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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 2  |  Issue : 2  |  Page : 116-119

Correlations of Sperm Mitochondrial Membrane Potential with Semen Parameters and Male Obesity


Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China

Date of Submission17-Nov-2017
Date of Web Publication4-Oct-2018

Correspondence Address:
Qi-Feng Lyu
Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.242759

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  Abstract 


Background: To investigate the correlations of sperm mitochondrial membrane potential (MMP) with semen parameters and body mass index (BMI) in males with obesity.
Methods: Semen samples were obtained by masturbation after 3–7 days of sexual abstinence from males who visited semen collect room of Shanghai Ninth People's Hospital. Conventional semen analyses were performed by computer-aided sperm analysis (CASA), and sperm morphology was analyzed by modified Papanicolaou staining. Spermatozoa were stained by JC-1 to evaluate MMP through flow cytometry.
Results: Sperm MMP of asthenozoospermia group (41.24% ± 9.71%) was significantly lower than that in control group (56.68% ± 11.13%). MMP was negatively correlated with BMI (r = −0.25, P < 0.01), but positively correlated with total sperm motility (r = 0.63, P < 0.01), motility of progressive sperm (r = 0.64, P < 0.01), and normal sperm morphology rate (r = 0.37, P < 0.01). In addition, MMP showed no significant correlations with age, volume of semen, sperm concentration, sperm count, and other indexes.
Conclusions: Sperm MMP is an important index in the evaluation of sperm function, and detection of MMP may provide references for the diagnosis and treatment of male infertility.

Keywords: Body Mass Index; Conventional Semen Analyses; JC-1; Mitochondrial Membrane Potential


How to cite this article:
Yang Y, Fan Y, Kuang YP, Lyu QF. Correlations of Sperm Mitochondrial Membrane Potential with Semen Parameters and Male Obesity. Reprod Dev Med 2018;2:116-9

How to cite this URL:
Yang Y, Fan Y, Kuang YP, Lyu QF. Correlations of Sperm Mitochondrial Membrane Potential with Semen Parameters and Male Obesity. Reprod Dev Med [serial online] 2018 [cited 2018 Dec 13];2:116-9. Available from: http://www.repdevmed.org/text.asp?2018/2/2/116/242759




  Introduction Top


Currently, about 15% of couples of childbearing age are suffering from infertility worldwide, and males and females contribute equally to this disease.[1],[2] Sperm motility is an important indicator of semen quality and male infertility, and asthenozoospermia as a common cause of male infertility is responsible for about 80% of this disease.[3]

Routine semen analyses as the major methods used in the diagnosis of male infertility mainly include sperm motility, concentration, and morphology. However, these indicators cannot be used to comprehensively evaluate male fertility and sperm quality. In the recent years, effects of sperm mitochondrial membrane potential (MMP) on sperm fertilization ability have attracted more and more attentions.[4],[5] Hoffmann-Berling[6] in 1955 first demonstrated that adenosine triphosphate (ATP) provides energy for sperm tail motility. Therefore, the functional state of mitochondria as an ATP-producing site is an important index for the evaluation of sperm quality. Sperm motility parameters such as average path velocity and curvilinear velocity are higher for sperm with higher MMP,[7] but this phenomenon still has not been well studied, especially the correlations between MMP and sperm parameters of untreated sperm. At the same time, body mass index (BMI) beyond normal range can cause male fertility decline or even infertility[8],[9],[10] and has become an important factor in male infertility. However, the correlation between MMP and BMI remains unclear. In this study, MMP was detected by flow cytometry, and its correlations with semen routine test indicators and BMI were analyzed to evaluate the application value of MMP in semen examination.


  Methods Top


Participants

Males who received semen analysis in our hospital from January 2015 to March 2015 were enrolled to serve as research subjects. Inclusion criteria: (1) Patients with 25–40 years of age; (2) with normal sexual function; (3) without traumatic and family history of genetic diseases; (4) without reproductive tract infection and obvious abnormalities of testis, epididymis, vas deferens, and other reproductive organs (including cryptorchidism, varicocele, and testicular torsion). Patients were divided into asthenozoospermia group and control group according to the conditions of semen. Patients were diagnosed as asthenozoospermia when motility of progressive sperm <32% and/or total motility <40% and sperm concentration ≥15 × 106/mL, but other indicators were normal. Patients who met the criteria of normal semen proposed by the World Health Organization (5th edition) were included in the control group.

