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Development of macaque secondary follicles exposed to neutral red prior to 3-dimensional culture

  • Fertility Preservation
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Neutral red (NR) may assist identification of preantral follicles in pieces of cortical tissue prior to cryopreservation in cancer patients requesting fertility preservation. This study is the first to analyze this effect by follicle growth rate after long-term culture in primates.

Methods

Ovarian cortex was obtained from adult rhesus macaques, was cut into fragments, and was incubated with NR. Secondary follicles were readily visualized following NR staining and then were encapsulated into alginate beads and cultured individually for 4 weeks in αMEM media supplemented with 10 ng/ml FSH at 5% O2.

Results

The survival rates of secondary follicles during culture were similar between those derived from control tissue (71 ± 13%) and those treated with NR (68 ± 9%). The proportion of surviving follicles that formed an antrum were also similar in both groups (70 ± 17% control; 48 ± 24% NR-treated). Follicle diameters were not different between control follicles (184 ± 5μm) and those stained with NR (181 ± 7 μm) on the day of isolation. The percentages of surviving follicles within three cohorts based on their diameters at week 4 of culture were similar between the control group and NR-stained tissue group, fast-grow follicles (24 ± 6% vs. 13 ± 10%), slow-grow follicles (66 ± 5% vs. 60 ± 9%), or no-grow (10 ± 9% vs. 27 ± 6%), respectively. There were no differences in follicle diameters between groups during the culture period. Pre-exposure of secondary follicles to NR diminished their capacity to produce both estradiol and androstenedione by week 4 of culture, when follicles are exhibiting an antrum. Inhibitory effects of NR on steroid production by slow-grow follicles was less pronounced.

Conclusions

NR does not affect secondary follicle survival, growth, and antrum formation during long-term culture, but steroid hormone production by fast-grow follicles is compromised. NR can be used as a non-invasive tool for in situ identification of viable secondary follicles in ovarian cortex before tissue cryopreservation without affecting follicle survival and growth in vitro. Whether maturation or developmental competence of oocytes derived from antral follicles in 3D culture that were previously isolated from NR-stained tissue is normal or compromised remains to be determined. Likewise, the functional consequences of pre-exposure to NR prior to ovarian cortical tissue cryopreservation and transplantation are unknown.

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Acknowledgements

We are grateful to the Division of Comparative Medicine for surgery and excellent animal care, and Maralee Lawson for her assistance.

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Authors and Affiliations

  1. Department of Obstetrics and Gynecology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, 14049-900, Brazil

    Daiane L. Bulgarelli & Ana Carolina Japur de Sá Rosa-e-Silva

  2. Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Sciences University, Beaverton, OR, USA

    Alison Y. Ting, Brenda J. Gordon & Mary B. Zelinski

  3. Cleveland High School, Portland, OR, USA

    Brenda J. Gordon

  4. Department of Obstetrics and Gynecology, Oregon Health & Science University, Portland, OR, USA

    Mary B. Zelinski

Authors
  1. Daiane L. Bulgarelli
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  2. Alison Y. Ting
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  4. Ana Carolina Japur de Sá Rosa-e-Silva
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  5. Mary B. Zelinski
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Contributions

All authors contributed to the study design, execution, analysis, critical discussion, and drafting or revising of the manuscript. All authors have approved the final version and submission of this manuscript.

Corresponding author

Correspondence to Mary B. Zelinski.

Ethics declarations

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Additional information

Supported by National Institute of Child Health and Human Development (NICHD)/National Institute of Health (NIH) Oncofertility Consortium UL1 RR024926, (RL1-HD058294, PL1-EB008542); ONPRC P51RR000163; NIH Fogarty International Center grant TW/HD-00668 to P. Michael Conn (DLB); M.J. Murdock Charitable Trust, Partners in Science 2010283 (BJG)

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Bulgarelli, D.L., Ting, A.Y., Gordon, B.J. et al. Development of macaque secondary follicles exposed to neutral red prior to 3-dimensional culture. J Assist Reprod Genet 35, 71–79 (2018). https://doi.org/10.1007/s10815-017-1043-y

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  • DOI: https://doi.org/10.1007/s10815-017-1043-y

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