Effects of myo-inositol plus folic acid on ovarian morphology and oocyte quality in PCOS mouse model

Published online by Cambridge University Press: 09 January 2023 Although the role of myo-inositol (MYO) in promoting the oocyte quality of PCOS patients has been documented in human studies; the cellular effects of this supplement on oocytes have not been directly examined due to ethical limitations. In the first phase of this study, MYO dosimetry was carried out simultaneously with the PCOS model development. An effective dose was obtained following the assessment of fasting insulin and testosterone levels using ELISA and ovarian morphology appraisal by histopathology. In the second phase, following the continuous administration of the effective dose of MYO and dehydroepiandrosterone (DHEA), cellular evaluation was performed. The quality of oocytes from superovulation was analyzed by examining maturity and normal morphology percentage using a stereomicroscope, intracellular reactive oxygen species (ROS) and glutathione (GSH) levels using fluorometry, and ATP count evaluation using ELISA. The results revealed that, among the four different MYO concentrations, the 0.36 mg/g dose compared with the DHEA group reduced testosterone levels and large atretic antral follicles (LAtAnF) diameter. This dose also increased the corpus luteum count and the granulosa:theca (G/T)layer thickness ratio in antral follicles. Furthermore, this dose increased mature oocytes and normal morphology percentage, ATP count, and GSH levels; however, it decreased ROS levels in mature oocytes. Our findings provide the grounds for further cellular and molecular studies on the PCOS mouse model, suggesting that the improvement in mitochondrial function and its antioxidant properties is probably one of the mechanisms by which MYO increases oocyte quality. - Type - Research Article - Information - Copyright - © The Author(s), 2023. Published by Cambridge University Press Akbari Sene, A., Tabatabaie, A., Nikniaz, H., Alizadeh, A., Sheibani, K., Mortezapour Alisaraie, M., Tabatabaie, M., Ashrafi, M. and Amjadi, F. (2019). The myo-inositol effect on the oocyte quality and fertilization rate among women with polycystic ovary syndrome undergoing assisted reproductive technology cycles: A randomized clinical trial. Archives of Gynecology and Obstetrics, 299(6), 1701–1707. doi: 10.1007/s00404-019-05111-1 CrossRefGoogle ScholarPubMed Amini, L., Tehranian, N., Movahedin, M., Ramezani Tehrani, F. R. and Soltanghoraee, H. (2016). Polycystic ovary morphology (PCOM) in estradiol valerate treated mouse model. International Journal of Women’s Health and Reproduction Sciences, 4(1), 13–17. doi: 10.15296/ijwhr.2016.04 CrossRefGoogle Scholar Aragno, M., Brignardello, E., Tamagno, E., Gatto, V., Danni, O. and Boccuzzi, G. (1997). Dehydroepiandrosterone administration prevents the oxidative damage induced by acute hyperglycemia in rats. Journal of Endocrinology, 155(2), 233–240. doi: 10.1677/joe.0.1550233 CrossRefGoogle ScholarPubMed Aragno, M., Mastrocola, R., Brignardello, E., Catalano, M., Robino, G., Manti, R., Parola, M., Danni, O. and Boccuzzi, G. (2002). Dehydroepiandrosterone modulates nuclear factor-κB activation in hippocampus of diabetic rats. Endocrinology, 143(9), 3250–3258. doi: 10.1210/en.2002-220182 CrossRefGoogle