Varicocele Time-dependently Affects DNA Integrity of Sperm Cells: Evidence for Lower In vitro Fertilization Rate in Varicocele-positive Rats

Abstract

Background: We designed this study to clarify how varicocele can time-dependently affect sperm morphological parameters and DNA integrity. In this study, we intend to estimate the effect of various periods of varicocele on the in vitro fertilization (IVF) rate in rats.

Materials and methods: In this experimental study, left varicocele were induced as the test group (n=18) which was further sub-divided into three groups based on the study termination time (4, 6 and 8 months after varicocele induction). The control-sham group (n=6) consisted of rats who received no treatment. Repopulation index (RI), tubular differentiation index (TDI), sperm viability and motility, morphological maturity, chromatin integrity and ability to undergo IVF were assessed. In addition, the potential impact of varicocele on serum total antioxidant capacity (TAOC) and total thiol molecules (TTM) were examined.

Results: Histological results showed that varicocele negatively influenced TDI and RI. All sperm morphological parameters were lower than those in the control-sham group. DNA damage was severely and time-dependently substantiated in all test groups. Varicocele significantly reduced the ability of sperm derived from varicocele rats to undergo IVF. Serum TAOC and TTM levels reduced in a time-dependent manner. Right testes varicocele-induced rats showed remarkably less damaged profile for all investigated parameters compared to the left testes varicocele.

Conclusion: Our data suggested that experimentally induced varicocele negatively impacted sperm maturation and chromatin integrity in a time-dependent manner. This consequently caused a remarkable reduction in IVF ability. The detrimental effect of varicocele may be attributed to the significant reduction of antioxidant capacity of the serum.

Keywords: DNA Damage; In vitro Fertilization; Oxidative Stress; Varicocele.

Fig 1

Photomicrograph of rat testis. A. Control-sham group. The seminiferous tubules with normal cellular junction (S) and interstitial connective tissue without any edema are seen (I). B. Left varicocelized testis. The dissociated germinal epithelium in seminiferous tubules (S) with edema in the interstitial connective tissue is observed (I). Iron-Weigert staining, (A × 100 and B × 400).

Fig 2

Photomicrograph of rat testis. A. Control-sham group. Note the seminiferous tubules with normal cellular junction (S) and the interstitial connective tissue with no edema (I). B. Right testes of varicocelized rats. Note the seminiferous tubules with negative tubular differentiation index (S). The spermatogenesis regions (arrowheads) are indicative of early maturation from the previous cycle. C. Left testis of varicocelized group. The seminiferous tubules (S) are completely depleted with no detectable spermatic maturation. Vascular thrombosis (T) is also observed in interstitial connective tissue. Iron-Weigert staining, (A × 100; B × 400 and C × 400).

Fig 3

Effect of varicocele on repopulation index in: (A) Left testis and (B) right testis. Repopulation index percentage of spermatogonia type A reduced over time in all varicoceleinduced rats and the spermatogonia type B increased by the time. Ø and stars indicate significant differences (p ≤ 0.05) between data for spermatogonia types A and B, respectively. All data are presented as mean ± SD.

Fig 4

Light microscopic architecture from sperm; A1; Abnormal sperm with dense blue stained mature nucleus, A2; Normal sperm with unstained cytoplasm in head section, B1; Normal sperm with light stained immature nucleus, and B2; Abnormal sperm; note the sperm in the left side of the figure with cytoplasmic droplet (arrows) and the dead sperm with eosin-stained cytoplasm (below, right hand side). Aniline-blue (A-1, B-1) and eosin-negrosin (A-2, B-2) stainings, (× 400).

Fig 5

Epi-fluorescent architecture of rat sperms by Comet assay. A. Sperms from control group; the green spots without any tails are normal sperm. B. Sperms from right testes; The green spots without tails (arrowhead) and spots with tails (arrow) indicate the DNA fragmentation. In panel B both normal and fragmented DNA are seen in sperms collected from the right testes of varicocelized rats, C. Sperms collected from the left testes of varicocelized rats with intensive DNA fragmentation; (Comet assay technique, × 100).

Fig 6

Effect of varicocele on double strand DNA in rat sperm. A. Epi-fluorescent architecture of the control-sham group. The light green stained nuclei are indicative of the normal double-strand DNA in this group. B. Light microscopic architecture of sperm from right testes of varicoceleinduced rats. The normal sperm with light green stained nucleolus (arrows) and sperm with damaged DNA with light yellow stained nucleolus (arrowheads) are presented. C. Light microscopic architecture of sperm from left testis of varicocele-induced rat, the sperm with light yellow stained nucleolus represent remarkable DNA damage (Acridineorange staining, × 400).

Fig 7

Effect of varicocele on IVF and 2-cell embryo after invitro fertilization in the control and varicocele-induced rats: A. Left testicle data, B. right testicle data. Stars indicate significant differences (p ≤ 0.05) between all data for different months following varicocele induction with each test group and the control group. All data are presented as mean ± SD.

Fig 8

Effect of varicocele on the serum level of TAOC (A) and TTM (B); Stars indicate significant differences (p ≤ 0.05) between control and test groups. All data are presented as mean ± SD.