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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 3  |  Page : 225-231

Evaluation of pro-inflammatory cytokine level in cases of idiopathic recurrent spontaneous miscarriage in Saudi Arabia


Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Prince Sattam Bin Abdulaziz University, Al-Kharj, Riyadh, KSA

Date of Submission14-May-2020
Date of Acceptance27-Jun-2020
Date of Web Publication12-Sep-2020

Correspondence Address:
Dr. Poonam Tyagi
Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Prince Sattam Bin Abdulaziz University, Al-Kharj, Riyadh
KSA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_71_20

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  Abstract 


Background: A balanced stimulation and action of T-helper type 2 (Th2) and type 1 (Th1) immune response is required for a successful embryo implantation and positive outcomes of pregnancy while specific pro-inflammatory cytokine levels either belong to Th1 or Th2 profile which may deteriorate the pregnancy causing miscarriage. Methods: This research study comprised two groups: Group I – pregnant women with a history of recurrent spontaneous miscarriage (RSM, n = 50) and Group II – healthy pregnant controls (n = 50). Blood was collected, and serum was analyzed for interleukin (IL)-6, IL-8, IL-12, IL-18, TNF-α, and interferon (IFN)-γ using ELISA kit. Results: Th1 activity (IL-8, IL-12, IL-18, TNF-α, and IFN-γ) was higher in study Group I irrespective of whether continuing their pregnancy or aborting in comparison to Group II controls (P < 0.001). On the other hand, Th2 activity (IL-6) was decreased in Group I patients (P < 0.001) as compared with normal control Group II. Conclusions: Patients with a history of RSM have significantly increased Th1 activity and decrease in Th2 activity in comparison to normal pregnant women without any history of spontaneous miscarriage in their past.

Keywords: Immunoregulatory pregnancy, interleukin profile, pro-inflammatory cytokines, recurrent spontaneous miscarriages, T-helper cells


How to cite this article:
Tyagi P, Alharthi NS. Evaluation of pro-inflammatory cytokine level in cases of idiopathic recurrent spontaneous miscarriage in Saudi Arabia. Biomed Biotechnol Res J 2020;4:225-31

How to cite this URL:
Tyagi P, Alharthi NS. Evaluation of pro-inflammatory cytokine level in cases of idiopathic recurrent spontaneous miscarriage in Saudi Arabia. Biomed Biotechnol Res J [serial online] 2020 [cited 2020 Sep 25];4:225-31. Available from: http://www.bmbtrj.org/text.asp?2020/4/3/225/294863




  Introduction Top


In the majority of women, the complexity and explanation of recurrent spontaneous miscarriage (RSM) is unidentified and still research work is being conducted to understand the substantial sources for this pathological condition. Recently, it is concluded that certain immune-genetic factors which initiate activate immune system abnormally may play a crucial role and might be responsible for recurrent pregnancy loss.[1] Researchers are paying more attention on decoding the immunobiological role and participation of cytokines in normal healthy pregnancy outcomes; subsequently, the expanding reports of complex cytokine impact within uteroplacental tissues.[2]

Cytokines are the proteins which are released by CD4+ type T-helper (Th) cells, CD8+ cytotoxic T-cells, NK cells, and NKT cells in humans[3] to restraint the inflammatory operation in the body. A gigantic pattern of chemical alterations starting from initiation of normal immune responses, dismissal of allografts, autoimmune diseases, and hypersensitive modulations has been proceeded by extremely potent and versatile cytokine mediators. These cytokines can be classified into two categories according to their function: pro-inflammatory (or TH1) and anti-inflammatory (TH2). Early response to pro-inflammatory cytokines is imperative in stimulating invasion of the blastocyst and the formation of new blood vessels during the process of implantation. Nonetheless, a delayed exposure resulting in a cell-mediated immune response that is harmful to the fetus and results in a failed implantation and early miscarriage.[4]

