Open Access

Choline supplementation influences ovarian follicular development

Xiaoshu Zhan1,2,Lauren Fletcher2,Serena Dingle2,Enzo Baracuhy2,Bingyun Wang1,*,Lee-Anne Huber2,Julang Li2,*
Department of Life Science and Engineering, Foshan University, 528231 Foshan, Guangdong, China
Department of Animal BioSciences, University of Guelph, Guelph, ON N1G 2W1, Canada
DOI: 10.52586/5046 Volume 26 Issue 12, pp.1525-1536
Submited: 15 August 2021 Revised: 24 November 2021
Accepted: 25 November 2021 Published: 30 December 2021
*Corresponding Author(s):  
Bingyun Wang
*Corresponding Author(s):  
Julang Li
Copyright: © 2021 The author(s). Published by BRI. This is an open access article under the CC BY 4.0 license (

Background: Female infertility is a health issue for both humans and animals and despite developments in medical interventions, there are still some conditions that cannot be treated successfully. It is important to explore other potential therapies or remedies that could improve reproductive health. Choline is an over-the-counter supplement and essential nutrient that has many health benefits. It has been suggested to be beneficial in various aspects of fertility, including fetal development and endocrine disorders like polycystic ovarian syndrome (PCOS). However, choline’s impact on ovarian function has not been explored. Methods: To study the effects of choline on ovarian development, 36 female Yorkshire ×× Landrace pigs were fed the following four supplemented diets between 90 and 186 days of age: (1) Control (corn and soybean meal-based diet that met estimated nutrient requirements, n = 9); (2) Choline (additional 500 mg choline per 1 kg of control diet, n = 8); (3) Omega-3 (additional 5556 mg Omega-3 per 1 kg control diet by introducing fish oil); (4) Choline + Omega-3 (500 mg choline + 5556 mg Omega-3 per 1 kg control diet). Pigs fed the choline-supplemented diet were compared to the control group and those fed diets supplemented with Omega-3 as fertility-promoting agent. Results: It was found that the number of corpus luteum per ovary in the Choline (16.25 ±± 2.88), Omega-3 (10.78 ±± 1.71) and Choline + Omega-3 (14.89 ±± 2.97) groups were all higher in comparison to that of the control group (5.56 ±± 1.72, p << 0.05). The percentage of antral follicles in the Choline + Omega-3 group were higher compared to the control group (p << 0.05). To elucidate the potential molecular mechanism of choline on these improved ovarian phenotypes, the expression of a group of genes that are involved in ovarian development, including cytochrome P450 family 11 subfamily A member 1 (CYP11A1), follicle stimulating hormone receptor (FHSR) and luteinizing hormone receptor (LHR), was analyzed using RT-qPCR. The expression of both LHR and CYP11A1 was significantly upregulated in the choline-supplemented group (p << 0.05), while there are no differences in FSHR expression among all the groups. Additionally, the expression of miR-21, -378, -574, previously found to be important in ovarian function, were examined. Our data showed that miR-574 was upregulated in the Choline group while miR-378 was upregulated in the Choline + Omega-3 group in comparison to the control group (p << 0.05). Further, serum metabolite analysis showed that 1-(5Z, 8Z, 11Z, 14Z, 17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, a form of phosphatidylcholine metabolite, was significantly increased in all the treatment groups (p << 0.05), while testosterone was significantly increased in both Omega-3 and Choline + Omega-3 groups (p << 0.05) and tended to be reduced in the choline-supplemented group (p = 0.08) compared to the control group. Conclusions: Our study demonstrated choline’s influence on ovarian function in vivo, and offered insights into the mechanisms behind its positive effect on ovarian development phenotype.

Key words

Infertility; Ovarian development; Female re-productive; Choline; Follicle maturation


[1] Esteves SC, Humaidan P, Roque M, Agarwal A. Female infertility and assisted reproductive technology. Panminerva Medica. 2019; 61: 1–2.

[2] Zegers-Hochschild F, Adamson GD, Dyer S, Racowsky C, de Mouzon J, Sokol R, et al. The International Glossary on Infertility and Fertility Care, 2017. Human Reproduction. 2017; 32: 1786–1801.

[3] Prevatt C, Lamb GC, Dahlen C, Mercadante VR, Waters K. What Is the Economic Impact of Infertility in Beef Cattle? EDIS. 2018; 2018.

