Research
HJOG 2026, 25 (1), 43-54 | doi: 10.33574/hjog.0614
Nedhal Mahmoud Kaleefah1, Bushra Jabbar Al-Tmimi2, Asmaa Kadhim Gatea3
1 Department of Medical Biotechnology, College of Science, Ashur University, Diyala, Iraq
2 Department of Microbiology, College of Medicine, University of Babylon, Babylon, Iraq
3 Department of Obstetrics and Gynaecology, College of Medicine, University of Babylon, Babylon, Iraq
Correspondence: Nedhal Mahmuod Kaleefah, Ph.D. Microbiology/ Department of Microbiology, College of Medicine, Diyala University, 32001, Iraq, e-mail: nedalalazzwy@gmail.com
Abstract
Background and Aim: This study investigates the influence of Cytomegalovirus (CMV) antibodies, specifically CMV IgG and IgM, on the expression of CD34+ and HLA-F in the peripheral blood of pregnant women. The research aims to assess the association of these immunological markers with risk factors for unexplained spontaneous miscarriage, with a particular focus on maternal health challenges in regions like Iraq.
Methods: A cross-sectional study conducted in Iraq, which consisted of 200 blood samples taken from 100 healthy pregnant women and 100 recent unexplained spontaneous miscarriage (USM) cases from two Iraqi hospitals, focusing on cases of USM and healthy pregnant women. It used ELISA for serological detection of CMV IgG and IgM and HLA-F and CD34 concentrations.
Results: A moderate positive correlation with statistical significance (r = 0.490, P ≤ 0.0001) between the levels of CD34+ and the HLA-F in both groups. Furthermore, a significant statistical relationship was found between CD34+ (p ≤ 0.028) and HLA-F (p ≤ 0.017) levels and the presence of CMV IgG in the miscarried group. The results indicated a critical association (p ≤ 0.001) between immune factor concentrations and the incidence of unexplained spontaneous abortion. The levels of CD34+ and HLA-F were lower in women who experienced miscarriages, measured at (1.54±0.34) and (2.65±0.56) ng/ml. Furthermore, the study found a significant association between the number of miscarriages and lower HLA-F concentration in miscarriages.
Conclusions: This study indicates a notable link between reduced expression of immune markers and the incidence of USM in women. Recurrent miscarriage, maternal age, and first trimester are also potentially associated risk factors. Further studies are needed to confirm the association with CMV IgG.
Keywords: Cytomegalovirus Antibodies, CD34+ levels, ELISA Technique, HLA-F Concentration, Unexplained Spontaneous Miscarriage (USM).
Introduction
Human Cytomegalovirus (HCMV) is among the most common herpes viruses, with an 83% seroprevalence worldwide. It is a double-stranded DNA virus [1]. Primary infection transmission rates increase with gestational age, with significant foetal consequences more common when the infection begins before 20 weeks of pregnancy [2]. Furthermore, it exhibits two phases: a productive phase involving virion production and a latent phase characterized by regulated gene transcription without virion production [3]. It possesses immunomodulatory properties that disrupt the host immune system and cytotrophoblast self-regulation, potentially leading to preterm birth or miscarriage [4]. The virus can infect various cell types, encompassing fibroblasts, hepatocytes, immune cells like macrophages and dendritic cells, and muscle cells [5].
Moreover, among the cells HCMV targets are the CD34+ hematopoietic progenitor cells (HPCs), the site for its latency [6]. Circulating endothelial progenitor cells (EPCs) may be necessary throughout pregnancy to ensure proper placentation and embryonic growth [7]. The CD34+ protein is a biomarker for hematopoietic stem cell progenitors with therapeutic potential in hematological diseases. It is also found in non-hematopoietic and cancer stem cells, contributing to proliferation, differentiation, lymphocyte adherence, and morphogenesis [8]. The risk of spontaneous miscarriage is enhanced when the peripheral blood CD34+ cell count is low [9]. The number of CD34+ cells in umbilical cord blood increases with maternal age, allowing the selection of cord blood in the cord blood bank based on maternal age [10]. Human leukocyte antigens (HLAs) are crucial for innate and adaptive immunity, functioning in antigen presentation, and being ligands for NK cell receptors. Specifically, HLA-F acts as a regulatory molecule that impacts immunological responses during and after pregnancy, capable of presenting various peptides [11]. Adaptation of NK cells to chronic viral infections improves their long-term viral control. However, it is critical to determine if CMV infection increases HLA-F open conformer expression, which might aid NK cell recruitment and activation via KIR3DS1 (KIR3DS1-activating NK receptor) [12].
