P-081 Effect of chemotherapy (cytarabine) and acute myeloid leukemia on the development of spermatogenesis, spermatogonial microenvironment and inflammatory factors at the adult age of immature-treated mice
Authors: Huleihel M , Khaleel B , Kapilushnik J , Lunenfeld E 
Published: June 2022

Abstract

Study question
How do AML and cytarabine affect the development of spermatogenesis, germ cell microenvironment and testicular inflammatory factors at the adult age of immature-treated mice?

Summary answer
AML and cytarabine significantly damaged the normal histology of the seminiferous tubules and impaired different stages of spermatogenesis, testicular growth/inflammatory factor in the adult age.

What is known already
The effect of different types of cancers on male fertility was reported. AML constitutes around 20% of the diagnosed childhood leukemia. Cytarabine is used as anti-AML treatment. Cytarabine is involved in reduction of testes growth and induces testicular atrophy in adults. In adult AML patients, it was shown that AML affects semen parameters even before chemotherapy treatment. Furthermore, a significant reduction in semen quality of thawed cryopreserved semen from leukemia patients compared to control patients was reported. However, the effect of AML and cytarabine before puberty on the development of spermatogenesis in adult age was not yet reported.

Study design, size, duration
Two-week-old males C57/BLACK mice were used. 1. Control group – injected with saline. 2. AML group – injected (intraperitoneal; i.p) with 3x104 AML cells/100 ml (murine C-1498 AML cell line). 3. Cytarabine group - three injections of cytarabine (140 mg/kg/100 ml) were performed every 12 hours. 4. AML+cytarabine group – cytarabine was i.p injected 24 hours after AML injection (injections were performed every 12 hours 3 times). Mice were sacrificed 1,2,3,4,5 weeks after AML injection.

Participants/materials, setting, methods
Testes were removed, weighted and fixed in Bouin’s solution for histological evaluation, or kept at -70°C for RNA extraction. The presence of premeiotic (SALL4, PLZF), meiotic (CREM-1) and postmeiotic (ACROSIN) cells were examined by immunofluorescence staining analysis and/or RNA expression by qPCR analysis.

Main results and the role of chance
We have successfully developed a system of AML in immature mice by i.p injection of C1498 cells. Cytarabine prolonged life of AML-treated mice from 2.5 weeks to 4 weeks. AML had no effect on both body and testicular weight. However, cytarabine significantly reduced testicular weight (around 50% of the control in the first 3 weeks after treatment, and around 30% of the control from 3-4.5 weeks after treatment; p &lt; 0.001). One and/or 2 weeks after the treatment showed that both AML and cytarabine alone or in combination significantly decreased seminiferous tubules normal histology compared to control, (30%, 20%, 10% respectively) (p &lt; 0.001). Cytarabine, but not AML significantly increased the expression of PLZF and SALL4 after 2 weeks compared to control (p &lt; 0.001). However, each treatment significantly increased the percentages of seminiferous tubules with apoptotic cells compared to control (p &lt; 0.05). AML or cytarabine significantly decreased the percentages of tubules with meiotic and postmeiotic markers compared to control (p &lt; 0.05). Also, AML or cytarabine significantly decreased the testicular protein levels of GDNF, CSF-1 (p &lt; 0.01) but did not affect LIF compared to control. Furthermore, AML or cytarabine significantly decreased the protein levels of IL-10. However, AML significantly increased IL-6 when cytarabine increased it compared to control (p &lt; 0.01).

Limitations, reasons for caution
Animal model of leukemia may behave different from human model.

Wider implications of the findings
We successfully developed AML disease in immature mice, and demonstrated its impairment the normal histology of the seminiferous tubules and spermatogenesis in the adult age. We showed that AML and cytarabine affect testicular microenvironmental and inflammatory factors. Our results may assist in development future therapeutic strategies for male fertility preservation.

Trial registration number
NON