Pediatric oncofertility care in limited versus optimum resource settings: results from 39 surveyed centers in Repro-Can-OPEN Study Part I & II.

PURPOSE: As a secondary report to elucidate the diverse spectrum of oncofertility practices for childhood cancer around the globe, we present and discuss the comparisons of oncofertility practices for childhood cancer in limited versus optimum resource settings based on data collected in the Repro-Can-OPEN Study Part I & II. METHODS: We surveyed 39 oncofertility centers including 14 in limited resource settings from Africa, Asia, and Latin America (Repro-Can-OPEN Study Part I), and 25 in optimum resource settings from the USA, Europe, Australia, and Japan (Repro-Can-OPEN Study Part II). Survey questions covered the availability of fertility preservation and restoration options offered in case of childhood cancer as well as their degree of utilization. RESULTS: In the Repro-Can-OPEN Study Part I & II, responses for childhood cancer and calculated oncofertility scores showed the following characteristics: (1) higher oncofertility scores in optimum resource settings than in limited resource settings for ovarian and testicular tissue cryopreservation; (2) frequent utilization of gonadal shielding, fractionation of anticancer therapy, oophoropexy, and GnRH analogs; (3) promising utilization of oocyte in vitro maturation (IVM); and (4) rare utilization of neoadjuvant cytoprotective pharmacotherapy, artificial ovary, in vitro spermatogenesis, and stem cells reproductive technology as they are still in preclinical or early clinical research settings. CONCLUSIONS: Based on Repro-Can-OPEN Study Part I & II, we presented a plausible oncofertility best practice model to help optimize care for children with cancer in various resource settings. Special ethical concerns should be considered when offering advanced and innovative oncofertility options to children.

A synopsis of global frontiers in fertility preservation.

Since 2007, the Oncofertility Consortium Annual Conference has brought together a diverse network of individuals from a wide range of backgrounds and professional levels to disseminate emerging basic and clinical research findings in fertility preservation. This network also developed enduring educational materials to accelerate the pace and quality of field-wide scientific communication. Between 2007 and 2019, the Oncofertility Consortium Annual Conference was held as an in-person event in Chicago, IL. The conference attracted approximately 250 attendees each year representing 20 countries around the world. In 2020, however, the COVID-19 pandemic disrupted this paradigm and precluded an in-person meeting. Nevertheless, there remained an undeniable demand for the oncofertility community to convene. To maintain the momentum of the field, the Oncofertility Consortium hosted a day-long virtual meeting on March 5, 2021, with the theme of "Oncofertility Around the Globe" to highlight the diversity of clinical care and translational research that is ongoing around the world in this discipline. This virtual meeting was hosted using the vFairs ® conference platform and allowed over 700 people to participate, many of whom were first-time conference attendees. The agenda featured concurrent sessions from presenters in six continents which provided attendees a complete overview of the field and furthered our mission to create a global community of oncofertility practice. This paper provides a synopsis of talks delivered at this event and highlights the new advances and frontiers in the fields of oncofertility and fertility preservation around the globe from clinical practice and patient-centered efforts to translational research.

Preserving fertility in female patients with hematological malignancies: a multidisciplinary oncofertility approach.

Oncofertility is a new interdisciplinary field at the intersection of oncology and reproductive medicine that expands fertility options for young cancer patients. The most common forms of hematological malignancies that occur in girls and young women and therefore necessitate oncofertility care are acute lymphocytic leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, and Hodgkin's lymphoma. Aggressive gonadotoxic anticancer regimens including alkylating chemotherapy and total body irradiation are used often in treating girls and young women with hematological malignancies. The risks of gonadotoxicity and subsequent iatrogenic premature ovarian insufficiency and fertility loss depend mainly on the type and stage of the disease, dose of anticancer therapy as well as the age of the patient at the beginning of treatment. To avoid or at least mitigate the devastating complications of anticancer therapy-induced gonadotoxicity, effective and comprehensive strategies that integrate different options for preserving and restoring fertility ranging from established to experimental strategies should be offered before, during, and after chemotherapy or radiotherapy. A multidisciplinary approach that involves strong coordination and collaboration between hemato-oncologists, gynecologists, reproductive biologists, research scientists, and patient navigators is essential to guarantee high standard of care.