A member of the HLA class I family, HLA-F has been associated with multiple physiological and pathological processes, such as autoimmune illnesses, viral infections, cancer, and pregnancy [13]. This marker can interact with various immune cells, including T cells and natural killer receptors, which are crucial for maintaining homeostasis and initiating an immune response against foreign or infectious antigens [14]. Their expression was upregulated and may play a vital role in the progression of viral infection [15]. Their role in maternal-fetal immune tolerance to placental tissue is associated with protecting the invading extravillous trophoblasts (EVT) from immunological attack because EVT cell surface HLA-F interacts with the decidua’s maternal immune cells [16].
Unexplained spontaneous abortion is a significant clinical issue, especially in areas with high maternal mortality. Cytomegalovirus (CMV) infection is a suspected but unproven cause of miscarriage. Emerging research highlights the importance of immunological markers for maternal-fetal tolerance and placental development, specifically HLA-F, which protects trophoblasts from immune rejection, and CD34+ angiogenic stem cells, vital for placental implantation and revascularization. Deficiencies in these markers can lead to pregnancy failure. The relationship between CMV infection, HLA-F and CD34+ expression, and the mechanisms of unexplained miscarriage requires further investigation, particularly in Iraq, where maternal health challenges are pronounced and miscarriage mechanisms are poorly understood.
Methodology
Ethical and Official Authorisations
The research project was submitted to the Ethics Committee of the College of Medicine Council at the University of Babylon for graduation requirements and received approval. The Medical Research Committee of the Diyala and Babylon Health Directorates within the Iraqi Ministry of Health was approached. After deliberation, the project was approved, and the letter was dispatched. The book, identified by 8199, is dated February 12, 2024.
Criteria for Sample Selection
All participants were between 20 and 40 years of age. The miscarriage group consisted of women who had recently experienced spontaneous miscarriages, whether complete or partial, without any surgical intervention, before 20 weeks of gestation. The selection criteria were determined by the lack of a known aetiology for the miscarriage, including congenital uterine abnormalities, fibroids, autoimmune disorders, and cervical insufficiency. Exclusions were made for cases of molar pregnancy, ectopic pregnancy, intrauterine foetal death, and absence of a foetal heartbeat. The control group comprised apparently healthy pregnant women without a history of miscarriage, foetal death, or any reproductive or general health issues.
Study Design
A cross-sectional study was conducted from February 15th, 2024, to September 15th, 2024, after obtaining verbal consent from the study participants and collecting all necessary data. One hundred women with USM of less than 20 weeks and 100 healthy pregnant women served as controls. A collection of emergency rooms belonging to the maternity department of Al-Batoul Hospital for Maternity and Children, and Babylon Hospital Maternity and Children.
Collection Samples
Following a diagnosis by specialist physicians through clinical examination of the cervix, ultrasound imaging, or observation of foetal tissue expelled from the uterus, samples are collected before the intervention. A volume of 2 millilitres of peripheral blood samples is necessary for each group, which was taken using disposable syringes (Shandong, China) after sterilisation and deposited in tubes with gel tubes (Arth Alrafidain, Iraq). The samples were centrifuged (Hettich, Germany) at 5000 RPM for 5 minutes, and the serum was transferred to a clean tube. The serum samples in gel tubes were preserved at -20°C until needed.
Processing Specimens and Methods
The samples were delivered to the ELISA laboratory at the Virus Detection Unit of Al-Batoul Hospital using an ELISA technique (washer and reader) (BioTek-USA). Following thawing, a subsequent centrifugation at 5000 RPM for 5 minutes was performed to confirm the absence of impurities. The washing and standard control solutions were prepared, ensuring they were all maintained at room temperature [17].
