Effects of cyclic adenosine monophosphate modulators on maturation and quality of vitrified-warmed germinal vesicle stage mouse oocytes.

BACKGROUND: It is still one of the unresolved issues if germinal vesicle stage (GV) oocytes can be successfully cryopreserved for fertility preservation and matured in vitro without damage after warming. Several studies have reported that the addition of cyclic adenosine monophosphate (cAMP) modulators to in vitro maturation (IVM) media improved the developmental potency of mature oocytes though vitrification itself provokes cAMP depletion. We evaluated whether the addition of cAMP modulators after GV oocytes retrieval before vitrification enhances maturation and developmental capability after warming of GV oocytes. METHODS: Retrieved GV oocytes of mice were divided into cumulus-oocyte complexes (COCs) and denuded oocytes (DOs). Then, GV oocytes were cultured with or without dibutyryl-cAMP (dbcAMP, cAMP analog) and 3-isobutyl-l-methylxanthine (phosphodiesterase inhibitor) during the pre-vitrification period for 30 min. RESULTS: One hour after warming, the ratio of oocytes that stayed in the intact GV stage was significantly higher in groups treated with cAMP modulators. After 18 h of IVM, the percentage of maturation was significantly higher in the COC group treated with dbcAMP. The expression of F-actin, which is involved in meiotic spindle migration and chromosomal translocation, is likewise increased in this group. However, there was no difference in chromosome and spindle organization integrity or developmental competence between the MII oocytes of all groups. CONCLUSIONS: Increasing the intracellular cAMP level before vitrification of the GV oocytes maintained the cell cycle arrest, and this process may facilitate oocyte maturation after IVM by preventing cryodamage and synchronizing maturation between nuclear and cytoplasmic components. The role of cumulus cells seems to be essential for this mechanism.

Allogeneic ovarian transplantation using immunomodulator preimplantation factor (PIF) as monotherapy restored ovarian function in olive baboon.

PURPOSE: Allogeneic ovarian transplantation may be an alternative in the future to oocyte donation in women with premature ovarian failure. The objectives of this study were to (a) evaluate allotransplantation feasibility for restoration of ovarian function and (b) assess efficacy of synthetic preimplantation factor (PIF) monotherapy as sole immune-acceptance regimen. METHODS: This is an experimental animal study using non-human primates (Papio anubis). Allogeneic orthotopic ovarian tissue transplantation was performed in two female olive baboons. PIF was administered as a monotherapy to prevent immune rejection and achieve transplant maintenance and function. Subjects underwent bilateral oophorectomy followed by cross-transplantation of prepared ovarian cortex. Postoperatively, subjects were monitored for clinical and biochemical signs of graft rejection and return of function. Weekly blood samples were obtained to monitor graft acceptance and endocrine function restoration. RESULTS: Postoperatively, there were no clinical signs of rejection. Laboratory parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine) did not indicate organ rejection at any stage of the experiment. Initially, significant loss of follicles was noticed after grafting and serum follicle-stimulating hormone (FSH) and E2 levels were consistent with ovarian failure. Seven months after transplantation, one animal exhibited recurrence of ovarian endocrine function (perineal swelling, E2 rise, FSH decrease, and return of menstruation). CONCLUSIONS: Organ rejection after allogeneic ovarian transplantation was prevented using PIF as monotherapy for the first time and no side effects were recorded. The study suggests the clinical feasibility of ovarian allotransplantation to obtain ovarian function.

The impact of vitrification on immature oocyte cell cycle and cytoskeletal integrity in a rat model.

PURPOSE: To test the effects of varying vitrification protocols on the cell cycle status and chromosomal integrity in cumulus-enclosed GV stage rat oocytes. METHODS: Vitrified and thawed rat oocytes were labeled with fluorescent markers for chromatin, cell cycle activation, and f-actin and analyzed by conventional and laser scanning confocal microscopy. RESULTS: In all vitrification groups, significant alterations in cumulus cell connectivity, cell cycle status, and cytoplasmic actin integrity were observed following warming compared to fresh control oocytes. Based on the protein phosphorylation marker MPM-2, it is clear that warmed oocytes rapidly enter M-phase but are unable to maintain chromosome integrity as a result of multiple chromatin fusions. A prominent reduction in f-actin is evident in both the ooplasm and at the cortex of vitrified oocytes. Finally, an irreversible but irregular retraction of TZPs occurs on the majority of oocytes subjected to any of the vitrification protocols. CONCLUSIONS: These findings draw attention to undesirable consequences of immature oocyte vitrification that compromise cell cycle status and chromatin and cytoskeleton integrity that may not be evident until after fertilization.

