|Year : 2020 | Volume
| Issue : 1 | Page : 1-7
CD62P and CD40 ligand in diabetic pregnant women
Nagwa I Okaily MD 1, Ashraf M Othman1, Reem Y Elgarhy1, Ahmed R Abdelraheem2
1 Department of Clinical Pathology, Faculty of Medicine, Minia University, Minia, Egypt
2 Department of Obstetrics and Gynecology, Faculty of Medicine, Minia University, Minia, Egypt
|Date of Submission||14-Jun-2019|
|Date of Acceptance||01-Oct-2019|
|Date of Web Publication||10-Sep-2020|
Nagwa I Okaily
Department of Clinical Pathology, Faculty of Medicine, Minia University, Minia, 61111
Source of Support: None, Conflict of Interest: None
Aim We evaluated the expression of CD62P and CD40 ligand in diabetic pregnant women to correlate between these parameters and hemostatic abnormalities and to evaluate the relationship of these parameters with the fetal outcome.
Settings and design This is a prospective case–control study that included 60 patients: 40 diabetic pregnant women (patient group), who were subdivided into group Ia (20 pregnant women with preexisting diabetes) and group Ib (20 with gestational diabetes), and 20 apparently healthy, age-matched and sex-matched pregnant women (control group).
Patients and methods Platelet activating markers (CD62P and CD40L) were measured using flow cytometry.
Results A significant increase in CD40L and CD62P percentage in group I compared with group II was present (P<0.001). There were significant correlations between the groups Ia and Ib in percentages of both CD40L and CD62P (P=0.001). There was a significant increase in maternal complications in those with preexisting diabetes mellitus (60%) more than in those with gestational diabetes mellitus (10%) and significant positive correlations between CD40L and CD62P.
Conclusion Expressions of CD62P and CD40 ligand are increased in diabetic pregnant women, and this means that platelet activation and aggregation play an important and possibly key role in fetal and maternal complications, whereas CD40L expression plays an important role in maternal complications.
Keywords: CD40 ligand, CD62P, diabetes, pregnancy
|How to cite this article:|
Okaily NI, Othman AM, Elgarhy RY, Abdelraheem AR. CD62P and CD40 ligand in diabetic pregnant women. Egypt J Haematol 2020;45:1-7
|How to cite this URL:|
Okaily NI, Othman AM, Elgarhy RY, Abdelraheem AR. CD62P and CD40 ligand in diabetic pregnant women. Egypt J Haematol [serial online] 2020 [cited 2020 Sep 19];45:1-7. Available from: http://www.ehj.eg.net/text.asp?2020/45/1/1/294780
| Introduction|| |
It is estimated that 28 million women of reproductive age experience diabetes mellitus (DM) worldwide . In pregnancy, DM can be either preexisting (type 1 or 2) or gestational diabetes mellitus (GDM) .
P-selectin (CD62P) is one of the several cellular adhesion molecules expressed on the platelet membrane immediately after activation  and easily detectable with flow cytometry .
CD40 ligand (CD40L) is a transmembrane protein expressed on the activated T cells and platelets. It may also be shed from cells and circulate in the blood in a soluble form involved in the pathophysiology of various thrombotic and inflammatory conditions .
| Patients and methods|| |
The present study was conducted on 40 diabetic pregnant women and 20 apparently healthy pregnant women as controls. Their age ranged from 20 to 40 years, and their gestational age ranged from 24 to 37 weeks. The study was approved by Ethical committee of Minia University hospital, Faculty of Medicine, Minia University.Written consents were taken from all patients participated in the study.
Patients were selected from outpatient clinics and those who were admitted in Obstetrics and Gynecology Department in Minia University Hospital from June 2016 to November 2016. All laboratory investigations were carried out in the Clinical Pathology Department of Faculty of Medicine, Minia University.
The selected patients were divided into two subgroups according to the onset of the disease:
- Group I (patients group):
- Group Ia: it included 20 women with preexisting (nongestational) diabetes.
- Group Ib: it included 20 women with gestational diabetes.
- Group II (control group): 20 apparently healthy pregnant women who were matched with patient population in terms of age and gestational age.
Acute or chronic inflammatory or infective diseases, acute or chronic liver disease, acute or chronic renal disease, history of malignancy, seizure disorders, and drug or alcohol abuse were the exclusion criteria.
All participants included in this study were subjected to the following:
- Complete history questionnaire.
- Thorough clinical examination.
- Routine laboratory investigations, after sample collection including.
A bout eight ml of venous blood were withdrawn from all subjects (cases and controls), under completely sterile conditions: Two ml of blood in EDTA containing tube for CBC (which was performed by an automated cell counter (Sysmex KX-21N (TAO Medical Incorporation, Japan) and for HbA1c using quantitative colorimetric determination using (Stanbio laboratory, Boerne, Texas), and 1.8 ml of blood on tube containing 0.2 ml trisodium citrate for prothrombin concentration using (STAGO COMPACT CT Coagulation Analyzer and used for APTT, and 2 ml on plain tube, left to clot, centrifuged and expressed serum for serum creatinine and liver enzymes and serum albumin and fasting blood sugar using (Thermo Electron Incorporation, Finland).