Primordial Follicle Transplantation within Designer Biomaterial Grafts Produce Live Births in a Mouse Infertility Model.

The gonadotoxic effects of chemotherapy and radiation may result in premature ovarian failure in premenopausal oncology patients. Although autotransplantation of ovarian tissue has led to successful live births, reintroduction of latent malignant cells inducing relapse is a significant concern. In this report, we investigated the design of biomaterial grafts for transplantation of isolated ovarian follicles as a means to preserve fertility. Primordial and primary ovarian follicles from young female mice were extracted and encapsulated into biomaterials for subsequent transplantation into adult mice. Among the formulations tested, aggregated follicles encapsulated within fibrin had enhanced survival and integration with the host tissue following transplantation relative to the fibrin-alginate and fibrin-collagen composites. All mice transplanted with fibrin-encapsulated follicles resumed cycling, and live births were achieved only for follicles transplanted within VEGF-loaded fibrin beads. The extent to which these procedures reduce the presence of metastatic breast cancer cells among the isolated follicles was evaluated, with significantly reduced numbers of cancer cells present relative to intact ovaries. This ability to obtain live births by transplanting isolated primordial and primary follicles, while also reducing the risk of re-seeding disease relative to ovarian tissue transplantation, may ultimately provide a means to preserve fertility in premenopausal oncology patients.

In vitro development of secondary follicles from pre-pubertal and adult goats cultured in two-dimensional or three-dimensional systems.

The aim of this study was to evaluate the influence of two-dimensional (2D) and three-dimensional (3D) alginate culture systems on in vitro development of pre-antral caprine follicles. In addition, the influence of the reproductive age of the ovary donor on the in vitro culture success was investigated. Pre-antral follicles from pre-pubertal or adult goats were isolated and cultured directly on a plastic surface (2D) or encapsulated in an alginate-based matrix (3D). After 18 days, the oocytes underwent in vitro maturation (IVM) and in vitro fertilization (IVF) to produce embryos. The 3D system showed higher rates of follicle survival, lower rates of oocyte extrusion, and a greater number of recovered oocytes for IVM and IVF (P < 0.05). Only pre-antral follicles from adult animals produced MII oocytes and embryos. The estradiol concentrations increased from day 2 to day 12 of culture in all groups tested (P < 0.05). Conversely, progesterone concentrations were lower in 3D-cultured follicles than in 2D-cultured follicles, with differences on days 2 and 6 of culture (P < 0.05). We provide compelling evidence that a 2D or 3D alginate in vitro culture system offers a promising approach to achieving full in vitro development of caprine pre-antral follicles to produce mature oocytes that are capable of fertilization and viable embryos.

Involvement of androgens in ovarian health and disease.

In women, ovary and adrenal gland produce androgens. Androgens are essential drivers of the primordial to antral follicle development, prior to serving as substrate for estrogen production in the later stages of folliculogenesis. Androgens play a crucial role in the follicular-stromal intertalk by fine tuning the extracellular matrix and vessel content of the ovarian stroma. Local auto-and paracrine factors regulate androgen synthesis in the pre-antral follicle. Androgen excess is a hallmark of polycystic ovary syndrome and is a key contributor in the exaggerated antral follicle formation, stromal hyperplasia and hypervascularity. Hyperandrogenaemia overrides the follicular-stromal dialog, resulting in follicular arrest and disturbed ovulation. On the other hand, androgen deficiency is likely to have a negative impact on fertility as well, and further research is needed to examine the benefits of androgen-replacement therapy in subfertility.

Rescue of platinum-damaged oocytes from programmed cell death through inactivation of the p53 family signaling network.

