Ovarian Tissue Transplantation Research Articles

028 Successful cryopreservation of avian germplasm: Why a multifaceted approach is required

The value of the ability to cryopreserve and store germplasm has long been recognized for indefinite preservation of genetic material, especially for “at-risk” populations. For domestic livestock species, options for preserving genetic material in frozen form range from sperm to oocytes to embryos. More than sixty years ago, the discovery of glycerol’s cryoprotective properties for rooster sperm pioneered the success of modern cryobiology and led to the development of semen cryopreservation for a wide range of species. Despite decades of research, the overall fertility rates of frozen/thawed poultry sperm are highly variable and not reliable enough for preservation of genetic stocks. The relatively low fertilizing ability most likely results from physiological sensitivity to the cryogenic process coupled with the requirement for prolonged sperm functionality in the hen reproductive tract. We have conducted systematic experiments to understand how and why poultry sperm lose functional competence after cryopreservation, and have shown that the glycocalyx, the carbohydrate-rich zone on the sperm surface, is altered by the cryogenic cycle. The amount of sialic acid, a terminal sugar critical for sperm transport in the female reproductive tract, was reduced after cryopreservation; however, we have shown that sperm cells can incorporate exogenous sialic acid, suggesting a strategy for sperm freezing protocols. Second, sperm lipids such as phosphotidylcholine also are altered during cold storage. The ratio of lipids in the sperm membrane determine the overall fluidity of the membrane and impact the ability of sperm to remain viable during the wide temperature fluctuations that are part of the cryogenic cycle. We have shown that supplemental lipids can circumvent the loss of phospholipids during cryopreservation. Finally, we have conducted the first proteomic analysis of poultry sperm and have identified three proteins not previously found in sperm from any species to date (dihydropyrimidinase, mitofilin and mitochondrial tri-functional protein). We currently are evaluating the proteomic profile of frozen/thawed sperm to determine the impact of cryopreservation on sperm proteins. Taken together, these data suggests innovative strategies for developing reliable poultry semen cryopreservation methods. For most species, sperm cryopreservation effectively captures the entire genome; however, in birds, the female is the heterogametic sex. Cryopreservation of avian oocytes and embryos is problematic because of the macrolecithal characteristics of the egg, where the surface-to-volume ratio poses a significant barrier to the movement of solutes/cryoprotectants and impedes effective protection of the cells from freeze/thaw damage. Recent advances for manipulation of poultry primordial germ cells and culture of embryonic stem cells suggests that cryopreservation of these cell types, followed by re-implantation in recipient embryos, could provide a mechanism to preserve the entire genome; albeit the efficiency rates of reconstitution associated with the current technologies are rather low. A novel innovation of cryopreservation and transplantation of ovarian tissue offers an alternative for whole genome preservation in birds (Song and Silversides, 2008); although chemical treatment to reduce the growth of host germ cells does not completely prevent the production of host-derived offspring. Reconstitution of poultry lines will most likely rely on more than one type of cryopreserved material. Source of funding: USDA ARS Project Number: 1245-31000-105-00. Conflict of interest: None declared. [email protected]

A model for cryobanking female germplasm in Japanese quail (Coturnix japonica)

Cryopreservation and transplantation of ovarian tissue can be used for cryobanking female germplasm of avian species. Here we report a model of cryobanking ovarian tissue of Japanese quail. Ovaries of 1-wk-old quail chicks from the recessive white-breasted (WB) line were cryopreserved using a vitrification method. The cryopreserved tissue was warmed and transplanted orthotopically into ovariectomized, 1-wk-old female quail chicks from the homozygous wild-type QO line. At sexual maturation, recipients were mated with WB males and the plumage coloration was used as a marker to determine the origin of their ovaries. Seven of the 15 recipients laid eggs and produced donor-derived offspring, and 5 produced 100% donor-derived offspring. No difference was seen in egg production between recipients and untreated birds. The high efficiency of the vitrification and transplantation procedures in preserving and recovering quail ovarian tissue shows that this model can be used for maintaining commercial and experimental quail strains and may benefit other poultry species and endangered avian species.

