Oncofertility Publications - All

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The Mouse Follicle Microenvironment Regulates Antrum Formation and Steroid Production: Alterations in Gene Expression Profiles

Abstract

Folliculogenesis is a coordinated process, and the genes that regulate development are difficult to investigate in vivo. In vitro culture systems permit the assessment of individual follicles during development, thereby enabling gene expression patterns to be monitored during follicle development. Mouse multilayered secondary follicles (150-180 microm in diameter) were cultured in three-dimensional matrices of varying physical properties for up to 8 days. During this period of follicle growth in vitro, antrum formation and steroid production were monitored, and mRNA was isolated. The expression levels of genes (Star, Cyp11a1, Cyp17a1, Hsd3b1, Cyp19a1, Fshr, Lhcgr, Aqp7, Aqp8, Aqp9, and Hif1a) were measured and correlated to follicle developmental status. Follicles that developed an antrum and produced appropriate levels of estrogen and progesterone had unchanging expression of Star, Aqp7, Aqp8, and Hif1a and a 34-fold increase in Cyp19a1 expression at Day 8 of culture and had elevated Lhcgr at Days 6 and 8 of culture. Follicles that were healthy but did not form an antrum or produce appropriate levels of steroids, however, demonstrated increasing levels of Star, Aqp7, Aqp8, and Hif1a and a 15-fold increase in Cyp19a1 at Day 8 of culture, and Lhcgr levels were not elevated until Day 8 of culture. To our knowledge, this study provides the first temporal analysis of gene expression using individual culture in alginate hydrogels that correlates growth and steroidogenesis during follicle development and identifies expression patterns in healthy follicles and in developmentally disadvantaged follicles.

Erin R. West-Farrell, Min Xu, Monica A. Gomberg, Yee Hoong Chow, Teresa K. Woodruff, and Lonnie D. Shea; BIOLOGY OF REPRODUCTION 80, 432–439 (2009)

 		Basic Science, Mouse Follicle Culture, Research Resources, Oncofertility-Related Publications
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Secondary Follicle Growth and Oocyte Maturation by Culture in Alginate Hydrogel Following Cryopreservation of the Ovary or Individual Follicles

Abstract

An option for fertility preservation for women facing a cancer diagnosis involves the cryopreservation of ovarian tissue for later re-transplantation or in vitro culture, with in vitro culture preferred to avoid reintroduction of the cancer. Small, immature follicles survive the freeze-thaw process, and can be matured through in follicle maturation (IFM) that involves an initial growth of the follicle and subsequent maturation of the oocyte. The ovarian tissue can be cryopreserved in two forms: (i) cortical strips consisting of follicles and surrounding stroma (Cryo-Ov) or (ii) individually isolated follicles (Cryo-In). The aim of this study was to assess the follicle growth and oocyte maturation for follicles that were cryopreserved either as strips or individually using a slow-freezing cryopreservation method. The two follicle groups, together with non-cryopreserved control follicles, were grown in an alginate-based three-dimensional culture system for 12 days. The overall survival, size increase and antrum formation rates were comparable among the three groups. At day 12 of culture, Androstenedione levels were decreased in the Cryo-Ov group relative to the other two, and the ratio of progesterone to estradiol was increased in the two cryopreserved groups relative to the control. Both Gja1 (known as connexin 43) and Gja4 (known as connexin 37) mRNA expression were decreased at day 6 in the cryopreserved groups relative to controls, and by day 12, Gja1 was similar for all three groups. Moreover, Cryo-In resulted in lower GVBD rate indicating some impaired oocyte development. Overall, the present study demonstrated that mouse preantral follicles, either within ovarian tissues or individually isolated, could be successfully cryopreserved by the slow-freezing method, as evidenced by post-thaw follicle development and steroidgenesis, oocyte maturation and molecular markers for oocyte and/or granulosa cells connection.

Min Xu, Anna Banc, Teresa K. Woodruff, Lonnie D. Shea; Biotechnology and Bioengineering, Vol. 103, No. 2, June 1, 2009

 		Basic Science, Mouse Follicle Culture, Research Resources, Oncofertility-Related Publications
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REVIEW Preserving Female Fertility Following Cancer Treatment: Current Options and Future Possibilities

Abstract

Children and women of reproductive age are increasingly surviving cancer diagnoses, and therefore long-term quality-of-life issues are of greater importance at the time of diagnosis. Cancer therapies including radiation and chemotherapy can be detrimental to fertility, and therefore many patients are motivated to preserve fertility prior to cancer treatment. The only highly successful method in preserving fertility to date is embryo cryopreservation, which may not be appropriate for some patients due to age, delay in treatment, cancer type and stage, as well as availability of an acceptable sperm donor. Alternative methods including oocyte cryopreservation and ovarian tissue banking may also preserve fertility while providing additional flexibility to patients. In vitro ovarian follicle maturation following tissue banking is one potential approach that would not require a delay in cancer therapy for ovarian stimulation, would not require an immediate sperm donor, and does not carry the risk of reintroducing malignant cells following tissue transplantation. In vitro follicle culture systems have resulted in successful live births in the mouse. However, many challenges must be addressed in translating the system to the human. This review summarizes current approaches to fertility preservation and discusses recent developments and future challenges in developing a human in vitro follicle culture system.

