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Obstetrics & Gynecology:
doi: 10.1097/01.AOG.0000124806.39111.ba
Original Research

Leukocyte Adhesion Molecules and Reactive Oxygen Species in Preeclampsia

Holthe, Mette Ree MD*; Staff, Anne Cathrine MD, PhD†; Berge, Lillian Nordboe MD, PhD†; Lyberg, Torstein MD, PhD†

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From the *Research Forum and the †Department of Obstetrics and Gynecology, Ullevaal University Hospital, Oslo, Norway.

This study was supported financially by grants from the Norwegian Foundation for Health and Rehabilitation through the Norwegian Health Association.

Reprints are not available. Address correspondence to: Mette Ree Holthe, Department of Pathology, Ullevaal University Hospital, 0407 Oslo, Norway; e-mail: m.r.holthe@ioks.uio.no.

Received October 17, 2003. Received in revised form December 30, 2003. Accepted January 15, 2004.

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Abstract

OBJECTIVE: The aim of our study was to compare the expression of leukocyte adhesion molecules, intracellular reactive oxygen species, and vasoactive substances in preeclampsia and matched normotensive pregnancies and to explore differences between pregnancy and the nonpregnant state regarding these parameters.

METHODS: Flow cytometry was used to analyze the monocyte and granulocyte expression of adhesion molecules from 20 matched pairs of preeclampsia/normotensive pregnancies and 12 nonpregnant subjects. Basal levels of CD11b, CD11c, CD62L, and CD14 were measured. In addition, expression of human lymphocyte antigen–DR, CD4, CD8, and CD4/CD8 ratio were assessed. Basal reactive oxygen species levels, as well as reactivity upon in vitro stimulation with phorbol 12-myristate 13-acetate, were measured in monocytes and granulocytes with the probes dihydroethidium, dichlorofluorescein-diacetate, and dihydrorhodamine-123. Further, the plasma levels of endothelin-1, the nitric oxide metabolites nitrite/nitrate, and total antioxidant status were analyzed.

RESULTS: Monocytes expressed significantly higher levels of CD11b and CD14 in preeclamptic patients compared with normotensive pregnant subjects, whereas CD11c was elevated on both monocytes and granulocytes in pregnancy compared with the nonpregnant state. Both monocytes and granulocytes displayed higher basal, as well as phorbol 12-myristate 13-acetate–stimulated, amounts of reactive oxygen species in the preeclampsia group compared with the normotensive group. We also found the endothelin-1 and antioxidant levels significantly elevated in preeclampsia patients compared with normotensive subjects, whereas no differences were seen between the groups regarding nitrite/nitrate levels.

CONCLUSION: These results show that the maternal blood leukocytes are activated in preeclampsia and support the view that oxidative stress is a contributing factor in the pathophysiology of preeclampsia.

LEVEL OF EVIDENCE: II-1

The preeclamptic syndrome is characterized by new-onset hypertension with proteinuria in the latter half of the pregnancy.1 Preeclampsia is worldwide a leading cause of morbidity and mortality in pregnancy. Poor placentation is a predisposing factor for the development of the syndrome, as well as several maternal factors.2 Disturbances of the vascular endothelium3 leading to vasoconstriction, increased endothelial permeability, and activation of thrombogenic factors are seen in preeclampsia.4 Many suggestions have been proposed to explain the origin of these changes, such as elevated plasma lipids5 or factors shed from the placenta. Placenta-derived factors that are suggested to be involved in the pathophysiology of the disease include oxidized lipids, such as 8-isoprostanes,6,7 interleukins,8 syncytiotrophoblast microvilli,9 and the soluble vascular endothelial growth factor receptor 1 (VEGF-R1, also designated sFlt1).10

It has been suggested by Redman et al2 that a normal pregnancy induces inflammatory changes in peripheral blood leukocytes, seen as augmented expression of reactive oxygen species and surface adhesion molecules, and that preeclampsia is associated with a further augmentation of these inflammatory responses. Stimulated leukocytes produce free radicals that can cause oxidative damage. Maternal cells are protected by both extracellular and intracellular antioxidants. Walker11 has proposed an imbalance between oxidant and antioxidant activity in preeclampsia. Changes in proportion of the lymphocyte T cell subsets (CD4- and CD8-positive cells) have been demonstrated in both graft rejection,12 sepsis,13 and pregnancies,14 suggesting common pathophysiological mechanisms.