Revisiting the role of heterotopic ovarian transplantation: futility or fertility.

Scepticism hovers over the future of heterotopic transplantation of cryopreserved human ovarian tissue, as its clinical efficacy and practicability for fertility preservation is still debatable. Despite its limitations, the potential advantages and roles of heterotopic transplantation should not be ignored. Indeed, restoration of ovarian function after heterotopic transplantation of cryopreserved ovarian tissue has been consistently demonstrated in humans. There are many unknowns with this technology, such as the optimal heterotopic site, environmental factors and oocyte quality. Hence, it will require further investigations before making a final verdict. Until then, rather than being considered prematurely as a futile technology, heterotopic ovarian transplantation should be viewed positively, with potential roles for restoration of ovarian function and fertility.

Clinical guidelines for sperm cryopreservation in cancer patients.

No clear clinical guidelines exist on how to counsel male cancer patients about fertility preservation. Detailed counseling is recommended before treatment when issues of collection and storage need to be highlighted. Concern about the quality of sperm collected before and/or after treatment in terms of assisted reproduction is needed, and the potential outcomes should be discussed early as part of cancer survivorship. The discussion should be sensitive and tailored to the ethical situation based on the age of the patient, the severity of the illness, the need to initiate treatment, and genetic risk. Cryopreservation should be attempted/achieved before cancer treatment is initiated. Cryopreservation should not be performed during treatment or for some time after treatment because of the chromosomal and structural damage to sperm from cancer treatment. Contraception should be instigated during this period.

Recommendations for fertility preservation in patients with lymphoma, leukemia, and breast cancer.

Fertility issues should be addressed to all patients in reproductive age before cancer treatment. In men, cryopreservation of sperm should be offered to all cancer patients in reproductive age regardless of the risk of gonadal failure. In women, the recommendation of fertility preservation should be individualized based on multiple factors such as the urgency of treatment, the age of the patient, the marital status, the regimen and dosage of cancer treatment.

Fertility preservation in young women with breast cancer.

When a young woman is diagnosed with breast cancer, there is often a sense of urgency by the patient and her providers to initiate treatment. This article provides guidelines for incorporating the discussion of fertility preservation with newly diagnosed young women with breast cancer.

Fertility considerations in young women with hematological malignancies.

The need for practice guidelines for fertility preservation in young women with hematological malignancies has been increased. To develop recommendations, publications relevant to fertility preservation and hematological cancers were identified through a PubMed database search and reviewed systematically, focusing on the effects of oncological treatments on fertility as well as on the efficacy, feasibility and risks of existing fertility preservation methods.

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

PURPOSE: To assess the longevity of ovarian grafts in five cancer patients who underwent heterotopic autotransplantation of frozen-thawed ovarian tissue. METHOD(S): 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). RESULT(S): 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. CONCLUSION(S): 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.

Breast cancer and fertility preservation.

OBJECTIVE: To review the benefits of adjuvant systemic therapy given to women with breast cancer of reproductive age, its effects on fertility, and options for fertility preservation. DESIGN: Publications relevant to fertility preservation and breast cancer were identified through a PubMed database search. CONCLUSION(S): Most women who develop invasive breast cancer under age 40 will be advised to undergo adjuvant chemotherapy with or without extended antihormonal therapy to reduce the risk of recurrence and death from breast cancer. Adjuvant chemotherapy particularly with alkylating agents such as cyclophosphamide is gonadotoxic and markedly accelerates the rate of age-related ovarian follicle loss. Although loss of fertility is an important issue for young cancer survivors, there is often little discussion about fertility preservation before initiation of adjuvant therapy. Greater familiarity with prognosis and effects of different types of adjuvant therapy on the part of infertility specialists and fertility preservation options such cryopreservation of embryos, oocytes, and ovarian tissue on the part of oncologists would facilitate these discussions. Establishment of rapid fertility consultation links within cancer survivorship programs can help ensure that every young woman who is likely to undergo gonadotoxic cancer treatment is counseled about the effects of therapy and options available to her to increase the likelihood of childbearing after cancer treatment.