Non-proliferating oocytes within avascular regions of the ovary are exquisitely susceptible to chemotherapy. Early menopause and sterility are unintended consequences of chemotherapy, and efforts to understand the oocyte apoptotic pathway may provide new targets for mitigating this outcome. Recently, the c-Abl kinase inhibitor imatinib mesylate (imatinib) has become the focus of research as a fertoprotective drug against cisplatin. However, the mechanism by which imatinib protects oocytes is not fully understood, and reports of the drug's efficacy have been contradictory. Using in vitro culture and subrenal grafting of mouse ovaries, we demonstrated that imatinib inhibits the cisplatin-induced apoptosis of oocytes within primordial follicles. We found that, before apoptosis, cisplatin induces c-Abl and TAp73 expression in the oocyte. Oocytes undergoing apoptosis showed downregulation of TAp63 and upregulation of Bax. While imatinib was unable to block cisplatin-induced DNA damage and damage response, such as the upregulation of p53, imatinib inhibited the cisplatin-induced nuclear accumulation of c-Abl/TAp73 and the subsequent downregulation of TAp63 and upregulation of Bax, thereby abrogating oocyte cell death. Surprisingly, the conditional deletion of Trp63, but not ΔNp63, in oocytes inhibited apoptosis, as well as the accumulation of c-Abl and TAp73 caused by cisplatin. These data suggest that TAp63 is the master regulator of cisplatin-induced oocyte death. The expression kinetics of TAp63, c-Abl and TAp73 suggest that cisplatin activates TAp63-dependent expression of c-Abl and TAp73 and, in turn, the activation of TAp73 by c-Abl-induced BAX expression. Our findings indicate that imatinib protects oocytes from cisplatin-induced cell death by inhibiting c-Abl kinase, which would otherwise activate TAp73-BAX-mediated apoptosis. Thus, imatinib and other c-Abl kinase inhibitors provide an intriguing new way to halt cisplatin-induced oocyte death in early follicles and perhaps conserve the endocrine function of the ovary against chemotherapy.

Imaging and elemental mapping of biological specimens with a dual-EDS dedicated scanning transmission electron microscope.

A dedicated analytical scanning transmission electron microscope (STEM) with dual energy dispersive spectroscopy (EDS) detectors has been designed for complementary high performance imaging as well as high sensitivity elemental analysis and mapping of biological structures. The performance of this new design, based on a Hitachi HD-2300A model, was evaluated using a variety of biological specimens. With three imaging detectors, both the surface and internal structure of cells can be examined simultaneously. The whole-cell elemental mapping, especially of heavier metal species that have low cross-section for electron energy loss spectroscopy (EELS), can be faithfully obtained. Optimization of STEM imaging conditions is applied to thick sections as well as thin sections of biological cells under low-dose conditions at room and cryogenic temperatures. Such multimodal capabilities applied to soft/biological structures usher a new era for analytical studies in biological systems.

In vitro growth and steroidogenesis of dog follicles are influenced by the physical and hormonal microenvironment.

The present study examined the influences of the physical and hormonal microenvironment on in vitro growth and steroidogenesis of dog follicles. Follicles were enzymatically isolated and individually encapsulated in 0.5% (w/v; n=17) or 1.5% (n=10) alginate and cultured with 0.5 IU/ml equine chorionic gonadotropin for 192 h. In a separate experiment, follicles were encapsulated in 0.5% alginate and cultured with 0 (n=22), 1 (n=23), 10 (n=20) or 100 (n=21) µg/ml FSH for 240 h. Follicle diameter and steroid production were assessed every 48 h in both studies. Follicles encapsulated in the 0.5% alginate grew faster (P<0.05) than those cultured in the 1.5% concentration. Oestradiol (E(2)) and progesterone (P(4)) increased consistently (P<0.05) over time, and follicles in the 1.5% alginate produced more (P<0.05) P(4) than those in the 0.5% solution. Follicles cultured in the highest FSH concentration (100 µg/ml) increased 100% in size after 240 h compared with 50 to 70% in lower dosages. E(2) concentration remained unchanged over time (P>0.05) across FSH dosages. However, P(4) increased (P<0.05) as culture progressed and with increasing FSH concentration. Results demonstrate that dog follicles cultured in alginate retain structural integrity, grow in size and are hormonally active. Lower alginate and increasing FSH concentrations promote in vitro follicle growth. However, the absence of an E(2) rise in follicles cultured in FSH alone suggests the need for LH supplementation to support theca cell differentiation and granulosa cell function.

Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation.

BACKGROUND Female cancer patients are offered 'banking' of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research.

Characterization of the ovarian and reproductive abnormalities in prepubertal and adult estrogen non-responsive estrogen receptor alpha knock-in (ENERKI) mice.

