Oncofertility Publications

Gracia, C R. Cancer and Treatment Research. 2010; 156: 3-9. PMID: 20811821.

PURPOSE:
Diminished reproductive capacity is a devastating consequence of life-sparing therapies for childhood malignancy. In 2006, the American Society of Clinical Oncology (ASCO) published fertility preservation recommendations (ASCOR) emphasizing the importance of early discussion and intervention for fertility preservation strategies. Using the Survey for Preservation of Adolescent REproduction (SPARE), we sought to determine fertility preservation attitudes and practice patterns post-ASCOR from pediatric oncology specialists nationwide.

MATERIALS and METHODS:
The SPARE survey consists of 22 questions assessing pediatric oncology specialists’ attitudes and practice patterns toward fertility preservation. Broad perspectives on fertility preservation, including a willingness to discuss fertility, knowledge of current fertility preservation methods and awareness of ASCOR, were assessed.

RESULTS:
The majority of respondents acknowledged that fertility threats are a major concern for them and agreed that all pubertal cancer patients should be offered a fertility consultation, but only 46% reported they refer male pubertal cancer patients to a fertility specialist prior to cancer treatment >50% of the time, and only 12% reported they refer female pubertal cancer patients to a fertility specialist prior to cancer treatment > 50% of the time. While 44% of respondents were familiar with the 2006 ASCOR, only 39% of those utilized them to guide decision-making in greater than half of their patients.

CONCLUSION:
Our study demonstrates pediatric oncologists’ motivation to preserve fertility in pediatric cancer patients; however, barriers to both gamete cryopreservation and referral to fertility specialists persist. Female pubertal patients are referred to fertility preservation specialists with much less frequency than are male pubertal patients, highlighting a disparity.

Köhler TS, Kondapalli LA, Shah A, Chan S, Woodruff TK, Brannigan RE. J Assist Reprod Genet. 2010 Nov 26.

“Review of Emily Monosson, Ed., Motherhood, The Elephant in the Laboratory: Women Scientists Speak Out *”, The American Journal of Bioethics, 10:11, 28 – 29

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

Campo-Engelstein, L. American Journal of Bioethics, 10:11, 32 – 34.

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

Abstract

BACKGROUND:
An alginate-based matrix supports the three-dimensional (3D) architecture of non-human primate follicles and, in the presence of FSH, permits the in vitro development of pre-antral follicles to the small antral stage, including the production of ovarian steroids and paracrine factors. The current study investigated the ability of gonadotrophins, fetuin and oxygen (O2) to improve primate follicle growth and oocyte maturation in vitro.

METHODS:
Macaque secondary follicles were isolated from the early follicular phase ovaries, encapsulated in a sodium alginate matrix and cultured individually for 40 days in supplemented medium. The effects of recombinant human (rh) FSH (15, 3 and 0.3 ng/ml for high, medium and low FSH, respectively), bovine fetuin (1 or 0 mg/ml) and O2 (5 or 20% v/v) were examined. Half of the follicles in each culture condition received rhLH on Day 30 –40. Follicles that reached antral stage were treated with rh chorionic gonadotrophin for 34 h to initiate oocyte meiotic maturation. Media were analyzed for ovarian steroids and anti-mu¨ llerian hormone (AMH).

RESULTS:
Improved culture conditions supported non-human primate, secondary follicle growth to the antral stage and, for the first time, promoted oocyte maturation to the MII stage. In the presence of fetuin at 5% O2, follicles had the highest survival rate if cultured with high or medium FSH, whereas follicles grew to larger diameters at Week 5 in low FSH. Oocyte health and maturation were promoted under 5% O2. High FSH stimulated steroid production by growing follicles, and steroidogenesis by follicles cultured with low FSH was promoted by LH. AMH biosynthesis was elevated with high compared with low FSH and for longer under 5% O2 than under 20% O2.

