Genotyping of the gene polymorphisms FASL 844C/T and FAS 670A/G in patients with idiopathic infertility in Iraq

SUMMARY

OBJECTIVE:  The failure to conceive following 12 months of unprotected intercourse is referred to as infertility. About 35% of the instances include women and 30% involve men.

AIM:  The aim of this study was to investigate the gene polymorphisms FASL-844C/T and FAS-670A/G in the promoter region to evaluate susceptibility to male infertility.

METHODS:  We have investigated single-nucleotide polymorphisms of FAS-670A/G gene and FASL-844C/T gene in 100 subjects. A total of 50 male patients with infertility constituted the infertility group and another group consisting of 50 apparently healthy individuals comprised the control group.

RESULTS:  The prevalence rate of FAS-670A/G gene polymorphism was statistically significant among both azoospermia and oligospermia subgroups, the homozygous mutant genotype GG (p=0.014 and p=0.043), and mutant allele G (p=0.001 and p=0.004), respectively. Also, FASL-844C/T gene polymorphism was statistically significant among both azoospermia and oligospermia subgroups, the homozygous mutant genotype TT (p=0.032 and p=0.032), and mutant allele T (p=0.007 and p=0.002), respectively.

CONCLUSIONS:  FAS homozygous mutant genotype (GG) and FASL homozygous mutant genotype (TT) act as etiologic factors in male infertility.

KEYWORDS:
FAS ; FASL ; Genotype; Male infertility; Polymorphism

INTRODUCTION

The World Health Organization defines infertility as the inability to get pregnant after 12 months of unprotected sexual ­activity¹. It now affects 10–15% of couples globally. Its incidence has increased over the years²,³.

FAS and FASL are known to participate in apoptosis. FASL plays a crucial role in the development of the normal immune system and the maintenance, regulation, and ­function of homeostasis⁴.

The FAS and FASL genes have been revealed to contain several polymorphism variations. More consideration has been given to these functional polymorphisms because of their functions in controlling gene expression⁵. Functional polymorphisms have been discovered within the FAS promoter region and FASL genes. These polymorphisms, which are −844C/T in the FASL gene and −670A/G in the FAS gene, may raise the risk of infertility and cancer⁶. The most prevalent single-­nucleotide polymorphism (SNP) within the FAS promoter region is FAS-670A/G polymorphism. The STAT1 transcription factor's binding site is altered when a G is substituted for an A at −670 base pairs, which lowers the level of this gene's transcription⁷.

The transcriptional factors can be impacted by the FASL-844C/T and FAS-670A/G polymorphisms and result in a considerable unusual apoptosis that is linked to the susceptibility of disease occurrence⁸. Recent research on male infertility revealed a substantial correlation between abnormal semen parameters and excessive or not enough germ cell apoptosis in ejaculated sperm⁹.

The current study aimed to investigate the gene polymorphisms FASL-844C/T and FAS-670A/G in the promoter region to evaluate susceptibility to male infertility.

METHODS

Subject

This study was conducted on 50 male infertile patients with age ranging between 20 and 43 years, who attended the Infertility and IVF Center in Al-Sader City Hospital for the period from November 2023 to May 2024. A total of 50 healthy subjects without any history of systemic disease were included as the control group. This study excluded any infertile men with reproductive system disorders, surgical histories, diagnosed varicocele, individuals with cryptorchidism and testicular tumors, or those receiving radiotherapy and chemotherapy. A volume of 2 mL of blood samples was used to extract the DNA, and amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was used to find polymorphisms in the promoter region of the FAS/FASL genes, including FASL-844C/T and FAS-670A/G polymorphisms.

Genotyping

The Tetra-ARMS-PCR technique was performed for the detection of FASL C-844T and FAS A-670G gene polymorphisms from patient and healthy control blood samples. The blood samples were used to extract genomic DNA using the gSYNCTM Genomic DNA extraction kit, and the purity and concentration of the DNA were checked using a NanoDrop ­spectrophotometer at 260/280 nm, where it was found to range between 1.7 and 2.1, with an average purity equal to 2 and a mean DNA concentration of 20 ng/μL (10–40 ng/μL). The T-ARMS-PCR master mix was performed for each sample using AccuPower PCR PreMix Kit according to manufacturer's instructions. The results were analyzed by loading the ARMS-PCR products on 2% agarose gel. Table 1 lists the ARMS-PCR primers for the genes FASL-844C/T and FAS-670A/G.

