This prospective longitudinal study aimed at comparing maternal immune response among naturally conceived (NC;
Maternal immunological tolerance is essential for successfully establishing and maintaining pregnancy [
The maternal immune system has to tolerate the semiallogeneic fetus in naturally conceived (NC) and
Maternal immune response in ED pregnancies is different compared with NC gestations. Actually, ED and IVF pregnancies at term display higher levels of both CD4+CD25bright regulatory T-cells and CD4+CD25dim activated T-cells in maternal peripheral blood mononuclear cells compared with NC gestations. In particular, the ratio of T-activated : T regulatory cells in ED pregnancies is significantly lower than that in NC pregnancies. The percentage of T-activated cells in ED pregnancies correlates positively with the number of HLA mismatches. Interestingly, peripheral blood mononuclear cells in ED pregnancies do not show a higher alloreactivity to the allogeneic fetus. Regarding the maternal humoral immune response, pregnant women by ED at term display higher levels of IL10 and IL6 and lower levels of TGF
This prospective study was carried out in two University Centers in Valencia, Spain: University & Polythecnic Hospital La Fe and Valencian Infertility Institute. We studied 75 consecutive women whose pregnancy was achieved by three different modes of conception: NC (
All women who achieved pregnancy by IVF or ED received vaginal progesterone, 400 mg/day, for the first 12 weeks of gestation. Patients were followed up at least quarterly until delivery, and pregnancy-related complications such as preeclampsia were gathered. The data of gestational age at delivery, weight of the newborn, and mode of delivery were also collected.
During pregnancy follow-up, 8 mL of peripheral blood was drawn from each patient between gestational weeks 11–14, 20–22, and 32–35 in one of the two hospitals. Two 4-mL EDTA tubes were stored at −20°C each time. Blood samples were centrifuged (3500 rpm for 10 min) at room temperature within the first 24 h after being taken, and the collected plasma was stored in 2-mL aliquots at −80°C.
This study was approved by the Ethics Review Boards of both participating hospitals. Written consent was obtained from each woman at the time of enrollment.
For cytokines detection in plasma samples, the MILLIPLEX MAP Technology (Merck Millipore, Germany) for Luminex 200 System (Luminex Corporation, USA) was used. The MILLIPLEX MAP Technology is a bead-based immunoassay capable of detecting up to 50 cytokines simultaneously in small sample volumes. The Luminex 200 System is a flow cytometry-based instrument equipped with two lasers, one for identifying cytokines and the other one for quantifying the concentration of the identified cytokine. The system uses Exponent 3.1 software to run and analyze samples (Luminex Corporation, USA).
Four different kits were used for the detection of 14 cytokines: Human Cytokine/Chemokine Magnetic Bead Panel (HCYTOMAG-60K) for RANTES, Human Cytokine/Chemokine Panel II (HCYP2MAG-62K) for SDF1
Samples were previously centrifuged at 1000 g for 1 min and the supernatant was collected to discard debris and excess lipids, which could interfere with the assay. For the Human Cytokine/Chemokine Panel II and Human High Sensitivity T-Cell kits, neat plasma samples were used. For the Human Cytokine/Chemokine kit, a 100-fold sample dilution was required. For the TGF
Numerical continuous data were expressed as the mean (standard deviation) and median (1st–3rd quartiles). Categorical data were shown as absolute and relative frequencies. A logarithmic transformation was applied to the concentration values of the different cytokines before any calculation. An exploratory heat map was built to view the evolution of the different cytokine concentration values throughout pregnancy in each group. To analyze our results, the inflammation status was summarized by performing two-group (high and low inflammation status) fuzzy clustering [
Women’s baseline characteristics are presented in Table
Baseline characteristics of the women studied. Data are expressed as
Natural conception group |
|
Egg donation group | |
---|---|---|---|
Maternal age (years) | 31.5 (4.7) | 34.6 (3.3) | 40.2 (2.7) |
Body mass index (kg/m2) | 24.4 (4.1) | 23.3 (2.6) | 25.2 (3.8) |
Nulliparity | 13 (52%) | 24 (96%) | 19 (76%) |
Previous pregnancy with the same partner | 13 (52%) | 1 (4%) | 3 (12%) |
Previous miscarriage with the same partner | 4 (16%) | 6 (24%) | 9 (36%) |
The studied women did not have medical diseases and did not report receiving any medication upon the first pregnancy control except for one woman in the NC group and one in the IVF group who occasionally took benzodiazepines for anxiety.
