Research Article | Open Access
Pratibha Kumari1 , S.P. Jaiswar1 , Pushplata Shankhwar1, Sujata Deo1, Kaleem Ahamad2, Waseem Ahamad3 and Abbas Ali Mahadi4
1Department of Obstetrics and Gynecology, King George Medical University, Lucknow – 226 003, India.
2Department of Biochemistry, King George Medical University, Lucknow, India.
3Department of Zoology, Lucknow University, India.
4Department of KGMU, Lucknow, India.
J Pure Appl Microbiol, 2019, 13 (3): 1711-1723 | Article Number: 5709
https://doi.org/10.22207/JPAM.13.3.46 | © The Author(s). 2019
Received: 20/06/2019 | Accepted: 07/08/2019 | Published: 28/09/2019
Abstract

To investigate whether LEP G2548A and LEPR Gln223Arg gene polymorphism associated with the pathogenesis of unexplained infertility in north Indian population. This investigation randomly selected 229 female subjects of age group between 18 to 40 years (120 cases and 109 controls). At first, the members were classified into fertile and infertile. Further, they are separated based on BMI, non-obese (BMI: 18.5 to 24.5) and obese (BMI ≥ 25). The selected gene polymorphisms LEP G2548A and LEPR Gln223Arg were analyzed by polymerase chain response (PCR) followed by restriction fragment length polymorphism (RFLP). The univariate analysis reveals that Leptin, LEP G2548A and LEPR Gln223Arg genotypes, the most significant predictor of unexplained infertility (all p<0.05). The logistic regression analysis found that these three variables significant in multivariate analysis (all p<0.05) suggesting these as significant and independent predictors of unexplained infertility. The allele frequency of both LEP G2548A and LEPR Gln223Arg was found significantly different and higher in unexplained infertile than fertile. Moreover, the phenotype frequency of both LEP G2548A and LEPR Gln223Arg was also found significantly different and higher in unexplained infertile than fertile. G2548A was more frequent than LEPR Gln223Arg. This study suggested that high Leptin level and risk genotype increases the risk of infertility. Further investigations in other geographical region of India are to substantiate our finding.

Keywords

Unexplained Infertility; Leptin; Leptin Receptor; Obesity.

Introduction

The obesity gene that encodes leptin was initially recognized by Freidman’s group in 1994 at Rocke Feller University1. Leptin is a 16kDa peptide hormone, found on human chromosome 7q21.3 which encoded  by leptin gene (LEP).  In the circulation system LEP is discharged by adipose tissue, that ties to leptin receptors that encoded by the leptin receptor gene (LEPR) in the hypothalamus and sign by means of the Jinus kinase begin activator of transcription(JAK-STAT) signal transduction pathway to censored food intake and enhanced energy expenditure2. In human LEPR maps to chromosome1p31 and the protein has a five long and short isoform, which have indistinguishable transmembrane and extracellular domains but have different cytoplasmic doman length3. Leptin is involved in food intake regulation, immune functions energy balance, and fertility4. Leptin can create furthermore by human ovarian follicles-both in cumulus and granulosa cells. It was certified at mRNA and protein levels, human preovulatory follicles express the leptin gene quality5. Besides, it has been recognized that leptin may play a significant role in menstrual cycle. Moreover, ladies with anomalous articulation of the ob gene product are prone to infertility and menstrual irregularities6. Normally infertility is described as year of unprotected coitus along with conception and it is basic issue among reproductive age of men and women7,8. Few less study have been carried out in infertile male and female population to investigate the status of serum leptin. Around 30% of infertile couples after the traditional diagnostic evaluation experience unexplained infertility9. Negative diagnostic test outcomes would be anticipated if female age were the reason for interruption of fecundity, or when a defect be present in spite of the fact that cannot be found at present available tests. Even if diagnostic test outcomes are normal, the prognosis for live birth is just marginally better than that with different causes of infertility9.

Leptin may affect on reproduction came from the insight ob/ob mice (lack functional leptin) or db/db mice (means leptin receptor lack) are fertile, and failed to undergo normal sexual development10. Recombinant leptin organization to these rodents diminished body weight and reestablished fertility  to ob/ob mouse11,12. Similar findings have also been reported in human13,14. In a reproductive medicine clinic up to 25% of patients who present for investigation are diagnosed to have unexplained infertility15. 5 to 37% couples are unexplained infertility of the total proportion 16-19. A significant difference between unexplained fertility and fertile female in serum leptin levels recommends that Leptin might be associated with pathophysiology of unexplained infertility20.

SNPs (Single Nucleotide Polymorphisms) is the most broadly recognized hereditary. SNPs are single base substitution of the one nucleotide with another all through the human genome, in both exon (coding region) and entron region (non coding region) variations among people21. Researchers also found that the genes LEP and LEPR have been linked for polymorphisms that could possibly identify with the pathophysiology of obesity and its difficulties22. For example Mammes et al. (1998) were the first to show that the G2548A variation in the promoter of LEP gene was associated with a reduction in BMI in obese women21.

