Leptin Receptor Gln223Arg Polymorphism of Human Spermatozoa Associated with Male Infertility in a Chinese Population

Background Leptin (LEP) is believed to play a crucial role in male reproduction, while the molecular mechanisms through which LEP affects the male reproductive system are unclear. LEP acts by binding to a leptin receptor (LEPR) which mediates its physiological action, but there are only limited studies on the function of LEPR in human sperm. Purpose This study aimed to determine the Gln223Arg polymorphisms of the LEPR gene in human spermatozoa and evaluate their possible relationship with semen variables. Methods The study was performed on Chinese men: 115 healthy subjects and 108 patients with primary and 98 with secondary infertility. Semen samples were obtained from all patients, and semen variables were analyzed. The genotypic and allelic frequencies of Gln223Arg polymorphism in spermatozoa were determined by PCR and restriction fragment length polymorphism (RFLP) analyses. Statistical analyses were performed using the chi-square test, the Kruskal–Wallis test, and the Mann–Whitney test. Results There were no significant differences in genotypic or allelic frequency distributions of Gln223Arg polymorphism among men with primary infertility, secondary infertility, and controls. Similarly, semen volume and sperm concentration did not differ with the different genotypes in all groups of men. The percentages of motile sperm for AA + AG genotypes in men with primary infertility (31.98%) were significantly lower than those in secondary infertility, and control men with GG genotypes were 34.41% and 59.36%, respectively. At the same time, the percentages of normal morphology sperm for AA + AG genotypes in men with primary infertility (2.93%) were significantly lower than those in secondary infertility and control men with GG genotypes 3.71% and 6.54%, respectively. Conclusion This study reveals a possible association between the Gln223Arg polymorphism of the LEPR gene in spermatozoa affecting spermatozoal membrane integrity and having a direct role in sperm motility.


Introduction
Infertility afects approximately 10% to 15% of couples, and male factors account for 40% of infertility cases [1].Despite an increasing efort to identify the causes of male infertility, infertility research has received much less attention than most common, complex diseases [2].LEP is believed to play a crucial role in male reproduction, as plasma concentrations were negatively correlated with a variety of reproductive indices.Te overexpression of LEP in hyperleptinemic mice decreased the weight and volume of the testicles, the diameter of the seminiferous tubules, and the numbers of the spermatocytes, spermatozoa, Leydig cells, and ofspring.After hyperleptinemia withdrawal, the testicular structure and function were partially recovered.Tese results indicated hyperleptinemia adversely afected testicular development, function, and fecundity [3].LEP defciency caused defects in spermatogenesis in Akita mice, resulting in infertility.After LEP treatment, the Akita mice revealed larger overall testes and seminal vesicles than untreated Akita sibling mice, their sperm motility was signifcantly increased, and spermatogenesis was restored.Te results showed that LEP treatment could prevent degeneration of the testes [4].LEP has been suggested to pharmacologically reduce the efects of obesity on male fertility, although data on its potential use in clinical practice are still limited [5].Te rs10244329 polymorphism in the LEP gene showed a statistically signifcant diference in fertility; it appeared that variability in the LEP gene might be associated with male infertility [6].Studies have suggested that LEP indirectly afects male reproductive function via the central neuroendocrine system and directly via the peripheral tissue membrane receptors [7].Te molecular mechanisms through which LEP afects the male reproductive system are elusive [8].In target tissues, LEP acts by binding to a LEPR which mediates its physiological action.Soluble LEPR has been found in human-seminal plasma, and plasmalemmal LEPR exists in the interstitium of testicular tissue, testis, and sperm [9][10][11].A 145 kDa isoform of LEPR was localized by immunofuorescence microscopy to the tail region of ejaculated spermatozoa [9,12].Te tail of spermatozoa contributes mainly to sperm motility.In this regard, studies suggested a possible role for LEP signaling in sperm motility.In cases of idiopathic asthenozoospermia, sperm motility was negatively associated with concentrations of LEP in seminal plasma but not in the serum.Tis also suggested that LEP exhibited certain local efects in the testis [13].LEPR is encoded by the LEPR gene, which has at least six diferent isoforms.Tese LEPR isoforms result in diferent biological activities of LEP.Te genetic variation and expression of the LEPR might afect male fertility; thus, the possible role requires further research [6].Knock-out LEPR mice have small testes, azoospermia, and multinucleated spermatids, while patients with LEPR gene mutations are infertile [14][15][16].Since 2009, as a novel approach, single nucleotide polymorphism (SNP) arrays have provided valuable data on rare genetic variations in men with impaired spermatogenesis [17].Trough peripheral blood samples for DNA extraction and genotypic analysis, the association between polymorphisms of LEP and LEPR genes and male infertility has been examined [6,18].Te mechanistic basis for the regulatory role of LEP signaling in male infertility, especially between LEPR polymorphisms of spermatozoa and male infertility, awaits further analysis.Te Gln223Arg (rs1137101) polymorphism of LEPR, the most studied in human subjects, lies within the frst domain in two putative LEP-binding regions and may afect all forms of the receptor.
Te present study was conducted to examine the Gln223Arg polymorphism of LEPR in human spermatozoa and evaluate its association with semen variables.

