Objectives
To study the influence of pregnancy and labor on the proportion and level of activation of monocyte subpopulations in human pregnancy.
Study Design
Peripheral blood samples were obtained from healthy nonpregnant women (n = 6); women in the third-trimester of healthy pregnancies (n = 18) and women with preterm premature rupture of membranes (n = 46), just before delivery for the last 2 groups. Monocyte subpopulations were characterized by flow cytometry using CD14, CD16, and activation level using macrophage chemoattractant protein-1 (MCP-1) and CCR2 antibodies.
Results
The relative proportion of each monocyte subset in nonpregnant women was similar to that in women with healthy or complicated pregnancies. However, pregnancy was associated with a significant decrease in MCP-1 expressing monocytes (79.5% ± 19.8% vs 9.3% ± 6.8% and 11.9% ± 8.3% for nonpregnant, healthy pregnancy, and preterm premature rupture of membranes (respectively, P < .05). Spontaneous labor was associated with a return to nonpregnant values for the proportion of MCP-1 expressing monocytes in both normal (74.4% ± 16.9) and preterm premature rupture of membranes pregnancy (68.4% ± 35.6), irrespective of the mode of delivery (vaginal or cesarean section). This was not observed in women who delivered without spontaneous labor onset. CCR-2 (MCP-1 receptor) expression was not modified in monocytes at the time of labor, but was significantly increased in granulocytes (3646 ± 1080 vs 7338 ± 2718 for nonlaboring and laboring preterm premature rupture of membranes, respectively, P < .05)
Conclusion
In light of previous reports of a role for MCP-1 in labor, our results suggest the downregulation of activation levels of monocytes, via MCP-1 expression might be involved in maternofetal immune tolerance. Monocyte reactivation might be associated with labor.
Pregnancy and delivery are both complex immune situations involving numerous processes. During pregnancy, maternal immunity is reduced, with both neutrophils and monocytes displaying decreased functionality and reactivity compared with those in nonpregnant women. This decrease in maternal immune response is necessary to prevent the fetal allograft from rejection and permits fetal cells to invade the uterine mucosa to establish the placenta. The role of endometrial monocytes/macrophages and dendritic cells has recently been suggested as pivotal for successful pregnancy establishment in cattle. Samstein and colleagues recently suggested that maternal–fetal tolerance to paternal alloantigens is an active process in which plasmatic Tregs specifically respond to paternal antigens to induce tolerance.
In contrast, human parturition, either at term or preterm, is increasingly recognized as a sterile inflammatory event. Several studies have shown a massive influx of both neutrophils and monocytes/macrophages from maternal blood into the major effector tissues of parturition, ie, myometrium, cervix, and fetal membranes during labor. Human parturition is a complex phenomenon with several factors, such as hormonal changes, increased production of prostaglandins and release of inflammatory cytokine, interacting together.
This recruitment of maternal monocytes and granulocytes from maternal blood is induced by the secretion of chemotactic proinflammatory cytokines, such as interleukin-8 (IL-8), MIP-1α or macrophage chemotracant protein-1 (MCP-1). MCP-1, also known as C-C motif chemokine ligand 2 (CCL-2), is a major mediator of monocyte/macrophage infiltration at inflammatory sites both under physiologic and pathologic conditions. The role of MCP-1 during delivery was suggested by its presence in the myometrium and gestational membranes during full-term labor and in amniotic fluid during preterm labor. Experimental studies conducted in pregnant mice demonstrated that the administration of lipopolysaccharide (LPS) induces parturition within 12 to 24 hours and that this event is preceded by a peak in MCP-1 expression in the maternal bloodstream, occurring 6 hours after LPS injection.
Beyond the contribution of cytokines to parturition, Yuan and colleagues demonstrated that full-term and preterm labor are also regulated by modifications in the proportion or number and activation status of peripheral blood monocytes and granulocytes. They concluded that monocyte and granulocyte priming precedes labor onset.