LEP gene SNP containing substitution of nucleotide G to A at nucleotide – 2548 upstream of the ATG begin site in this gene promoter. The connection among G2548A (rs7799039) and with regards to the LEPR polymorphism, the A to G change in exon 6 at 668 nucleotides from the begin codon 223 LEPR Q223R (rs1137101) was related with impeded leptin-binding activity22, 23, 24-28. The LEPR Q223R polymorphism has been related to reduced BMI, blood pressure, leptin levels, and fat mass29, however, some different examinations are in strife with these findings30, 31. There are a couple of studies dealing with the connection between LEP G2548A and LEPR Gln223Arg polymorphisms, obesity and daily energy intake. Significantly more important, these linked are showing  up in a racial dependent design32-34. Likewise, the relationship of G2548A and Gln223Arg gene polymorphism with unexplained infertility among Indian has not been examined till date. Leptin plays vital role in reproductive biology of humans, especially in ovulation, and spermatogenesis. An alteration in the leptin flow may demonstrate to be a significant connection between body fat stores and status of fertility among childbearing women and men35. Such foundation may give new information into the cause of infertility and might be prompt better treatment modalities in child bearing females.

As to the link between LEP G2548A and LEPR Gln223Arg gene polymorphism with leptin levels, the previous study advice a common gene polymorphism in the promoter LEP G2548A influencing secretion of adipose tissue and expression of Leptin26 might be affecting the unexplained infertility in females. Hence, objective of the present study was to evaluate the link between LEP G2548A and LEPR Gln223Arg gene polymorphism with unexplained infertility. Moreover, we are also looking at whether LEP G2548A and LEPR Gln223Arg polymorphism may be associated with the pathogenesis of unexplained infertility in north Indian population.

Cyperus rotundus L. (family: Cyperaceae) is regarded as a traditionally medicinal herb that has been used for treating numerous clinical ailments such as diarrhea, diabetes, inflammation, malaria, and belly and bowel disorders. Phytochemical and pharmacological research printed that C. rotundus includes an excessive concentration of vital oils, ascorbic acids, phenolic acids, and flavonoids demonstrating its antibacterial, antioxidant, anti-inflammatory, anti-cancerous, and antimalarial4.

Nuclear magnetic resonance (NMR) considered the most advanced technique for the profiling and identifying the metabolites in complex matrices such as plant extracts. The technique is broadly used due to its ease in pattern instruction and the outcomes received are reproducible5. The NMR technique in combination with multivariate statistics evaluation (MVA) has been employed appreciably in recent studies for metabolites profiling for the discrimination of samples of same origin subjected to different processes6. Therefore, this study objective was to determine the antibacterial and antioxidant activities of C. rotundus L Fermented by lactic acid bacteria. Moreover, the metabolites in charge for the antibacterial and antioxidant activities of C. rotundus were identify by using 1H-NMR spectroscopy.

Materials and Methods

For smoothening this research work and earlier ethical approval were obtained from the institutional ethical committee vide letter no-2214/R-Cell-11. Ref. code: 53 ECMIIB/P1 from the King George’s Medical University (K.G.M.U.) of Lucknow, India. The present case-control study was performed at the division of Obstetrics and Gynecology, KGMU, Lucknow. The study includes 229 female participants (120 cases and 109 controls females) which were arbitrarily chosen, aged 18 to 40 years. After writing concern, a 5 ml blood samples were collected. At the time of blood collection, information about high and weight (Waist: WC); hip circumference (HC) was recorded (only females) by trained researchers. All volunteers were asked for some information, for example family history of obesity and hereditary diseases. Patients who are on hormonal treatment and pregnant were excluded from this study. The case group (unexplained infertile female) was compared with control group (normal fertile female) for finding the contribution of serum leptin level in the causation of unexplained fertility. Subjects were categorized first into unexplained  infertile and fertile group and then further subdivided into subcategories on the basis of BMI, non obese (BMI range 18.5 to 24.5) and obese (BMI more than 25). Leptin level was quantified by Active human leptin ELISA kit unit and BMI was calculated as weight in kg/height in m2.

Genetic analysis
Blood DNA was isolated and purified by using the classical phenol chloroform Method.  Samples were collected in EDTA vials. Approx. 40 ng of the DNA was used for each PCR reaction. DNA was quantified by measuring the optical density at 260nm. 1µl of stock genomic DNA was placed on the plate and the OD was taken at 260nm (Thermo Scientific). DNA concentration of the samples ranged between 100ng to 250ng. The DNA was further diluted to 40ng for using in the PCR reaction. The purity was determined by calculation of absorbance at 260nm to absorbance at 280 nm. DNA samples were aliquoted and
one aliquot which was in regular use was kept at 4°C while the rest of the samples were stored at -20°C.

Amplification of LEP G2548A and LEPR Gln223Arg gene
The genetic polymorphism was analyzed by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) method. Isolated genomic DNA was amplified by PCR thermo cycler (Applied Biosystem, Germany) using PCR condition 94 °C for 4 minutes, 35 cycles at 94 °C for 30 second, 58 °C for 40 second, 72 °C for 40 second and lastly 72 °C for 8 minutes. The forward and reversed primers for LEP gene were 5’-TTTCCTGTAATTTTCCCGTGAG-3’ and 5’-AAAAGCAAAGACAGGCATAAA-3’repectively. For LEPR gene 5’-AGT TCA AAT AGA GGT CCA AAT CA-3’ and 5’ –TTC TGA GGT TGT GTC ACT GGC A-3’respectively. Reaction mixture 100 ng of DNA, 4nM of each primer, 2.5 mM of dNTPs, 2.5 mM of MgCl2, 0.025 U Taq polymerase, and 1xPCR buffer (Invirtogen). Initial melting step of 2 minute at 94 °C, followed by 35 cycle of 30 second at 94 °C, 45 second at 60 °C (for LEP gene), 45 second at 56 °C (for LEPR gene), and 45 second at 72 °C, and final elongation was 6 minute at 72 °C. After PCR, amplified product (242bp for LEP and 367bp for LEPR ) was digested with restriction enzyme Cfo I for LEP gene and MspI for LEPR gene for 2 hours at 37 °C. The digested PCR products (181bp, 61bp and 242bp, 125 bp, respectively) were separated on 3 % Agarose gel.

