@2024 Afarand., IRAN

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ISSN: 2251-8215    Sarem Journal of Reproductive Medicine 2018;2(3):99-103

Background
Spontaneous abortion is one of the most common complications of pregnancy. Three or more ‎spontaneous pregnancy terminations before the 20th week or even the 28th week is called recurrent ‎spontaneous abortion (RSA) [1].‎
Previous Studies
The causes of recurrent spontaneous abortion are multifactorial, but it can be divided into two major ‎embryonic and maternal categories. ... [2, 3]. In general, the cause of about half of the cases of repeated ‎abortions is unknown, in which the role of immunologic factors is more remarkable than other factors ‎‎[4]. Human leukocyte antigen (HLA-G) is a non-classic molecule of HLA-class I, which, unlike classical ‎HLA, has a low polymorphism. One of the polymorphisms known in this gene is 14-bp polymorphism ‎in Exon 8, which is associated with the stability of the mRNA in HLA-G gene and its different ‎processing pattern. This has an effect on the expression and function of HLA-G during pregnancy. The ‎HLA-G molecule was initially observed on aggressive trophoblast cells and is thought to have ‎mediating role in relation to mother and fetus [4].‎ Human leukocyte antigen (HLA-G) is a ligand for a natural lethal cell receptor (NK) and can prevent the ‎activity of NK cells. In the case of the lack of limitation, it can potentially damage the fetus [5]. The ‎promoter of HLA-G is highly associated with the risk of spontaneous abortion, and the imbalance in the ‎promoter prevents this causal relationship [6]. ... [7-11]. ‎ It has been suggested that insertion / removal of 14 pairs (14bp / -14bp) in the unregistered region of ‎the 3rd HLA-G gene in exon 8 affects the transcription of the gene. Studies show that insertion of 14 ‎pairs in this gene is associated with low levels of HLA-G solution, and this increases the risk of ‎spontaneous abortion. Some studies have shown that homozygous women have a higher risk of ‎abortion for insertion of 14 buffer pair, and that their plasma levels of HLA-G were significantly lower ‎than that of heterozygous or non-14bp individuals [11-14].‎
Aim(s)
The aim of this study was to investigate the association between + 14bp / -14bp polymorphism of ‎HLA-G gene and RSA history among Iranian women.‎
Research type
This is a case-control study.
Research society, place and time
This research was conducted on Iranian women referring to Repetitive Abortion Clinic of Sarem ‎Hospital in Tehran during the second half of 2012.‎
Sampling method and number
Blood samples were taken From 50 women with recurrent spontaneous abortion, whose results of ‎karyotype, hysteroscopy conditions, hormonal values, infections and blood coagulation were normal, ‎and 50 women with a normal pregnancy with at least two children.‎
Used devices&materials
DNA extraction: To extract the DNA from the blood sample of people, Fermentas extraction kit, the ‎K0512 catalog number (Thermo Fisher Scientific, USA) was used. At first, 200 μl blood sample was ‎mixed with 400 μL of lysing solution and incubated for 5 minutes at 65 ° C. Then 600 μl of chloroform ‎was added to the samples and suspended and then centrifuged at 10000 RPM for 2 minutes. The ‎supernatant was transferred to a fresh tube and 800 μl of the sediment solution was added and mixed at ‎room temperature for 1-2 minutes. In the next step, the sample was centrifuged at 10000 rpm for 2 ‎minutes. After discharging the supernatant, the DNA precipitation was solved in 100 μl of 1/2 molar ‎NaCl solution. By adding 300 μL of cold ethanol, the DNA was deposited for 10 minutes at -20 ° C. Then ‎it was centrifuged for 3 to 4 minutes (10,000 rpm) and the purified DNA was solved in 100 μl of non-‎nucleic acid water and it was used to carry out PCR.‎ Polymerase Chain Reaction (PCR): PCR reaction was performed using GE14 HLAG 5' ‎GTGATGGGCTGTTTAAAGTGTCACC-3 and RHG4: 5'-GGAAGGAATGCAGTTCAGCATGA-3 ' primers [11, ‎‎15, 16]. Reaction compounds for each sample included 25 μL of 2X Fermentas main mix, catalog ‎number: K0171 (Thermo Fisher Scientific, USA). One microgram of DNA pattern and 10 Pico moles of ‎each primer and distilled water was as needed value to a volume of 50 μL. The PCR process was ‎carried out in a thermo cycler device including the start of denaturation, a triple cycle at 95 ° C, ‎followed by 30 cycles of 30 seconds denaturation at 90 ° C, 60 seconds connection at 64 ° C, 120 ‎seconds reproduction at 72 ° C, and finally 10 minutes at 72 ° C. ‎ PCR products were analyzed using 10% polyacrylamide gel electrophoresis using a gel-documentation ‎system. The size of the replicated part was 224 bp in cases with 14 nucleotide sequences, and it was ‎‎210 bp in cases without 14 nucleotides, which appeared in polyacrylamide gel, either homozygous or ‎heterozygote. The sequence for the 14 nucleotides was reported as 5'-ATTTGTTCATGCCT-3 '(Fig. 1) ‎‎[14].‎ Statistical analysis of data was done using chi-square test by SPSS software.‎
