Clomiphene Citrate Induces ROS-Mediated Apoptosis in Mammalian Oocytes


The clomiphene citrate (CC), a nonsteroidal triphenylethylene compound, is a first line of medicine used for the induction of ovulation in anovulatory women worldwide. In spite of high ovulation induction with the use of CC, the pregnancy rate is much lower. Such a discrepancy could be due to the peripheral anti-estrogenic effect of CC, particularly at the level of ovary, endometrium and cervical mucus. CC induces ovulation by binding to the estrogen receptors and generates hypoestrogrnic state in hypothalamus leading to release of pituitary gonadotropins. CC may have a direct effect at the level of ovary but the molecular mechanism remains unclear. Animal studies suggest that the CC induces apoptosis in granulosa cells and results hypoestrogenic state in the ovary. Reduced estradiol 17β level in the ovary affects development and maturation of oocyte leading to oocyte apoptosis. Further, CC increases hydrogen peroxide (H2O2) level and thereby bax protein expression and DNA fragmentation in cumulus-granulosa cells as well as in oocytes. The exogenous supplementation of either estradiol 17β or melatonin reduces H2O2 level in ovary, delays meiotic cell cycle progression in oocyte and protects oocyte apoptosis. Hence, supplementation of estradiol 17β or melatonin along with CC could be beneficial to protect granulosa cell as well as oocyte apoptosis and inhibit deterioration of oocyte quality. Thus, maintenance of oocyte quality may overcome the adverse effect caused due to CC treatment during infertility management.

Share and Cite:

Chaube, S. , Shrivastav, T. , Prasad, S. , Tiwari, M. , Tripathi, A. , Pandey, A. and Premkumar, K. (2014) Clomiphene Citrate Induces ROS-Mediated Apoptosis in Mammalian Oocytes. Open Journal of Apoptosis, 3, 52-58. doi: 10.4236/ojapo.2014.33006.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Agarwal, A., Gupta, S. and Sharma, R. (2005) Oxidative Stress and Its Implications in Female Infertility—A Clinician's Perspective. Reproduction Biomedicine Online, 11, 641-650.
[2] Fujii, J., Iuchi, Y. and Okada, F. (2005) Fundamental Role of Reactive Oxygen Species and Protective Mechanism in the Female Reproductive Systems. Reproduction Biology and Endocrinology, 3, 43-52.
[3] Sugino, N. (2005) Reactive Oxygen Species in Ovarian Physiology. Reproductive Medicine and Biology, 4, 31-44.
[4] Chaube, S.K., Khatun, S., Misra, S.K. and Shrivastav, T.G. (2008) Calcium Ionophore-Induced Egg Activation and Apoptosis Are Associated with the Generation of Intracellular Hydrogen Peroxide. Free Radical Research, 42, 212- 220.
[5] Tripathi, A., Khatun, S., Pandey, A.N., Mishra, S.K., Chaube, R., Shrivastava, T.G. and Chaube, S.K. (2009) Intracellular Levels of Hydrogen Peroxide and Nitric Oxide in Oocyte at Various Stages of Meiotic Cell Cycle and Apoptosis. Free Radical Research, 43, 287-294.
[6] Tripathi, A., Kumar, K.V.P. and Chaube, S.K. (2010) Meiotic Cell Cycle Arrest in Mammalian Oocytes. Journal of Cellular Physiology, 223, 592-600.
[7] 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, 280-287.
[8] Tripathy, S., Mohapatra, S., Muthulakshmi, M. and Chandrasekhar, A. (2013) Induction of Ovulation with Clomiphine citrate Versus Clomiphene with Bromocriptine in PCOS Patients with Normal Prolactin: A Comparative Study. Journal of Clinical and Diagnostic Research, 7, 2541-2543.
[9] Practice Committee of the American Society of Reproductive Medicine (2013) Use of Clomiphine Citrate in Infertile Women: A Committee Opinion. Fertility Sterility, 100, 341-348.
[10] Practice Committee of the American Society for Reproductive Medicine (2003) Use of Clomiphene Citrate in Women. Fertility Sterility, 80, 1302-1308.
[11] Barbieri, R.L. (1999) Infertility. In: Yen, S.S.C., Jaffe, R.B. and Barbieri, R.L., Eds., Reproductive Endocrinology-Physiology, Pathophysiology, and Clinical Management, 4th Edition, WB Saunders,
Philadelphia, 563-593.