Statistical analysis
Discrete (categorical) data were summarized in numbers and percentage.  Categorical data were compared with the help of Chi square test. The proportions were compared with Z test after corrections for continuity (ZC). Univariate and multivariate logistic regression analysis was done to predict independent predictors of unexplained infertility, considering group as variable (dependent) and clinical characteristics like Age, WHR, BMI and Leptin, and LEP G2548A and LEPR Gln223Arg genotypes the independent variables. The clinical characteristics were sub grouped into low and high on the basis of median value of both fertile and unexplained groups except BMI. For BMI the standard cutoff of normal (BMI: <25 kg/m2) and overweight (BMI: ³25 kg/m2) was used.  The control (fertile) genotypes data of both SNPs LEP G2548A and LEPR Gln223Arg are in HWE (Hardy-Weinberg equilibrium) tested by goodness of fit chi squire. The two tailed p<0.05 was set for the entire experiment. All the statistical analysis was done by Statistical package for social sciences (SPSS, version 16, SPSS inc, Chicago, IL, U.S.A)36.

RESULTS

Table 1 summarized the clinical characteristics of case (unexplained infertile) and control (fertile) group. Evaluate the frequency (%) distribution of discrete (low and high) variables of two groups, χ2 test showed Leptin frequency in higher subgroup compared to lower of the case than the fertile group. On the other hand, the distribution of age, BMI & WHR not differed among case and control groups. The association of LEP G2548A genotypes with basic clinical characteristics of selected groups is shown in Table 1. Infertile groups, LEP G2548A genotypes did not show a significant association with any of the clinical characteristics.

Table (1):
Relationship of LEP G2548A gene polymorphism with clinical parameters of fertile and unexplained infertile group

Variables Fertile (n=109) Unexplained infertile (n=120) Fertile vs. Infertile

G/G

(n=39) (%)

G/A

(n=51) (%)

A/A

(n=19) (%)

p

value

G/G

(n=22) (%)

G/A

(n=59) (%)

A/A

(n=39) (%)

p

value

p

value

Age (yrs):

≤ 30

> 30

 

26 (66.7)

13 (33.3)

 

39 (76.5)

12 (23.5)

 

12 (63.2)

7

(36.8)

 

0.439

 

13 (59.1)

9

(40.9)

 

44 (74.6)

15 (25.4)

 

26 (66.7)

13 (33.3)

 

0.373

 

0.008*

0.285

BMI (kg/m2):

< 25

≥ 25

 

27 (69.2)

12 (30.8)

 

32 (62.7)

19 (37.3)

 

11 (57.9)

8

(42.1)

 

0.669

 

20 (90.9)

2

(9.1)

 

39 (66.1)

20 (33.9)

 

18 (46.2)

21 (53.8)

 

0.002*

 

0.213

0.002*

WHR:

< 1

≥ 1

 

29 (74.4)

10 (25.6)

 

38 (74.5)

13 (25.5)

 

13 (68.4)

6

(31.6)

 

0.864

 

15 (68.2)

7

(31.8)

 

49 (83.1)

10 (16.9)

 

27 (69.2)

12 (30.8)

 

0.191

 

0.007*

0.232

TSH (mIU/ml):

< 3

≥ 3

 

13 (33.3)

26 (66.7)

 

14 (27.5)

37 (72.5)

 

5

(26.3)

14 (73.7)

 

0.790

 

15 (68.2)

7

(31.8)

 

34 (57.6)

25 (42.4)

 

21 (53.8)

18 (46.2)

 

0.545

 

0.089

0.015

LH (mIU/ml):

< 7

≥ 7

 

17 (43.6)

22 (56.4)

 

19 (37.3)

32 (62.7)

 

6

(31.6)

13 (68.4)

 

0.656

 

19 (86.4)

3

(13.6)

 

41 (69.5)

18 (30.5)

 

27 (69.2)

12 (30.8)

 

0.273

 

0.036

0.021

FSH (mIU/ml):

< 8

≥ 8

 

22 (56.4)

17 (43.6)

 

32 (62.7)

19 (37.3)

 

12 (63.2)

7

(36.8)

 

0.804

 

10 (45.5)

12 (54.5)

 

19 (32.2)

40 (67.8)

 

15 (38.5)

24 (61.5)

 

0.524

 

0.142

0.010

PRL (mg/mL):

<15

≥ 15

 

25 (64.1)

14 (35.9)

 

19 (37.3)

32 (62.7)

 

10 (52.6)

9

(47.4)

 

0.040

 

11 (50.0)

11 (50.0)

 

34 (57.6)

25 (42.4)

 

21 (53.8)

18 (46.2)

 

0.816

 

0.002

0.122

Leptin (pg/ml):

< 5

≥ 5

 

27 (69.2)

12 (30.8)

 

30 (58.8)

21 (41.2)

 

14 (73.7)

5

(26.3)

 

0.408

 

11 (50.0)

11 (50.0)

 

24 (40.7)

35 (59.3)

 

5 (12.8)

34 (87.2)

 

0.003*

 

0.196

0.003*

1st and 2nd p value is the 2×3 chi squire comparisons between

However, in the unexplained infertile group it showed significant association with BMI (p=0.002) as well as Leptin level (p=0.003). The higher concentration of TSH and lower PRL is significantly false positive associated with in unexplained infertile in this study.