[4] Gaskins AJ, Chavarro JE. Diet and fertility: a review. American Journal of Obstetrics and Gynecology. 2018; 218: 379–389.

[5] Chavarro JE, Rich-Edwards JW, Rosner BA, Willett WC. Diet and Lifestyle in the Prevention of Ovulatory Disorder Infertility. Obstetrics & Gynecology. 2007; 110: 1050–1058.

[6] Wise LA, Wesselink AK, Tucker KL, Saklani S, Mikkelsen EM, Cueto H, et al. Dietary Fat Intake and Fecundability in 2 Preconception Cohort Studies. American Journal of Epidemiology. 2018; 187: 60–74.

[7] Simopoulos AP. The importance of the ratio of omega-6/Omega-3 essential fatty acids. Biomedicine & Pharmacotherapy. 2002; 56: 365–379.

[8] Nehra D, Le HD, Fallon EM, Carlson SJ, Woods D, White YA, et al. Prolonging the female reproductive lifespan and improving egg quality with dietary Omega-3 fatty acids. Aging Cell. 2012; 11: 1046–1054.

[9] Mumford SL, Schisterman EF, Dasharathy S, Pollack AZ, Zhang C, Wactawski-Wende J. Omega-3 fatty acids and ovulatory function. Fertility and Sterility. 2011; 96: S15.

[10] Calder PC. Omega-3 Fatty Acids and Inflammatory Processes. Nutrients. 2010; 2: 355–374.

[11] Barsky M, Blesson CS. Oocytes, obesity, and Omega-3 fatty acids. Fertility and Sterility. 2020; 113: 71–72.

[12] Zeisel SH, Da Costa KA, Franklin PD, Alexander EA, Lamont JT, Sheard NF, et al. Choline, an essential nutrient for humans. The FASEB Journal. 1991; 5: 2093–2098.

[13] Zeisel SH, Blusztajn JK. Choline and Human Nutrition. Annual Review of Nutrition. 1994; 14: 269–296.

[14] Troen AM, Chao WH, Crivello NA, D’Anci KE, Shukitt-Hale B, Smith DE, et al. Cognitive Impairment in Folate-Deficient Rats Corresponds to Depleted Brain Phosphatidylcholine and is Prevented by Dietary Methionine without Lowering Plasma Homocysteine. The Journal of Nutrition. 2008; 138: 2502–2509.

[15] Moore LD, Le T, Fan G. DNA Methylation and its Basic Function. Neuropsychopharmacology. 2013; 38: 23–38.

[16] Sweiry JH, Page KR, Dacke CG, Abramovich DR, Yudilevich DL. Evidence of saturable uptake mechanisms at maternal and fetal sides of the perfused human placenta by rapid paired-tracer dilution: studies with calcium and choline. Journal of Developmental Physiology. 1986; 8: 435–445.

[17] Shaw GM, Carmichael SL, Yang W, Selvin S, Schaffer DM. Periconceptional Dietary Intake of Choline and Betaine and Neural Tube Defects in Offspring. American Journal of Epidemiology. 2004; 160: 102–109.[18] Albright CD, Tsai AY, Friedrich CB, Mar MH, Zeisel SH. Choline availability alters embryonic development of the hippocampus and septum in the rat. Developmental Brain Research. 1999; 113: 13–20.

[19] Sun L, Hu W, Liu Q, Hao Q, Sun B, Zhang Q, et al. Metabonomics Reveals Plasma Metabolic Changes and Inflammatory Marker in Polycystic Ovary Syndrome Patients. Journal of Proteome Research. 2012; 11: 2937–2946.

[20] Ersahin AA, Çalışkan E. Clomiphene citrate changes metabolite content of follicular fluid of PCOS women. European Review for Medical and Pharmacological Sciences. 2018; 22: 4359–4362.

[21] Meurens F, Summerfield A, Nauwynck H, Saif L, Gerdts V. The pig: a model for human infectious diseases. Trends in Microbiology. 2012; 20: 50–57.

[22] Mordhorst BR, Prather RS. Pig Models of Reproduction. In Heide Schatten, Gheorghe M. Constantinescu (eds.) Animal Models and Human Reproduction (pp. 213–234). John Wiley & Sons: Hoboken, New Jersey. 2017.

