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.

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.

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.

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.

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.

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.

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.

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.

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.

Heterotopic autotransplantation of cryobanked human ovarian tissue as a strategy to restore ovarian function.

OBJECTIVE: To investigate ovarian function after heterotopic autotransplantation of human ovarian tissue banked at -196 degrees C. DESIGN: A clinical case study. SETTING: University medical center. PATIENT(S): A 37-year-old woman with cervical cancer (stage Ib). INTERVENTION(S): Frozen-thawed human ovarian tissue was transplanted to two different heterotopic sites. MAIN OUTCOME MEASURE(S): Ovarian function of the grafts was monitored sequentially by blood sampling for the hormonal profiles and by ultrasound. RESULT(S): The hormonal profile remained at the postmenopausal level until 10 weeks after transplantation. By 14 weeks, the return of ovarian function was evidenced by the elevation of the serum E(2) level (57.5 pg/mL). While monitoring hormonal profiles every 2 days for 5 weeks, we observed the LH surge (69.8 IU/L) followed by the elevation of the P(4)concentration (9.6 ng/mL), suggesting presumptive ovulation. The ultrasound revealed a dominant follicle on the rectus muscle in the abdominal site. However, there was no sign of follicle development in the breast site. Ovarian function ceased around 28 weeks after transplantation. CONCLUSION(S): Heterotopic autotransplantation of cryobanked human ovarian tissue can be a practical strategy for restoration of ovarian function after cancer treatment. As a site for transplantation, a space between the rectus sheath and the rectus muscle appeared to be effective.

Follicular development, ovulation, and corpus luteum formation in cryopreserved human ovarian tissue after xenotransplantation.

OBJECTIVE: To assess the competency of human frozen/thawed ovarian follicles matured in xenografts to form functioning corpora luteae after human chorionic gonadotropin (hCG) administration. DESIGN: Prospective controlled animal study. SETTING: University research laboratory. PATIENT(S): Three women (19, 28, and 36 years) who underwent oophorectomy. ANIMAL(S): Nineteen female severe combined immunodeficient (SCID) mice. INTERVENTION(S): Cryopreserved human ovarian tissues were grafted into the s.c. space of bilaterally oophorectomized SCID mice. All the animals were stimulated with pregnant mare's serum gonadotropin (PMSG) for 4 weeks starting from 16 weeks after transplantation. Twelve animals were injected with hCG at the end of gonadotropin stimulation. MAIN OUTCOME MEASURE(S): [1] The rate of grafts with growing follicles, with antral follicles, and/or with corpora luteae. [2] The histologic assessment of follicles and corpora luteae. [3] The serum progesterone and estradiol level in animals with corpus luteum in the grafts. RESULT(S): [1] The rate of grafts with growing follicles and with corpora luteae was 33% to 100%, and 28% to 50%, respectively. [2] Corpora luteae in xenografts were all morphologically normal. [3] The progesterone levels were all above 3.0 ng/mL. CONCLUSION(S): This study showed that the cryopreserved human ovarian follicles can be matured to a stage at which they can form functioning corpora luteae in the host animal.

Early and late hormonal responses to the microdose gonadotropin-releasing hormone agonist in normal menstruating women.

OBJECTIVE: To assess the effect of a microdose gonadotropin-releasing hormone (GnRH) agonist on the LH, FSH, and E2 secretion in normal menstruating women. DESIGN: Prospective study. SETTING: Tertiary teaching hospital. PATIENT(S): Five normal menstruating women. INTERVENTION(S): Five microg of triptorelin was injected daily in 5 women for 7 days beginning from the cycle day 3. In the next cycle, the same amount of triptorelin was injected into the same women daily for 3 days. MAIN OUTCOME MEASURE(S): Serial serum FSH, LH, and E2 levels. RESULT(S): The FSH levels peaked (27.53 +/- 6.34 IU/L) after 5 hours, and the LH levels peaked (34.35 +/- 7.81 IU/L) by 4 hours. The increased gonadotropin levels persisted even after the second and third day of the GnRH-agonist injections, although the peak levels were not as high as observed with the first injection (19.56 IU/L in the second day, 9.15 IU/L in the third day for FSH; 32.18 IU/L in the second day, 13.59 IU/L in the third day for LH). The down-regulation of gonadotropins was established in 4 days. When the GnRH-agonist was administered for 7 days, the E2 level began to increase 6 days after the last injection. When the GnRH-agonist was administered for 3 days, the E2 level began to increase 3 days after the last injection. CONCLUSION(S): Pituitary down-regulation could be achieved even with a microdose of GnRH agonist. The increased level of gonadotropins persisted for 3 days at this dose. The duration of the down-regulation was influenced by the duration of GnRH-agonist administration.

Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment.

OBJECTIVE: To estimate ischemic tissue damage in ovarian cortex and to evaluate the effectiveness of ascorbic acid, an antioxidant, to protect ovarian tissue from apoptosis caused by ischemia. DESIGN: In vitro laboratory experiments. SETTING: Academic research institute. INTERVENTION(S): Fresh and frozen/thawed cortical sections of bovine ovaries were incubated in MEM medium with or without ascorbic acid for a duration of 3, 24, and 48 hours at 37 degrees C. MAIN OUTCOME MEASURE(S): Oxygen consumption rates, lactate dehydrogenase concentrations, apoptosis rates determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and DNA fragmentation analysis. RESULT(S): The oxygen consumption rates were correlated inversely with the duration of incubation. When the rates of apoptosis in primordial follicles with or without ascorbic acid treatment were compared, there was no statistically significant difference between the two groups. However, the ascorbic acid treatment group showed significantly decreased apoptosis in ovarian cortex (stromal cells) with 24 hours of incubation. CONCLUSION(S): The correlation between ischemic tissue damage and the duration of ischemia was verified. Ovarian cortex could tolerate ischemia at least for 3 hours. Ascorbic acid treatment reduced apoptosis in ovarian cortex up to 24 hours of incubation in vitro. It appeared that stromal cells were more vulnerable to ischemia compared to primordial follicles.

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.

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.