Detection of CMV IgG and IgM by Qualitative ELISA
The ELISA kits include separate components for CMV IgM and IgG, which Demeditec of Germany manufactures. Standard, control, and diluted samples were carefully transferred to appropriate containers and vials. The substrate sample was then placed in position A1. After making the additions described in the kit’s package insert, the standard plate was covered with a protective cover, and the incubation was set at 37°C for one hour. After the time was up, the covers were removed, and the standard plate was washed by the ELISA washer for five times using the kit’s buffer, after having been diluted to the required ratio. The conjugated substrate solution (TMB) was added to each well. The plate was left to incubate for 15 minutes without light. The stop solution, which changes color from blue to yellow, was then added. The absorbance at 450 and 620 nm was adjusted for 30 minutes. The ELISA reader was used to determine the absorbance values and compare the results with the positive and negative control values. The acceptable range for absorbance values should be between 0.150 and 1.300. Positive and negative samples were identified by comparison with the Cut-off equation.
Measurement of Concentration of CD34+ and HLA-F by Quantitative ELISA
The ELISA sandwich method was used to determine the concentrations of CD34+ (MyBioSource, USA) and human HLA class I histocompatibility antigen, alpha chain F (MyBioSource, USA), following the manufacturer’s standard operating procedure and the work scale provided in the book. The experiment for each CD34+ and HLA-F marker involves preparing reagents, standard solutions, and samples; inserting strips; introducing standard solutions; transferring sample diluent and serum; applying sealant; incubating for 60 minutes; and transferring streptavidin-HRP. Substrate solutions A and B are then dispensed into wells. The plate is incubated at 37°C for 10 minutes, then 50 µl of stop solution is dispensed. Each well’s optical density (Optical Densitometry value) is measured using a microplate reader, calibrated to 450 nm, within 10 minutes. Create a standard curve by plotting optical density against concentration using computer-based curve-fitting software and regression analysis for optimal alignment [18].
Statistical Analysis
The statistical analysis used version 28 of the Statistical Package for the Social Sciences (SPSS Inc., Chicago, USA). Continuous variables were articulated as the mean ± standard deviation. Specifically, an independent T-test for CD34+ and HLA-F concentrations and a chi-square test for CMV IgG and IgM were employed. A p-value below 0.05, or 0.001, was used to determine statistical significance [19].
Results
Seroprevalence of CMV IgG and IgM between Both Groups
A statistically significant difference exists between the miscarriage and control groups, with IgM observed in 22% of the miscarriage group and 1% of the control group (P-value ≤ 0.001). Regarding the prevalence of IgG, 93% of the SM group exhibited a latent infection, while only 20% of the control group did, with a P-value ≤0.001. This is demonstrated in Figure 1.
Figure 1. Results of the Prevalence of Serum CMV IgM and IgG in Miscarried and Pregnant Groups
Comparison of Immune Levels in Miscarriage Cases Versus Normal Pregnancy, Considering Maternal Age and Gestational Age
The independent T-test showed a statistically significant relationship (p-value ≤ 0.001) between the levels of the two markers and gestational age in both groups. Furthermore, the same is true for the concentrations of markers CD34+ (ng/ml) and HLA-F (ng/ml) between pregnant and aborted groups, as shown in Table 1.
Correlation between concentrations of CD34+ and HLA-F Markers
The Pearson correlation test for both study groups showed a moderate positive correlation between the levels of CD34+ and HLA-F in aborted and pregnant women. The results showed an r-value of 0.490 and a p-value ≤ 0.0001 (2-tailed) for the peripheral blood CD34+ marker and a similar positive correlation for the HLA-F marker, as shown in Table 2.
Marker Levels According to Gestational Age in Pregnant and Abortion Women
The study found a significant association between CD34+ levels and gestational age in both groups (P-value ≤ 0.037) and (P-value ≤ 0.005), respectively. Its concentration was higher in healthy women during the first trimester (9.77 ± 1.26) and decreased in those with miscarriages (1.58 ± 0.35). However, HLA-F concentration did not reach statistical significance, as in Table 3.
Relationship between Concentrations of CD34+ and HLA-F Markers and the Number of Miscarriages in the Aborted Group
The research identified a significant association (p-value ≤ 0.001) between HLA-F concentrations and the incidence of miscarriages, revealing elevated levels in initial miscarriages (3.09 ± 0.45) compared to multiple miscarriages (2.28 ± 0.40). No substantial correlation was identified between the CD34+ marker and the incidence of miscarriage, as presented in Table 4.