Further, comparing the genotypes among the case and control group, the genotype showed significant association with lower age (p=0.008), WHR (p=0.007), while significant association with higher BMI (p=0.002), and Leptin (p=0.003). In contrast, LEPR Gln223Arg did not show a significant association with any of the clinical characteristics in both the groups (Table 2). However, between the two groups, it showed significant association with lower age (p=0.020), WHR (p=0.048) and while a significant link with higher BMI (p=0.020), and Leptin (p=0.002).

Table (2):
Relationship of LEPR Gln223Arg gene polymorphism with clinical parameters of fertile and unexplained infertile groups.

Variables Fertile (n=109) Unexplained infertile (n=120) Fertile vs. Infertile

Gln/Gln

(n=67) (%)

Gln/Arg

(n=341) (%)

Arg/Arg

(n=8) (%)

p

value

Gln/Gln

(n=52)(%)

Gln/Arg

(n=46) (%)

Arg/Arg

(n=22) (%)

p

value

p

value

Age (yrs):

≤ 30

> 30

 

50 (74.6)

17 (25.4)

 

22 (64.7)

12 (35.3)

 

5 (62.5)

3 (37.5)

 

0.510

 

37 (71.2)

15 (28.8)

 

32 (69.6)

14 (30.4)

 

14 (63.6)

8 (36.4)

 

0.813

 

0.020*

0.333

BMI (kg/m2):

< 25

≥ 25

 

46 (68.7)

21 (31.3)

 

17 (50.0)

17 (50.0)

 

7 (87.5)

1 (12.5)

 

0.065

 

36 (69.2)

16 (30.8)

 

29 (63.0)

17 (37.0)

 

12 (54.5)

10 (45.5)

 

0.475

 

0.069

0.020*

WHR:

< 1

≥ 1

 

49 (73.1)

18 (26.9)

 

25 (73.5)

9 (26.5)

 

6 (75.0)

2 (25.0)

 

0.993

 

40 (76.9)

12 (23.1)

 

36 (78.3)

10 (21.7)

 

15 (68.2)

7 (31.8)

 

0.643

 

0.048

0.133

TSH (mIU/ml):

< 3

≥ 3

 

20 (29.9)

47 (70.1)

 

9 (26.5)

25 (73.5)

 

3 (37.5)

5 (62.5)

 

0.819

 

31 (59.6)

21 (40.4)

 

27 (58.7)

19 (41.3)

 

12 (54.5)

10 (45.5)

 

0.920

 

0.219

0.030

LH (mIU/ml):

< 7

≥ 7

 

24 (35.8)

43 (64.2)

 

16 (47.1)

18 (52.9)

 

2 (25.0)

6 (75.0)

 

0.393

 

38 (73.1)

14 (26.9)

 

33 (71.7)

13 (28.3)

 

16 (72.2)

6 (27.3)

 

0.989

 

0.089

0.104

FSH (mIU/ml):

< 8

≥ 8

 

39 (58.2)

28 (41.8)

 

22 (64.7)

12 (35.3)

 

5 (62.5)

3 (37.5)

 

0.814

 

17 (32.7)

35 (67.3)

 

20 (43.5)

26 (56.5)

 

7 (31.8)

15 (68.2)

 

0.473

 

0.088

0.075

PRL (mg/mL):

<15

≥ 15

 

33 (49.3)

34 (50.7)

 

19 (55.9)

15 (44.1)

 

2 (25.0)

6 (75.0)

 

0.290

 

26 (50.0)

26 (50.0)

 

29 (63.0)

17 (37.0)

 

11 (50.0)

11 (50.0)

 

0.377

 

0.018

0.265

Leptin (pg/ml):

< 5

≥ 5

 

40 (59.7)

27 (40.3)

 

24 (70.6)

10 (29.4)

 

7 (87.5)

1 (12.5)

 

0.215

 

21 (40.4)

31 (59.6)

 

12 (26.1)

34 (73.9)

 

7 (31.8)

15 (68.2)

 

0.321

 

0.505

0.002*

1st and 2nd p value is the 2×3 chi squire comparisons between lower to higher subgroup within fertile and infertile group separately. Third p value is the 2×3 comparison between lower to lower and higher to higher subgroup between fertile and infertile group of respective parameters.  : *Asterisk indicates p< 0.05

