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The Effect of Clomiphene Citrate and Letrozole in Apoptotic Pathways and Cell Cycle in Human Primary Cumulus Cells and the Protective Effect of Estradiol

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Abstract

Clomiphene citrate (CC) and letrozole are ovulatory stimulants that, despite high ovulation rates, achieve low pregnancy rates. This study aimed to investigate the in vitro effects of CC and letrozole, alone or in combination with estradiol, on apoptosis in human cumulus cells. We performed a controlled prospective study using primary cumulus cell cultures from patients undergoing in vitro fertilization (n=22). Alpha-inhibin immunocytochemistry was used to assess cell culture purity and morphology. Cell viability was evaluated by MTT assay, cell cycle status by flow cytometry, and Caspase-3, Bax and SOD-2, and S26 gene expression by qPCR. Cells were treated for 24 hours in 5 conditioned media: CC, CC + estradiol, letrozole, letrozole + estradiol and control. None of the treatments affected cell viability, but letrozole reduced the mean percentage of cells in the S phase compared to control (24.79 versus 21.70, p=0.0014). Clomiphene treatment increased mRNA expression of Bax (4 fold) and SOD-2 (2 fold), which was reversed by co-treatment with estradiol. SOD-2 expression increased in cells treated with letrozole compared to control (4 fold), which was also reversed by estradiol. These findings suggest that clomiphene citrate and letrozole do not significantly affect the viability of human cumulus cells. Still, the expression of genes involved in apoptosis was modulated by these drugs alone and in association with estradiol, suggesting that CC and letrozole may have direct effects on cumulus cells beyond their known mechanisms of action.

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Acknowledgments

The authors are grateful to the Fertibaby clinic work team, Dr. Iara Couy, Dr. Fernando Reis and all patients enrolled in this study.

Funding

Study was supported by Federal University of Minas Gerais Women’s Health Post Graduation Program and received funding from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Federal University of Minas Gerais, Belo Horizonte, Brazil

    Camila O. Silveira & Marcia C. F. Ferreira

  2. Fertibaby Clinic, Belo Horizonte, Brazil

    Camila O. Silveira, Leonardo M. Moraes, Gabriella G. Oliveira & Luciana P. T Aguiar

  3. Department of General Pathology, Federal University of Minas Gerais, Belo Horizonte, Brazil

    Rafaela M. Oliveira, Felipe H. S. Souza, Santuza S. Coelho, Pedro H. D. M. Prazeres & Helen L. Del Puerto

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  1. Camila O. Silveira
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  2. Rafaela M. Oliveira
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  4. Gabriella G. Oliveira
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Contributions

All authors contributed to this study. Conceptualization, patient recruitment, oocyte retrieval, data curation, formal analysis, investigation, writing—original draft, review & editing were performed by Camila Silveira. Patient’s recruitment, oocyte retrieval and oocyte denudation were performed by Leonardo Moraes, Gabriella Oliveira and Luciana Aguiar. Cells sample’s collection, primary cell culture experiments, drugs treatment, flow cytometry and RNA isolation were performed by Rafaela Oliveira and Santuza Coelho. Flow cytometry and molecular biology experiments and analysis were performed by Felipe Souza and Pedro Prazeres. Conceptualization, formal analysis, funding acquisition, methodology, project administration, resources, supervision, writing—review & editing were performed by Marcia Ferreira and Helen Del Puerto.

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Correspondence to Camila O. Silveira.

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This study was approved by IRB of Federal University of Minas Gerais, number 1.979.648, on March 23, 2017.

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Silveira, C.O., Oliveira, R.M., Moraes, L.M. et al. The Effect of Clomiphene Citrate and Letrozole in Apoptotic Pathways and Cell Cycle in Human Primary Cumulus Cells and the Protective Effect of Estradiol. Reprod. Sci. 29, 2272–2281 (2022). https://doi.org/10.1007/s43032-022-00961-8

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