transplantation (OTT) with frozen/thawed tissue (OTC) with respect to age, diagnosis, transplantation site, fertility outcome, and potential side effects, including data from all women in the Danish program. A systematic review of the literature was performed in PubMed combined with results from all patients who had received OTT in Denmark up to December 2017. OTT has been reported from 21 different countries comprising a total of 360 OTT procedures in 318 women. In nine women, malignancy was diagnosed after OTT; none were considered to be directly caused by the OTT. Despite a potential under reporting of cancer recurrence, there is currently no evidence to suggest that OTT causes reseeding of the original cancer. Renewed ovarian endocrine function was reported in 95% of the women. Half of all children born following OTT resulted from natural conception, and newborns were reported to be healthy except for one neonate with a chromosome anomaly with a family disposition. Women who conceived after OTT were significantly younger than those who failed. This study found no indications of sufficient numbers of malignant cells present in the ovarian tissue to cause recurrence of cancer after OTT. Further, it is unlikely that OTC affects the well-being of children born. OTC is now an established method of fertility preservation in Denmark with public reimbursement. The current data encourage that women who require gonadotoxic treatment should be offered an individual evaluation considering fertility preservation.

Robot-assisted Laparoscopic Transplantation of Frozen-thawed Ovarian Tissue

Study ObjectiveTo show a new approach for orthotopic human ovarian tissue transplantation via robot-assisted laparoscopic surgery. DesignA step-by-step video explanation of the surgical technique (Canadian Task Force classification III). SettingAcademic medical center. InterventionsThe robot-assisted transplantation approach consisted of 3 steps: (1) reconstruction of the ovarian tissue graft, (2) preparation of the contralateral menopausal ovary as the recipient site, and 3) transplantation of the reconstructed graft to the bivalved contralateral ovary. Institutional review board approval was obtained. Measurements and Main ResultsAlthough still experimental, cryopreservation and subsequent transplantation of frozen-thawed ovarian tissue are currently the only available methods for prepubertal girls and young women with cancer who are not eligible for established fertility preservation options such as oocyte or embryo cryopreservation [1]. We performed the first reported autologous ovarian transplantation with a conventional laparoscopic technique [2]. To date, over 60 babies have been born after the orthotopic transplantation of cryopreserved ovarian tissue, and this number is growing [3,4]. Until recently, all of these children were born from ovarian transplants that were performed via laparotomy or conventional laparoscopy [5]. We have recently developed a robot-assisted ovarian transplantation procedure that uses an extracellular matrix scaffold to facilitate ovarian reconstruction, handling, and revascularization. Both of the procedures resulted in robust ovarian function and births [6]. The purpose of this video reports the surgical technique in detail, which uses the da Vinci Xi (Intuitive Surgical Inc, Sunnyvale, CA) robotic system for transplantation, and a decellularized human extracellular tissue matrix (Alloderm; LifeCell Corp, Branchburg, NJ) for graft reconstruction. ConclusionRobotic ovarian transplantation may have several advantages, which include precision, more delicate graft handling, and reduced time from tissue thawing to transplantation. The collective usefulness of the extracellular tissue matrix may enhance this technique by enabling a niche for ovarian reconstruction and potentially enhanced revascularization. The feasibility and comparative advantages of this technique are currently being studied in ongoing trials.

Alginate: A Versatile Biomaterial to Encapsulate Isolated Ovarian Follicles.

In vitro culture of ovarian follicles isolated or enclosed in ovarian tissue fragments and grafting of isolated ovarian follicles represent a potential alternative to restore fertility in cancer patients who cannot undergo cryopreservation of embryos or oocytes or transplantation of frozen-thawed ovarian tissue. In this regard, respecting the three-dimensional (3D) architecture of isolated follicles is crucial to maintaining their proper follicular physiology. To this end, alginate hydrogel has been widely investigated using follicles from numerous animal species, yielding promising results. The goal of this review is therefore to provide an overview of alginate applications utilizing the biomaterial as a scaffold for 3D encapsulation of isolated ovarian follicles. Different methods of isolated follicle encapsulation in alginate are discussed in this review, as its use of 3D alginate culture systems as a tool for in vitro follicle analysis. Possible improvements of this matrix, namely modification with arginine-glycine-aspartic acid peptide or combination with fibrin, are also summarized. Encouraging results have been obtained in different animal models, and particularly with isolated follicles encapsulated in alginate matrices and grafted to mice. This summary is designed to guide the reader towards development of next-generation alginate scaffolds, with enhanced properties for follicle encapsulation.

