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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 3  |  Page : 146-155

Supplementation with L-carnitine rescues sperm epigenetic changes in asthenospermic male semen with altered acetyl-L-carnitine levels


1 Human Sperm Bank, West China Second University Hospital of Sichuan University; Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu 610041, China
2 Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second University Hospital, Sichuan University; Reproductive Endocrinology and Regulation Laboratory, West China Second University Hospital, Sichuan University, Chengdu 610041, China
3 Human Sperm Bank, West China Second University Hospital of Sichuan University, Chengdu 610041, China
4 Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu 610041, China
5 Reproductive Endocrinology and Regulation Laboratory; Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu, 610041, China
6 Northeast Pharmaceutical Group, Shenyang 110023; Department of Sports Medicine, Shenyang Sports University, Shenyang 110101, China
7 Human Sperm Bank, West China Second University Hospital of Sichuan University; Reproductive Endocrinology and Regulation Laboratory, West China Second University Hospital, Sichuan University, Chengdu 610041, China

Date of Submission12-Feb-2020
Date of Decision06-May-2020
Date of Acceptance26-May-2020
Date of Web Publication04-Sep-2020

Correspondence Address:
Fu-Ping Li
Human Sperm Bank, West China Second University Hospital of Sichuan University, 1416th Chenglong Road Avenue, Chengdu 610011
China
Wen-Ming Xu
Reproductive Endocrinology and Regulation Laboratory, West China Second University Hospital, Sichuan University, 20th 3 Section of South Renmin Road, Chengdu 610041
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.294314

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  Abstract 


Objective: To investigate the relationship between the concentration of L-carnitine in semen and sperm parameters and investigate the epigenetic profile in sperm cell after L-carnitine usage.
Methods: From February 2017 to February 2018, 46 semen samples from asthenospermic males and 41 semen samples from healthy donors were acquired. Motility parameters were assessed using computer-assisted sperm analysis (CASA, n = 78) and the DNA fragmentation index (DFI) was evaluated through flow cytometry (n = 86), %DFI = % cells outside main population. Other oxidative stress markers, such as reactive oxygen species (ROS) levels (n = 86) and the mitochondria DNA copy numbers, were detected (n = 78). The concentration of L-carnitine and acetyl-L-carnitine was detected (n = 82), and methylation was analyzed (n = 30). After that, we collected 13 fresh semen samples from asthenospermic males and 23 fresh semen samples from healthy donors. These samples were used in a freeze-thaw model that was used to determine whether adding L-carnitine could change sperm progressive motility (n = 23), apoptosis index (n = 9), and methylation analysis (n = 7). In total, we have done 13 asthenospermia samples for Western blot, and except for the poor Western result, we analyzed 6 samples for H3K9ac detection, 7 samples for H3K9m3 and H3K27m3 detection, and immunofluorescence (n = 3). Finally, we had recruited 30 volunteers, and they were given oral administration of L-carnitine for 3 months and then collected semen samples at different time points for methylation analysis.
Results: The concentration of acetyl-L-carnitine is negatively correlated with the %DFI value (r2 = 0.1090; P = 0.0026), and the concentration of acetyl-L-carnitine is positively correlated with sperm forward motility (r2 = 0.0543; P = 0.0458) and ROS (r2 = 0.1854;P < 0.0001), and the acetyl-L-carnitine level is negatively correlated with %DFI in asthenospermia (r2 = 0.1701; P = 0.0066), and the level of acetyl-L-carnitine in asthenospermic semen is significantly lower than the normal group (P = 0.0419). In addition, this study indicates that adding L-carnitine significantly improved sperm motility (P = 0.0325) and reduced sperm apoptosis (P = 0.0032). Importantly, Western blotting (P = 0.0429) and immunofluorescence staining results showed that the addition of L-carnitine reduced H3K9Me3 methylation level in sperm, respectively. Furthermore, semen samples from asthenospermic patients had reduced methylation levels in a specific region (16th P = 0.0003; 17th P = 0.0016) of the brain-derived neurotrophic factor (BDNF) promoter. The 16th methylation decreased with age (r2 = 0.1564; P = 0.0306), and the 17th methylation was decreased after treatment with L-carnitine for 28 days (P = 0.0341).
Conclusion: L-carnitine can reduce the %DFI and also affect the methylation of the histone modification marker in sperm as a possible epigenetic regulator.