First reported clinical pregnancy following heterotopic grafting of cryopreserved ovarian tissue in a woman after a bilateral oophorectomy

Ovarian tissue cryopreservation and transplantation is a form of fertility preservation offered to young women at high risk of losing ovarian function after cancer treatment. While there have been successful births resulting from orthotopic site grafts, we report the first case of an ongoing pregnancy from a heterotopic graft in a patient who had previously undergone bilateral oopherectomy for a granulosa cell tumour. Frozen-thawed ovarian tissue was transplanted to the anterior abdominal wall. Subsequent ovarian stimulation and transabdominal ultrasound-guided oocyte retrieval from the grafts resulted in two oocytes. These were fertilized with ICSI and two embryos were transferred. Serial ultrasounds have confirmed an ongoing 26-week intrauterine twin pregnancy. Thus, this first demonstration of a pregnancy from a heterotopic graft site provides unequivocal evidence that cryopreservation preserves complete follicle development and that normal ovarian function can occur at a non-ovarian site. This provides optimism for further efforts to assist women who have had oophorectomy and pelvic surgery or radiotherapy, without an appropriate orthotopic site for grafting.

Is transplantation of cryopreserved ovarian tissue from patients with advanced-stage breast cancer safe? A pilot study

To assess the safety of reimplantation of cryopreserved ovarian tissue from advanced-stage breast cancer patients. Cryopreserved ovarian cortical fragments were obtained from 13 advanced-stage breast cancer patients aged 17-35years. After thawing, part of the ovarian cortical tissue was grafted to severe combined immunodeficient mice for 6months. The presence of malignant mammary cells in ovarian tissue was evaluated after thawing as well as after grafting by 1) histology and immunohistochemistry (epithelial membrane antigen, Her2/neu and gross cystic disease fluid protein 15 identification), and 2) detection of the MGB2 gene by qPCR. No malignant cells were evidenced by histology and immunohistochemistry. None of the mice died during the 6-month grafting period, nor developed macroscopically visible masses. MGB2 gene expression was detected by qPCR and confirmed by sequencing in frozen-thawed ovarian tissue in 4 cases and in grafts in 1 case. This pilot study is the first to evaluate the risk of contamination of cryopreserved ovarian tissue from advanced-stage breast cancer patients by xenotransplantation for 6months to immunodeficient mice, associated with more conventional screening methods. Our xenografting results are reassuring, but caution needs to be exercised, as MGB2 gene expression was detected in some cases. Larger numbers of ovarian tissue samples from patients with advanced-stage breast cancer are required to confirm our findings before ovarian tissue transplantation can be contemplated in these patients.

Does vascular endothelial growth factor (VEGF) and inhibition of apoptosis improve neovascularization in nonhuman primate, ovarian heterotopic transplants?

While ovarian tissue banking with subsequent transplantation is an effective strategy for preserving fertility, the process is relatively inefficient. We hypothesize that VEGF and inhibition of apoptosis with FTY720 may improve neovascularization and survival of ovarian tissue transplants in the rhesus macaque, and that the abdomen may be a better site for heterotopic transplantation than the arm.

Ovarian Tissue Transplantation: Current State and Research Advancement

During the past decade, babies born from cryopreservation and transplantation of ovarian tissue have emerged as a promising alternative to restore ovarian function and fertility in cancer patients. Despite successful results obtained with conventional freezing protocols to preserve ovarian tissue, attempt for whole ovary transplantation is still under-propelling in large animal and human studies. Patient selection to avoid reimplanting cancer cells in ovarian grafts as a prerequisite safety concern is as important as advanced transplantation technique. Multidisciplinary strategies for the combined isolation immature follicle for culture or in vitro activation of ovarian follicle with the frozen-thawed ovarian tissue raised the diversity for female fertility preservation along with the egg-embryo freezing or in vitro maturation of immature eggs. The purpose of this updated review of fertility preservation techniques is to develop a network among health care professional interested in this challenging field for those who are threatened the reproductive function.