Second children born after autotransplantation of cryopreserved ovarian tissue in a young patient previously treated with chemotherapy for Askin's diseasethe successful of fertility preservation program

Oncological treatment was associated with a reduced ovarian reserve, the chance of a successful pregnancy is currently around 1/3 for those with ovarian-tissue autotransplantationOvarian tissue cryopreservation is an effective option,gaining ground as a valid method for fertility preservation especially for pre-pubertal patients and patients who have a short time between diagnosis of a disease and gonadotoxic treatment.More than 62 children worldwide have now been born following this procedure. Case report and a retrospectively analyse, of patients whose underwent ovarian tissue cryopreservation at IFER, Argentina between 1999-2015.One of these women, had orthotopic transplantation, after chemotherapy premature ovarian failure (POF) has now conceived again following natural conception. She gave birth to a healthy girl on 7/2015 and is therefore the second woman in the world to have had two children, from separate pregnancies, born as a result of transplanting frozen/thawed ovarian-tissue. 68 patients referred from private oncological units underwent cryopreservation of ovarian tissue before gonadotoxic therapy for malignant disease. 7 patients (10,3%) died during follow-up due to recurrence of disease. 24 post-pubertal patients without the need for grafts of cryopreserved tissue. 4 patients had undergone ovarian tissue transplantation in order to restore their fertility after remission of the disease and one of these women became pregnant twice and has given birth two healthy babies. In 2005 a 28 year old patient was referred for fertility preservation before undergoing aggressive chemotherapy for Askin tumor. Several strips of ovarian cortical tissue were obtained by laparoscopy and cryopreserved . The patient underwent surgical treatment, and chemotherapy (CVEA), ovarian failure was established. In 2009 orthotopic transplantation of frozen-thawed ovarian tissue was carried out by laparoscopy in the right ovary,.Three months after transplantations she resumed regular menses. Four IVF cycles were performed with no pregnancy.9/2012 POF was established. In 2/2013 another laparoscopy was performed with replacement of ovarian tissue this time in the left ovary. In 5/2013 spontaneous menses began. An Intrauterine Insemination resulted in pregnancy. In 2/2014, and after an uneventful pregnancy, a healthy baby boy was born.The patient was breastfeeding her first baby and returned to our fertility clinic in 9/2014 a pregnancy test revealed that she had already conceived naturally,after an uneventful pregnancy a normal healthy girl was born in 7/2015. The present result supports cryopreservation of ovarian tissue as a valid method of fertility preservation to be used in women facing gonadotoxic treatment. However alternative procedures such as oocyte or embryo cryopreservation should be considered as first options especially for older patients.

Transplantations of frozen-thawed ovarian tissue demonstrate high reproductive performance and the need to revise restrictive criteria

To report the single-center results of orthotopic retransplantations of cryopreserved ovarian tissue in cancer survivors and evaluate the validity of commonly accepted procedure limitations. Prospective cohort study. Tertiary university-affiliated assisted reproduction technology (ART) and oncology centers. Twenty cancer survivors who underwent ovarian transplantation of frozen-thawed ovarian tissue with the aim to conceive. Ovarian tissue cryopreservation (OTCP) and transplantation, endocrine monitoring, invitro fertilization (IVF). Endocrine profile, IVF, pregnancies, live births. The patient ages at tissue harvesting ranged from 14 to 39years. Fifteen women had hematologic malignancies, and two had leukemia (chronic myelogenous leukemia and acute myelogenous leukemia). Ten patients were exposed to nonsterilizing chemotherapy before OTCP. After transplantation, the endocrine recovery rate was 93%. Fourteen patients underwent IVF treatments with a fertilization rate of 58%. Sixteen pregnancies were achieved (10 after IVF, 6 spontaneous), resulting in 10 live births, two (twins) after harvesting from the mother at the age of 37. Two pregnancies are currently ongoing. After transplantation, 53% of patients conceived, and 32% delivered at least once. One patient conceived four times. Preharversting chemotherapy exposure was not associated with inferior outcomes. All patients, including two leukemia survivors, remained cancer free. Orthotopic transplantation of thawed ovarian tissue is a highly effective measure to restore fertility in sterilized cancer patients. Chemotherapy exposure before harvesting and age >35 is a realistic option in selected patients. Retransplantation in leukemic patients is possible after application of maximal safety measures. These results have led the national ethical and professional authorities to decide for the first time not to consider OTCP as an experimental modality for fertility preservation. NCT02659592.