Erin R. West, PhD, Mary B. Zelinski, PhD, Laxmi A. Kondapalli, MD, Clarisa Gracia, MD, Jeffrey Chang, MD, Christos Coutifaris, MD, PhD, John Critser, PhD, Richard L. Stouffer, PhD, Lonnie D. Shea, PhD, and Teresa K. Woodruff, PhD; Pediatr Blood Cancer 2009;53:289–295

 		Oncofertility and Fertility Preservation, Clinical Science and Medicine, Research Resources, Oncofertility-Related Publications
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Oncofertility: Fertility Preservation for Cancer Survivors TABLE OF CONTENTS

Part I. Introduction

  1. The Emergence of a New Interdiscipline: Oncofertility by Teresa K. Woodruff, PhD

Part II. Fertility Risk and Treatment Options

  1. Fertility Management for Women with Cancer by Sanjay K. Agarwal, MD and R. Jeffrey Chang, MD
  2. Fertility Preservation in Adult Male Cancer Patients by Robert E. Brannigan, MD
  3. Managing Fertility in Childhood Cancer Patients by Kimberley J. Dilley, MD, MPH
  4. Fertility Risk in Pediatric and Adolescent Cancers by Clarisa R. Gracia, MD, MSCE and Jill P. Ginsberg, MD

Part III. Oncofertility Techniques and Research

  1. Bioengineering and the Ovarian Follicle by Min Xu, PhD, Teresa K. Woodruff, PhD, and Lonnie D. Shea, PhD
  2. The Science of Cryobiology by Steven F. Mullen, PhD and John K. Critser, PhD
  3. Ovarian Tissue Cryopreservation and Transplantation: Banking Reproductive Potential for the Future by David Lee, MD
  4. Progress, History and Promise of Ovarian Cryopreservation and Transplantation for Pediatric Cancer Patients by Yasmin Gosiengfiao, MD

Part IV. Health Care Decision-Making

  1. Oncofertility and the Social Sciences by Karrie Ann Snyder, PhD
  2. Shared Decision Making:  Fertility and Pediatric Cancers by Marla L. Clayman, PhD, MPH, Kathleen M. Galvin, PhD, and Paul Arntson, PhD

Part V. Ethical and Psychosocial Impact of Cancer-Related Infertility

  1. Today's Research, Tomorrows Cures:  The Ethical Implications of Oncofertility by Leilah E. Backhus, MD, MS and Laurie Zoloth, PhD
  2. The Psychosocial Context of Cancer-Related Infertility by Matthew J. Loscalzo, MSW and Karen L. Clark, MS
  3. Childhood Cancer: Fertility and Psychosocial Implications by Karen E. Kinahan, MS, RN, APRN, Aarati Didwania, MD, and Carrie L. Nieman, BS
  4. Fertility Preservation and Adolescent Cancer Patients: Lessons from Adult Survivors of Childhood Cancer and Their Parents by Carrie L. Nieman, BS, Karen E. Kinahan, MS, RN, APRN, Susan E. Yount, PhD, Sarah K. Rosenbloom, PhD, Kathleen J. Yost, PhD, Elizabeth A. Hahn, MA, Timothy Volpe, Kimberley J. Dilley, MD, MPH, Laurie Zoloth, PhD, and Teresa K. Woodruff, PhD

Part VI. Training in a New Medical Discipline and Medical Guidelines

  1. Oncofertility: A New Medical Discipline and the Emerging Scholar by Laxmi A. Kondapalli, MD, MS
  2. Oncofertility Consortium® Consensus Statement:  Guidelines for Ovarian Tissue Cryopreservation by Leilah E. Backhus, MD, MS, Laxmi A. Kondapalli, MD, MS, R. Jeffrey Chang, MD, Christos Coutifaris, MD, PhD, Ralph Kazer, MD, and Teresa K. Woodruff, PhD

Part VII.  Patient Stories and Oncofertility

  1. Personal Accounts of Cancer and Infertility provided by Fertile Hope
Oncofertility and Fertility Preservation, Clinical Science and Medicine, Research Resources, Oncofertility-Related Publications
Encapsulated Three-Dimensional Culture Supports Development of Nonhuman Primate Secondary Follicles