Various kinds of cytokines such as tumor necrosis factor (TNF)-α, TNF-β, interferon (IFN)-γ, and interleukin (IL)-2 are secreted by Th1 cells. Some Th2 cells and cytotoxic T-cells also released a small amount of TNF-α. However, the maximum amount is secreted during Th1 stimulation reactions and committed to cellular immunity. All Th-1 related cytokines are recruited to be performing various functions like to stimulate macrophages causing cell mediated modifications, in elaboration of resisting infections by intracellular progress of pathogens and also trigger cytotoxic and delayed type of inflammation reactions. The Th2 cells are responsible for stimulating B-cells and eosinophils. The class of ILs such as IL-4, IL-5, IL-6, IL-10, and IL-13 is directly linked with strong antibody reactions to proceed infections with extracellular organisms and categorized as Th2-type cytokines designed for humoral reactions.[5]

The specialized cells found in the uterus and placenta released a massive multitude of cytokines which are moderately responsible for the alteration of the immune response from Th1 to Th2. This shifting and increased production of Th1 cytokines may create embryonic condition more susceptible to inflammation and has been directly linked to spontaneous abortion. This sequence of cytokines contributes to cross-regulation between immune responses and other systems, as observed during the interplay between infection and pregnancy. Recently, In some cases of miscarriages, the altered level of cytokines and their production, accumulated or diminished level in association with reproductive failure is being analyzed and discussed by the scientists in some research studies.[6] The successful outcome is associated with the downregulation of Th1 activity and enhancement of Th2 activity.

The production of cytokines regulates development, growth, and differentiation of the placenta in pregnancy during implantation and proper growth of embryo. The total uterine leukocyte cell population and cytokine receptor expression in placental tissues altered all over the period of gestation. The complex interrelationship between trophoblast and uterine decidual cells, participate and regulate the diversification of cytokine profile, cytokine receptors, adhesion molecules, enzymes and hormones during the length of gestation. Few cytokines such as IL-1 and TNF are directly involved in alteration of embryonic development and plantation process and therefore may threaten the reproductive process and fetal well-being significantly. The successful and positive reproductive outcomes at different phases of pregnancy such as implantation, placental growth and development, maintenance of pregnancy and delivery, depends on complex, gestational age-related interplay between cells of developing embryo origin and the maternal immune system.[7]

The leading immune-regulatory pro-inflammatory cytokines are IL-1α, IL-1β, IL-6, and TNF-α which favor inflammation and initiate quick responses. Alternatively, pro-inflammatory mediators including in this category are Leukemia inhibitory factor (LIF), IFN-γ, oncostatin-M, ciliary neurotrophic factor, transforming growth factor-beta, granulocyte-macrophage colony-stimulating factor, IL-11, IL-12, IL-17, and IL-18. IL-8 and other chemokines that fascinate inflammatory cells are involved in multidirectional processes such as development of endogenous pyrogens (IL-1, IL-6, TNF-α), readjust the integration of pro-inflammatory cytokines and various secondary mediators by the action of both macrophages and mesenchymal cells like fibroblasts, epithelial and endothelial cells and reassure the production of acute phase proteins. The neutrophil chemoattractant IL-8 is the primitive typical chemokine, and it stimulates neutrophils to degranulate and induce tissue damage. On the other hand, IL-6 is associated with humoral immunity (Th2 response).

Considering all these points, this research study was designed to measure the pro-inflammatory cytokine level, mainly IL-6, IL-8, IL-12, IL-18, IFN-α, and TFN-γ, in pregnant women having a history of recurrent miscarriage without any knowing reason and compared levels with healthy pregnant women who had a childbirth.


  Methods Top


This research study enrolled 100 females with an age group of 21–41 years at King Saud Medical City, Riyadh. Blood samples were collected after getting written informed consent from the patients. An approval from the university ethics committee was obtained for this research study. All patients included in this research study were divided into two groups.

  1. Study group: Fifty pregnant females having a history of RSM and treated/admitted in the hospital were selected as a study group. Idiopathic recurrent miscarriage was considered by the exclusion of autoimmune, anatomic, infectious, genetic, endocrine, and infectious disorders
  2. Control group: Fifty normal healthy pregnant women who attended obstetrics and gynecology outpatient clinic for a routine checkup, having <20 weeks of gestation with at least one live birth, and without any history of the treatment of abortion or infertility and normal ultrasonographic evidence were recruited as a control group.