[12] Wassarman, P.M. and Albertini, D.F. (1994). The Mammalian Ovum In: Knobil, E. and Neill, J.D., Eds., The physiology of Reproduction, Raven Press, New York, 79-122.
[13] Acosta, T.J., Miyamoto, A., Ozawa, T., Wijayagunawardane, M.P. and Sato, K. (1998) Local Release of Steroid Hormones, Prostaglandin E2, and Endothelin-1 from Bovine Mature Follicles in Vitro: Effects of Luteinizing Hormone, Endothelin-1 and Cytokines. Biology of Reproduction, 59, 437-443.
[14] Chaube, S.K. (2001) Role of Meiotic Maturation Regulatory Factors in Developmental Competency of Mammalian oocytes. Health and Population Perspect Issues, 24, 218-231.
[15] Duffy, D.M. and Stouffer, R.L. (2003) Luteinizing Hormone Acts Directly at Granulose Cells to Stimulate Periovulatory Processes: Modulation of Luteinizing Hormone Effects by Prostaglandins. Endocrine, 22, 249-256.
[16] Peters, M.W., Pursley, J.R. and Smith G.W. (2004) Inhibition of Intrafollicular PGE2 Synthesis And ovulation Following Ultrasound-Mediated Intrafollicular Injection of the Selective Cyclooxygenease-2 Inhibitor NS-398 in Cattle. Journal of Animal Science, 82, 1656-1662.
[17] Duffy, D.M., Dozier, B.L. and Seachord, C.L. (2005) Prostaglandin Dehydrogenase (PGDH) and Prostaglandin Levels in Preovulatory Follicles: Implications for Control of Primate Ovulation by PGE2. Journal of Clinical Endocrinology and Metabolism, 90, 1021-1027.
[18] Homburg, R. (2005) Clomiphine Citrate: End of an Era? A Mini Review. Human Reproduction, 20, 2043-2051.
[19] Clark, J.H. and Markaverich, B.M. (1981) The Agonist-Antagonistic Properties of Clomiphene: A Review. Pharmacology and Therapeutics, 15, 467-519.
[20] Mikkelson, T.J., Kroboth, P.D., Cameron, W.J., Dittert, L.W., Chungi, V. and Manberg, P.J. (1986) Single-Dose Pharmacokinetics of Clomophine Citrate in Normal Volunteers. Fertility and Sterility, 46, 392-396.
[21] Van, C.J., Borreman, E., Wyman, H. and Antaki, A. (1973) Induction of ovulation with Cisclomiphine. American Journal of Obstetrics Gynecology, 115, 321-327.
[22] Young, S.L., Opsahl, M.S. and Fritz, M.A. (1999) Serum Concentrations of Enclomiphine and Zuclomiphine across Consecutive Cycles of Clomiphine Citrate Therapy in Anovulatory Infertile Woman. Fertility and Sterility, 71, 639-644.
[23] Reber, R., Judd, H.L., Yen, S.S.C., Rakoff, J., Vaden Berg, G. and Naftolin, F. (1976) Characterization of the Inappropriate Gonadotropin Secretion in Polycystic Ovary Syndrome. Journal of Clinical Investigation, 57, 1320-1329.
[24] Dodge, S.T., Strikler, R.C. and Keller, D.W. (1986) Ovulation Induction with Low Doses of Clomiphine Citrate. Obstetrics and Gynecology, 67, 635-655.
[25] Gysler, M., March, C.M., Mishell Jr., D.R. and Bailey, E.J. (1982) A Decade’s Experience with an Individualized Clomiphine Treatment Regime Including IT’s Effect on the Postcoital Test. Fertility and Sterility, 37, 161-167.
[26] Schwartz, L.B., Brezinski, A. and Laufer, N. (1993) The Effects of Clomiphene Citrate Isomers on Human Granulosa- Lutein Cells in Culture. Gynecological Endocrinology, 7, 229-233.
[27] Mitwally, F.M. and Casper, R.F. (2002) Aromatase Inhibition for Ovarian Stimulation: Future Avenues for Infertility Management. Current Opinion in Obstetrics and Gynecology, 14, 255-263.
[28] Goidfarb, M., Morales, A., Rakoff, A.E. and Portos, P. (1968) Critical Review of 160 Clomiphene-Related Pregnancies. Obstetrics and Gynecology, 31, 342-345.
[29] Gonen, Y. and Casper, R.F. (1990) Sonographic Determination of a Possible Adverse Effect of Adverse Effect of Clomiphene Citrate on Endometrial Growth. Human Reproduction, 5, 670-674.
