Seido Takae

Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.

Significance Human ovaries hold follicles containing oocytes. When follicles mature, they release eggs for fertilization. Patients with primary ovarian insufficiency develop menopausal symptoms at less than 40 y of age. They have few remaining follicles and their only chance for bearing a baby is through egg donation. Kawamura et al. demonstrated that Hippo and Akt signaling pathways regulate follicle growth. Using an in vitro activation approach, they first removed ovaries from infertile patients, followed by fragmentation to disrupt Hippo signaling and drug treatment to stimulate Akt signaling. After grafting ovarian tissues back to patients, they found rapid follicle growth in some patients and successfully retrieved mature eggs. After in vitro fertilization and embryo transfer, a live birth is now reported. Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation–in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.

Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency

STUDY QUESTIONnIs ovarian tissue cryopreservation using vitrification followed by in vitro activation (IVA) of dormant follicles a potential approach for infertility treatment of patients with primary ovarian insufficiency (POI)?nnnSUMMARY ANSWERnOur vitrification approach followed by IVA treatment is a potential infertility therapy for POI patients whose ovaries contain residual follicles.nnnWHAT IS KNOWN ALREADYnAkt (protein kinase B) stimulators [PTEN (phosphatase with TENsin homology deleted in chromosome 10) inhibitor and phosphatidyinositol-3-kinase (PI3 kinase) stimulator] activate dormant primordial follicles in vitro and ovarian fragmentation disrupts the Hippo signaling pathway, leading to the promotion of follicle growth. We treated POI patients with a combination of ovarian vitrification, fragmentation and drug treatment, followed by auto-transplantation, and reported successful follicle growth and pregnancies.nnnSTUDY DESIGN, SIZE, DURATIONnProspective clinical study of 37 infertile women with POI between 12 August 2011 and 1 November 2013. We enrolled 10 new patients since the previous publication.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnPOI patients were originally selected based on a history of amenorrhea for more than 1 year and elevated serum FSH levels of >40 mIU/ml (n = 31) but this was later changed to >4 months, age <40 years and serum FSH levels of >35 mIU/ml (n = 6) (mean 71.8 ± 30.8, range 35.5-197.6) so as to include patients with a shorter duration of amenorrhea. Under laparoscopic surgery, ovariectomy was performed and ovarian cortices were dissected into strips for vitrification. Some pieces were examined histologically. After warming, two to three strips were fragmented into smaller cubes before culturing with Akt stimulators for 2 days. After washing, ovarian cubes were transplanted beneath the serosa of Fallopian tubes under laparoscopic surgery. Follicle growth was monitored by ultrasound and serum estrogen levels. After oocyte retrieval from mature follicles, IVF was performed.nnnMAIN RESULTS AND THE ROLE OF CHANCEnAmong 37 patients, 54% had residual follicles based on histology. Among patients with follicles, 9 out of 20 showed follicle growth in auto-grafts with 24 oocytes retrieved from six patients. Following IVF and embryo transfer into four patients, three pregnancies were detected based on serum hCG, followed by one miscarriage and two successful deliveries. For predicting IVA success, we found that routine histological analyses of ovarian cortices and shorter duration from initial POI diagnosis to ovariectomy are valid parameters.nnnLIMITATIONS, REASONS FOR CAUTIONnAlthough our findings suggest that the present vitrification protocol is effective for ovarian tissue cryopreservation, we have not compared the potential of vitrification and slow freezing in follicle growth after grafting. We chose the serosa of Fallopian tubes as the auto-grating site due to its high vascularity and the ease to monitor follicle growth. Future studies are needed to evaluate the best auto-grafting sites for ovarian tissues. Also, future studies are needed to identify biological markers to indicate the presence of residual follicles in POI to predict IVA treatment outcome.nnnWIDER IMPLICATIONS OF THE FINDINGSnIn POI patients, ovarian reserve, namely the pool of residual follicles, continues to diminish with age. If one ovary is cryopreserved at an earlier stage of POI, patients could undergo additional non-invasive infertility treatments before the final decision for the IVA treatment. Furthermore, in the cases of unmarried POI patients, cryopreservation of ovarian tissues allows their fertility preservation until they desire to bear children.nnnSTUDY FUNDING/COMPETING INTERESTSnThis work was supported by Grant-In-Aid for Scientific Research (Research B: 24390376, Challenging Exploratory Research: 24659722, and Innovative Areas, Mechanisms regulating gamete formation in animals: 26114510) and by research funds from the Smoking Research Foundation, and the Takeda Science Foundation. None of the authors has a conflict of interest.nnnTRIAL REGISTRATION NUMBERnUMIN000010828.