Estrogen non-responsive estrogen receptor alpha (ERalpha) knock-in (ENERKI) mice have a mutation (glycine 525 to leucine, G525L) in the ligand-binding domain of ERalpha. The mutant ERalpha protein has a significantly lower affinity and response to endogenous estrogens, while not altering growth factor activated ligand-independent pathways. ENERKI females demonstrated signs of early follicle development as determined by a significant increase in antral follicle formation by 20 days of age. Adult ENERKI females were infertile, had hemorrhagic ovarian follicular cysts, and failed to develop corpora lutea in response to a superovulation regimen. These results illustrate the importance of ERalpha ligand-induced signaling for ovarian development and for estrogen feedback on the hypothalamus and pituitary. Although ERalpha ligand-induced signaling by endogenous estrogens is lost in ENERKI females, the ERalpha selective agonist propyl pyrazole triol (PPT), a synthetic nonsteroidal compound, is still able to activate G525L ERalphain vivo to increase uterine weight. To test whether PPT could restore ligand-dependent receptor activation, ENERKI females were treated with PPT and evaluated for spontaneous ovulation, ovarian hemorrhagic cysts, and LH serum levels. Daily PPT treatments beginning on day 4 of life prevented formation of ovarian hemorrhagic cysts in adult ENERKI animals. In accordance with this result, preputial gland weight and LH levels were also lowered in these animals, indicating PPT treatments most likely led to restoration of ERalpha negative feedback of the hypothalamic-pituitary axis.

Photoperiod-dependent regulation of inhibin in Siberian hamsters: II. Regulation of inhibin production and secretion by pregnant mare serum gonadotropin.

Inhibin production differs in ovaries of Siberian hamsters (Phodopus sungorus) exposed to long days (LD) or short days (SD). We believe that seasonal differences in serum follicle-stimulating hormone contribute to this difference. However, given the profound photoperiodic differences in follicle maturation, serum gonadotropins alone may not account for all of the observed differences in inhibin processing. To test this hypothesis, we challenged LD and SD female hamsters with exogenous gonadotropins. While both groups responded with increased inhibin expression, the effects were muted in ovaries of SD females and there was no evidence of ovulation in these animals. These data indicate that the ovaries of SD females are not immediately equipped to respond to gonadotropin stimulation. More generally, these data suggest that photoperiodic history affects ovarian inhibin production and secretion in response to gonadotropins.

Mechanisms of inhibin signal transduction.

Inhibin was first identified as a gonadal hormone that potently inhibits pituitary follicle-stimulating hormone (FSH) synthesis and secretion. Although the notion of a nonsteroidal, gonadally derived inhibitory substance was realized in the early 1930s (McCullagh, 1932), identification of the hormone was not accomplished until more than 50 years later. At that time, inhibin was purified from bovine and porcine follicular fluid and was shown to be produced in two forms through dimeric assembly of an alpha subunit (18 kDa) and one of two closely related beta subunits (betaA and betaB, approximately 14 kDa) (Ling et al., 1985; Miyamoto et al., 1985; Rivier et al., 1985; Robertson et al., 1985). Dimers of alpha and betaA and alpha and betaB subunits form inhibin A and inhibin B, respectively. In the process of purifying inhibin, two groups also identified homo- and heterodimers of the inhibin beta subunits (Ling et al., 1986; Vale et al., 1986). These hormones, the activins, were shown to potently stimulate FSH secretion from primary pituitary cultures and are now known to play important roles in growth and development (Woodruff, 1998; Pangas and Woodruff, 2000). Inhibins and activins are considered members of the transforming growth factor-beta (TGF-beta) superfamily of growth and differentiation factors, based on a pattern of conserved cysteine residues in the alpha and beta subunits, similar to other ligands in the family. Identification of the subunit proteins led to the cloning of their cDNAs and subsequently to their chromosomal mapping in several species (Mason et al, 1985,1986; Forage et al., 1986; Mayo et al., 1986; Esch et al., 1987; Woodruff et al., 1987; Barton et al., 1989; Hiendleder et al., 2000). Three additional activin-related beta subunits (betaC and betaE in mammals and betaD in Xenopus laevis) also have been identified but do not appear to play a role in FSH regulation (Hotten et al., 1995; Oda et al., 1995; Fang et al., 1996, 1997; Loveland et al., 1996; Schmitt el al., 1996; O'Bryan et al., 2000; Lau et al., 2000). To date, only one alpha subunit has been reported. The inhibin subunits are expressed in various tissues (Meunier et al., 1988a, 1988b) but the gonads are clearly the primary source of circulating inhibins (Woodruff et al., 1996). While inhibins act in a paracrine role in some tissues (Hsueh et al., 1987), their best-understood roles are as endocrine regulators of pituitary FSH. Activins also were purified from follicular fluid but because circulating activin levels generally are low, most actions of the hormones are likely to be paracrine in nature (Woodruff, 1998). Several reviews in the past decade have clearly and thoroughly addressed the characterization and regulation of the inhibins and activins and their roles in reproductive function (Vale et al., 1988; Ying, 1988; Woodruff and Mayo, 1990; Mayo, 1994; Woodruff and Mather, 1995). In this chapter, we focus our attention on more-recent developments in inhibin research. First, we discuss differential regulation of inhibin isoforms. Specifically, we describe patterns of inhibin A and B secretion in the context of the female reproductive cycle. Second, we review molecular mechanisms of inhibin subunit regulation. Third, while inhibins are best known for their role in pituitary FSH regulation, other functions of the ligands are becoming better understood. We review the animal and human literature addressing the possible role of inhibins in gonadal cancers. While we know "what" inhibins do in various contexts, we have a very limited understanding of "how" the ligands have their effects on target cells. Recently, candidate inhibin receptor molecules have been identified (Draper et al., 1998; Hertan et al., 1999; Lewis et al., 2000; Chung et al., 2000). Next, we detail our current understanding of inhibin signal transduction. Finally, in light of the data reviewed here, we pose questions and outline future directions for inhibin research. While this review is concerned primarily with expression and function of inhibin, activin function and mechanisms of action are described where necessary to shed light on inhibin function. Several reviews of activin's role in reproductive and other processes can be found elsewhere (Woodruff, 1998; Pangas and Woodruff, 2000).

Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions.

The activins (dimers of betaA or betaB subunits, encoded by the genes Inhba and Inhbb, respectively) are TGF-beta superfamily members that have roles in reproduction and development. Whereas mice homozygous for the Inhba-null allele demonstrate disruption of whisker, palate and tooth development, leading to neonatal lethality, homozygous Inhbb-null mice are viable, fertile and have eye defects. To determine if these phenotypes were due to spatiotemporal expression differences of the ligands or disruption of specific ligand-receptor interactions, we replaced the region of Inhba encoding the mature protein with Inhbb, creating the allele Inhbatm2Zuk (hereafter designated InhbaBK). Although the craniofacial phenotypes of the Inhba-null mutation were rescued by the InhbaBK allele, somatic, testicular, genital and hair growth were grossly affected and influenced by the dosage and bioactivity of the allele. Thus, functional compensation within the TGF-beta superfamily can occur if the replacement gene is expressed appropriately. The novel phenotypes in these mice further illustrate the usefulness of insertion strategies for defining protein function.

Activin A-induced HepG2 liver cell apoptosis: involvement of activin receptors and smad proteins.

A balance between cell proliferation and apoptosis is important for regulating normal liver function. Proteins of the transforming growth factor-beta superfamily are known to be important mediators of apoptosis in the liver. In this study we demonstrate that activin A potently induces apoptotic cell death in a hepatoma cell line, HepG2 cells. To determine the roles of activin receptors and downstream signaling proteins in activin A-induced apoptosis in these cells, the activin signaling pathway was analyzed using the transcription of an activin-responsive reporter gene, p3TP-Lux, as an assay. Although individual activin receptors had little effect on transcriptional activity, coexpression of an activin type I receptor and a type II receptor significantly increased both basal and activin-induced transcriptional activation, with the combination ofreceptors IB and IIB being the most potent. Similarly, expression of individual Smad proteins had only a modest effect on reporter gene activity, but the combination of Smad2 and Smad4 strongly stimulated transcription. Activin signaling induced a rapid relocation of Smad2 to the nucleus, as determined using a green fluorescence protein-Smad2 fusion protein. In contrast, green fluorescence protein-Smad4 remained localized to the cytoplasm unless it was coexpressed with Smad2. In agreement with the transcriptional response assays, overexpression or suppression of activin signaling components in HepG2 cells altered apoptosis. Overexpression of receptors IB and IIB or Smad proteins 2 and 4 stimulated apoptosis, whereas dominant negative mutant forms of the activin type IIB receptor or Smad2 blocked activin-stimulated apoptosis. These studies suggest that signaling from the cell surface to the nucleus through Smad proteins is a required component of the activin A-induced cell death process in liver cells.

