Home » Key Scientific Articles » Fibrin promotes development and function of macaque primary follicles during encapsulated three-dimensional culture.

Fibrin promotes development and function of macaque primary follicles during encapsulated three-dimensional culture.

Xu J, Lawson MS, Yeoman RR, Molskness TA, Ting AY, Stouffer RL, Zelinski MB.

Hum Reprod. 2013 ;28(8):2187-200.

Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006, USA. [email protected]



Does fibrin introduced into the extracellular matrix affect the growth and maturation of individual primate follicles duringencapsulated three-dimensional (3D) culture?


While not altering follicle survival, fibrin-alginate (FIBRIN) improves macaque primary, but not secondary, follicle developmentduring encapsulated 3D culture in terms of growth, steroidogenesis, anti-Müllerian hormone (AMH)/vascular endothelial growth factor (VEGF) production and oocyte maturation.


Efforts to grow non-human primate ovarian follicles from the secondary to the antral stage during encapsulated 3Dculture have been successful. However, the growth and maturation of primary follicles in vitro has not been reported in primates, especially in chemically defined conditions.


In vitro follicle maturation was investigated using the rhesus macaque (Macaca mulatta). Ovaries (n = 7 pairs) were obtained during the early follicular phase of the menstrual cycle (cycle day 1-4). Primary (80-120 µm diameter) and secondary (125-225 µm diameter) follicles were isolated mechanically, randomly assigned to experimental groups, encapsulated into alginate (0.25% w/v) or FIBRIN (25 mg/ml fibrinogen-0.25% alginate) and cultured for 13 and 5 weeks, respectively.


Individual follicles were cultured in alpha minimum essential medium supplemented with FSH. Follicle survival and growth were assessed by microscopy. Follicles that reached the antral stage were treated with recombinant hCG. Metaphase II (MII) oocytes were inseminated via ICSI. Follicle morphology was evaluated by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed for cytochrome P450 family 17 subfamily A polypeptide 1 (CYP17A1) and 19 subfamily A polypeptide 1 (CYP19A1). Culture medium was analyzed for estradiol (E2) and progesterone by chemiluminescence, androstenedione (A4) by radioimmunoassay, as well as anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay.


A total of 105 primary and 133 secondary follicles were collected. The presence of fibrin in the alginate matrix had no effect on either primary or secondary follicle survival. Growing primary and secondary follicles formed an antrum at Weeks 9 and 3, respectively. The percentage of growing follicles was higher (P < 0.05) for primary follicles cultured in FIBRIN than alginate at Week 13. The diameters were larger for the growing secondary follicles cultured in alginate than FIBRIN at Week 5 (P < 0.05). H&E staining revealed the typical morphology for small antral follicles. CPY17A1 immunostaining was detected in theca cells, while CYP19A1 was observed in granulosa cells. E2 increased (P < 0.05) during antrum formation in growing follicles at Week 9 for primary and Week 3 for secondary follicles. AMH levels in medium from growing primary follicles increased (P < 0.05) after Week 4 with peak levels at Weeks 9-11. AMH increased (P < 0.05) in growing secondaryfollicles at Weeks 3-5. VEGF levels in medium were elevated (P < 0.05) in growing primary follicles at Week 9. VEGF increased (P < 0.05) in medium from growing secondary follicles at Weeks 3-5. E2, AMH and VEGF production was higher (P < 0.05) in primary follicle culture with FIBRIN than alginate alone. One primary follicle cultured in FIBRIN (1 of 5 follicles harvested) and a secondary follicle cultured in alginate alone (1 of 15 folliclesharvested) yielded an MII oocyte. The fertilized oocyte from primary follicle culture arrested without cell division after fertilization, while the oocyte from secondary follicle culture cleaved and reached the morula stage.


The study reports on in vitro development and function of individual macaque follicles, that is limited to the interval from the primary and secondary stage to the small antral stage. The findings await translation to human ovarian follicles.


The 3D model for primate follicle development offers a unique opportunity to investigate the growth and regulation of primate primary, as well as secondary follicles, and their enclosed oocytes, as they grow to the antral stage by monitoring and manipulating factors or signaling pathways in vitro. Since primate primary follicles, in addition to secondary follicles, can be cultured to the antral stage to provide mature oocytes, they represent an additional source of pre-antral follicles for in vitro follicle maturation with the potential to provide gametes for assisted reproductive technology as an option for fertility preservation in women, including patients with cancer.


This work was supported by The Oncofertility Consortium (NIH U54 RR024347-HD058294, PL1-EB008542), NIH U54-HD18185 (Eunice Kennedy Shriver Specialized Cooperative Centers Program in Reproduction and Infertility Research), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), Oregon National Primate Research Center 8P51OD011092. There are no conflicts of interest.


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Additional Information

Cancer rates have been rising for the past few decades. But the good news is that advances in cancer detection and therapy have greatly increased survival rates of patients, which include females prior to or during reproductive age. The important question now is what major impacts cancer treatment can have on future life quality for these women. One of the side effects of chemo- or radiotherapy for female patients is the destruction of oocytes in the ovary. The ovary contains follicles, which are the basic units of female reproductive biology, each including an oocyte surrounded by follicular cells. As a follicle grows, the enclosed oocyte also grows and matures to the metaphase II stage of meiosis for fertilization. After radio- or chemo-therapy, the ovary shrinks with remarkably reduced numbers of follicles and their oocytes. This premature ovarian failure causes early menopause and infertility.

Strategies are under development to preserve the ovary for fertility after eradicating the cancer. The ovary can be removed and ovarian tissue cryopreserved before cancer treatment. Once the patient is cancer-free after treatment, ovarian tissue can be thawed when the patient is ready for pregnancy. The patient can receive the tissue through transplantation. However, there is the risk of transferring cancer cells back to the patient. Another option would be isolating follicles and growing them in vitro. In theory, preantral follicles can develop to the antral stage and produce mature oocytes during culture. The oocyte could then be fertilized by in vitro fertilization, and an embryo transferred to the patient. Successful pregnancy would result in a baby who is the biological child of the mother. Our research focuses on improving and validating this culture system, so called in vitro follicle maturation (IFM), using a nonhuman primate model for future clinical application. Besides the clinical relevance, IFM also provides a unique opportunity to monitor and manipulate molecular signaling pathways and related factors (e.g., hormones, local growth factors or components of the extracellular matrix) to obtain knowledge of their roles and importance on follicle growth, steroid and local factor production, as well as oocyte maturation, in intact individual follicles precluding complex follicle-follicle interactions that may occur in vivo.


Fibrin promotes development and function