ScholarPubMed Artini, P. G., Di Berardino, O. M., Papini, F., Genazzani, A. D., Simi, G., Ruggiero, M. and Cela, V. (2013). Endocrine and clinical effects of myo-inositol administration in polycystic ovary syndrome. A randomized study. Gynecological Endocrinology, 29(4), 375–379. doi: 10.3109/09513590.2012.743020 CrossRefGoogle ScholarPubMed Azhary, J. M. K., Harada, M., Takahashi, N., Nose, E., Kunitomi, C., Koike, H., Hirata, T., Hirota, Y., Koga, K., Wada-Hiraike, O., Fujii, T. and Osuga, Y. (2019). Endoplasmic reticulum stress activated by androgen enhances apoptosis of granulosa cells via induction of death receptor 5 in PCOS. Endocrinology, 160(1), 119–132. doi: 10.1210/en.2018-00675 CrossRefGoogle ScholarPubMed Bizzarri, M., Cucina, A., Dinicola, S., Harrath, A. H., Alwasel, S. H., Unfer, V. and Bevilacqua, A. (2016). Does myo-inositol effect on PCOS follicles involve cytoskeleton regulation? Medical Hypotheses, 91, 1–5. doi: 10.1016/j.mehy.2016.03.014 CrossRefGoogle ScholarPubMed Cardozo, E., Pavone, M. E. and Hirshfeld-Cytron, J. E. (2011). Metabolic syndrome and oocyte quality. Trends in Endocrinology and Metabolism, 22(3), 103–109. doi: 10.1016/j.tem.2010.12.002 CrossRefGoogle ScholarPubMed Chiu, T. T., Rogers, M. S., Law, E. L., Briton-Jones, C. M., Cheung, L. P. and Haines, C. J. (2002). Follicular fluid and serum concentrations of myo-inositol in patients undergoing IVF: Relationship with oocyte quality. Human Reproduction, 17(6), 1591–1596. doi: 10.1093/humrep/17.6.1591 CrossRefGoogle ScholarPubMed Ciotta, L., Stracquadanio, M., Pagano, I., Carbonaro, A., Palumbo, M. and Gulino, F. (2011). Effects of myo-inositol supplementation on oocyte’s quality in PCOS patients: A double blind trial. European Review for Medical and Pharmacological Sciences, 15(5), 509–514.Google ScholarPubMed Croze, M. L., Vella, R. E., Pillon, N. J., Soula, H. A., Hadji, L., Guichardant, M. and Soulage, C. O. (2013). Chronic treatment with myo-inositol reduces white adipose tissue accretion and improves insulin sensitivity in female mice. Journal of Nutritional Biochemistry, 24(2), 457–466. doi: 10.1016/j.jnutbio.2012.01.008 CrossRefGoogle ScholarPubMed Eini, F., Novin, M. G., Joharchi, K., Hosseini, A., Nazarian, H., Piryaei, A. and Bidadkosh, A. (2017). Intracytoplasmic oxidative stress reverses epigenetic modifications in polycystic ovary syndrome. Reproduction, Fertility, and Development, 29(12), 2313–2323. doi: 10.1071/RD16428 CrossRefGoogle ScholarPubMed Genazzani, A. D., Prati, A., Santagni, S., Ricchieri, F., Chierchia, E., Rattighieri, E., Campedelli, A., Simoncini, T. and Artini, P. G. (2012). Differential insulin response to myo-inositol administration in obese polycystic ovary syndrome patients. Gynecological Endocrinology, 28(12), 969–973. doi: 10.3109/09513590.2012.685205 CrossRefGoogle ScholarPubMed Gerli, S., Papaleo, E., Ferrari, A. and Di Renzo, G. C. (2007). Randomized, double blind placebo-controlled trial: Effects of myo-inositol on ovarian function and metabolic factors in women with PCOS. European Review for Medical and Pharmacological Sciences, 11(5), 347–354.Google ScholarPubMed Gleicher, N., Weghofer, A. and Barad, D. H. (2011). The role of androgens in follicle maturation and ovulation induction: Friend or foe of infertility treatment? Reproductive Biology