Findings by Text
The mean age of patients was 31.42 ± 4.60 years, the highest and lowest age was 40 and 22 years, ‎respectively. On average, these patients had 2.82 ± 1.26 abortions, with a maximum of 10 cases and a ‎minimum of 2 cases. The mean age of puberty in these individuals was 13.00 ± 1.10. The highest and ‎lowest age of maturity was 10 and 16 years old, respectively. The menstruation condition was normal ‎and regular in 78% of these patients, and 22% had irregular menstruation, that most of them had either ‎abortion recently or had abnormal menstruation after experience of abortion. A doctor was consulted ‎in this regard, so that this issue does not reject the recurrent spontaneous abortion of these people. ‎Homozygote genotype was observed in 60% of people with recurrent spontaneous abortion, while in ‎the control group, only 34% of the homozygote genotypes were identified which was statistically ‎significant (p = 0.034, Table. 1). Odds ratio (OR) obtained from the comparison of homozygote with ‎heterozygote was 2.91.‎
Main comparison to the similar studies
‎.... [17-19]. In Hviid et al. [11], 61 couples with more than 3 spontaneous abortions were compared ‎with 47 healthy couples as controls with gene sequencing and RFLP method. There was no statistically ‎significant difference in the genotype of these genes in these individuals. However, the polymorphism ‎of removing 14bp of exon 8 in patients was sporadic. There was homozygous allele eliminated 14bp in ‎‎15% of patients, while this polymorphism was present in 2% of control subjects and 14bp ‎polymorphism was more in form of heterozygous in the control group. In their view, the chance of a ‎pregnancy success in heterozygous mothers was higher than that of homozygous mothers, as ‎heterozygote individuals may have a better regulation of concentration of mRNA isoforms and, ‎consequently, protein levels. Pandey et al. [12], according to a study in the United States, have suggested that the 14Bp / -14bp ‎polymorphism in the 3 'untranslated HLA-G region in exon 8 affects the transcription of this gene. ‎Immunotherapy studies using a probe indicate that the + 14bp in this gene is associated with low ‎levels of HLA-G solution and increases the risk of spontaneous abortion. Some studies have shown that ‎homozygote + 14bp women have an increased risk of spontaneous abortion and that their plasma ‎levels of HLA-G are also significantly lower than heterozygote individuals or the persons lack 14-bp ‎sequences. 14bp polymorphism in Exon 8 is associated with the stability mRNA of the HLA-G gene and ‎its different processing pattern, thus affecting the expression and function of HLA-G during pregnancy ‎‎[10, 15]. In Tripathi et al., In India, the frequency of heterozygote -14bp / + 14bp genotype in RSA women was ‎statistically increased. Another study by Yan et al. in China did not show the significant difference ‎between the genotype of 109 fertile female as a control group and 79 women with a history of RSA in ‎terms of frequency of 14bp or 14bp alleles between the control and RSA subjects. However, the 14bp ‎allele in RSA women was more than the control group, and the sick patients often had heterozygote -‎‎14bp / + 14bp genotype. Another study in China was performed by Xue et al. [10] and the review of 24 ‎RSA and 88 normal individuals showed that the frequency of 14bp / + 14bp heterozygote genotype was ‎significantly higher in RSA patients compared to normal people. These results were not consistent with ‎the results obtained in Denmark [11]. ‎
Suggestions
More studies are needed using more samples and a variety of ethnic races in the country to investigate ‎this relationship as a predisposing factor for RSA. ‎
Limitations

Conclusions
The + 14bp / -14bp polymorphism of the HLA-G gene differs among Iranian women in terms of the ‎frequency of heterozygous and homozygous genotypes with a history of RSA between healthy and ‎unhealthy women. ‎
Acknowledgments

Conflict of interest

Ethical Permissions
Informed consent was obtained from the women participating in this study, according to medical ‎ethics codes.
Funding Sources
This article is the result of a master's degree dissertation of the Islamic Azad University of Ahar ‎Branch, which was carried out using the credits of the Sarem Cell Research Center.‎

TABLES and CHARTS

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