rs4889 polymorphism in KISS1 gene, its effect on polycystic ovary syndrome development and anthropometric and hormonal parameters in Saudi women

rs4889 polymorphism in KISS1 gene, its effect on polycystic ovary syndrome development and... Background: Kisspeptin is involved in female reproduction. This study was designed to i- estimate kisspeptin levels in women with polycystic ovary syndrome (PCOS), in comparison with controls, ii- study the correlations between kisspeptin and PCOS-related reproductive hormones, and iii- investigate the relation between KISS1 gene polymorphisms and hormone levels in women suffering from PCOS. Methods: The investigation was a clinically designed study on 28 women with PCOS, and 30 normal, healthy women with no signs of PCOS as controls. Blood samples were collected between day 3 and day 6 of the menstrual cycle in both groups at 8:00 a.m., and circulating levels of LH, FSH and kisspeptin were estimated. DNA was extracted from whole blood and all coding exons of KISS1 gene were sequenced. Results: Women with PCOS had higher LH levels and BMI compared to controls. Plasma kisspeptin levels were positively correlated with LH levels. There was no statistically significant difference between the groups in terms of kisspeptin and FSH levels. The SNP rs4889 C/G, a non-synonymous SNP, was investigated in the PCOS group. The frequency of GG genotype was significantly higher in the PCOS compared to the controls. These patients were more obese, had higher kisspeptin and FSH levels. Conclusion: The results of the study show that the genetic variation of KISS1 gene may be a factor contributing to PCOS development. The association between the gene and the gene variation and PCOS need further validation in large-scaled and functional studies. Keywords: KISS1 gene, Kisspeptin, LH, FSH, GPR54, PCOS Background in the stimulation of gonadotropin release, which in turn Kisspeptin is a 54-amino-acid peptide encoded by the binds to the GnRH receptors in the pituitary and influences KISS1 gene, and is also known as metastin [1, 2], which the release of LH and FHS [7]. These hormones act on the was first isolated from the human placenta in 2001 [3]. gonads and influence estrogen, testosterone and pro- Kisspeptinactionisexerted by a trans-membrane G-protein gesterone release. Hence, kisspeptin has been shown coupled receptor, named GPR54, AXOR12, or HoT7T175 to regulate the secretion of luteinizing hormone (LH) [4]. Loss of KISS1 gene function is reported to be associated during the promotion of ovulation [8]bystimulating with hypogonadotropic hypogonadism in humans and gonadotropin releasing hormone (GnRH) from the animal models [5, 6]. Binding of kisspeptin to its receptor hypothalamus [9, 10]. (GPR54) in the GnRH neurons in the hypothalamus, results The polycystic ovary syndrome (PCOS) is a common het- erogeneous disorder affecting women, with a prevalence of 6–12% in women of reproductive age [11]. It is of complex * Correspondence: mdaghestani@ksu.edu.sa pathogenesis, characterized by hypothalamic-pituitary dis- Department of Zoology, Center for Scientific and Medical Female Colleges, King Saud University, P.O. Box 22455, Riyadh 11495, Saudi Arabia turbances in gonadotropin secretion, specifically increased Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Albalawi et al. Journal of Biomedical Science (2018) 25:50 Page 2 of 6 LH levels [12, 13], chronic anovulation, and polycystic In addition, 5 mL of blood was collected in EDTA-coated ovaries (PCO) on ultrasound. In addition, patients fre- tubes for DNA extraction. All blood samples for each quently suffer from hyperinsulinemia, insulin resistance subject were immediately centrifuged, and plasma, serum [14], type 2 diabetes mellitus (DM), cardio-vascular and buffy coat were stored at − 80 °C until analysis. disease and infertility [15, 16]. Given the complex rela- Kisspeptin levels were measured with an enzyme-linked tionship between kisspeptin and hypothalamic-pituitary immunoassay kit (ELISA kit, Phoenix Pharmaceuticals Inc., gonadal axis, the present study aimed to investigate the Belmond, CA), after extraction with Phoenix Peptide relation between KISS1, BMI, FSH and LH and the in- sep-columns (RK-Sepcol-2). FSH and LH levels were fluence of KISS1 gene polymorphisms on these parame- measured with an enzyme-linked immunoassay kit (ELISA ters in normal women and those suffering from PCOS. kit, Human, cat.No.65205.GER). Genotyping of KISS1 gene polymorphism Methods Genomic DNA was extracted from peripheral blood Subjects leucocytes using commercially available Puregene Blood The samples were collected from Al-Hira Hospital in Kit (QIAGEN, cat. No. 158389, USA). Polymorphism Makkah Al Mukarama, from 58 Saudi women volunteers was determined by sequencing following polymerase aged 19–36 after obtaining Ethical Committee approval, chain reaction (PCR), using forward and reverse primers. from the Institutional Review Board. Each participant The primers were designed using PRIMER 3 program gave a written informed consent prior to enrolment in and the sequence of each primer was as follows: the study. A total of 58 Saudi women volunteers aged F: 5′- ACCTGCCGAACTACAACTGG-3′;R: 5’-TGAA 19–36 years were recruited. Cases were 28 Saudi women GGAACAGGCGGTTAGT -3’. attending the outpatient clinics and fulfilling the follow- The PCR conditions consisted of initial denaturation ing criteria: step