Molecular Human Reproduction, Vol. 6, No. 1, 19-25,
January 2000
© 2000 European Society of Human Reproduction and Embryology
Endocrinology |
Superoxide dismutase expression in the human corpus luteum during the menstrual cycle and in early pregnancy
Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Minamikogushi 1-1-1, Ube 755-8505, Japan
Abstract
To investigate the possible role of the superoxide radical and its scavenging system in the human corpus luteum, superoxide dismutase (SOD) values and lipid peroxide concentrations were analysed in the corpora lutea during the menstrual cycle and in early pregnancy. Copper–zinc SOD (Cu,Zn-SOD) activities increased from the early to mid-luteal phase, and gradually decreased thereafter and were the lowest in the regression phase. In pregnant corpus luteum, Cu,Zn-SOD activities were significantly higher than those in the mid-luteal phase. In contrast, manganese SOD (Mn-SOD) activities were low in the mid-luteal phase and increased toward the regression phase. Changes in mRNA expression of both types of SOD were similar to changes in their activities. Lipid peroxide concentrations were the highest in the regression phase whereas they were remarkably low in pregnant corpus luteum. The effects of human chorionic gonadotrophin (HCG) on luteal SOD were studied in vitro. HCG significantly increased Cu,Zn-SOD expression in mid-luteal phase corpora lutea, but not in late luteal phase corpora lutea. In conclusion, the present study suggests that the superoxide radical and its scavenging system, especially Cu,Zn-SOD, play important roles in the regulation of human luteal function. The stimulation of luteal Cu,Zn-SOD expression by HCG may be important in maintaining luteal cell integrity when pregnancy occurs.
corpus luteum/human/HCG/superoxide dismutase/superoxide radical
Introduction
Reactive oxygen species including superoxide radicals are well known to cause cell damage. They are increased in the corpus luteum during the regression phase (Riley and Behrman, 1991
; Sawada and Carlson, 1991, 1994; Sugino et al., 1993a; Shimamura et al., 1995) and inhibit progesterone production in rats (Behrman and Preston, 1989; Behrman and Aten, 1991; Sugino et al., 1993a,b, 1999; Kodaman et al., 1994), suggesting the involvement of reactive oxygen species in corpus luteum regression (Kato et al., 1997). In contrast, the corpus luteum has specific enzymes to scavenge superoxide radicals: copper–zinc superoxide dismutase (Cu,Zn-SOD), located in the cytosol, and manganese SOD (Mn-SOD), located in the mitochondria. Both types of SOD belong to a first enzymatic step that protects cells against toxic oxygen radicals. It has been reported that SOD acts protectively against superoxide radicals to stimulate progesterone production by the corpus luteum in rats (Laloraya et al., 1988; Sugino et al., 1993b, 1999; Sawada and Carlson, 1996). We previously reported that both Cu,Zn-SOD and Mn-SOD activities in the corpus luteum increased until day 9 and decreased thereafter in pseudopregnant rats (Shimamura et al., 1995), while they further increased until day 12 of pregnancy in pregnant rats, in a manner similar to the change in serum progesterone concentrations (Sugino et al., 1993a). In addition, Cu,Zn-SOD and Mn-SOD mRNA expression were stimulated by rat placental lactogens in rat luteal cells (Sugino et al., 1998a). These findings strongly suggest that SODs may play important roles in the maintenance of luteal function and possibly in the rescue of the corpus luteum when pregnancy occurs. In human, there are few reports showing that hydrogen peroxide, one of the reactive oxygen species, inhibits progesterone production by luteal cells (Endo et al., 1993; Vega et al., 1995) and that SOD is detected in luteal cells by immunohistochemistry (Shiotani et al., 1991; Suzuki et al., 1999). However, the regulatory mechanism of SOD expression and its roles in the human corpus luteum are poorly understood. In the present study, to investigate the possible role of SOD in the human corpus luteum, changes in activities and mRNA expression of both Cu,Zn-SOD and Mn-SOD and lipid peroxide values were examined in the human corpus luteum throughout the menstrual cycle and in early pregnancy. We further examined the regulation of SODs in the human corpus luteum by human chorionic gonadotrophin (HCG).
