Acute Myeloid Leukemia Affects Mouse Sperm Parameters, Spontaneous Acrosome Reaction, and Fertility Capacity

Abstract

Leukemia is one of the most common cancers in patients of reproductive age. It is well known that chemotherapy, used as anti-cancer therapy, adversely affects male fertility. Moreover, the negative effect of leukemia on sperm quality, even before chemotherapy treatment, has been reported. However, the mechanisms behind this disease's effect on sperm quality remains unknown. In this study, we examine the direct effect of leukemia and chemotherapy alone and in combination on sperm parameters and male fertility. For this, we developed an acute myeloid leukemia (AML) mouse model (mice were treated with AML cells C1498 and developed leukemia); these mice then received cytarabine chemotherapy. Our findings reveal a significant reduction in sperm concentration and motility and a significant increase in abnormal morphology and spontaneous acrosome reaction of the sperm following AML and chemotherapy treatment, alone and in combination. We also found a reduction in male fertility and the number of delivered offspring. Our results support previous findings that AML impairs sperm parameters and show for the first time that AML increases spontaneous acrosome reaction and decreases male fertility capacity and number of offspring.

Keywords: acrosome reaction; acute myeloid leukemia; male infertility; sperm parameters; testis.

Figure 1

Effect of AML cells, cytarabine, and the combination of both on mice survival: Adult C57/black mice were injected with PBS (control), C1498 cells (C1498), cytarabine (Cyt), or with a combination of both (Cyt + C1498). The survival of mice was evaluated 2–8 weeks after injection (see the Methods) in intervals of one week. The results are representative of four independent experiments with 10 mice in each group.

Figure 2

Effect of AML cells, cytarabine, and the combination of both on sperm concentration: Mice were treated as described in Figure 1. Sperm were extracted from the epididymis 1–4 weeks post-treatment. Sperm concentration was evaluated using a Makler counting chamber and determined according to WHO criteria. The results are representative of three independent experiments with 8–10 mice in each group. * Significant compared to control. @ Significant for Cyt + C1498 compared to Cyt. *, @ p < 0.05; **, @@ p < 0.01; ***, @@@ p < 0.001.

Figure 3

Effect of AML cells, cytarabine, and the combination of both on sperm motility: Mice were treated as described in Figure 1. Sperm were extracted from the epididymis 1–4 weeks post-treatment. Sperm motility/immotility was evaluated using a Makler counting chamber and determined as a percentage of total sperm according to WHO criteria. The results are representative of three independent experiments with 8–10 mice in each group. * Significant compared to control. $ Significant for Cyt + C1498 compared to C1498. *, $ p < 0.05; **, $$ p <0.01; ***, $$$ p < 0.001.

Figure 4

Effect of AML cells, cytarabine, and the combination of both on sperm morphology: Mice were treated as described in Figure 1. Sperm were extracted from the epididymis three weeks post-treatment. Sperm morphology was evaluated following staining with Diff-Quick stain (magnification of ×1000) (A). Cells were divided into different types of morphology: (I) normal morphology, (II) abnormal neck, (III) abnormal tail, (IV) abnormal head according to WHO criteria. The percentage of sperm with normal morphology was calculated (B). The results are representative of three independent experiment with three mice in each group. * Significant compared to control. $ Significant of Cyt + C1498 compared to C1498. $$ p < 0.01; *** p < 0.001.

Figure 5

Effect of AML cells, cytarabine, and the combination of both compared to control (CT) on sperm spontaneous acrosome reaction: Mice were treated as described in Figure 1. Three weeks post-treatment, sperm were extracted from the epididymis and stained by fluorescein (FITC) staining (A). Acrosome reacted (without green staining) and non-reacted sperm (with green staining) were counted (A), and the percent of sperm that underwent spontaneous acrosome reaction was calculated (B). The results are representative of three independent experiment with four mice in each group. * Significant compared to control. *** p < 0.001.

Figure 6

Effect of AML cells, cytarabine, and the combination of both on mice fertility and number of offspring: Mice were treated as described in Figure 1. Two weeks post-treatment, a single male from each group was mated with two females. After two weeks, the females were separated each to a single cage. The number of pregnant females (A) and number of offspring from each female were counted after five weeks (B). The results are representative of three independent experiments with four mice in each group. * Significant compared to control. * p < 0.05; ** p < 0.01; *** p < 0.001.