About 1.8 ml of blood on tube containing 0.2 ml trisodium citrate for flow cytometric assessment of CD40 ligand and CD62p using (BD-FACS flow Argon laser, USA).
- For each sample, two tubes were required (test tube, negative controls).
- Platelet-rich plasma (PRP) was isolated by centrifugation at room temperature.
- Five micron of PRP was added to test and control tubes containing 100 micron of HEPES buffer
- Ten micron of fluorescein isothiocyanate (FITC) Conjugated anti CD40 L - monoclonal antibody, and ten micron of phycoerythrin (PE) conjugated anti-CD62p monoclonal antibody were added to test tube Then samples were incubated in dark for 10 minutes at room temperature
- Both tubes are diluted and fixed with 300 micron PBS containing 1% paraformaldehyde. There after mixed and immediately analyzed by flow cytometry after fixation to avoid further activation. Ten thousand cells were measured by flow cytometry and data processing by XL software.
All statistical analyses were performed using Statistical Package for Social Science, version 21 (SPSS). Results were expressed as means±SD for parametric data and by number and percentage for nonparametric data. Comparisons between the groups were conducted by Student’s t test for parametric data and by χ2 test or Mann–Whitney test for nonparametric data. Correlations were carried out by Pearson’s correlation coefficients. P value was considered significant if less than 0.05. Data were statistically analyzed using the SPSS software package, version 16 (SPSS Inc., Chicago, IL, USA) on a personal computer.
| Results|| |
A significant increase in CD40L and CD62P percentage in group I compared with group II was present (P<0.001). There were significant correlations between the groups Ia and Ib in percentages of both CD40L and CD62P (P=0.001). There was a significant increase in maternal complications in those with preexisting diabetes mellitus (60%) more than in those with gestational diabetes mellitus (10%) and significant positive correlations between CD40L and CD62P. [Table 1],[Table 2],[Table 3],[Table 4] and [Figure 1],[Figure 2],[Figure 3].
|Table 3 Correlation between CD40L and CD62P, baby weight, hemoglobin, platelet, mean platelet volume, fasting blood sugar, glycated hemoglobin, fetal complications, and maternal complication in group Ia|
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|Table 4 Correlation between CD62P and CD40L, baby weight, hemoglobin, platelet, mean platelet volume, fasting blood sugar, glycated hemoglobin, fetal complications, and maternal complications in group Ia|
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|Figure 1 Correlation between CD40L and MPV in GDM. GDM, gestational diabetes mellitus; MPV, mean platelet volume.|
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|Figure 3 Correlation between CD40L and CD62P in GDM. GDM, gestational diabetes mellitus.|
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| Discussion|| |
Diabetes with pregnancy is one of the most common medical conditions encounter during pregnancy (preexisting diabetes − type 1 or type 2–which antedates pregnancy or is first identified during testing in the index pregnancy); the majority (84%) of the cases are owing to GDM .
GDM is defined as impaired glucose tolerance with onset or first recognition during pregnancy . Women with GDM are at high risk for pregnancy and delivery complications including infant macrosomia, neonatal hypoglycemia, and cesarean delivery .
Diabetes in pregnancy is associated with increased risk to the woman and to the developing fetus . Hyperglycemia during the critical period of organogenesis may lead to a high risk of spontaneous abortions and congenital anomalies .
Infants born to mothers with GDM are also more likely to develop impaired glucose tolerance. Risk factors for GDM include higher parity, advanced maternal age, family history of DM, nonwhite race, overweight, and obesity .
DM is associated with an increased risk of atherothrombotic complications. Platelet activation, endothelial dysfunction, and inflammation appear to be involved in all stages of diabetic microangiopathy and macroangiopathy .
CD154 (CD40 ligand) is a trimeric, type II transmembrane protein of the tumor necrosis factor family. CD154 is cryptic in unstimulated platelets but is rapidly presented to the platelet surface after platelet stimulation by agonists such as APP, thrombin, or collagen .
P-selectin, also known as lysosomal membrane protein (CD62P) or granular membrane glycoprotein (GMP140), belongs to the selectin family of cell adhesion molecules. CD62P is rarely expressed during the quiescent stage. Platelet activation leads to the rapid fusion of CD62P with the plasma membrane, and subsequent expression on the platelet surface. The increase in CD62P is not reversed with the passage of time; therefore, CD62P is considered to be a major indicator (gold standard) of platelet activation .
The expression of CD62P (P-selectin) at the surface of the platelet is taken by many workers as a clinical marker of activation, easily delectable with flow cytometry .
In the present work, we tried to evaluate CD62P as platelet activation marker and CD40L as an inflammatory marker with a predictive value for hemostatic and fetal complications.
All participants included in the study were subjected to clinical examination and laboratory investigations: routine investigations and special investigations, such as flow cytometric analysis, for CD62P and CD40L.
Our study showed that there was no significant difference in white blood cells of the three groups; this was in agreement with other studies , which showed that there was no significant relation between diabetes and white blood cells.
This result was in disagreement with other studies , which reported that leukocytosis is mentioned as a common feature of diabetic pregnancy, and this may be owing to different number of studied patients and control of their study.
