Volume 110, Issue 6, Pages 1045–1057.e3
Jose V. Medrano, Ph.D., Teresa Vilanova-Pérez, M.Sc., Victoria Fornés-Ferrer, M.Sc., Ana Navarro-Gomezlechon, B.Sc., María L. Martínez-Triguero, M.D., Sofía García, M.D., Javier Gómez-Chacón, M.D., Ivan Povo, M.D., Antonio Pellicer, M.D., María M. Andrés, M.D., Edurne Novella-Maestre, Ph.D.
To study how temperature, serum, and gonadotropin supplementation affect the organotypic culture of human immature testicular tissue (ITT) in vitro.
Experimental basic science study.
Reproductive biology laboratory.
ITT from 4 boys with cancer that had testicular tissue cryopreserved as part of their fertility preservation treatment.
In vitro organotypic culture of ITT, exposed to different temperatures (37°C vs. 34°C), serum (fetal bovine serum [FBS] vs. Knockout Serum Replacement [KOS]), and gonadotropin supplementation (with and without FSH and LH).
Main Outcome Measure(s)
Characterization of the tissue was performed at days 0, 14, and 70 with the use of reverse-transcription quantitative polymerase chain reaction, terminal deoxynucleotide transferase–mediated dUTP nick-end labeling, histologic analysis by means of hematoxylin-eosin staining, and immunohistochemical staining. Hormonal secretion was determined at days 3, 14, 28, and 70 by means of immunofluorescent assay.
The 37°C conditions showed an accelerated loss of tubular morphology and higher intratubular apoptosis. KOS supplementation triggered the up-regulation of STAR, SOX9, DAZL, DDX4, PLZF, and UTF1, the percentage of SOX9+/androgen receptor (AR)–positive mature Sertoli cells at day 14, and testosterone secretion. Gonadotropin supplementation increased the numbers of both undifferentiated UTF1+ spermatogonia and premeiotic VASA+/SYCP3+ spermatogonia at day 14, and the number of SOX9+ Sertoli cells at day 70. The low SOX9+/AR+ colocalization, the disorganized pattern of ZO-1, and the progressive decrease of antimüllerian hormone secretion indicated inefficient Sertoli cell maturation in vitro.
The 34°C condition in KOS showed the best results for the survival of both spermatogonia and Sertoli cells. FSH/LH supplementation also improved long-term survival of Sertoli cells and the maturation of spermatogonia up to meiotic initiation in short-term culture.