Materials and methods
This project was reviewed and approved by the committee on investigations involving human subjects of Yamaguchi University School of Medicine, Japan. Informed consent from the patient was obtained before collection of any tissue samples for this study.
Reagents
Roswell Park Memorial Institute (RPMI) 1640 medium was from Flow Laboratories Inc (McLean, VA, USA). Streptomycin, penicillin, deoxynucleotide triphosphates and Moloney murine leukaemia virus reverse-transcriptase were from Life Technologies Inc (Grand Island, NY, USA). HCG was from Sigma Chemical Co (St Louis, MO, USA). Random hexamer and Taq DNA polymerase were from Perkin-Elmer Co (Foster City, CA, USA). [
-32P]-deoxycytidine triphosphate (dCTP) was from Amersham (Arlington Heights, IL, USA). Isogen was from Wako Pure Chemical Industries Ltd (Osaka, Japan).
Tissue samples
Human corpora lutea were obtained at hysterectomy from normally cycling women, aged 39–49 years, who underwent surgery for myoma uteri or cervical cancer. The menstrual history and endometrial histology were used to determine the age of the corpus luteum. Corpora lutea of the cycle were classified into four different groups according to their age; the early luteal phase (days 1–5 of luteal phase), mid-luteal phase (days 6–11), late luteal phase (days 12–15) with day 1 being the day of ovulation, and regression phase (after onset of menstruation, days 3–7 of follicular phase). Corpora lutea of early pregnancy (6–8 weeks of pregnancy) were obtained from the patients, aged 24–30 years, with ectopic pregnancy. Tissue samples were washed with saline to remove blood, and immediately frozen in liquid nitrogen and stored at –80°C until SOD activity assay, lipid peroxide assay and RNA isolation. In some patients, blood samples were obtained at surgery for determination of serum progesterone concentrations.
Incubation of corpora lutea
Corpora lutea obtained from the mid-luteal phase or the late luteal phase were sliced into small pieces and incubated in serum-free RPMI 1640 medium (35–60 mg wet weight/ml/tube) at 37°C for 1 h under an atmosphere of 95% O2:5% CO2 in a shaking water bath. The medium was then changed to the test medium containing HCG (1 or 10 IU/ml), and the incubation was continued for 6 h under the same atmosphere as described above. After incubation, the medium was collected and stored at –20°C for progesterone assay and the corpus luteum tissue was immediately frozen in liquid nitrogen and stored at –80°C until SOD activity assay or RNA isolation. The incubation was run in triplicate.
SOD assay and lipid peroxide assay
Corpora lutea were homogenized with Tris–HCl buffer (0.01 mol/l, pH 7.4) using glass homogenizers and centrifuged at 800 g for 10 min at 4°C, and the supernatant was used for SOD assay. Cu,Zn-SOD and Mn-SOD activities were determined as reported previously (Sugino et al., 1993a), based on the nitrite method (Oyanagui, 1984). The amount of protein required for 50% inhibition in the absorbance at 550 nm was defined as one unit (nitrite unit; NU) of SOD activity. All data were expressed in NU of SOD activity per mg protein. Protein concentrations were determined by Lowry's method (Lowry et al., 1951). The intra- and inter-assay coefficients of variation were 3.8 and 9.6%, for the Cu,Zn-SOD assay, and 4.7 and 6.4% for the Mn-SOD assay respectively.
Concentrations of lipid peroxide in corpora lutea were measured by the thiobarbituric acid method (Ohkawa et al., 1979). The results were expressed as nmol of malondialdehyde (MDA) per g wet weight.
