Molecular Human Reproduction vol.4 no.1 pp. 87–91, 1998
Apoptosis in human chorionic villi and decidua in normal and
ectopic pregnancy
Katsuji Kokawa, Toshihiko Shikone and Ryosuke Nakano1
Department of Obstetrics and Gynaecology, Wakayama Medical College, Wakayama 640, Japan
1To
whom correspondence should be addressed
To investigate possible effects of implantation on apoptosis, we examined the cleavage of DNA in human
chorionic villi and decidua in intrauterine and ectopic pregnancy. Very limited but detectable cleavage of DNA
was recognized in the chorionic villi and decidua in normal pregnancy. A ladder pattern, characteristic of the
apoptotic breakdown of DNA, was present in the villi in tubal pregnancy. High molecular weight DNA was
predominant in the decidua in tubal pregnancy. Quantitative analysis of low molecular weight fragments of
DNA revealed a significant increase in the villous tissue, together with a significant decrease in the decidual
tissue, in tubal pregnancy as compared to those in normal pregnancy. An analysis in situ revealed that
apoptotic cells were predominant in the syncytiotrophoblast in tubal pregnancy. In decidual tissue, labelled
cells were occasionally seen in normal pregnancy, and their numbers decreased in tubal pregnancy. The
present study demonstrates that apoptosis occurs in the villi, but not in the decidua in tubal pregnancy,
unlike the situation in normal pregnancy. Our results suggest that the implantation site might affect the
occurrence of apoptotic changes in early pregnancy of humans.
Key words: apoptosis/chorionic villi/decidua/early pregnancy/ectopic pregnancy
Introduction
It is commonly accepted that apoptotic cell death is an
important phenomenon not only for the removal of pathologically injurious tissue but also for the survival and development of physiologically normal organisms (Glücksmann, 1951;
Kerr et al., 1972). The biochemical event unique to apoptosis
is the internucleosomal cleavage of DNA upon activation of
a Ca21/Mg21-dependent endogenous endonuclease (Zeleznik
et al., 1989; Arends et al., 1990). The cleavage of DNA results
in the appearance on agarose gels of a ladder-like pattern of
fragments, that consist of multiples of 185–200 base pairs
(Wyllie, 1980; Tilly and Hsueh, 1993). In recent years,
apoptotic cells have become detectable in histological sections
using in-situ labelling of DNA (Gavrieli et al., 1992).
Many studies have demonstrated that apoptosis occurs in
the reproductive organs of several mammals, e.g. in the ovary
of the female rat (Palumbo and Yeh, 1994; Hsueh et al., 1994),
the oviduct of the female monkey (Slayden and Brenner,
1994), the uterus of the female rat (Rottello et al., 1989; Gu
et al., 1994), and the testis of the male rat (Shikone et
al., 1994). In some rodents, uterine endometrium undergoes
apoptotic cell death during early pregnancy (Parr et al., 1987).
In humans, it has been reported that the cyclic expression
of tumour necrosis factor-α and BCL-2 is associated with
endometrial apoptosis (Tabibzadeh et al., 1995). We have
previously demonstrated that apoptotic cell death occurs in the
corpus luteum in the mid- and late luteal phases (Shikone
et al., 1996) and in the endometrium in the early proliferative,
late secretory and menstrual phases (Kokawa et al., 1996).
However, only very limited studies (Koh et al., 1995; Shikone
© European Society for Human Reproduction and Embryology
et al., 1996) have been performed on apoptosis during early
pregnancy in humans.
Ectopic pregnancy is the consequence of an anomaly of
implantation of the ovum and accounts for 1–2% of all
pregnancies. More than 95% of ectopic gestations are tubal
pregnancies. The tube is enlarged and placental villi progressively erode into the wall (Gompel and Silverberg, 1994b). The
uterus undergoes some of the changes which are defined as the
Arias-Stella reaction (Arias-Stella, 1954). These endometrial
changes may occur during an intrauterine pregnancy, and are
not specific for ectopic pregnancy. However, the mechanisms
of these changes are not entirely understood. The objective of
the present investigation was, therefore, to determine whether
apoptosis occurs in the villous and decidual tissues in the
intrauterine and ectopic pregnancies using an end-labelling of
DNA gel fractionation and an in-situ labelling of DNA.
Materials and methods
Subjects
Human chorionic villi and decidual tissues were obtained from nine
normal pregnant women (6–8 weeks gestation), who were undergoing
an elective abortion, and from seven patients with ectopic pregnancy
(6–8 weeks gestation), who were undergoing a salpingectomy for
tubal pregnancy. In all cases of ectopic pregnancy, embryonic heart
beats were recognized by transvaginal ultrasonography in the adnex
mass, but not in the uterus. Cases in which heart beats could not be
detected were excluded. The project was approved by the committee
on investigations involving human subjects of Wakayama Medical
College. Informed consent was obtained from each subject after the
purpose and nature of the study had been fully explained. The
87
K.Kokawa, T.Shikone and R.Nakano
menstrual cycle was used to determine the gestational age with day 1
being taken as the first day of the last menstrual period. After three
washes in saline, each sample was rapidly frozen and stored at 270°C
or fixed in Bouin’s solution.
Isolation and analysis of the apoptotic fragmentation of
DNA
DNA was isolated from each sample and quantified spectrophotometrically at 260 nm. Autoradiographic analysis of apoptotic fragmentation of DNA was performed as described previously (Tilly and
Hsueh, 1993; Shikone et al., 1996). One microgram of DNA from
each sample was labelled at 39-ends with [α-32P]dideoxy-ATP (ddATP;
3000 Ci/mmol; Amersham, Arlington Heights, IL, USA) and terminal
transferase (TdT; 25 U; Boehringer Mannheim, Indianapolis, IN,
USA). After autoradiography, gels were cut between bands of high
and low molecular weight (mol. wt; 15 kb) fragments and radioactivity
was determined in a β-counter for quantitative analysis.