Hypertension could occur as a result of an imbalance between vasoconstrictors and vasodilators. Maternal plasma levels of endothelin-1, a very potent vasoconstrictor, is elevated in preeclampsia,15 whereas nitrate, a metabolite of the vasodilator nitric oxide, is also found to be increased in the syndrome, but not fully able to compensate for the vasoconstriction caused by the elevated endothelin-1 concentration.16

The aim of this study was to test the hypothesis that leukocytes from preeclamptic pregnancies are in an excessive inflammatory state compared with leukocytes from normotensive pregnant and nonpregnant women, measured by altered expression of surface adhesion molecules and intracellular reactive oxygen species. In addition, we wanted to compare the plasma levels of the vasoactive substances endothelin-1 and nitric oxide metabolites and also the antioxidant status in the respective groups of women.

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MATERIALS AND METHODS

Between January 2001 and March 2002, 20 preeclamptic patients admitted to the Department of Obstetrics and Gynecology at Ullevaal University Hospital in Oslo were included in the study. The preeclamptic patients were identified by a rise in blood pressure after 20 weeks of gestation to above 140/90 mm Hg on more than 2 occasions, 6 hours apart, in previously normotensive women, combined with proteinuria, more than +1 on a dipstick for 2 occasions, 6 hours apart, in the absence of urinary tract infection. To promote fetal lung maturation, 12 mg of betamethasone (Celeston Chronodose; Schering-Plough, Kenilworth, NJ) had been given to 4 of the preeclamptic women on admission to the hospital and thus before blood sampling. The patients were carefully matched (one-to-one matching; parity 0 or 1, body mass index [BMI] ± 3 kg/m2, age ± 5 years, and gestation length ± 2 weeks) with 20 normotensive pregnant women. The latter women were recruited from general practices outside the hospital performing ordinary pregnancy controls. No women included in the study had a history of preexisting hypertension, renal disease, diabetes, or hemostatic disease. The 12 healthy nonpregnant women were recruited among female hospital employees and were included during March 2002. They were in the same age range as the pregnant women and matched for calculated nonpregnant BMI ± 3 kg/m2. All women included in the study were Caucasian and of Scandinavian ethnicity. The study was approved by the Regional Committee for Medical Ethics in eastern Norway, and informed written consent was given by all participants.

An antecubital vein was used for venipuncture and blood was sampled into Vacutainer tubes with ethylenediaminetetraacetic acid (EDTA) (Vacutainer System; Becton-Dickinson Europe, Meyland, France). The blood samples were kept at 4°C and antibody binding performed within 1 hour after blood collection. The leukocyte adhesion molecules were measured incubating 50 μL whole blood with the antibodies (10 or 20 μL) for 30 minutes at 4°C. Thereafter, a hypotonic lysis of erythrocytes was performed by adding 30 volumes of lysing solution (156 mmol/L ammonium chloride [NH4CL], 10 mmol/L sodium bicarbonate [NaHCO3], 0.12 mmol/L EDTA) at room temperature in the dark for 15 minutes followed by centrifugation at 300g for 5 minutes at 4°C. The supernatant was discarded, and the leukocyte pellet was washed once in 2 mL phosphate buffered saline (pH 7.4). Finally, the leukocytes were resuspended in 0.5 mL 1% (wt/vol) paraformaldehyde in phosphate buffered saline and stored in the dark at 4°C until flow cytometry was performed the next day. The basal levels of reactive oxygen species in leukocytes were measured by incubating 50 μL of whole blood with 5 μmol/L (final concentration) of the reactive oxygen species–sensitive probes for 15 minutes at 37°C. Probes used for reactive oxygen species measurements were dihydroethidium (oxidized to ethidium, mainly reflecting superoxide anion, O2−), dichlorodihydrofluorescein-diacetate (oxidized to 2′,7′-dichlorofluorescein, mainly reflecting hydrogen peroxide, H2[r]O2), and dihydrorhodamine 123 (oxidized to cationic rhodamine 123, mainly reflecting peroxynitrite, ONOO, and hypochlorous acid, HOCl), all from Sigma Chemical Co (St. Louis, MO). After incubation, the blood was lysed and washed according to the procedure described earlier for the adhesion molecules, except that the leukocytes were finally resuspended in 0.5 mL phosphate buffered solution and kept in the dark at 4°C. In preliminary experiments, fluorescence was found to be stable between 1 and 3 hours after phosphate buffered saline suspension; therefore, the samples were always read in the flow cytometer within this time span. The in vitro leukocyte stimulation with phorbol 12-myristate 13-acetate (final concentration 100 ng/mL) was carried out for 60 minutes with dihydroethidium and 90 minutes with dihydrorhodamine 123 and dichlorodihydrofluorescein-diacetate at 37°C in an atmosphere of humidified air with 5% carbon dioxide (CO2). Thereafter, the same procedure as described for the basal samples of reactive oxygen species was followed.