Inclusion criteria for the study group patients were as follows:

  1. At least two consecutive miscarriages
  2. Female karyotype in normal range
  3. Normal pelvic ultrasonography and hysterosalpingography
  4. Negative values for antiphospholipid antibodies
  5. Any positive inherited and/or acquired coagulopathies
  6. Negative chronic disorders
  7. Normal fertility and thyroid hormonal profile
  8. No previous live births
  9. No uterine pathologies.


Exclusion criteria for both group patients were as follows:

  1. Noticeable previous medical (hypertension, diabetes, and asthma) problems
  2. Noticeable previous gynecological-related issues
  3. Medication intake.


Sampling

Blood samples were collected from both selected group patients and processed generally within 1 h. Samples were centrifuged at 3,000 rpm for 10 min at 4°C and stored at −80°C after aliquot ting the serum until the ELISA analysis performed.

Determination of interleukin-6, interleukin-8, interleukin-12, interleukin-18, TFN-α, and interferon-γ

At the time of analysis process, all previously stored frozen serum samples, thawed properly and centrifuged at 10,000 rpm for 5 minute at 40 °C to remove out any debris.

The expression level of IL-8, IL-12, IL-18, and IFN-γ was determined in triplicate using ELISA (MyBioSource) and IL-6 cytokines (RayBiotech Inc., USA). The TFN-α level was analyzed using ELISA kit that was brought from Cusabio Biotech Co., Ltd. The absorbance value was evaluated using ELISA reader. Exact concentrations of cytokines were analyzed by comparing their respective absorbencies with those obtained for the reference standards plotted on a standard curve using reference recombinant cytokines. All procedures and reagent preparation were done according to the instruction of manufacturer. The assays are based on a solid-phase enzyme immunoassay employing the dual-antibody sandwich principle and were performed according to the manufacturer's procedures, and recombinant reference cytokine samples served as positive controls for calibration. No cross-reactions against other standard cytokines were observed.

Statistical analysis

Data were presented as mean ± standard deviation (SD). To compare the differences of the studied parameters (IL-6, IL-8, IL-12, IL-18, IFN-γ, and TNF-α) between women with RSM and age-matched fertile controls, Student's t-test was used. Statistical significance was calculated using the Statistical Package for Social Sciences version 20.0 (SPSS, NY, USA), and P < 0.05 was considered statistically significant.


  Results Top


There were 100 patients included in this research study: 50 pregnant women having a history of miscarriage without any known reason and 50 healthy pregnant women recruited as controls.

The mean ± SD level of various clinical characteristics, including age, body mass index, hemoglobin percentage, number of abortions, and time since last pregnancy of the women participating in this study, is summarized in [Table 1]. There is no statistically significant difference in all these characteristics except number of abortions in case of study group patients (P < 0.001).
Table 1: Comparative clinical characteristics of study group and control group patients

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Hormonal levels including fertility profile (follicle-stimulating hormone [FSH], luteinizing hormone', and prolactin), estrogen, and thyroid-stimulating hormone are also estimated and tabulated in [Table 2]. FSH level was statistically significantly different (6.14 ± 1.32) in case of women having a history of miscarriage as compared with pregnant control group women (9.13 ± 1.10) (P < 0.001).
Table 2: Hormonal level in study and control group patients

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As presented in [Table 3], a statistically significant difference was observed in cytokine production between study group pregnant patients having a history of idiopathic recurrent miscarriage and normal healthy pregnant controls.
Table 3: Cytokine level (pg/ml) in serum samples among the study group

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The mean ± SD level of IL-6 cytokine level was found significantly low (79.63 ± 1.49) in women having a history of miscarriage in comparison to pregnant control group females (93.26 ± 1.63). The P value was calculated as < 0.001.

The increased level of IL-8 cytokines (72.97 ± 1.67 pg/ml) was observed in pregnant females with a history of idiopathic recurrent miscarriage as compared with normal healthy pregnant controls (64.67 ± 1.36) (P < 0.001)

The highly elevated level of IL-12 cytokines (51.51 ± 1.97) in serum samples of study group patients with a history of miscarriage was identified as compared to healthy pregnant controls (18.23 ± 3.13). The result was significant statistically (P < 0.001).

