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Technical Briefs |
1 Department of Obstetrics and Gynecology, Showa University School of Medicine, 1-5-8 Hatonodai, Shinagawa, Tokyo 142-8666, Japan
aauthor for correspondence: fax 81-33784-8355, e-mail sekizawa{at}med.showa-u.ac.jp
The fetal circulation contains abundant nucleated erythrocytes (NRBCs). The PO2 of fetal blood is an estimated one-third to one-fourth of the PO2 of adult blood, and fetal hypoxemia stimulates fetal erythropoietin production and consequently increases the number of hematopoietic progenitor cells or NRBCs in fetal circulation (1). Severe hypoxia stimulates the proliferation of hematopoietic progenitor cells (2). When neonates are born with abundant NRBCs in their peripheral blood, the number of NRBCs rapidly decreases, and NRBCs become undetectable within 1 week after birth. Together, these facts suggest that differences in oxygen concentration between fetal and maternal blood play an important role in the clearance of NRBCs from maternal circulation.
In a previous study, we isolated fetal NRBCs from maternal blood by fluorescence-activated cell sorting (FACS). We used an antibody to the 
-chain of fetal hemoglobin and confirmed the fetal origin of the NRBCs by fluorescence in situ hybridization analysis with chromosome-specific probes (3). Microscopic analysis of these fetal cells by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), which detects DNA fragmentation, showed that apoptotic changes occurred in 47% of fetal NRBCs circulating in maternal blood (3). This suggests that apoptosis is the mechanism by which fetal cells are cleared from the maternal circulation. Apoptosis is an active, physiologic form of cell death that is mediated by the internal machinery of certain cells. Apoptosis leads to rapid recognition, uptake, and degradation of intact cells by phagocytes, thus preventing the release of noxious contents (4). We speculated that apoptosis in fetal NRBCs may play an important role in preventing immunization of the mother against the fetus.
The purpose of the present study was to clarify the mechanism by which apoptotic changes are induced in fetal NRBCs transferred into the maternal circulation. We focused on oxygen concentration as a factor in the induction of apoptotic changes in NRBCs. The effects of oxygen concentration on the fate of NRBCs were studied in vitro.
Blood samples from the umbilical vein were obtained immediately after birth from five women who delivered preterm infants (32–35 weeks of gestation); NRBCs are more abundant in preterm deliveries than in term deliveries. In all cases, no fetomaternal complications were detected other than preterm labor, and all Apgar scores were 8 or 9 at 1 min after delivery.
Immediately after the umbilical cord was clamped, 7 mL of umbilical cord blood was collected into an EDTA tube. From each sample, 150 µL was added to 7 mL of PB-MaxTM Karyotyping Medium (Life Technologies). Cells were then incubated under two different oxygen concentrations: 1% O2 (1% O2, 5% CO2, 94% N2) and 20% O2 (20% O2, 5% CO2, 75% N2) at 37 °C. Incubation was performed in a personal multigas incubator (APM-30D; Astec Inc.). To equilibrate the oxygen concentration in the medium, the medium was preincubated in the incubator for 2 h. For each sample, cell culture was performed in triplicate at each oxygen concentration. After 12 h of incubation, cells were washed twice with phosphate-buffered saline (PBS), and analyses were then performed. Approval for this study was obtained from the ethics committee of Showa University, and written informed consent was received from all patients.
We investigated the effect of oxygen concentration on the number of NRBCs. Preincubated and incubated blood cells were used to form thin smears on plain slide glasses. After the smears were dried, they were fixed with absolute methanol and stained with Giemsa. NRBCs were then identified morphologically and counted (5). The relative number of NRBCs was calculated as the number of NRBCs per 100 leukocytes. We counted cells in three areas of each slide, and the average of these three areas was used to calculate the relative NRBC number for the slide.
To detect apoptotic NRBCs before and after incubation, we performed two-color flow cytometry (FCM) using anti--hemoglobin antibody conjugated with phycoerythrin (PE) and TUNEL staining with fluorescein isothiocyanate (FITC). Briefly, a suspension of cells in PBS was layered onto Percoll solution (density = 1.077 kg/L; Amersham Pharmacia Biotech) and centrifuged at 1600g for 30 min. NRBCs enriched in the mononuclear cell layer were transferred to a new tube and then washed twice with PBS, containing 10 g of bovine serum albumin per liter, at 4 °C. These cells were fixed with freshly prepared paraformaldehyde (40 mL/L in PBS, pH 7.4) for 1 h at room temperature. They were then centrifuged at 800g for 10 min, and the fixative was removed. After being washed with PBS, the cells were resuspended in permeabilization solution (1 g/L Triton X-100 in 1 g/L sodium citrate) for 2 min on ice, and were again washed with PBS. We then added 50 µL of TUNEL-FITC reaction mixture (In Situ Cell Death Detection Fluorescein Kit; Boehringer Mannheim) and 2 µL of PE-conjugated anti--hemoglobin antibody (Cortex Biochem) to the cell suspension. This mixture was incubated at 37 °C in a dark, humidified atmosphere for 1 h.