The following murine antibodies designated by their corresponding CD (cluster of differentiation) antigen number were used: fluorescein isothiocyanate (FITC)-conjugated anti-CD11b and anti-CD11c; these and corresponding isotype controls were from Sigma. FITC-conjugated anti-CD4 and anti-CD14 and phycoerythrin-conjugated anti-CD62L (L-selectin), anti–human lymphocyte antigen (HLA)-DR, and anti-CD8 with their corresponding isotypes were from Becton-Dickinson (San José, CA). Paraformaldehyde was from Merck (Darmstadt, Germany), and phosphate buffered saline and phorbol 12-myristate 13-acetate were from Sigma.

The samples were analyzed in a FACSort flow cytometer equipped with a FACStation and CellQuest software, all from Becton-Dickinson. A window of analysis was set with QC Windows calibration beads (Flow Cytometry Standards Corporation, San Juan, Puerto Rico). Daily control of fluorescence intensity was done with Flow-Set fluorescence microspheres (Coulter Corporation, Miami, FL) according to the manufacturer’s instructions. A calibration was performed weekly with Quantum 26 Fluorescein and Quantum 27 R-Phycoerythrin standards (Bangs Laboratories Inc, Fishers, IN), allowing conversion of the fluorescence intensity into molecules of equivalent soluble fluorochrome units. The QC Windows and Quantum beads were suspended in 1% paraformaldehyde solution. The mean fluorescence intensity (indicating surface antigen expression) for adhesion molecules is given in molecules of equivalent soluble fluorochrome units after subtraction of the isotype values. The CD4 and CD8 results are expressed both as percentage of positive lymphocytes and as amounts expressed on the lymphocyte surface in quantitative molecules of equivalent soluble fluorochrome units. Reactive oxygen species values are presented as difference mean fluorescence intensity, subtracting the background fluorescence (leukocytes with phosphate buffered saline only) from the measurements with the specific probes. The reactivities upon stimulation are presented as the rise in levels of reactive oxygen species upon phorbol 12-myristate 13-acetate stimulation, subtracting the measurements done with a negative control containing probe and leukocytes but no phorbol 12-myristate 13-acetate added.

The EDTA plasma endothelin-1 levels were measured by using an enzyme-linked immunosorbent assay (ELISA) kit (R&D System Europe Ltd, Abingdon, UK) with intra- and interassay coefficients of variation below 10%. Nitric oxide levels were estimated by measuring the sum of the stable end products nitrite (NO2−) and nitrate (NO3) with a fluorometric assay originally described by Misko et al17 and modified by Saetre et al.18 The results are reported as NOx, ie, the sum of converted nitrate and the very small amount of nitrite found in serum.

Total antioxidant status was measured in serum with a kit from Randox (Crumling, UK) described by Miller et al.19

The hematology analyses were performed with a Sysmex SE 9500 TOA (Medical Electronics, Kobe, Japan) at the Ullevaal University Hospital, Department for Clinical Chemistry.