The mean ± SD level of IL-18 type of cytokine level was found significantly increased (596.18 ± 99.16 pg/ml) in patients having a history of recurrent miscarriage in comparison to controls (329 ± 101.18). The results were found significant statistically (P < 0.001).

Mean ± SD level of IFN-±was also observed elevated significantly (216.86 ± 59.86) in serum of women suffering from idiopathic recurrent miscarriage as compared with pregnant controls (123.12 ± 61.81) (P < 0.001).

Increased production of TNF- α (335.13 ± 4.97) was measured in case of study group patients with a history of recurrent miscarriage as compared to normal healthy pregnant controls (180.79 ± 4.12). The results were significant statistically (P < 0.001).


  Discussion Top


Although the fetus is partially sensitive to the mother, however, generally, it is not rejected by the maternal immune system. Recently, it has been concluded in some research studies that contained an immune cell approach which is an important component of the fetomaternal alliance and is possible to play a unique function in the immune cellular regulation and shelter of the fetus from a maternal immune system reaction. The systemic maternal immune hypersensitive responses are partial in favor of a Th2 cytokine profile. Our analyzed research data provide support and confirm previously published data.[8],[9],[10],[11]

The pattern of cytokines has been suggested to be muddled in a positive or negative progression of ongoing pregnancies.[9],[12],[13] Various cytokines have been established to have a detrimental effect on conceptus. These cytokines are representative of Th1 cells which promote several cytotoxic and inflammatory responses. Th-2 cells, on the other hand, released cytokines IL- 6, which is related with humoral immunity and highly responsible in preserving a normal pregnancy.[14]

Previous studies confirmed that cytokines and related inflammatory reactions during implantation and progression of pregnancy portray a significant role in cases of recurrent miscarriage. Embryotoxic activity was detected in supernatants collected from trophoblast-activated peripheral blood mononuclear cells in women having a history of recurrent miscarriage. Unlikely, normal women neither were embryotoxic nor contained Th1 type of cytokines but mostly contained Th2-type cytokines such as IL-6, IL-10, and IL-4.[15] Th2-type cytokines are released immediately from placental tissue in culture, as discussed in a research study previously.[16] This has been shown in animal studies, jointly with the perception that pregnancy loss can be induced by Th1 cytokines[17] and positively regulated by Th2 cytokines.[18]

In this research study, we also presumed that the best way to understand the pathological events, mainly the participation and action of pro-inflammatory cytokines, is the observation of the level of cytokines in women having a history of idiopathic recurrent miscarriage during their pregnancy in comparison to normal healthy pregnant controls.

In this study, we observed that in the serum sample of women with a history of recurrent miscarriage, the level of IL-6 cytokines was lower as compared with control samples. The results are significant statistically (P < 0.001). However, the blood level of IL-8, IL-12, IL-18, IFN-γ, and TNF-α was significantly higher statistically in women with a history of RSM in comparison to normal healthy controls (P < 0.001). If we would have been able to recognize the embryo implantation and development controlling process, the cases of RSM might be handled more precisely to inhibit pregnancy failure. The cases of RSM might be handled more precisely to inhibit pregnancy failure. Assessment and identification of such parameters with blood samples may be useful to review implantation process.[19] For a long time, scientists are paying more attention seriously toward the relationship between the reproductive immunological process and the performance of cytokines during embryo implantation process and pregnancy outcomes. Recent research studies have further explained this theory by disputing that reproductive failure is an intensified TH1-type immunity.[13],[20],[21] Nonetheless, recurrent miscarriage in cases of TH2 dominant immunity was also identified and reported in women in few research studies.[20],[22] In our research study, patients with RSM conferred increased levels of cytokines secreted by TH1 cells and lower levels of cytokines produced by TH2 cells. In addition, there might be a persistent imbalance of TH1/TH2 in patients with RSM. The levels of pro-inflammatory cytokines were significantly different in patients with RPL than those of the control group in our study. The results were found significant statistically.