Use of hormonal protection for chemotherapy-induced gonadotoxicity.

It is still controversial that GnRH agonist (GnRHa) protects ovarian function from chemotherapy-induced gonadotoxicity. Indeed, the results of many studies related to this issue are neither consistent nor convincing because of the weak study design and the inadequate sample size. We identified 11 prospective controlled studies (8 nonrandomized and 3 randomized) for the systemic review and meta-analysis. The meta-analysis showed that GnRHa cotreatment during chemotherapy can protect ovarian function. However, it is worthy to note that the result of this meta-analysis is influenced by nonrandomized studies. The protective effect of GnRHa will remain elusive until the currently ongoing large, prospective, randomized studies are completed. In addition, tamoxifen, a selective estrogen receptor modulator, may have the protective effect against loss of follicles and ovarian function, which was caused by chemotherapy.

Time to re-think: ovarian tissue transplantation versus whole ovary transplantation.

Transplantation of cryobanked ovarian tissue is a promising strategy to restore fertility in cancer patients. However, ischaemia following ovarian tissue grafting can lead to significant follicular loss. Transplantation of the whole ovary by vascular anastomosis has been considered as a method of preventing ischaemic damage that occurs with avascular transplantation of ovarian tissue. Even so, the unavailability of the cryotechnology for whole organs can be a major barrier to whole ovary transplantation. Severe cryoinjury will cause not only follicular death but also irreversible damage to the vascular system of the ovary. Damaged ovarian vasculatures can induce thromboembolism after transplantation which leads to severe tissue ischaemia and follicular loss. As a consequence, follicular loss after the frozen-thawed whole ovary was transplanted with microsurgical vascular anastomosis has been shown to be as severe as that which occurred after ovarian tissue was grafted. In addition, the risk of cancer cell reintroduction can be potentially higher with whole ovary transplantation with vascular anastomosis. The safety and efficacy of the new procedure should be proven before any further clinical applications take place. Nevertheless, research on whole ovary cryopreservation should not be discouraged.

Long-term ovarian function and fertility after heterotopic autotransplantation of cryobanked human ovarian tissue: 8-year experience in cancer patients.

OBJECTIVE: To assess the long-term ovarian function and fertility after heterotopic autotransplantation of frozen-thawed ovarian tissue in cancer patients. DESIGN: Prospective clinical case series. SETTING: Academic medical center PATIENT(S): Four young cancer patients who completed cancer treatment. INTERVENTION(S): Cryopreserved ovarian tissue (2000-2002) was thawed and transplanted to the heterotopic site (between the rectus muscle and fascia) between 2002 and 2005. MAIN OUTCOME MEASURE(S): [1] Serial blood tests (FSH, LH, estradiol, progesterone, testosterone) and ultrasound examinations. [2] Oocyte retrieval and in vitro fertilization. RESULT(S): The hormonal profiles were consistent with the postmenopausal level before transplantation. The return of the ovarian function was evidenced by hormonal profiles between 12 and 20 weeks after transplantation. Three patients underwent a second transplantation, as restored ovarian function lasted only 3 to 5 months. After the second transplantation, long-term ovarian function (lasting for 15-41 months) was established in all three patients. Six oocytes (one GV, four MI, one MII) were retrieved from the grafts. Three MI oocytes were developed to full maturity in vitro. Four MII oocytes were fertilized and developed to the cleavage stage embryos (up to six-cell). CONCLUSION(S): Autotransplantation of frozen-thawed ovarian tissue to a heterotopic site restored long-term ovarian function (for >40 months), and showed a potential to restore fertility in cancer patients.