Two Marker Levels in Both Studied Groups According to CMV IgG Presence
A significant difference (P ≤ 0.017*) was found in the presence of latent infection in the aborted group compared to the healthy pregnant group with CD34+ and HLA-F immunity markers in peripheral blood, according to the independent T-test analysis in CMV IgG-positive pregnant women, as in Table 5.
Correlation of CMV IgM Presence with CD34+ and HLA-F Concentrations in Pregnant and Abortion Groups
An independent T-test reveals no significant difference (p-value > 0.05) in the presence of CMV IgM between aborted and healthy pregnant women regarding two CD34+ (ng/ml) and HLA-F (ng/ml) levels, as shown in Table 6.
Discussion
Human cytomegalovirus, a herpesvirus, infects 60% of adults in developed countries and 90% in developing countries. A robust immune response regulates symptoms [20]. Pregnancy-induced IgG presence indicates CMV infection or low avidity, while positive IgG and negative IgM during pregnancy suggest a previous secondary maternal infection [21]. This study, illustrated in Figure 1, shows a high prevalence of CMV IgG and IgM in both groups; however, the abortion group had a higher prevalence of IgM at 22%, compared to 1% in the control group. However, IgG shows 93% in the aborted group and 20% in the control group. A literature review revealed a disparity in results worldwide, particularly in Iraq, due to differences in detection methods, including serological methods such as ELISA and molecular techniques such as PCR [22]. This study agrees with a study that indicates a CMV prevalence of 13.53%, highlighting significant correlations between CMV infection and sexually transmitted diseases, multiple sexual partners, recurrent miscarriages, and headache/fever. The sensitivity and specificity for IgG in detecting CMV infection were 100% and 6.12%, whereas for IgM, the values were 78.26% and 100%, respectively [23].
The study reveals a strong correlation (P value 0.001) between immune markers CD34+ and human HLA class I histocompatibility antigen and alpha chain F concentration levels, highlighting their impact on immune regulation, as demonstrated in Table 1. HLA-F expression levels are crucial for understanding pregnancy dynamics and regulating maternal immune responses, and they are associated with adverse pregnancy outcomes, indicating their potential as predictive biomarkers [24]. The interaction between markers and CMV infection raises questions about their collective impact on pregnancy risks, necessitating extensive research for predictive models for improved maternal health management [25]. Upon examining the existing literature, this study found no published studies that connect these two immune receptors. We have identified this link as pertinent to our investigation, which requires further research and validation.
The independent T-test results in the current study reveal a significant association between the concentrations of CD34+ (1.54±0.34 ng/ml) and HLA-F (2.65±0.56 ng/ml) in the aborted group. In comparison, CD34+ (8.05±1.77 ng/ml) and HLA-F (4.54±1.50 ng/ml) were observed in the control, as illustrated in Table 2. This suggests that women with healthy pregnancies have elevated levels of both CD34+ and HLA-F. In contrast, their levels significantly decline in women who experience miscarriages, underscoring the significance of these two markers in preserving pregnancy viability. Nonetheless, the question is whether their levels diminished before the miscarriage or if they declined concurrently with the miscarriage at the time of sampling. This necessitates future research for validation. These findings suggest their involvement in immune regulation during pregnancy or their potential as predictive markers for miscarriage [26].
Our results indicate that gestational age is significantly associated with CD34+ levels in pregnant women and the aborted group. The levels of CD34+ (9.77±1.26 ng/ml) and HLA-F (4.42±1.46 ng/ml) in the first trimester in the control group, while their levels in the aborted group are (1.58±0.35 ng/ml) and (2.64±0.56 ng/ml) in the first trimester, as shown in Table 3. This signifies that the concentration of two immunological markers is elevated during the first trimester of pregnancy, surpassing levels observed in the second and third trimesters. This study agrees with a study that found that women with congenital renal malformations had significantly fewer CD34+ cells in their maternal peripheral blood than in normal pregnancies and congenital renal malformations [8, 27]. Additionally, endothelial progenitor cells (EPCs) increase during pregnancy, which is crucial for placentation; however, lower levels can lead to preeclampsia and miscarriage, unlike MS-related autoimmune conditions [28]. The CD34+ levels vary during pregnancy, possibly due to cellular metabolism or immune function changes, with women with miscarriages having lower levels [29]. According to Table 4 of the independent T-test results, the study found a significant disparity in the relationship between HLA-F concentration and miscarriage frequency, with lower concentrations (2.28±0.40 ng/ml) observed in successive miscarriages but higher in the first miscarriage (3.09±0.45 ng/ml). The study confirms that HLA-F expression decreases in women with multiple miscarriages, with a lower percentage of immunogenic antigens after a single successful pregnancy followed by a miscarriage [30].