[23] Lorenzen E, Follmann F, Jungersen G, Agerholm JS. A review of the human vs. porcine female genital tract and associated immune system in the perspective of using minipigs as a model of human genital Chlamydia infection. Veterinary Research. 2015; 46: 116.

[24] Shahidi F, Ambigaipalan P. Omega-3 Polyunsaturated Fatty Acids and their Health Benefits. Annual Review of Food Science and Technology. 2018; 9: 345–381.

[25] Tuz R, Schwarz T, Małopolska M, Nowicki J. The Use of Vagina-Cervix Length Measurement in Evaluation of Future Reproductive Performance of Sows: A Preliminary Study under Commercial Conditions. Animals. 2019; 9: 158.

[26] Huynh E, Penney J, Caswell J, Li J. Protective Effects of Protegrin in Dextran Sodium Sulfate-Induced Murine Colitis. Frontiers in Pharmacology. 2019; 10: 156.

[27] Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods. 2001; 25: 402–408.

[28] Yan K, Cheng L, Liu P, Liu Z, Zhao S, Zhu W, et al. Polyinosinic–Polycytidylic Acid Perturbs Ovarian Functions through Toll-Like Receptor 3-Mediated Tumor Necrosis Factor a Production in Female Mice1. Biology of Reproduction. 2015; 93: 11.

[29] Zeisel SH. Choline: Critical Role during Fetal Development and Dietary Requirements in Adults. Annual Review of Nutrition. 2006; 26: 229–250.

[30] Martin Rillo S, de Alba Romero C, Romero Rodriguez A, Cidoncha R, Ziecik AJ. Litter Size and Vagina-cervix Catheter Penetration Length in Gilts. Reproduction in Domestic Animals. 2001; 36: 297–300.

[31] McIntosh J, Feltovich H, Berghella V, Manuck T. The role of routine cervical length screening in selected high- and low-risk women for preterm birth prevention. American Journal of Obstetrics and Gynecology. 2016; 215: B2–B7.

[32] Ridgway ND. The role of phosphatidylcholine and choline metabolites to cell proliferation and survival. Critical Reviews in Biochemistry and Molecular Biology. 2013; 48: 20–38.

[33] Ridgway ND. Phospholipid Synthesis in Mammalian Cells. In Ridgway ND, McLeod RS (eds.) Biochemistry of Lipids, Lipoproteins and Membranes (pp. 209–236). 6th edn. Elsevier: Boston. 2016.

[34] McKay RM. Preweaning losses of piglets as a result of index selection for reduced backfat thickness and increased growth rate. Canadian Journal of Animal Science. 1993; 73: 437–442.

[35] Roongsitthichai A, Tummaruk P. Importance of Backfat Thickness to Reproductive Performance in Female Pigs. The Thai Journal of Veterinary Medicine. 2014; 44: 171.

[36] Houde AA, Méthot S, Murphy BD, Bordignon V, Palin MF. Relationships between backfat thickness and reproductive efficiency of sows: a two-year trial involving two commercial herds fixing backfat thickness at breeding. Canadian Journal of Animal Science. 2010; 90: 429–436.

[37] Kuda O, Rossmeisl M, Kopecky J. Omega-3 fatty acids and adipose tissue biology. Molecular Aspects of Medicine. 2018; 64: 147–160.

[38] Frisch RE. Body fat, menarche, fitness and fertility. Human Reproduction. 1987; 2: 521–533.

[39] Farquhar CM, Bhattacharya S, Repping S, Mastenbroek S, Kamath MS, Marjoribanks J, et al. Female subfertility. Nature Reviews Disease Primers. 2019; 5: 7.

[40] García-Guerra A, Motta JCL, Melo LF, Kirkpatrick BW, Wiltbank MC. Ovulation rate, antral follicle count, and circulating anti-Müllerian hormone in Trio allele carriers, a novel high fecundity bovine genotype. Theriogenology. 2017; 101: 81–90.

[41] Echternkamp SE, Gregory KE, Dickerson GE, Cundiff LV, Koch RM, Van Vleck LD. Twinning in cattle: II. Genetic and environmental effects on ovulation rate in puberal heifers and postpartum cows and the effects of ovulation rate on embryonic survival. Journal of Animal Science. 1990; 68: 1877–1888.