Table 1
Sequence of primers and the size of their products.

Primer	Sequence (5’-3’)		Product size
FAS-670A/G gene	FO	"GGG GCT ATG CGA TTT GGC TTA AGT TGTT"	A allele 158 bp G allele 207 bp
	RO	"GTA GTT CAA CCT GGG AAG TTG GGG AGGT"
	FI	"TTT TTC ATA TGG TTA ACT GTC CAT TCC CGG"
	RI	"GCA ACA TGA GAG GCT CAC AGA CGG TT"
FASL-844C/T gene	FO	"TGC AGT TAA CTA CGA TAG CAC CAC TGCA"	C allele 192 bp T allele 145 bp
	RO	"AGG AGG AAA ACA TCT GTT GCC ATC ATCT"
	FI	"GGC AAA CAA TGA AAA TGA AAA CAT GGC"
	RI	"AAC CCA CAG AGC TGC TTT GTA TTG CA"

• FO: forward outer; RO: reverse outer; FI: forward inner; RI: reverse inner.

Statistical analysis

Data were either normally distributed or not normally distributed and were recorded in a Microsoft Excel spreadsheet. Statistical analysis was carried out with the SPSS v. 0.26 ­software (chi-square test, independent sample t-test, and Spearman's and Pearson's correlation coefficients) after translating data into codes. A p<0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Demographic and clinical parameters

When controls and infertile patients were compared in terms of age, the largest rate of infertility was found among young adult patients, especially those under the age of 30 years. The mean age of the infertile patients and controls were 31.3±11.23 and 32.6±12.11 years, respectively. Age did not significantly differ across the research groups (p=0.133). Family history is a significant contributing factor to infertility. In addition, the present study's findings revealed that the prevalence of oligospermia was higher in patients with a positive family history (68%) than in those with no family history (32%; p=0.05).

This agrees with a study conducted by Attia et al.¹⁰ who discovered no statistically significant differences in the ages of oligospermic and azoospermic patients in comparison to controls (33±4.8, 37±5.4, and 33±5.3 years, respectively; p>0.24). Also, Ejzenberg et al.¹¹ discovered no statistically significant differences between controls and infertile patients in terms of age. Shabani et al.¹² found that testosterone levels diminish over time as men age, but this loss varies greatly across men and may be influenced by variables, including drugs, obesity, genetics, alcoholism, and chronic illnesses.

In a previous study, 13.5% of the patients that were included had a positive family history of male infertility, suggesting that male infertility may be caused by hereditary factors¹³. Given the genetic foundation of infertility, family history is a portion of the patient's history that is becoming more significant¹⁴.

Genotype and allele distribution in patient and control groups for FAS-670A/G and FASL-844C/T gene polymorphisms

Tetra-ARMS-PCR technique was used to identify the FAS A-670G gene polymorphism distribution. At this locus, there are three genotypes for FAS-670A/G such as AA, AG, and GG with band sizes of 158 pb, 158/207 pb, and 207 pb, respectively. In this study, we genotyped the FAS-670A/G gene polymorphism in infertile patients to establish a link between their clinical features and the genotypes/allotypes.

The genotypes’ relative frequency of the FAS-670A/G gene polymorphism in the azoospermia subgroup and control group was as follows: AA (28 and 54%), AG (24 and 26%), and GG (48 and 20%), respectively, as shown in Table 2.

Table 2
Comparison between azoospermia subgroup and control group by genotype.

