Follicular fluid hormonal profile and cumulus cell gene expression in controlled ovarian hyperstimulation with recombinant FSH: effects of recombinant LH administration

Abstract

Purpose: Down-regulation with gonadodropin-releasing agonist (GnRH-a) protocol during IVF stimulation leads to a severe endogenous LH suppression, which may affect the follicular development. The aim of the study was to evaluate the effects of recombinant LH (r-LH) administration, during late follicular development stages, in recombinant FSH (r-FSH) stimulated cycles on follicular fluid (FF) parameters and on cumulus cell quality.

Methods: Twenty patients undergoing IVF were stimulated in a long GnRH agonist protocol with r-FSH alone or with r-LH supplementation when the leading follicle reached diameter of 14 mm. FF was collected at the time of oocyte retrieval from 32 follicles ≥ 18 mm. Serum FSH, LH, estradiol (E(2)), and progesterone (P(4)) were evaluated on the day of hCG administration. Intra-follicular E(2), P(4), AMH and TGF-β were assayed. Total RNA from 18 individual cumuli was isolated for gene expression analyses.

Results: R-LH increased FF P(4) levels. FF TGF-β levels and PTGS2 and HAS2 expression in cumulus cells (CCs) positively correlated with increased P(4) levels observed in FFs, while a negative correlation was found between P(4) and AMH levels.

Conclusions: FF positive correlation between P(4) and TGF-β levels and CC expression of PTGS2 and HAS2 suggest an association with a better follicle quality. In addition, our data suggest that late follicular phase r-LH supplementation leads to a more advanced stage of follicular maturation.

Fig. 1

a Progesterone and b Estradiol levels in FFs obtained from single follicles at oocyte retrieval after stimulation with r-FSH (FSH), or r-FSH plus r-LH administered in the late follicular phase (F+L). c ratio of estradiol/progesterone (E/P). Each point represents the value obtained from a single FF. Dots on the rigth represent the mean ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001. n = follicle numbers

Fig. 2

a TGF-β and c AMH levels in FFs. FFs were collected from single follicles at oocyte retrieval after stimulation with r-FSH (FSH; n = 19), or r-FSH plus r-LH administered in the late follicular phase (F+L; n = 13). Relationships between TGF-β (b), AMH (d) and Progesterone (P₄) levels in FFs. There is a positive and statistically significant relationship between TGF-β and follicular P₄ levels while there is a negative and statistically significant relationship between AMH and P₄. ● r-FSH ▼ r-FSH + r-LH; *P < 0.05

Fig. 3

Upper panel: relative expression of PTGS2, HAS2 and GREM1 obtained by multiplex-PCR. Total RNA was extracted from CCs obtained from single follicles and subjected to multiplex RT-PCR using primers for PTGS2, HAS2 and GREM1, as well as for β-actin used as internal control. An aliquot of each PCR product was electrophoresed onto 2 % agarose gel and stained with ethidium bromide. Expression levels were quantified by densitometric evaluation of the bands with a chemiluminescence detection system (raytest) and values were normalised by their respective β-actin values and represent the mean ± S.E.M. In the right panel is represented the mean ± S.E.M. of the levels of P₄ in the FFs corresponding to the CCs examined. *P < 0.05; **P < 0.01 vs. respective FSH. Lower panels: relationship between PTGS2 and HAS2 expression in CCs and the progesterone (P₄) levels in the corresponding FFs. There is a positive and statistically significant relationship between PTGS2 and HAS2 and follicular P₄ levels ● r-FSH (n = 8)▼ r-FSH+r-LH (n = 10)

Fig. 4

Patients with more then 1 follicle retrieved. (Upper panel) Relationship between PTGS2 and HAS2 expression in CCs and the levels of progesterone (P₄), TGF-β and AMH in the corresponding FFs. Two patients (5 follicles) treated with r-FSH (●), and two patients (4 follicles) treated with r-FSH+r-LH (▼). Symbols with the same colour belong to the same patient. The same positive or negative correlation observed for the entire group was observed in CCs coming from single patients. (Lower panel) Representative images of multiplex RT-PCR from one patient of the r-FSH group (● red) and one patient of the r-FSH+r-LH group (▼ yellow)