Data were analyzed with SPSS 10.0 software (SPSS Inc, Chicago, IL). Sample size was calculated according to Pocock.20 Data from the preeclamptic patients were not normally distributed; therefore, nonparametric statistics were used, and all data are presented as medians with interquartile ranges. Women with preeclampsia were matched with the normotensive pregnant controls, and the Wilcoxon signed rank test was performed. When testing the nonpregnant against the normotensive pregnant women, Mann-Whitney U test was used. Because of the many parameters studied only P values less than .01 were considered significant.

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RESULTS

As expected from the matching criteria, the preeclampsia group did not differ from the control group at blood sampling with regard to maternal age, pregnancy length, BMI, or parity (17 nulliparous and 3 uniparous matched pairs of preeclamptic and control patients) (Table 1). The final median pregnancy length at delivery was 5 weeks shorter for the preeclampsia group than for the matched control group. Also, there was a statistically significant lower (1,096 g) median neonatal weight at delivery in the preeclampsia group than in the control group, as well as a lower mean neonatal weight percentile in the preeclamptic group (Table 1).

Table 1
Table 1
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Hemoglobin levels and the red cell counts were significantly lower in the 2 pregnancy groups compared with the nonpregnant situation, whereas the white blood cell counts were significantly increased in the 2 pregnancy groups compared with the nonpregnant controls (Table 2).

Table 2
Table 2
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The median monocyte expression of CD11b was markedly and statistically significantly increased (38%) in the preeclamptic compared with the normotensive pregnant women, whereas CD14 showed lesser, although significant, elevation (9%) in the preeclampsia compared with the normotensive pregnancy group. Monocytes in the normotensive pregnant women showed a significantly higher (91%) expression of CD11c than the nonpregnant controls (Figure 1). An even larger increase (138%) was seen when the expression of CD11c on granulocytes was compared in the 2 groups (Table 3). The HLA-DR measurement showed a decreasing tendency from nonpregnant to pregnant controls, with lowest values in the women with preeclampsia, without reaching statistical differences. Monocyte HLA-DR levels were 41,200 (34,000–49,000), 38,000 (31,900–43,600), and 36,200 (29,500–43,200) in nonpregnant, normotensive pregnant, and preeclamptic women, respectively (medians and 75th percentiles in molecules of equivalent soluble fluorochrome units). None of the other parameters on granulocytes or lymphocytes showed statistically significant differences among the 3 groups tested (Tables 3 and 4).

Figure 1
Figure 1
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Table 3
Table 3
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Table 4
Table 4
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The 4 patients receiving corticosteroid injections before blood sampling did not differ from the rest of the preeclampsia group with respect to monocyte CD11b and CD14 expression (data not shown).

In monocytes, median basal intracellular reactive oxygen species measured with the probes dihydroethidium and dichlorodihydrofluorescein-diacetate were significantly elevated (84% and 25%, respectively) in preeclampsia compared with the normotensive pregnancy group. Upon in vitro phorbol 12-myristate 13-acetate stimulation, the levels of monocyte intracellular reactive oxygen species were significantly elevated by 90% (dihydroethidium) and by 35% (dichlorodihydrofluorescein-diacetate) in preeclampsia compared with the normotensive pregnancy group. The group of normotensive pregnant women had significantly higher levels of monocyte reactive oxygen species (58%, dihydroethidium) after phorbol 12-myristate 13-acetate stimulation compared with the group of nonpregnant women. Likewise, the levels of basal reactive oxygen species (measured with dichlorodihydrofluorescein-diacetate) in monocytes were 60% elevated in the normotensive pregnancy group compared with the nonpregnant group (Figure 2).

Figure 2
Figure 2
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In granulocytes, the preeclampsia group had a significantly elevated (86%) expression of basal reactive oxygen species (dihydroethidium) compared with the group of normotensive pregnant women. Also, using the dichlorodihydrofluorescein-diacetate probe, levels of basal granulocyte reactive oxygen species were significantly higher (48%) in the preeclampsia group compared with the normotensive pregnancy group. After in vitro phorbol 12-myristate 13-acetate stimulation, a 26% higher reactivity of reactive oxygen species was seen in the preeclampsia group compared with the normotensive pregnant control group when using the dichlorodihydrofluorescein-diacetate probe. No differences were seen in granulocytes comparing normotensive pregnant and nonpregnant women (Figure 3).