CONCLUSIONS:
This encapsulated 3D culture model permits further studies on the endocrine and local factors that influence primate follicle growth and oocyte maturation, with relevance to enhancing fertility preservation options in women.

Xu J, Lawson MS, Yeoman RR, Pau KY, Barrett SL, Zelinski MB, Stouffer RL. Secondary follicle growth and oocyte maturation during encapsulated three-dimensional culture in rhesus monkeys: effects of gonadotrophins, oxygen and fetuin. Hum Reprod. 2011 Feb 28. [Epub ahead of print]

Please see below IRB template for TESTICULAR TISSUE CRYOPRESERVATION (Manual of Operations). Use this document as a guideline when submitting to your own IRB.

Please see below IRB template for TESTICULAR TISSUE CRYOPRESERVATION (Protocol). Use this document as a guideline when submitting to your own IRB.

Please see below IRB template for TESTICULAR TISSUE CRYOPRESERVATION (Adult Consent). Use this document as a guideline when writing your documents. Contact Kathrin Gassei, PhD at gasseik@mwri.magee.edu for consent review prior to submitting to your own IRB.

Please see below IRB template for TESTICULAR TISSUE CRYOPRESERVATION (Pediatric Consent). Use this document as a guideline when writing your documents. Contact Kathrin Gassei, PhD at gasseik@mwri.magee.edu for consent review prior to submitting to your own IRB.

Please see below IRB template for TESTICULAR TISSUE CRYOPRESERVATION (18 year old Re-consent). Use this document as a guideline when writing your documents. Contact Kathrin Gassei, PhD at gasseik@mwri.magee.edu for consent review prior to submitting to your own IRB.

Please see below IRB template for TESTICULAR TISSUE CRYOPRESERVATION (Adult HIV Consent). Use this document as a guideline when submitting to your own IRB. IMPORTANT NOTE: Applicable to institutions within the state of Pennsylvania

IMPORTANT NOTE: Applicable to institutions within the state of Pennsylvania

Please see below IRB template for TESTICULAR TISSUE CRYOPRESERVATION (Pediatric HIV Consent). Use this document as a guideline when submitting to your own IRB. IMPORTANT NOTE: Applicable to institutions within the state of Pennsylvania

IMPORTANT NOTE: Applicable to institutions within the state of Pennsylvania

Kim J, Steiner AZ, Fritz M, Mersereau JE. J Assist Reprod Genet. 2012 Feb;29(2):127-9. Epub 2011 Nov 17. PMID: 22089265

Abstract

A three-dimensional culture system supports development of primate preantral follicles to the antral stage with appreciable steroid production. The present study assessed: (1) whether in vitro developmental competence of follicles is age-dependent, (2) the role of gonadotropins and insulin in supporting folliculogenesis, and (3) anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) production by growing follicles. Ovaries were obtained from prepubertal, young and older adult, rhesus macaques. Secondary follicles were encapsulated into alginate beads and cultured individually for 40 days in media containing 0.05 or 5 microg/ml insulin, with or without recombinant human (rh) follicle stimulating hormone (FSH, 500 mIU/ml). No follicles survived in culture without rhFSH. In the presence of rhFSH, survival was lower for follicles from older animals, whereas growth, i.e., follicle diameter, was less by day 40 for follicles from prepubertal animals. The surviving follicles were categorized as no-grow (</= 250 microm), slow-grow (250-500 microm), and fast-grow (>/= 500 microm) according to their diameters. Slow-grow follicles cultured with 5 microg/ml insulin produced more ovarian steroids compared with those with 0.05 microg/ml insulin by week 5. Slow- and fast-grow follicles produced more AMH and VEGF than the no-grows, and levels peaked at week 2 and 5, respectively. After 100 ng/ml rh chorionic gonadotropin treatment for 34 hrs, more healthy oocytes were retrieved from young adults whose follicles were cultured with 5 microg/ml insulin. This culture system offers an opportunity to characterize the endocrine and paracrine function of primate follicles that influence follicle growth and oocyte maturation.