Equipment and reagents

Experimental equipment mainly includes computer-aided sperm analysis (CASA, Hamilton Thorne, USA) and flow cytometry (Beckman Coulter, USA). Reagents include fluorescent dye JC-1 kit (BD Pharmingen, USA) and modified Papanicolaou reagent (Shanghai Hongqiao Lexiang Medical Reagent Technology Co., Ltd., Shanghai, China).

Specimen collection

All participants were subjected to 3–7 days of sexual abstinence, and semen samples were obtained by masturbation after washing their hands with disinfectant. Semen samples were stored in a sterile disposable semen collection cup and kept at 37°C for 30 min to reach liquefaction.

Semen routine analysis

Volume and pH of the semen samples were measured after semen were completely liquefied. Semen sample (10 μL) was transferred to a prewarmed Marker plate (37°C), and the routine analysis was performed using a CASA instrument to record sperm concentration, total sperm motility (percentage), and motility of progressive sperm (percentage).

Morphological observation

Semen sample (20 μL) was transferred to slides and smeared. After air-dry, sperm were fixed with 4% paraformaldehyde, and then stained with modified Papanicolaou staining reagent and finally counted under an optical microscope. At least 200 sperm per slide were counted and the counting was repeated three times to calculate the average.

Mitochondrial membrane potential detection

According to the manufacturer's instruction, semen samples were centrifuged at 845 g for 5 min, and sperm were resuspended with phosphate-buffered saline (PBS) to adjust sperm density to 1 × 106/mL. Then, JC-1 was added to a final concentration of 5 mg/L and mixed. After incubation at 37°C for 10 min, the samples were washed with PBS to remove dye followed by detection using flow cytometry. A total of 10,000 cells were collected from each sperm suspension sample, and fluorescence intensity was detected using fluorescence channels FL1 and FL2. The ratio of cells expressing red fluorescence, that is, cells in R2, was used to indicate the proportion of sperm with normal MMP.

Statistical analysis

SAS8.2 software (SAS Institute Inc, Tervuren, Belgium) was used for all data analyses. Measurement data were expressed as mean ± standard deviation ( ± s), and comparisons between two groups were performed by t-test. Pearson's correlation analysis was performed to investigate the correlations between sperm MMP and other parameters. P < 0.05 indicated that the difference was statistically significant.


  Results Top


Basic characteristics of participants

A total of 203 males, including 127 in control group and 76 in asthenozoospermia group, were enrolled in this study. As shown in [Table 1], total motility, progressive motility, and ratio of sperm with normal morphology in asthenozoospermia group were significantly lower than those of control group (P < 0.01). No significant differences in age, BMI, sperm pH, semen volume, and sperm concentration were found between the two groups (P > 0.05).
Table 1: Basic characteristics of participants

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Sperm MMP value

JC-1 monochromatic labeling was performed to detect changes in sperm MMP (R2). Results showed that compared with the control group (56.68% ± 11.13%), MMP in the asthenozoospermia group were significantly decreased (41.24% ± 9.71%, P < 0.01) [Figure 1].
Figure 1: MMP expression levels between control group and asthenozoospermic group. The percentage of cells in R2 indicates the percentage of normal MMP sperm. (a) MMP of control group. (b) MMP of asthenospermia group. (c) Comparison of MMP between control group and asthenospermia group. MMP: Mitochondrial membrane potential.

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Correlation analysis between sperm MMP and various indicators

Pearson's correlation analysis was performed to investigate the correlations between sperm MMP and various indicators of 203 participants. Results showed that there was a significant negative correlation between MMP and BMI (r = −0.25, P < 0.01), but MMP was positively correlated with sperm motility (r = 0.63, P < 0.01), progressive motility (r = 0.64, P < 0.01), and normal sperm morphology rate (r = 0.37, P < 0.01). No significant correlations were found between MMP and other indicators (P > 0.05) [Table 2].
Table 2: Pearson's correlation coefficients and the corresponding P values between various parameters and the sperm MMP

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  Discussion Top


As an important indicator of semen quality, sperm motility has been considered to be the main factor that affects fertilization. Sperm motility can be reduced by many factors, such as genetic factors, inflammation, immune function, and physical and chemical factors,[11] of which, obstacles of energy synthesis are an important factor, and energy synthesis in sperm depends on functional state of the mitochondria within the tail flagella.