Reverse transcription–polymerase chain reaction (RT–PCR)
Total RNA was isolated from the corpora lutea with Isogen by the method provided by the manufacturer. For mRNA analysis, RT–PCR was performed as reported previously (Sugino et al., 1998b). The oligonucleotide primers for Cu,Zn-SOD (5'-CGAGCAGAAGGAAAGTAATG-3' and 5'-TAGCAGGATAACAGATGAGT-3') and for Mn-SOD (5'-AGTTCAATGGTGGTGGTCATA-3' and 5'-CAATCCCCAGCAGTGGAATAA-3') were designed on the basis of the human Cu,Zn-SOD (Hallewell et al., 1985) and Mn-SOD cDNA sequences (Gene Bank, accession NO. E01408). Two oligonucleotide primers (5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-GGACAGAGTCTTGATGATCTC-3') were also used to amplify ribosomal protein L19 as an internal control (Chan et al., 1987). Briefly, 3 µg of total RNA were reverse transcribed at 42°C in a reaction mixture (single-strength PCR buffer, 2.5 mmol/l deoxynucleotide triphosphates, 5 µmol/l random hexamer primer, 1.5 mmol/l MgCl2, and 200 IU Moloney murine leukaemia virus reverse-transcriptase). The RT product was divided into equal aliquots and placed into two tubes for SOD primers and L19 primers, and PCR was performed. For PCR amplification, a mixture containing the oligonucleotide primers (50 pmol), [-32P]-dCTP (2 µCi at 3000 Ci/mmol), and Taq DNA polymerase (2.5 IU) was added to each reaction. Amplification was carried out for 25 cycles consisting of 95°C (1 min), 52°C (1 min) and 72°C (1 min) for Cu,Zn-SOD and 25 cycles consisting of 95°C (1 min), 54°C (1 min) and 72°C (1 min) for Mn-SOD, followed by 10 min of final extension at 72°C in a programmed temperature control system PC-800 (ASTEC, Fukuoka, Japan). The predicted sizes of the PCR-amplified products were 455 bp for Cu,Zn-SOD, 282 bp for Mn-SOD, and 194 bp for L19. A linear curve was plotted using number of cycles of amplification versus densitometric values of the PCR products, measured with a BAS2000 (Fuji Photo Film Co, Tokyo, Japan). The optimal number of cycles for amplification that fit within the linear range was chosen for each set of primers of SODs and L19 (data not shown). Reaction products were electrophoresed on an 8% polyacrylamide non-denaturing gel. After autoradiography, data were quantified using a bioimaging analyser BAS2000. To validate that the amplified cDNAs were Cu,Zn-SOD and Mn-SOD, the PCR products were cloned with TA cloning kit (Invitrogen Co, San Diego, CA, USA). Then direct sequence analyses of the PCR products were performed. The cDNA sequences of the amplified cDNA with primer sets for Cu,Zn-SOD and Mn-SOD were consistent with the previously reported sequences of human Cu,Zn-SOD (Hallewell et al., 1985) and Mn-SOD (Gene Bank, accession NO. E01408).
Progesterone assay
Progesterone concentrations in the serum and the medium were determined by a specific radioimmunoassay as reported previously (Kato et al., 1982). The sensitivity of the assay was 100 pg/ml, and the intra- and inter-assay coefficients of variation were 7.0 and 14.4% respectively. Progesterone concentrations in the medium were expressed as ng of progesterone per mg wet weight of the corpus luteum.
Statistical analysis
Data were examined by analysis of variance and Duncan's multiple range test. P < 0.05 was considered to be significant.
Results
In this study, serum progesterone concentrations (mean ± SEM) were higher in the mid-luteal phase (13.1 ± 1.8 ng/ml, n = 5) than in the early luteal phase (4.8 ± 1.5 ng/ml, n = 4) and the late luteal phase (3.2 ± 0.6 ng/ml, n = 5), whereas the progesterone concentrations of all patients in the regression phase were <1.0 ng/ml.
Cu,Zn-SOD activities in the corpus luteum increased from the early to mid-luteal phase, gradually decreased thereafter and were the lowest in the regression phase (Figure 1A). In the pregnant corpus luteum, Cu,Zn-SOD activities were significantly higher than those in the mid-luteal phase (Figure 1A). In contrast, Mn-SOD activities increased from the early-to-mid luteal phase to late luteal phase, and further increased in the regression phase (Figure 1B). In the pregnant corpus luteum, Mn-SOD activity was the same as those in the early luteal phase (Figure 1B). Activities of Cu,Zn-SOD and Mn-SOD were not statistically correlated.