Table 3 summarized the genotype frequency distribution of the LEP G2548A gene (Wild: G/G, Heterozygous: G/A and Homozygous mutant: A/A) and gene LEPR Gln223 Arg (Wild: Gln/Gln, Heterozygous: Gln/Arg  and Homozygous mutant: Arg/Arg). The χ2 test showed significantly different and higher homozygous mutant frequency of both LEP G2548A (A/A: 17.4% vs. 32.5%, p=0.003) and LEPR Gln223Arg (Arg/Arg: 7.3% vs. 18.3%, p=0.008) in unexplained infertile than the fertile group. Further, the allele frequency of both LEP G2548A (A: 40.8% vs. 57.1%, p<0.001) and LEPR Gln223Arg (Arg: 22.9% vs. 37.5%, p<0.001) was also found significantly different and higher in unexplained infertile than fertile. Moreover, the phenotype frequency of both LEP G2548A (A+: 64.2% vs. 81.7%, p=0.001) and LEPR Gln223Arg (Arg+: 38.5% vs. 56.7%, p=0.016) was also found significantly different and higher in unexplained infertile than fertile. Further , using dominant model, genotype was also observed tobe associated with unexplained infertility as compaired to their wild type genotype for both polymorphism (table 4,and 5). Unfortunately, some calculation such as BMI>25;LH<7 in LEPG2548A gene polymorphism in dominant model and age >30;BMI>25;WHR>1;TSH<3;LH< and >7; FSH, PRL and Leptin in recessive model was restricted by small number of participants (table 4 and 5)

Table (3):
Frequency distribution of LEP G-2548A and LEPR Gln223Arg polymorphisms between two groups (Wild: G/G or Gln/Gln, Heterozygous: G/A or Gln/Arg, Homogygous mutant: A/A or Arg/Arg).

SNPs
Characteristics
Fertile (n=109) (%)
Unexplained infertile(n=120) (%)
PValue
LEP G-2548A

Genotypes:

G/G

G/A

A/A

Allele:

G

A

Phenotype:

A+

 

39 (35.8)

51 (46.8)

19 (17.4)

 

129 (59.2)

89 (40.8)

 

70 (64.2)

 

22 (18.3)

59 (49.2)

39 (32.5)

 

103 (42.9)

137 (57.1)

 

98 (81.7)

 

0.003

 

 

 

<0.001

 

 

0.001

LEPR Gln223Arg

Genotypes:

Gln/Gln

Gln/Arg

Arg/Arg

Allele:

Gln

Arg

Phenotype:

Arg+

 

67 (61.5)

34 (31.2)

8 (7.3)

 

168 (77.1)

50 (22.9)

 

42 (38.5)

 

52 (43.3)

46 (38.3)

22 (18.3)

 

150 (62.5)

90 (37.5)

 

68 (56.7)

 

0.008

 

 

 

<0.001

 

 

0.016

*Asterisk indicates p< 0.05; A+: sum of G/A and A/A; Arg+= sum of Gln/Arg and Arg/Arg.

Table (4):
Relationship of LEP G2548A gene polymorphism with clinical parameters of fertile and unexplained infertile group using dominant and recessive model

Variables Fertile (n=109) Unexplained infertile (n=120) Fertile vs. Infertile
GG GA AA p value GG GA AA

p

value

P- value
Age (yrs): Dom GG vs GA+AA Dom GG vs GA+AA
≤ 30 26 (66.7) 51 (139.7)

 

0.49

13 (59.1) 70 (141.3)

 

0.27

0.007*
> 30 13 (33.3) 19 (60.3) 9 (40.9) 28 (58.6) 0.14
Rec GG+ GA vs AA Rec GG+ GA vs AA
≤ 30 65 (143.2) 12 (63.2)

 

0.43

57 (133.7) 26 (66.7)

 

0.68

0.01*
> 30 25 (56.8) 7 (36.8) 24 (66.3) 13 (33.3) 0.22
BMI (kg/m2): Dom GG vs GA+AA Dom GG vs GA+AA
< 25 27 (69.2) 43 (120.6)

 

0.42

20 (90.9) 57 (112.3)

 

0.008

0.1
≥ 25 12 (30.8) 27 (79.4) 2 (9.1) 41 (87.7) NA
Rec GG+ GA vs AA Rec GG+ GA vs AA
< 25 59 (131.9) 11 (57.9)

 

0.52

59 (157) 18 (46.2)

 

0.004

0.24
≥ 25 31 (68.1) 8 (42.1) 22 (43) 21 (53.8) 0.007*
WHR: Dom GG vs GA+AA Dom GG vs GA+AA
< 1 29 (74.4) 51 (142.9

 

0.86

15 (68.2) 76 (152.3)

 

0.35

0.003*
≥ 1 10 (25.6) 19 (57.1) 7 (31.8) 22 (47.7) 0.38
Rec GG+ GA vs AA Rec GG+ GA vs AA
< 1 67 (148.9) 13 (68.4)

 

0.59

64 (151.3) 27 (69.2)

 

0.24

0.05
≥ 1 23 (51.1) 6 (31.6) 17 (48.7) 12 (30.8 0.08
TSH (mIU/ml): Dom GG vs GA+AA Dom GG vs GA+AA
< 3 13 (33.3) 19 (53.8)

 

0.49

15 (68.2) 55 (111.4)

 

0.29

0.05
≥ 3 26 (66.7) 51 (146.2) 7 (31.8) 43 (88.6) 0.01*
Rec GG+ GA vs AA Rec GG+ GA vs AA
< 3 27 (60.8) 5 (26.3)

 

0.75

49 (125.8) 21 (53.8)

 

0.48

0.122
≥ 3 63 (139.2) 14 (73.7) 32 (74.2) 18 (46.2) 0.23
LH (mIU/ml): Dom GG vs GA+AA Dom GG vs GA+AA
< 7 17 (43.6) 25 (68.9)