Xu J, Bernuci MP, Lawson MS, Yeoman RR, Fisher TE, Zelinski MB, Stouffer RL. Reproduction, Aug. Epub 2010. PMID 20729335

Laurritzen P. Cancer Treatment and Research. 2010; 156: 295-306. PMID: 20811843.

Gwendolyn P. Quinn, Caprice A. Knapp, and Devin Murphy

Synder K A., Thazin M K., Pearse W B., Moinuddin M. Cancer Treatment and Research. 2010; 156: 403-12. PMID: 20811851.

Abstract

Facing a cancer diagnosis at any age is devastating. However, young cancer patients have the added burden that life-preserving cancer treatments, including surgery, chemotherapy, and radiotherapy, may compromise their future fertility. The possibility of reproductive dysfunction as a consequence of cancer treatment has a negative impact on the quality of life of cancer survivors. The field of oncofertility, which merges the clinical specialties of oncology and reproductive endocrinology, was developed to explore and expand fertility preservation options and to better manage the reproductive status of cancer patients. Fertility preservation for females has proved to be a particular challenge because mature female gametes are rare and difficult to acquire. The purpose of this article is to provide the gynecologist with a comprehensive overview of how cancer treatments affect the female reproductive axis, delineate the diverse fertility preservation options that are currently available or being developed for young women, and describe current measures of ovarian reserve that can be used pre- and post-cancer treatment. As a primary care provider, the gynecologist will likely interact with patients throughout the cancer care continuum. Thus, the gynecologist is in a unique position to join the oncofertility team in providing young cancer patients with up-to-date fertility preservation information and referrals to specialists.

Duncan FE, Jozefik JK, Kim AM, Hirshfeld-Cytron J, Woodruff TK. US Obstet Gynecol. 2012 Jan 1;6(1):24-34.

Fleetwood, A and Campo-Engelstein, L. Cancer Treatment and Research. 2010; 156: 237-48. PMID: 20811838.

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

Abstract

The murine primordial follicle pool develops largely within 3 days after birth through germline nest breakdown and enclosure of oocytes within pregranulosa cells. The mechanisms that trigger primordial follicle formation likely are influenced by a transition from the maternal to fetal hormonal milieu at the time of birth. High levels of maternal estrogen maintain intact germline nest in fetal ovary, and decrease of estrogen after birth is permissive of follicle formation. In the present study, we measured an increase in neonatal serum follicle-stimulating hormone (FSH), which corresponded to falling estradiol (E(2)) levels during the critical window of primordial follicle formation (Postnatal Days 1-3). To determine whether fetal hormones contribute in an active manner to primordial follicle formation, mouse fetal ovaries (17.5 days postcoitus) were cultured in vitro at two concentrations of E(2) (meant to reflect maternal and fetal levels of E(2)) and FSH for 6 days. High levels of E(2) (10(-6) M) inhibited germline nest breakdown, and this effect was significantly reduced when fetal ovaries were cultured in the low E(2) concentration (10(-10) M). FSH facilitated germline nest breakdown and primordial follicle formation under both high and low E(2) culture conditions. Low E(2) was identified as being more permissive for the effects of FSH on primordial follicle formation by stimulating the up-regulation of Fshr and activin beta A subunit (Inhba) expression, pregranulosa cell proliferation, and oocyte survival. The decrease of E(2) plus the presence of FSH after birth are critical for primordial follicle formation and the expression of oocyte-specific transcription factors (Figla and Nobox) in that inappropriate exposure to FSH or E(2) during follicle formation resulted in premature or delayed primordial folliculogenesis. In conclusion, with the drop of E(2) level after birth, FSH promotes primordial follicle formation in mice by stimulating local activin signaling pathways and the expression of oocyte-specific transcription factors.

Lei Lei, Shiying Jin, Kelly E. Mayo, and Teresa K. Woodruff. Biology of Reproduction, 82, 13-22 (2010).