Among diabetic pregnant women, this study showed significant differences between diabetic and normal pregnant women in fasting blood sugar level, but significance is higher in preexisting diabetes than GDM. These results agreed with a study  which showed that mean platelet volume (MPV) values have been found to be significantly increased in GDM, with mean±SD 7.34±1.31, when compared with normal controls, with mean±SD 8.05±1.21. This result agreed with the result of Turhan et al.  who reported higher MPV levels in patients with GDM compared with healthy pregnant women when measured in the third trimester (with mean±SD 8.8±1.0 vs. 8.1±0.7).
This was against Çeltik et al.  who proved that MPV was not significantly different between women with GDM, with mean±SD 8.69±1.07, and women with previous normal pregnancies, with mean±SD 8.69±0.93, and this may be owing to different number of cases.
Among diabetic pregnant women, 60% with preexisting diabetes had maternal complications and 10% with GDM had maternal complications. This was near to the results of Sultana et al.  who found maternal complications in 42% of preexisting diabetes cases and 34% in GDM cases.
This study showed that there is a significant difference between baby birth weight of preexisting DM and GDM cases, as it is higher in babies of mothers with preexisting DM than GDM, and this is in approval with a study by Patel et al.  who reported that infants of mothers with preexisting DM experience double the risk of serious injury at birth and triple the likelihood of cesarean delivery as a cause of macrosomia.
Concerning mode of labor, the present study showed that 85% of patient group required cesarean section in both preexisting diabetes and GDM cases. This agreed with the study done by Tutino et al.  who found in their study on 100 diabetic women that there were 84% of preexisting diabetes cases and 68% of GDM cases that required cesarean section.
Wahabi et al. , also showed that there was an increase in the incidence of cesarean sections in diabetic pregnant women when compared with normal pregnant women.
Regarding the percentage of recurrence of gestational diabetes in previous pregnancies in this study, it was 35% of gestational diabetes group. This was in agreement with a previous study done by Heon et al.  who reported that the frequency of recurrent GDM in subsequent pregnancies was 45%.
These results are also in agreement with Permanente  who proved that there is an increased risk of recurring gestational diabetes in pregnant women who developed gestational diabetes during their first and second pregnancies.
In our study, we found that HbA1C levels were increased in both GDM, with mean±SD 6.71±0.65, and preexisting diabetes, with mean±SD 7.61±0.77, versus controls, with mean±SD 5.02±0.58 .This finding was expected and was compatible, with near results of Turhan et al.  who showed that the mean HbA1C of the patients with GDM was significantly higher than the control group (with mean±SD 6.2±0.6 vs. 4.89±0.34%).
Hov et al. , showed that there was a significant increase in HBA1C in preexisting diabetic pregnant women, and this was in agreement with our results.
In our flow cytometric study, we found that CD62P percentages are higher in women with diabetic pregnancy, both GDM (with mean±SD 23.83±3.61) and PGDM (with mean±SD 28.32±2.76), compared with women with healthy pregnancies (3.49±0.59), and this was in agreement with other studies , which found that the expression of CD62P (P-selectin) was significantly increased in DM compared with normal participants.
Soma et al. , also agreed with these results when they proved increased expression of CD62P in diabetic patients, and enhanced platelet activation and aggregation are viewed as one of the major causes of atherosclerosis and thrombosis in diabetes.
Our study showed that there was a significant correlation between CD62P and blood glucose level. This was in agreement with other reports , which showed that neither platelet aggregation nor CD62P expression correlated with the blood glucose.On the contrary, we have shown that diabetic pregnant women are associated with significant increased platelet surface expression of CD40L, in both GDM (with mean±SD 25.3±3.74; P<0.001) and PGDM (with mean±SD 29.43±2.88; P<0.001) compared with control group (mean±SD 3.82±0.63; P<0.001). This was in agreement with a study by Mohamed et al.  who showed that type 1 diabetes is associated with elevation of platelet surface expression of CD40L.
Our results showed that there was a significant positive correlation between expression percentages of both CD40L and CD62P in both GDM and preexisting diabetes. These results agreed with others  who showed that both CD40L and CD62P are expressed on activated platelets with diabetes.
Linna et al. , showed that inflammatory markers such as CD40L may play an important role in both the initiation and progression of atherosclerosis as well as reflect the progression of complications in patients with type 2 diabetes. This was in agreement with our results as we showed that there was a significant positive correlation between maternal complications and expression percentage of CD40L in both PGDM and GDM.
Our study showed that there was a significant positive correlation between CD40L and MPV in PGDM, and this agreed with Hwa et al.  who proved that there was a correlation between MPV and CD40L, as a clinical marker of platelet hyperactivity in DM.
| Conclusion|| |
Expressions of platelets activating markers (CD62P and CD40 ligand) are increased in diabetic pregnant women, and this means that platelets activation and aggregation play an important and possibly key role of fetal and maternal complications. CD40L expression play an important role in maternal complications.
The authors wish to thank all staff members of the Gynecology & Obstetrics, Minia University Hospital, for their assistance during the data collection and follow-up of patients.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]