 

0.42

19 (86.4) 68 (138.7)

 

0.17

0.02*
≥ 7 22 (56.4) 45 (131.1) 3 (13.6) 30 (61.3) NA
Rec GG+ GA vs AA Rec GG+ GA vs AA
< 7 36 (80.9) 6 (31.6)

 

0.49

60 (155.9) 27 (69.2)

 

0.57

0.05
≥ 7 54 (119.1) 13 (68.4) 21 (44.1) 12 (30.8) 0.06
FSH (mIU/ml): Dom GG vs GA+AA Dom GG vs GA+AA
< 8 22 (56.4) 44 (125.9)

 

0.5

10 (45.5) 34 (70.7)

 

0.34

0.23
≥ 8 17 (43.6) 26 (74.1) 12 (54.5) 64 (129.3) 0.003*
Rec GG+ GA vs AA Rec GG+ GA vs AA
< 8 54 (101.4) 12 (63.2)

 

0.79

29 (77.7) 15 (38.5)

 

0.77

0.06
≥ 8 36 (80.9) 7 (36.8) 52 (122.3) 24 (61.5) 0.07
PRL (mg/mL): Dom GG vs GA+AA

 

0.02

Dom GG vs GA+AA

 

0.6

<15 25 (64.1) 29 (89.9) 11 (50.0) 55 (111.4) 0.004*
≥ 15 14 (35.9) 41 (110.1) 11 (50.0) 43 (88.6) 0.52
Rec GG+ GA vs AA Rec GG+ GA vs AA
<15 44 (101.4) 10 (52.6)

 

0.76

45 (107.6) 21 (53.8)

 

0.86

0.09
≥ 15 46 (98.6) 9 (47.4) 36 (92.4) 18 (46.2) 0.05
Leptin (pg/ml): Dom GG vs GA+AA Dom GG vs GA+AA
< 5 27 (69.2) 44 (100)

 

0.002

11 (50) 29 (53.5)

 

0.06

0.26
≥ 5 12 (30.8) 26 (67.5) 11 (50) 69 (146.5) 0.02*
Rec GG+ GA vs AA Rec GG+ GA vs AA
< 5 57 (128) 14 (73.7) 0.12 35 (90.7) 5 (12.8) 0.001 0.33
≥ 5 53 (72) 5 (26.3) 46 (109.3) 34 (87.2) 0.001*

1st and 2nd p value is the 2x2chi squire comparisons between lower to higher subgroup within fertile and infertile group separately. Third p value is the 2x2comparison between lower to lower and higher to higher subgroup between fertile and infertile group of respective parameters.  : Asterisk indicates p< 0.05; Dom: Dominant; Rec: Recessive; (*p<0.05); NA: not applicable

Table (5):
Relationship of LEPR Gln223Arg gene polymorphism with clinical parameters of fertile and unexplained infertile groups using dominant and recessive model

Variables Fertile (n=109) Unexplained infertile (n=120) Fertile vs. Infertile
Gln/Gln Gln/Arg Arg/Arg p value Gln/Gln Gln/Arg Arg/Arg

p

value

P- value
Age (yrs): Dom Gln/Gln vs Gln/Arg+Arg/Arg Dom GG vs GA+AA
≤ 30 50(74.6) 27(127.2)

 

0.24

37(71.2) 46(133.2

 

0.68

0.009*
> 30 17 (25.4) 15(72.8) 15(28.8) 22(66.8) 0.29
Rec Gln/Gln +Gln/Arg Vs Arg/Arg Rec Gln/Gln +Gln/Arg Vs Arg/Arg
≤ 30 72(139.3) 5(62.5)

 

NA

69(140.8) 14(63.6)

 

0.55

0.04
> 30 29(60.7) 3(37.5) 29(59.2) 8(36.4) NA
BMI (kg/m2): Dom Gln/Gln vs Gln/Arg+Arg/Arg Dom Gln/Gln vs Gln/Arg+Arg/Arg
< 25 46(68.7) 24(137.5)

 

0.22

36(69.2) 46(68.7)

 

1.0

0.007*
≥ 25 21(31.3) 18(62.5) 16(30.8) 21(31.3) 0.35
Rec Gln/Gln +Gln/Arg Vs Arg/Arg Rec Gln/Gln +Gln/Arg Vs Arg/Arg
< 25 63(118.7) 7(87.5) NA 65(132.2) 12(54.5) 0.29 0.31
≥ 25 38(81.3) 1(12.5) 33(67.8) 10(45.5) NA
WHR: Dom Gln/Gln vs Gln/Arg+Arg/Arg Dom Gln/Gln vs Gln/Arg+Arg/Arg
< 1 49(73.1) 31(148.5)

 

0.92

40(76.9) 51(146.5)

 

0.81

0.02*
≥ 1 18(26.9) 11(51.5) 12(23.1) 17(53.5) 0.11
Rec Gln/Gln +Gln/Arg Vs Arg/Arg Rec Gln/Gln +Gln/Arg Vs Arg/Arg
< 1 74(146.6) 6(75)

 

NA

76(155.2) 15(68.2)

 

0.35

0.07
≥ 1 27(53.4) 2(25) 22(44.8) 7(31.8) NA
TSH (mIU/ml): Dom Gln/Gln vs Gln/Arg+Arg/Arg Dom Gln/Gln vs Gln/Arg+Arg/Arg
< 3 20(29.9) 12(64)

