Association of Ovarian Steroids in Follicular Fluid with Pregnancy Rates in Patients with or without Polycystic Ovary during in vitro Fertilization

Document Type : Research Article


Department of Biology, Faculty of Basic Science, Ale Taha Institute of Higher Education, Tehran, Iran


The point of this consideration is to examine the possible influence of progesterone (P4), estradiol (E2), and testosterone (T) concentrations in follicular fluid (FF) on fertilization, implantation, and biochemical pregnancy rates in patients with polycystic ovary (PCO) and normal women during in vitro fertilization (IVF) treatment. 30 normal-ovulatory women between 24-42 years old and 26 women with the PCO, between 21-37 years old under assisted reproductive therapy were recruited from two university hospital IVF centers during this study. The fluid from more than one preovulatory follicle was obtained on the day of ovulation for hormone measurement using the Elisa method. Association between the mentioned steroid levels and fertilization, implantation, and biochemical pregnancy rates were assessed by using spearman, Mann-Whitney, and regression tests. Among the assessed outcomes, in the PCO group, the FF, E2, and T levels appeared to have a noteworthy reverse relationship with fertilization rate (P= 0.050). In both groups, intrafollicular levels of the E2/T ratio correlated positively with implantation rate (P=0.024 in control vs. P= 0.009 in PCO) and biochemical pregnancy (P= 0.020 in control vs. P= 0.012 in PCO). Furthermore, intrafollicular E2/P4 levels showed a positive relationship with both implantation (P= 0.047) and biochemical pregnancy (P= 0.050) rates in the control group. Our results for the first time suggest that high intrafollicular levels of E2 and T may influence reduced fertilization rate. The E2/T ratio would be a good predictor of successful implantation and pregnancy rates regardless of infertility causes. Moreover, the E2/P4 ratio could be a valuable prescient marker for implantation and biochemical pregnancy rates in normal-ovulatory women. Beyond the mentioned results, the underlying pathological processes, and pathways, including hyperandrogenism and interruptions in the hypothalamic-pituitary-ovarian (HPO) axis could be the potent concepts in polycystic ovary syndrome (PCOS) etiology and an alteration in genetic variants of SHBGs and catalytic enzymes responsible in steroidogenesis pathways. Furthermore, possible correlations between the mentioned steroid hormones and the functions of genes related to gonadotropins and steroid hormones in the pathophysiological pathways involved in PCOS were explored.


Andersen CY, Byskov AG. 2006. Estradiol and regulation of anti-mullerian hormone, Inhibin-A, and Inhibin-B secretion: analysis of small antral and preovulatory human follicle’s fluid. J Clin Endocrinol Metab 91(10): 4064-4069.
Aghajani S, Salehzadeh A, Ghasemian F, Mehrafza M, Hosseini A. 2021. The impact of maternal age on chromosomal aneuploidy, blastocyst quality, and pregnancy outcomes during intracytoplasmic sperm injection cycles.  J Adv Biomed Sci 11(4): 4107-4117.
Artini PG, Obino MER, Sergiampietri C, Pinelli S, Papini F, Casarosa E. 2018. PCOS and pregnancy: a review of available therapies to improve the outcome of pregnancy in women with polycystic ovary syndrome. Expert Rev Endocrinol Metab 13(2): 87-98.
Azziz R. 2018. Polycystic ovary syndrome. Obstet Gynecol 132(2): 321-336.
Baculescu N. 2013. The role of androgen receptor activity mediated by the CAG repeat polymorphism in the pathogenesis of PCOS. J Med Life 6(1): 18-25.
Barzegar MH, Khazali H, Kalantar SM, Khoradmehr A. 2018. The comparative effect of Citrullus colocynthis hydro-alcoholic extract (CCT) and metformin on morphometric ovarian follicles disorders in estradiol valerate induced PCOS rats. Galen Med J 7: e1045.
Ben-Rafael Z, Meloni F, Strauss JF, Blasco L, Mastroianni L, Flickinger GL. 1987. Relationships between polypronuclear fertilization and follicular fluid hormones in gonadotropin-treated women. Fertil Steril 47(2): 284-288.