[42] Clutter AC, Kirby YLK, Nielsen MK. Uterine capacity and ovulation rate in mice selected 21 generations on alternative criteria to increase litter size. Journal of Animal Science. 1994; 72: 577–583.

[43] Kemp B, Da Silva CLA, Soede NM. Recent advances in pig reproduction: Focus on impact of genetic selection for female fertility. Reproduction in Domestic Animals. 2018; 53: 28–36.

[44] Wallace WH, Kelsey TW. Human Ovarian Reserve from Conception to the Menopause. PLoS ONE. 2010; 5: e8772.

[45] McClatchie T, Meredith M, Ouédraogo MO, Slow S, Lever M, Mann MRW, et al. Betaine is accumulated via transient choline dehydrogenase activation during mouse oocyte meiotic maturation. Journal of Biological Chemistry. 2017; 292: 13784–13794.

[46] Lai FN, Liu XL, Li N, Zhang RQ, Zhao Y, Feng YZ, et al. Phosphatidylcholine could protect the defect of zearalenone exposure on follicular development and oocyte maturation. Aging. 2018; 10: 3486–3506.

[47] Puett D, Angelova K, da Costa MR, Warrenfeltz SW, Fanelli F. The luteinizing hormone receptor: Insights into structurefunction relationships and hormone-receptor-mediated changes in gene expression in ovarian cancer cells. Molecular and Cellular Endocrinology. 2010; 329: 47–55.

[48] Davis JS, LaVoie HA. Molecular Regulation of Progesterone Production in the Corpus Luteum. In Leung PCK, Adashi EY (eds.) The Ovary (pp. 237–253). 3rd edn. Academic Press: Cambridge, MA. 2019.

[49] Narayan P, Puett D. Luteinizing Hormone Receptor Signaling. In Henry HL, Norman AW (eds.) Encyclopedia of Hormones (pp. 612–616). Academic Press: New York. 2003.

[50] Bo Pan, Julang Li. MicroRNA-21 up-regulates metalloprotease by down-regulating TIMP3 during cumulus cell-oocyte complex in vitro maturation. Molecular and Cellular Endocrinology. 2018; 477: 29–38.[51] Sun XF, Li YP, Pan B, Wang YF, Li J, Shen W. Molecular regulation of miR-378 on the development of mouse follicle and the maturation of oocyte in vivo. Cell Cycle. 2018; 17: 2230–2242.

[52] Pan B, Toms D, Shen W, Li J. MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells. American Journal of Physiology-Endocrinology and Metabolism. 2015; 308: E525–E534.

[53] Zi X, Lu J, Ma L. Identification and comparative analysis of the ovarian microRNAs of prolific and non-prolific goats during the follicular phase using high-throughput sequencing. Scientific Reports. 2017; 7: 1921.

[54] Ma T, Jiang H, Gao Y, Zhao Y, Dai L, Xiong Q, et al. Microarray analysis of differentially expressed microRNAs in non-regressed and regressed bovine corpus luteum tissue; microRNA-378 may suppress luteal cell apoptosis by targeting the interferon gamma receptor 1 gene. Journal of Applied Genetics. 2011; 52: 481–486.

[55] Kelly DM, Jones TH. Testosterone: a metabolic hormone in health and disease. Journal of Endocrinology. 2013; 217: R25–R45.

[56] Atiomo W, Khalid S, Parameshweran S, Houda M, Layfield R. Proteomic biomarkers for the diagnosis and risk stratification of polycystic ovary syndrome: a systematic review. BJOG: an International Journal of Obstetrics abd Gynaecology. 2009; 116: 137–143.

[57] Wallace M, Cottell E, Gibney MJ, McAuliffe FM, Wingfield M, Brennan L. An investigation into the relationship between the metabolic profile of follicular fluid, oocyte developmental potential, and implantation outcome. Fertility and Sterility. 2012; 97: 1078–1084.e8.

Landmark/articles/materials/Supplementary Table 1.xlsx
Landmark/articles/materials/Supplementary Table 2.xlsx
Share and Cite
Xiaoshu Zhan, Lauren Fletcher, Serena Dingle, Enzo Baracuhy, Bingyun Wang, Lee-Anne Huber, Julang Li. Choline supplementation influences ovarian follicular development. Frontiers in Bioscience-Landmark. 2021. 26(12); 1525-1536.