Improvement in the quality of heterotopic allotransplanted mouse ovarian tissues with basic fibroblast growth factor and fibrin hydrogel

Does basic fibroblast growth factor (bFGF) in combination with fibrin hydrogel improve follicle development and revascularization of heterotopically transplanted mouse ovarian tissues? Treatment of transplanted ovarian tissues with higher concentrations (75, 100 and 150 μg/ml), but not lower concentrations (25 and 50 μg/ml), of bFGF significantly improved primordial follicle survival and angiogenesis, while apoptosis of follicles and stromal cells was significantly decreased. Use of transplanted ovarian tissues in female fertility preservation is limited by the massive loss of follicles and ischemia-reperfusion injury due to the expected delay in revascularization. Ovarian tissues from 18-day-old ICR mice were encapsulated in fibrin hydrogel mixed with different concentrations of bFGF, then transplanted under the skin of adult female mice for 1 week. The ovarian tissues treated without fibrin hydrogels and bFGF were designated as Control group I, and the ovarian tissues treated with fibrin hydrogels but without bFGF were designated as Control group II. The ovarian tissues treated with 25 and 50 μg/ml bFGF were designated as the lower concentration group, and the ovarian tissues treated with 75, 100 and 150 μg/ml bFGF were designated as the higher concentration group. Assessment of follicular quantity and follicle classification was carried out by histologic analysis. Follicle proliferation was evidenced by immunostaining with proliferating cell nuclear antigen and apoptosis was verified by anti-active caspase-3 staining. Epithelial cells of new blood vessels were stained using CD31 antibody to evaluate neoangiogenesis, and the blood vessel density was analyzed by immunohistochemistry. The ovarian tissues were recovered 1 week post-transplantation. Compared with the control group, the survival and proliferation of the follicles was significantly increased, the apoptosis of follicles and stromal cells was significantly decreased, and angiogenesis was significantly enhanced when the transplanted ovarian tissues were treated with a higher concentration of bFGF. Treatment with a lower concentration of bFGF did not improve follicle survival and blood revascularization. The results obtained may not be fully extrapolated to humans because of the physiologic differences between mice and humans. For the first time, the present study investigated the role of bFGF in transplanted ovarian tissues and demonstrated that bFGF might significantly improve the quality of transplanted ovarian tissues by increasing follicle quantity and promoting neoangiogenesis. This study sets the stage for further study and application of ovarian tissue transplantation in clinics, and may eventually benefit females for fertility preservation. This work was partially supported by the Ministry of Science and Technology of China Grants (973 Program; 2011CB944503 to Q.J.), the Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (30825038 to Q.J.), and the National Natural Science Funds for Young Scholar (81200470 to Y.J. and 81000275 to Y.L.Y.). None of the authors have any conflicts of interest.

Restauration de l’infertilité après congélation de tissu ovarien, données expérimentales

In recent decades, reproductive surgery and medicine have entered a new era of prevention. Oocyte freezing is at the heart of this new era, along with the preservation offemale fertility. Several options are being evaluated, and time will tell which one will prove most suitable for routine use. Currently, only the freezing and transplantation of ovarian tissue seems to have entered clinical practice. Animal studies demonstrated its effectiveness, with pregnancies and births in several species. These studies showed that both slow and rapid freezing (vitrification) allows the survival of more than 80 % of primordial follicles after thawing. Similar results have since been obtained in humans. Freezing allows satisfactory follicular survival Reimplantation of frozen ovarian tissue has helped to restore menstrual cycling and, more importantly, to obtain pregnancies and births, either spontaneously or by IVF This restoration of fertility offers great hopefor female patients having to confront not only their disease but also the prospect of permanent infertility. Related research has led to major advances in biology and reproductive medicine.

Динаміка морфогенезу і функціонального розвитку алотрансплантата оваріальної тканини щурів

We examined the dynamics of folliculogenesis in ovarian tissue allograft and its endocrine function in an organism of castrated recipient rats during 100 days. Growth and development of ovarian tissue and its steroidogenic function in heterotopic transplantation were determined by its initial morphological organization. Allotransplantation of mature ovarian tissue was found to cause preservation of its morphological structure, whereas the transplantation of neonatal ovarian tissue caused formation of follicular cysts. At 100th day of observation, we observed a recovery of the levels of sex hormones to the physiological norm after transplantation of both mature ovarian tissue and neonatal tissue of 10-days of postnatal development.

