DNA damaging effect of paclitaxel in the epididymal sperms as a chemotherapeutic agent and possible remedies to prevent this effect: A study on reproductive potential of male cancer patients of reproductive age

Highlights

• Paclitaxel induced the level of DNA damage in the epididymal sperm samples.

• Resveratrol ameliorated the DNA damaging effect of paclitaxel.

• Supplementing the extender with l-glutamine and basal medium eagle solution prevented DNA damage after cryopreservation.

• Sperm cryopreservation before chemotherapy for male cancer patients of reproductive age is suggested to preserve fertility.

Abstract

Cancer is a major public health problem, young cancer patients therefore undergo chemotherapy, and most of them may lose their fertility. DNA damage level provides important clues about the quality and reproductive potential of spermatozoa. In this study, we evaluated the levels of both DNA fragmentation and abnormal DNA integrity in the epididymal sperms of New Zealand rabbit (Oryctolagus cuniculus) after cryopreservation using the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay and the toluidine blue (TB) staining methods and assessed the effects of paclitaxel, resveratrol, l-glutamine (LG), and basal medium eagle (BME) solution on DNA damage. Paclitaxel induced the levels of both DNA damages in the sperms, but resveratrol ameliorated this effect. LG and BME supplementation to the extender prevented the sperm samples from DNA fragmentation after cryopreservation. Chemotherapy drugs containing paclitaxel can cause the sperm DNA to be damaged, and hence adversely affect the fertility of male cancer patients of reproductive age. The administration of resveratrol together with paclitaxel may ameliorate the DNA damage inducing effect of paclitaxel. Sperm banking and cryopreservation with the appropriate cryoprotectants such as LG and BME prior to cancer treatment can also be suggested to all male cancer patients of reproductive age facing cancer treatment for fertility preservation.

Introduction

In general, sperms are maturated in the epididymis, stored in cauda region of epididymis until they ejaculated, and they are then able to fertilise oocytes. In addition, epididymal spermatozoa are physiologically protected by various antioxidant enzymes such as catalase and superoxide dismutase in their environment [1,2].

Cancer is a major public health problem in many parts of the world [3]. For instance, the incidence of testicular cancer in males from 15 to 45 years old nowadays reaches up to 70% [4]. Cancer itself can adversely affect the male fertility by impairing spermatogenesis, and all cancer therapies (chemotherapy, radiation, and surgery) are also potential threats to reproductive potential of males [5]. A lot of young cancer patients undergo chemotherapy, and most of them may lose their fertility; therefore, future fertility is also an area of remarkable concern for young cancer survivors [6]. Sperm recovery from the caudal epididymis of young cancer patients with microsurgical epididymal sperm aspiration as well as ejaculates and the cryopreservation of these sperms prior to initiating cancer treatment may enable these patients to father biologically related children [5].

Paclitaxel is a member of the taxane class of anticancer drugs and one of the most common chemotherapeutic agents used against many forms of cancer [7]. It causes reactive oxygen species (ROS) production and DNA damages, and DNA damages induced by paclitaxel are used to kill the cancer cells [8,9]. Resveratrol (3,4′,5-trihydroxystilbene), which is a phytoalexin, is found in many plants including grapes, peanuts, and berries [10] and it shows antioxidant and radical scavenging activities [11,12].

Cryopreservation is a long-term storage technique with very low temperatures to preserve the sperm of various animal species for extended period of time at a low cost. Therefore, cryopreservation of semen has been of great importance in agriculture, conservation of wild animals and endangered species, and treatment of human infertility [13]. However, it is well known that cryopreservation induces sublethal damage to the spermatozoa, which may result in loss of motility, viability, in vivo fertilising capacity, deterioration of acrosomal and plasma membrane integrity, and DNA damage [[14], [15], [16]].

The freezing-thawing process in cryopreservation results in high concentrations of ROS [[17], [18], [19]]. ROS can cause damage in sperm plasma membranes and in DNA [20], and it is related with defective spermatozoa and male infertility [21,22]. In some cases, sperms carrying significant levels of oxidatively damaged DNA do not lose their fertilisation capacity and can fertilise oocyte both in vivo and in vitro [23]. As a consequence of this, poor implantation and pregnancy rates, unhealthy offspring, and de novo mutations in germ line and post fertilisation may occur [24]. Both enzymatic and nonenzymatic antioxidants are highly found in seminal plasma [25,26], but spermatozoa do not contain protective cytoplasmic antioxidant enzymes due to their limited cytoplasm [26]. Various cryoprotectants supplemented with different antioxidants are used in sperm freezing protocols in order to protect sperm from the detrimental effects of the seminal ROS caused by the imbalance between the scavenging activity of antioxidant systems and the ROS generation [[27], [28], [29], [30], [31], [32]].

Amino acids look like to be crucial components for sperm freezing extender due to their antioxidant properties. The most abundant amino acid in the body is l-glutamine (LG) which is the precursor of glutathione known as the most important antioxidant [33]. LG plays an important role in regulating the protein synthesis/degradation and the acid–base balance and in improving the adaptation to stress [34,35]. Basal medium eagle (BME) essential amino acid solution is classical cell culture solution supplemented with the minimal components including 13 amino acids and eight vitamins [36]. Both additives have been documented to protect the sperm against the adverse effects of cryopreservation and to improve the spermatozoa quality in various animal species [[37], [38], [39], [40], [41], [42]], since both of them have been reported to have antioxidant activities [37,41,43].

In this study, we evaluated the levels of DNA damage in the epididymal sperms of New Zealand rabbit (Oryctolagus cuniculus) after cryopreservation. The objectives of this study were (i) to evaluate the effects of intravenous (IV) injections of paclitaxel and resveratrol before collection of epididymal sperms on the levels of DNA damage, (ii) to test whether addition of BME and LG to extender has a reducing effect on DNA damage after cryopreservation, and (iii) to compare the results of the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay and the toluidine blue (TB) staining methods used for examination of DNA damage in sperm samples.