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BOR - Papers in Press, published online ahead of print January 8, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.008029
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BIOLOGY OF REPRODUCTION 68, 1968–1974 (2003)
DOI: 10.1095/biolreprod.102.008029
© 2003 by the Society for the Study of Reproduction, Inc.

Pregnancy

Apoptosis and Related Proteins in Placenta of Intrauterine Fetal Death in Prostaglandin F Receptor-Deficient Mice1

Junwu Mu2,3, Toru Kanzaki3, Xiaoe Si4, Takuji Tomimatsu3, Hirotsugu Fukuda3, Mitsnori Shioji3, Yuji Murata3, Yukihiko Sugimoto5, and Atsushi Ichikawa5

Department of Obstetrics and Gynecology,3 Radiation Biology,4 Osaka University, Faculty of Medicine, Osaka 565-0871, Japan Department of Physiological Chemistry,5 Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study investigated whether the increase of apoptosis in the placenta is associated with intrauterine fetal death in prostaglandin F receptor-deficient mice. Apoptosis was demonstrated within placental and decidual tissue by the TUNEL method. The majority of apoptosis was found in syncytiotrophoblast tissues. Enhanced TUNEL-positive staining in the syncytiotrophoblast layer was scattered in the placental tissues in clusters of apoptotic cells in the death group. Marked TUNEL-positive cells were identified in decidua of both groups. The rate of apoptosis in the placenta and decidua in the death group was higher than that in the survival group (P < 0.05). Immunohistochemical analysis showed that the level of active caspase-3 protein expression in the placenta in the death group was much higher than that in the survival group. The level of Bcl-2 protein expression in the placenta in the death group was much lower than that in the survival group. Western blot analysis demonstrated that increased expression of the active form of caspase-3 was detected in the placenta and decidua in the death group compared with that in the survival group. In contrast, a decrease in the expression of Bcl-2 was detected in the placenta and decidua in the death group compared with that in the survival group. Enhanced expression of Bax:Bcl-2 ratio was detected in placenta and decidua in the death group compared with that in the survival group. Thus, significantly increased apoptosis in the mouse placenta and decidua might be involved in the pathophysiologic mechanism of intrauterine fetal death.

apoptosis, placenta, pregnancy, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intrauterine fetal death is an alarming event in the practice of obstetrics. Postterm pregnancy is associated with an increased risk of perinatal mortality and morbidity [1, 2]. However, the mechanisms leading to fetal and neonatal death in postterm pregnancies or term pregnancies are unclear.

Clearly, apoptosis is important in many aspects of reproduction. Apoptosis occurs in all placental cell types, increases from first to third trimester, and is believed to be physiologically important for normal placental growth and development [3, 4]. Aberrant placental apoptosis may affect placental function, resulting in complicated pregnancies. Increased trophoblast apoptosis has been documented in the placentas of growth-restricted fetuses, and maternal smoking has been shown to be associated with decreased placental apoptosis at term [5, 6]. Placental apoptosis is also increased during spontaneous abortion in the first trimester [7], preeclamptic pregnancy [8, 9], and postterm pregnancy [10].

Apoptotic signaling is processed by highly regulated and specific proteolysis via caspase. Caspases are synthesized as inactive precursors that must be cleaved autocatalytically or by other caspases for activation. Triggering of apoptosis results in a cascade of caspase activation, in which the last caspases to be activated are those that digest cellular substrates, resulting in morphological changes and death of the cell. Bcl-2 of antiapoptotic regulators was first discovered in human follicular lymphoma and was regarded as a proto-oncogene [11]. It is reported that Bcl-2 has the function of inhibiting apoptosis [12, 13]. Bcl-2 plays an important role in preventing apoptosis in the syncytiotrophoblast [14]. Bax of proapoptotic regulators appears to accelerate the cell-death signal. Overexpression of Bax promotes apoptotic cell death, and conversely, Bcl-2 promotes this type of cell survival [15, 16]. The ratio of Bax to Bcl-2 is an important determinant of cell death or survival [17].