Pre-ovulatory LH/hCG surge decreases C-type natriuretic peptide secretion by ovarian granulosa cells to promote meiotic resumption of pre-ovulatory oocytes

BACKGROUNDnIn mammalian follicles, oocytes are arrested at the diplotene stage of prophase I until meiotic resumption following the LH surge. Recently, C-type natriuretic peptide (CNP), encoded by natriuretic peptide precursor type C (NPPC) was found to suppress mouse oocyte maturation by promoting cyclic guanosine 5-monophospate (cGMP) production in cumulus cells. However, regulation of NPPC/CNP expression during the pre-ovulatory period and their regulation by the LH surge have not been investigated.nnnMETHODS AND RESULTSnBased on genome-wide analysis of DNA microarray data sets using samples from periovulatory ovaries, we found increases in NPPC transcripts in granulosa cells during pre-ovulatory follicle growth in mice and a rapid decline induced by the pre-ovulatory LH/hCG stimulation. Treatment of pre-ovulatory animals with hCG decreased ovarian CNP content. In isolated ovarian cells, NPPC mRNA was predominantly expressed in mural granulosa cells exhibiting similar regulation following gonadotrophin treatment. In cultured mouse pre-ovulatory follicles, meiosis resumption in oocytes by hCG treatment was accompanied by decreases in NPPC transcript levels. In cultured mouse cumulus cell-oocyte complexes, CNP treatment inhibited the resumption of meiosis with increases in cGMP levels in both cumulus cells and oocytes. In human ovaries, CNP levels in ovarian follicular fluid were also decreased following treatment of patients with an ovulatory dose of hCG.nnnCONCLUSIONSnOur findings demonstrate gonadotrophins regulation of NPPC/CNP expression in mouse and human ovaries and confirm the role of CNP as a potent paracrine oocyte maturation inhibitor.

Assessment of long-term function of heterotopic transplants of vitrified ovarian tissue in cynomolgus monkeys

BACKGROUNDnOvarian tissue cryopreservation by rapid cooling (vitrification) is a convenient fertility preservation option. However, the progress of vitrified ovarian tissue after transplantation is not well understood in primates.nnnMETHODSnFor tissues from cynomolgus monkeys, we used closed straw vitrification and open cryosupport vitrification in which tissues are immersed directly into liquid nitrogen. Following warming, ovarian cortical pieces were autotransplanted and their function was monitored by computed tomography (CT), hormone assays and oocyte recovery, ICSI and embryo transfers (ETs).nnnRESULTSnHormone cycles were restored in 6 of 7 animals in a mean of 126 days with no significant difference between the two vitrification regimens. The presence of new blood vessels supplying the grafted ovarian tissue was confirmed by contrast-enhanced CT. Oocyte retrieval from two monkeys after transplantation of the ovarian cortex vitrified by cryosupport vitrification yielded a total of nine oocytes of which six fertilized after ICSI, but ETs did not lead to any pregnancies.nnnCONCLUSIONSnThis work shows that CT can give insight into ovarian function after heterotopic transplantation, and that heterotopic autografts of vitrified ovarian cortex can give rise to long-term ovarian function and embryos in a primate model. It remains to be established how outcomes following rapid vitrification compared with outcomes following conventional slow cooling procedures.

C-Type Natriuretic Peptide Stimulates Ovarian Follicle Development

C-type natriuretic peptide (CNP) encoded by the NPPC (Natriuretic Peptide Precursor C) gene expressed in ovarian granulosa cells inhibits oocyte maturation by activating the natriuretic peptide receptor (NPR)B (NPRB) in cumulus cells. RT-PCR analyses indicated increased NPPC and NPRB expression during ovarian development and follicle growth, associated with increases in ovarian CNP peptides in mice. In cultured somatic cells from infantile ovaries and granulosa cells from prepubertal animals, treatment with CNP stimulated cGMP production. Also, treatment of cultured preantral follicles with CNP stimulated follicle growth whereas treatment of cultured ovarian explants from infantile mice with CNP, similar to FSH, increased ovarian weight gain that was associated with the development of primary and early secondary follicles to the late secondary stage. Of interest, treatment with FSH increased levels of NPPC, but not NPRB, transcripts in ovarian explants. In vivo studies further indicated that daily injections of infantile mice with CNP for 4 d promoted ovarian growth, allowing successful ovulation induction by gonadotropins. In prepubertal mice, CNP treatment alone also promoted early antral follicle growth to the preovulatory stage, leading to efficient ovulation induction by LH/human chorionic gonadotropin. Mature oocytes retrieved after CNP treatment could be fertilized in vitro and developed into blastocysts, allowing the delivery of viable offspring. Thus, CNP secreted by growing follicles is capable of stimulating preantral and antral follicle growth. In place of FSH, CNP treatment could provide an alternative therapy for female infertility.