FSH prevents depletion of the resting follicle pool by promoting follicular number and morphology in fresh and cryopreserved primate ovarian tissues following xenografting

BackgroundCryopreservation and transplantation of ovarian tissue is one option for re-establishing ovarian function, but optimal conditions for graft sustainment and follicular survival are still considered experimental. The present study aims to analyze the effect of FSH treatment on the resting follicle pool in fresh and cryopreserved primate ovarian tissues following xenografting.MethodsOvarian tissues from adult marmosets were grafted freshly or following cryopreservation to ovarectomized nude mice treated with FSH 25 IU twice daily post transplantation or left untreated as controls. Grafts were retrieved 2 or 4 weeks after transplantation to evaluate the number and morphological appearance of follicles.ResultsEarly start of FSH treatment within 1 week following transplantation partly prevents primordial follicle loss in fresh and frozen-thawed tissues, whereas after a 3 weeks time interval this effect is present only in fresh tissues. A similar positive effect of early, but not later FSH treatment on primary follicles is seen in fresh tissues compared to only marginal effects in frozen-thawed tissues. The percentage of morphologically normal follicles is generally increased in FSH treated tissues, whereas the percentage of primary follicles over all primordial and primary follicles is increased by FSH only in freshly-grafted tissues.ConclusionsFSH treatment alleviates depletion of the resting follicle pool and promotes normal follicular morphology both in freshly and frozen-thawed grafted tissues. In previously cryopreserved tissues, applying to most of the tissues intended for clinical use in fertility preservation attempts, its positive effect on primordial follicle numbers and potential graft sustainment is dependent on an early start of treatment within one week of transplantation.

Long-term function of ovarian tissue transplants

The long term duration of function of ovarian cortical tissue grafts is reviewed in this paper by describing cases of restoration of fertility by transplant of ovarian cortical tissue from matching donors where the recipient was in need of the transplant due to sterilizing effects of leukemia, premature ovarian failure (POF), and to reflect that it may be possible to postpone the normal time of menopause or to alleviate its symptoms.

Fertility issues of breast cancer survivors.

In the United States, more women are diagnosed with breast cancer than any other type of cancer. More than 11,000 of these women will be younger than 40, and many of these women will want to have children in the future. A significant number of these young breast cancer patients will require treatment that can cause ovarian failure or premature menopause. Several options do exist for fertility preservation, both standard and investigational. Embryo cryopreservation is the most established intervention. Investigational interventions include oocyte cryopreservation, ovarian tissue transplantation, ovarian suppression with a gonadotropin-releasing hormone agonist, and harvesting of immature follicles with in vitro maturation and cryopreservation. Although pregnancy during cancer treatment is not recommended, pregnancies occurring after completion of therapy have not been linked to increased cancer recurrence. Young women diagnosed with breast cancer need evidence-based information presented in a timely manner in order to make decisions regarding fertility preservation prior to the initiation of treatment. The oncology advanced practitioner must be knowledgeable about fertility preservation options available to these women as well as comfortable with ethical and financial concerns that can arise. The informed practitioner can effectively counsel patients and refer to fertility specialists when appropriate.