Prostaglandin F2{alpha} (PGF2{alpha}), one of the most abundant prostaglandins, is particularly involved in reproductive functions, such as ovulation, luteolysis, and parturition [18]. Sugimoto et al. [19] developed mice lacking the prostaglandin F receptor. These mice do not deliver fetuses at term and, instead, continue their pregnancy, although these mice are normal with respect to other aspects of reproductive physiology. No abnormalities in the weight or histology of placentas at term were observed in the PGF2{alpha} receptor-deficient mice. These fetuses gradually died in uterus with prolonged pregnancy and were reabsorbed. Therefore, this PGF2{alpha} receptor-deficient mouse provides a good animal model for studying intrauterine fetal death in postterm pregnancy [10, 19]. The present study investigated whether the increase of apoptosis in the placenta and decidua is associated with intrauterine fetal death in PGF2{alpha} receptor-deficient mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All animals were maintained in accordance with institutional guidelines for the care and use of laboratory animals. Mice were housed under standard photoperiod (12L:12D) and temperature (23 ± 1°C). Mice were also given free access to a nutritionally balanced diet and tap water. The PGF2{alpha} receptor-deficient mice were obtained as described previously [19]. Normal adult female mice with the +/- genotype bred in our animal facility were mated with either PGF2{alpha} receptor-deficient male mice or male mice with the +/- genotype, and the resulting female PGF2{alpha} receptor-deficient mice were used in the present experiment. Mouse genotypes were identified by polymerase chain reaction.

The day that a copulation plug was found was considered to be Day 0 of pregnancy. Placentas were removed from fetuses on Days 18–23 of pregnancy. At the same time, the survival and weight of fetuses were recorded. At least three pregnant mice were used for each day of pregnancy. All fetuses were divided into the survival and death groups. Fetal death was determined by lack of movement, respiratory activity, or reaction to painful stimuli. The placentas from fetuses in the death group were excluded when partial or complete body maceration, skin wrinkling, and discoloration of skin were noted. The placentas on Days 21 and 22 of pregnancy were used for analysis of apoptosis, caspase-3, Bcl-2, and Bax. Half the placentas from the same mice were fixed in a 10% formaldehyde neutral buffer solution and embedded in paraffin. Five sections were cut for each sample. The other placentas were isolated by gently separating placental and decidual tissues. The tissues were flash-frozen and stored at -80°C until processed for Western blot analysis.

In Situ Detection of DNA Nicking

Detection of DNA fragmentation was performed using the TUNEL technique. The TUNEL procedure was performed using an Apop Tag kit (Oncor, Gaithersburg, MD) according to the manufacturer's instructions. The tissue sections were deparaffinized, and protein was digested with 20 µg/ml of proteinase K for 15 min at room temperature. Endogenous peroxidase activity was quenched with 3% H2O2 in PBS. After washing with PBS, an equilibration buffer was applied directly to the specimen. Terminal deoxynucleotidyl transferase (TdT) enzyme and dUTP-digoxigenin were added and incubated at 37°C for 1 h in a humidified chamber. The reaction was then stopped with a stop/wash buffer supplied with the kit, and the slides were incubated with an anti-digoxigenin-peroxidase solution for 30 min at room temperature, colorized with diaminobenzidine (DAB)/H2O2, and counterstained with methyl green. Negative controls were processed with labeled dUTP in the absence of the TdT enzyme. Sections of normal rodent mammary gland were used as positive controls. For each section, six fields of view were examined, and the number of TUNEL-positive stained nuclei was expressed as a percentage of the total number of nuclei counted.