at 95 °C for 15 min, followed by 34 cycles of denaturation at 95 °C for 1 min, annealing at 60 °C for ○ Oligomenorrhoea (cycle duration between 35 days to 1 min, and extension at 72 °C for 1 min, with final 3 months). extension of 10 min at 72 °C. A PCR product of 353 bp ○ Transvaginal ultrasound image of enlarged ovaries was obtained. Nucleotide sequencing was carried out with > 10 cysts in the largest plane; each measuring using the ABI Big Dye Terminator protocol on ABI 3100 <I0mm in diameter scattered around an echodense Avant Genetic Analyzer. thickened stroma [17] ○ None had received any drugs known to interfere with Statistical analyses hormonal concentrations for at least 3 months before The descriptive characteristics of the group variables the study. were expressed as mean ± SEM. The comparisons be- tween PCOS patients and their matched controls were Controls were 30 Saudi women volunteers representing carried out using the independent t-test and ANOVA cross section of Saudi society. All subjects were healthy, test with respect to all variables. Pearson Correlation had spontaneous onset of puberty and sexual development, Coefficients was used to study the correlation between with regular menstrual cycle, and no history of gastrointes- Kisspeptin, BMI and other studied variables. Genotype tinal or endocrine disorders. and allele frequencies were calculated manually https:// ihg.gnf.de/cgi-bin/hw/hwa1.pl. Significance of the differ- Anthropometric measurements ence in the result of POCS cases and controls was ob- Anthropometric measurements included body weight, tained using Fisher’s Exact test (two-tailed) and odds height and body mass index (BMI), (weight in Kg ratios, 95% confidence intervals, χ2and p value were ob- divided by height in m ). tained. All statistical analyses were performed by using SPSS for Windows (version 9.3). Laboratory methods Blood samples were collected between day 3 and day 6 Results of a menstrual cycle in the PCOS and control groups, all Basic anthropometrics and hormonal features of the blood samples were obtained at 8:00 a.m. (This ensured study group are summarized in Table 1. Women with obtaining the true level of the reproductive hormones, PCOS had significantly higher weight, BMI and LH level as these hormones show variations with the days of the compared to the control group (p < .0001). No signifi- menstrual cycle). Five milliliter of blood was drawn by cant differences in FSH and kisspeptin level between venipuncture in red top tube for kisspeptin, FSH and LH groups were observed. Kisspeptin level were higher in estimations. PCOS women compared to control group, but not Albalawi et al. Journal of Biomedical Science (2018) 25:50 Page 3 of 6 Table 1 Comparisons of clinical parameters amongst PCOS and Table 3 Frequencies of allele and genotypes of rs4889 C/G in control group KISS1 gene in PCOS and control groups Variable PCOS Group Control Group P-value Variation Control Case OR CI χ P-value (n = 28) (n = 30) (N = 30) (28) Mean ± SD)) Mean ± SD)) CC 1 (3.3) 4 (14.3) 8.0 0.79–80.4 3.96 0.046 Age (years) 29.4 ± 3.93 26.7 ± 3.6 0.008 CG 7 (23.3) 13 (46.4) 3.714 1.15–11.96 5.04 0.024 Weight (Kg) 72.63 ± 12.8 59.87 ± 10.8 0.0001 GG 22 (73.3) 11 (39.4) 0.125 0.012–1.26 3.96 046 Height (cm) 155.9 ± 6.9 161.5 ± 4.1 0.001 CG + GG 29 (0.48) 24 (0.43) 0.207 0.02–1.98 2.21 0.137 BMI (Kg\m ) 46.04 ± 7.3 36.6 ± 8.17 0.0001 C 9 (0.15) 21 (0.375) 3.4 1.4–8.3 7.65 0.006 FSH (IU\I) 6.81 ± 5.4 5.7 ± 1.95 0.325 G 51 (0.85) 35 (0.625) 0.294 0.12–0.72 7.65 0.006 LH (IU\I) 15.12 ± 3.7 8.73 ± 1.16 0.0001 OR odds ratio, CI confidence intervals, χ chi square, P significance Kisspeptin 0.43 ± .15 0.39 ± .07 0.178 (pg\mL) in BMI (wt/ht ) (48.32 ± 6.73 vs 35.01 ± 8.04, p < .0001). Statistically significant difference were observed in the significantly. Kisspeptin level were found to be positively heterozygous CG in LH level (13.73 ± 3.35 vs. 8.741 ± correlated with LH level in the PCOS patients (r = 0.604; 1.28, P < .0001). The effect of the different genotypes of p = 0,005, control: r = 0.409; p = 0.007). Correlations rs4889 on the level of of kisspeptin was studied and as between kisspeptin level, hormonal and anthropometric shown in the Table 4. The kisspeptin level did not differ measurement of women studied are summarized in significantly in individuals with the CC, CG and GG ge- Table 2. Direct sequencing of KISS1 gene revealed notypes in PCOS group and GG and CG in control several SNPs. The SNP rs4889 (C/G) was detected more group. Only one control had CC genotype and was not frequently in PCOS group than in control, and this non- included in the calculations. synonymous SNP results in the substitution of P81R. Distribution of rs4889 C/G in KISS1 gene and the allele Discussion and genotype frequencies in PCOS and control groups Thepresent studyinvestigatedplasmakisspeptinlevelsin are summarized in Table 3. Statistically significant differ- Saudi women with and without PCOS and studied the ences were observed for the alleles (p = 0.0057), and nature of correlation between kisspeptin, anthropometric genotype frequency between PCOS and controls. Allele parameters and PCOS-related reproductive hormones. It C showed higher frequency in PCOS group (37.5%) than also evaluated the sequence of exons in the KISS-1 control group (15%). The individuals with the different gene andcomparedthe frequencyofrs4889C/G, a genotypes were separated