Blaschka C, Sánchezā€Guijo A, Wudy SA, Wrenzycki C. 2020. Profile of bile acid subspecies is similar in blood and follicular fluid of cattle. Vet Med Sci 6(2): 167-176.
Brower MA, Jones MR, Rotter JI, Krauss RM, Legro RS, Azziz R, Goodarzi MO. 2015. Further investigation in Europeans of susceptibility variants for polycystic ovary syndrome discovered in genome-wide association studies of Chinese individuals. J Clin Endocrinol Metab 100(1): E182-E186.
Carpintero NL, Suárez OA, Mangas CC, Varea CG, Rioja RG. 2014. Follicular steroid hormones as markers of oocyte quality and oocyte development potential. J Hum Reprod Sci 7(3): 187-193.
Carrasco RA, Leonardi CE, Hutt K, Singh J, Adams GP. 2020. Kisspeptin induces LH release and ovulation in an induced ovulatory. Biol Reprod 103(1): 49-59.
Chen ZJ, Zhao H, He L, Shi Y, Qin Y, Shi Y, …, Zhao Y. 2011. Genome-wide association study identifies susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3, 2p21 and 9q33.3. Nat Genet 43(1): 55-59.
Day FR, Hinds DA, Tung JY, Stolk L, Styrkarsdottir U, Saxena R, ..., Perry J. 2015. Causal mechanisms and balancing selection inferred from genetic associations with polycystic ovary syndrome. Nat Commun 6:8464.
De Leo V, Musacchio MC, Cappelli V, Massaro MG, Morgante G, Petraglia F. 2016. Genetic, hormonal and metabolic aspects of PCOS: an update. Reprod Biol Endocrinol 14(1):38-54. 
Deligeoroglou E, Kouskouti C, Christopoulos P. 2009. The role of genes in the polycystic ovary syndrome: predisposition and mechanisms. Gynecol Endocrinol 25(9): 603-609.
El-Shal AS, Zidan HE, Rashad NM, Abdelaziz AM, Harira MM. 2016. Association between genes encoding components of the Leutinizing hormone/Luteinizing hormone-choriogonadotrophin receptor pathway and polycystic ovary syndrome in Egyptian women. IUBMB life 68(1):23-36.
Enien WM, el Sahwy S, Harris CP, Seif MW, Elstein M. 1995. Human chorionic gonadotrophin and steroid concentrations in follicular fluid: the relationship to oocyte maturity and fertilization rates in stimulated and natural in-vitro fertilization cycles. Hum Reprod 10(11): 2840-2844.
Fenichel P, Rougier C, Hieronimus S, Chevalier N. 2017. Which origin for polycystic ovaries syndrome: Genetic, environmental, or both? Ann Endocrinol 78(3):176-185.
Forde N, O'Gorman A, Whelan H, Duffy P, O'Hara L, Kelly AK, …, Lonergan P. 2016. Lactation-induced changes in metabolic status and follicular-fluid metabolomic profile in postpartum dairy cows. Reprod Fertil Dev 28(12):1882-1892.
Fujibe Y, Baba T, Nagao S, Adachi S, Ikeda K, Morishita M, …, Saito T. 2019. Androgen potentiates the expression of FSH receptor and supports preantral follicle development in mice. J Ovarian Res 12(1): 31.
Gervásio CG, Bernuci MP, Silva-de-Sá MF, Rosa-e-Silva AC. 2014. The role of androgen hormones in early follicular development. ISRN obstetrics and gynecology 2014: 818010.
Hickey T, Chandy A, Norman RJ. 2002. The androgen receptor CAG repeats polymorphism and X-chromosome inactivation in Australian Caucasian women with infertility related to polycystic ovary syndrome. J Clin Endocrinol Metab 87(1): 161-165.
Jin JL, Sun J, Ge HJ, Cao YX, Wu XK, Liang FJ, …, Wang Y. 2009. Association between CYP19 gene SNP rs2414096 polymorphism and polycystic ovary syndrome in Chinese women. BMC Med Genet 10: 139.
