Correlation of Sperm DNA Fragmentation Index With Seminal Plasma Biochemical Index and Semen Parameters in Infertile Men

In general, routine semen analysis makes only limited predictions about a man's reproductive potential and is not always able to explain why he is infertile. In fact, many male infertility cases are caused by sperm DNA defects ,which routine semen quality analyses fail to detect. The relationship between sperm DFI, sperm parameters and their diagnostic value were analyzed and evaluated by observing the seminal parameters of infertile patients without accessory gonadal infection. Specimens of 151 cases were collected from infertile patients who visited the male department of STD and reproductive specialty clinic of our hospital from August 2018 to September 2019. SCD test was performed to measure the DNA fragmentation in native. The routine semen analysis was performed with a semen quality detection system (CFT-920, Jiangsu Ruiqi Life Science & Tech Dev .Co.Ltd) and supporting reagents. Seminal plasma malondialdehyde (MDA), and total antioxidant capacity (TAC) were assessed. Fructose(Fru) (cid:0) a-glucosidase (a-glu), and zinc (Zn) levels are quantitatively detected by kehua-310, a fully automated biochemical tester provided by Nanjing Xindibio. According to DFI level, there were 31 cases in group I (DFI ≤ 15%), 81 cases in group II (15% < DFI < 30%), and 39 cases in group III (DFI ≥ 30%). Compared with group II, there were signicant differences in sperm survival rate, PR% and Fru by non-parametric test (Z = -2.16 -2. 43. − 2. 20,respectively,P < 0. 05). There were signicant differences in sperm survival rate and PR% between group I and group III (t = 4. 32, 4.25, respectively, P < 0.01). Compared with group III, there were signicant differences in sperm survival rate and PR% by non-parametric test (Z= -3. 26, -3. 50, respectively).Sperm DFI was negatively correlated with sperm survival rate and PR%(R=-0.56,-0.46,P < 0.01). DFI was positively correlated with MDA content (R = 0.42, P < 0.01) and negatively correlated with TAC (r=-0.40, P < 0.01).There was no correlation with age ,abstinence days, semen volume, sperm concentration, percentage of normal form sperm, Fru, a-Glu, Zn (R = 0. 15, 0. 05,0.03,-0.03, -0.2, -0.16,-0.20, 0.03, 0.15, p > 0.05).The survival rate and PR% of sperm decreased signicantly with the increase of DFI level, antioxidant can decrease the rate of DNA fragmentation, antioxidant can decrease the rate of DNA fragmentation. Semen volume and sperm concentration were mainly related to the secretion volume of accessory gonads and total sperm count, but no correlation was found between them and DFI.


Introduction
Male infertility, a rising concern in reproductive medicine, is caused by a wide variety of spermatogenic abnormalities, including sperm concentration, motility, morphology, and so on. Up to 50% of infertility is male-factor, which ratio was only 30% in 2013 [1] . Nowadays, DNA fragmentation index (DFI) has been becoming an adjunct test for the standard semen analysis because lower rates of conception always accompany with the elevated sperm DFI and up to 8% of infertile men with normal semen analysis results may have a high abnormal sperm DFI [2][3] . The damaged sperm DNA may lead to unsuccessful pregnancy outcomes which could not be evaluated by the routine semen analysis. Thus, the sperm DFI has been introduced into the clinical laboratory to diagnose the male infertility [4] . The simultaneous detection of the above-mentioned semen parameters can provide a comprehensive understanding of the patient's semen status, sperm motility, sperm morphology, defect degree of genetic material in sperm head nucleus and accessory gonadal secretion function, which has important guiding signi cance for the diagnosis and treatment of infertility.
In this paper, the relationship and diagnostic value between sperm DFI and various sperm parameters were analyzed and evaluated by observing the semen parameters of infertile patients without accessory gonadal infection.

Materials And Methods
The general information The selected 151 infertile patients (ages from 22 to 53 years, mean age 31.6 ± 3.2 years)who visited at the male department and reproductive Medicine Center at our hospital in August 2018 to September 2019 were included in the present study. Excluded were those with family diseases, genital trauma and organic lesions, urological and reproductive diseases (like varicocele, cryptorchidism, prostatitis, epididymitis, leukocytopenia, hematospermia and azoospermia) .