and the levels of the anthropo- non-synonymous SNP, in PCOS and controls. Our metric and hormonal parameters and kisspeptin were investigation showed that the level of kisspentin was separately analyzed for each genotype. Table 4 presents slightly higher in females with PCOS, though the the results of the study variables in the different rs4889 results compared to the control group were not genotypes (CC, CG and GG) in Saudi women with PCOS significantly different. Three previous studies [18–20] and Control groups. Statistically significant difference were reported higher kisspeptin levels in women with observed in the wild type GG in women with PCOS in PCOS. Another study [21] reported lower levels in weight (Kg) (75.83 ± 11.42vs. 57.59 ± 10.07, p < .0001), and women with PCOS as compared to controls. Yerlikaya et al., [22] study and our study did not support these Table 2 Correlations between kisspeptin level, hormonal and findings. This may be the result of the obesity and anthropometric measurement of PCOS patients and control insulin resistance that may have negative impact on group kisspeptin levels. Correlation PCOS Group Controls Group The Kiss-1 system has emerged in the recent years as between (n = 28) (n = 30) a fundamental player in the control of the reproductive kisspeptin rp Sig rP Sig and axis, with essential roles in differentiation and pubertal activation of the reproductive system as well as key Age (years) 0.182 0.355 NS 0.083 0.663 NS functions in the regulation of ovulation and fertility [18]. Weight (Kg) 0.111 0.575 NS 0.152 0.422 NS Kisspeptin has been recently associated with increased Height (cm) −0.118 0.549 NS −0.177 0.351 NS GnRH and regulates the secretion of LH during the BMI (Kg/m ) 0.165 0.401 NS 0.109 0.567 NS promotion of ovulation [23, 24]. PCOS is a condition as- FSH (IU/I) 0.049 0.804 NS 0.225 0.231 NS sociated with disordered hypothalamic-pituitary-gonadal LH (IU/I) 0.604 0.005 S 0.409 0.007 S axis, higher LH levels, compared to ovulatory women NS non-significant = p > 0.05, S significant = p ≤ 0.05, r correlation coefficient without the syndrome [25, 26]. The present study Albalawi et al. Journal of Biomedical Science (2018) 25:50 Page 4 of 6 Table 4 Level of anthropometric parameters and hormones in different genotypes of rs4889 C/G in KISS1 gene in Saudi women with PCOS and Control groups Variable PCOS Group Control Group P-value (n = 28) (n = 30) (Mean ± SD) (Mean ± SD) GG CG CC GG CG GG CG CC n =11 n =13 n =4 n =22 n =7 ** Weight (Kg) 75.8 ± 11 74.1 ± 13 58.8 ± 4.1 57.5 ± 10 68.4 ± 9.6 0.0001 0.362 0.157 Height (cm) 156. ± 6.8 156 ± 7.5 152 ± 5.91 162 ± 3.9 158.8 ± 3.5 0.007 0.660 0.468 2 ** BMI (Kg/m ) 48.3 ± 6.7 47.2 ± 7.5 38 ± 1.76 35 ± 8 42.6 ± 6.43 0.0001 0.219 0.157 FSH (IU/I) 6.52 ± 5.6 7.64 ± 5.8 4.9 ± 4.02 5.8 ± 2 5.5 ± 1.4 0.879 0.475 1.00 ** ** LH (IU/I) 13.7 ± 3.3 16.2 ± 3.8 15.3 ± 3.7 8.7 ± 1.2 8.6 ± 0.89 0.0001 0.0001 0.147 kisspeptin (pg\mL) 0.38 ± 0.2 0.43 ± 0.1 0.35 ± 0.18 0.37 ± .06 0.45 ± 0.06 0.349 0.968 1.00 There was only 1 sample with a CC genotype in the control group, and was not used in the comparison * ** p-value< 0.05; p-value< 0.001 evaluated the possible role of kisspeptin in the pathophysi- similar to the findings of our study. Some researchers ology of PCOS and showed the correlation between levels have demonstrated that kisspeptin may play a key role of kisspeptin and LH. Several groups have now reported in the activation of the gonadotropic axis at puberty that kisspeptin administered either centrally or peripher- [35, 36]. So elevated kisspeptin levels of adolescent ally, stimulate gonadotropin secretion. Similar observa- PCOS may play a role as a marker to recognize PCOS tions were reported in the rat [27, 28], sheep [29], monkey in adolescents more clearly and sometimes at an earlier [30, 31] and more recently, the human male [32]. Our stage. In the study of Chen and coworkers [19], the study showed that LH levels were higher in PCOS women number of the controls was relatively small and no compared to controls, and plasma kisspeptin levels were adult controls were involved. Therefore, it needs further positively correlated with LH levels. It was consistent with research to draw any conclusions. Plasma kisspeptin the idea that kisspeptin may stimulate LH secretion, levels were measured by Jeon et al. [20]in54women although the way of direct pituitary effects of kisspeptin in with PCOS and 36 controls. Their results suggested the control of gonadotropin secretion remains controver- that plasma kisspeptin levels were significantly higher sial [33, 34]. In another study conducted by Panidis et al., in the PCOS group than in controls; however, they did [21], it was found that LH levels were significantly higher not find correlations between kisspeptin and any of the in PCOS groups compared to controls. This finding was hormones. The data of Yilmaz et al., [18] claimed that in line with the results of the present study. However, no women with PCOS exhibited higher kisspeptin levels significant correlation between plasma kisspeptin and LH than controls. Furthermore, kisspeptin concentrations levels was observed in their study, and they also found were found to be in a positive correlation with LH that obese and overweight women with PCOS had signifi- levels. cantly lower kisspeptin levels compared to normal weight In this study, polymorphism in SNP rs4889 C/G was women