Abstract

In vitro ovarian follicle cultures may provide fertility-preserving options to women facing premature infertility due to cancer therapies. An encapsulated three-dimensional (3-D) culture system utilizing biomaterials to maintain cell-cell communication and support follicle development to produce a mature oocyte has been developed for the mouse. We tested whether this encapsulated 3-D system would also support development of nonhuman primate preantral follicles, for which in vitro growth has not been reported. Three questions were investigated: Does the cycle stage at which the follicles are isolated affect follicle development? Does the rigidity of the hydrogel influence follicle survival and growth? Do follicles require luteinizing hormone (LH), in addition to follicle-stimulating hormone (FSH), for steroidogenesis? Secondary follicles were isolated from adult rhesus monkeys, encapsulated within alginate hydrogels, and cultured individually for </=30 days. Follicles isolated from the follicular phase of the menstrual cycle had a higher survival rate (P < 0.05) than those isolated from the luteal phase; however, this difference may also be attributed to differing sizes of follicles isolated during the different stages. Follicles survived and grew in two hydrogel conditions (0.5% and 0.25% alginate). Follicle diameters increased to a greater extent (P < 0.05) in the presence of FSH alone than in FSH plus LH. Regardless of gonadotropin treatment, follicles produced estradiol, androstenedione, and progesterone by 14-30 days in vitro. Thus, an alginate hydrogel maintains the 3-D structure of individual secondary macaque follicles, permits follicle growth, and supports steroidogenesis for </=30 days in vitro. This study documents the first use of the alginate system to maintain primate tissue architecture, and findings suggest that encapsulated 3-D culture will be successful in supporting the in vitro development of human follicles.

 

Min Xu, Erin R. West-Farrell, Richard L. Stouffer, Lonnie D. Shea, Teresa K. Woodruff, and Mary B. Zelinski; Biology of Reproduction(3):587-94 Sep.8, 2009

 		Basic Science, Research Resources, Nonhuman Primate Follicle Culture, Oncofertility-Related Publications
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In Vitro Grown Human Ovarian Follicles From Cancer Patients Support Oocyte Growth

Abstract

BACKGROUND: Young female adult and adolescent cancer patients facing life-preserving but fertility-threatening chemo- or radiation-therapy are increasingly seeking options to protect their reproductive potential. Ovarian tissue cryopreservation with transplantation is a promising technique to safeguard fertility in cancer patients. However, this method may risk re-introduction of the original cancer to the survivor of the disease. Thus, developing a method for in vitro growth of immature follicles may provide a method for fertility restoration in the future.

METHODS: Human secondary follicles were isolated from ovarian tissues obtained from cancer patients and grown in vitro within a bio-engineered culture system for 30 days.

RESULTS: Human ovarian follicles became steroidogenically active, and developed from the early secondary to antral stage in vitro. The follicles contained healthy, growing oocytes that were connected by transzonal projections between the somatic cells and oocyte.

CONCLUSIONS: Our data support the notion that human follicle development can be achieved in vitro in a bio-engineered culture system. More studies are required to investigate whether the fully sized oocytes obtained from in vitro grown follicle are competent to resume meiosis and be fertilized.

Min Xu, Susan L. Barrett, Erin West-Farrell, Laxmi A. Kondapalli,, Sarah E. Kiesewetter, Lonnie D. Shea, and Teresa K. Woodruff; Human Reproduction Vol.24, No.10 2531-40 Oct. 24, 2009

 		Basic Science, Research Resources, Human Follicle Culture, Oncofertility-Related Publications
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Interpenetrating Fibrin–Alginate Matrices For In Vitro Ovarian Follicle Development

Abstract

In this report, we investigate the fibrin-alginate interpenetrating network (FA-IPN) to provide dynamic cell-responsive mechanical properties, which we apply to the in vitro growth of ovarian follicles. The mechanical properties and polymerization rate of the gels were investigated by rheology, and the fiber structure was imaged by electron microscopy. Using a mouse model, two-layered secondary follicles were encapsulated in FA-IPNs, and growth, morphology, hormone production, fibrin degradation rate and the numbers of competent eggs were assessed. The initial mechanics of the FA-IPN are determined by the composite material, and subsequent degradation of fibrin by the encapsulated cells would produce a material with mechanical properties due to the alginate alone. The rate of meiotically competent oocytes produced by culture in FA-IPN was 82%, which was significantly greater than in alginate alone. This increase in oocyte quality is an important step in identifying 3D culture systems that can provide a fundamental tool to investigate follicle maturation, and may be applied to promote the growth of human follicles, which can be used to provide reproductive options for women facing a cancer diagnosis.