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Lipid peroxide concentrations in the corpus luteum were significantly higher in the regression phase compared with the other phases, and were remarkably low in the pregnant corpus luteum (Figure 2
).
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Cu,Zn-SOD mRNA expression in the corpus luteum decreased from the mid-to-late luteal phase to regression phase (Figure 3A
). In the pregnant corpus luteum, Cu,Zn-SOD mRNA expression was significantly higher than those in the mid-luteal phase (P < 0.05; Figure 3A). In contrast, Mn-SOD mRNA expression increased from the mid-luteal to late luteal phase (P < 0.01), and further increased in the regression phase (P < 0.01; Figure 3B). In the pregnant corpus luteum, Mn-SOD mRNA expression was the same as that in the mid-luteal phase (Figure 3B).
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To study whether HCG is involved in the change in activities and mRNA expression of both SODs shown in the pregnant corpus luteum, corpora lutea of the mid-luteal phase were incubated with HCG. HCG significantly increased progesterone concentrations in the medium, Cu,Zn-SOD activities and Cu,Zn-SOD mRNA expression in a dose-dependent manner (Figure 4A
). To study whether the responsiveness of the corpus luteum to HCG is dependent on the age of the corpus luteum, corpora lutea of the late luteal phase were also incubated with HCG. As shown in Figure 4B, HCG significantly increased progesterone production (P < 0.01) but caused no significant effect on activities and mRNA expression of Cu,Zn-SOD in the late luteal phase corpus luteum. HCG had no effect on activities and mRNA expression of Mn-SOD in the corpus luteum of both mid-luteal phase and late luteal phase (data not shown).
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Discussion
To our knowledge, this is the first report showing the change in activities and mRNA values of both Cu,Zn-SOD and Mn-SOD and lipid peroxide concentrations in the human corpus luteum during the menstrual cycle and in early pregnancy. Cu,Zn-SOD expression increased from the early to mid-luteal phase and gradually decreased toward the regression phase. These changes were similar to the well-known pattern of progesterone production by the corpus luteum and also consistent with our previous findings in rats that changes in Cu,Zn-SOD expression in the corpus luteum paralleled changes in serum progesterone concentrations during pregnancy and pseudopregnancy (Sugino et al., 1993a, 1998b; Shimamura et al., 1995). It is, therefore, likely that Cu,Zn-SOD is related to progesterone production by the human corpus luteum. We have recently found that the suppression of intracellular Cu,Zn-SOD activity by antisense oligonucleotides to Cu,Zn-SOD cDNA inhibited progesterone production in rat luteal cells (Sugino et al., 1999). Since superoxide radicals are generated during normal metabolic activity including steroidogenesis (Carlson et al., 1995), Cu,Zn-SOD may, at least in part, contribute to the maintenance of luteal function by protecting luteal cells from toxic oxygen radicals. It has been reported that the corpus luteum also has antioxidants in the form of ascorbate and carotenoids, which have been suggested to scavenge free radicals generated during steroidogenesis (Behrman et al., 1993).
In contrast to Cu,Zn-SOD, Mn-SOD expression was low in the mid-luteal phase and increased during the late-to-regression phase. It is thought that Cu,Zn-SOD is a constitutive type and Mn-SOD is an inducible type that can be responsive to inflammatory reaction or cytokines (Sugino et al., 1998b). We reported that Cu,Zn-SOD and Mn-SOD are differently expressed and regulated in the rat corpus luteum during the regression phase, and that Mn-SOD, but not Cu,Zn-SOD, is highly induced by inflammatory reaction and cytokines in the rat corpus luteum (Sugino et al., 1998b). Recently, it was reported in human that Mn-SOD, but not Cu,Zn-SOD, was immunohistochemically detected in non-functioning luteal cells and in a large number of macrophages in the regression phase corpus luteum (Suzuki et al., 1999). Macrophages are the source of cytokines and are increased in number in the corpus luteum during the late-to-regression phase in human (Takaya et al., 1997; Suzuki et al., 1998). Therefore, the high expression of Mn-SOD in the corpus luteum undergoing luteolysis shown in the present study may be due to both the selective induction of Mn-SOD in luteal cells by inflammatory reactions or cytokines and the increase in number of Mn-SOD positive macrophages. The difference in expression of Cu,Zn-SOD and Mn-SOD in the human corpus luteum may suggest that they play different roles in regulating luteal function. Further studies are needed to clarify the different roles of Cu,Zn-SOD and Mn-SOD.