Recently, few research studies reported increased levels of cytokines such as IL-2, IFN-γ, and TNF-α and decreased levels of IL-4, IL-5, IL-6, and IL-10 in women having a history of recurrent miscarriage while compared with normal pregnant women. The level of cytokines was analyzed in activated mitogen peripheral blood mononuclear cells. It proved a higher Th1 tendency in patients having a history of recurrent miscarriage and increased Th2 shifting in normal healthy pregnancy.[15] It has been concluded that there is a pro-Th2 shift of cytokine profile at the maternal–fetal coherence in patients with normal pregnant women as compared with recurrent miscarriage suffering pregnant women.[23],[24]

Interleukin-6

Although extensive studies have been made on the role of several cytokines in different stages of pregnancy, the inconclusive results make the interpretation of their effect on pregnancy difficult.[25] One of the well-studied cytokine is IL-6 and reduction of this protein in the mid- secretary phase inside endometrial tissue is prone to recurrent pregnancy loss in women.[26] As compared with normal women, patients with the history of RPL have significantly lower serum concentrations of Th2 cytokines including IL-6 and IL-10.[27] The production of pro-inflammatory and anti-inflammatory cytokines is partly under genetic control. In the present research, the emphasis is laid on IL-6 measurement in serum as this cytokine supposed to have a crucial role on the implanting embryo. There are few scientific reports concerning the role of IL-6 in miscarriages, and the related results vary significantly. The role of IL-6 in regulating trophoblast invasion is currently not very clear. In a research study, IL-6 was observed to enhance trophoblast invasion.[28] Yamada et al., 2004, revealed that the fetal karyotype is affected by the cytokine level present in the maternal circulation in cases of recurrent miscarriages.[29] The low level of IL-6 was observed in the present study which is similar to the results of some other scientific studies.[30],[31] Researchers concluded in their study that the low level of IL-6 in threatened abortions who aborted during the study and the reason for this negative outcome is due to lack of balance of cytokines that stimulate placental growth and rapid invasion of trophoblastic cells within the maternal endometrium. Communication between trophoblastic and decidual cells is mediated by cytokines and cell-mediated receptors. In an another research study, scientists analyzed highly elevated serum IL-6 levels in the women with a history of recurrent miscarriage in comparison to normal pregnant controls, which is showing and confirming a lethal effect on the maturation and progression of embryo.[16],[32] However, our data is not similar as reported in the earlier studies[16] and showing a decrease in plasma levels of IL-6 in women with miscarriage as compared with normal pregnant control group.

Interleukin-8

As cytokines are pleiotrophic and multipotent by nature and at different concentrations, can intercede various actions during time periods of gestation.[33] IL-8 is a chemokine and prognostic of dynamic inflammatory and growth-controlling properties. This IL is a robust captivator of neutrophil cells and angiogenic agent.[34] During pregnancy, human trophoblast cells released IL-8 spontaneously and defend against chorioamnionitis condition.[35] It has also been indicated that IL-8 elaborated inaction of cervical stimulation, growth, and/or burst of fetal membranes at maturation.[36] Presently, not much research work has been done and few data is available showing the role of IL-8 during first trimester of pregnancy and their relation with spontaneous recurrent fetal loss.

We also analyzed a statistically significant higher level of IL-8 cytokines in pregnant women with a history of RPL as compared with normal healthy women during their pregnancy. In a research study contrast data was presented by Koumantaki et al. in 2001. These researchers reported decreased IL-8 level in women plasma with spontaneous abortion as compared with normal pregnancy. There are no data available in literature, showing the role of IL-8 in relation to recurrent miscarriage.[37] Laham et al. in 1993 also found no significant changes of IL-8 level in peripheral plasma sample of the recurrent miscarriage woman group collected during pregnancy as compared with normal pregnant controls. Our results are more compatible and providing a direction with confirmed involvement of the immune system, subsequently inflammatory pathogenesis in recurrent pregnancy loss.[36]