Genotype/allele	Azoospermia subgroup (n=25)			Control group (n=50)			p-value	Odds ratio	95%CI	Etiologic fraction	Preventive fraction
	No.	Frequency	%	No.	Frequency	%
AA	7	0.28	28	27	0.54	54	0.036	0.331	0.117–0.932	***	0.292
AG	6	0.24	24	13	0.26	26	0.851	0.898	0.294–2.739	***	0.034
GG	12	0.48	48	10	0.2	20	0.014	3.692	1.296–10.517	0.397	***
A	20	0.4	40	67	0.67	67	0.001	0.328	0.162–0.663	***	0.318
G	30	0.6	60	33	0.33	33	0.001	3.045	1.508–6.149	0.973	***

• CI: confidence interval.
• ^***
  Etiologic fraction for genotype AA is the source (reference), its value must be 1.

The corresponding frequency in the oligospermia subgroup and the control group was as follows: AA (32 and 58%), AG (28 and 24%), and GG (40 and 18%), respectively, as shown in Table 3.

Table 3
Comparison between oligospermia subgroup and control group by genotype.

Genotype/allele	Oligospermia subgroup (n=25)			Control group (n=50)			p-value	Odds ratio	95%CI	Etiologic fraction	Preventive fraction
	No.	Frequency	%	No.	Frequency	%
AA	8	0.32	32	29	0.58	58	0.036	0.34	0.124–0.936	***	0.294
AG	7	0.28	28	12	0.24	24	0.707	1.231	0.414–3.655	0.069	***
GG	10	0.4	40	9	0.18	18	0.043	3.037	1.034–8.920	0.352	***
A	23	0.46	46	70	0.7	70	0.004	0.365	0.181–0.736	***	0.299
G	27	0.54	54	30	0.3	30	0.004	2.739	1.358–5.524	0.3	***

• CI: confidence interval.
• ^***
  Etiologic fraction for genotype AA is the source (reference), its value must be 1.

The distribution of the FASL-844C/T gene polymorphism was detected by the Tetra-ARMS-PCR technique. At this locus, there are three genotypes for FASL-844C/T such as CC, CT, and TT with band sizes of 192 pb, 192/145 pb, and 145 pb, respectively. In this study, we genotyped the FASL-844C/T gene polymorphism in infertility patients to determine the relationship between their clinical features and the genotypes/allotypes.

The genotypes’ relative frequency of the FASL-844C/T gene polymorphism in the azoospermia subgroup and control group was as follows: CC (32 and 54%), CT (24 and 26%), and TT (44 and 20%), respectively, while the corresponding frequency in the oligospermia subgroup and control group was as follows: CC (28 and 56%), CT (28 and 24%), and TT (44 and 20%), respectively, as shown in Table 4.

Table 4
Comparison between azoospermia and oligospermia subgroups and control group by genotype.

Genotype/allele	Azoospermia subgroup (n=25)			Control group (n=50)			p-value	Odds ratio	95%CI	Etiologic fraction	Preventive fraction
	No.	Frequency	%	No.	Frequency	%
CC	8	0.32	32	27	0.54	54	0.075	0.4	0.146–1.098	***	0.235
CT	6	0.24	24	13	0.26	26	0.851	0.898	0.294–2.739	***	0.034
TT	11	0.44	44	10	0.2	20	0.032	3.142	1.099–8.986	0.356	***
C	22	0.44	44	67	0.67	67	0.007	0.387	0.192–0.776	***	0.28
T	28	0.56	56	33	0.33	33	0.007	2.584	1.287–5.187	0.281	***
Oligospermia subgroup
CC	7	0.28	28	28	0.56	56	0.024	0.305	0.108–0.861	***	0.313
CT	7	0.28	28	12	0.24	24	0.707	1.231	0.414–3.655	0.069	***
TT	11	0.44	44	10	0.2	20	0.032	3.142	1.099–8.986	0.356	***
C	21	0.42	42	68	0.68	68	0.002	0.34	0.169–0.687	***	0.313
T	29	0.58	58	32	0.32	32	0.002	2.934	1.455–5.917	0.313	***

• CI: confidence interval.
• ^***
  Etiologic fraction for genotype CC is the source (reference), its value must be 1.

The current study revealed that there was a considerable difference in the control group compared to the patient group when both groups were compared with azoospermia and oligospermia subgroups, with p<0.05 for genotype and allele frequencies. Statistical analysis showed that FASL-844C/T and FAS-670A/G were risk factors for infertility, and there was an association between their polymorphism and infertility.

