FSH Stimulation promotes progesterone synthesis and output from human granulosa cells without luteinization.
Can granulosa cells produce progesterone (P) in response to FSH stimulation?



FSH actively promotes P synthesis and output from granulosa cells without luteinization by up-regulating the expression and increasing enzymatic activity of 3β-hydroxysteriod dehydrogenoase (3β-HSD), which converts pregnenolone to P.



Serum P level may rise prematurely prior to ovulation trigger in stimulated IVF cycles and adversely affect implantation and clinical pregnancy rates by impairing endometrial receptivity.



A translational research study.



Human ovarian cortical samples (n = 15) and non-luteinizing FSH-responsive human mitotic granulosa cell line (HGrC1) were stimulated with rec-FSH at 12.5, 25 and 50 mIU/ml concentrations for 24 and 48 h. FSH receptor expression was knocked-down and up-regulated in the granulosa cells using short hairpin RNA (shRNA) technology and activin-A administration, respectively. The expressions of the steroidogenic enzymes were analyzed at mRNA level by real-time quantitative RT-PCR, and protein level by western blot and immunoprecipitation assay. The enzymatic activity of 3β-HSD was measured using a spectrophotometric method. In vitro estradiol (E2) and P productions of the cells before and after FSH stimulation were measured by electro-chemiluminescence immunoassay method.



Stimulation of the HGrC1 cells with FSH resulted in a dose-dependent increase in the mRNA and protein level of 3β-HSD. Overall, when all time points and FSH doses were analyzed collectively, FSH significantly up-regulated the mRNA expression of its own receptor (3.73 ± 0.06-fold, P < 0.001), steroidogenic acute regulatory protein (stAR, 1.7 ± 0.03-fold, P < 0.01), side-chain cleavage enzyme (SCC, 1.75 ± 0.03-fold, P < 0.01), aromatase (4.49 ± 0.08-fold, P < 0.001), 3β-HSD (1.68 ± 0.02-fold, P < 0.01) and 17β-hydroxy steroid dehydrogenase (17β-HSD, 2.16 ± 0.02-fold, P < 0.01) in the granulosa cells. Expression of 17α-hydroxylase (17α-OH, 1.03 ± 0.01-fold P > 0.05) did not significantly change. Similar changes were observed in the protein expression analysis of these enzymes on western blotting after FSH stimulation. FSH significantly increased 3β-HSD, 17β-HSD and aromatase in a dose-dependent manner but did not affect 17α-OH. Protein expression of P was increased along with 3β-HSD after FSH stimulation, which was further evidenced by immunoprecipitation assay. Enzymatic activity of 3β-HSD was significantly enhanced by FSH administration in the HGrC1 cells in a dose-dependent manner. In line with these findings P output (1.05 ± 0.3 vs. 0.2 ± 0.1 ng/ml, respectively, P < 0.001) from the samples stimulated with FSH were significantly increased along with E2 (1918 ± 203 vs. 932 ± 102 pg/ml, respectively, P < 0.001) compared to unstimulated controls. FSH-induced increase in 3β-HSD expression was amplified and reversed in the HGrC1 cells when FSH receptor expression was up-regulated by activin-A and down-regulated with shRNA, respectively.



As only the effect of FSH was studied we cannot extrapolate our findings to the potential effects of HMG and recombinant LH.



This data provides a molecular explanation for the largely unexplained phenomenon of P rise during the follicular phase of gonadotropin stimulated IVF cycles. Our findings may progress the research to uncover potential mechanisms for preventing premature P rise that appears to be associated with inferior outcomes in women undergoing IVF.



Funded by the School of Medicine and the Graduate School of Health Sciences of Koc University. All authors declare no conflict of interest.



None.
Department of Obstetrics and Gynecology, Koc University School of Medicine, Davutpasa Cad. No:4, 34010 Topkapi Istanbul, Turkey.|American Hospital Women's Health Center, Assisted Reproduction Unit, Guzelbahce Sok, Nisantasi, Istanbul, Turkey.
Ozgur
O
Oktem
The Graduate School of Health Sciences, Koc University, Rumelifeneri yolu, Sariyer, Istanbul, Turkey.
Nazli
N
Akin
The Graduate School of Health Sciences, Koc University, Rumelifeneri yolu, Sariyer, Istanbul, Turkey.
Gamze
G
Bildik
Department of Obstetrics and Gynecology, Koc University School of Medicine, Davutpasa Cad. No:4, 34010 Topkapi Istanbul, Turkey.|American Hospital Women's Health Center, Assisted Reproduction Unit, Guzelbahce Sok, Nisantasi, Istanbul, Turkey.
Kayhan
K
Yakin
American Hospital Women's Health Center, Assisted Reproduction Unit, Guzelbahce Sok, Nisantasi, Istanbul, Turkey.
Ebru
E
Alper
American Hospital Women's Health Center, Assisted Reproduction Unit, Guzelbahce Sok, Nisantasi, Istanbul, Turkey.
Basak
B
Balaban
Department of Obstetrics and Gynecology, Koc University School of Medicine, Davutpasa Cad. No:4, 34010 Topkapi Istanbul, Turkey.|American Hospital Women's Health Center, Assisted Reproduction Unit, Guzelbahce Sok, Nisantasi, Istanbul, Turkey.
Bulent
B
Urman
D015096:3-Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D005260:Female; D005640:Follicle Stimulating Hormone / Q000627:therapeutic use*; D006107:Granulosa Cells / Q000187:drug effects*; D006801:Humans; D034461:Luteinization / Q000187:drug effects*; D010062:Ovulation Induction / Q000379:methods; D011284:Pregnenolone / Q000378:metabolism; D011374:Progesterone / Q000096:biosynthesis* / Q000097:blood; D012333:RNA, Messenger / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D012333:RNA, Messenger; D011374:Progesterone; D011284:Pregnenolone; D005640:Follicle Stimulating Hormone; D015096:3-Hydroxysteroid Dehydrogenases
3β-HSD; FSH stimulation; granulosa cells; ovary; progesterone; steroidogenic enzymes