[30] Randall, J.M. and Templeton, A. (1991) Cervical Mucus Score and in Vitro Sperm Mucus Interaction in Spontaneous and Clomiphene Citrate Cycles. Fertility and Sterility, 56, 465-468.
[31] Li, T.C., Warren, M.A. and Murphy, C. (1999) A Prospective, Randomized, Cross-Over Study Comparing the Effects of Clomiphene Citrate and Cytophenil on Endometrial Morphology in the Luteal Phase of Normal Fertile Women. Fertility and Sterility, 71, 639-644.
[32] Chaube, S.K., Prasad, P.V., Thakur, S.C. and Srivastav, T.G. (2005) Estradiol Protects Clomiphene Citrate-Induced Apoptosis in Ovarian Follicular Cells and Ovulated Cumulus-Oocyte Complexes. Fertility and Sterility, 84, 1163- 1172.
[33] Chaube, S.K., Prasad, P.V., Tripathi, V. and Shrivastav, T.G. (2006) Clomiphene Citrate Inhibits Gonadotropin-Induced Ovulation by Reducing Cyclic Adenosine 3’,5’-Cyclic Monophosphate and Prostaglandin E2 Levels in Rat Ovary. Fertility and Sterility, 86, 1106-1111.
[34] Olsson, J.H. and Granberg, S. (1990) Effect of Clomiphene Isomers on Oestradiol Synthesis in Cultured Human Granulosa Cells. Human Reproduction, 5, 928-932.
[35] Tripathi, A., Prem Kumar, K.V., Pandey, A.N., Khatun, S., Mishra, S.K., Shrivastava, T.G. and Chaube, S.K. (2011) Melatonin Protects against Clomiphine Citrate-Induced Generation of Hydrogen Peroxide and Morphological Apoptotic Changes in Rat Eggs. European Journal of Pharmacology, 667, 419-424.
[36] Marut, E.L. and Hodgen, G.D. (1982) Anti-Estrogenic Action of High-Dose Clomiphene in Primates: Pituitary Augmentation but with Ovarian Attenuation. Fertility and Sterility, 38, 100-104.
[37] Laufer, N., Reich, R., Braw, R., Shenker, J.G. and Tsafriri, A. (1982) Effect of Clomiphene Citrate on Preovulatory Rat Follicles in Culture. Biology of Reproduction, 27, 463-471.
[38] Yashimura, Y., Hosoi, Y., Atlas, S.J. and Wallac, E. (1986) Effect of Clomiphene Citrate on in Vitro Ovulated Ova. Fertility and Sterility, 45, 800-804.
[39] Schmidt, G.E., Sites, C., Mansour, R., Friedman, C.I. and Kim, M.H. (1985) Embryo Toxicity of Clomiphene Citrate on Mouse Embryos Fertilized in Vitro and in Vivo. American Journal of Obstetrics Gynecology, 153, 679-684.
[40] Pena, J.E., Chang, P.L., Zeitoun, K., Thornton II, M.H. and Sauer, M.V. (2002) Supraphysiological Estradiol Levels Do Not Affect Oocyte and Embryo Quality in Oocyte Donation Cycles. Human Reproduction, 17, 83-87.
[41] Bateman, B.G., Nunley Jr., W.C. and Kolp, L.A. (1990) Exogenous Estrogen Therapy for Treatment of Clomiphene Citrate-Induced Cervical Mucus Abnormalities: Is It Effective? Fertility and Sterility, 54, 577-579.
[42] Yagel, S., Ben-Chetrit, A., Anteby, E., Zacut, D., Hochner-Celnikier, D. and Ron, M. (1992) The Effect of Ethinyl Estradiol on Endometrial Thickness and Uterine Volume during Ovulation Induction by Clomiphene Citrate. Fertility and Sterility, 57, 33-36.
[43] Ben-Ami, M., Geslevich, Y., Matilsky, M., Battino, S., Weiner, E. and Shlev, E. (1994) Exogenous Estrogen Therapy Concurrent with Clomiphene Citrate—Lack of Effect of Serum Sex Hormones Levels and Endometrial Thickness. Gynecologic and Obstetric Investigation, 37, 180-182.
[44] Shimoya, K., Tomiyama, K., Hashimoto, K., Moriyama, A., Kawamoto, A., Tokugawa, Y., Ohashi, K., Saji, F. and Murata, Y. (1999) Endometrial Development Was Improved by Transdermal Estradiol in Patients Treated with Clomiphene Citrate. Gynecologic and Obstetric Investigation, 47, 251-254.