with the syndrome. These conclusions are contra- detected more frequently in PCOS group than in control dictory to our study, possibly due to differences in the (p-value = 0.0057). Allele C showed higher frequeny in BMI in these studies as well as methodological differences. PCOS group (37.5%) than control group (15%). When the Additionally, small sample size and the heterogeneity of results in different genotypes were compared, the weight the study groups are the limitations of these previous and BMI were higher, while the LH level was lower in the studies. Plasma kisspeptin levels were measured by GG genotype, in the PCOS patients and the results were Chen et al. [19] in 42 women with PCOS (23 adult, 19 significantly different. Kisspeptin was higher in the GG adolescents) and 20 adolescent controls to investigate genotype but the results were not significantly different. the possible correlations between kisspeptin and PCOS When compared with the control group, the weight, related reproductive and metabolic disturbances. Their height and BMI were significantly higher in PCOS carry- results suggested that plasma kisspeptin levels were ing GG genotype, compared to the controls with the same increased in lean adolescent and adult women with genotype. LH was higher in all genotypes in the PCOS, PCOS compared with lean adolescent control group. compared to the results in the control group. Our study did not support these findings. Moreover, rs4889 polymorphism introduced a substitution of LH levels of both adult and adolescent PCOS women proline for arginine at the 81 position. This is a substi- were higher than that of adolescent controls and tution that was observed in kisspeptin-54, but not in kisspeptin showed a positive correlation with LH the other three forms of kisspeptin (kisspeptin-14, − 13, Albalawi et al. Journal of Biomedical Science (2018) 25:50 Page 5 of 6 and − 10). This SNP is found in the coding region and Authors’ contributions FSA and MHD designed the study, obtained all the needed chemicals, kits results in the substitution of an imino acid Pro by a and other requirements, conducted all analysis and contributed to the basic amino acid Arg. Change in the DNA sequence as 2 writing of the manuscript. MHD enrolled the patients, obtained informed a result of this polymorphisms could alter the structure, consent, and collected all the clinical data. AME conducted statistical analysis and contributed to the writing of the paper. ASW performed the genetics function and binding capacity of kisspeptin to its recep- data analysis and contributed to the writing of the paper. All authors read tor GPR54.. Previous studies in Korean and Chinese and approved the final manuscript. populations found no relationship between this SNP Ethics approval and consent to participate and CPP [37, 38]. From the results of this study, it Ethical approval was obtained from the Institutional Review Board (IRB), appears that rs4889 influences the mechanism by which Umm Al-Qura University, Makkah, Kingdom of Saudi Arabia (IRB No. 235) and kisspeptin activates secretion of LH but not FSH. each female was required to sign an informed consent form. All participants gave a written informed consent prior to inclusion in the study. Further studies are necessary to investigate the possible mechanisms involved in kisspeptin function and mode Competing interests of action. The authors declare that they have no competing interests. Conclusion Publisher’sNote In conclusion, this study has shown significant rela- Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. tionship between the rs4889 C/G polymorphisms in KISS1 gene and PCOS in Saudi females, where the Author details mutant G allele is highly protective (OR = 0.294; 95% Department of Zoology, Center for Scientific and Medical Female Colleges, King Saud University, P.O. Box 22455, Riyadh 11495, Saudi Arabia. CI = 0.12–0.72, χ2 = 7.65; p = 0.006). The LH levels Department of Obstetrics & Gynecology, Umm-Al-Qura University, P.O. Box were higher in the women with PCOS, compared to 424, Makkah 21955, Saudi Arabia. Department of Biostatistics, Epidemiology the controls, and plasma kisspeptin levels positively &Scientific Computing, King Faisal Specialist Hospital and Research Center, P.O. Box 3354, Riyadh 11211, Saudi Arabia. Central Laboratory, Center for correlated with LH levels. Within the genotypes of Scientific and Medical Female Colleges, King Saud University, P.O. Box 22455, rs4889 the LH levels were significantly lower in the Riyadh 11495, Saudi Arabia. GG compared to the CG and CC genotypes in the Received: 8 January 2018 Accepted: 22 May 2018 PCOS females. The mechanism of kisspeptin for regulating gonadotropin secretion remains unknown. Discrepant findings among the results of the published References studies may be attributed to the design and sample 1. Hori A, Honda S, Asada M, Ohtaki T, Oda K, Watanabe T, et al. Metastin suppresses the motility and growth of CHO cells transfected with its size and the demographic and genetic characteristics receptor. Biochem Biophys Res Commun. 2001;286:958–63. of the different populations. 2. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, et al. KiSS-1, The major limitation of our study was the small num- a novel human malignant melanoma metastasis-suppressor gene. 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rs4889 polymorphism in KISS1 gene, its effect on polycystic ovary syndrome development and anthropometric and hormonal parameters in Saudi women

