Engineering the ovarian cycle using in vitro follicle culture

Abstract

Study question: Can cultured follicles model the ovarian cycle, including follicular- and luteal-phase hormone synthesis patterns and ovulation?

Summary answer: Under gonadotrophin stimulation, murine follicles grown in an encapsulated three-dimensional system ovulate in vitro and murine and human follicle hormone synthesis mimics follicular and luteal phases expected in vivo.

What is known already: Studies of the human ovary and follicle function are limited by the availability of human tissue and lack of in vitro models. We developed an encapsulated in vitro follicle growth (eIVFG) culture system, which preserves 3D follicular structure. Thus far, the alginate system has supported the culture of follicles from mice, dog, rhesus macaque, baboon and human. These studies have shown that cultured follicles synthesize steroid hormones similar to those observed during the follicular phase in vivo.

Study design, size, duration: Cultured murine follicles were treated with human chorionic gonadotrophin (hCG) and epidermal growth factor (EGF) and either assayed for luteinization or removed from alginate beads and assayed for ovulation. Human follicles were also cultured, treated with follicle-stimulating hormone (FSH), hCG and EGF to mimic gonadotrophin changes throughout the ovarian cycle, and culture medium was assayed for hormone production.

Participants/materials, setting, methods: Murine and human follicles were cultured in alginate hydrogel and hormone production [17β-estradiol, progesterone, inhibin A, inhibin B, activin A and anti-Müllerian hormone (AMH)] was quantified in medium by enzyme-linked immuno assay (ELISA). Human ovarian tissue was acquired from females between 6 and 34 years of age with a cancer diagnosis. These participants were undergoing ovarian tissue cryopreservation at National Physicians Cooperative sites as part of the Oncofertility Consortium.

Main results and the role of chance: When grown in this system, 96% of mouse follicles ovulated in response to hCG and released meiotically competent eggs. Ovulated follicles recapitulated transcriptional, morphologic and hormone synthesis patterns post-luteinizing hormone (LH/hCG). In addition to rodent follicles, individual human follicles secreted steroid and peptide hormones that mimicked the patterns of serum hormones observed during the menstrual cycle.

Limitations, reasons for caution: This was a descriptive study of an in vitro model of ovulation and the ovarian hormone cycle. The ovulation studies were limited to murine tissue and further studies are needed to optimize conditions using other species.

Wider implications of the findings: The eIVFG system reliably phenocopies the in vivo ovarian cycle and provides a new tool to study human follicle biology and the influence of cycling female hormones on other tissue systems in vitro.

Study funding/competing interests: This work was supported by NIH U54 HD041857, NIH U54 HD076188, NIH UH2 E5022920, NIH UH3 TR001207 and F30 AG040916 (R.M.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Keywords: alginate; in vitro follicle growth; luteinization; ovarian cycle; ovulation.

Figure 1

An ovulatory stimulus elicits follicular rupture in vitro. Murine follicles cultured in alginate rupture and release metaphase II (MII) eggs in response to hCG. (A) On the day of hCG administration, follicles contained an eccentric germinal vesicle (GV)-stage oocyte (arrowhead) and fluid-filled antrum (asterisk); scale bar = 50 µm. (B) Shown are representative images of follicles in the hours following hCG treatment (0, 4, 8 and 12 h). In some follicles, cumulus expansion was visible by 4 h (arrow). Extruded oocytes (arrowheads) and cumulus cells were visualized 12 h post-hCG; scale bar = 50 µm. (C) Image of a representative ruptured follicle 14 h post-hCG. The oocyte (arrowhead) is seen within the expanded cumulus matrix; scale bar = 50 µm. (D) Image of a representative MII egg collected from ruptured follicles with its extruded first polar body (asterisk); scale bar = 50 µm. (E) Overall, 96% of all follicles ruptured (n = 70). (F) Of the ruptured follicles (n = 36), 92% released MII stage eggs, while 8% were in the GVBD stage. Unruptured follicles were those that required mechanical isolation of the COC.

Figure 2

Effects of protease inhibitors and emergency contraceptives on ovulation in vitro. To assess the effects of the matrix metalloproteinases (MMPs) and PA/plasmin system on follicular rupture, inhibitors were added concomitant to hCG and rupture rates were determined. Similarly, the effects of emergency contraceptives (RU-486 and UPA) were assayed. (A) Schematic representation of putative proteases involved in ovulation and targeted action of protease inhibitors, see Materials and Methods. (B) Rupture rates of follicles treated with hCG and protease inhibitors (PIs) or emergency contraceptives (ECs). Control follicles are those treated with hCG only. Data are expressed as mean ± SEM, n = 3–4 cultures, 30–56 follicles; *P < 0.05, **P < 0.01, ***P < 0.001 according to one-way ANOVA followed by Bonferroni's Multiple Comparison Test.

Figure 3

eIVFG supports murine follicle luteinization. Murine secondary follicles were cultured in alginate for 10 days, luteinized by hCG administration and cultured for an additional 5 days. (A) Follicles were fixed and stained with hematoxylin–eosin before and after hCG treatment (culture days 10 and 13, respectively). Follicles showed morphological signs of luteinization, marked by an increased granulosa cell cytoplasmic to nuclear ratio (scale bars = 10 µm). (B) Progesterone and estradiol and (C) inhibin A and inhibin B were quantified in the medium by enzyme-linked immuno assay (ELISA) (n = 3 cultures). Data are expressed as mean ± SEM.

Figure 4

eIVFG supports the complete hormone cycle in human follicles. Secondary human follicles were isolated and cultured in alginate from 40 to 65 days. Steroid and peptide hormones were quantified in the medium throughout the culture period. The culture time on the x-axis indicates the time of hCG addition (day 0, dotted line); follicles were cultured for 14–15 days post-hCG. The concentration of (A) estradiol, (B) progesterone, (C) inhibin A, (D) inhibin B, (E) AMH and (F) activin A are reported throughout culture. Data are expressed as mean ± SD for n = 5 follicles. Follicles were collected from the ovaries of three women and cultured individually.