Keywords: Acetyl-L-Carnitine; Asthenospermia; Epigenetic; Sperm DNA Damage


How to cite this article:
Jiang XH, Jiang C, Yu L, Li XL, Zuo T, Gu PF, Li FP, Xu WM. Supplementation with L-carnitine rescues sperm epigenetic changes in asthenospermic male semen with altered acetyl-L-carnitine levels. Reprod Dev Med 2020;4:146-55

How to cite this URL:
Jiang XH, Jiang C, Yu L, Li XL, Zuo T, Gu PF, Li FP, Xu WM. Supplementation with L-carnitine rescues sperm epigenetic changes in asthenospermic male semen with altered acetyl-L-carnitine levels. Reprod Dev Med [serial online] 2020 [cited 2020 Oct 22];4:146-55. Available from: https://www.repdevmed.org/text.asp?2020/4/3/146/294314


  Introduction Top


L-carnitine is commonly used in the treatment of primary systemic L-carnitine deficiency, which is the inability to properly synthesize or use L-carnitine from food sources,[1] and secondary L-carnitine deficiency caused by metabolism-related factors, such as methyl malonate, propionic acid, or fatty coenzyme A dehydrogenase deficiency of long-chain fatty acids.[1] L-carnitine has certain preventive and therapeutic effects on L-carnitine deficiency in patients with secondary L-carnitine deficiencies, which are often associated with disorders such as chronic renal failure.[2]

Converging evidence indicates that L-carnitine and acetyl-L-carnitine could be effective in the treatment of idiopathic asthenospermia[3] and that it plays an important role in male and female reproduction.[4] Sperm cells, which are highly specialized cells that are produced in the testes, lack motility and have to mature in the epididymis to acquire motility.[5] Fatty acid oxidation serves as the main source of energy for sperm cells in the epididymis. The main functions of L-carnitine are to transfer long-chain fatty acids from the mitochondrial membrane to the inner membrane in the form of lipid acyl carnitine and to perform beta oxidation in the mitochondria.[6] Furthermore, accumulating evidence shows that L-carnitine levels are altered in the semen samples of asthenospermic patients.[7],[8],[9] Therefore, as one of the most commonly prescribed drugs in male clinics, L-carnitine has a significant curative effect in the treatment of male infertility disease.[1],[3],[7],[10] L-carnitine can be metabolized to acetyl-L-carnitine in vivo, and it has been recently shown that altered acetyl-L-carnitine levels are associated with depression through epigenetic regulation. Furthermore, acetyl-L-carnitine has been shown to be critically involved in the etiology of various diseases, such as depression, autism, and other developmental diseases.[2],[6],[11]

Epigenetic change is critically involved in sperm functional regulation.[12],[13],[14] Both DNA methylation and histone modifications, such as H3K9 methylation and acetylation, have been shown to be critically involved in sperm function, as well as embryo development.[15],[16] For example, it has been shown that H3K9 methylation possesses bivalent regulation during the preimplantation stage of embryo development. Furthermore, significant genomic methylation changes were found in biopsy samples from azoospermia, indicating that methylation change is also associated with spermatogenesis defects.[17] Although the function of L-carnitine in sperm has been widely investigated, most studies have focused on its effect on oxidative stress.[18],[19] Few studies have investigated the correlation of L-carnitine and acetyl-L-carnitine with human sperm DNA damage parameters, such as the sperm chromatin structure assay (SCSA) and other sperm parameters, such as apoptosis (except in rats).[5] Importantly, no study has investigated the epigenetic profile in sperm after L-carnitine usage. Therefore, the current study investigated the effect of L-carnitine on sperm epigenetic profiles.

We systemically determined whether the L-carnitine level was altered in asthenospermic patients and whether the oxidative stress parameter was related to L-carnitine levels. We also determined whether L-carnitine affected sperm parameters after a freeze-thaw treatment. To further examine whether L-carnitine could affect the epigenetic profile of human sperm, major epigenetic markers, including H3K9ac, H3K9me3, and H3K27me3, were detected in asthenospermic patients after the addition of L-carnitine. Previous studies have shown that the marker H3K9me3 plays a critical role in regulating methylation and transcription of key developmental genes, such as BDNF. Interestingly, a recent study has shown that BDNF promoter methylation is regulated by acetyl-L-carnitine in neurons.[20] Therefore, in the current study, the methylation level of BDNF was determined in sperm of asthenospermic patients. Pyrosequencing of the BDNF promoter was used to assess whether adding L-carnitine could affect BDNF promoter methylation levels in the freeze-thaw model.