In NC pregnant women, there was a clear trend that the values of all the quantified cytokines, except RANTES, TNF
Evolution of the levels of each cytokine throughout pregnancy in the three study groups. Cytokine concentrations were expressed as standardized values (mean = 0, SD = 1). RANTES, regulated upon activation normal T-cell expressed and secreted; TNF
Heat map representing the cytokine and chemokine concentration values for each woman in all three groups throughout pregnancy. Whereas the three trimesters of pregnancy are represented on the first upper horizontal line, the three study groups in all three trimesters of pregnancy are represented on the second horizontal line. It can be observed that most cytokines and chemokines follow a similar pattern during pregnancy; their concentration rises in the second trimester and remains high in the third trimester of pregnancy. RANTES, regulated upon activation normal T-cell expressed and secreted; TGF
Subtle differences were found in the cytokine patterns for the three study groups. IL8 levels were higher in the three trimesters in ED and IVF pregnancies (
Values of IL8, RANTES, and SDF1
Chemokine | Conception method | 1st trimester | 2nd trimester | 3rd trimester |
---|---|---|---|---|
IL8 | NC | 0.18 (0.34) | 0.29 (0.70) | 0.61 (1.13) |
IVF | 0.45 (1.18) | 0.34 (0.59) | 0.57 (0.54) | |
ED | 0.40 (0.68) | 1.00 (1.77) | 1.22 (1.29) | |
|
||||
RANTES | NC | 59005.1 (41368.2) | 48423.2 (28486.7) | 43258.1 (30534.9) |
IVF | 47438.1 (31725.0) | 30195.4 (19065.6) | 27216.9 (16902.2) | |
ED | 35255.8 (27131.5) | 18625.9 (17928.7) | 21275.1 (17626.3) | |
|
||||
SDF1 |
NC | 1424.0 (492.3) | 1449.5 (377.9) | 3329.7 (1007.8) |
IVF | 1304.4 (511.5) | 1194.0 (441.6) | 3057.4 (1027.5) | |
ED | 1480.7 (375.2) | 1089.8 (502.9) | 2577.8 (2299.2) |
NC: natural conception; IVF:
The results of the fuzzy clustering performed to summarize inflammatory status are depicted in Figure
Heat map with the fuzzy clustering results. Individuals were ordered according to their GoM (grade of membership) score; a higher GoM score (red) denotes a higher inflammation status. TGF
Effect plots for our beta regression mixed model results. (a) The three groups showed the same trend: their GoM (grade of membership) values increased in the second trimester and remained high in the third trimester. (b) A positive association between maternal age and GoM values.
Pregnancy-related complications are shown in Table
Comparison of pregnancy outcome of the three study groups. Data expressed as
Natural conception group |
|
Egg donation group |
| |
---|---|---|---|---|
First trimester vaginal bleeding | 4 (16%) | 6 (24%) | 15 (60%) | 0.001 |
Preeclampsia | 1 (4%) | 2 (8%) | 2 (8%) | 0.81 |
HELLP syndrome | 0 (0%) | 1 (4%) | 1 (4%) | 0.60 |
Gestational diabetes | 0 (0%) | 3 (12%) | 4 (16%) | 0.075 |
Gestational age at delivery (weeks) | 39.7 ( |
39.7 ( |
39.0 ( |
0.14 |
Preterm births | 0 (0%) | 0 (0%) | 2 (8%) | 0.13 |
Vaginal delivery | 23 (92%) | 12 (48%) | 6 (24%) | 0.004 |
Cesarean section | 2 (8%) | 13 (52%) | 19 (76%) | 0.001 |
Birth weight (g) | 3286.8 ( |
3401.4 ( |
3282.3 ( |
0.62 |
Low birth weight (<2500 g) | 2 (8%) | 1 (4%) | 1 (4%) | 0.94 |
Given the small sample size, incidence of preeclampsia did not statistically differ among the three study groups (Table
No differences were recorded among the groups for gestational age at delivery and birth weight. Only one fetus displayed intrauterine growth restriction whose mother was in the NC group and had preeclampsia. One ED pregnancy ended with a preterm birth, and the mother had preeclampsia and HELLP syndrome.