Ariella Shikanov, Min Xu, Teresa K. Woodruff, Lonnie D. Shea; Biomaterials Vol. 29 5476-85 Oct 30, 2009

 		Basic Science, Biomaterials, Research Resources, Oncofertility-Related Publications
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The Primordial Pool of Follicles and Nest Breakdown In Mammalian Ovaries

Abstract

The creation of the pool of follicles available for selection and ovulation is a multi-faceted, tightly regulated process that spans the period from embryonic development through to the first reproductive cycle of the organism. In mice, this development can occur in mere weeks, but in humans, it is sustained for years. Embryonic germ cell development involves the migration of primordial germs cells to the genital ridge, and the mitotic division of germ cell nuclei without complete cytokinesis to form a multi-nucleated syncytia, or germ cell nest. Through combined actions of germ cell apoptosis and somatic cell migration, the germ cell nuclei are packaged, with surrounding granulosa cells, into primordial follicles to form the initial follicle pool. Though often dismissed as quiescent and possibly uninteresting, this initial follicle pool is actually quite dynamic. In a very strictly controlled mechanism, a large portion of the initial primordial follicles formed is lost by atresia before cycling even begins. Remaining follicles can undergo alternate fates of continued dormancy or selection leading to follicular growth and differentiation. Together, the processes involved in the fate decisions of atresia, sustained dormancy, or activation carve out the follicle pool of puberty, the pool of available oocytes from which all future reproductive cycles of the female can choose. The formation of the initial and pubertal follicle pools can be predictably affected by exogenous treatment with hormones or molecules such as activin, demonstrating the ways the ovary controls the quality and quantity of germ cells maintained. Here, we review the biological processes involved in the formation of the initial follicle pool and the follicle pool of puberty, address the alternate models for regulating germ cell number and outline how the ovary quality-controls the germ cells produced.

Candace Tingen, Alison Kim, and Teresa K. Woodruff; Molecular Human Reproduction Vol. 15 No. 12 795-803 Dec 15, 2009

 		Basic Science, Research Resources, Mammalian Folliculogenesis, Oncofertility-Related Publications
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Fertility Preservation For Female Cancer Patients: Early Clinical Experience

Abstract

OBJECTIVE: To characterize the early experience of a clinical program designed to provide strategies for fertility preservation to female cancer patients about to undergo chemotherapy or radiation therapy.

DESIGN: Retrospective chart review; case-control study.

SETTING: Academic medical center.

PATIENT(S): Sixty-five female cancer patients and 57 age-matched infertility patients.

INTERVENTION: Enrollment in a program for fertility preservation.

MAIN OUTCOME MEASURE(S): Choice of active participation, fertility preservation option selection, clinical outcomes of patients undergoing oocyte retrieval, attitudes regarding embryo disposition.

RESULT(S): Of 65 patients referred to the program, 18 declined to undergo embryo, oocyte, or tissue cryopreservation. Six were found not to be eligible for medical reasons. Of the remaining 41 patients, 35 chose to cryopreserve embryos, four chose to cryopreserve oocytes, and two chose to undergo ovarian tissue freezing. Fewer oocytes were recovered from the embryo cryopreservation group when compared with an age-matched control group, but the mean number of zygotes generated was similar. Attitudes regarding embryo disposition were different between the two groups. No serious clinical sequelae resulted from participation.

CONCLUSION(S): Fertility preservation techniques employing available technology may provide safe and practical options to female cancer patients facing chemotherapy or radiation therapy. A significant number of otherwise appropriate participants decline active management. Cancer patients display different attitudes regarding embryo disposition when compared with infertility patients without cancer.

Susan C. Klock, John X. Zhang, and Ralph R. Kazer; Fertility and Sterility, In Press 2009

 		Oncofertility and Fertility Preservation, Clinical Science and Medicine, Research Resources, Oncofertility-Related Publications
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A Novel Two-Step Strategy For In Vitro Culture Of Early-Stage Ovarian Follicles In The Mouse

Abstract

OBJECTIVE: To develop an in vitro strategy to support the growth of early-stage follicles and produce mature oocytes competent for fertilization.

DESIGN: Whole ovaries from 8-day-old mice were cultured for 4 days, and then secondary follicles were isolated and cultured for 12 days in a three-dimensional alginate or fibrin-alginate (FA) hydrogel matrix.

SETTING: University-affiliated laboratory.

ANIMALS: Mice.

INTERVENTION(S): None.

MAIN OUTCOME MEASURES: Histologic evaluation of follicle development, steroid hormone production, and rates of oocyte maturation, oocyte fertilization, and embryo formation.

RESULT(S): Culture of 8-day-old mouse ovaries for 4 days resulted in transition of the follicle population from primordial and primary follicles to secondary follicles, similar to that seen in a 12-day-old ovary. Isolated secondary follicles cultured for 12 days showed larger increases in oocyte diameter and more frequent antrum formation and theca cell differentiation in the FA-hydrogel matrix compared with the alginate matrix. Steroid hormone secretion patterns were consistent with the changes in follicle morphology and cell differentiation observed in the cultured follicles. Compared with oocytes from alginate follicle cultures, a greater number of oocytes retrieved from the FA-based follicle cultures progressed to metaphase I, reached metaphase II, and could be fertilized and cleaved to two-cell embryos. The organ culture plus FA-hydrogel follicle culture strategy produced a very high rate of oocyte progression to metaphase II (88 +/- 8.7% [mean +/- SEM]) and formation of two-cell embryos (54 +/- 4%).