 

 

0.88

31(59.6)

 

39(113.2)

 

 

0.80

0.08
≥ 3 47(70.1) 30(136) 21(40.4) 29(86.8) 0.03
Rec Gln/Gln +Gln/Arg Vs Arg/Arg Rec Gln/Gln +Gln/Arg Vs Arg/Arg
<3 29(56.4) 3(37.5)

 

NA

58(118.3) 12(54.5)

 

0.68

NA
≥ 3 72(143.6) 5(62.5)   40(81.7) 10(45.5) 0.02
LH (mIU/ml): Dom Gln/Gln vs Gln/Arg+Arg/Arg Dom Gln/Gln vs Gln/Arg+Arg/Arg
< 7 24(35.8) 18(72.1)

 

0.46

38(73.1) 49(143.9)

 

0.88

0.15
≥ 7 43(64.2) 24(127.9) 14(26.9) 19(55.6) 0.03
Rec Gln/Gln +Gln/Arg Vs Arg/Arg Rec Gln/Gln +Gln/Arg Vs Arg/Arg
< 7 40(82.9) 2(25)

 

NA

71(144.8) 16(72.2)

 

1.0

NA
≥ 7 61(117.1) 6(75 27(55.2) 6(27.3) NA
FSH (mIU/ml): Dom Gln/Gln vs Gln/Arg+Arg/Arg Dom Gln/Gln vs Gln/Arg+Arg/Arg
< 8 39(58.2) 27(127.2)

 

0.53

17(32,7) 27(75.3)

 

0.43

0.035
≥ 8 28(41.8) 15(72.8) 35(67.3) 41(124.7) 0.04
Rec Gln/Gln +Gln/Arg Vs Arg/Arg Rec Gln/Gln +Gln/Arg Vs Arg/Arg
< 8 61 (122.9 5 (62.5)

 

NA

37 (76.2) 7 (31.8)

 

0.60

NA
≥ 8 40 (77.1) 3 (35.3) 61 (123.6) 15 (68.2) NA
PRL (mg/mL): Dom Gln/Gln vs Gln/Arg+Arg/Arg Dom Gln/Gln vs Gln/Arg+Arg/Arg
<15 33 (49.3) 21 (80.9)

 

0.92

26 (50) 40 (113)

 

0.33

0.01
≥ 15 34 (50.7) 21 (119.1) 26 (50) 28 (87) 0.17
Rec Gln/Gln +Gln/Arg Vs Arg/Arg Rec Gln/Gln +Gln/Arg Vs Arg/Arg
<15 52 (105) 2 (25)

 

NA

55 (113) 11 (50)

 

0.60

NA
≥ 15 49 (94.8) 6 (75) 43 (87) 11 (50) NA
Leptin (pg/ml): Dom Gln/Gln vs Gln/Arg+Arg/Arg Dom Gln/Gln vs Gln/Arg+Arg/Arg
< 5 40 (59.7) 31 (158.1)

 

0.13

21 (26.1) 21  (57.9)

 

0.23

0.5
≥ 5 27 (40.3) 11 (41.9) 31 (73.9) 49 (142.1) 0.01*
Rec Gln/Gln +Gln/Arg Vs Arg/Arg Rec Gln/Gln +Gln/Arg Vs Arg/Arg
< 5 64 (130) 7 (87.5)

 

NA

33 (66.5) 7 (31.8)

 

0.86

NA
≥ 5 37 (69.7) 1 (12.5) 65 (133.5) 15 (68.2) NA

1st and 2nd p value is the 2x2chi squire comparisons between lower to higher subgroup within fertile and infertile group separately. Third p value is the 2x2comparison between lower to lower and higher to higher subgroup between fertile and infertile group of respective parameters.  : Asterisk indicates p< 0.05; Dom: Dominant; Rec: Recessive; (*p<0.05); NA: not applicable

The univariate and multivariate regression analysis was done in evaluating out independent predictors of unexplained infertility the clinical characteristics and genotypes of both the SNPs were regressed against fertile and unexplained infertile groups which are summarized in Table 6. The univariate analysis found that the level of leptin, gene LEP G2548A as well as LEPR Gln223Arg genotypes the significant (p<0.05 or p<0.01 or p<0.001) predictors of unexplained infertility. This univariate statistical analysis further found these variables significant (p<0.05 or p< 0.01 or p<0.001) in the further multivariate analysis suggesting these as significant and independent predictors of unexplained infertility.

Table (6):
Identification of predictors of unexplained infertility among clinical characteristics and gene polymorphisms using logistic regression analysis

Predictors Univariate Analysis Multivariate analysis
Odds Ratio (OR), 95% CI P value Odds Ratio (OR), 95% CI P value

Age (yrs):

≤ 30

> 30

 

Ref

1.07 (0.61-1.89)

 

 

0.808

 

Ref

0.93 (0.43-2.00)

 

 

0.848

BMI (kg/m2):

< 25

≥ 25

 

Ref

1.00 (0.58-1.72)

 

 

0.993

 

Ref

0.43 (0.19-0.96)

 

 

0.038*

WHR:

< 1

≥ 1

 

Ref

0.88 (0.48-1.60)

 

 

0.672

 

Ref

1.06 (0.48-2.34)

 

 