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Abstract

Background: Kisspeptin is involved in female reproduction. This study was designed to i- estimate kisspeptin levels in women with polycystic ovary syndrome (PCOS), in comparison with controls, ii- study the correlations between kisspeptin and PCOS-related reproductive hormones, and iii- investigate the relation between KISS1 gene polymorphisms and hormone levels in women suffering from PCOS. Methods: The investigation was a clinically designed study on 28 women with PCOS, and 30 normal, healthy women with no signs of PCOS as controls. Blood samples were collected between day 3 and day 6 of the menstrual cycle in both groups at 8:00 a.m., and circulating levels of LH, FSH and kisspeptin were estimated. DNA was extracted from whole blood and all coding exons of KISS1 gene were sequenced. Results: Women with PCOS had higher LH levels and BMI compared to controls. Plasma kisspeptin levels were positively correlated with LH levels. There was no statistically significant difference between the groups in terms of kisspeptin and FSH levels. The SNP rs4889 C/G, a non-synonymous SNP, was investigated in the PCOS group. The frequency of GG genotype was significantly higher in the PCOS compared to the controls. These patients were more obese, had higher kisspeptin and FSH levels. Conclusion: The results of the study show that the genetic variation of KISS1 gene may be a factor contributing to PCOS development. The association between the gene and the gene variation and PCOS need further validation in large-scaled and functional studies. Keywords: KISS1 gene, Kisspeptin, LH, FSH, GPR54, PCOS Background in the stimulation of gonadotropin release, which in turn Kisspeptin is a 54-amino-acid peptide encoded by the binds to the GnRH receptors in the pituitary and influences KISS1 gene, and is also known as metastin [1, 2], which the release of LH and FHS [7]. These hormones act on the was first isolated from the human placenta in 2001 [3]. gonads and influence estrogen, testosterone and pro- Kisspeptinactionisexerted by a trans-membrane G-protein gesterone release. Hence, kisspeptin has been shown coupled receptor, named GPR54, AXOR12, or HoT7T175 to regulate the secretion of luteinizing hormone (LH) [4]. Loss of KISS1 gene function is reported to be associated during the promotion of ovulation [8]bystimulating with hypogonadotropic hypogonadism in humans and gonadotropin releasing hormone (GnRH) from the animal models [5, 6]. Binding of kisspeptin to its receptor hypothalamus [9, 10]. (GPR54) in the GnRH neurons in the hypothalamus, results The polycystic ovary syndrome (PCOS) is a common het- erogeneous disorder affecting women, with a prevalence of 6–12% in women of reproductive age [11]. It is of complex * Correspondence: mdaghestani@ksu.edu.sa pathogenesis, characterized by hypothalamic-pituitary dis- Department of Zoology, Center for Scientific and Medical Female Colleges, King Saud University, P.O. Box 22455, Riyadh 11495, Saudi Arabia turbances in gonadotropin secretion, specifically increased Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Albalawi et al. Journal of Biomedical Science (2018) 25:50 Page 2 of 6 LH levels [12, 13], chronic anovulation, and polycystic In addition, 5 mL of blood was collected in EDTA-coated ovaries (PCO) on ultrasound. In addition, patients fre- tubes for DNA extraction. All blood samples for each quently suffer from hyperinsulinemia, insulin resistance subject were immediately centrifuged, and plasma, serum [14], type 2 diabetes mellitus (DM), cardio-vascular and buffy coat were stored at − 80 °C until analysis. disease and infertility [15, 16]. Given the complex rela- Kisspeptin levels were measured with an enzyme-linked tionship between kisspeptin and hypothalamic-pituitary immunoassay kit (ELISA kit, Phoenix Pharmaceuticals Inc., gonadal axis, the present study aimed to investigate the Belmond, CA), after extraction with Phoenix Peptide relation between KISS1, BMI, FSH and LH and the in- sep-columns (RK-Sepcol-2). FSH and LH levels were fluence of KISS1 gene polymorphisms on these parame- measured with an enzyme-linked immunoassay kit (ELISA ters in normal women and those suffering from PCOS. kit, Human, cat.No.65205.GER). Genotyping of KISS1 gene polymorphism Methods Genomic DNA was extracted from peripheral blood Subjects leucocytes using commercially available Puregene Blood The samples were collected from Al-Hira Hospital in Kit (QIAGEN, cat. No. 158389, USA). Polymorphism Makkah Al Mukarama, from 58 Saudi women volunteers was determined by sequencing following polymerase aged 19–36 after obtaining Ethical Committee approval, chain reaction (PCR), using forward and reverse primers. from the Institutional Review Board. Each participant The primers were designed using PRIMER 3 program gave a written informed consent prior to enrolment in and the sequence of each primer was as follows: the study. A total of 58 Saudi women volunteers aged F: 5′- ACCTGCCGAACTACAACTGG-3′;R: 5’-TGAA 19–36 years were recruited. Cases were 28 Saudi women GGAACAGGCGGTTAGT -3’. attending the outpatient clinics and fulfilling the follow- The PCR conditions consisted of initial denaturation ing criteria: step at 95 °C for 15 min, followed by 34 cycles of denaturation at 95 °C for 1 