A threshold concentration of FSH is needed during IVM of ex vivo collected human oocytes

Abstract

Purpose: To investigate the effect of different FSH concentrations on human oocyte maturation in vitro and its impact on gene expression of key factors in the surrounding cumulus cells.

Methods: The study included 32 patients who underwent unilateral oophorectomy for ovarian tissue cryopreservation (OTC) (aged 28 years on average). Immature oocytes were collected from surplus medulla tissue. A total of 587 immature oocytes were divided into three categories according to the size of the cumulus mass: large (L-COCs), small (S-COCs), and naked oocytes (NOs), and submitted to 44-h IVM with one of the following concentrations of recombinant FSH: 0 IU/L, 20 IU/L, 40 IU/L, 70 IU/L, or 250 IU/L. After IVM, oocyte nuclear maturation stage and diameter were recorded. The relative gene expression of FSHR, LHCGR, and CYP19A1 in cumulus cells before (day 0; D0) and after IVM were evaluated.

Results: Addition of 70 or 250 IU/L FSH to the IVM medium improved oocyte nuclear maturation compared to 0, 20, and 40 IU/L FSH by upregulating LHCGR and downregulating FSHR in the cumulus cells.

Conclusion: FSH improved oocyte nuclear maturation at concentrations above 70 IU/L suggesting a threshold for FSH during IVM of ex vivo collected human oocytes from small antral follicles. Moreover, current results for the first time highlight that FSH function in vitro is mediated via cumulus cells by downregulating FSHR and upregulating LHCGR, which was also observed when the immature oocytes progressed in meiosis from the GV to the MII stage.

Keywords: Ex vivo collected oocytes; FSH; Fertility preservation; IVM; Small antral follicles.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Immature cumulus-oocyte complexes (COCs) collected from the surplus medulla tissue during OTC. a Naked oocyte (NO). b COC with small cumulus mass (S-COC). c COC with large cumulus mass (L-COC). Scale bars: 50 μm

Fig. 2

a–d Oocyte classification after IVM: GV (a), MI (b), MII (c), or DEG (d). Scale bars: 50 μm

Fig. 3

Scatter dot plot representation of maturation rates in naked oocytes (NOs), and cumulus-oocyte complexes with small cumulus mass (S-COCs) and large cumulus mass (L-COCs). (Error bars: mean ± SEM). Each circle of the graph represents one single patient. ***P < 0.001

Fig. 4

Relative expression of FSHR, LHCGR, and CYP19A1 in cumulus cells from L-COCs before (D0, n = 4) and after IVM in the presence of increasing concentrations of FSH (0 IU/L, n = 6; 20 IU/L, n = 6; 40 IU/L, n = 7; 70 IU/L, n = 6; and 250 IU/L, n = 6) (upper figures), and according to maturation stage before IVM (GV (D0), n = 4) and after IVM (GV, n = 6; MI, n = 13; and MII, n = 12) (lower figures) (error bars: mean ± SEM). All samples were run in duplicates and normalized to GAPDH and RPLP0. *P = 0.01; **P < 0.001