Safety Assessment of Ovarian Cryopreservation and Transplantation in Nude Mice Bearing Human Epithelial Ovarian Cancer

Nude mice with orthotopic transplantation of human ovarian epithelial cancer were used to investigate screening criteria for paraneoplastic normal ovarian tissue and the security of the freezing and thawing for ovarian tissue transplantation. Expression of CK-7, CA125, P53, survivin, MMP-2/TIMP- 2 in paraneoplastic normal ovarian tissues were detected by RT-PCR as well as immunohistochemistry. The tissues of the groups with all negative indicators of RT-PCR, all negative indicators of immunohistochemistry, negative expression of CK-7, CA125 and survivin, positive expression of CK-7, CA125 and survivin, cancer tissues and normal ovarian tissues of nude mice were used for freezing and thawing transplantation, to analyze overt and occult carcinogenesis rates after transplantation. When all indicators or the main indicators, CK-7, CA125 and survivin, were negative, tumorigenesis did not occur after transplantation. In addition the occult carcinogenesis rate was lower than in the group with positive expression of CK-7, CA125 and survivin (P<0.01). After subcutaneous and orthotopic transplantation of ovarian tissues, rates did not change (P>0.05). There was no statistical significance among rates after transplantation of ovarian tissues which were obtained under different severity conditions (P>0.05). Negative expression of CK-7, CA125 and survivin can be treated as screening criteria for security of ovarian tissues for transplantation. Immunohistochemical methods can be used as the primary detection approach. Both subcutaneous and orthotopic transplantation are safe. The initial severity does not affect the carcinogenesis rate after tissue transplantation. Freezing and thawing ovarian tissue transplantation in nude mice with human epithelial ovarian carcinoma is feasible and safe.

Preservation of fertility

Cryopreservation of ovarian tissue is widely performed to preserve fertility in cancer patients, but its efficacy has been difficult to assess because most patients do not request transplantation of the thawed tissue for over a decade. Our objective was to evaluate the long-term results of transplantation of fresh ovarian versus frozen ovarian tissue.

Erratum to: Assessment of long term endocrine function after transplantation of frozen-thawed human ovarian tissue to the heterotopic site: 10 year longitudinal follow-up study

Erratum to: Assessment of long term endocrine function after transplantation of frozen-thawed human ovarian tissue to the heterotopic site: 10 year longitudinal follow-up study

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.

Preserving integrity, along with fertility

Preserving integrity, along with fertility

Extreme infertility

New germ cells and new organs open new areas in the approach to treating infertility.

On Ovarian Tissue Transplantation and the Metaphysics of Self-Recognition: A Response to Paul Lauritzen and Andrea Vicini

To resolve questions regarding ovarian tissue transplantation, the author proposes a metaphysical theory positing that the common ensoulment shared by all the cells in a woman's body is manifested primarily in her immune system's ability to distinguish her own cells from those of another. The author therefore suggests that human procreation, to be morally licit, has to involve the giving of one's gametes, defined, not by their genetic constitution, but by their being part of the immunologically defined self given in the mutual self-gift that is conjugal love.

Assessment of long term endocrine function after transplantation of frozen-thawed human ovarian tissue to the heterotopic site: 10 year longitudinal follow-up study

To assess the longevity of ovarian grafts in five cancer patients who underwent heterotopic autotransplantation of frozen-thawed ovarian tissue. Five cancer survivors underwent heterotopic ovarian transplantation between 2001 and 2011. Stored ovarian tissue (for 1-10 years) was rapidly thawed and transplanted into the space between the rectus sheath and the rectus muscle (8-20 cortical sections per patient). Endocrine function was assessed by monthly blood tests (FSH, LH, E2, progesterone and testosterone) and ultrasound after transplantation. The monitoring was continued until the cessation of endocrine function by consecutive blood tests (E2 < 20 pg/ml; FSH ≥ 35 IU/L). Endocrine function was restored in all patients between 12-20 weeks after transplantation. Four patients required the second transplantation one to two years after the first transplantation. The duration of endocrine function after the second transplantation was much longer (9 months-84 months). The longest duration of endocrine function was seen in a woman who underwent ovarian transplantation in 2003 and 2004 after radiotherapy for cervical cancer. Even more than seven years after transplantation, endocrine function has not ceased (FSH 9.5, E2 108, on July 1, 2011). Of note, this patient underwent three IVF cycles in 2004 which resulted in four embryos. Long-term endocrine function lasting for seven years can be established with heterotopic transplantation of cryobanked human ovarian tissue. Re-establishment of long-term endocrine function after ovarian transplantation will benefit young cancer survivors with premature ovarian failure.