Immunohistochemistry

Tissue samples were embedded in paraffin, and sections (thickness, 5 µm) were cut with a microtome and placed on coated slides. Paraffin was removed from the tissue sections with xylene, and the sections were rehydrated in graded ethanol solutions. Antigen retrieval was performed by heating in 10 mM citrate buffer (pH 6.0) for 10 min. Endogenous peroxidase activity was blocked with 3% H2O2 for 10 min. After blocking with 5% normal rabbit serum, sections were incubated with the primary antibody for Bcl-2 (polyclonal antibody N-19 at 1:200 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and for caspase-3 (polyclonal antibody D-175 at 1:100 dilution; Cell Signaling Technology, Beverly, MA) at 4°C overnight. They were rinsed in PBS and incubated with biotinylated goat anti-rabbit immunoglobulin G (Vector Laboratories, Burlingame, CA), followed by avidin-biotin-peroxidase solution (Vectastain ABC Elite kit; Vector Laboratories). Next, DAB with 0.003% H2O2 in PBS was added to each slide. The tissues were lightly counterstained with hematoxylin and examined by light microscopy. Negative controls were performed with normal rabbit serum instead of primary antibody with all other steps unchanged.

Western Blot Analysis

Each sample was homogenized in a lysis buffer (0.01 M Tris [pH 7.8], 0.1 M NaCl, 0.1 mM EDTA, 1 mM PMSF, 2 µg/ml of pepstatin, 1 µg/ml leupeptin, and 2 µg/ml of chymostatin) and centrifuged at 12 000 x g for 10 min at 4°C, and the supernatants were stored at -80°C. Then, the protein concentration of the lysates was determined with a BCA protein assay kit (Pierce, Rockford, IL), and samples (25 µg) were denatured in a gel loading buffer at 100°C for 3 min and loaded on a 4%–20% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, the proteins were transferred to nitrocellulose membranes. Blocking was performed with PBST (PBS containing 0.1% Tween 20) containing 5% nonfat dried milk solution for 1 h. The transferred membrane was incubated with the primary antibody for caspase-3 (polyclonal antibody D-175 at 1:500 dilution; Cell Signaling Technology) and for Bcl-2 and Bax (polyclonal antibody N-19 and P-19, respectively, at 1:1000 dilution; Santa Cruz Biotechnology, Inc.) overnight at 4°C. After washing, horseradish peroxidase-conjugated anti-rabbit immunoglobulin G was applied at a 1:1000 dilution for 1 h at room temperature. The blot was washed in PBST three times and visualized with an enhanced chemiluminescence Western blotting detection system (Amersham, Arlington Heights, IL) and exposed to x-ray film. The same membranes were reprobed with anti-glyceraldehyde phosphate dehydrogenase antibody and used as a control to ascertain that equivalent amounts of proteins had been transferred. Quantitative densitometry of Western blots was performed using the Scion Image Program (Eastman Kodak, Rochester, NY).

Statistical Analysis

Values are expressed as the mean ± SEM. Statistical analysis between the groups was performed using the Student t-test and Mann-Whitney U-test. Values were considered to be significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To assess reproductive function in PGF2{alpha} receptor-deficient mice, we evaluated the number of pups, survival, weight, and amniotic fluid in the previous studies [10, 19]. In the present study, wild-type mice delivered normal fetuses at term, but PGF2{alpha} receptor-deficient mice were unable to deliver the fetuses. The fetuses gradually died in the uterus in PGF2{alpha} receptor-deficient mice after Day 18 of pregnancy. The rate of intrauterine fetal death was lower on Days 19 and 20 of pregnancy but increased to 31.5% on Day 21 and to 60.7% on Day 22 of pregnancy. All fetuses died in the uterus by Day 23 of pregnancy. No significant differences were observed in the weight of the fetus, placenta, and decidua on Days 21 and 22 of pregnancy between the survival and death groups.

Apoptosis

Apoptosis was demonstrated within placental and decidual tissue by the TUNEL method. The TUNEL-positive cells were found in the trophoblast and stromal cells. The majority of apoptosis was found in syncytiotrophoblast tissues in the placenta. Increased TUNEL-positive staining in the syncytiotrophoblast layer was shown in the placenta in the death group (Fig. 1b) compared with that in the survival group (Fig. 1a). Elevated TUNEL-positive staining in the syncytiotrophoblast layer was scattered throughout the placenta, showing clusters of apoptotic cells in the death group. Marked TUNEL-positive cells were identified in the decidua in both groups (Fig. 1, c and d). The rate of apoptosis in the placenta and decidua in the death group was higher than that in the survival group (P < 0.05) (Fig. 2).