Interleukin-12

A macrophage and B-cell-derived cytokine is IL-12 which is responsible for biological activity and also plays a critical role for Th1 initiation process.[38] It is highly predominating and perceptive indicator of the Th1-type reactions and their regulation process and also act as inhibitor of the Th2-type response as studied in few research studies in vivo.[39] This cytokine was shown to act as an immune enhancer as involved in various mechanisms during pregnancy, but the majority of the researches work done in “in vitro” condition. This cytokine was also observed in the decidua of miscarried women with Th immunity to trophoblast.[40] IL-12 cytokine level was also found in a significantly higher amount in the endometrium of women suffering from unexplained RSM as compared with the healthy women selected as controls.[38] In other research studies, the role and level of IL-12 in the first-trimester pregnancy and in pregnancy loss were concluded as very low and in threatened abortion totally missing. In contrast with our presented data, some studies[38],[41] show that there was no difference in IL-12 concentrations between normal pregnant women and women with spontaneous pregnancy loss. On the other hand, a research study done by Wilson et al. in 1997 observed a statistically significant higher level of IL-12 in recurrent pregnancy loss patients than in normal pregnant women. Results are similar, as reported in our present study.[38]

Interleukin-18

Both IL-18 and IL-12 are liable and provoke the synthesis of IFN-γ.[42] and have similar functions in relation of participation is successful pregnancy outcomes. However, a mutual equivalence among IL-12 and IL-18 is required for the protection of the maternal uterus from immunologic alterations, which has been recommended by the scientists in few research studies.[43] In a systematic review, analysis researchers appraised the expressions of IL-12, IL-18, and other cytokines during pregnancy. Their results confirmed that simultaneously these two cytokines possessed symbolic controlling functions during pregnancy; an extremely high or low level of any one or both of these cytokines may result in spontaneous pregnancy loss and implantation failure or preeclampsia.[44],[45],[46] Our findings also suggested that, along with IL-18 and IL-12, levels in blood were higher in women with a history of RPL.

Interferon-γ

In the present study, IFN-γ levels were significantly higher in patients with recurrent miscarriage as compared with normal healthy pregnant controls. The research data provided in literature confirm that the higher production of TH1 cytokines including IFN-γ has been directly associated with pregnancy loss.[11],[47] Our results also approved this theory. In a research study, the diminished level of IFN-γ cytokines was observed in the first-trimester serum samples of pregnant study group females having RPL history as compared with nonpregnant controls, however, results were not statistically significant. No disparity was observed between the levels of IFN-γ in those RPL patients who successfully completed their pregnancy compared with those who went on to miscarry.

Tumor necrosis factor-α

In our study, the level of TNF-α cytokines was significantly higher in pregnant women having a history of miscarriage as compared with normal pregnant healthy women. Th1- or Th2-type responses released cytokine TNF-α which is an important immune-modulating cytokine. This higher amount of these cytokines is identified as implying various effects into the immature placenta, such as inflammation, survival, apoptosis, cell migration, proliferation, and differentiation. Along with other inflammatory cytokines, TNF-α may play critical roles in causing pregnancy complications, such as recurrent miscarriage, premature rupture of membranes (PROM), preeclampsia, and IUGR. Depending on gestational period during pregnancy, TNF-α generation by PBMC is restrained at the mRNA level during the initial phase of pregnancy, and a significantly higher and maximum level increases at the time of the third trimester and close to full time of gestation as was described in a study.[48] The maximum level of TNF-α amount is required and critically related to the initiation of labor pain in mice[17] and women.[49] It has also been reported that the production of soluble receptors in the placenta is also related to restraining TNF-α function during pregnancy.


  Conclusion Top


The pro-inflammatory cytokine level related to Th1 immune response was significantly higher, and Th2-related cytokines were significantly low in women having a history of recurrent miscarriage in comparison to normal pregnant controls. A positive relationship between Th cells and pregnancy loss has been established in our present study. These data might help to adopt the preventive and therapeutic methods for solving the recurrent miscarriage issues in patients having no other reason for their problem. This observation would also throw light for the development of methods to manipulate the maternal immune system toward a Th2-type situation that would favor the continuation of pregnancy to patients who are seeking for a hope for their problems. It is recommended that mainly pro-inflammatory cytokine profile might be recommended as an indirect marker for monitoring problems of recurrent miscarriage, which needs further confirmations.

Acknowledgment

The authors are grateful to the Administration Department of the College of Applied Medical Sciences and Deanship of Scientific Research, Prince Sattam Bin Abdulaziz University, Al Kharj, Saudi Arabia, for providing their help and support for completing this scientific research study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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