min, annealing at 60 °C for ○ Oligomenorrhoea (cycle duration between 35 days to 1 min, and extension at 72 °C for 1 min, with final 3 months). extension of 10 min at 72 °C. A PCR product of 353 bp ○ Transvaginal ultrasound image of enlarged ovaries was obtained. Nucleotide sequencing was carried out with > 10 cysts in the largest plane; each measuring using the ABI Big Dye Terminator protocol on ABI 3100 <I0mm in diameter scattered around an echodense Avant Genetic Analyzer. thickened stroma [17] ○ None had received any drugs known to interfere with Statistical analyses hormonal concentrations for at least 3 months before The descriptive characteristics of the group variables the study. were expressed as mean ± SEM. The comparisons be- tween PCOS patients and their matched controls were Controls were 30 Saudi women volunteers representing carried out using the independent t-test and ANOVA cross section of Saudi society. All subjects were healthy, test with respect to all variables. Pearson Correlation had spontaneous onset of puberty and sexual development, Coefficients was used to study the correlation between with regular menstrual cycle, and no history of gastrointes- Kisspeptin, BMI and other studied variables. Genotype tinal or endocrine disorders. and allele frequencies were calculated manually https:// ihg.gnf.de/cgi-bin/hw/hwa1.pl. Significance of the differ- Anthropometric measurements ence in the result of POCS cases and controls was ob- Anthropometric measurements included body weight, tained using Fisher’s Exact test (two-tailed) and odds height and body mass index (BMI), (weight in Kg ratios, 95% confidence intervals, χ2and p value were ob- divided by height in m ). tained. All statistical analyses were performed by using SPSS for Windows (version 9.3). Laboratory methods Blood samples were collected between day 3 and day 6 Results of a menstrual cycle in the PCOS and control groups, all Basic anthropometrics and hormonal features of the blood samples were obtained at 8:00 a.m. (This ensured study group are summarized in Table 1. Women with obtaining the true level of the reproductive hormones, PCOS had significantly higher weight, BMI and LH level as these hormones show variations with the days of the compared to the control group (p < .0001). No signifi- menstrual cycle). Five milliliter of blood was drawn by cant differences in FSH and kisspeptin level between venipuncture in red top tube for kisspeptin, FSH and LH groups were observed. Kisspeptin level were higher in estimations. PCOS women compared to control group, but not Albalawi et al. Journal of Biomedical Science (2018) 25:50 Page 3 of 6 Table 1 Comparisons of clinical parameters amongst PCOS and Table 3 Frequencies of allele and genotypes of rs4889 C/G in control group KISS1 gene in PCOS and control groups Variable PCOS Group Control Group P-value Variation Control Case OR CI χ P-value (n = 28) (n = 30) (N = 30) (28) Mean ± SD)) Mean ± SD)) CC 1 (3.3) 4 (14.3) 8.0 0.79–80.4 3.96 0.046 Age (years) 29.4 ± 3.93 26.7 ± 3.6 0.008 CG 7 (23.3) 13 (46.4) 3.714 1.15–11.96 5.04 0.024 Weight (Kg) 72.63 ± 12.8 59.87 ± 10.8 0.0001 GG 22 (73.3) 11 (39.4) 0.125 0.012–1.26 3.96 046 Height (cm) 155.9 ± 6.9 161.5 ± 4.1 0.001 CG + GG 29 (0.48) 24 (0.43) 0.207 0.02–1.98 2.21 0.137 BMI (Kg\m ) 46.04 ± 7.3 36.6 ± 8.17 0.0001 C 9 (0.15) 21 (0.375) 3.4 1.4–8.3 7.65 0.006 FSH (IU\I) 6.81 ± 5.4 5.7 ± 1.95 0.325 G 51 (0.85) 35 (0.625) 0.294 0.12–0.72 7.65 0.006 LH (IU\I) 15.12 ± 3.7 8.73 ± 1.16 0.0001 OR odds ratio, CI confidence intervals, χ chi square, P significance Kisspeptin 0.43 ± .15 0.39 ± .07 0.178 (pg\mL) in BMI (wt/ht ) (48.32 ± 6.73 vs 35.01 ± 8.04, p < .0001). Statistically significant difference were observed in the significantly. Kisspeptin level were found to be positively heterozygous CG in LH level (13.73 ± 3.35 vs. 8.741 ± correlated with LH level in the PCOS patients (r = 0.604; 1.28, P < .0001). The effect of the different genotypes of p = 0,005, control: r = 0.409; p = 0.007). Correlations rs4889 on the level of of kisspeptin was studied and as between kisspeptin level, hormonal and anthropometric shown in the Table 4. The kisspeptin level did not differ measurement of women studied are summarized in significantly in individuals with the CC, CG and GG ge- Table 2. Direct sequencing of KISS1 gene revealed notypes in PCOS group and GG and CG in control several SNPs. The SNP rs4889 (C/G) was detected more group. Only one control had CC genotype and was not frequently in PCOS group than in control, and this non- included in the calculations. synonymous SNP results in the substitution of P81R. Distribution of rs4889 C/G in KISS1 gene and the allele Discussion and genotype frequencies in PCOS and control groups Thepresent studyinvestigatedplasmakisspeptinlevelsin are summarized in Table 3. Statistically significant differ- Saudi women with and without PCOS and studied the ences were observed for the alleles (p = 0.0057), and nature of correlation between kisspeptin, anthropometric genotype frequency between PCOS and controls. Allele parameters and PCOS-related reproductive hormones. It C showed higher frequency in PCOS group (37.5%) than also evaluated the sequence of exons in the KISS-1 control group (15%). The individuals with the different gene andcomparedthe frequencyofrs4889C/G, a genotypes were separated and the levels of the anthropo- non-synonymous SNP, in PCOS and controls. Our