The present study showed that lipid peroxide concentrations remarkably increased in the human corpus luteum of the regression phase. Reactive oxygen species inhibit progesterone production by luteal cells in rats (Behrman and Preston, 1989; Behrman and Aten, 1991; Sugino et al., 1993a,b, 1999; Kodaman et al., 1994) and in human (Endo et al., 1993; Vega et al., 1995). These findings suggest that the accumulation of reactive oxygen species is involved in luteolysis in the human corpus luteum. This increase in lipid peroxides may be, at least in part, due to the decline in Cu,Zn-SOD expression, and other possible mechanisms may include phagocytic leukocytes (Sugino et al., 1996a) or a decline in ovarian blood flow (Sugino et al., 1993b; Sugino and Kato, 1994).
Interestingly, in the pregnant corpus luteum, activities and mRNA values of Cu,Zn-SOD were significantly higher than those in the corpus luteum of the mid-luteal phase, and lipid peroxide concentrations were remarkably low, suggesting that the pregnant corpus luteum has a high ability to scavenge superoxide radicals. The present study also demonstrated in vitro that HCG stimulated Cu,Zn-SOD expression of the mid-luteal phase corpus luteum. Therefore, the high expression of Cu,Zn-SOD in the pregnant corpus luteum may be due to the HCG stimulation. Sugino et al. (1998a) reported that placental lactogens up-regulated SOD mRNA expression in rat luteal cells. We also recently found in vivo that injection of placental luteotrophins increased SOD expression in the corpus luteum with the prolongation of luteal function in pseudopregnant rats (S.Takiguchi et al., unpublished data). Recent evidence from studies using knock-out mice has shown that Cu,Zn-SOD plays important roles in the maintenance of early pregnancy (Ho et al., 1998). Taken together, it is suggested that one facet of the luteotrophic effect of HCG, when pregnancy occurs, is to stimulate the expression of molecules that protect luteal cells from toxic oxygen radicals.
The mechanism by which HCG rescues the corpus luteum is poorly understood although extended studies have indicated that ability of HCG to maintain human luteal function may be dependent on the age of the corpus luteum (Baird et al., 1991; Benyo et al., 1993; Zeleznik, 1998). Baird et al. (1991) reported that there is a pivotal stage of the luteal phase beyond which successful rescue of the corpus luteum cannot be achieved in the human. The present study showed that corpora lutea of the mid-luteal phase responded to HCG with increased Cu,Zn-SOD expression, whereas corpora lutea of the late luteal phase did not, despite progesterone production being increased. These results suggest that with advancing age of the human corpus luteum there is a lack of the responsiveness to HCG in terms of Cu,Zn-SOD induction. As we described above, pregnant corpus luteum has a higher ability to scavenge superoxide radicals. Therefore, the increase in ability to scavenge superoxide radicals may be associated with the maintenance of luteal cell integrity and the prolonged life span of the corpus luteum. It is difficult to clearly explain why progesterone production was still stimulated by HCG in the corpus luteum of the late luteal phase although Cu,Zn-SOD was not increased. The late luteal phase corpus luteum may still have had an ability to scavenge superoxide radicals for producing progesterone because Cu,Zn-SOD levels were maintained at control levels under the HCG stimulation. We also found in rat luteal cells that progesterone production was stimulated by HCG without the increase in Cu,Zn-SOD activities, and that HCG-stimulated progesterone production was inhibited when Cu,Zn-SOD activities were decreased (Sugino et al., 1999). Also, the present finding that progesterone production was still stimulated by HCG in the corpus luteum of the late luteal phase may suggest that superoxide radicals were not accumulated as much as progesterone production was inhibited. However, further studies are needed regarding the relationship between Cu,Zn-SOD concentrations and luteal progesterone production.