and Endocrinology: RBandE, 9(1), 116. doi: 10.1186/1477-7827-9-116 CrossRefGoogle ScholarPubMed Goud, P. T., Goud, A. P., Van Oostveldt, P. and Dhont, M. (1999). Presence and dynamic redistribution of type I inositol 1, 4, 5-trisphosphate receptors in human oocytes and embryos during in-vitro maturation, fertilization and early cleavage divisions. Molecular Human Reproduction, 5(5), 441–451. doi: 10.1093/molehr/5.5.441 CrossRefGoogle ScholarPubMed Hakimpour, S., Jelodar, G., Shabani, R., Pourheydar, B., Ajdary, M. and Mehdizadeh, M. (2022). Study of vitamin D3 formulation prepared by phytosolve technique and its effect on CTRP6 gene expression in PCOS model. Journal of Drug Delivery Science and Technology, 73, 103489. doi: 10.1016/j.jddst.2022.103489 CrossRefGoogle Scholar Hayes, M. G., Urbanek, M., Ehrmann, D. A., Armstrong, L. L., Lee, J. Y., Sisk, R., et al. (2015). Genome-wide association of polycystic ovary syndrome implicates alterations in gonadotropin secretion in European ancestry populations. Nature Communications, 6, 7502. doi: 10.1038/ncomms8502 CrossRefGoogle ScholarPubMed Henmi, H., Endo, T., Nagasawa, K., Hayashi, T., Chida, M., Akutagawa, N., Iwasaki, M., Kitajima, Y., Kiya, T., Nishikawa, A., Manase, K. and Kudo, R. (2001). Lysyl oxidase and MMP-2 expression in dehydroepiandrosterone-induced polycystic ovary in rats. Biology of Reproduction, 64(1), 157–162. doi: 10.1095/biolreprod64.1.157 Google ScholarPubMed Huang, Y., Yu, Y., Gao, J., Li, R., Zhang, C., Zhao, H., Zhao, Y. and Qiao, J. (2015). Impaired oocyte quality induced by dehydroepiandrosterone is partially rescued by metformin treatment. PLOS ONE, 10(3), e0122370. doi: 10.1371/journal.pone.0122370 CrossRefGoogle ScholarPubMed Ikeda, K., Baba, T., Morishita, M., Honnma, H., Endo, T., Kiya, T. and Saito, T. (2014). Long-term treatment with dehydroepiandrosterone may lead to follicular atresia through interaction with anti-Müllerian hormone. Journal of Ovarian Research, 7(1), 46. doi: 10.1186/1757-2215-7-46 CrossRefGoogle ScholarPubMed Kamenov, Z. and Gateva, A. (2020). Inositols in PCOS. Molecules, 25(23), 5566. doi: 10.3390/molecules25235566 CrossRefGoogle ScholarPubMed Karuputhula, N. B., Chattopadhyay, R., Chakravarty, B. and Chaudhury, K. (2013). Oxidative status in granulosa cells of infertile women undergoing IVF. Systems Biology in Reproductive Medicine, 59(2), 91–98. doi: 10.3109/19396368.2012.743197 CrossRefGoogle ScholarPubMed Kim, H. H., Shaipanich, M., Hasselblatt, K. and Yeh, J. (2003). Induction of apoptosis and ovarian cyst formation in the mouse ovary by dehydroepiandrosterone (DHEA). Journal of Medicine, 34(1–6), 101–112.Google ScholarPubMed Kockskämper, J., Zima, A. V., Roderick, H. L., Pieske, B., Blatter, L. A. and Bootman, M. D. (2008). Emerging roles of inositol 1,4,5-trisphosphate signaling in cardiac myocytes. Journal of Molecular and Cellular Cardiology, 45(2), 128–147. doi: 10.1016/j.yjmcc.2008.05.014 CrossRefGoogle ScholarPubMed Kokosar, M., Benrick, A., Perfilyev, A., Fornes, R., Nilsson, E., Maliqueo, M., Behre, C. J., Sazonova, A., Ohlsson, C., Ling, C. and Stener-Victorin, E. (2016). Epigenetic and transcriptional alterations in human adipose tissue of polycystic ovary syndrome. Scientific Reports, 6, 22883. doi: 10.1038/srep22883 CrossRefGoogle ScholarPubMed