Ovarian follicular concentrations of activin, follistatin, inhibin, insulin-like growth factor I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), IGFBP-3, and vascular endothelial growth factor in spontaneous menstrual cycles of normal women of advanced reproductive age.

Previous studies indicate that the menstrual cycles of older reproductive age women are characterized by a selective elevation of FSH associated with early development and ovulation of a dominant follicle. Several intraovarian hormones and growth factors have been identified that appear to serve important paracrine roles. The purpose of this study was to examine follicular fluid (FF) hormones and growth factors in the dominant follicle of unstimulated cycles of older, ovulatory women. We aspirated FF from the preovulatory dominant follicle in natural menstrual cycles of older subjects (age, 40-45 yr; n = 20) and younger controls (age, 20-25 yr; n = 19). FF was analyzed for estradiol, progesterone, testosterone, androstenedione, inhibin A and B, total activin A, total follistatin, insulin-like growth factor I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), IGFBP-3, and vascular endothelial growth factor (VEGF) concentrations. We found that the dominant follicles from older women contain normal concentrations of steroids, inhibin A and B, IGF-II, IGFBP-2, and IGFBP-3; increased concentrations of follistatin, activin A, and VEGF; and decreased concentrations of IGF-I. Therefore, under the influence of elevated FSH, the dominant follicle in older women is highly competent in terms of hormone and growth factor secretion. We postulate that elevated FF activin may be related to the early ovulation observed in older women, whereas elevated VEGF may be related to the meiotic spindle abnormalities observed in the oocytes of older reproductive age women.

Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis.

The role of FSH in gonadal tumorigenesis and, in particular, in human ovarian cancer has been debated. It is also unclear what role the elevated FSH levels in the inhibin-deficient mouse play in the gonadal tumorigenesis. To directly assess the role of FSH in gonadal growth, differentiation, and gonadal tumorigenesis, we have generated both gain-of-function and loss-of-function transgenic mutant mice. In the gain-of-function model, we have generated transgenic mice that ectopically overexpress human FSH from multiple tissues using a mouse metallothionein-1 promoter, achieving levels far exceeding those seen in postmenopausal women. Male transgenic mice are infertile despite normal testicular development and demonstrate enlarged seminal vesicles secondary to elevated serum testosterone levels. Female transgenic mice develop highly hemorrhagic and cystic ovaries, have elevated serum estradiol and progesterone levels, and are infertile, mimicking the features of human ovarian hyperstimulation and polycystic ovarian syndromes. Furthermore, the female transgenic mice develop enlarged and cystic kidneys and die between 6-13 weeks as a result of urinary bladder obstruction. In a complementary loss-of-function approach, we have generated double-homozygous mutant mice that lack both inhibin and FSH by a genetic intercross. In contrast to male mice lacking inhibin alone, 95% of which die of a cancer cachexia-like syndrome by 12 weeks of age, only 30% of the double-mutant male mice lacking both FSH and inhibin die by 1 yr of age. The remaining double-mutant male mice develop slow-growing and less hemorrhagic testicular tumors, which are noted after 12 weeks of age, and have minimal cachexia. Similarly, the double-mutant female mice develop slow-growing, less hemorrhagic ovarian tumors, and 70% of these mice live beyond 17 weeks. The double-mutant mice demonstrate minimal cachexia in contrast to female mice lacking only inhibin, which develop highly hemorrhagic ovarian tumors, leading to cachexia and death by 17 weeks of age in 95% of the cases. The milder cachexia-like symptoms of the inhibin and FSH double-mutant mice are correlated with low levels of serum estradiol and activin A and reduced levels of aromatase mRNA in the gonadal tumors. Based on these and our previous genetic analyses, we conclude that elevated FSH levels do not directly cause gonadal tumors. However, these results suggest FSH is an important trophic modifier factor for gonadal tumorigenesis in inhibin-deficient mice.

Preoperative serum level of inhibin A is an independent prognostic factor for the survival of postmenopausal women with epithelial ovarian carcinoma.