Study Population and Sample Collection.
A total of 321 males participated in this study and were referred to Guangxi Research Center for Family Planning (Nanning, China).Samples of patients were collected from June 2017 to December 2018.Te study population was selected based on the following criteria: (1) body mass index (BMI), folliclestimulating hormone (FSH), luteinizing hormone (LH), testosterone, and prolactin levels were normal; (2) volunteers with infertility causes, such as thyroid disorder, infammation, and infectious disease, varicocele, and abnormal karyotype were excluded; (3) those with female factor infertility were excluded.All participants were assigned to one of three groups: primary infertility (n = 108), secondary infertility (n = 98), and controls (n = 115).Primary infertility patients (aged 26-40 years) referred to a man for whom the culprit factor cannot identify and who had no child after at least a year of unprotected intercourse.Secondary infertility patients (aged 25-40 years) are defned as having the inability to have a child for at least 12 months after having had children.Te healthy donors (aged 24-39 years) in the control group gave birth to children by natural conception with their female partners, and the criteria for selecting healthy fertile individuals included normal genital examinations and normozoospermia in addition to the following criteria: sperm concentration ≥15 × 10 6 /ml, total motility ≥40%, progressive motility ≥32%, and normal forms ≥4%.Each participant signed informed consent to participate in the study, which was approved by the Ethical Committee of the Guangxi Research Center for Family Planning.Semen samples were collected in the laboratory of the Guangxi Research Center for Family Planning and from all participants by masturbation after 3-5 days of sexual abstinence.

Semen Analysis and Spermatozoa Preparation.
Semen variables were examined after liquefaction according to the World Health Organization laboratory manual [19].Semen volume was measured with a 10 mL serological pipet.Te Sperm Class Analyzer system (Microptic S.L., Barcelona, Spain) was employed to determine sperm concentration (×10 6 /mL), sperm motility (%), and straight-line velocity (VSL) (μm/s).Semen smears stained with Berg's stain (carbol fuchsin and methylene blue) were used to detect sperm abnormalities.After semen analysis, the remaining raw semen was fltered through glass wool to remove gelatinous material, and then, sperm was obtained by centrifugation twice at 1000 g for 10 minutes (min) and resuspension in Earle's balanced salt solution (EBSS).Finally, spermatozoa were purifed by the swim-up method (SUM).

DNA Extraction and Genotyping of LEPR Gln223Arg
Polymorphisms.Sperm genomic DNA was extracted using the standard phenol-chloroform method, and its quality was determined by the ratio of A260 : A280.Te extracted DNA was stored at 4 °C until used for amplifcation of a 416 bp 2 International Journal of Clinical Practice fragment bracketing the LEPR Gln223Arg polymorphism and genotyped by RFLP analyses.For PCR amplifcation, the following primers, based on Khosropour et al. [18], were used (Table 1).DNA was amplifed on a Bio-Rad S1000 PCR machine (Hercules, CA) according to the following protocol: initial denaturation at 95 °C for 3 min, followed by 35 cycles of 95 °C for 30 seconds (sec), 53 °C for 30 sec, and elongation at 72 °C for 30 sec, and a fnal extension at 72 °C for 5 min.After the size of PCR products (416 bp) was confrmed by electrophoresis on 2% agarose gels, PCR products were subjected to restriction enzyme (Msp 1) digestion at 37 °C overnight, according to the manufacturer's protocol (Termo Scientifc, Waltham, MA).Restriction fragments containing ethidium bromide were electrophoresed on 3% agarose gels and detected visually under ultraviolet light.Tus, to evaluate the accuracy of genotyping, a random double-typing approach was used on 20% of the restriction fragments derived from PCR products.Te verifcation process resulted in a 100% match, indicating high accuracy in the genotyping analysis.Te restriction fragments revealed three genotypes (Figure 1).Te AA genotype (absence of restriction site) indicated a single 416 bp fragment, and the GG genotype exhibited two fragments of 291 and 125 bp, while the AG genotypes exhibited all three fragments, including 416, 291, and 125 bp.