Figure 3
Figure 3
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No differences were seen among the 3 groups in either monocytes or granulocytes when applying the dihydrorhodamine 123 probe for reactive oxygen species measurements (Figures 2 and 3).

The median total antioxidant status was significantly higher in the preeclampsia group compared with the group of normotensive pregnant women, but was not different from the group of nonpregnant women (Table 2).

The median plasma endothelin-1 level was significantly higher (80%) in the preeclampsia group compared with the normotensive pregnancy group. There were no significant differences between plasma endothelin-1 levels in normotensive pregnant and nonpregnant women. Serum NOx measurements showed no differences among the 3 study groups (Table 2).

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DISCUSSION

The main findings of our study were that monocytes showed higher membrane expression of CD11b and CD14 in the preeclampsia group than in the normotensive pregnancy group. Both monocytes and granulocytes displayed higher basal and phorbol 12-myristate 13-acetate–stimulated amounts of reactive oxygen species in the preeclampsia group than in the normotensive pregnancy group. The differences found between the normotensive pregnant and the nonpregnant women were that both monocytes and granulocytes expressed a higher amount of CD11c on their surface in pregnancy, whereas only monocytes revealed elevated intracellular reactive oxygen species basally and upon in vitro phorbol 12-myristate 13-acetate stimulation in the pregnant versus the nonpregnant state. For all measurements provided, there is a considerable overlap among the groups, and a single value cannot be used to predict/distinguish if the actual patient belongs to one group or another.

The integrins CD11b and CD11c combine with the CD18 integrin to form the Mac-1 and p150,95 adhesion molecules, respectively. Intercellular adhesion molecule-1 is one of their ligands on the endothelium and is important for the adhesion and transendothelial migration of monocytes and neutrophils in inflammatory processes. The finding of elevated CD11b levels on preeclamptic monocytes supports the notion that monocytes are activated and can adhere more easily to the activated vascular endothelium in preeclampsia, or alternatively, that the monocytes could be involved in the activation of endothelial cells in preeclampsia. Both the leukocyte integrins and CD14 are receptors for the bacterial lipopolysaccharide (LPS), and our findings of an elevated level of these adhesion molecules on monocytes in the preeclampsia group compared with normotensive pregnancy group might suggest a role for bacterial LPS in the development of preeclampsia. Naccasha et al21 found that pregnant women express higher levels of granulocyte CD14 than do nonpregnant controls, whereas they found monocyte CD11b to be elevated only in pregnant women with an ongoing infection (compared with normal pregnant women).

Our findings of elevated leukocyte expression of adhesion molecules in preeclampsia is in accordance with the proposition of von Dadelszen and Magee,22 who in a review summarize the evidence, although indirect, linking infection as a potential trigger of the immune system in the pregnant women who develop preeclampsia. Other investigators have found conflicting results; Barden et al23 and Gervasi et al24 found elevated levels of CD11b on granulocytes, whereas Crocker et al25 and Sacks et al26 found no differences between neutrophilic granulocytes from normotensive pregnant and those from preeclamptic women in this respect. Sacks et al26 focused on the similarity between the preeclampsia and the sepsis syndromes regarding activation of peripheral leukocytes.

In our study we found no significant differences between the study groups as regards the lymphocyte CD4/CD8 ratio. This ratio can be altered in certain autoimmune diseases and immune reactions, such as transplant rejections.27 The relative percentages of helper lymphocytes decline, and the suppressor lymphocytes increase also in septic patients.13 Matthiesen et al28 found a lower CD4/CD8 ratio in preeclampsia than in normal pregnancies and also a lower ratio in nonpregnant compared with normal pregnant controls. The ratio values they found in preeclampsia (n = 63) and the nonpregnant state (n = 5) were in accordance with our findings, but the ratios in normal pregnant controls were much higher than in our study and might be due to differences in the numbers of persons tested. In a longitudinal study, Bardeguez et al29 found no change in the CD4/CD8 ratio in 46 normal pregnancies from week 16 up to week 40. Also women who later developed preeclampsia (n = 10) showed no significant differences in the ratio between CD4 and CD8.