  Methods Top


The study and experimental procedures were approved by the Ethics Committee of West China Second University Hospital (K2018089). All participants provided written informed consent, and all experiments were performed in accordance with the guidelines and regulations of the hospital's Ethics Committee. From February 2017 to February 2018, 46 semen samples from asthenospermic males and 41 semen samples from healthy donors were acquired from the Andrology clinic of West China Second University Hospital, Sichuan, China. The inclusion criteria for asthenozoospermia sample is progressive motility (A + B) below 32% in computer-assisted sperm analysis (CASA) analysis, and other parameters including semen volume, pH, viability and sperm number are in normal range, while the inclusion criteria for healthy sample is progressive motility above 32%, with all other parameters are normal. Other data including the occupations and health states were all obtained and they were matched to exclude the effect of occupations and health states to the semen parameters.

Motility parameters were assessed using CASA. DNA fragmentation index (DFI), reactive oxygen species (ROS) levels, and methylations level were also evaluated. Mitochondria DNA copy numbers and the concentration of L-carnitine and acetyl-L-carnitine were detected; the relevant measuring methods to L-carnitine and acetyl-L-carnitine were described in [Supplementary File 1]. After that, we collected 13 fresh semen samples from asthenospermic males and 23 fresh semen samples from healthy donors again. These samples were used in a freeze-thaw model that was used to determine whether adding L-carnitine could change sperm progressive motility, apoptosis index, methylation level, and histone methylation caused by the freezing protocol. For human clinical study, we had recruited 30 volunteers from asthenospermia patients who have been prescribed for L-carnitine for the treatment; the detailed protocol of human clinical study was described in the part of “Human Clinical study.”



Sperm progressive motility

The sperm progressive motility analysis was conducted in the Androloy Department of Sichuan University West China Second Hospital with CASA. The hardware used was SSA-II Suiplus semen analysis system (Beijing, China), and the data were analyzed using Suijia software, which is in compliance with the World Health Organization laboratory manual for the examination and processing of human semen, 5th ed.

Sperm freeze-thaw model

For sperm freeze-thaw model, each semen sample was divided into three parts: A, B, and C. A was untreated, B was treated with 0.5 mg/mL L-carnitine (Northeast Pharmaceutical Group Co. Ltd., CN), and C was treated with 1.5 mg/mL L-carnitine. One milliliter of semen and 500 μL of cryoprotectant were mixed in the freezing tube (NEST, CN). The tube was placed at 4°C for 10 min and then held above the liquid nitrogen surface at a height of 10 cm for 5 min. Finally, the freezing tube was plunged into liquid nitrogen. After 24 h, the freezing tube was submerged in a 37°C water bath for 10 min. Various sperm parameters were tested, and the following molecular biology experiments were performed: Western blotting, SCSA, ROS production, apoptosis index, and immunofluorescence.

Mitochondria DNA copy numbers

Mitochondria DNA (mtDNA) copy numbers of the samples were determined by real-time polymerase chain reaction. DNA was extracted using DNA Extract Kit (Axygen). The mtDNA primers are forward: 3'-CGAAAGGACAAGAGAAATAAGG-5'; reverse: 3'-CTGTAAAGTTTTAAGTTTTATGCG-5'; beta-tubulin as reference primers ares forward: 3'-CAACTTCATCCACGTTCACC-5', reverse: 3'-GAAGA GCCAAGGACAGGTAC-5'.

Methylation level assessed using bisulfite sequencing polymerase chain reaction

The protocol for methylation is described in our previous publication.[21] The CpG-rich region of the BDNF Exon IXa between 1 and 471 bp (accession number: EF125690.1) includes six CpG sites. The primers for amplification of specific target sequences are listed as follows: 5'-ATGTGTGATTGTGTTTTTGGTG-3' (forward), 5'-CCTACAAAAAACACAAATCAATATC-3' (reverse). Following amplification and treatment, the products were purified and subjected to direct sequencing at Sangon Biotech (Shanghai, China).