Our findings supported those of research done by Hassan et al.¹⁵, who showed that the proportions of the GA and AA genotypes were statistically significantly greater in the group of patients with severe oligozoospermia (p<0.01) than in the group of patients with normozoospermia (30 vs. 20% and 60 vs. 0%, respectively).

The current findings were in line with those of research done by Balkan et al.⁵, who showed a correlation between the high propensity for idiopathic azoospermia and the homozygous genotypes GG and AA in the FAS-670A/G gene polymorphism. Furthermore, it was shown that the heterozygous AG genotype was considerably lower in patients compared to controls, suggesting that heterozygous genotypes in this ­variation may have had a protective effect against idiopathic azoospermia. The FAS-670 A/G gene polymorphism is in fact supported by these findings as a risk factor for male infertility in the Turkish community.

Ji et al.¹⁶ revealed a strong correlation between sperm apoptosis and semen quality and functional polymorphisms in the FAS-670G/A gene promoter (OR 0.48; 95%CI 0.28–0.83). When compared to individuals with the AA genotype, those with the FAS-670 GG genotype exhibited lower rates of apoptosis, which were associated with lower sperm concentration and poorer sperm motility (mean: 90.15×10⁶/mL; 72.44×10⁶/mL; and 58.14×10⁶/mL, respectively, p=0.001).

Asgari et al.⁸ discovered that the presence of the TT, CT, and CT+TT genotypes was related to an elevated risk of infertility. The TT genotype was also associated with a 2.02-fold higher risk of infertility that was statistically significant (p=0.03). Furthermore, the −844T allele was more common in infertile males (59.8%) compared to controls (50.9%). As a result, this difference might be regarded as a risk factor for male infertility in the Western Iranian community.

Idiopathic azoospermia has been discovered to be highly associated with the polymorphism in FASL-844C/T. In other words, Han Chinese men may be predisposed to severe oligozoospermia or idiopathic azoospermia due to a familial predisposition⁹.

CONCLUSION

FAS homozygous mutant genotype (GG) and FASL homozygous mutant genotype (TT) acted as risk factors for male infertility.

• Funding:
  The source of funding for this work was personal finance.
• ETHICAL APPROVAL
  The current study obtained ethical approval from Sawa University, and written consent was taken from all participants in the research (patient group and control group).

ACKNOWLEDGMENTS

We are grateful to Sawa University, College of Health and Medical Technology, and the infertility and IVF center in Al-Sader City Hospital for meeting all of our study needs.

