Effects Of Non-alcoholic Fatty Liver Disease On Male Reproductive Function And Its Mechanism

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liverdisease not only in western countries but also in Asia. Approximately15to30%of adults inthe general population in Asia have NAFLD. Emerging evidence suggests that NAFLD isstrongly associated with a number of important co-morbidities, including obesity, diabetes,hypertension, cardiovascular disease and chronic kidney disease. Early studies mainlyfocused on the interaction mechanisms between NAFLD and its co-morbidities, whereaswith less attention to the endocrine system. Recently, some studies reported that NAFLDmay cause sex hormone disorder. As the sex hormone levels are closely related withreproductive function, whether NAFLD impairs male reproductive function or paternalNAFLD affects offspring is still unknown. Therefore, it is necessary to study the impact ofNAFLD on male reproductive function and its mechanism.In the first part of our study, we screen the NAFLD patients by questionnaires andultrasound examination in Chongqing, and detect their hormone levels and sperm quality, toassess the reproductive function of the patients with NAFLD. In the second part, weinduced NAFLD in a male rat model and evaluated the reproductive function of theseanimals. The following parameters were observed: serum and testicular testosterone levels,sperm quality, morphological changes in testis, germ cell differentiation, apoptosis, andmale fertility. In the third part, we explored the mechanisms involved in NAFLD, such asthe expression differences in StAR, P450scc, P45017βand AC-cAMP-PKA pathway relatedgenes and proteins.Materials and MethodsPartⅠEpidemiological survey of male reproductive function in NAFLD patients.1. Cross-sectional investigationThe study was performed with consecutive recruitment of NAFLD patients and healthycontrols in Chongqing including Yuzhong, Jiangbei and Fuling district. The studypopulation consisted of513adult men. The questionnaire included general demographic characteristics, fertility, lifestyle, medical history, etc. All participants completed abdominalultrasound examination, according to NAFLD diagnosis criteria, the study population weredivided into control group, NAFLD and NASH group.2. Clinical and laboratory evaluationThe semen and blood samples from all of the participants were collected. Semenanalysis was performed according to the guidelines of the WHO (1996), and spermmorphology was assessed using the tygerberg strict criteria. Serum testosterone, LH, FSHand E2were measured by radioimmunoassay kit. All other biochemical tests wereperformed using Synchron Clinical System LX20.PartⅡ the impairment of reproductive function in a male rat model of non-alcoholicfatty liver disease1. Animal model5-weeks-old male SD rats (n=60) were randomly divided into3groups (n=20),control group (normal diet), NAFLD group (high-fat diet for12weeks) and N-3treatmentgroups (high fat diet12weeks+N-3for4weeks). After12weeks, rats (10per group) wereeuthanized; blood, liver and male reproductive organs were removed. Another10male and20female rats in each group were selected for cohabitation. On day20of gestation, half ofthe females were euthanized and caesarean-sectioned. The remaining females were allowednatural birth. At postnatal day (PND)1and PND7, the gender, body weight, and survivalstatus of each pup were recorded.2. Detection of reproductive indicatorsThe male reproductive organs were collected and weighed. Morphological changes intestes were observed under light microscope and electron microscopy. Sperm counts andsperm motility were analyzed by CASA system, sperm malformation rate were examined byeosin staining. Leydig cells were stained with periodic acid Schiff reagent. The apoptoticgerm cells in testes were measured by TUNEL. Serum testosterone, LH, FSH and E2weremeasured by ELISA kit, and testicular testosterone was measured by125I-basedradioimmunoassay kit.Part Ⅲ the mechanism of NAFLD in male reproductive damageThe expression of testicular LHR and testosterone biosynthesis enzymes (StAR,P450scc, P45017α,17β-HSD) in tests was measured by semi-quantitative polymerase chain reaction (RT-PCR) and Western blot. Testicular AC activity and cAMP levels weremeasured by using a [3H] cAMP assay kit and ELISA kit, respectively.ResultsPartⅠEpidemiological study1. Baseline characteristics of study cohortsAmong513recruited adult men, a total of285participants completed all steps of thestudy,103controls,97with NAFLD and85with NASH. The three groups did not differ interms of age, education, occupation, marital status, income level, BMI, smoking anddrinking. Patients with NAFLD and NASH had higher WBC, RBC, HGB, HCT, TBIL,AST, ALT, GGT, CHOL, TG, CREA and URIC than the control group.2. Sexual hormone levelsCompared with the control group, the testosterone, LH and SHBG levels in the NASHgroup significantly decreased, while FSH increased significantly. The SHBG level inNAFLD group was significantly lower than the control group, while the other parameterswere not significantly differences.3. Sperm quality analysisIn NAFLD group, the median was2.9ml for semen volume,65.3×106/mL for semenconcentration,124.8×106for total sperm count,33.8%for Grade a sperm motility,53.1%for Grade a+b sperm motility, and27.9%for normal morphlogy. The sperm count and thesperm motility in NAFLD group were decreased than that in the control group, while theother parameters were not significantly differences.4. Multivariate analysisAbstinence duration and NAFLD were strongly associated with semen quality(P<0.05). Other factors such as age, BMI, smoking and alcohol use had little or no effect onsemen parameters. With the increase in liver fat content, sperm concentration, sperm countand sperm activity were significantly reduced.PartⅡ Animal experimental study1. A male rat model of NAFLDThe NAFLD group