0.885

Leptin (pg/ml):

< 5

≥ 5

 

Ref

3.74 (2.16-6.46)

 

 

<0.001

 

Ref

5.24 (2.47-11.11)

 

 

<0.001*

LEP G-2548A:

G/G

G/A

A/A

 

Ref

2.05 (1.08-3.90)

3.64 (1.71-7.76)

 

 

0.029

0.001

 

Ref

4.71 (1.86-11.97)

6.00 (2.08-17.31)

 

 

0.001*

0.001*

LEPR Gln 223Arg

Cln/Gln

Gln/Arg

Arg/Arg

 

Ref

1.74 (0.98-3.09)

3.54 (1.46-8.60)

 

 

0.057

0.005

 

Ref

2.07 (0.88-4.89)

7.21 (2.26-23.00)

 

 

0.097

0.001*

CI: confidence interval ; *Asterisk indicates p< 0.05

 

DISCUSSION

Apparently, this is the first Indian study to look at the association of polymorphism of LEP G2548A and LEPR Gln223Arg with unexplained infertility among North Indian population. The key findings of this work were that leptin levels were higher in UI (unexplained infertility) group as compared to control, and that the LEP G2548A and LEPR Gln223Arg polymorphism demonstrated a critical relationship between leptin focus and UI among North Indians.

In the present investigation, no difference was found with weight, height and BMI, screened crosswise genotypes in the obese compared with no obese group.  Likewise, when the subject was divided into female and male, the difference was found to be null in BMI and others parameters. This result is consistent with two studies, which identified no relationship between Gln223Arg, BMI and leptin levels29,37.

In the present study, the χ2 test showed significantly different and higher homozygous mutant frequency, the allele frequency and phenotype frequency of both LEP G2548A and LEPR Gln223Arg in unexplained infertile than the fertile group. Infertile groups, LEP G2548A genotypes did not show any kind of association with clinical parameters. Moreover, in the unexplained infertile group it showed significant association with BMI and Leptin. In addition, in population, no association was found between the LEP-2548G/A polymorphism and obese and their related variables. On the other hand, higher level of leptin was acknowledged in-2548GG carriers38. In other research, workers found that there was no association between LEP-2548G/A polymorphism and obesity in Tunisian obese and their control group26. But, same author like Le et al. (2000) stated that there was a relationship of this polymorphism with extremely obese subjects in North American, Caucasian ladies, in whom the G allele was more frequent25. 

Sahin Daniz Say et al. (2013) reveals that gene LEP 2548 AA or AG genotype are key predictor for increased leptin concentration and BMI in obese subjects and reveals that it might be a useful marker for risk of obesity 32. However in this present study show, in unexplained infertile group it showed a link with BMI (p=0.002) and Leptin (p = 0.003) and genotype GA & AA (p-value =0.001 both) is more significant.

Present study shows a significant association of Gly223Arg with higher BMI (p=0.020), and Leptin (p=0.002) where AA (Arg/Arg) is more significant than GA (Gln/Arg)(p value 0.001 vs 0.097). This study is supported by a Boumaiza Imen et al which showed Q223R (Gln223Arg) polymorphism is associated with Weight and obesity risk 33. Boumaiza et al (2012) also showed that G2548A has significant association with obesity 33. Zayani et al 2018  were also found a significant association between  Q223R and G2548A SNPs and obesity39.

Suryaprom et al 2014 likewise demonstrated that there was a relationship between LEPR Gln223Arg polymorphism and Leptin Level and metabolic disorder34,35. Farooq et al similarly demonstrated that leptin assumes a basic role in particularly, ovulation and spermatogenesis. A fluctuation in circulating leptin also presumed that obesity is related with infertility in men as well as women. In addition, Sex hormonal irregularity may likewise be related BMI and serum leptin in infertility35. From our present study it might conceivable that there is role of Serum Leptin, G2548A and Gln223Arg polymorphism in UI.

CONCLUSION

This present study showed an association between G2548A and Gln223Arg polymorphism and serum leptin with UI. Statistical analysis reveals that these parameters are significant and independent predicators of UI. The allele frequency of both LEP G2548A and LEPR Gln223Arg was also found significantly different and higher in unexplained infertile than fertile. Moreover, the phenotype frequency of both LEP G2548A and LEPR Gln223Arg was also found significantly different and higher in unexplained infertile than fertile. G2548A was more frequent than LEPR Gln223Arg. Recommended to conduct, more studies with large sample size may be helpful for the knowing the association and impact of these Gene in unexplained female infertility in Indian population.

Declarations

Acknowledgements
None.

Conflicts of Interest
The authors declares that there is no conflict of interest.

Authors’ contribution
PK did design study, review and substantially participated in all other work. SPJ guided Infertility in Assays and Data compilation. PS and SD supervised in sample collection of review and sample. AAM and KA guided in all the Molecular and Biochemical studies. WA helped in statistical analysis.

Funding
DST-INSPIRE Fellowship (DST/2012/30 dated August 17, 2012).

Data Availability
The datasets are available from the corresponding author on reasonable request.

Ethical Statement
A prior approval was obtained from the King George’s Medical University (K.G.M.U.) of Lucknow, India ethics committee vide letter no-2214/R-Cell-11. Ref. code: 53 ECMIIB/P1 to conduct this research.
Participants: human Participant
Consent: Informed consent were taken from all the patients.

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