BACKGROUND: The aim of this study was to determine the prognostic significance of preoperative serum inhibin and activin levels in postmenopausal women with epithelial ovarian carcinoma (EOC) by correlating serum levels with disease parameters, including tumor stage and grade and patient age. METHODS: Serum levels of inhibin A, inhibin B, pro-alpha C, activin A, and activin B were quantitated with sensitive and specific two-site enzyme-linked immunosorbent assays (ELISAs) in samples collected from 44 postmenopausal women diagnosed with EOC. Serum was obtained within 14 days prior to primary tumor reductive surgery and stored at -55 degrees C. All patients underwent definitive surgical staging and cytoreduction at Mayo Clinic and were followed for at least 5 years or until death. Postoperative adjuvant therapy was selected based on stage of disease. Demographics included 5 Stage I, 2 Stage II, 33 Stage III, and 4 Stage IV tumors, and the predominant histology was serous subtype and poorly differentiated grade. RESULTS: Inhibin A was detected in 98% of the serum samples (range, 0-12.18 pg/mL). Univariate analysis was used to demonstrate an association between patients with serum inhibin A levels exceeding the median (1.21 pg/mL) and compromised disease free (P = 0.025) and overall (P = 0.006) survival. While the 5 year disease free survival (DFS) for the entire population was 32%, the corresponding DFS rates for patients with inhibin A levels above and below the median were 10% and 43%, respectively. Similarly, the 5-year overall survival (OS) for the entire population was 35%, compared with 16% for patients above and 47% for patients below the median inhibin A level. Stepwise regression analysis that incorporated age, stage, grade, and inhibin A levels identified serum inhibin A levels above the median to be the most cogent predictor of DFS and OS. CONCLUSIONS: Preoperative serum inhibin A levels provided valuable prognostic information independent of age, stage, and grade in a postmenopausal cohort given standardized treatment for EOC.

Regulation of cellular and system function by activin.

Activin is an important molecule that regulates hormonogenesis, cellular homeostasis (divide or die pathways), and differentiation programs (developmentally and in adult cells). The cellular mechanisms that integrate an activin signal into a physiological response include a binary receptor complex and tandem serine threonine kinases, intracellular signal mediators, and nuclear transcription factors. Activin antagonists (inhibins) and bioneutralizing binding proteins (follistatins) act as gating molecules to ensure accurate delivery of activin signals to cellular machinery. Correct execution of an activin cue intracellularly permits actions as fundamental as embryonic mesoderm development, neuronal survival, hematopoietic function, and reproductive cyclicity. Absent or incorrect activin signaling results in phenotypes as catastrophic as embryonic lethality, tumor formation, and infertility. The general ways in which a cell senses and responds to an activin signal will be reviewed in the first part of this paper. The role of this ligand in reproductive function will also be examined as a specific example of activin activity.

Identification of an inhibin receptor in gonadal tumors from inhibin alpha-subunit knockout mice.

Inhibins and activins are dimeric proteins that are functional antagonists and are structurally related to the transforming growth factor-beta (TGFbeta) family of growth and differentiation factors. Receptors for activin and TGFbeta have been identified as dimers of serine-threonine kinase subunits that regulate cytoplasmic proteins known as Smads. Despite major advances in our understanding of activin and TGFbeta receptors and signaling pathways, little is known about inhibin receptors or the mechanism by which this molecule provides a functionally antagonistic signal to activin. Studies described in this paper indicate that an independent inhibin receptor exists. Numerous tissues were examined for inhibin-specific binding sites, including the developing embryo, in which the spinal ganglion and trigeminal ganglion-bound iodinated inhibin A. Sex cord stromal tumors, derived from male and female inhibin alpha-subunit-deficient mice, were also identified as a source of inhibin receptor. Abundant inhibin and few activin binding sites were identified in tumor tissue sections by in situ ligand binding using iodinated recombinant human inhibin A and 125I-labeled recombinant human inhibin A. Tumor cell binding was specific for each ligand (competed by excess unlabeled homologous ligand and not competed by heterologous ligand). Based on these results and the relative abundance and homogeneity of tumor tissues versus the embryonic ganglion, tumor tissues were homogenized, membrane proteins were purified, and putative inhibin receptors were isolated using an inhibin affinity column. Four proteins were eluted from the column that bind iodinated inhibin but not iodinated activin. These data suggest that inhibin-specific membrane-associated proteins (receptors) exist.