Recent studies have highlighted the heterogeneity of human peripheral blood monocytes. Based on the recently proposed nomenclature, monocyte subsets can be discriminated by their differential expression of CD14 and CD16. Monocytes are thus characterized as CD14 high CD16 − classical (a major subset accounting for 70% to 90% of peripheral blood monocytes), intermediate CD14 high CD16 + , and nonclassical CD14 low CD16 + monocytes. Monocytes also differ phenotypically by chemokine-receptor expression such as CCR2 (receptor of MCP-1) and CX3CR1 (fractalkine receptor). Classical CD14 high CD16 − monocytes mainly express CCR2, whereas nonclassical CD14 low CD16 + mainly express CX3CR1.
Although the proportion of each monocyte subset has only been scarcely studied in pregnancy, it may vary with the onset of inflammatory events. Monocytes appear to play a central although complex role during pregnancy, as they are involved in both tolerance to the fetal allograph and in delivery. Thus this study aimed to investigate (1) the changes induced by pregnancy in the relative proportions and activation level of each monocyte subset, (2) the influence of preterm premature rupture of membranes (pPROM) on this proportion and activation level, and (3) the influence of intrauterine infection and labor onset in women with pPROM on the activation status of monocytes via MCP-1 and CCR-2 expression and that of granulocytes via CCR-2 expression.
Materials and Methods
Study design and patient recruitment
The study was designed as a prospective, single center, observational study. Peripheral blood samples were obtained between January 2009 and April 2010, from healthy nonpregnant female volunteers (nonpregnant, NP, n = 6), in the third-trimester of a healthy pregnancy (healthy pregnancies, HP, n = 18), and from women with preterm premature rupture of membranes (pPROM, n = 46).
Inclusion criteria were as follows: pPROM defined by either vaginal pooling of amniotic fluid during the speculum examination or a positive Actim PROM TEST (Medix Biochemica, Kauniainen, Finland). Normal pregnancy: women admitted to our institution for full-term delivery following a healthy pregnancy. Nonpregnant women with childbearing potential: age between 18 and 40, regular cycle, during the first phase of the cycle, and without oral contraceptive use at the time of blood sampling.
Exclusion criteria were as follows: for the pPROM group: hospitalization before 24 or after 34 weeks of gestation, delivery within 12 hours after admission, hemorrhagic placenta previa, HIV, HBV, or HCV infection, consent refusal.
Diagnosis of chorioamnionitis: chorioamnionitis was assessed in all of the pregnancies including pPROM in the present study by a positive culture of the placenta and/or a histologic assessment of the placenta by a single pathologist (Dr Laurent) using validated criteria.
All clinical and biologic data were collected prospectively and reported in the case report form.
This study was approved by the French Medicines Agency (registration no. 2008-A00119-46), and local ethics committee (CPP-Est1, Dijon, France). Written informed consent was obtained from all volunteers.
Blood sampling and fluorescence-activated cell sorting for the analysis of blood monocyte subsets
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Fresh blood samples were collected by venipuncture, either during the first phase of the cycle for nonpregnant women or before delivery during spontaneous labor or before labor induction or cesarean delivery for pregnant women. Women who had their blood sampling performed before labor induction were logically included in the nonlaboring group irrespective of the final mode of delivery (ie, vaginal or cesarean delivery). Blood was collected in EDTA separator tubes and promptly subjected to antigen characterization. Monocyte subpopulations were analyzed with monoclonal antibodies against CD14-FITC, CD16-APC-Cy7, CCR2-Alexa 647, and MCP1-PE (all from BD Bioscience). Whole blood (100 μL) was first subjected to erythrocyte lysis with ACK lysing buffer (ACK; BioWhittaker, Walkersville, MD) followed by centrifugation at 240 g, 4°C for 10 minutes. Pelleted leukocytes were then washed with phosphate buffered saline (PBS), centrifuged at 240 g, 4°C for 10 minutes and stained for 20 minutes at 4°C with CD14-FITC, CD16-APC-Cy7, and CCR2-Alexa 647 antibodies. Leukocytes were then fixed in PBS-0.1% paraformaldehyde for 10 minutes at room temperature, washed with PBS and subjected to MCP1-PE intracellular staining in PBS-Tween 0.1%-BSA 1% for 1 hour at room temperature. Staining with the corresponding isotype control was performed for each staining.