Recent evidence has shown that accumulation of superoxide radicals and a decrease in SOD concentrations are involved in apoptotic cell death (Buttke and Sandstrom, 1994; Rothstein et al., 1994; Troy and Shelanski, 1994; Rueda et al., 1995), whereas antioxidants including SOD can inhibit apoptosis (Hockenbery et al., 1993; Greenlund et al., 1995; Tilly and Tilly, 1995). It has been also reported that apoptosis is involved in the regulation of luteal function in human (Shikone et al., 1996; Sugino et al., 1996b). Shikone et al. (1996) reported that human luteal regression may be mediated by apoptosis and the corpus luteum of early pregnancy may be rescued from luteolysis through the anti-apoptotic effect of HCG. It will be of interest to investigate the relationship between HCG-stimulated SOD expression and apoptosis inhibition in the rescue of the human corpus luteum.
In conclusion, the present study indicates that the superoxide radical and its scavenging system, especially Cu,Zn-SOD, play important roles in the regulation of human luteal function. The phenomenon that HCG stimulates luteal Cu,Zn-SOD expression may be important in maintaining luteal cell integrity when pregnancy occurs.
Acknowledgments
This work was supported in part by a grant from the UBE Foundation and Grant-in-Aid 11671623 from the Ministry of Education, Science, and Culture, Japan.
Notes
1 To whom correspondence should be addressed 
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Submitted on June 30, 1999; accepted on September 30, 1999.
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 K. H Al-Gubory, P. Bolifraud, G. Germain, A. Nicole, and I. Ceballos-Bicot Antioxidant enzymatic defence systems in sheep corpus luteum throughout pregnancy Reproduction, December 1, 2004; 128(6): 767 - 774. [Abstract] [Full Text] [PDF]
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 N. Sugino, A. Karube-Harada, A. Sakata, S. Takiguchi, and H. Kato Different mechanisms for the induction of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone in human endometrial stromal cells Hum. Reprod., July 1, 2002; 17(7): 1709 - 1714. [Abstract] [Full Text] [PDF]
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 N. Sugino, A. Karube-Harada, S. Kashida, S. Takiguchi, and H. Kato Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone withdrawal in human endometrial stromal cells Mol. Hum. Reprod., January 1, 2002; 8(1): 68 - 74. [Abstract] [Full Text] [PDF]
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A. Karube-Harada, N. Sugino, S. Kashida, S. Takiguchi, H. Takayama, Y. Yamagata, Y. Nakamura, and H. Kato Induction of manganese superoxide dismutase by tumour necrosis factor-{alpha} in human endometrial stromal cells Mol. Hum. Reprod., November 1, 2001; 7(11): 1065 - 1072. [Abstract] [Full Text] [PDF]
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 S. Kashida, N. Sugino, S. Takiguchi, A. Karube, H. Takayama, Y. Yamagata, Y. Nakamura, and H. Kato Regulation and Role of Vascular Endothelial Growth Factor in the Corpus Luteum During Mid-Pregnancy in Rats Biol Reprod, January 1, 2001; 64(1): 317 - 323. [Abstract] [Full Text]
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 N. Sugino, T. Suzuki, S. Kashida, A. Karube, S. Takiguchi, and H. Kato Expression of Bcl-2 and Bax in the Human Corpus Luteum during the Menstrual Cycle and in Early Pregnancy: Regulation by Human Chorionic Gonadotropin J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4379 - 4386. [Abstract] [Full Text]
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N. Sugino, S. Kashida, S. Takiguchi, A. Karube, and H. Kato Expression of Vascular Endothelial Growth Factor and Its Receptors in the Human Corpus Luteum during the Menstrual Cycle and in Early Pregnancy J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3919 - 3924. [Abstract] [Full Text]
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N. Sugino, M. Nakata, S. Kashida, A. Karube, S. Takiguchi, and H. Kato Decreased superoxide dismutase expression and increased concentrations of lipid peroxide and prostaglandin F2{alpha} in the decidua of failed pregnancy Mol. Hum. Reprod., July 1, 2000; 6(7): 642 - 647. [Abstract] [Full Text] [PDF]
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