Laganà, A. S., Rossetti, P., Buscema, M., La Vignera, S., Condorelli, R. A., Gullo, G., Granese, R. and Triolo, O. (2016). Metabolism and ovarian function in PCOS women: A therapeutic approach with inositols. International Journal of Endocrinology, 2016, 6306410. doi: 10.1155/2016/6306410 CrossRefGoogle ScholarPubMed Lewin, L. M., Szeinberg, A. and Lepkifker, E. (1973). Gas chromatographic measurement of myo-inositol in human blood, cerebrospinal fluid and seminal fluid. Clinica Chimica Acta, 45(4), 361–368. doi: 10.1016/0009-8981(73)90036-3 CrossRefGoogle Scholar Maurya, V. K., Sangappa, C., Kumar, V., Mahfooz, S., Singh, A., Rajender, S. and Jha, R. K. (2014). Expression and activity of Rac1 is negatively affected in the dehydroepiandrosterone induced polycystic ovary of mouse. Journal of Ovarian Research, 7(1), 32. doi: 10.1186/1757-2215-7-32 CrossRefGoogle ScholarPubMed Mehlmann, L. M. and Kline, D. (1994). Regulation of intracellular calcium in the mouse egg: Calcium release in response to sperm or inositol trisphosphate is enhanced after meiotic maturation. Biology of Reproduction, 51(6), 1088–1098. doi: 10.1095/biolreprod51.6.1088 CrossRefGoogle ScholarPubMed Mikaeili, S., Rashidi, B. H., Safa, M., Najafi, A., Sobhani, A., Asadi, E. and Abbasi, M. (2016). Altered FoxO3 expression and apoptosis in granulosa cells of women with polycystic ovary syndrome. Archives of Gynecology and Obstetrics, 294(1), 185–192. doi: 10.1007/s00404-016-4068-z CrossRefGoogle ScholarPubMed National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. (2011). Guide for the Care and Use of Laboratory Animals. 8th edition. Washington (DC): National Academies Press (US). Available from: https://www.ncbi.nlm.nih.gov/books/NBK54050/ doi: 10.17226/12910 Google Scholar Nikmard, F., Hosseini, E., Bakhtiyari, M., Ashrafi, M., Amidi, F. and Aflatoonian, R. (2017). Effects of melatonin on oocyte maturation in PCOS mouse model. Animal Science Journal, 88(4), 586–592. doi: 10.1111/asj.12675 CrossRefGoogle ScholarPubMed Papaleo, E., Molgora, M., Quaranta, L., Pellegrino, M. and De Michele, F. (2011). myo-inositol products in polycystic ovary syndrome (PCOS) treatment: Quality, labeling accuracy, and cost comparison. European Review for Medical and Pharmacological Sciences, 15(2), 165–174.Google ScholarPubMed Patel, S. S. and Carr, B. R. (2008). Oocyte quality in adult polycystic ovary syndrome. Seminars in Reproductive Medicine, 26(2), 196–203. doi: 10.1055/s-2008-1042958 CrossRefGoogle ScholarPubMed Qiao, J. and Feng, H. L. (2011). Extra- and intra-ovarian factors in polycystic ovary syndrome: Impact on oocyte maturation and embryo developmental competence. Human Reproduction Update, 17(1), 17–33. doi: 10.1093/humupd/dmq032 CrossRefGoogle ScholarPubMed Ravanos, K., Monastra, G., Pavlidou, T., Goudakou, M. and Prapas, N. (2017). Can high levels of d-chiro-inositol in follicular fluid exert detrimental effects on blastocyst quality? European Review for Medical and Pharmacological Sciences, 21(23), 5491–5498. doi: 10.26355/eurrev_201712_13940 Google ScholarPubMed Safiulina, D., Peet, N., Seppet, E., Zharkovsky, A. and Kaasik, A. (2006). Dehydroepiandrosterone inhibits complex I of the mitochondrial respiratory chain and is neurotoxic in vitro and in vivo at high concentrations. Toxicological Sciences, 93(2), 