Cytometric analysis was performed on an LSRII cytometer (Becton Dickinson) using fluorescence-activated cell sorting Diva 6.1.2 software (Becton Dickinson). Monocytes were gated in a forward scatter/side scatter dot plot and 4-color staining was analyzed within this primary gate. Monocyte subsets were defined according to their CD14/CD16 pattern and classified into classical, intermediates, and nonclassical as described above. 18 The proportions of monocyte subsets were expressed as percentage of total monocytes. The percentage of each subset expressing CCR2 and MCP-1 were obtained by appropriate gating and expressed as a percentage of CCR2+ and MCP-1+ monocytes within these gates. CCR2 and MCP-1 expression in each monocyte subset was also determined by mean fluorescence intensity minus the respective isotype control (MFI−MFI isotype).
Plasma levels of MCP-1
MCP-1 was measured in the plasma of women who by ELISA test using Quantikine ELISA kit (R&D systems, ref. DCP00). Analyses were performed in duplicate, according to manufacturer’s instruction. Quantification was done according to internal standard, without normalization to protein level.
Statistical analysis
Sample size calculation: as no data were available on the proportion of MCP-1 expressing monocyte at the time of study design, preliminary results were used to assess sample size. With a type 1 error of 0.025, 7 patients per group gave us a 0.80 power to detect a 65% decrease in the proportion of MCP-1 expressing monocytes in pregnant women. This MCP-1 expressing monocyte assessment took also part of a prospective study of women with pPROM, and it explain why the number of pPROM pregnancy is much higher than 7.
Results were expressed as the percentage (± standard error to the mean, SEM) of each subpopulation (classical, CD4 high CD6 − ; intermediates CD4 high CD6 + ; and nonclassical, CD4 low CD6 ++ ) among total monocytes and as median fluorescence intensity (MFI) for MCP-1 and CCR-2 in arbitrary units. Several analyses were performed according to our study objectives. First, the different subsets of monocytes in nonpregnant and women with healthy pregnancies were compared. They were then compared in healthy and pPROM pregnancies. Thereafter, the proportion and MFI of MCP-1 expressing monocytes was compared in women with healthy or with pPROM pregnancies, and then in those who delivered spontaneously after labor onset and those who underwent cesarean section performed before labor onset or labor induction. Mean and SEM for percentages of each subpopulation were generated on each of the variables. Tests were performed using both parametric and nonparametric methods ( t tests and Mann-Whitney U tests for continuous variables, and Fisher exact tests for ordinal variables). All analyses were performed using GraphPad Instat 3 (GraphPad Software Inc., La Jolla, CA).
Results
Baseline characteristics of the population are presented in the Table . Mean maternal age was similar in the different groups. The average gestational age at delivery and subsequently birthweight were significantly lower in the pPROM group than in the normal pregnancy group. Most of the women with pPROM (18/46, 56.5%), and almost all of the women with an uncomplicated pregnancy (17/18, 94.4%) had a vaginal delivery.
| Characteristic | Nonpregnant | Healthy pregnancy | pPROM | P value |
|---|---|---|---|---|
| Number of women | 6 | 18 | 46 | |
| Age, y | 32 (± 5.9) | 29.71 (± 5.38) | 30.04 (± 6.10) | |
| Birthweight, g | 3138.24 (± 404.80) | 1803.4 (± 760.46) | ||
| Parity (± SD) | 0.5 (± 1.22) | 2.30 (± 1.30) | ||
| Gravidity (± SD) | 1.5 (± 1.10) | 1.15 (± 1.37) | ||
| Tobacco | 3/6 | 3/18 | 10/46 | |
| Gestational age at delivery, wks | 39.3 (± 1.3) | 31.2 (± 2.46) | .049 | |
| Mode of delivery, n (%) | ||||
| Vaginal (natural) | 14 (77.7) | 16 (34.8) | ||
| Vaginal (induced) | 3 (16.7) | 10 (21.7) | ||
| Cesarean section, during labor | 0 (0) | 5 (10.8) | ||
| Cesarean section, not during labor | 1 (5.6) | 15 (32.6) | ||
| Confirmed chorioamnionitis | 0 (0) | 22 (47.8) |
Among the women with a healthy pregnancy, 14 delivered after labor onset (14 natural vaginal delivery, no cesarean section during labor), 4 delivered before spontaneous labor onset (3 with induced vaginal delivery, and 1 with cesarean section not during labor). In the pPROM group, 21 women delivered after labor onset (16 natural vaginal deliveries and 5 cesarean sections during labor), and 25 delivered before spontaneous labor onset (10 induced vaginal deliveries and 15 cesarean sections before labor onset). Twenty-two (47.8%) women in the pPROM group had a confirmed chorioamnionitis vs none in the healthy pregnancy group.