CONCLUSION(S): A strategy combining whole ovary culture of early-stage follicles and subsequent FA hydrogel in vitro follicle culture produced a high percentage of oocytes competent for fertilization; this might provide new options for fertility preservation in women and prepubertal girls facing fertility-threatening diseases or treatments.

Shi Ying Jin, Lei Lei, Ariella Shikanov, Lonnie D. Shea, and Teresa K. Woodruff; Fert. Ster. ePub ahead of print. 2010

 		Basic Science, Mouse Follicle Culture, Research Resources, Oncofertility-Related Publications
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Novel Approach for the Three-Dimensional Culture of Granulosa Cell–Oocyte Complexes

Abstract

The in vitro culture of immature ovarian follicles is used to examine the factors that regulate follicle development and may ultimately provide options for reproductive infertility. The objective of this study was to develop a three-dimensional in vitro culture system for the growth and development of individual granulosa cell-oocyte complexes. An alginate hydrogel was used to encapsulate immature mouse granulosa cell-oocyte complexes (GOCs) that were subsequently maintained in a serum-free in vitro culture. An overall incorporation efficiency of 50% was achieved. The complexes were assessed by transmission electron microscopy for changes in ultrastructure during in vitro growth. The architecture of the follicular complex was maintained during the encapsulation and the subsequent culture. The granulosa cells proliferated, and the oocytes also grew in volume and obtained the structural characteristics of mature oocytes including cortical granule formation, a well-developed zona pellucida with microvilli, normal mitochondria, and lattice-like structures in the cytoplasm. Oocytes retrieved and matured were able to resume meiosis, a necessary step for proper development. Thus, this system represents a new in vitro methodology for growth of individual granulosa cell-oocyte complexes.

Stephanie A. Pangas, Hahhad Saudye, Lonnie D. Shea, and Teresa K. Woodruff; Tissue Engineering Vol. 9 No. 5 1013-1021 2003

 		Basic Science, Biomaterials, Research Resources, Oncofertility-Related Publications
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Regulation of Mouse Follicle Development by Follicle-Stimulating Hormone in a Three-Dimensional In Vitro Culture System Is Dependent on Follicle Stage and Dose

Abstract

The developmental requirements of ovarian follicles are dependent on the maturation stage of the follicle; in particular, elegant studies with genetic models have indicated that FSH is required for antral, but not preantral, follicle growth and maturation. To elucidate further the role of FSH and other regulatory molecules in preantral follicle development, in vitro culture systems are needed. We employed a biomaterials-based approach to follicle culture, in which follicles were encapsulated within matrices that were tailored to the specific developmental needs of the follicle. This three-dimensional system was used to examine the impact of increasing doses of FSH on follicle development for two-layered secondary (100-130 microm; two layers of granulosa cells surrounding the oocyte) and multilayered secondary (150-180 microm, several layers of granulosa cells surrounding the oocyte) follicles isolated from mice. Two-layered secondary follicles were FSH responsive when cultured in alginate-collagen I matrices, exhibiting FSH dose-dependent increases in follicle growth, lactate production, and steroid secretion. Multilayered secondary follicles were FSH dependent, with follicle survival, growth, steroid secretion, metabolism, and oocyte maturation all regulated by FSH. However, doses greater than 25 mIU/ml of FSH negatively impacted multilayered secondary follicle development (reduced follicle survival). The present results indicate that the hormonal and environmental needs of the follicular complex change during the maturation process. The culture system can be adapted to each stage of development, which will be especially critical for translation to human follicles that have a longer developmental period.

Pamela K. Kreeger, Nisha N. Fernandes, Teresa K. Woodruff, and Lonnie D. Shea; Biology of Reproduction Vol 73 No 5 942-50 June 29 2005

 		Basic Science, Mouse Follicle Culture, Research Resources, Oncofertility-Related Publications
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The In Vitro Regulation Of Ovarian Follicle Development Using Alginate-Extracellular Matrix Gels