348–356. doi: 10.1093/toxsci/kfl064 CrossRefGoogle ScholarPubMed Song, X., Shen, Q., Fan, L., Yu, Q., Jia, X., Sun, Y., Bai, W. and Kang, J. (2018). Dehydroepiandrosterone-induced activation of mTORC1 and inhibition of autophagy contribute to skeletal muscle insulin resistance in a mouse model of polycystic ovary syndrome. Oncotarget, 9(15), 11905–11921. doi: 10.18632/oncotarget.24190 CrossRefGoogle Scholar Tamura, H., Takasaki, A., Miwa, I., Taniguchi, K., Maekawa, R., Asada, H., Taketani, T., Matsuoka, A., Yamagata, Y., Shimamura, K., Morioka, H., Ishikawa, H., Reiter, R. J. and Sugino, N. (2008). Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate. Journal of Pineal Research, 44(3), 280–287. doi: 10.1111/j.1600-079X.2007.00524.x CrossRefGoogle ScholarPubMed Ubaldi, F. and Rienzi, L. (2008). Morphological selection of gametes. Placenta, 29, Suppl. B, 115–120. doi: 10.1016/j.placenta.2008.08.009 CrossRefGoogle ScholarPubMed Unfer, V., Carlomagno, G., Papaleo, E., Vailati, S., Candiani, M. and Baillargeon, J. P. (2014). Hyperinsulinemia alters myoinositol to d-chiroinositol ratio in the follicular fluid of patients with PCOS. Reproductive Sciences, 21(7), 854–858. doi: 10.1177/1933719113518985 CrossRefGoogle ScholarPubMed Unfer, V., Dinicola, S., Laganà, A. S. and Bizzarri, M. (2020). Altered ovarian inositol ratios may account for pathological steroidogenesis in PCOS. International Journal of Molecular Sciences, 21(19), 7157. doi: 10.3390/ijms21197157 CrossRefGoogle ScholarPubMed Unfer, V., Facchinetti, F., Orrù, B., Giordani, B. and Nestler, J. (2017). myo-inositol effects in women with PCOS: A meta-analysis of randomized controlled trials. Endocrine Connections, 6(8), 647–658. doi: 10.1530/EC-17-0243 CrossRefGoogle ScholarPubMed US Food and Drug Administration. (2005). Guidance for industry: estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers. Center for Drug Evaluation and Research (CDER), Rockville, MD. Available from: http://www.fda.gov/downloads/drugs/guidances/ucm078932.pdf.Google Scholar Vitale, S. G., Rossetti, P., Corrado, F., Rapisarda, A. M. C., La Vignera, S., Condorelli, R. A., Valenti, G., Sapia, F., Laganà, A. S. and Buscema, M. (2016). How to achieve high-quality oocytes? The key role of myo-inositol and melatonin. International Journal of Endocrinology, 2016, 4987436. doi: 10.1155/2016/4987436 CrossRefGoogle ScholarPubMed Wang, F., Tian, X., Zhang, L., He, C., Ji, P., Li, Y., Tan, D. and Liu, G. (2014). Beneficial effect of resveratrol on bovine oocyte maturation and subsequent embryonic development after in vitro fertilization. Fertility and Sterility, 101(2), 577–586. e571. doi: 10.1016/j.fertnstert.2013.10.041 CrossRefGoogle ScholarPubMed Wesson, D. E. and Elliott, S. J. (1995). The H2O2-generating enzyme, xanthine oxidase, decreases luminal Ca2+ content of the IP3-sensitive Ca2+ store in vascular endothelial cells. Microcirculation 2(2), 195–203.CrossRefGoogle ScholarPubMed Zacchè, M. M., Caputo, L., Filippis, S., Zacchè, G., Dindelli, M. and Ferrari, A. (2009). Efficacy of myo-inositol in the treatment of cutaneous disorders in young women with polycystic ovary syndrome. Gynecological Endocrinology, 25(8), 508–513. doi: 10.1080/09513590903015544 CrossRefGoogle ScholarPubMed Zeng, L. and Yang, K. (2018). Effectiveness of myoinositol for