Assessment of vascular endothelial growth factor expression and apoptosis in the ovarian graft: can exogenous gonadotropin promote angiogenesis after ovarian transplantation?

OBJECTIVE: To evaluate the effects of gonadotropin on angiogenesis by assessing vascular endothelial growth factor (VEGF) expression in rat ovaries transplanted after freezing and thawing. DESIGN: In vitro laboratory experiments. SETTING: Academic research institute. ANIMAL(S): Sixty immature female rats. INTERVENTION(S): Frozen-thawed ovaries were autotransplanted into the SC tissue of 60 rats (ages between 21 and 28 days). After transplantation, either pregnant mare's serum gonadotropin (PMSG) or saline was administered. The grafted ovaries were collected 2, 7, and 30 days after transplantation for evaluation. MAIN OUTCOME MEASURE(S): Assessment of the morphology and number of follicles, evaluation of apoptosis, and analysis of VEGF expression in the grafted ovaries. RESULT(S): Most follicles in the grafts were apoptotic on day 2 but recovered by day 7. The proportion of antral follicles and corpora lutea in the graft correlated with the duration after transplantation. A significant increase in the expression of VEGF188 mRNA was noticed in the grafted ovaries on day 2. Moreover, the mRNA expression in the PMSG group was higher than that in the control group. The increased VEGF protein production in the graft was confirmed by Western blot analysis. CONCLUSION(S): In ovariectomized animals, gonadotropin treatment may not provide any added benefits for folliculogenesis and angiogenesis. Nevertheless, a significant increase in the VEGF188 isoform in the gonadotropin-treated group may suggest the positive effect of exogenous gonadotropin therapy in the early stages of angiogenesis.

Fertility preservation in female cancer patients: current developments and future directions.

OBJECTIVE: To review the current advances in fertility preservation strategies and to discuss future directions with an emphasis on ovarian tissue cryobanking. DESIGN: The publications related to fertility preservation in cancer patients were identified through Medline and other bibliographic databases, focusing on the most recent developments. CONCLUSION(S): There are several options for fertility preservation in cancer patients. Even though most of them are still experimental and their efficacy and reliability have not been determined, the future of fertility preservation in women with cancer is promising. In particular, the recent report of a live birth after transplantation of human ovarian tissue has reinforced the clinical potential of ovarian tissue banking for fertility preservation. Many exciting studies are underway to improve the efficacy and solve the problems with current fertility preservation strategies. It is inevitable that we will see the emergence of more complex ethical problems with the application of new technologies to humans. However, continuous efforts to improve current strategies and to develop new strategies will benefit many women and children who are facing premature ovarian failure and sterility.

Assessment of the integrity of human oocytes retrieved from cryopreserved ovarian tissue after xenotransplantation.

BACKGROUND: Previous studies showed that immature oocytes stored in ovarian tissue could develop to the mature stage after transplantation. However, the quality and competency of the oocytes developed in xenografted ovarian tissue have never been investigated. As a pilot study to investigate this uncharted issue, we evaluated microtubule organization and chromatin configuration of human oocytes harvested from xenografted frozen-thawed ovarian tissue. METHODS: Frozen-thawed human ovarian tissue was transplanted into severe combined immunodeficient mice. All animals were stimulated with gonadotrophin from 20 weeks after transplantation. Grafts were recovered 36 h after hCG administration. The oocytes were retrieved from the antral follicles (>2 mm diameter), cultured in vitro, stained for microtubule and chromatin localization. RESULTS: Five oocytes from 21 female mice and seven oocytes from nine male mice were retrieved. Immunocytochemical examinations of these oocytes after in vitro maturation revealed only two developed to the metaphase II stage. Most oocytes were between prophase and metaphase with abnormal microtubule organization and chromatin configuration. CONCLUSIONS: Immature oocytes in stored human ovarian tissue can grow to maturity in host animals after xenotransplantation. Retrieval of oocytes from the xenograft can be carried out and is reproducible. However, many oocytes, grown in host animals and further matured in vitro, showed aberrant microtubule organization and chromatin patterns.