Creating a Global Community of Practice for Oncofertility

Fertility preservation in the cancer setting, known as oncofertility, is a field that requires cross-disciplinary interaction between physicians, basic scientists, clinical researchers, ethicists, lawyers, educators, and religious leaders. Funded by the National Institutes of Health, the Oncofertility Consortium (OC) was formed to be a scientifically grounded, transparent, and altruistic resource, both intellectual and monetary, for building this new field of practice capable of addressing the unique needs of young patients with cancer. The OC has expanded its attention to include other nonmalignant conditions that can threaten fertility, and the work of the OC now extends around the globe, involving partners who together have created a community of shared effort, resources, and practices. The OC creates materials that are translated, disseminated, and amended by all participants in the field, and local programs of excellence have developed worldwide to accelerate the pace and improve the quality of oncofertility research and practice. Here we review the global oncofertility programs and the capacity building activities that strengthen these research and clinical programs, ultimately improving patient care.

Oocyte-derived R-spondin2 promotes ovarian follicle development

R‐spondin proteins are adult stem cell growth factors capable of stimulating gut development by activating LGR4, 5, and 6 receptors to promote Wnt signaling. Although multiple Wnt ligands and cognate Frizzled receptors are expressed in the ovary, their physiological roles are unclear. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R‐spondin2 in oocytes of ovarian follicles. In cultured somatic cells from preantral follicles, R‐spondin2 treatment (ED50: 3 ng/ml) synergized with Wnt3a to stimulate Wnt signaling. In cultured ovarian explants from prepubertal mice containing preantral follicles, treatment with R‐spondin2, similar to follicle stimulating hormone, promoted the development of primary follicles to the secondary stage. In vivo administration of an R‐spondin agonist stimulated the development of primary follicles to the antral stage in both immature mice and gonadotropin releasing hormone antagonist‐treated adult mice. Subsequent treatment with gonadotropins allowed the generation of mature oocytes capable of undergoing early embryonic development and successful pregnancy. Furthermore, R‐spondin agonist treatment of immune‐deficient mice grafted with human cortical fragments stimulated the development of primary follicles to the secondary stage. Thus, oocyte‐derived R‐spondin2 is a paracrine factor essential for primary follicle development, and R‐spondin agonists could provide a new treatment regimen for infertile women with low responses to the traditional gonadotropin therapy.—Cheng, Y., Kawamura, K., Takae, S., Deguchi, M., Yang, Q., Kuo, C., Hsueh, A. J. W. Oocyte‐derived R‐spondin2 promotes ovarian follicle development. FASEB J. 27, 2175–2184 (2013). www.fasebj.org

Intraovarian Thrombin and Activated Protein C Signaling System Regulates Steroidogenesis during the Periovulatory Period

In addition to its role in blood coagulation, thrombin directly stimulates protease-activated receptors (PAR) or interacts with thrombomodulin (THBD) to activate membrane-bound protein C which stimulates PAR1 and PAR4 receptors to promote downstream pleiotropic effects. Our DNA microarray, RT-PCR, and immunostaining analyses demonstrated ovarian expression of THBD, activated protein C (APC) receptor [endothelial protein C receptor (EPCR)], as well as PAR1 and PAR4 receptors in mice. After treatment of gonadotropin-primed immature mice with an ovulatory dose of human chorionic gonadotropin (hCG) (a LH surrogate), major increases in the expression of THBD, EPCR, PAR1, and PAR4 were detected in granulosa and cumulus cells of preovulatory follicles. Immunoassay analyses demonstrated sustained increases in ovarian prothrombin and APC levels after hCG stimulation. We obtained luteinizing granulosa cells from mice treated sequentially with equine CG and hCG. Treatment of these cells with thrombin or agonists for PAR1 or PAR4 decreased basal and forskolin-induced cAMP biosynthesis and suppressed hCG-stimulated progesterone production. In cultured preovulatory follicles, treatment with hirudin (a thrombin antagonist) and SCH79797 (a PAR1 antagonist) augmented hCG-stimulated progesterone biosynthesis, suggesting a suppressive role of endogenous thrombin in steroidogenesis. Furthermore, intrabursal injection with hirudin or SCH79797 led to ipsilateral increases in ovarian progesterone content. Our findings demonstrated increased ovarian expression of key components of the thrombin-APC-PAR1/4 signaling system after LH/hCG stimulation, and this signaling pathway may allow optimal luteinization of preovulatory follicles. In addition to assessing the role of thrombin and associated genes in progesterone production by the periovulatory ovary, these findings provide a model with which to study molecular mechanisms underlying thrombin-APC-PAR1/4 signaling.

