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Recombinant Anti-Müllerian Hormone (rAMH) for Stalling In Vitro Granulosa Cell Replication

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Abstract

To investigate whether recombinant AMH (rAMH) is able to decrease cellular proliferation/apoptosis in luteinized granulosa cells (GCs) through hormonal regulation, a primary culture of GCs was established from GCs obtained at time of oocyte retrieval from follicular fluid of 3 patients. Cells were seeded in well cell culture plates at a density of 100,000 cells/well in medium and treated with rAMH 20 ng/ml (rAMH group), or phosphate-buffered saline (PBS-control group), for 24 h. Total RNA was extracted from all cells, followed by cDNA synthesis and real-time RT-PCR to quantify the expression levels of AMH, AMH-R2, FSH-R, inhibin B, cell proliferation (Ki67), and apoptosis (Caspase 3). We used independent sample t test (SPSS v25) and a p < 0.05 significance. Cellular expressions of AMH, AMH-R2, FSH-R, and inhibin B were reduced greater than 50% in the rAMH group, compared with that of the the control group (p ≤ 0.005 for all). Ki67 and Caspase3 were also reduced greater than 30% in the rAMH group (p ≤ 0.001 for both). Our findings show a direct inhibitory effect of AMH on luteinized GCs’ expression of the major regulatory hormones, in addition to a significant decrease in markers of cell proliferation and apoptosis. These results confirm the inhibitory effects of AMH on follicular development.

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Funding

This study was supported by an institutional grant (E07-3225-001) from the University of Tennessee Health Science Center, Memphis, TN.

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Authors and Affiliations

  1. Department of Obstetrics and Gynecology, University of Tennessee Health Science Center, Memphis, TN, USA

    Laura Detti, Irene Peregrin-Alvarez, Mary E. Christiansen, Pouran Malekzadeh & Jennifer Sledge

  2. Department of Obstetrics and Gynecology Subspecialties, Women’s Health Institute, Cleveland Clinic, Cleveland, OH, USA

    Laura Detti

  3. IVF Michigan Rochester Hills and Flint, Rochester Hills, MI, USA

    Mustafa I. Abuzeid

  4. The C. S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, MI, USA

    Ghassan M. Saed

Authors
  1. Laura Detti
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  2. Mustafa I. Abuzeid
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  3. Irene Peregrin-Alvarez
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  4. Mary E. Christiansen
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  5. Pouran Malekzadeh
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  6. Jennifer Sledge
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  7. Ghassan M. Saed
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Correspondence to Laura Detti.

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Detti, L., Abuzeid, M.I., Peregrin-Alvarez, I. et al. Recombinant Anti-Müllerian Hormone (rAMH) for Stalling In Vitro Granulosa Cell Replication. Reprod. Sci. 27, 1873–1878 (2020). https://doi.org/10.1007/s43032-020-00206-6

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  • DOI: https://doi.org/10.1007/s43032-020-00206-6

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