Ethics
The research was approved by the Ethics Committee of Women's Hospital of Nanjing Medical University, and was conducted in accordance with the Declaration of Helsinki. An information sheet was provided to all participants. Written informed consent was obtained from all participants. The relevant guidelines and regulations of the local institute were strictly followed when conducting the study. Participants were informed that they could withdraw from the trial without giving a reason.

Semen collection
Semen samples were collected by masturbation after 2 to 5 days of ejaculatory abstinence (WHO, 2010) [5] . The duration of abstinence was recorded. Each semen sample was directed into a sterile plastic cup, lique ed in an incubator at 37 ℃. After the semen was completely lique ed, at least 2.6 ml of semen was taken for the test (1.5 ml of the semen was used for routine semen analysis, the left for DNA fragmentation analysis and oxidative stress parameters)..

Rutine semen analysis
According to Laboratory Manual of the WHO for the Examination and Processing of Human Semen (5th edition ) and WHO Manual for the Standardized Investigation, Diagnosis and Management of the Infertile Male [5] , the routine semen analysis was performed with a semen quality detection system (CFT-920, Jiangsu Ruiqi Life Science & Tech Dev .Co.Ltd) and supporting reagents. Main parameters were as follows [6] . Image acquisition frame: low and middle sperm concentration collected at 20 Hz, and high sperm concentration at 7 Hz; acquisition interval: 3 ms; maximum sperm motile velocity: 200 um s − 1 ; area range of spermatozoa head detected at 7-60 um 2 . Index of sperm motility: straight line velocity (VSL). Grayscale thresholds were set to collect spermatozoa and exclude nonsperm granules. According to the thresholds set for sperm analysis, sperm images were collected and analyzed.

Sperm morphology assessment
For morphological evaluations, seminal smears were stained with Diff-Quik (MICROPTIC S.L. Co., Barcelona, Spain) [8] . Approximately 10 µl of sperm was smeared into a thin and homogeneous layer on a clean glass slide and was air-dried at room temperature for at least 10 min. The slides were stained and observed under a bright eld microscope (BH-2; Olympus, Tokyo, Japan) at 1000 × magni cation. According to WHO guidelines, a sperm with deformed head, or midpiece, or principal piece was counted as. SDI (sperm deformity index) = number of deformed sperm/number of total sperm. For each semen sample, at least 200 sperms (or the whole sperm if the slide had less than 200 sperm) were counted via a double-blinded method. Then, the percentage of sperm with normal morphology was calculated [9] .

DFI(SCD test)
Using the SpermFunc™ DNAf kit (BRED Life Science, Shenzhen, China), SCD test was performed to measure the DNA fragmentation in native and DGC-separated semen. Gelled aliquots of low-melting-point agarose in the kit were provided for semen sample processing in Eppendorf tubes. Eppendorf tubes were placed in a water bath at 80 °C for 20 min to melt the agarose and then transferred to a water bath at 37 °C for 5 min for temperature equilibration. A total of 60 µL of sampled semen was added to and mixed with the agarose in the Eppendorf tubes. Then, 30 µL of semen-agarose mixture was pipetted onto precoated slides in the kit that were covered with a 22 × 22-mm coverslip. The slides were placed on a cold plate in the refrigerator (4 °C) for 5 min, allowing the agarose to produce microgel in which the sperm cells were embedded. The coverslips were gently removed, and the slides were immediately immersed horizontally in solution A and incubated for 7 min. Next, the slides were horizontally immersed in solution B for 25 min. After being washed for 5 min in a tray with abundant distilled water, the slides were dehydrated in gradient concentrations of ethanol (70%, 90%, 100%; respectively) for 2 min, air-dried, and stored at room temperature in opaque closed boxes [10] .
For bright-eld microscopy, the slides were horizontally covered with a mixture of Wright's staining solution (BRED Life Science, Shenzhen, China) and phosphate buffer solution (BRED Life Science, Shenzhen, China) (1:2) for 15 min with continuous air ow. Then, the slides were washed in running water for 10 s and allowed to dry. Strong staining was recommended to allow the periphery of the dispersed DNA loop halos more visible. A minimum of 500 sperm were counted on each sample under the 100 × magni cation [11] .
Normal spermatic DNA presented radiate halos and damaged spermatic DNA presented no or small halos. Fragmented sperm referred to those having a small or no halo. The thickness of the halo on one side was less than the 1/3 diameter of the head's thinnest part [12] . The rate of sperm DNA fragmentation (%) = the number of sperm with fragmented DNA ÷ the total number of sperm × 100%, and 25% was considered normal.