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  		FIG. 1. TUNEL staining in placenta and decidua of the survival and death groups. Dark-brown staining indicates a positive reaction. Tissues were counterstained with 1% methyl green. The TUNEL-positive cells (arrows) in the placenta (a) and decidua (c) at Day 22 of pregnancy in the survival group are shown. Increased TUNEL-positive staining (arrows) was seen in the syncytiotrophoblast layer (b) and decidual tissues (d) at Day 21 of pregnancy in the death group. Bars = 50 µm



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  		FIG. 2. Comparison of incidences of apoptosis in the placenta (P) and decidua (D) from the survival group and death group at Days 21–22 of pregnancy. The data represent the mean ± SEM of 8–12 samples from different fetuses. *P < 0.05, compared with survival group

Caspase-3, Bcl-2, and Bax Expression

Immunohistochemical analysis of active caspase-3 protein expression was demonstrated in the syncytiotrophoblasts in the mouse placenta in both the survival and death groups. Active caspase-3 protein in the placenta in the death group (Fig. 3b) was much more abundant than in the survival group (Fig. 3a). Immunohistochemical analysis demonstrated Bcl-2 protein expression in the cytoplasm of syncytiotrophoblasts. The nuclei were never immunostained for Bcl-2. No immunostaining for Bcl-2 protein was detected in fetal capillaries. Bcl-2 protein in the placenta in the death group (Fig. 3d) was much less abundant than in the survival group (Fig. 3c). Bax protein in the placenta in the death group was more abundant than in the survival group (data not shown).



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  		FIG. 3. Immunohistochemical staining for active caspase-3 and Bcl-2 protein in sections of the placentas from the survival and death groups at Day 22 of pregnancy. Active caspase-3 positive immune reactivity (arrows) was seen in the survival group (a) and the death group (b). Bcl-2-positive immune reactivity was seen in the survival group (c) and the death group (d). Brown staining indicates a positive reaction. Tissues were counterstained with hematoxylin. Bars = 50 µm

Western blot analysis demonstrated specific bands for caspase-3, Bcl-2, and Bax in placental and decidual tissues. Increased expression of the active form of caspase-3 was detected in the placenta and decidua in the death group compared with the survival group (Fig. 4). In contrast, a decrease in the expression of Bcl-2 was detected in the placenta and decidua in the death group compared with the survival group (Fig. 5). The high levels of Bax expression were clearly observed in both placenta and decidua in the survival and death groups. The enhanced expression of Bax was detected in placenta in the death group compared with that in the survival group (Fig. 6). The expression of the Bax:Bcl-2 ratio increased approximately 3.4-fold on placenta and 2.3-fold on decidua in the death group compared with the survival group (Fig. 7). These results are in agreement with the findings of direct immunostaining of the placenta in the survival and the death groups.



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  		FIG. 4. Expression of active caspase-3 in the placental and decidual tissues on Days 21–22 of pregnancy. Top) A representative Western blot of active caspase-3 expression. This antibody detects only the large fragment of activated caspase-3 (17–19 kDa); it does not recognize endogenous levels of full-length caspase-3 or other caspases. Data were representative of five independently performed experiments. Bottom) A summary of densitometric values normalized to glyceraldehyde phosphate dehydrogenase (GAPDH). The density of the placenta from survival group bands was set arbitrarily at 1.0. Values shown represented the mean ± SEM from five separate experiments. D, Decidua; P, placenta. *P < 0.05 compared with the survival group