Statistical Analysis.
All data were analyzed using the Statistical Analysis System (SAS) software (version 9.2).Te gene counting method was used to calculate the allelic frequencies, and the distribution of genotypes for Hardy-Weinberg equilibrium was tested in all groups.Te Chisquare test (χ 2 ) was used to analyze the signifcance of the association between allelic and genotypic frequencies and infertility status.After applying one-sample Kolmogorov-Smirnov tests, the data of semen variables showed nonnormal distribution; therefore, an equivalent nonparametric test was used.Te signifcance of the association between semen parameters and genotypes was analyzed using the Kruskal-Wallis test, and the descriptive characteristics of the group variables were expressed as median with an interquartile range.Tus, when statistical signifcance was demonstrated (P < 0.05), comparisons between groups were made with the Mann-Whitney test.Tis study revealed more than 80% power to detect the genotypic association of LEPR polymorphism with a signifcance level of 0.05 using online tools, such as "https://www.power.com"and "https://sample.size.com."

Leptin Receptor Gln223Arg Polymorphism Analysis.
Te genotypic distribution and allelic frequencies of the Gln223Arg polymorphism are presented (Table 2).Genotypic distribution in all groups was in accordance with the Hardy-Weinberg equilibrium.Allelic and genotypic frequency distributions of G1n223Arg polymorphisms among men with primary infertility, secondary infertility, and controls were compared using Chi-square tests (Table 3).Te results indicate that no signifcant diferences in genotypic or allelic frequency distributions were detected for Gln223Arg polymorphism in the three groups.

Semen Variables in Genotypes of LEPR Gln223Arg.
Semen variables in men with primary infertility, secondary infertility, and controls distributed by genotypes of LEPR Gln223Arg polymorphisms are presented (Table 4).Tere were no signifcant diferences in semen volume and sperm numbers.Sperm motility of AA + AG genotypes in primary infertility was signifcantly lower than that of the GG genotype in the three groups.Te sperm motility of AA + AG genotypes was signifcantly lower than that of the GG genotype in controls.Sperm normal morphology of AA + AG genotypes in primary infertility was signifcantly lower than that of the GG genotype in all three groups.Sperm normal morphology of AA + AG genotypes in secondary infertility and controls was signifcantly lower than that of the GG genotype in controls.

Discussion
Te LEPR gene includes 20 exons and exists as several common variants, including 2 nonconservative changes at codon 223 in exon 6, the glutamine to arginine substitution (CAG to CGG) (Q223R), and at codon 656 in exon 14, the lysine to asparagine substitution (AAG to AAC) (K656N).Tere is a conservative change at codon 109 in exon 4, the lysine to arginine substitution (AAG to AGG) (K109R), and  International Journal of Clinical Practice 3 2 silent changes in exon 1 at codon 1019 (CGT to CAT, Pro1019Pro) and at codon 343 (GTG to GCG, Ser343Ser) [20].Among these, the A to G substitution at codon 223 (Gln223Arg polymorphism) is within the extracellular domain of LEPR and afects all forms of the receptor.Tis substitution could afect LEPR function and change its signaling capacity [21].Te present study examined the relationship between allelic and genotypic frequencies of the Gln223Arg polymorphism with male infertility in a Chinese population and analyzed semen variables to determine whether Gln223Arg polymorphism of LEPR was directly associated with male infertility.In the present study, the LEPR 223Arg allelic frequencies of primary infertility, secondary infertility, and controls were 87.04%, 84.69%, and 83.48%, respectively.Although the LEPR 223Arg allelic frequencies were much higher than fgures reported for Caucasians (44%), Pima Indians (32%), and Brazilians of European descent (40%), these estimates were comparable to others (85%) measured in Asians [22].Tese tests and the regenotyping of 20% of the samples indicate the reliability of the data.Te frequencies of GG, AG, and AA genotypes in Chinese men in these three groups difered from those of other populations, but the lack of diference in allelic and genotypic frequencies related to fertility status was consistent with fndings in Iranian, Slovenian, and Macedonian men [6,18].Meanwhile, the Gln223Arg polymorphism and genotypic or allelic frequency distributions did not difer among individuals with primary infertility, secondary infertility, and controls.
For the semen variables, semen volume and sperm concentration in the diferent genotypes did not vary signifcantly with fertility status.In contrast, sperm motility and sperm normal morphology of AA + AG genotypes in primary infertility were signifcantly lower than those of GG genotypes; male primary infertility is more likely in carriers of the Gln (A) allele.Te percentage of progressively motile sperm in Iranian men carrying the AA and AG genotypes was lower than that of those with the GG genotype of LEPR [18], whereas another study involving Slovenians and Macedonians [6] found no diferences between genotypes with semen variables.Tese varied results undoubtedly refect the studied populations having diferent ethnicities and genetic backgrounds.
As codon 223 in exon 6 of the LEPR gene lies within the frst of two putative LEP-binding regions, an A to G transition mutation (Gln223Arg) could afect the afnity of LEPbinding afnity and impair the signaling capacity of the LEPR [23].Serum LEP-binding afnity was afected by LEPR Gln223Arg polymorphism; it is lower in individuals carrying the Gln (A) allele than in those who were homozygous for the Arg (G) allele [24].Tus, Western blot and fuorescence microscopy indicated that LEPR in spermatozoa was signifcantly related to the integrity of the spermatozoal membranes.Te quantity of LEPR in spermatozoa with deteriorated membranes was signifcantly lower than in those with intact membranes [9].In addition, some studies showed that the (A) allele of LEPR Gln223Arg gene polymorphism might be associated with type 2 diabetes mellitus (T2DM) [25].As the leading cause of T2DM, obesity may induce testicular oxidative stress (OS), while fatty acid oxidation produces large amounts of ROS due to adipose tissue accumulation.Tus, the amounts of ROS may mediate oxidative damage in the sperm membrane and afect sperm DNA integrity [26].In the present study, the BMI of the selected study population was normal, and oxidative damage is less likely to damage the sperm membrane.Te A to G   Genes encode proteins, and proteins are the embodiment of specifc genes.Alternative splicing of the human LEPR mRNA can generate several isoforms of the protein: the full-length form (OB-Rb), soluble form (OB-Re), and short forms (OB-Ra, OB-Rc, OB-Rd, and OB-Rf ) [27][28][29].Te activation of these receptors can convey diferent biological activities of LEP.Te various isoforms are not uniform across all tissues, and their presence and position, as well as function in human sperm, need further studies [30].It is planned to determine which forms of the LEPR protein exist in spermatozoa of the stated Chinese individuals with the Gln223Arg polymorphism and examine their relationship with sperm motility and morphology.

