Oncofertility Publications

The stage at which follicle-enclosed cumulus-oocyte complexes achieve developmental competence in primates is unknown. Therefore, studies were designed to characterize the ability of oocytes in small antral follicles present during the menstrual cycle to spontaneously resume meiosis, fertilize, and support early embryo development. Ovaries were removed from adult rhesus monkeys (n = 12) during the early follicular phase (Days 3-4) of spontaneous cycles. Small antral follicles were divided into five groups according to their diameter; group I: <0.5 mm; group II: 0.5-0.99 mm; group III: 1.0-1.49 mm; group IV: 1.5-1.99 mm; and group V: 2.0-2.5 mm. The cumulus-oocyte complex from healthy small antral follicles (devoid of dark oocytes or granulosa cells) were extracted (n = 199) and cultured for 48 h under different conditions: in TALP (tyrode, albumin, lactate, pyruvate) medium alone, SAGE medium alone, or plus gonadotropins. At 48 h, oocyte meiotic status and diameter were measured after treatment of cumulus-oocyte complexes with hyaluronidase. Cumulus-oocyte complexes derived from follicles of 0.5- to 2-mm diameter contain oocytes that typically reinitiate meiosis in the absence or presence of gonadotropins and fertilize via in vitro fertilization or intracytoplasmic sperm injection. Moreover, the inseminated oocytes can reach the morula stage but arrest. Thus, the ability of these oocytes to complete maturation, as monitored from subsequent embryonic development after fertilization, is suboptimal. Further studies on primate IVM of oocytes from SAFs are warranted in order for them to be considered as an additional, novel source of gametes for fertility preservation in cancer patients. Peluffo MC, Barrett SL, Stouffer RL, Hennebold JD, Zelinski MB. Biol Reprod 83:525-532, 2010. PMID 20519694.

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

Abstract

OBJECTIVE:
To investigate the effect of slow cryopreservation on the morphology and function of primate primordial follicles within ovarian tissue slices.

DESIGN:
Fresh monkey ovarian tissue was frozen by slow cryopreservation and thawed for analysis of morphologic and functional parameters.

SETTING:
University-affiliated laboratory.

ANIMALS:
Rhesus monkey ovarian tissue.

INTERVENTION(S):
None.

MAIN OUTCOME MEASURE(S):
Histologic analysis, follicle counting, assessment of protein abundance and localization.

RESULT(S):
After freezing and thawing, 89% of the primordial follicles maintained their laminar-based architecture, with sizes close to those of fresh fixed follicles. Molecular markers of early follicle health (activin subunits and the phosphorylated form of the signaling protein Smad2 [pSmad2]) were present in fresh and frozen-thawed primordial follicles. Stroma cells, but not follicles, had a higher level of TUNEL staining. Granulosa cells within the follicles of frozen-thawed ovarian tissue cultured for 48 hours had the capacity to proliferate and sustained expression of the activin subunits and nuclear pSmad2.

CONCLUSION(S):
This study provides evidence that markers of early follicle growth and development are preserved after slow cryopreservation and thaw, with little effect on follicle morphology and function.

Shiying Jin, PhD, Lei Lei, PhD, Lonnie D. Shea, PhD, Mary B. Zelinski, PhD, Richard L. Stouffer, PhD, and Teresa K. Woodruff, PhD. Fertility and Sterility, 2010.

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]

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