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  		FIG. 5. Expression of active Bcl-2 in the placental and decidual tissues on Days 21–22 of pregnancy. Top) A representative Western blot of Bcl-2 expression. Data are representative of five independently performed experiments. Bottom) A summary of the densitometric values normalized to glyceraldehyde phosphate dehydrogenase (GAPDH). The density of the placenta from survival group bands was set arbitrarily at 1.0. Values shown represent the mean ± SEM from five separate experiments. D, Decidua; P, placenta. *P < 0.05 compared with survival group



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  		FIG. 6. Expression of Bax in the placental and decidual tissues on Days 21–22 of pregnancy. Top) A representative Western blot of Bax expression. Data are representative of three independently performed experiments. Bottom) A summary of the densitometric values normalized to glyceraldehyde phosphate dehydrogenase (GAPDH). The density of the placenta from survival group bands was set arbitrarily at 1.0. Values shown represent the mean ± SEM from three separate experiments. D, Decidua; P, placenta. *P < 0.05 compared with survival group



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  		FIG. 7. Expression of Bax:Bcl-2 ratio in the placental (P) and decidual (d) tissues on Days 21–22 of pregnancy. Values were determined from Figures 5 and 6 and represent the mean ± SEM from three to five separate experiments. *P < 0.05 compared with survival group


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The many possible causes of fetal death generally can be categorized as fetal, placental, and maternal causes. The clinical history is often unremarkable, and often no detectable, preexisting antepartum medical or obstetric complication can be directly associated with the human fetal death. Obvious abnormalities are uncommon on gross inspection of the fetus or placenta [20]. Little is known regarding the environmental and genetic controls that regulate programmed cell death in the placental trophoblast. The trophoblast regulates maternal-fetal gas, nutrient, and waste product exchange. Homeostasis of syncytiotrophoblast is crucial to the maintenance of normal transport and secretary functions of the placental villous. Increases in trophoblast apoptosis were found in placentas from women with pregnancies complicated by preeclampsia and intrauterine growth restriction compared to controls [5, 21]. Therefore, the identification of apoptosis in this key cellular interface highlights the importance of understanding what controls apoptosis in the placenta [22]. However, to our knowledge, no reports concerning apoptosis and related proteins in intrauterine fetal death have been published.

We previously reported that increases in apoptosis and the apoptotic staining of the syncytiotrophoblast layer were found in a few postterm placentas in cases in which the fetus died within 24 h [10]. In the present study, we investigated this phenomenon and demonstrated that a significant increase of apoptosis in the syncytiotrophoblast layer of the mouse placenta might be involved in intrauterine fetal death. Increased TUNEL-positive staining in the syncytiotrophoblast layer was shown in the placenta of the death group compared with that of the survival group. Syncytiotrophoblast apoptosis has also been suggested to precede the breaks in the trophoblast covering villi and the loss of integrity in the syncytium [23]. A recent study demonstrated increased apoptosis in syncytiotrophoblast cells in either severe preeclamptic term placentas or in term placentas of intrauterine growth-restricted fetuses compared with normal term placentas [24]. Dysregulation of the apoptotic process probably causes a variety of diseases [25, 26]. An obvious question raised by the present study is what happens to placental function when placental apoptosis is increased in term pregnancy of preeclamptic placentas or placentas from intrauterine growth-restricted fetuses [5, 21]. The elevation of apoptosis in syncytiotrophoblast cells of the postterm pregnant placenta may be the cause of the damage of the syncytiotrophoblast layer. The syncytiotrophoblast plays a key role throughout pregnancy, being the site of many placental functions that are required for growth and development. Placental dysfunction leads to the loss of nutrition of the fetuses and contributes to the increased morbidity associated with postterm pregnancies.