Limitations
Tis study has several potential limitations which should be acknowledged.First, a small sample size of our study is not enough to get the optimal statistical power to detect a correlation with the size of the weak efect; thus, the results of our research should be further verifed in a larger cohort.Second, social and psychological data of patients were not obtained, such as success in the history of marriage or childbirth, lack of sexual knowledge, and disharmony of married life; these issues could lead to a heavy psychological burden that causes infertility.Finally, information on patients' lifestyles, such as work environment, career, and dietary habits, was not available; these factors would also impact male fertility status.

Future Perspectives
Based on this study, we are expected to expand the sample size and verify the corresponding results.Ten, we consider whether LEPR Gln223Arg polymorphism in spermatozoa could be used as the biomarker included in the screening program for male infertility, especially in obese men.However, the mechanism of how the LEPR Gln223Arg mutation in spermatozoa may infuence the integrity of the membranes from spermatozoa should be explored in future studies.

Conclusion
Result reveals lower percentages of motility sperm and those with normal morphology in AA + AG genotypes than in GG genotypes in Chinese men with primary infertility, regardless of fertility status.Our fndings suggest that the LEPR Gln223Arg polymorphism in spermatozoa may be associated with the integrity of the membranes from spermatozoa and play a direct role in sperm motility to cope with the limits of LEPR, also with less unintended results.

Figure 1 :
Figure 1: A representative 3% agarose gel shows RFLP products of LEPR after digestion with MspI: lane 1 is the AA genotype (416 bp); lane 2 is the AG genotype (416, 291, and 125 bp); lane 3 is the GG genotype (291 and 125 bp); lane M is DNA ladder.

Table 1 :
Primers are used for PCR amplifcation.

Table 2 :
Genotype distribution and allelic frequencies of Gln223Arg polymorphism in the LEPR gene.Only proportions are given for the alleles and genotypes.* P value <0.05 for diferences after the Hardy-Weinberg equilibrium test.

Table 3 :
Allelic and genotypic frequency distributions of G1n223Arg polymorphisms among subjects with primary infertility, secondary infertility, and controls.Te number of cases and proportions (in parentheses) is given for the genotypes.Only proportions are given for the alleles.Te P value represents the Chi-square analysis of genotypes (AA + AG and GG) and alleles (A and G) by groups.P < 0.05 for diferences among primary infertility, secondary infertility, and controls.

Table 4 :
Semen variables of primary infertility, secondary infertility, and controls partitioned by genotypes of LEPR Gln223Arg polymorphisms.Signifcant diference between AA + AG and GG among primary infertility, secondary infertility, and controls.Results are presented as median (interquartile range).Gln223Arg) of LEPR in this Chinese population might reduce LEP-binding afnity in individuals carrying the Gln (A) allele and thus lower the percentage of motility sperm.Meanwhile, spermatozoal LEPR in individuals with AA + AG genotypes was signifcantly related to the intactness of the spermatozoal membranes, likely reducing the percentage of sperm with normal morphology.