Jones MR, Mathur R, Cui J, Guo X, Azziz R, Goodarzi MO. 2009. Independent confirmation of association between metabolic phenotypes of polycystic ovary syndrome and variation in the type 6 17β-hydroxysteroid dehydrogenase gene. J Clin Endocrinol Metab 94(12): 5034-5038.
Ju R, Wu W, Fei J, Qin Y, Tang Q, Wu D, …, Wang X. 2015. Association analysis between the polymorphisms of HSD17B5 and HSD17B6 and risk of polycystic ovary syndrome in Chinese population. Eur J Endocrinol 172(3): 227-233.
Kielkopf  CL, Bauer W, Urbatsch IL. 2020. Bradford Assay for Determining Protein Concentration. Cold Spring Harb Protoc.136-138.
Kohl TO, Ascoli CA. 2017. Immunometric Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay. Cold Spring Harb.458-462.
Lee EB, Chakravarthi VP, Wolfe MW, Rumi M. 2021. ERβ regulation of gonadotropin responses during folliculogenesis. Int J Mol Sci 22(19), 10348.
Lv Y, Du S, Huang X, Hao C. 2020. Follicular fluid estradiol is an improved predictor of in vitro fertilization/intracytoplasmic sperm injection and embryo transfer outcomes. Exp Ther Med 20(6): 131.
Marchiani S, Tamburrino L, Benini F, Pallecchi M, Bignozzi C, Conforti A, …, Baldi E. 2020. LH supplementation of ovarian stimulation protocols influences follicular fluid steroid composition contributing to the improvement of ovarian response in poor responder women. Sci Rep 10(1):12907.
Mehdizadeh A, Kalantar SM, Sheikhha MH, Aali BS, Ghanei A.2017. Association of SNP rs 2414096 CYP19 gene with polycystic ovarian syndrome in Iranian women. Int J Reprod Biomed 15(8):491-496.
Moore AM, Campbell RE. 2016. The neuroendocrine genesis of polycystic ovary syndrome: a role for arcuate nucleus GABA neurons. J Steroid Biochem Mol Biol 160:106-117.
Moran LJ, Teede HJ, Noakes M, Clifton PM, Norman RJ, Wittert GA. 2013. Sex hormone binding globulin, but not testosterone, is associated with the metabolic syndrome in overweight and obese women with polycystic ovary syndrome. J Endocrinol Invest 36(11): 1004-1010.
Mostafa RA, Al-Sherbeeny MM, Abdelazim IA, Fahmy AA, Farghali MM, Abdel-Fatah MA, Mahran MZ. 2016. Relation between aromatase gene CYP19 variation and hyperandrogenism in polycystic ovary syndrome Egyptian women. J Infertil Reprod Biol 4(1):1-5.
Nagy B, Poto L, Farkas N, Koppan M, Varnagy A, Kovacs K, …, Bodis J. 2019. Follicular fluid progesterone concentration is associated with fertilization outcome after IVF: a systematic review and meta-analysis. Reprod Biomed Online 38(6): 871-882.
Niu Z, Ye Y, Xia L, Feng Y, Zhang A. 2017. Follicular fluid cytokine composition and oocyte quality of polycystic ovary syndrome patients with metabolic syndrome undergoing in vitro fertilization. Cytokine 91: 180-186.
O'Brien Y, Wingfield M, O’Shea LC. 2019. Anti-Müllerian hormone and progesterone levels in human follicular fluid are predictors of embryonic development. Reprod Biol Endocrinol 17(1): 47.
Petry CJ, Ong KK, Michelmore KF, Artigas S, Wingate DL, Balen AH, …, Dunger DB. 2005. Association of aromatase (CYP 19) gene variation with features of hyperandrogenism in two populations of young women. Hum Reprod 20(7):1837-1843.
Revelli A, Delle Piane L, Casano S, Molinari E, Massobrio M, Rinaudo P. 2009. Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol 7: 40.
Roland AV, Moenter SM. 2014. Reproductive neuroendocrine dysfunction in polycystic ovary syndrome: insight from animal models. Front Neuroendocrinol 35(4): 494-511.