tended to develop obesity and had higher liver weight. In the liverhistological analysis, the NAFLD group showed microvesicular steatosis and scattered ofpronounced periportal macrovesicular steatosis. With the extension of the high-fat diet time, the degree of steatosis was increased. Serum ALT, TG and TCL levels were alsosignificantly higher in NAFLD group than that in ND group.2. Serum hormones and histology in testesIn NAFLD group, serum testosterone, SHBG, LH, and IGF-1levels were significantlylower than that in the control group, and testicular testosterone level also decreasedsignificantly. The weights of the testes and epididymides were lower in the NAFLD groupcompared with the ND group. Pathological lesions observed in NAFLD group such asseminiferous tubules markedly increased diameters, and structural disturbance ofspermatogenic cells with prominent vacuolization.3. Semen characteristicsThe sperm count and sperm motility were was significantly lower in NAFLD groupthan that in the ND group. The rates of morphological abnormalities were not significantlydifferent among the three groups. In the NAFLD group, the percentage of HC cells wassignificantly higher than that in the ND group. However, the number of4C cells in theNAFLD group was decreased compared with the ND group. In the TUNEL assay, thenumber of apoptotic cells in the NAFLD group was significantly higher than that in the NDand N-3PUFA+NAFLD groups.4. Breeding experimentIn the breeding experiment, the number of cohabitation days in the NAFLD group wassignificantly higher than that of the ND and N-3PUFA+NAFLD groups. However, nosignificant differences were observed in the three groups in regard to the fertility index, orthe number of implantation site, fetal resorptions, and live or dead fetuses. In addition, inthe mated female rats, no differences were found in the three groups after spontaneousdelivery about the number of gestation days, birth weight, survival of pups, male/femaleprogeny ratio, survival rate and body weight at PND7.5. Beneficial effect of N-3fatty acid supplementationAfter4weeks of N-3PUFA treatment, the serum hormone level in NAFLD group wassignificantly increased, while the number of lipid droplets in the testis and vacuoles werereduced. The sperm count and sperm motility were recovered at different degrees, whichindicated that N-3PUFA treatment may have a beneficial therapeutic effect on reproductivedamage. Part Ⅲ The mechanism1. Testosterone synthetic enzymesNAFLD markedly reduced mRNA and protein levels of StAR, P450scc,17β-HSD andP45017αin testes. There were no significant differences in the testicular mRNA and proteinlevels of LHR in the three groups. However, the NAFLD rats had a significantly lesstesticular AC activity and cAMP level than that in the ND group. It is tempting to considerthat the decrease in serum LH in rats with NAFLD might cause testosterone biosynthesisdeterioration by affecting the expressions of testosterone synthetic enzymes throughAC-cAMP-PKA pathway.2. Beneficial effect of N-3fatty acid supplementationAfter4weeks of N-3PUFA treatment, the AC activity and cAMP level were increasedin N-3PUFA+NAFLD group, while testosterone synthetic enzymes were no changes inthe three groups, which indicated that N-3PUFAs treatment may have a beneficialtherapeutic effect on AC-cAMP-PKA pathway.Conclusion1. This study reported the reproductive function in NAFLD patient for the first time.We found the hepatic lipid content was closely correlated with the sperm quality andhormone levels. Moreover, NAFLD is a risk factor for sperm quality, which needs greaterattention.2. NAFLD could induce the morphology changes in testis, such as increased apoptosisin germ cells and reduction of interstitial cells, which leading to reduction of the synthesisof testosterone. NAFLD lead to a decline in sperm counts and sperm activity, while it doesnot affect the sperm morphology and F1rats growth.3. The decrease of serum LH in rats with NAFLD might cause testosteronebiosynthesis deterioration by affecting the expressions of testosterone synthetic enzymesthrough AC-cAMP-PKA pathway.4. Administration of N-3PUFA not only improved NAFLD, but also ameliorated theimpairment in reproductive function of rats with NAFLD. N-3PUFA administration couldimprove the life quality of patients with NAFLD.In summary, our study indicates that paternal diet-induced NAFLD leads to significantimpairment in reproductive function, such as decreases in the organ coefficients of reproductive organs, sperm quality, and serum and testicular testosterone levels. It appearsthat inhibition of testosterone biosynthesis may be associated with the reduction in thenumber of Leydig cells, serum LH levels and expressions of StAR or testosterone syntheticenzymes. Furthermore, treating NAFLD rats with N-3PUFAs not only reduced hepaticsteatosis but also improved reproductive function. After literature search, there are nosimilar studies have been reported.At the same time, our study had some limitations. First, the size of our participant wasrelatively small, limited the applicability of our conclusions to NAFLD patients in thegeneral population. Second, in the breeding experiment, we only observed7days of F1ratsgrowth, which may have underestimated the effect of NAFLD on the growth of F1rats;Finally, for further validated the serum LH measurements, we only detect AC activity,cAMP production and LHR mRNA, without detection of combination of LH and LHR, itmay affect the data integrity.In future studies, we could delineate the underlying mechanisms whereby NAFLDaffects reproductive function, such as how NAFLD influence serum LH level or howNAFLD regulate the hypothalamic-pituitary-gonadal axis. In addition, we could detect thechanges of mitochondrial pathway, endoplasmic reticulum stress, fatty acid metabolism andsugar metabolism pathway in testicular tissue of the animal model of NAFLD.