Anti-Müllerian hormone and progesterone levels produced by granulosa cells are higher when derived from natural cycle IVF than from conventional gonadotropin-stimulated IVF

Abstract

Background: The study was designed to compare the effect of in vitro FSH stimulation on the hormone production and gene expression profile of granulosa cells (GCs) isolated from single naturally matured follicles obtained from natural cycle in vitro fertilization (NC-IVF) with granulosa cells obtained from conventional gonadotropin-stimulated IVF (c-IVF).

Methods: Lutein granulosa cells from the dominant follicle were isolated and cultured in absence or presence of recombinant FSH. The cultures were run for 48 h and six days. Messenger RNA (mRNA) expressions of anti-Müllerian hormone (AMH) and FSH receptor were measured by quantitative polymerase chain reaction (qPCR). AMH protein and progesterone concentration (P4) in cultured supernatant were measured by ELISA and RIA.

Results: Our results showed that the mRNA expression of AMH was significantly higher in GCs from NC- than from c-IVF on day 6 after treatment with FSH (1 IU/mL). The FSH stimulation increased the concentration of AMH in the culture supernatant of GCs from NC-IVF compared with cells from c-IVF. In the culture medium, the AMH level was correlated significantly and positively to progesterone concentration.

Conclusions: Differences in the levels of AMH and progesterone released into the medium by cultured GC as well as in AMH gene expression were observed between GCs obtained under natural and stimulated IVF protocols. The results suggest that artificial gonadotropin stimulation may have an effect on the intra-follicular metabolism. A significant positive correlation between AMH and progesterone may suggest progesterone as a factor influencing AMH secretion.

Figure 1

Staining for Granulosa cells and Immunofluorescence staining for FSH receptor. (A) Aggregated granulosa cells stained with Papanicolaou (20x magnification). Bar = 20 μm. Localisation of FSH receptor (FSHR) in human granulosa cells (GC) after six days in culture, Immunofluorescence FSHR staining with monoclonal mouse anti-human FSHR (primary), chicken anti-mouse (Alexa, secondary), counterstained with DAPI for nuclei. (B) GCs in control culture (without FSH) showing cytoplasmic vesicles; (C) GC culture stimulated with FSH 0.1 IU/mL showing outgrowth with fibroblast-like morphology; (D) GC culture stimulated with FSH 1 IU/mL showing an intracellular signal for FSHR which could indicate de novo production (40x magnification). Bar = 40 μm.

Figure 2

Effect of FSH on the expression of FSHR- and of AMH- mRNA in GCs from NC-IVF and c-IVF. Gene expression of AMH mRNA (panel A) and FSHR mRNA (panel B) levels by qPCR in the granulosa cell lysate after six days of culture in 28 GC preparations (NC-IVF, N = 14, open bars; c-IVF, N = 14, closed bars).

Figure 3

Effect of FSH on AMH concentrations in the culture media of GCs from NC-IVF and c-IVF. Comparison of AMH concentrations in the culture medium of granulosa cells (GCs) isolated from conventionally stimulated IVF (c-IVF, N = 20) and from natural cycle IVF (NC-IVF, N = 21), cultured in absence (control, open bars) and presence of FSH (1.0 IU/mL, closed bars). Incubation in culture was 48 h (Graph A) and 6 days (Graph B). Data are presented as the mean +/− SEM.

Figure 4

Effect of FSH on progesterone concentration in the culture media of GCs from NC-IVF and c-IVF. Concentration of progesterone (P4) in the culture medium of granulosa cells (GCs) isolated from conventionally stimulated (c-IVF, N = 20) and from natural cycle IVF (NC-IVF, N = 21), cultured in absence (control) and presence of FSH (0.1 IU/mL, 1.0 IU/mL). Incubation in culture was 48 h (Graph A) and 6 days (Graph B). Data are presented as the mean +/− SEM.