Western blotting

In the freeze-thaw model, one milliliter of fresh semen from the patients or healthy volunteers were treated following the freeze-thaw model protocol and then diluted with 500 μL of phosphate-buffered saline (PBS) and centrifuged at 500 ×g for 10 min at 25°C. Spermatozoa were washed with PBS and added to 30 μL of radioimmunoprecipitation assay buffer (P0013B, Beyotime, CN) containing protease inhibitors (Cat. No: 04693159001, Roche, CH) and phosphatase inhibitors (Cat. No: 04906845001, Roche, CH). Samples were sonicated for 5 min (on/off cycles of 10 s/10 s) and then placed on ice for 30 min. All samples were centrifuged at 14,000 ×g at 4°C for 20 min. The supernatant contained total protein was collected, and protein concentrations were determined using the Thermo BCA Protein Assay Kit (23227, Thermo, USA). The lysates were mixed with 5 × SDS-PAGE loading buffer at 95°C for 10 min.

For Western blot analysis, 30 μg of total protein from each sample was separated on a 12.5% gel by SDS-PAGE (PG112, BAIHE, CN). After being electrotransferred onto an Immobilon-P polyvinylidene difluoride membrane (ISEQ00010, Millipore, USA), proteins were detected using the appropriate primary and secondary antibodies and visualized with the ECL reagent (WBKLS0500, Millipore, USA) according to the manufacturer's instructions. The primary antibodies were diluted with bovine serum albumin (BSA) blocking buffer (containing 5% BSA, 0.1% Tween 20, 1 0 mmol/L Tris, and 100 mmol/L NaCl, pH 7.4) and were as follows: anti-H3K9ac (ab32129, Abcam, UK, MW = 17 KDa) diluted 1:500; anti-H3K9me3 (ab32521, Abcam, UK; MW = 17 KDa) diluted 1:1,000; anti-H3K27me3 (ab6147, Abcam, UK; MW = 17 KDa) diluted 1:1,000; anti-Histone H3 (EM30605, HuaBio, MW = 17 KDa) diluted 1:5,000; and anti-GAPDH (ZenBio, China) diluted 1:5,000, and appropriate secondary antibodies were diluted 1:10,000.

Flow cytometry to measure sperm DNA damage

Sperm DNA damage was measured using a modified method based on these previous studies.[22],[23] The excitation wavelength of flow cytometry (FCM) was adjusted to 488 nm using dichroic filter, and the FCM sample channel was balanced using acridine orange (AO)-balanced buffer (0.4 mL of low-pH detergent solution [0.08 mol/L HCl, 0.15 mol/L NaCl, 0.1% Triton X‒100, pH = 1.2] and 1.2 mL Dye buffer [370 mL 0.1 mol/L citrate buffer; 630 mL 0.2 mol/L NaH2PO4 buffer; 372 mg EDTA·Na2(1 mol/L); 8.77 g NaCL [0.15 mol/L] mixed over night to ensure that EDTA·Na2 completely dissolved, pH adjusted to 6.0 with NaOH]). Sperm cells were counted and then diluted to 1–2 × 106/mL using TEN (0.01 mol/L Tris-HCl; 0.1 mmol/L EDTA·Na2; 0.15 mol/L NaCl) buffer and 50 uL sperm diluent was added to 100 μL of a low-pH detergent solution. After 30 s, 300 μL staining solution (6 μg/mL AO in 0.1 mol/L citric acid monohydrate, 0.2 mol/L Na2 HPO4, 1 mmol/L disodium EDTA, 0.15 mol/L NaCl, pH = 6.0) was added, and the stained sample was placed into the FCM sample chamber. The sample was stained with AO and measured using FCM, which utilizes the metachromatic properties of AO to distinguish between low-pH-denatured (red fluorescence = single-stranded under blue laser light [488 nm]) and native (green fluorescence = double-stranded under blue laser light [488 nm]) DNA in sperm chromatin.

Twenty samples were analyzed in each FCM batch, and a reference sample was used as internal control for every batch to ensure quality control. The result was analyzed using FlowJo version 10 software. For each experiment batch, we first analyzed the internal control, and the protocol was then used for analysis of the other samples. All the parameters were calibrated for every batch of data and pooled for analysis.

The linear parameters of green fluorescence value were ordinate, and the linear parameters of red fluorescence value were abscissa. The percent of sperm with measurable DNA strand breaks is shown as %DFI.[21],[22] The method of statistical treatment for %DFI is shown in [Supplementary Figure 1].