Quantitative detection of seminal plasma biochemical indexes
Spectrophotometry was used to test the level of TAC (U/L) and MDA (nmol/mL). MDA levels were determined using the thiobarbituric acid (TBA) method. Semen samples were centrifuged at 4 °C for 15 min with a speed of 2000 r/min. The supernatant was mixed with the reagents supplied in an MDA Assay Kit (Nanjing Jiancheng Bioengineering Corporation, China, A003-2) and incubated at 95 °C for 40 min. Having been cooled at room temperature, the mixture was centrifuged at 4000 g for 10 min. The absorbance of the supernatant was measured at 530 nm. All operations were performed according to the manufacturer's instructions. The MDA concentrations were expressed as nmol/mL [13] .

Statistical analysis
The software(SPSS 22.0)analyzed the data, and the measurement data description was expressed as x ± s in accordance with the normal distribution, and the skewed distribution was expressed as M(P25, P75).Independent sample T test was used to test the homogeneity of normal distribution and variance, and mann-Whitney test was used to test skewed distribution. The correlation was analyzed by Pearson, and the correlation coe cient was expressed by p < 0.05 indicates that the difference is statistically signi cant.

Results
Basic information description of the research object Comparison of semen parameters between different sperm DFI levels  Table  3.
MDA content and TAC levels were compared between the two groups  Table 4.
Correlation analysis between sperm DNA integrity and sperm oxidative stress index Pearson correlation analysis showed that DFI was positively correlated with MDA content (r =0.42, P<0.01) and negatively correlated with TAC(r=-0.40, P<0.01).See Table 5.