metric and hormonal parameters and kisspeptin were investigation showed that the level of kisspentin was separately analyzed for each genotype. Table 4 presents slightly higher in females with PCOS, though the the results of the study variables in the different rs4889 results compared to the control group were not genotypes (CC, CG and GG) in Saudi women with PCOS significantly different. Three previous studies [18–20] and Control groups. Statistically significant difference were reported higher kisspeptin levels in women with observed in the wild type GG in women with PCOS in PCOS. Another study [21] reported lower levels in weight (Kg) (75.83 ± 11.42vs. 57.59 ± 10.07, p < .0001), and women with PCOS as compared to controls. Yerlikaya et al., [22] study and our study did not support these Table 2 Correlations between kisspeptin level, hormonal and findings. This may be the result of the obesity and anthropometric measurement of PCOS patients and control insulin resistance that may have negative impact on group kisspeptin levels. Correlation PCOS Group Controls Group The Kiss-1 system has emerged in the recent years as between (n = 28) (n = 30) a fundamental player in the control of the reproductive kisspeptin rp Sig rP Sig and axis, with essential roles in differentiation and pubertal activation of the reproductive system as well as key Age (years) 0.182 0.355 NS 0.083 0.663 NS functions in the regulation of ovulation and fertility [18]. Weight (Kg) 0.111 0.575 NS 0.152 0.422 NS Kisspeptin has been recently associated with increased Height (cm) −0.118 0.549 NS −0.177 0.351 NS GnRH and regulates the secretion of LH during the BMI (Kg/m ) 0.165 0.401 NS 0.109 0.567 NS promotion of ovulation [23, 24]. PCOS is a condition as- FSH (IU/I) 0.049 0.804 NS 0.225 0.231 NS sociated with disordered hypothalamic-pituitary-gonadal LH (IU/I) 0.604 0.005 S 0.409 0.007 S axis, higher LH levels, compared to ovulatory women NS non-significant = p > 0.05, S significant = p ≤ 0.05, r correlation coefficient without the syndrome [25, 26]. The present study Albalawi et al. Journal of Biomedical Science (2018) 25:50 Page 4 of 6 Table 4 Level of anthropometric parameters and hormones in different genotypes of rs4889 C/G in KISS1 gene in Saudi women with PCOS and Control groups Variable PCOS Group Control Group P-value (n = 28) (n = 30) (Mean ± SD) (Mean ± SD) GG CG CC GG CG GG CG CC n =11 n =13 n =4 n =22 n =7 ** Weight (Kg) 75.8 ± 11 74.1 ± 13 58.8 ± 4.1 57.5 ± 10 68.4 ± 9.6 0.0001 0.362 0.157 Height (cm) 156. ± 6.8 156 ± 7.5 152 ± 5.91 162 ± 3.9 158.8 ± 3.5 0.007 0.660 0.468 2 ** BMI (Kg/m ) 48.3 ± 6.7 47.2 ± 7.5 38 ± 1.76 35 ± 8 42.6 ± 6.43 0.0001 0.219 0.157 FSH (IU/I) 6.52 ± 5.6 7.64 ± 5.8 4.9 ± 4.02 5.8 ± 2 5.5 ± 1.4 0.879 0.475 1.00 ** ** LH (IU/I) 13.7 ± 3.3 16.2 ± 3.8 15.3 ± 3.7 8.7 ± 1.2 8.6 ± 0.89 0.0001 0.0001 0.147 kisspeptin (pg\mL) 0.38 ± 0.2 0.43 ± 0.1 0.35 ± 0.18 0.37 ± .06 0.45 ± 0.06 0.349 0.968 1.00 There was only 1 sample with a CC genotype in the control group, and was not used in the comparison * ** p-value< 0.05; p-value< 0.001 evaluated the possible role of kisspeptin in the pathophysi- similar to the findings of our study. Some researchers ology of PCOS and showed the correlation between levels have demonstrated that kisspeptin may play a key role of kisspeptin and LH. Several groups have now reported in the activation of the gonadotropic axis at puberty that kisspeptin administered either centrally or peripher- [35, 36]. So elevated kisspeptin levels of adolescent ally, stimulate gonadotropin secretion. Similar observa- PCOS may play a role as a marker to recognize PCOS tions were reported in the rat [27, 28], sheep [29], monkey in adolescents more clearly and sometimes at an earlier [30, 31] and more recently, the human male [32]. Our stage. In the study of Chen and coworkers [19], the study showed that LH levels were higher in PCOS women number of the controls was relatively small and no compared to controls, and plasma kisspeptin levels were adult controls were involved. Therefore, it needs further positively correlated with LH levels. It was consistent with research to draw any conclusions. Plasma kisspeptin the idea that kisspeptin may stimulate LH secretion, levels were measured by Jeon et al. [20]in54women although the way of direct pituitary effects of kisspeptin in with PCOS and 36 controls. Their results suggested the control of gonadotropin secretion remains controver- that plasma kisspeptin levels were significantly higher sial [33, 34]. In another study conducted by Panidis et al., in the PCOS group than in controls; however, they did [21], it was found that LH levels were significantly higher not find correlations between kisspeptin and any of the in PCOS groups compared to controls. This finding was hormones. The data of Yilmaz et al., [18] claimed that in line with the results of the present study. However, no women with PCOS exhibited higher kisspeptin levels significant correlation between plasma kisspeptin and LH than controls. Furthermore, kisspeptin concentrations levels was observed in their study, and they also found were found to be in a positive correlation with LH that obese and overweight women with PCOS had signifi- levels. cantly lower kisspeptin levels compared to normal weight In this study, polymorphism in SNP rs4889 C/G was women with the syndrome. These conclusions are contra- detected more frequently in