Influence of temperature, serum, and gonadotropin supplementation in short- and long-term organotypic culture of human immature testicular tissue

Organotypic culture of human immature testicular tis- sue at 34 C in Knockout Serum Replacement with FSH/LH showed the best results in terms of tissue integrity, cell survival, Sertoli cell maturation, testosterone secretion, and maturation of spermatogonia up to meiotic initiation.

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 STARSOX9DAZLDDX4PLZF, 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.

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