In our study the levels of reactive oxygen species in monocytes displayed a larger difference between preeclamptic and normotensive pregnant women than did these levels in granulocytes. In addition, only monocytes revealed reactive oxygen species differences between normotensive pregnant and nonpregnant women upon testing. These findings indicate that granulocytes and monocytes are differently modulated in normal pregnancy compared with preeclampsia. Our results are in accordance with the findings of Gervasi et al,24 who found that basal intracellular reactive oxygen species were increased in monocytes, but not in granulocytes, in preeclampsia patients compared with normal pregnant women. Basal intracellular reactive oxygen species were found to be increased in granulocytes, monocytes, and lymphocytes by Sacks et al26 in preeclampsia compared with normal pregnant and nonpregnant subjects. In contrast, Crocker at al25 could not find basal neutrophilic superoxide anion production to differ when comparing preeclamptic, normal pregnant, and nonpregnant women. They measured neutrophil extracellular superoxide anion production, and this methodological distinction could possibly explain the discrepancy in findings.

The reactive oxygen species superoxide anion and nitric oxide react to form peroxynitrite (ONOO ). The stable end products of the nitric oxide pathway are nitrite (NO2−) and nitrate (NO3). We measured the sum of nitrite/nitrate (NOx) finding no differences among the 3 groups tested; neither could we find differences among the 3 groups when using the dihydrorhodamine 123 probe (mainly reflecting peroxynitrite) to measure leukocyte reactive oxygen species levels.

Sagol et al30 found antioxidant activity and the amount of ascorbic acid to be significantly decreased in preeclampsia compared with normal pregnancies, a result we could not confirm by measuring the total antioxidant capacity with the total antioxidant status assay. The discordance is probably due to the differences in analytical methods.

The finding of elevated plasma endothelin-1 in patients with preeclampsia compared with normotensive pregnant and nonpregnant women in our study is in agreement with the findings of Bussen et al31 and Nishikawa et al.16 We found no association between plasma endothelin-1 and the sum of nitrite/nitrate in the preeclamptic women. This is not in accordance with the findings of Nishikawa et al,16 who found a positive correlation between endothelin-1 and nitrate levels in the preeclampsia group. This discrepancy could possibly be caused by differences in analytical and study design. We measured the serum content of total nitrite and nitrate in 20 pairs of nonfasting matched preeclamptic and control pregnant women. Nishikawa collected serum after 12 hours fasting and measured nitrite and nitrate separately in 17 preeclamptic and 16 nonmatched pregnant control women.

Altogether, our findings support the concept of a modulation of the maternal immune system taking place during the course of uneventful pregnancies, as summarized by Pearson32: “with preeclampsia, in some cases, to be linked to immunological abnormalities.”

Our study indicates that more parameters of leukocyte activation are affected in preeclampsia compared with normotensive pregnancies, with a higher degree of monocyte than granulocyte activation. We conclude that preeclampsia is associated with a form of leukocyte activation that differs both qualitatively and quantitatively from the leukocyte activation seen in uneventful pregnancies.

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Lack of proinflammatory effects of free fatty acids on human umbilical cord vein endothelial cells and leukocytes
Holthe, MR; Andersson, Y; Lyberg, T
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Acta Obstetricia Et Gynecologica Scandinavica
Calprotectin plasma level is elevated in preeclampsia
Holthe, MR; Staff, AC; Berge, LN; Fagerhol, MK; Lyberg, T
Acta Obstetricia Et Gynecologica Scandinavica, 84(2): 151-154.

International Journal of Fertility & Sterility
Oxidative Stress and its Role in Female Infertility and Assisted Reproduction: Clinical Implications
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Veterinary Immunology and Immunopathology
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© 2004 The American College of Obstetricians and Gynecologists

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