REFERENCES

• ¹ WHO. Laboratory manual for the examination of human semen and semen cervical mucus interaction. 3rd ed. Cambridge University Press; 2013.
• ² Smits RM, Mackenzie-Proctor R, Yazdani A, Stankiewicz MT, Jordan V, Showell MG. Antioxidants for male subfertility. Cochrane Database Syst Rev. 2019;3(3):CD007411. https://doi.org/10.1002/14651858.CD007411.pub4
  » https://doi.org/10.1002/14651858.CD007411.pub4
• ³ Ejzenberg D, Gomes TJO, Monteleone PAA, Serafini PC, Soares-Jr JM, Baracat EC. Prognostic factors for pregnancy after intrauterine insemination. Int J Gynaecol Obstet. 2019;147(1):65-72. https://doi.org/10.1002/ijgo.12898
  » https://doi.org/10.1002/ijgo.12898
• ⁴ Panzan MQ, Mattar R, Maganhin CC, Simões Rdos S, Rossi AG, Motta EL, et al. Evaluation of FAS and caspase-3 in the endometrial tissue of patients with idiopathic infertility and recurrent pregnancy loss. Eur J Obstet Gynecol Reprod Biol. 2013;167(1):47-52. https://doi.org/10.1016/j.ejogrb.2012.10.021
  » https://doi.org/10.1016/j.ejogrb.2012.10.021
• ⁵ Balkan M, Atar M, Erdal ME, Rustemoğlu A, Yildiz I, Gunesacar R, et al. Possible association of FAS and FASLG polymorphisms with the risk of idiopathic azoospermia in southeast Turkey. Genet Test Mol Biomarkers. 2014;18(6):383-8. https://doi.org/10.1089/gtmb.2013.0454
  » https://doi.org/10.1089/gtmb.2013.0454
• ⁶ Synowiec E, Wojcik KA, Izdebska J, Blasiak J, Szaflik J, Szaflik JP. Polymorphisms of the apoptosis-related FAS and FAS ligand genes in keratoconus and Fuchs endothelial corneal dystrophy. Tohoku J Exp Med. 2014;234(1):17-27. https://doi.org/10.1620/tjem.234.17
  » https://doi.org/10.1620/tjem.234.17
• ⁷ Sibley K, Rollinson S, Allan JM, Smith AG, Law GR, Roddam PL, et al. Functional FAS promoter polymorphisms are associated with increased risk of acute myeloid leukemia. Cancer Res. 2003;63(15):4327-30. PMID: 12907599
• ⁸ Asgari R, Yari K, Mansouri K, Bakhtiari M. Association analysis of FAS-670A/G and FASL-844C/T polymorphisms with risk of generalized aggressive periodontitis disease. Biomed Rep. 2017;8(4):391-5. https://doi.org/10.3892/br.2018.1060
  » https://doi.org/10.3892/br.2018.1060
• ⁹ Wang W, Lu N, Xia Y, Gu A, Wu B, Liang J, et al. FAS and FASLG polymorphisms and susceptibility to idiopathic azoospermia or severe oligozoospermia. Reprod Biomed Online. 2009;18(1):141-7. https://doi.org/10.1016/s1472-6483(10)60436-1
  » https://doi.org/10.1016/s1472-6483(10)60436-1
• ¹⁰ Attia H, Finocchi F, Orciani M, Mehdi M, Zidi Jrah I, Lazzarini R, et al. Pro-inflammatory cytokines and microRNAs in male infertility. Mol Biol Rep. 2021;48(8):5935-42. https://doi.org/10.1007/s11033-021-06593-6
  » https://doi.org/10.1007/s11033-021-06593-6
• ¹¹ Ejzenberg D, Jesus Simões M, Pinheiro W, Soares Júnior JM, Serafini PC, Chada Baracat E. Blind aspiration biopsy versus a guided hysteroscopic technique for investigation of the endometrium in infertile women. Histol Histopathol. 2016;31(9):981-6. https://doi.org/10.14670/HH-11-725
  » https://doi.org/10.14670/HH-11-725
• ¹² Shabani K, Hosseini S, Moghbelinejad S. The effects of semen parameters and age on sperm motility of Iranian men. Glob J Fertil Res. 2017;2(1):024-9. https://doi.org/10.17352/gjfr.000008
  » https://doi.org/10.17352/gjfr.000008
• ¹³ Al-Kandari AM, Al-Enezi AN, Ibrahim H, Alkandari O. A population-based study of the epidemiology and the risk factors for male infertility in Kuwait. Urol Ann. 2020;12(4):319-23. https://doi.org/10.4103/UA.UA_50_20
  » https://doi.org/10.4103/UA.UA_50_20
• ¹⁴ Pathak UI, Gabrielsen JS, Lipshultz LI. Cutting-edge evaluation of male infertility. Urol Clin North Am. 2020;47(2):129-38. https://doi.org/10.1016/j.ucl.2019.12.001
  » https://doi.org/10.1016/j.ucl.2019.12.001
• ¹⁵ Hassan GM, Abdul-Hassan IA. FAS and FASL genes polymorphisms and their relationship with the incidence of severe oligozoospermia in a sample of Iraqi patients. Iraqi J Biotechnol. 2017;16(4);60-8.
• ¹⁶ Ji G, Gu A, Hu F, Wang S, Liang J, Xia Y, et al. Polymorphisms in cell death pathway genes are associated with altered sperm apoptosis and poor semen quality. Hum Reprod. 2009;24(10):2439-46. https://doi.org/10.1093/humrep/dep223
  » https://doi.org/10.1093/humrep/dep223

Publication Dates

• Publication in this collection
  16 June 2025
• Date of issue
  2025

History

• Received
  15 Aug 2024
• Accepted
  17 Jan 2025