Influence of pregnancy and pregnancy-related complications on the relative proportion of each monocyte subset. Figure 1 , A, shows the gating strategy used to determine the relative proportion of each monocyte subpopulation presented in Figure 1 , B and C. Figure 1 , B, shows that nonpregnant women (NP), and pregnant women, with either a healthy (HP) or pPROM pregnancy had similar relative proportions of the classical (CD14 high CD16 − ), intermediate (CD14 high CD16 + ), and nonclassical monocytes (CD14 low CD16 ++ ) (classical: 95.0 ± 3.2%, 92.4 ± 5.3% and 94.0 ± 3.3%; intermediate: 3.2 ± 0.9%, 5.4 ± 3.7% and 4.4 ± 2.5%; and nonclassical; 1.7 ± 1%, 1.6 ± 1.7% and 1.5 ± 1.2% for NP, HP, and pPROM women, respectively, P = NS).
Figure 1 , C, shows that in women with pPROM, the presence of confirmed (see Method section) chorioamnionitis (CA) did not affect the relative proportion of each monocyte subset (classical: 93.5 ± 3.2% vs 94.8 ± 3.3%; intermediate: 4.8 ± 2.6% vs 3.5 ± 2.4%; and nonclassical: 1.7 ± 1.5% vs 1.2 ± 0.8%, respectively, in women with pPROM with or without CA, respectively, Figure 1 , C).
MCP-1 expression in monocytes is reduced in normal and pPROM pregnancies
Figure 2 shows that, compared with nonpregnant women ( Figure 2 , A), nonlaboring pregnant women, with either a healthy or pPROM pregnancy ( Figure 2 , B), had a significantly lower proportion of MCP-1 expressing monocytes (MCP-1 + ) (79.5% ± 19.75, n = 6; 9.3% ± 6.8, n = 4 and n = 25 for NP, nonlaboring HP, and nonlaboring pPROM, respectively; P < .001) ( Figure 2 , D, black bars ). When blood sampling was performed in pregnant women before the onset of spontaneous labor, mean fluorescence intensity was also decreased, compared with nonpregnant women ( Figure 2 , D, red, green and blue lines ).
Labor induces MCP-1 expression in both classical and intermediate monocytes
Moreover, Figure 2 , C and D, also shows that spontaneous labor was associated with a marked and statistically significant increase in the percentage of MCP-1-expressing monocytes; proportions returned to a nonpregnant state in both HP women (73.4% ± 23.3% vs 9.3% ± 6.8%, for laboring and nonlaboring HP, respectively, P < .001) and women with pPROM (68.4% ± 35.6% vs 11.9% ± 8.3% for laboring and nonlaboring pPROM, respectively, P < .001).
In women with pPROM, the proportion of MCP-1 expressing monocytes, according to the presence or absence of spontaneous labor, was not statistically different according to the mode of delivery (nonlaboring women: 13.41% ± 8.03 vs 10.86% ± 5.63 [ P = .429]; and laboring women 50.8% ± 31.04 vs 75.15% ± 33.63 [ P = .276] for cesarean or vaginal delivery, respectively). No such comparison was feasible for women with healthy pregnancies as all of them had a vaginal delivery.
In laboring (n = 21) and nonlaboring (n = 25) women with pPROM, we further investigated the contribution of each monocyte subset to MCP-1 expression. Figure 3 , B, shows that labor onset was associated with an increase in the proportion of MCP-1–expressing intermediates (73.7% ± 16.8% vs 10.6% ± 8.0% in laboring and nonlaboring pPROM, respectively, P < .001) and classical monocytes (74.4% ± 19.9% vs 9.3% ± 6.8% in laboring and nonlaboring pPROM, respectively, P < .001). No significant differences between laboring and nonlaboring women with pPROM were observed in the very low proportions of nonclassical MCP-1−expressing monocytes (0.8% ± 0.5%, vs 0.3% ± 0.2%, in laboring and nonlaboring pPROM, respectively, P = NS).
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