Reactive oxygen species

One hundred microliters of each semen sample was diluted in 100 μL nitro blue tetrazolium (NBT) buffer, gently mixed and placed at 37°C in dark conditions. After 1 h, the mixture was centrifuged for 10 min at 20°C at 3,000 ×g. After discarding the supernatant, the mixture was washed with PBS, centrifuged for 10 min at 20°C at 3,000 ×g, and the resulting supernatant was discarded. After adding 120 μL of 2 mol/L KOH and 140 μL DMSO, the resulting mixture was vortexed until no pellets were present and incubated for 15 min in the dark. Individual samples were transferred into a 96-well plate, and absorbance was measured at 630 nm.

Apoptosis

Apoptosis of sperm cells was determined using a Sizhengbo Annexin V-FITC/PI Apoptosis Detection Kit (Cat: FXP018). One-hundred microliters of individual semen samples were centrifuged for 10 min at room temperature at 500 ×g, and the supernatant was removed. Then, 500 μL 1 × binding buffer, 5 μL annexin V, and 10 μL PI buffer was added to the pellet. The samples were stored in the dark prior to detection by FCM at an excitation wavelength of 488 nm.

Immunofluorescence

Sperm samples were diluted with two volumes of PBS, centrifuged at 500 ×g for 5 min, resuspended with 50 μL PBS, and then placed onto microscope slides for immunostaining. The cells were air-dried and then fixed in 10% formaldehyde for 5 min to maintain intact structures. Sperm head decondensation was performed as described in a previous study.[24] Briefly, the decondensation was performed at in 10 mmol/L dithiothreitol and 0.5 mg/mL heparin for 30 min on ice. The cells were then washed in PBS, permeabilized with 0.5% Triton X-100 in PBS, and incubated in a blocking solution consisting of 4% BSA in PBS with Tween 20. They were then stained with the primary Ab (H3K9me3, 1:50) and the secondary Ab. Cells were counterstained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich) at room temperature to label the nuclei. Images were acquired using a laser scanning confocal microscope (Olympus).

Human clinical study of L-carnitine treatment

In the cohort study, 30 patients were recruited and every patient was administered 1 g L-carnitine twice daily (Northeast Pharmacy, Shenyang, China) for 3 months. The semen samples were stored after treatment, and the DNA was analyzed and methylation level was determined using the pyrosequencing method.

Statistical analysis

Data were expressed as mean ± standard deviation. Differences between two groups were evaluated by t-test. One-way analysis of variance and post hoc analysis (Tukey's test) were adopted to assess the significance of difference between three subgroups. Pearson's correlation test was used for correlation analysis. A two-sided P < 0.05 was considered statistically significant for all tests.


  Results Top


Association of L-carnitine/acetyl-L-carnitine with sperm DNA fragmentation index, progressive motility, oxidative stress, and mitochondrial DNA copy number

We measured L-carnitine and acetyl-L-carnitine levels in both asthenospermic patients and healthy controls. Correlation analysis indicated that while L-carnitine levels showed no correlation with %DFI, acetyl-L-carnitine levels did show a significant negative correlation (P = 0.0026) [Figure 1]a. Interestingly, the acetyl-L-carnitine level was negatively correlated with sperm %DFI in asthenospermic patients, but not in healthy controls [Figure 1]b. In addition, the acetyl-L-carnitine level was positively correlated with sperm progressive motility (P = 0.0458), whereas the L-carnitine level was not [Figure 1]c. Oxidative stress parameters were also examined and the results showed that both L-carnitine and acetyl-L-carnitine levels were positively correlated with ROS production [Figure 1]d. Furthermore, L-carnitine/acetyl-L-carnitine was not correlated with mitochondrial DNA copy number [Supplementary Figure 2]. Meanwhile, the acetyl-L-carnitine level was higher in the healthy group than in the asthenospermia group [Figure 1]e. Thus, the above results showed that the acetyl-L-carnitine level was negatively correlated with DNA fragmentation, and positively correlated with progressive motility and ROS production, but the L-carnitine level showed no significance with these phenotypes, and the mitochondrial DNA copy number showed no correlation with L-carnitine/acetyl-L-carnitine.
Figure 1: The concentration of L-carnitine and acetyl-L-carnitine in seminal plasma was determined, and were correlated with the parameters of semen samples, including sperm DNA damage, sperm motility, sperm reactive oxygen species, mitochondria DNA copy numbers. (a) The concentration of L-carnitine and acetyl-L-carnitine is correlated to the %DFI. The results show that the concentration of acetyl-L-carnitine is negatively correlated with the %DFI value, while the L-carnitine level shows no statistical relationship. (b) The concentration of acetyl-L-carnitine is positively correlated with sperm forward motility, while the L-carnitine level shows no statistical relationship. (c) The L-carnitine and acetyl-L-carnitine levels are positively correlated with ROS production. (d) The acetyl-L-carnitine level is negatively correlated with %DFI in asthenospermia. (e) The level of acetyl-L-carnitine in asthenospermic semen is significantly lower than the normal group. DFI: DNA fragmentation index.