Rosenfield RL, Ehrmann DA. 2016. The pathogenesis of polycystic ovary syndrome (PCOS): the hypothesis of PCOS as functional ovarian hyperandrogenism revisited. Endocr Rev 37(5): 467-520.
Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group. 2004. consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Hum Reprod 19(1): 41-47.
Salehnia M, Zavareh S. 2013. The effects of progesterone on oocyte maturation and embryo development. Int J Fertil Steril 7(2): 74-81.
Salehzadeh A, Aghajani Sh, Ghasemian F, Mehrafza M, A. 2022. Tracking of single embryo blastomere biopsy: Development potential, chromosomal aneuploidy, and embryo transfer outcomes. Cell J.
Schüring AN, Welp A, Gromoll J, Zitzmann M, Sonntag B, Nieschlag E, ..., Kiesel L. 2012. Role of the CAG repeat polymorphism of the androgen receptor gene in polycystic ovary syndrome (PCOS). Exp Clin Endocrinol Diabetes 120(2): 73-79.
Shaaban Z, Jafarzadeh Shirazi MR, Nooranizadeh MH, Tamadon A, Rahmanifar F, Ahmadloo S, …, Koohi Hosseinabadi O. 2018. Decrease in arginine-phenylalanine-amide-related peptide-3 gene expression of dorsomedial hypothalamic nucleus in constant light exposure model of polycystic ovarian syndrome. Int J Fertil Steril 12(1): 43-50
Sheikhha MH, Kalantar SM, Ghasemi N. 2007. Genetics of polycystic ovary syndrome. IJRM 5(2): 1-6.
Shi Y, Zhao H, Shi Y, Cao Y, Yang D, Li Z, …, Chen ZJ. 2012. Genome-wide association study identifies eight new risk loci for polycystic ovary syndrome. Nat Genet 44(9): 1020-1025.
Sun Z, Song J, Zhang X, Wang A, Guo Y, Yang Y, …, Deng J. 2018. SWATHHM-Based Metabolomics of Follicular Fluid in Patients Shows That Progesterone Adversely Affects Oocyte Quality. Biomed Res Int 2018: 1780391.
Tian Y, Zhao H, Chen H, Peng Y, Cui L, Du Y, …, Chen ZJ. 2016. Variants in FSHB are associated with polycystic ovary syndrome and luteinizing hormone level in Han Chinese women. J Clin Endocrinol Metab 101(5): 2178-2184.
Ubuka T, Morgan K, Pawson AJ, Osugi T, Chowdhury VS, Minakata H, …, Bentley GE. 2009. Identification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis. PLoS ONE 4(12): e8400.
Velasco M, Alexander C, King J, Zhao Y, Garcia J, Rodriguez A.  2006. Association of lower plasma estradiol levels and low expression of scavenger receptor class B, type I in infertile women. Fertil Steril 85(5): 1391-1397.
Wen X, Li D, Tozer AJ, Docherty SM, Iles RK. 2010. Estradiol, progesterone, testosterone profiles in human follicular fluid and cultured granulosa cells from luteinized pre-ovulatory follicles. Reprod Biol Endocrinol 8: 117.
Welt CK, Styrkarsdottir U, Ehrmann DA, Thorleifsson G, Arason G, Gudmundsson JA, …, Stefansson K. 2012. Variants in DENND1A are associated with polycystic ovary syndrome in women of European ancestry. J Clin Endocrinol Metab 97(7): E1342-E1347.
Xia P, Younglai EV. 2000. Relationship between steroid concentrations in ovarian follicular fluid and oocyte morphology in patients undergoing intracytoplasmic sperm injection (ICSI) treatment. J Reprod Fertil 118(2): 229-233.
Xita N, Lazaros L, Georgiou I, Tsatsoulis A. 2010. CYP19 gene: a genetic modifier of polycystic ovary syndrome phenotype. Fertil Steril 94(1): 250-254.
Xu P, Zhang XL, Xie GB, Zhang CW, Shen SM, Zhang XX, ..., Wang Y. 2013. The (TTTA) n polymorphism in intron 4 of CYP19 and the polycystic ovary syndrome risk in a Chinese population. Mol Biol Rep 40(8): 5041-5047.