Abstract

The extracellular matrix (ECM) provides a three-dimensional structure that promotes and regulates cell adhesion and provides signals that direct the cellular processes leading to tissue development. In this report, synthetic matrices that present defined ECM components were employed to investigate these signaling effects on tissue formation using ovarian follicle maturation as a model system. In vitro systems for follicle culture are being developed to preserve fertility for women, and cultures were performed to test the hypothesis that the ECM regulates follicle maturation in a manner that is dependent on both the ECM identity and the stage of follicle development. Immature mouse follicles were cultured within alginate-based matrices that were modified with specific ECM components (e.g., laminin) or RGD peptides. The matrix maintains the in vivo like morphology of the follicle and provides an environment that supports follicle development. The ECM components signal the somatic cells of the follicle, affecting their growth and differentiation, and unexpectedly also affect the meiotic competence of the oocyte. These effects depend upon both the identity of the ECM components and the initial stage of the follicle, indicating that the ECM is a dynamic regulator of follicle development. The development of synthetic matrices that promote follicle maturation to produce meiotically competent oocytes may provide a mechanism to preserve fertility, or more generally, provide design principles for scaffold-based approaches to tissue engineering.

Pamela K. Kreeger, Jason W. Deck, Teresa K. Woodruff, and Lonnie D. Shea; Biomaterials Vol 27 No 5 714-23 2006

 		Basic Science, Biomaterials, Research Resources, Oncofertility-Related Publications
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Tissue-Engineered Follicles Produce Live, Fertile Offspring

Abstract

Oocytes grown in vitro are of low quality and yield few live births, thus limiting the ability to store or bank the ova of women wishing to preserve their fertility. We applied tissue engineering principles to the culture of immature mouse follicles by designing an alginate hydrogel matrix to maintain the oocyte's 3- dimensional (3D) architecture and cell-cell interactions in vitro. A 3D culture mimics the in vivo follicle environment, and hydrogel-encapsulated follicles develop mature oocytes within the capacity for fertilization similar to that of oocytes matured in vivo. Embryos derived from cultured oocytes fertilized in vitro and transferred to pseudopregnant female mice were viable, and both male and female offspring were fertile. Our results demonstrate that alginate hydrogel-based 3D in vitro culture of follicles permits normal growth and development of follicles and oocytes. This system creates new opportunities for discovery in follicle biology and establishes a core technology for human egg banks for preservation of fertility.

Min Xu, Pamela K. Kreeger, Lonnie D. Shea, and Teresa K. Woodruff; Tissue Engineering Vol 10 2739-46 Oct 13 2006

 		Basic Science, Mouse Follicle Culture, Research Resources, Oncofertility-Related Publications
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Identification of a Stage-Specific Permissive In Vitro Culture Environment for Follicle Growth and Oocyte Development

Abstract

The availability of viable oocytes is the limiting factor in the development of new reproductive techniques. Many attempts have been made to grow immature oocytes in vitro during recent decades. Recently, a modified alginate-based three-dimensional culture system was designed to support the growth and maturation of multilayered secondary follicles. This system was able to produce oocytes that successfully completed meiosis, fertilization, and development to the blastocyst stage. Subsequent attempts to culture two-layered secondary follicles were unsuccessful under the original conditions. Herein, we investigated the effect of alginate consistency on two-layered follicle growth and oocyte developmental competence by encapsulating follicles into alginate scaffolds of various concentrations. Although there were no significant differences in survival rates, 0.25% and 0.5% alginate supported more rapid growth of follicles and antrum formation compared with 1.5% and 1.0% alginate after 8 days of culture. Alginate scaffold concentration also affected the proliferation and differentiation of somatic cells (theca and granulosa cells), measured in terms of morphological changes, steroid profiles (androstenedione, estradiol, and progesterone), and specific molecular markers (Fshr, Lhcgr, and Gja1). Theca cell proliferation and steroid production were hindered in follicles cultured in 1.5% alginate. In vitro fertilization and embryo culture revealed that oocytes obtained from 0.25% alginate retained the highest developmental competence. Overall, the present study showed that the alginate scaffold consistency affects folliculogenesis and oocyte development in vitro and that the alginate culture system can and should be tailored to maximally support follicle growth depending on the size and stage of the follicles selected for culture.

Min Xu, Erin West, Lonnie D. Shea, and Teresa K. Woodruff; Biology of Reproduction Vol 75 916-923 Sep 6 2006

 		Basic Science, Biomaterials, Research Resources, Oncofertility-Related Publications
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Physical Properties of Alginate Hydrogels and Their Effects On In Vitro Follicle Development

Abstract

The mechanical properties and density of natural and synthetic extracellular matrices are known to affect cellular processes and regulate tissue formation. In this report, these factors were independently investigated for their role in ovarian follicle development. The matrix composition was controlled through decreasing the solids concentration or the molar mass of the encapsulating biomaterial, alginate. Decreasing matrix stiffness and solids concentration enhanced follicle growth and coordinated differentiation of the follicle cell types, as evidenced by antral cavity formation, theca cell differentiation, oocyte maturation, and relative hormone production levels. While a stiff environment favored high progesterone and androgen secretion, decreasing alginate stiffness resulted in estrogen production which exceeded progesterone and androgen accumulation. These studies reveal, for the first time, a direct link between the biomechanical environment and follicle function, and suggest a novel non-hormonal mechanism regulating follicle development.