Discussion And Conclusions
SDF was produced in sperm cells during spermatogenesis and maturation periods, which can re ect the damage status of chromosome and integrity of impaired DNA [12][13] . The SDF index (DFI) is used to assess the degree of sperm DNA impairment. Damage on sperm chromatin can directly affect the sperm's normal physiological functions [14] , and also leads to transmit the incorrect genetic information to the offspring. Currently, three factors are considered to be the main cause of sperm damage, including abnormal sperm chromatin assemble, aberrant sperm cell apoptosis, and excessive oxidative stress [15] . During sperm maturation, histones are gradually replaced by the arginine-rich, cysteine-rich and smaller protamine (HP), and this process reduces the self-repair ability of sperm DNA in response to internal changes and external environment. Furthermore, under the effect of torsion-tension that is generated by the double-stranded DNA helix, the misfolding of DNA supercoiling structures in the chromosome can also lead to aberrant DNA repair, which result in abnormalities of the chromatin structures abnormalities and SDF increase [16]. In addition, in ammation in the external genital tract and varicocele can also increase the production of SDF by inducing free radical-mediated oxidative stress in the sperm [17] .
Many studies have proposed hypotheses on the mechanism of sperm DNA damage. The main possible reasons are as follows: 1) Antioxidants in semen can protect the oxidative damage of sperm DNA, and a small amount of appropriate ROS can help sperm capacitation and acrosomal reaction. Excessive ROS removal ability and defensive ability, more than itself lead to sperm DNA double chain rupture, produce single, its principle is ROS may through direct oxidation of sperm DNA bases, may through covalent binding of lipid peroxidation products and DNA, causing the sperm DNA chain rupture, leading to a biological structure of sperm, sperm DNA damage 2)Abnormal sperm chromatin assembly can lead to sperm DNA double strand break. The main link leading to sperm nuclear DNA damage is the abnormal appearance of protosomal proteins when they replace histones [18][19][20][21][22]. During the sperm formation process, nucleocapsid proteins of chromatin undergo the transformation from histones to transition proteins to protosomal proteins to concentrate chromatin and maintain the normal morphology and DNA integrity of sperm. In damaged or immature sperm, a large amount of sulfhydryl (SH) of protamine cannot be oxidized into disul de bonds, so it cannot bind closely with DNA. As a result, loose chromatin is formed and its structure is unstable. Its DNA is denatured into single strand under the action of acid, which eventually leads to sperm DNA damage. Therefore, sperm DFI can be detected to re ect the defect degree of genetic material in sperm nucleus and sperm DNA maturity status.
In this study, The low DFI group had an obviously lower seminal MDA level and an obviously higher seminal TAC level than high DFI groups, indicating that too much MDA was produced during seminal lipid peroxidation and that the drop of TAC level triggered oxidative stress reaction and destroyed the spermatic membranes [21] . According to Ni et al. [23] and Fu et al. [24] , sperm DNA damage could be caused by ROS in patients with varicocele. Shang et al . [25] and Greco et al . [26] have reported that antioxidant can decrease the rate of DNA fragmentation, suggesting that the seminal ROS participates in the process of sperm DNA damage.
The results of this study showed that sperm DFI level was signi cantly negatively correlated with sperm survival rate and PR%, which was consistent with relevant research reports [27][28][29] , and there were statistical differences among groups I, II and III. The survival rate and PR% of sperm decreased signi cantly with the increase of DFI level, but this does not mean that the lower percentage of the former means that sperm DNA is damaged. However, damaged sperm DNA can affect sperm fertilization ability and even inhibit embryo development. This study showed that there was a statistically signi cant difference in Fru content between group II and III and group I, while there was no statistically signi cant difference between group II and group III. However, most PR% of these two groups was lower than the lower limit of WHO reference value [30] , which was classi ed as asthenospermia. However, spermatozoa Fru is closely related to sperm motility. Fru provides energy source for sperm motility, and the reduced content of Fru can reduce sperm motility [31] . The seminal plasma Fru originates from the seminal vesicle and mainly re ects its secretion function. In this study, in ammation and infection have been excluded, which may be related to the endocrine function of the seminal vesicle and other factors. In summary, sperm DFI was low, sperm Fru was high, and sperm PR% was high. The results also showed that the percentage of normal sperm in group III was statistically different from that in group II, which was consistent with some reports [32] , and the former was signi cantly lower than the latter. Although there was no statistical difference between group I and the other two groups, the percentage of normal sperm was higher than that of group III and lower than that of group II .In this study, no correlation was found between patient age, abstinence days, semen volume, sperm concentration, spermatozoal G-glu and spermatozoal Zn and sperm DFI. However, it can be seen from table 2 that DFI increases with age, and it has been reported that age is positively correlated with DFI With the aging of male organs such as testis, prostate and epididymis, it leads to the increase of reactive oxygen species (ROS) and the decline of antioxidant capacity. Too much ROS produces a large amount of lipid peroxides, which attack the sperm cell membrane, causing the sperm DNA strand to break and destroy it integrity [33] , which may be due to insu cient sample size in this study. Semen volume and sperm concentration were mainly related to the secretion volume of accessory gonads and total sperm count, but no correlation was found between them and DFI. About 60% of the seminal plasma is from the seminal vesicle, 30% from the prostate, and the rest from epididymis, paraurethral gland, paraurethral gland, testis, etc. The spermatic a-Glu and spermatic Zn are mainly from the secretion of epididymis and prostate. It has been reported that too high free Zn has toxic effects on sperm, and its oxide inhibits sperm fermentation and oxidation process [34][35][36] .
It has also been suggested that Zn can delay cell membrane lipid oxygenation. In this study, the level of spermatic Zn in each group was basically within the normal reference range, and it was not clear whether it affected sperm quality [37] .
In summary. In future studies, the relationship between a-Glu and DFI can be further explored through large sample analysis of normal group and abnormal group based on the content levels of sperm a -Glu and sperm Zn. As an increasingly common technology in clinical testing for reproduction, sperm DFI has proven to be very valuable in male fertility evaluation.In future studies, the sample size should be expanded to allow more accurate conclusions.

Fru Fructose
A-glu a-glucosidase MDA Seminal plasma malondialdehyde TAC total antioxidant capacity