In normal sperm mitochondria, energy generated by the tricarboxylic acid cycle is delivered to electrons, which are transported through the respiratory chain and they pump protons from the stromal side of the mitochondrial inner membrane to the intima side, forming a transmembrane potential difference, i.e., MMP. It has been reported that mitochondria play a pivotal role in the process of apoptosis.[12] Decreased MMP accompanied by changes in membrane permeability ultimately leads to the appearance of typical apoptotic phenotypes in cells. JC-1 is a cyanide-based cationic fluorescent dye that can be translocated into living cells and located in mitochondrial membrane. Aggregation of JC-1 can be promoted by the increase of MMP. When MMP is low, JC-1 exists in the form of monomer, showing green fluorescence. In contrast, when MMP is high, JC-1 is in the form of polymer, showing orange-red fluorescence. Therefore, JC-1 signals can be used to accurately reflect the functional status of sperm mitochondria.[13] This study showed that compared with the control group, sperm MMP in asthenospermia group decreased significantly, and total sperm motility and progressive motility were positively correlated with MMP. In accordance with this, the sperm motility after swimming and living test showed positive correlation with sperm motility parameters and fertilization potential.[7] The main function of mitochondria is to generate ATP through the oxidative phosphorylation, and ATP is the energy source for sperm tail swing. Therefore, loss of MMP may lead to obstacles of energy synthesis, thereby reducing sperm motility. In addition, results of our study also showed that normal sperm morphology was also positively correlated with MMP, which is consistent with the findings obtained from sperm swimming and living test in a previous study.[7] The mechanism may be related to early programmed death of sperm cells caused by loss of MMP,[14] destroyed cell membrane structure, and increased sperm deformity rate.

Interestingly, Pearson's correlation analysis showed that there was a significant negative correlation between male BMI and sperm MMP. As a complex chronic metabolic disease that leads to the accumulation of body fat due to overnutrition, more and more studies have shown that obesity can cause male fertility decline or even infertility.[8],[9],[10] Reactive oxygen species (ROS) is the product of normal cellular metabolism, and ROS in semen is mainly from sperm and leukocytes. Obese patients often show oxidative stress reactions and increased levels of ROS due to the increased metabolic rate.[15],[16] Excessive ROS can attack sperm cell membranes, resulting in oxidation of unsaturated fatty acids, so as to disrupt the lipid bilayer structure of sperm membranes.[17] Study has also shown that ROS could increase sperm apoptosis.[18] Therefore, BMI beyond normal range can cause accumulation of ROS and superoxide anion in seminal plasma, and sperm mitochondria will be attacked, resulting in a reduction of sperm MMP, eventually damaging male fertility.

Dong et al.[19] reported that MMP was significantly correlated with sperm motility, deformity, and other functional indicators in males with infertility. Bai et al.[20] observed increased levels of free fatty acids in overweight and obese male infertility patients, and excessive free fatty acids caused increased levels of ROS and decreased MMP, which was responsible for the decreased sperm motility. The former study did not group patients, while grouping in the latter study is based on BMI. In this study, sperm motility was used as a grouping criterion to reveal that sperm MMP in male infertility is an important index for the evaluation of sperm function. Studies have shown that with the raise of sperm MMP, either sperm motility parameters or fertilization potential increased.[7] When sperm MMP is low, intracytoplasmic sperm injection should be considered in the clinical treatment of infertile couples with unexplained reasons.[7]

In summary, reduced sperm MMP reflects the decline in energy supply, thereby reflecting reduced sperm motility and motor ability. At the same time, decrease of MMP can also reflect the increased number of sperm with abnormal morphology which related with mitochondrial factor. In other words, in addition to sperm concentration and the total number of sperm, MMP is also significantly correlated with another two major indicators of semen including sperm motility and morphology. This study also showed that the sperm MMP was negatively correlated with BMI, which means sperm MMP can reflect the effects of obesity on sperm fertility. However, the application values of weight loss and anti-oxidative drug treatment in improving the sperm motility, morphology, and fertilization potential by improving MMP remain unclear. In conclusion, sperm MMP is closely related to BMI, sperm motility, and morphology. Detection of sperm MMP may provide new insights for analysis of factors related to male infertility and clinical treatment of this disease. However, given that the spouse of males in the control group did not have a history of pregnancy and the sample size was small, clinical application of sperm MMP assay as an indicator of infertility should be further tested.

Financial support and sponsorship

This study was supported by the project of Shanghai Municipal Commission of Health and Family Planning (20164Y0137) and the National Natural Science Foundation of China (81571486).

Conflicts of interest

There are no conflicts of interest.



 
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