Erin R. West, Min Xu, Teresa K. Woodruff, Lonnie D. Shea; Biomaterials Vol 30 4439-48 Oct 2007

 		Basic Science, Biomaterials, Research Resources, Oncofertility-Related Publications
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Extracellular Matrix Functions in Follicle Maturation

Abstract

The extracellular matrix (ECM) promotes and/or inhibits many cellular processes, including but not limited to proliferation, differentiation, and survival, which must occur for follicle growth and oocyte maturation. The ECM regulation of cellular processes in ovarian cells is being investigated in many animal models, including avian, rat, bovine, porcine, rabbit, sheep, human, and mouse. Granulosa cells are more frequently employed; however, the culture of intact follicles and ovaries has been developed and enables ECM functions in folliculogenesis to be studied. ECM components that have been examined are used individually (collagen, laminin, fibronectin) or collectively (Matrigel, isolated basal lamina, and ECM produced by cell lines) in both two- and three-dimensional model systems. In granulosa cell cultures, ECM affects morphology, aggregation and communication, survival, proliferation, and steroidogenesis; whereas follicle and ovary cultures demonstrate a regulation of folliculogenesis. This article describes the ECM functionality on ovarian cells throughout development, and highlights the potential of developing technologies to identify structure-function relationships in follicle maturation.

Courtney B. Berkholtz, Lonnie D. Shea, and Teresa K. Woodruff; Semin Reproducion Med Vol 24 No 4 262-9 Sep 2006

 		Basic Science, Research Resources, Mammalian Folliculogenesis, Oncofertility-Related Publications
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The Structures that Underlie Normal Reproductive Function

Abstract

The mechanisms and physiology of reproductive function have fascinated scientists throughout time. Recent cellular and molecular level structural studies have provided unprecedented insights into reproductive systems and signaling networks. This 'cutting edge' editorial provides a recent example in each of these areas, namely, the anatomical integrity of the follicle, the molecular structure of activin with its binding partners and the molecular regulation of inhibin. These three examples of structure informing function help explain reproductive health and may provide solutions to reproductive disease.

Thomas F. Lerch, Min Xu, Theodore S. Jardetzky, Kelly E. Mayo, Ishwar Radhakrishnan, Ralph Kazer, Lonnie D. Shea, and Teresa K. Woodruff; Molecular Cell Endocrinol 267 1-5 2006

 		Basic Science, Research Resources, Mammalian Folliculogenesis, Oncofertility-Related Publications
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Engineering the Follicle Microenvironment

Abstract

In vitro ovarian follicle culture provides a tool to investigate folliculogenesis, and may one day provide women with fertility-preservation options. The application of tissue engineering principles to ovarian follicle maturation may enable the creation of controllable microenvironments that will coordinate the growth of the multiple cellular compartments within the follicle. Three-dimensional culture systems can preserve follicle architecture, thereby maintaining critical cell-cell and cell-matrix signaling lost in traditional two-dimensional attached follicle culture systems. Maintaining the follicular structure while manipulating the biochemical and mechanical environment will enable the development of controllable systems to investigate the fundamental biological principles underlying follicle maturation. This review describes recent advances in ovarian follicle culture, and highlights the tissue engineering principles that may be applied to follicle culture, with the ultimate objective of germline preservation for females facing premature infertility.

Erin R. West, Lonnie D. Shea, and Teresa K. Woodruff; Semin Reproduction Med. Vol 25 No 4 287-99 July 2007

 		Basic Science, Biomaterials, Research Resources, Oncofertility-Related Publications
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Prepubertal Primordial Follicle Loss in Mice Is Not Due to Classical Apoptotic Pathways

Abstract

More than half of the primordial follicles that are formed by Day 6 of postnatal life in the mouse will be eliminated from the ovary by the time of puberty. Apoptosis, a form of programmed cell death, is one mechanism by which these follicles could be actively lost. To investigate whether apoptosis is responsible for the loss of primordial follicles, follicular atresia was examined during the prepubertal period, when follicles die and are cleared from the ovary at an extremely high rate. Four hallmarks of classical apoptosis were measured in follicles present in prepubertal ovaries. The primordial follicle cohort was not positively associated with nuclear condensation or cell shrinkage, activation of caspase 3, cleavage of poly(ADP ribose) polymerase 1 (PARP1), or fragmentation of DNA. These data are consistent with a nonapoptotic pathway that is responsible for small follicle death.