Transplantation of ovarian tissue

Since the first live birth after orthotopic transplantation of frozen-thawed ovarian tissue, >40 babies have been born. It is time to consider fertility preservation in women as one of the foremost challenges of the next decade and to offer women facing the risk of induced or iatrogenic premature menopause the best chances of becoming mothers. Heterotopic transplantation has also been attempted, with consistent restoration of endocrine function; nonetheless, its clinical value remains questionable as it may not provide an optimal environment for follicular development, possibly because of differences in temperature, pressure, paracrine factors and blood supply. Finally, orthotopic allo-transplantation of fresh human ovarian tissue has been successfully attempted between monozygotic twins and also between genetically different sisters. The next step in this field will be the development of an artificial ovary, using, as a support, a biodegradable scaffold made of an alginate matrigel matrix onto which isolated preantral follicles and ovarian cells can be grafted.

Ovarian tissue cryopreservation in young cancer patients for fertility preservation.

Several options are currently available to preserve fertility and give female cancer survivors a chance to have children at a later date, including the cryopreservation of embryos, oocytes, and ovarian tissue. Selection of the most suitable strategy to preserve fertility depends on the type and timing of anticancer therapy, the cancer, the patient's age, and the presence of the patient's partner. Several studies have shown that the ovarian tissue can be successfully frozen and later grafted in the human womb. To date, approximately 30 live births have been achieved after the transplantation of frozen-thawed ovarian tissue. At present, the standard procedure for cryopreservation of ovarian tissue is the slow-cooling method. The slow-cooling method uses an optimal cooling rate for the target cells, and relies on extracellular ice crystals to gradually dehydrate and equilibrate the tissue. Several groups reported that slow cooling is more efficient than vitrification for the cryopreservation of human ovarian tissue. However, vitrification can be performed under a variety of conditions, and therefore, the choice of methods is important. In addition, vitrification traps aqueous solutions in an amorphous, "vitreous" solid phase that prevents ice crystal formation in tissues. Vitrification methods that were developed using mice and monkey have recently been shown to improve the viability of vitrified ovarian tissues. In this review article, recent topics of ovarian tissue cryopreservation are described.

Live birth after transplantation of frozen-thawed ovarian tissue after bilateral oophorectomy for benign disease

To report the restoration of ovarian function and pregnancy in a woman after bilateral oophorectomy for benign disease after autotransplantation of cryopreserved ovarian cortex. Case report. Gynecology research unit in a university hospital. A 28-year-old woman who underwent bilateral adnexectomy for ovarian abscesses at the age of 18 years. We performed ovarian cortex autotransplantation to a peritoneal pocket in the broad ligament. Restoration of ovarian activity and pregnancy. Restoration of ovarian function began at 20 weeks and was achieved 24 weeks after transplantation. After the fifth stimulation attempt, two mature oocytes were obtained and microinjected. One embryo (seven cells) was obtained and transferred, leading to a normal pregnancy. The patient delivered a healthy baby boy weighing 2,370 g at 38 weeks of gestation. Ovarian cortex cryopreservation can be performed at the time of surgery for benign diseases when fertility is impaired. We report the first pregnancy to occur after ovarian tissue cryopreservation for benign ovarian pathology after bilateral oophorectomy.