The present study also showed the expression of apoptosis and related proteins in decidual tissues. A higher level of apoptosis was detected in the death group compared with the survival group. Increased active caspase-3, increased Bax:Bcl-2 ratio, and decreased Bcl-2 were found in the decidua of the death group compared to that of the survival group. The decidua is thought to provide nutrition to the developing embryo, to protect the embryo from the immunologic responses of the mother, and to regulate trophoblast invasion into the uterine stroma. Apoptosis was detected in decidua of early pregnancy in several studies [7, 27]. Apoptosis in the decidua increased with gestational day in mice [10]. The decidual weight begins to decrease during later pregnancy in mice [10], and the histological regression of decidual basics begins at approximately Day 14 of pregnancy in rats [28]. Increased apoptosis with advancing gestational age suggested that apoptosis is a normal placental aging process [3]. Increased apoptosis in decidua of postterm pregnancy represents an acceleration of the aging process. The increased apoptosis in the decidua in cases of spontaneous abortion might cause apoptosis in the syncytiotrophoblast [7]. The increase in trophoblast apoptosis in postterm pregnancy possibly is a consequence of decidual apoptosis; thus, both trophoblast cell death and fetal mortality may relate to increased dysfunction of the decidua.

We found that the expression of Bcl-2 was decreased in the placenta in the death group compared with that in the survival group. High expression of Bcl-2 in the syncytiotrophoblast would protect this key layer of placental villi from apoptosis [29]. The lower Bcl-2 expression in the placenta of the death group might be related to the induction of apoptosis by increasing the expression of active caspase-3. Active caspase-3 has been demonstrated in human and mouse placentas [30, 31]. Caspase activity is regulated by the Bcl-2 family of proteins [32–35]. Lea et al. [36] examined the immunostaining of Bcl-2, a proto-oncogene thought to inhibit apoptosis, in biopsy specimens from first-trimester failing pregnancies. Immunostaining of Bcl-2 was less intense in failing pregnancies. The decreased Bcl-2 expression in complete hydatidiform mole, severe preeclamptic, and intrauterine growth-retarded placentas was considered to be a biological change that precedes apoptosis [24, 37]. Smith et al. [3] showed ultrastructural evidence of apoptosis in the syncytiotrophoblast, suggesting that Bcl-2 does not completely protect the syncytium from self-destruction. Therefore, the lack of protection by Bcl-2 in the syncytiotrophoblast layer leads to a great increase in apoptosis in the placenta at fetal death.

In the present study, a high Bax:Bcl-2 ratio in placental and decidual tissues was found in the death group compared with the survival group. These results provide evidence that the Bax:Bcl-2 "rheostat" may be a critical factor in regulating apoptosis in the placenta of postterm pregnancy. These findings are in agreement with those of Qiao et al. [37], who reported that an increased Bax:Bcl-2 ratio in complete hydatidiform mole might contribute to the high level of apoptosis. Bax is a likely candidate for regulating apoptosis to the effector stage in syncytiotrophoblast [38]. No significant difference was found in Bax of decidua between groups. Therefore, the Bax:Bcl-2 ratio and/or Bcl-2 may be more useful in assessing cell death via apoptosis.

Hormones have been implicated in the stimulation or inhibition of apoptosis. To our knowledge, little PGF{alpha}-receptor signaling was detected in trophoblasts, although it was found in myometrium (unpublished data). As reported previously, no significant differences were found in apoptosis of the placenta and decidua between PGF2{alpha} receptor-deficient and wild-type mice [10, 31]. Therefore, trophoblast apoptosis in this mouse model was unlikely to have been caused by the lack of prostaglandin signaling. We did not measure the levels of progesterone and estrogen to directly assess their effect on apoptotic expression in the present study. However, some studies have demonstrated that the level of progesterone and estrogen was associated with apoptosis in decidua of rat [39, 40]. Gu et al. [41] suggested that progesterone did not play a role in preventing decidual apoptosis in the rat. In the present study, the maternal plasma concentration of progesterone and estrogen was similar for the fetuses in the two groups, because they were from same maternal mouse. The difference of the apoptotic expression in the survival and death groups was not affected by the change of endocrine capacity in maternal plasma levels. It is unknown whether the local endocrine capacity on the trophoblast or decidual component influences apoptosis in fetal death.