After incubation, the cells were washed twice with PBS, resuspended in 500 µL of PBS, and subjected to FCM (FACS Calibur HG flow cytometer; Becton Dickinson). NRBCs were identified by positive PE staining of -hemoglobin. Apoptotic cells were identified by positive FITC staining. Thus, PE-positive/FITC-negative cells were identified as nonapoptotic NRBCs, and PE-positive/FITC-positive cells were identified as apoptotic NRBCs. As negative controls for FCM, antibody-free cells, peripheral blood from healthy adults, and commercially available TUNEL-negative cells (Boehringer Mannheim) were used. As positive controls for FCM, we used cord blood and commercially available TUNEL-positive cells (Boehringer Mannheim). The percentage of total NRBCs (PE-positive) that were apoptotic (PE-positive/FITC-positive) was calculated. Statistical evaluation of the effects of oxygen concentration on apoptosis of NRBCs was performed by comparing results for the five samples.
We used the Wilcoxon sign-rank test to analyze differences between 1% and 20% oxygen. A P value 
0.05 was considered to indicate statistical significance.
In preliminary studies, we measured PO2 in the culture medium during incubation under 1% oxygen. The PO2 value was between 15 and 25 mmHg and was concordant with the value obtained for the umbilical artery. We therefore used 1% oxygen for our studies to simulate the fetal environment.
With the preincubation NRBC number designated as 100%, the median number of NRBCs after incubation under 20% oxygen was 30% (range, 15–44%). The median number of NRBCs after incubation under 1% oxygen was 89% (range, 46–102%). The number of NRBCs decreased significantly after incubation under 20% oxygen (P <0.05), whereas the number of NRBCs did not decrease significantly after incubation under 1% oxygen (Fig. 1A
).
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The median percentages of total NRBCs (PE-positive) that were TUNEL-positive (PE-positive/FITC-positive) under 1% and 20% oxygen were 14% (range, 4.3–38%) and 20% (range, 9.5–57%), respectively (Fig. 1B
). Thus, the percentage of NRBCs undergoing apoptosis was significantly greater under 20% oxygen than under 1% oxygen (P <0.05).
The present results suggest that, because the maternal circulation is higher in oxygen concentration than fetal circulation, the oxygen environment of maternal circulation induces apoptotic changes in fetal NRBCs transferred to maternal circulation, leading to clearance of NRBCs from the maternal circulation. The fact that approximately one-half of the NRBCs in our previous study showed apoptotic change indicates that fetomaternal cell traffic is more common than we had expected. It may also explain the difference between amounts of fetal cells and cell-free fetal DNA detectable in maternal plasma. It is generally assumed that some portion of the cell-free fetal DNA in maternal plasma originates from fetal cells in maternal circulation.
The identification of NRBCs is relatively easy by common methods of morphologic observation (5), but such methods do not yield sufficiently precise quantitative results. Therefore, we used two-color FCM to identify NRBCs and assess apoptotic changes in NRBCs. However, initially, we were not able to obtain exact numbers of NRBCs and apoptotic cells by FCM analysis. In a previous study using FACS (6)(7), we isolated NRBCs by staining with anti--hemoglobin antibody and identified NRBCs by morphology and staining. Because the purity of NRBCs separated by FACS analysis with this antibody is reasonably stable and reproducible (6)(7), we used the same antibody for staining in the present study. The positive controls (TUNEL-positive cells and cord blood) and negative controls (TUNEL-negative cells and mononuclear cells from nonpregnant adults) that we used in the present study helped to ensure the reliability of TUNEL staining results. Thus, the present methods used to quantify NRBCs and evaluate apoptosis in NRBCs appear to be reliable.
We conclude that the high oxygen concentration in maternal blood induces apoptotic changes in fetal NRBCs transferred into maternal blood. We speculate that this phenomenon may be important to maintain the pregnancy because persistence of fetal cells in maternal circulation would stimulate the maternal immune system.
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sport, and Culture of Japan (Grants 12770933 and 13770940) and by the Kanzawa Medical Research Foundation.
References
The following articles in journals at HighWire Press have cited this article:
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  M. CSETE Stem cell biology: development and plasticity. Proceedings of the Thirteenth Annual Growth Factor and Signal Transduction Conference. September 16-19, 2004. Ames, Iowa, USA. Ann. N.Y. Acad. Sci., May 1, 2005; 1049: 1 - 200. [Abstract] [Full Text] [PDF]
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 S. Hristoskova, W. Holzgreve, and S. Hahn Fetal Nucleated Erythrocytes in Maternal Circulation Do Not Display a Classic Membrane-associated Apoptotic Characteristic (Phosphatidylserine Exposure) Despite Being Positive by Terminal dUTP Nuclear End Labeling Clin. Chem., November 1, 2003; 49(11): 1934 - 1937. [Full Text] [PDF]
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