This study found a significant difference (P-value=0.028) in CD34+ and HLA-F concentrations (1.51±0.31 ng/ml and 2.6±0.56 ng/ml, respectively) among aborted women with CMV IgG-positive latent infection, lower than their levels in the control group and in the absence of IgG. In contrast, no significant difference was observed in healthy pregnant women. Refer to Table 5 for details. HCMV persistence in hosts is regulated by latency and reactivation processes within infected progenitor cells, with latent infection established in CD34+ cells, involving US28 and UL7 genes [31]. The importance of immunological homeostasis at the maternal-foetal interface has been well-documented; it promotes healthy embryo implantation and guarantees normal placental vascular architecture development [32]. HCMV enters CD34+ cells through micropinocytosis, establishing latency. Low CD34+ cells and HLA-F concentration in the latent CMV virus impact pregnant women’s immune systems and stem cell functions, leading to increased CMV replication and transmission [33]. Furthermore, HLA-F triggers a stress response in virus-infected cells, inhibiting viral replication. When interacting with KIR3DS1, the immune system produces cytokines, increasing NK cell cytolysis [34]. These results agree with a study that shows that genetic predisposition to anti-cytomegalovirus IgG levels elevates the risk of coronary artery disease, peripheral artery disease, and deep vein thrombosis [35].
The study found no significant difference in the presence of primary infection CMV IgM in levels of CD34+ and HLA-F immunity markers between aborted and healthy pregnant women, as detailed in Table 6. Upon reviewing the pertinent published research, we did not identify a study correlating the concentrations of the immune markers used in the investigation to the initial infection. Consequently, the primary infection with the virus does not substantially influence the expression of these markers. The peril resides in the latent infection and reactivation caused by the virus persisting in progenitor cells for an extended duration, resulting in cellular damage and diminished expression levels [36].
Limitation: The study design for investigating immune parameters in relation to viral infection and spontaneous abortions should include infected individuals with spontaneous abortions and a control group without infection. Participants from both groups should be at the same gestational age for accurate immune parameter concentration calculation. The considerations are cross-sectional design, CMV DNA molecular testing, and controlling for gestational age variability.
Conclusion
In conclusion, a study found a moderate positive correlation between peripheral blood CD34+ and the human HLA class I antigen, alpha F chain, which was significantly associated with spontaneous abortion. This suggests a potential risk factor for latent CMV IgG infection in recurrent miscarriage. The study also found a significant association between CD34+ concentration and gestational age in pregnant women, with levels being highest in healthy individuals during the first trimester and lowest in women who had miscarried. Furthermore, this study found a significant association between HLA-F levels and recurrent miscarriage. A higher number of miscarriages was associated with lower HLA-F concentrations, which poses a potential risk to pregnancy safety and increases the likelihood of additional miscarriages. The study recommends further research to explore the possible use of these two immunological markers as pre-miscarriage predictive tests.
Acknowledgements
The authors wish to acknowledge the management of Al-Batool Teaching Hospital for Maternity and Children for facilitating the sample collection process and for providing the essential laboratory and experimental equipment. We also acknowledge the management of Babylon Maternity and Children’s Hospital for streamlining the sample collection process. We extend our gratitude to all individuals who participated in the study.
Conflict of Interest
There is no conflict of interest to declare.
Author Contributions
Professor Dr. Bushra Jabbar Hmood and Professor Dr. Asmaa Kadhim Gatea contributed to the study’s design and previewing all results; Nedhal Mahmoud Khaleefah prepared the manuscript. All authors read and approved this manuscript.
Financial support
No external funding was received for this research.
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