The question is whether the increased apoptosis in the placenta and decidua is a cause or a result of intrauterine fetal death. We found increased apoptosis in placenta and decidua of the death group compared with the survival group in postterm pregnancy. A higher level of apoptosis was found in placentas from pregnancies complicated by preeclampsia and intrauterine growth restriction compared to placentas from pregnancies with normal fetal growth [5, 21]. The dysregulation of the apoptotic process probably causes a further increase in apoptosis in the placenta and decidua of fetal death, resulting in the extreme placental and decidual senescence. In this situation, the placenta may be hard-pressed to meet the metabolic requirements of fetal growth and development. The compensatory process is known to occur in an area of relatively poor placental perfusion, resulting in hypoxic ischemia of the placental circulation. Hypoxia is a known trigger of apoptosis in different tissues, and hypoxia may trigger apoptosis in the placenta as a possible mechanism of pregnancy-related complication [42, 43]. Again, placenta may respond to hypoxic stress by increasing apoptosis; thus, the increased apoptosis aggravates the senescence. This process may be a vicious circle in placenta of fetal death. The dysregulated apoptosis may lead to dysfunction of placenta, resulting in intrauterine fetal death. If so, then apoptosis in such conditions might be occurring as a cause of the pathologic processes leading to fetal death. Alternatively, if the increase in apoptosis is an intrinsic component of trophoblast and decidual differentiation that is programmed to begin at the "end" of the trophoblast differentiation program, then the increased trophoblast apoptosis in postterm pregnancy could be a result of the finalization of the differentiation program and could not be a direct contributor to fetal death.

The design of the present study had a few limitations. First, we could not precisely identify the time of fetal death in the mouse uterus. The placentas of fetal death which included body maceration, skin wrinkling, and skin discoloration were excluded from the death group. Therefore, the remaining placentas included in the death group were considered to have been collected shortly after fetal death. In a future study, we will attempt to evaluate the condition of the mouse fetuses early, using high-frequency flow imaging [44]. The second limitation was the use of the TUNEL method for identifying apoptosis. Although it has been reported that necrosis might cause false-positive staining in this method [45, 46], it appeared to be a reliable and highly sensitive method for showing apoptosis [40, 47]. Our previous results showed that findings using the TUNEL method were similar to those using DNA fragmentation assay [10, 31]. It has also been demonstrated that this method involves less false-positive staining from necrosis after fetal death [48]. Edwards et al. [48] reported that a large number of brain cells undergo apoptosis in infants who suddenly die after intrauterine insults but that a significantly lower number undergo necrosis. The third limitation was the possibility that the increase in placental apoptosis in the death group occurred after fetal death. In general, placental cells do not die soon after fetal death. Furthermore, no further increase in apoptosis was shown in placentas after fetal death at later time points compared with the level in the death group in the present study (data not shown). Because placental apoptosis increases with gestation [3, 10, 49], the increases in apoptosis that we observed were unlikely to have been the result of placental changes after fetal death. Our group will attempt to further identify the relationship between fetal death and placental apoptosis in future studies.

In conclusion, apoptosis, active caspase-3, Bcl-2, and Bax were found in the mouse placentas and decidua of both the survival and death groups. Apoptosis has a positive relationship with active caspase-3 and the Bax:Bcl-2 ratio and a negative relationship with Bcl-2 in the placenta and decidua. Increased apoptosis, active caspase-3, and Bax:Bcl-2 ratio in the placenta and decidua were found in the death group compared with the survival group. In contrast, a decrease in the expression of Bcl-2 was detected in the death group compared with the survival group. A significant increase in apoptosis in the mouse placenta and decidua might be involved in the pathophysiologic mechanism of intrauterine fetal death.


   FOOTNOTES
 
1 Supported in part by grant 13671711 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Back

2 Correspondence: Junwu Mu, Department of Obstetrics and Gynecology, Osaka University, Faculty of Medicine, 2-2 Yamadaoka Suita, Osaka, 565-0871 Japan. FAX: 81 6 6879 3359; mujunwu{at}hotmail.com Back

Received: 4 June 2002.

First decision: 27 June 2002.

Accepted: 26 December 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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