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Figure 2: Each semen samples with cryoprotectant was divided into three parts: a, b, c. Part a was none treated, part b was treated with 0.5 mg/mL L-carnitine, part c was treated with 1.5 mg/mL L-carnitine. (a) After freeze-thaw treatment, left panel: the sperm forward motility of part a was decreased compared with the value of before treatment; middle panel: the sperm forward motility of part b was increased compared with part a (P=0.0325); right panel shows that the treatment with L-carnitine in 1.5 mg/mL has no significant change of the sperm forward motility. (b) Treatment with L-carnitine (0.5 mg/mL) can decrease the percentage of apoptosis in semen. The results of apoptosis percentage of part b was decreased compared with part a (P = 0.0032). There were no significant between part a and part b. (c) is the statistical result of semen apoptosis after treatment with L-carnitine. N is the number of individuals. Part a = 0 mg/mL L-carnitine; part b = 0.5 mg/mL L-carnitine; part c = 1.5 mg/mL L-carnitine.

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L-carnitine effectively increases sperm motility and inhibits apoptosis in the freeze-thaw model

We established anin vitro sperm freeze-thaw model and examined whether adding L-carnitine could protect the sperm from freeze-thaw damage. Our results show that while the forward motility of the freeze-thaw sperm was significantly decreased compared with that of fresh sample, adding L-carnitine could partially rescue the motility. Interestingly, only low concentrations of L-carnitine significantly increased the motility [Figure 2]a. We then used FCM to check whether L-carnitine could effectively rescue the apoptosis associated with freeze-thaw damage. The results show that freeze-thaw damage effectively promoted apoptosis. Adding 0.5 mg/mL L-carnitine inhibited apoptosis, but adding 1.5 mg/mL L-carnitine had no significant effect [Figure 2]b and [Figure 2]c.

Adding L-carnitine changes H3K9me3 methylation in sperm

In addition to the ability of L-carnitine to rescue motility in asthenospermic patients, recent studies[12],[13],[15],[16] indicate that histone modifications, such as H3K9ac, H3K9me3, and H3K27me2/3, are present in human sperm and play critical roles in fertilization and embryo development. Therefore, we measured the levels of these epigenetic markers in asthenospermia patients and determined whether L-carnitine could change the level of methylation of these epigenetic markers in our freeze-thaw model. The asthenospermia sperm samples showed increased methylation of H3K9me3, and only the low L-carnitine concentration (0.5 mg/mL) inhibited H3K9me3 methylation in our freeze-thaw model [Figure 3]a and [Figure 3]b. The other two markers, H3K9ac and H3K27me3, showed no significant change after L-carnitine treatment [Figure 3]a.
Figure 3: The results of Histone methylation. (a) After freeze-thaw the H3K9Me3 methylation of part b is significantly decreased compared with part a (P=0.0429), but there were no significant between part a and part c. While there were no significant among part a, part b, part c in H3K9Me27 and H3K9ac. (b) The statistics of the relative density of the western blotting for H3K9ac. (c) The statistics of the relative density of the western blotting for H3K9me3 (P=0.0429). (d) The statistics of the relative density of the western blotting for H3K27me3. N is the number of individuals. Part a = 0 mg/mL L-carnitine; part b = 0.5 mg/mL L-carnitine; part c = 1.5 mg/mL L-carnitine.

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We used immunofluorescence to examine their localization after L-carnitine treatment. Adding L-carnitine to the samples resulted in reduced methylation in the nucleus of the sperm [Figure 4]. Furthermore, immunofluorescence staining indicated that H3K9me3 was methylated in the centrosome and the addition of L-carnitine to the frozen solution could inhibit the methylation of H3K9me3 in the centrosome of the sperm.
Figure 4: Sperm are stained by antibody and 4,6-diamidino-2-phenylindole after treatment with 0.5 mg/mL or 1.5 mg/mL L-carnitine during cryopreservation in liquid nitrogen for 24 h. Immunofluorescence results are as follows: H3K9ac (red), H3K9me3 (red), H3K27me3 (red), and 4,6-diamidino-2-phenylindole (blue).

