Is sperm telomere length altered in teratozoospermia specimens? A case-control study

Abstract Background Male factor infertility is a multifactorial defect, and many of its etiologies are unknown. Teratozoospermia is determined by the existence of over 85% morphologically abnormal spermatozoa in semen which are almost incompetent in fertilization function. One of the most novel issues in genetic alterations studies is the variation of sperm telomere lengths (STL) and its collaboration with male infertility. The present study has been focused on STL alterations in teratozoospermia. Objective Investigation of differences in telomere length of teratozoospermia specimens and sperms with normal parameters. Materials and Methods In this case-control study, 60 men referred to Arak Fertility Clinic, Markazi province, Iran from November 2017 to February 2018 were categorized into teratozoospermia and normozoospermic groups. Sperm genomic DNA extraction was conducted, and STL were evaluated using quantitative polymerase chain reaction. Results Statistical evaluation of relative telomere length was calculated by the ratio of telomere to single-copy gene for teratozoospermia and normal specimens. Results significantly demonstrated that relative telomere length in teratozoospermia samples is nearly 3 times shorter than in normal samples (p > 0.001). Conclusion Our results represent the reduction of telomeres length in teratozoospermia and suggest that this alteration might be one of the factors contributing to the sperm fertility potential of this kind of specimen. However, defining relevant molecular processes requires further detailed investigations.


Introduction
Fertility is one of the major challenges in the modern era and afflicts almost 8-12% of couples worldwide, with even higher prevalence in industrialized countries (1,2). Male infertility is responsible for 40-50% of these cases, and the percentage of infertile men is approximately 2.5-12% worldwide (1). Infertility of male origin has numerous and multifactorial causes such as anatomic, endocrine, metabolic, and genetic problems, which in some cases can lead to numerical and morphological disorders of sperms and low semen quality (3). Among these abnormalities, teratozoospermia is described by the presence of more than 85% spermatozoa with irregular morphology in semen (4,5). This heterogeneous category includes a broad range of sperm phenotypes that affect structures of the spermatozoa (6). It seems sperm maturation in testis is defected in teratozoospermia patients and abnormal sperms are functionally incompetent.
In vitro fertilization and intracytoplasmic sperm injection are useful methods for obtaining live births in patients with teratozoospermia. Although the outcomes seem poor in most cases, thus clarifying molecular aspects of this defect is of great importance (7).
Various causes such as abnormal sperm parameters and sperm DNA damage are known to decrease the chance of fertility. Lately, studies in reproductive fields have increasingly focused on telomere length (8,9). Telomeres are composed of single-stranded DNA and DNA-protein complexes located at the end of chromosomes and are well known for their protective role against chromosome degradation and fusion. The singlestranded DNA is a noncoding tandem repeat sequence with hexa nucleotide 5′-TTAGGG-3′, which play a key role in maintaining chromosomal stability and cell viability (10,11). This sequence extends for 5-10 kilobases in human somatic cells, while in male germ cells, the average length is doubled. Although the length of telomeres decreases with each cell division, it has been observed that in male germ cells, the length of telomeres increases with age indicating the importance of preserving genomic integrity between generations (12,13).
Over recent years, telomere biology has attracted significant attention in human reproduction, leading researchers to focus on the relation between sperm telomere length (STL) and male fertility and spermatogenesis.
The studies assessing telomere length in sperms suggest that in low-quality semen samples and even in infertile men with normal semen indices, the telomere is shortened compared to fertile men (14). Studies show that reduced telomere length in spermatozoa may be a marker of abnormal spermatogenesis, which can represent feasible problems (15,16).
The direct relation between teratozoospermia and telomere length had not been evaluated in previous studies. Thus, this study aimed to assess any differences between telomere length with normal and teratozoospermia sperm in men of the same age range.

Study population
In this case-control study conducted from In addition, 30 healthy donors with proven normal semen profiles were used as controls.
Case and control groups were similar in terms of age and absence of illnesses and therapies.

Genomic DNA extraction and quantitative polymerase chain reaction (qPCR)
Genomic DNA was extracted directly from semen specimens using the GeneAll kit (General Biosystems, Seoul, Korea). Almost 100 µl of semen was used for extraction according to the manufacturer's instructions. Quality assessment was then performed by measuring the concentration and purity of extracted DNA.
STL was assessed using qPCR, according to Cawthon. In this method the signal intensity received during qPCR was used to quantify the amount of telomeric DNA as copy number variation relative to the amount of a single copy gene which result in a ratio called as telomere/single copy gene (T/S) ratio. The 36B4 gene that encodes acidic ribosomal phosphoprotein was used as the single-copy reference gene (17).

Results
Q-PCR curves were evaluated to verify the accuracy of the test, and melt curves showed sharp and singular peaks while negative controls had not shown any amplification ( Figure   1).
To evaluate the relative telomere length, the T/S ratio was calculated for each specimen using the

Discussion
In the present study, relative telomere length of teratozoospermia patients were compared with standard samples and the results indicated that telomere lengths of teratospermic specimens were significantly shortened. This alteration might be one factor contributing incompetency of this kind of sperms. To our knowledge, teratospermic specimens were not evaluated separately but were included in some studies as a part of an abnormal group (18). Several studies have investigated the relationship between telomere length and male infertility and mostly reported the suggestive relationship between STL and male infertility (16,19,20

Conclusions
Amongst different biological factors that affect semen characteristics, genetic alterations are known as remarkable actors, and telomeres length is one of the issues which has been highlighted in this field lately. Our results represent the reduction of telomeres length in teratozoospermia and suggest that this alteration might be one of the factors contributing to the sperm fertility potential of this kind of specimens.
However, defining relevant molecular processes requires further detailed investigations.