polycystic ovary syndrome: A systematic review and meta-analysis. Endocrine, 59(1), 30–38. doi: 10.1007/s12020-017-1442-y CrossRefGoogle ScholarPubMed Zhang, J., Bao, Y., Zhou, X. and Zheng, L. (2019). Polycystic ovary syndrome and mitochondrial dysfunction. Reproductive Biology and Endocrinology: RBandE, 17(1), 67. doi: 10.1186/s12958-019-0509-4 CrossRefGoogle ScholarPubMed Zhu, J. Q., Zhu, L., Liang, X. W., Xing, F. Q., Schatten, H. and Sun, Q. Y. (2010). Demethylation of LHR in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome. Molecular Human Reproduction, 16(4), 260–266. doi: 10.1093/molehr/gap089 CrossRefGoogle ScholarPubMed Zimmermann, M. (1983). Ethical guidelines for investigations of experimental pain in conscious animals. Pain, 16(2), 109–110. doi: 10.1016/0304-3959(83)90201-4 CrossRefGoogle ScholarPubMed - 10 - Cited by Cited by Crossref Citations Guo, Zhenhua Lv, Lei Liu, Di Ma, Hong and Radovic, Cedomir 2023. A meta-analysis: Effect of androgens on reproduction in sows. Frontiers in Endocrinology, Vol. 14, Issue. , Khoshvaghti, Ameneh and Rahbari, Raha 2024. The effect of ellagic acid on sex hormones and miRNA-21 expression in rats with polycystic ovary syndrome. Naunyn-Schmiedeberg's Archives of Pharmacology, Vol. 397, Issue. 6, p. 4263. Anderson, George 2024. Polycystic Ovary Syndrome Pathophysiology: Integrating Systemic, CNS and Circadian Processes. Frontiers in Bioscience-Landmark, Vol. 29, Issue. 1, Albadri, Haider M Badea Alrubaye, Yasir Sj and Abdulamir, Haidar A 2025. Inositol role in polycystic ovary syndrome (PCOS) and the awareness of Iraqi doctors regarding this role. Journal of Research in Pharmacy, Vol. 29, Issue. 5, p. 1972. Cheng, Xin Ma, Dan Wang, Xiuhong Li, Meiling and Jiang, Jinpeng 2025. Causal analysis of dietary preferences and the risk of endometriosis using large-scale population data. Scientific Reports, Vol. 15, Issue. 1, Zhao, Yifan Zhang, Gaochen Pan, Jiexue and Huang, Hefeng 2025. One-carbon metabolism and risk of PCOS: insights from Mendelian randomization. Journal of Ovarian Research, Vol. 18, Issue. 1, Yang, Han Xu, Chunyue Lai, Lanfeng Luo, Bingyi Zhang, Wenwen and Yi, Wei 2025. Reconsidering antral follicle changes in the DHEA-induced PCOS model: the overlooked role of AMH and methodological variability. Journal of Ovarian Research, Vol. 18, Issue. 1, Zhang, Yanli Shi, Baichao Tian, Yuan Xu, Shujun and Chang, Hui 2026. Nutrients and bioactive compounds in polycystic ovary syndrome: updated insights into effects and underlying mechanisms. Frontiers in Nutrition, Vol. 13, Issue. , Subakathulla, Sumayyah Manoj, Norwin Patanwala, Azima Muzzammil Premvignesh, Prasanna Appiya Maher Alobaid, Yamen Majie, Ankit Gorain, Bapi Dutta, Sulagna Sengupta, Pallav Rosas, Israel Maldonado and Roychoudhury, Shubhadeep 2026. Redox-endocrine triad in PCOS: can vitamin D, myo-inositol, and melatonin synergize as bioactive cocktails?. Frontiers in Endocrinology, Vol. 17, Issue. , Wei, Yaochang Yang, Yifan Li, Yifan Wang, Zhuangsheng Deng, Tingting Hu, Xiaojiao Jiang, Xuefeng Xiao, Rui Wu, Yanfang Qing, Suzhu and Wang, Yongsheng 2026. Folic acid alleviates heat stress-induced ovarian dysfunction in dairy cows via modulation of follicular fluid metabolism and ABC transporter activity. Animal Reproduction Science, Vol. 292, Issue. , p. 108240.