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Asthenospermic patients have altered methylation levels in the promoter of brain-derived neurotrophic factor

We then analyzed whether L-carnitine could change BDNF methylation in sperm samples of asthenospermic patients. Pyrosequencing of BDNF CpG sites showed that the BDNF promoter was differentially methylated in asthenospermic patients compared with the normal controls [Figure 5]a and [Figure 5]b. Specifically, the CpG sites 16 and 17 in the BDNF promoter were significantly changed in asthenospermic patients [Figure 5]c, while CpG sites 1, 2, and 18 showed no significant change [Supplementary Figure 3]a. We then examined whether adding L-carnitine changes the methylation level of BDNF in asthenospermic patients. The addition of L-carnitine had no significant effect on CpG methylation in the promoter region of BDNF in semen samples from asthenospermic patients [Supplementary [Figure 3]b. Interestingly, correlation analysis indicated that there was a negative correlation between the age of all groups and the methylation level of the 16th locus [Figure 5]d. There was no correlation between the other methylation locations and age [Supplementary Figure 3]c.
Figure 5: (a) The CpG island of the BDNF promotor. The upper panel shows the CpG rich region of the BDNF promoter and middle panel shows the sequence of the BDNF promoter. The bottom panel shows the pyrosequenced CpG sites of the promoter region. (b) The pyrosequencing results show that the 16th and 17th CpG loci were hypomethylated in samples from asthenospermia patients compared with normal donors. (c) The statistical result of methylation of the 16th and 17th CpG loci. (d) The ratio of methylation on the BDNF locus and the age of all groups are negatively correlated. (e) Clinical study shows that the BDNF methylation is significantly increased after 28 days of L-carnitine treatment. N is the number of individuals and the P value is labeled in the figures. BDNF: Brain-derived neurotrophic factor.

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Methylation level of specific brain-derived neurotrophic factor promoter in sperm samples was increased in clinical samples

After determining that the asthenospermia patients have decreased BDNF methylation, we then examined if the use of L-carnitine could affect the methylation level after the 3 months of treatment. Our result showed that after L-carnitine was added, BDNF showed variable change in methylation level, as determined by pyrosequencing analysis. The results also showed that the methylation level increased after 28 days of treatment, and the methylation level of the 17th locus increased significantly [Figure 5]e, P = 0.0341, Supplementary [Figure 3]d. Collectively, the results showed that L-carnitine could increase the BDNF methylation level in sperm, indicating that L-carnitine indeed affects the methylation of BDNF in sperm; thus, the L-carnitine usage could affect the methylation level in asthenospermia patients.


  Discussion Top


One of the key findings of the current study is that acetyl-L-carnitine, instead of L-carnitine, is correlated with %DFI parameters in semen samples of asthenospermic patients, indicating that the acetyl-L-carnitine levels are closely related to sperm DNA damage in these patients. Most studies have shown that L-carnitine levels are changed in male infertile patients.[8] In addition, L-carnitine supplements not only decrease oxidative stress and sperm membrane integrity,[25],[26] but also decrease sperm aneuploidy levels in subfertility patients.[25] Therefore, the relationship between L-carnitine and the %DFI[21] is of interest. In accordance with a previous study,[8] we found that acetyl-L-carnitine levels were significantly decreased in asthenospermic patients. Interestingly, acetyl-L-carnitine, but not L-carnitine, was correlated with the %DFI, indicating that acetyl-L-carnitine may play a more important role regulating DNA integrity inside cells.

It is clear that L-carnitine could also be beneficial for increasing the motility of frozen-thawed human sperm,[26] and a recent study indicates that L-carnitine could promote thein vitro fertilization rate in mice and other models.[27] A previous study showed that L-carnitine protected the plasma membrane and acrosomal integrity[26] of the sperm, and our study further shows that it can inhibit apoptosis and promote motility in sperm. Our results suggest the possibility that L-carnitine can be used as a protection agent in the freezing protocol of sperm, oocytes, or embryos in the future.