Heterotopic autotransplantation of ovarian cortex in cynomolgus monkeys.

In recent years, removal of ova or ovaries before chemotherapy or radiation therapy has been investigated in young female cancer patients to avoid the adverse effects of treatment. Orthotopic autotransplantation of ovarian cortex has advantages such as easy collection of ova and the possibility of spontaneous pregnancy. Although children have been born after successful orthotopic autotransplantation into the residual ovaries, some patients cannot undergo this procedure such as those who need bilateral ovariectomy or pelvic radiation therapy, therefore it is still necessary to investigate suitable heterotopic autotransplantation sites. The present study was performed in primates (cynomolgus monkeys) with the objective of determining the optimum site for heterotopic autotransplantation of ovarian cortex to enhance the clinical application of this method. The retroperitoneal iliac fossa and omentum were selected as sites for heterotopic autotransplantation. Two cynomolgus monkeys were subjected to laparotomy under anesthesia. After resection of the bilateral adnexae, the ovaries were cut into 0.5 cm cubes that were transplanted. Blood levels of follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone were monitored, and monkeys with a regular estrus cycle underwent superovulation and egg collection. In both monkeys studied, recovery of a regular estrus cycle was confirmed after heterotopic autotransplantation of ovarian tissue. Mll phase ova were successfully collected from tissues transplanted into the retroperitoneal iliac fossa or omentum. Development to the early blastocyst stage was confirmed after microfertilization. We established an appropriate method of heterotopic autotransplantation using ovarian cortex into the retroperitoneal iliac fossa or omentum in primates.

Survey of Fertility Preservation Options Available to Patients With Cancer Around the Globe

Purpose Oncofertility focuses on providing fertility and endocrine-sparing options to patients who undergo life-preserving but gonadotoxic cancer treatment. The resources needed to meet patient demand often are fragmented along disciplinary lines. We quantify assets and gaps in oncofertility care on a global scale. Methods Survey-based questionnaires were provided to 191 members of the Oncofertility Consortium Global Partners Network, a National Institutes of Health–funded organization. Responses were analyzed to measure trends and regional subtleties about patient oncofertility experiences and to analyze barriers to care at sites that provide oncofertility services. Results Sixty-three responses were received (response rate, 25%), and 40 were analyzed from oncofertility centers in 28 countries. Thirty of 40 survey results (75%) showed that formal referral processes and psychological care are provided to patients at the majority of sites. Fourteen of 23 respondents (61%) stated that some fertility preservation services are not offered because of cultural and legal barriers. The growth of oncofertility and its capacity to improve the lives of cancer survivors around the globe relies on concentrated efforts to increase awareness, promote collaboration, share best practices, and advocate for research funding. Conclusion This survey reveals global and regional successes and challenges and provides insight into what is needed to advance the field and make the discussion of fertility preservation and endocrine health a standard component of the cancer treatment plan. As the field of oncofertility continues to develop around the globe, regular assessment of both international and regional barriers to quality care must continue to guide process improvements.Purpose Oncofertility focuses on providing fertility and endocrine-sparing options to patients who undergo life-preserving but gonadotoxic cancer treatment. The resources needed to meet patient demand often are fragmented along disciplinary lines. We quantify assets and gaps in oncofertility care on a global scale. Methods Survey-based questionnaires were provided to 191 members of the Oncofertility Consortium Global Partners Network, a National Institutes of Health–funded organization. Responses were analyzed to measure trends and regional subtleties about patient oncofertility experiences and to analyze barriers to care at sites that provide oncofertility services. Results Sixty-three responses were received (response rate, 25%), and 40 were analyzed from oncofertility centers in 28 countries. Thirty of 40 survey results (75%) showed that formal referral processes and psychological care are provided to patients at the majority of sites. Fourteen of 23 respondents (61%) stated that some fertility preservation services are not offered because of cultural and legal barriers. The growth of oncofertility and its capacity to improve the lives of cancer survivors around the globe relies on concentrated efforts to increase awareness, promote collaboration, share best practices, and advocate for research funding. Conclusion This survey reveals global and regional successes and challenges and provides insight into what is needed to advance the field and make the discussion of fertility preservation and endocrine health a standard component of the cancer treatment plan. As the field of oncofertility continues to develop around the globe, regular assessment of both international and regional barriers to quality care must continue to guide process improvements.