No congenital malformations were reported. Cesarean sections were more common among ART pregnancies, particularly in the ED group (Table
The principal findings of this study are illustrated as follows. (1) The maternal cytokine and chemokine profile is preserved in ED pregnancies compared with the gestations with a semiallogeneic fetus. (2) The small number of patients who developed preeclampsia displayed higher plasma SDF1
The maternal humoral immune response throughout pregnancy was similar in pregnancies with a semiallogeneic fetus (NC and IVF) compared with those with a fully allogeneic fetus (ED pregnancies). All the quantified cytokines, except RANTES, TNF
RANTES levels were similar throughout pregnancy but showed lower values in the three trimesters in ED and IVF pregnancies compared with NC pregnancies. RANTES is the chemokine most synthesized by uNK cells at the human maternal-fetal interface. uNK cells play a key role in placental development as they regulate trophoblast invasion and vascular growth [
Like RANTES, the TNF
The IL8 levels constantly increased from the beginning to the end of pregnancy. In addition, ED and IVF pregnancies showed significantly higher IL8 levels throughout gestation. uNK cells are potent secretors of IL8 [
SDF1
Finally, TGF
One unexpected finding in the three study groups was the high levels of most of the studied pro- and anti-inflammatory cytokines in the second half of pregnancy. The maternal immune response needs to be more intense at onset of pregnancy for proper fetal immune recognition and initial tolerance of pregnancy. However, this initial maternal immune response may take place locally in the basal decidua, which may not be associated with a systemic immune response in maternal blood. Even if this assumption was correct, it poses the question of why maternal plasma cytokines increase in the second half of pregnancy in uneventful gestations and which role this may play if there is any.
It has been suggested that pregnancies with a completely allogeneic fetus require an enhanced maternal immune tolerance in order to achieve a successful pregnancy [
Regarding pregnancy-related disorders, the incidence of first trimester vaginal bleeding was significantly higher in ED pregnancies compared with NC and IVF gestations. This is consistent with knowledge about the higher incidence of bleeding complications in the first trimester as a result of the more frequent placental pathology in ED pregnancies [
Compared with healthy pregnant women, it is known that preeclamptic women present similar plasma RANTES levels [
In spite of the small simple size, the ED group did not have worse pregnancy outcomes. This supports previous knowledge that incidence of perinatal complications in ED pregnancies is comparable to conventional IVF pregnancies [
The strength of the present study is the fact that, to the best of our knowledge, this is the first study to compare the maternal plasma cytokine profile among NC, IVF, and ED pregnancies. The limited sample size of the three study groups can be considered as study’s main weakness. Moreover, although the maternal peripheral immune response throughout pregnancy provides prospective information about the tolerogenic response in both pregnancies with a semiallogeneic fetus and with an allogeneic fetus, further studies of the placenta are required to unravel the local maternal-fetal immune response particularly in ED pregnancies.
The maternal plasmatic cytokine profile of naturally conceived pregnancies was greatly conserved in IVF and ED pregnancies. Despite the limited sample size, the SDF1
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to thank Mercedes Monterdre, Ana Sanz, Antonio Abad-Carrascosa, Vicente Jorge, Elena Martín, Inés Olmos, Vicente Diago, and Vicente Martínez-Molina for their help in recruiting the studied women and would also like to thank all those who kindly collaborated in the project. The authors are particularly thankful to all the pregnant women who participated in this research project.