[45] Gerli, S., Gholami, H. and Manna, A. (2000) Use of Ethinyl Estradiol to Reverse the Anti-Estrogenic Effects of Clomiphene Citrate in Patients Undergoing Intrauterine Insemination: A Comparative, Randomized Study. Fertility and Sterility, 73, 85-89.
[46] Hayon, T., Bvilansky, A., Oriev, L. and Nathan, I. (1999) Non-Steroidal Antiestrogens Induce Apoptosis in HL60 and MOLT3 Luekemic Cells: Involvement of Reactive Oxygen Radicals and Protein Kinase C. Anticancer Research, 19, 2089-2093.
[47] Tan, D.X., Manchester, L.C., Terron, M.P., Flores, L.J. and Reiter, R.J. (2007) One Molecule, Many Derivatives: A Never-Ending Interaction of Melatonin with Reactive Oxygen and Nitrogen Species? Journal of Pineal Research, 42, 28-42.
[48] Liu, F. and Ng, T.B. (2000) Effect of Pineal Indoles on Activities of the Antioxidant Defense Enzymes Superoxide Dismutase, Catalase, and Glutathione Reductase, and Levels of Reduced and Oxidized Glutathione in Rat Tissues. Biochemistry and Cell Biology, 78, 447-453.
[49] Ozturk, G., Coskun, S., Erbas, D. and Hasanoglu, E. (2000) The Effect of Melatonin on Liver Superoxide Dismutase Activity, Serum Nitrate and Thyroid Hormone Levels. Japanese Journal of Physiology, 50, 149-153.
[50] Reiter, R.J., Tan, D.X., Manchester, L.C. and Qi, W. (2001) Biochemical Reactivity of Melatonin with Reactive Oxygen and Nitrogen Species: A Review of the Evidence. Cell Biochemistry and Biophysics, 34, 237-256.
[51] Reiter, R.J., Tan, D.X., Manchester, L.C., Paredes, S.D., Mayo, J.C. and Sainz, R.M. (2009) Melatonin and Reproduction Revisited. Biology of Reproduction, 81, 445-456.
[52] Feng, Z. and Zhang, J.T. (2005) Long-Term Melatonin or 17β-Estradiol Supplementation Alleviates Oxidative Stress in Ovariectomized Adult Rats. Free Radical Biology and Medicine, 39, 195-204.
[53] Berlinguer, F., Leoni, G.G., Succu, S., Spezzigu, A., Madeddu, M., Satta, V., Bebbere, D., Contreras-Solis, I., Gonzalez-Bulnes, A. and Naitana, S. (2009) Exogenous Melatonin Positively Influences Follicular Dynamics, Oocyte Developmental Competence and Blastocyst Output in a Goat Model. Journal of Pineal Research, 46, 383-391.
[54] Kang, J.T., Koo, O.J., Kwon, D.K., Park, H.J., Jang, G., Kang, S.K. and Lee, B.C. (2009) Effects of Melatonin on in Vitro Maturation of Porcine Oocyte and Expression of Melatonin Receptor RNA in Cumulus and Granulosa Cells. Journal of Pineal Research, 46, 22-28.
[55] Tsiligianni, T., Valasi, I., Cseh, S., Vainas, E., Faigl, V., Samartzi, F., Papanikolaou, T., Dovolou, E. and Amiridis, G.S. (2009) Effects of Melatonin Treatment on Follicular Development and Oocyte Quality in Chios Ewes-Short Communication. Acta Veterinaria Hungarica, 57, 331-335.
[56] Vazquez, M., Forcada, F., Casao, A., Abecia, J., Sosa, C. and Palacin, I. (2010) Under Nutrition and Exogenous Melatonin Can Affect the in Vitro Developmental Competence of Ovine Oocytes on a Seasonal Basis. Reproduction in Domestic Animals, 45, 677-684.
[57] Adriaens, I., Smitz, J. and Jacquet, P. (2009) The Current Knowledge on Radiosensitivity of Ovarian Follicle Development Stages. Human Reproduction Update, 15, 359-377.
[58] Forcada, F., Abecia, J.A., Cebrian-Perez, J.A., Muino, T., Valares, J.A., Palacin, I. and Casao, A. (2006) The Effect of Melatonin Implants during the Seasonal Anestrous on Embryo Production after Superovulation in Aged High-Prolificacy Rasa Aragonesa Ewes. Theriogenology, 65, 356-365.

Copyright © 2021 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.