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Fertil Steril. 2018 Nov;110(6):1045-1057.e3. doi: 10.1016/j.fertnstert.2018.07.018.

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

Author information

1
Instituto de Investigación Sanitaria La Fe, Valencia, Spain. Electronic address: jomepla@gmail.com.
2
Instituto de Investigación Sanitaria La Fe, Valencia, Spain.
3
Hospital Universitario y Politécnico La Fe, Valencia, Spain.
4
Instituto de Investigación Sanitaria La Fe, Valencia, Spain; Hospital Universitario y Politécnico La Fe, Valencia, Spain.
5
Instituto de Investigación Sanitaria La Fe, Valencia, Spain; Fundación IVI, Valencia, Spain.

Abstract

OBJECTIVE:

To study how temperature, serum, and gonadotropin supplementation affect the organotypic culture of human immature testicular tissue (ITT) in vitro.

DESIGN:

Experimental basic science study.

SETTING:

Reproductive biology laboratory.

PATIENT(S):

ITT from 4 boys with cancer that had testicular tissue cryopreserved as part of their fertility preservation treatment.

INTERVENTION(S):

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.

RESULT(S):

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.

CONCLUSION(S):

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.

KEYWORDS:

Fertility preservation; human immature testicular tissue; in vitro spermatogenesis; organotypic culture

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