Complete follicular development and recovery of ovarian function of frozen-thawed, autotransplanted caprine ovarian cortex

Frozen-thawed ovarian cortical fragments (1 mm(3)) were autotransplanted to the uterus of completely ovariectomized goats. The grafts developed preovulatory follicles, accompanied by estrous behavior and a rise in plasma E(2) levels, demonstrating successful cryopreservation and transplantation.

Developmental potential of oocytes fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) after cryopreservation and mesometrial autotransplantation of rabbit ovarian tissue

Objective: To evaluate mesometrial transplantation of frozen-thawed ovarian tissue in rabbit and to choose the optimized fertilization method for oocytes retrieved from grafts by investigating the capability of oocyte fertilization and further development. Forty rabbits were divided into three groups randomly: control group, fresh tissues transplantation group and frozen-thawed tissues transplantation group. Three months after the transplantation, rabbits were stimulated with FSH and oocytes were retrieved 13 h after human chorionic gonadotropin (HCG) injection. Oocytes matured in vivo or in vitro were then fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), followed by observation and evaluation of fertilization rate and blastocyst formation rate. Blastocytes embryos were transferred to pseudopregnancy rabbits to observe pregnancy rate and birth rate. There were no significant differences in the percentage of oocytes matured either in vivo or in vitro among the three groups. The fertilization rate, cleavage rate and blastocyst formation rate of in vivo-matured oocytes had no difference among the three groups, whether they were fertilized by IVF or ICSI. Significantly higher fertilization rates of in vitro-matured oocytes were observed with ICSI compared with IVF in each group. The blastocyst formation rate of in vitro-matured oocytes was significantly lower than that of in vivo-matured oocytes in each group. The birth rate of in vivo-matured oocytes was significantly higher than that of in vitro-matured oocytes, although the pregnancy rate was similar between them. Mesometrial transplantation of frozen-thawed ovarian tissue may provide favorable conditions for follicle development. Oocytes retrieved from mesometrial grafts can develop to the blastocyst stage and produce live offspring. ICSI can optimize the fertilization rate of in vitro-matured oocytes retrieved from grafts.

Embryo Development After Heterotopic Transplantation of Cryopreserved Ovarian Tissue

Patients with cancer might want to avoid infertility and premature menopause as sequelae of chemotherapy and other treatments. Most patients with cancer do not have time for ovarian stimulation as a prelude to in vitro fertilization (IVF). An alternative approach, which could also be useful for single women who do not want to use donor sperm and for children, is to cryopreserve oocytes, but this also requires several weeks of ovarian stimulation. Ovarian function has been preserved experimentally after orthotopic transplantation of frozen-thawed ovarian tissue and heterotopic grafting of fresh human ovarian cortical pieces. This study intended to restore fertility by subcutaneously transplanting frozen, banked ovarian tissue. One ovary was removed from a woman aged 30 years with breast cancer before starting gonadotoxic chemotherapy. Cortical pieces were cryopreserved using a slow freezing technique and dimethylsulfoxide as a cryoprotectant. The tissue was transplanted beneath the abdominal skin 6 years later, after the patient had entered menopause. There were an estimated 11,000 follicles that could potentially last for 12 months. All 15 ovarian cortical pieces were transplanted in the lower abdominal wall. Tissue was suppressed with a combination of follicle-stimulating hormone and human menopausal gonadotropin before every cycle of ovarian stimulation. Oocyte maturity was induced by recombinant human chorionic gonadotropin. Oocytes were retrieved 36 to 40 hours later and intracytoplasmic sperm injection was carried out. A lump was felt at the transplant site 85 days after transplantation, and ovarian endocrine function resumed (Fig. 1). Eight percutaneous oocyte retrievals were carried out over 8 months, 6 of them after ovarian stimulation. Eight of 20 oocytes taken from transplanted tissue were suitable for IVF. One of them fertilized normally and developed into a high-grade 4-cell-stage embryo 24 hours later that was transferred to the patient's uterus. This experience indicates that endocrine function and fertility could be restored by long-term cryopreservation of ovarian tissue.