PCOS group than in control dictory to our study, possibly due to differences in the (p-value = 0.0057). Allele C showed higher frequeny in BMI in these studies as well as methodological differences. PCOS group (37.5%) than control group (15%). When the Additionally, small sample size and the heterogeneity of results in different genotypes were compared, the weight the study groups are the limitations of these previous and BMI were higher, while the LH level was lower in the studies. Plasma kisspeptin levels were measured by GG genotype, in the PCOS patients and the results were Chen et al. [19] in 42 women with PCOS (23 adult, 19 significantly different. Kisspeptin was higher in the GG adolescents) and 20 adolescent controls to investigate genotype but the results were not significantly different. the possible correlations between kisspeptin and PCOS When compared with the control group, the weight, related reproductive and metabolic disturbances. Their height and BMI were significantly higher in PCOS carry- results suggested that plasma kisspeptin levels were ing GG genotype, compared to the controls with the same increased in lean adolescent and adult women with genotype. LH was higher in all genotypes in the PCOS, PCOS compared with lean adolescent control group. compared to the results in the control group. Our study did not support these findings. Moreover, rs4889 polymorphism introduced a substitution of LH levels of both adult and adolescent PCOS women proline for arginine at the 81 position. This is a substi- were higher than that of adolescent controls and tution that was observed in kisspeptin-54, but not in kisspeptin showed a positive correlation with LH the other three forms of kisspeptin (kisspeptin-14, − 13, Albalawi et al. Journal of Biomedical Science (2018) 25:50 Page 5 of 6 and − 10). This SNP is found in the coding region and Authors’ contributions FSA and MHD designed the study, obtained all the needed chemicals, kits results in the substitution of an imino acid Pro by a and other requirements, conducted all analysis and contributed to the basic amino acid Arg. Change in the DNA sequence as 2 writing of the manuscript. MHD enrolled the patients, obtained informed a result of this polymorphisms could alter the structure, consent, and collected all the clinical data. AME conducted statistical analysis and contributed to the writing of the paper. ASW performed the genetics function and binding capacity of kisspeptin to its recep- data analysis and contributed to the writing of the paper. All authors read tor GPR54.. Previous studies in Korean and Chinese and approved the final manuscript. populations found no relationship between this SNP Ethics approval and consent to participate and CPP [37, 38]. From the results of this study, it Ethical approval was obtained from the Institutional Review Board (IRB), appears that rs4889 influences the mechanism by which Umm Al-Qura University, Makkah, Kingdom of Saudi Arabia (IRB No. 235) and kisspeptin activates secretion of LH but not FSH. each female was required to sign an informed consent form. All participants gave a written informed consent prior to inclusion in the study. Further studies are necessary to investigate the possible mechanisms involved in kisspeptin function and mode Competing interests of action. The authors declare that they have no competing interests. Conclusion Publisher’sNote In conclusion, this study has shown significant rela- Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. tionship between the rs4889 C/G polymorphisms in KISS1 gene and PCOS in Saudi females, where the Author details mutant G allele is highly protective (OR = 0.294; 95% Department of Zoology, Center for Scientific and Medical Female Colleges, King Saud University, P.O. Box 22455, Riyadh 11495, Saudi Arabia. CI = 0.12–0.72, χ2 = 7.65; p = 0.006). The LH levels Department of Obstetrics & Gynecology, Umm-Al-Qura University, P.O. Box were higher in the women with PCOS, compared to 424, Makkah 21955, Saudi Arabia. Department of Biostatistics, Epidemiology the controls, and plasma kisspeptin levels positively &Scientific Computing, King Faisal Specialist Hospital and Research Center, P.O. Box 3354, Riyadh 11211, Saudi Arabia. Central Laboratory, Center for correlated with LH levels. Within the genotypes of Scientific and Medical Female Colleges, King Saud University, P.O. Box 22455, rs4889 the LH levels were significantly lower in the Riyadh 11495, Saudi Arabia. GG compared to the CG and CC genotypes in the Received: 8 January 2018 Accepted: 22 May 2018 PCOS females. The mechanism of kisspeptin for regulating gonadotropin secretion remains unknown. Discrepant findings among the results of the published References studies may be attributed to the design and sample 1. Hori A, Honda S, Asada M, Ohtaki T, Oda K, Watanabe T, et al. Metastin suppresses the motility and growth of CHO cells transfected with its size and the demographic and genetic characteristics receptor. Biochem Biophys Res Commun. 2001;286:958–63. of the different populations. 2. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, et al. KiSS-1, The major limitation of our study was the small num- a novel human malignant melanoma metastasis-suppressor gene. 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Journal of Biomedical ScienceSpringer Journals

Published: May 30, 2018

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