Candace M. Tingen, Sarah K. Bristol-Gould, Sarah E. Kiesewetter, Jason Tyler Wellington, Lonnie Shea, and Teresa K. Woodruff; Biology Reproduction Vol 81 No 1 16-25 July 2009

 		Basic Science, Research Resources, Mammalian Folliculogenesis, Oncofertility-Related Publications
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The Regulatory Role of Dicer In Folliculogenesis In Mice

Abstract

Dicer is the ribonuclease III for synthesis of mature functional microRNAs (miRNAs), which play an important role in regulating cell development. In the mouse ovary, the Dicer1 protein was expressed in both oocyte and granulosa cells of the follicle. In the present study, the role of miRNAs in mouse ovarian development was explored by using Dicer1 conditional knockout (cKO) mouse ovarian tissue (Amhr2 Cre/-; Dicer flox/flox), in which Dicer1 is deleted specifically in follicular granulosa cells. The morphology and gene expression profile of cKO and wild type (WT) mouse ovaries at various stages of development (day 4, day 8, 8 weeks and 8 months) were examined. Comparative analysis of the follicle number indicated that conditional inactivation of Dicer1 in the follicular granulosa cells led to an increased primordial follicle pool endowment, accelerated early follicle recruitment and more degenerate follicles in the cKO ovaries. In addition, significant differences were noted in the expression of some follicle development-related genes between cKO and WT mouse ovaries, such as Amh, Inhba, Cyp17a1, Cyp19a1, Zps, Gdf9 and Bmp15, suggesting the function of miRNAs in regulating gene expression is time- and gene-dependent. With the Dicer1 inactivation, mmu-mir-503, a miRNA that is more abundant in mouse ovary than in other tissues, was down-regulated significantly. Meanwhile, the expression of mmu-mir-503 decreased notably with follicle development in the gonadotropin-primed mouse ovary. Up-regulation of mmu-mir-503 in primary cultured granulosa cells resulted in the decreased expression of both the target gene and non-target gene at the transcriptional level, which involve genes related to granulosa cell proliferation and luteinization. In conclusion, Dicer1 plays important roles in follicular cell development through the differential regulation of gene expression.

Lei Lei, Shiying Jin, Gabriel Gonzalez, Richard R. Behringer, Teresa K. Woodruff; Mol Cell Endocrinol Epub ahead of print Sep 30 2009

 		Basic Science, Research Resources, Mammalian Folliculogenesis, Oncofertility-Related Publications
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Preservation of Fertility in Patients with Cancer

Jacqueline S. Jeruss and Teresa K. Woodruff; New England J. Med. Vol 360 No 9 902-911 Feb 26 2009.

The unique duality involved in confronting a life-threatening diagnosis while simultaneously considering the deeply human desire to have a child presents a struggle both for patients with cancer and for clinicians. Yet with improved survival rates among young patients with cancer, recent bench to-bedside translation of new techniques to preserve fertility, and increased awareness of choices for the preservation of fertility, options for family planning are now being offered to patients who have received a diagnosis of cancer. Concerns about fertility are similar for men and women; however, their opportunities for intervention differ considerably. This review describes current and emerging options for the preservation of fertility in patients with cancer and provides a conceptual framework for managing concerns about fertility at the time of diagnosis

 		Oncofertility and Fertility Preservation, Clinical Science and Medicine, Oncofertility and the Humanities and Social Sciences, Research Resources, Social Science/Humanities, Oncofertility-Related Publications
application/pdf iconPreservation_of_Fertility_in_Patients_with_Cancer.pdf
Tips for Living and Coping

Trainer, J.; The American Brain Cancer Foundation Fertility Preservation March 24, 2009

 		Oncofertility and Fertility Preservation, Clinical Science and Medicine, Research Resources, Oncofertility-Related Publications
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The Role of the Extracellular Matrix in Ovarian Follicle Development

Abstract

Regulation of ovarian follicle development depends on endocrine- and paracrine-acting hormones, the 3-dimensional architecture of the follicle, and the physical rigidity of the surrounding tissue. These 3 forces are integrated throughout the life cycle of the follicle to ensure appropriate hormone secretion, differentiation of the somatic cells, and maturation of the oocyte. The process of in-follicle maturation provides a new tool for understanding ovarian follicle development under the influence of these factors.

Teresa K. Woodruff and Lonnie D. Shea; Reprod Sci. Vol 14 No 8 Suppl 6-10 Dec 2007

 		Basic Science, Research Resources, Mammalian Folliculogenesis, Oncofertility-Related Publications
application/pdf icon33.pdf
Oncofertility Consortium Consensus Statement: Guidelines for Ovarian Tissue Cryopreservation

Leilah E. Backhus, MD, MS, Laxmi A. Kondapalli, MD, MS, R. Jeffrey Chang, MD, Christos Coutifaris, MD, PhD, Ralph Kazer, MD, and Teresa K. Woodruff, PhD

 		Oncofertility-Related Publications
application/pdf icononco_chapter_17.pdf
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