Sperm methylation changes and histone modification are increasingly thought of as epigenetic markers,[13] and our study and other studies show that they contribute significantly to embryo development[15],[24] and the disease risk of offspring.[13],[28] Some epigenetic markers, such as DNA methylation, can be transmitted to offspring and therefore are important to disease risk in the next generation.[13] L-carnitine deficiency has been increasingly related to neurologic disease, such as depression, cancer, and cardiac disease.[10] BDNF is a neurotrophic factor critical for neurogenesis and a recent study shows that early adverse exposure, such as BPA exposure, can alter BDNF methylation in rat models as well as human samples.[20] It has been recently shown that BDNF promoter methylation was regulated by acetyl-L-carnitine.[29] The methylation of BDNF is regulated by MeCP2, as well as HDAC and H3K9me during neurogenesis. Since our results showed that L-carnitine could alter the H3K9 methylation in the semen freeze-thaw model, we then determined whether L-carnitine could alter the methylation of the BDNF promoter. Our results indicated that samples from asthenospermic patients show dysregulated methylation in the BDNF promoter. Although thein vitro model does not show significant changes of methylation levels after the addition of L-carnitine, thein vivo effect of L-carnitine on the BDNF methylation in sperm warrants further investigation. Interestingly, a recent study shows that paternal obesity could change the methylation of the BDNF promoter in sperm, as well as the hippocampus in offspring.[30] Although the exact mechanism needs further investigation, the impact of acetyl-L-carnitine on methylation regulation warrants investigation.

Our study is the first to show that L-carnitine can alter the major histone modification in sperm using anin vitro model and in clinic study. It is possible that consumption of a L-carnitine supplement could alter the disease risk of offspring and even have a transgenerational effect.[31] One recent study has shown that supplementation of carnitine during pregnancy could reduce oxidative stress in offspring in animal models.[32] Considering that environmental factors have been shown to affect the epigenetic profile, which can be transmitted to offspring,[13],[16],[30] and the broad impact of the drug for use in male patients, it is critical to investigate the direct and possible transgenerational effect and the underlying mechanism of L-carnitine on offspring.

Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.

Acknowledgments

The authors would like to thank Min Hu and Ruizhen Jia for operation of the flow cytometer. We would like to thank Editage (www.editage.cn) for English language editing.

Financial support and sponsorship

This work was funded by the National Key Research and Development Program of China (SQ2018YFC1003603), the Natural Science fund of Liaoning Province (project number: 201601424), and the Northeast Pharmaceutical Group Co., Ltd (NEPG).

Conflicts of interest

There are no conflicts of interest.


  Supplementary File 1: LC-MS/MS Method to Measure L-Carnitine and Acetyl-L-Carnitine Levels in Seminal Plasma Top



  Sample Pretreatment Technology Top


Fifty microliter of seminal plasma was mixed with 10 μL internal reference (100 μmol/L d9-L-carnitine, d3-acetyl-L-carnitine dissolved in 5% acetonitrile), vortexed 15 s. SPE column chromatography (C18SCX) was activated by 500 μL methanol, then it was added 500 μL pure water for balance, after that the sample was added in the column, successively eluted by 200 μL pure water, methanol, 0.1% methanol and acetonitrile, 5% acetonitrile. Collected elution of acetonitrile 150 μL to sample bottle, 1 μL was taken to analyze by LC-MS/MS.

The following LC conditions were used:

Mobile phase: A – 0.2% methanol, B – Acetonitrile.



Flow rate: 0.6 mL/min.

Chromatographic column: Waters Cortecs® T3 4.6 × 50 mm, 2.7 μm, S.N.: 01013612716015.

Column temperature: 40°C.

Injection volume: 1 μL.

Automatic sampler temperature: 4°C.

The following MS conditions were used:

Ion detection method: Multi reaction monitoring.

  • L-carnitine: 162 → 85; Cone = 40V; Callsion = 18V
  • d9-L-carnitine: 171 → 103; Cone = 24V; Callsion = 16V
  • Acetyl-L-carnitine: 204 → 85; Cone = 40V; Callsion = 16V
  • d3-Acetyl-L-carnitine: 207 → 85; Cone = 66V; Callsion = 18V.


Ionization mode: Electrospray ionization mass spectrometry.

The other parameters used were as following:

  • Capillary: 3.5 KV;
  • Velocity of solvent removal gas: 1000 KV;
  • Desolvation temperature: 500°C;
  • Source temperature: 150°C.




 
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