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Letters to the Editor |
Departments of1
Obstetrics and Gynecology, and 2
Human Genetics, Nagasaki University Graduate School, of Biomedical Sciences, Nagasaki, Japan
3 CREST, Japan Science and Technology Agency, Kawaguchi, Japan
aAddress correspondence to this author at: Department of Obstetrics and Gynecology, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-7-1, Nagasaki 852-8501, Japan. Fax 81-95-849-7365; e-mail kiyonori{at}net.nagasaki-u.ac.jp.
To the Editor:
Fetal and/or placental mRNA in maternal plasma has been detected during pregnancy, and such mRNA tends to be stable against degradation (1). A quantitative study of plasma mRNA for 
-globin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed significantly higher concentrations in pregnant women than in nonpregnant women (2). These findings suggest that the quantitative analysis of placental mRNA in maternal plasma may be a useful method to monitor placental status.
In this study, we measured placental mRNA in maternal plasma to evaluate residual placenta in a pregnant woman with placenta previa-percreta (PPP) and bladder invasion that was diagnosed by both magnetic resonance imaging and pathology examination. Although a supravaginal hysterectomy just after the cesarean section (the first surgery) was done at 37 weeks of gestation, a 16-cm placental mass close to the internal os of the uterus could not be removed. Therapy with methotrexate (MTX) was therefore initiated on days 1–4, days 14–17, and days 33–36 after the first surgery (on day 0) to aid resorption of the placental residue, and a second surgery was performed to complete the hysterectomy on day 75. The study protocol was approved by the Committee for the Ethical Issues on Human Genome and Gene Analysis in Nagasaki University, and written informed consent was obtained. Data regarding plasma mRNA concentrations did not influence clinical management.
Blood samples (10 mL) were collected at intervals and before and after the surgeries. Plasma mRNA was extracted as described by Ng et al. (1). Human chorionic gonadotropin-ß (hCG-ß) and human placental lactogen (hPL) were selected as representative placental mRNAs, and GAPDH mRNA was measured as a housekeeping gene. A one-step real-time reverse transcription (RT)-PCR assay (EZ rTth RNA PCR Kit; Applied Biosystems) was performed on an Applied Biosystem 7900T Sequence Detector (Perkin-Elmer) to measure the mRNA concentration in maternal plasma. Primer sets and TaqMan probes for the 3 genes selected were prepared as described previously (1). Calibration curves for the quantification of each mRNA were prepared by assaying serial dilutions of HPLC-purified single-strand synthetic DNA oligonucleotides from each PCR amplicon (R2 = 0.99; 95% confidence interval for the slope of the calibration curve for the real-time RT-PCR, –2.9 to 3.3). The absolute concentration of each mRNA was expressed as copies/mL of maternal plasma, based on the formula described by Farina et al. (3). Calibration curves for hCG-ß and hPL mRNA ranged from 1 x 107 to 1 x 101 copies/mL, and the curve for GAPDH mRNA from 1 x 1010 to 1 x 104 copies/mL. Each sample was analyzed in triplicate with thermal cycling as described previously (1).
The hCG-ß mRNA showed a decreasing tendency similar to the pattern for hCG protein concentrations measured by an IRMA, but exhibited a transient increase to 1.90 x 103 copies/mL on day 12 after the first MTX therapy and then decreased again to <10 copies/mL after the second surgery (Fig. 1A
). Interestingly, plasma hPL mRNA concentrations also showed a decreasing tendency but increased again after the second MTX therapy from 7.88 x 102 copies/mL on day 15 to 2.68 x 103 copies/mL on day 19, whereas the hCG-ß mRNA concentration decreased (Fig. 1B
). This discrepancy may be attributable to different expression patterns of the hPL and hCG-ß genes in the placental cell. hCG-ß mRNA is produced primarily in proliferating cytotrophoblasts and to a lesser extent in mature syncytiotrophoblasts, whereas the hPL gene is expressed mainly in the mature syncytiotrophoblast (4). Because MTX strongly inhibits DNA synthesis, the hCG-ß mRNA concentration may reflect the MTX-induced apoptotic activity in dividing cytotrophoblasts directly and the hPL mRNA concentration may reflect the resorption of syncytiotrophoblasts. Because the placenta at term has many mature syncytiotrophoblasts, combined monitoring of hCG-ß and hPL mRNA concentrations in maternal plasma can be used for evaluation of chemotherapeutic efficacy of MTX for PPP.
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The GAPDH mRNA concentrations in plasma also increased after both surgeries and after the first MTX therapy (Fig. 1C
). Thus, cell/tissue damage by both surgery and chemotherapy may be associated with increased concentrations of GAPDH mRNA. Because circulating GAPDH mRNA in cancer patients was thought to originate in apoptotic cancer cells (2)(5), the increased concentrations of GAPDH mRNA detected may be caused by apoptosis in the placental residue in our case.
We conclude that real-time quantitative RT-PCR is a sensitive method to monitor changing mRNA concentrations resulting from apoptotic effects in the placenta and to evaluate invading conditions of the trophoblastic villus.
Acknowledgments
We thank Dr. Joseph Wagstaff for help and valuable advice. This work was supported in part by Grants-in-Aid for Scientific Research (15591761, 16591670, and 13854024) from the Ministry of Education, Sports, Culture, Science and Technology of Japan (to H.M, K.M., and N.N.) and CREST from Japan Science and Technology Agency (JST; to N.N.). H. Masuzaki and K. Miura contributed equally to this work.
References
-globin gene expression suggests that fetal hematopoietic cells contribute to the pool of circulating cell-free fetal nucleic acids during pregnancy. Clin Chem 2004;50:689-693.
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) and serum hCG protein (
; measured by IRMA); (B), plasma hCG-ß (
); (C), plasma hCG-ß (
) mRNA. * day 0 (day of first surgery); **, days 34–74 when the woman was discharged and serial sampling was unavailable; ***, day of the second surgery.