In-situ analysis of DNA fragmentation in histological
sections
Human chorionic villi and decidual tissues were fixed in Bouin’s
solution and embedded in paraffin. Analysis in situ was performed
as described previously (Gavrieli et al., 1992; Kokawa et al., 1996).
Deparaffinized and hydrated samples on slides were treated with
proteinase K (10 µg/ml). Samples were then subjected to 39-endlabelling of the DNA with digoxigenin-dideoxyUTP (dig-ddUTP;
Boehringer Mannheim). TdT, dig-ddUTP and ddATP were used at
concentrations of 1 U/µl, 1 µM and 49 µM, respectively. The standard
substrates (337.5 µg/ml nitroblue tetrazolium and 175 µg/ml 5-bromo4-chloro-3-inolyl-phosphate) were used for staining. After the colour
reaction, sections were counterstained with eosin. Positive and negative controls for the technique were performed: the addition of
deoxyribonuclease I caused the positive staining of all nuclei, whereas
the omission of either TdT or dig-ddUTP led to completely negative
staining (data not shown).
Analysis of data
For analysis of the internucleosomal cleavage of DNA, representative
autoradiograms were examined. Quantitative comparisons between
groups were made on the basis of the radioactivity in low mol. wt
fractions (ø15 kb) of DNA from every sample (mean percentage
change 6 SEM versus the value in the normal pregnancy). For
analysis in situ, cells in three microscopic fields per slide of each
sample were enumerated by two of the authors. The number of
positive cells was not significantly different between the two authors
and was used to define a mean apoptotic index, defined as: (the
number of apoptotic cells/the total cell number)3100. Statistical
analysis was performed using ANOVA and Student’s t-test, and
P , 0.05 was considered to reflect a significant difference from the
corresponding control group.
Results
In this study, urinary concentrations of human chorionic
gonadotrophin (HCG; mean 6 SEM IU/l) in patients who
experienced ectopic pregnancy (7400 6 2300, n 5 7) were
significantly lower than those of normal pregnant women
(22000 6 3200, n 5 9). Autoradiographic analysis revealed
very limited but detectable fragmentation of DNA both in
the villous and decidual tissues of normal pregnancy (Figures
1A and 2A). In tubal pregnancy, a ladder pattern, characteristic of the apoptotic cleavage of DNA into small fragments
88
(ø15 kb), was generated in the villi (Figure 1A), whereas
high mol. wt DNA was predominant in the decidua (Figure
2A). Quantitative analysis of the small DNA fragments
(ø15 kb) revealed a significant increase (290 6 37.4%,
n 5 7, P , 0.05) in the villi in tubal pregnancy as compared
to normal pregnancies (100 6 9.0%, n 5 9; Figure 1B).
By contrast, a significant decrease (28.6 6 5.3%, n 5 7,
P , 0.05) was noted in the decidua in tubal pregnancy as
compared to normal pregnancy (100 6 24.7%, n 5 9;
Figure 2B).
To identify the cells involved in the apoptotic fragmentation
of DNA, we carried out an in-situ labelling of DNA with digddUTP. Labelled cells were detected focally in the cytotrophoblast and decidual cells of normal pregnancy (Figure 3A
and C). In contrast, dense labelling was predominant in the
syncytiotrophoblast in tubal pregnancy (Figure 3B), while
labelled cells were rarely seen among the decidual cells
(Figure 3D). Comparative counting of positive cells revealed
a significant increase in the syncytiotrophoblast, but a decrease
in the decidual cells in tubal pregnancy as compared to normal
pregnancy (P , 0.05; Table I).
Discussion
Apoptosis has been used to define a type of cell death that is
distinct from necrosis. Recently, it has become recognized as
an essential feature of the development of tissues (Raff, 1992).
In the present study, autoradiographic analysis revealed that
very limited but detectable cleavage of DNA was observed in
the villi and decidua in normal pregnancy. However, apoptosis
in normal pregnancy does not show any significant quantitative
changes from 6 to 8 weeks gestation (data not shown).
By contrast, a ladder pattern that is characteristic of the
apoptotic cleavage of DNA was recognized in the villi, but
not in the decidua, in ectopic pregnancy. Our analysis in
situ resulted in dense labelling in a small portion of the
cytotrophoblast in normal pregnancy, with heavy labelling of
the syncytiotrophoblast in ectopic pregnancy. In the decidual
tissue, labelled cells were rarely seen in the decidual cells in
normal pregnancy, and their numbers decreased in ectopic
pregnancy. In some systems, apoptosis occurs without the
appearance of DNA ladders, and the method for labelling
DNA in situ does not discriminate between apoptotic and
necrotic cells. However, our results suggest that apoptotic cell
death occurs in the chorionic villi, but not in the decidua, in
ectopic pregnancy.
The villous trophoblast consists of the cytotrophoblast and
the syncytiotrophoblast. The former consists of single, polygonal to oval cells, in which mitotic activity is brisk during
the first trimester of pregnancy. The latter represents the
differentiation form of cytotrophoblast into a large and multinucleated cell (Gompel and Silverberg, 1994a), which is
incapable of cell division but has metabolic activity and the
ability to produce HCG. Our study revealed that apoptotic
cells were scattered in the cytotrophoblast, but not in the
syncytiotrophoblast, in normal pregnancy. It seems likely that
apoptosis in the cytotrophoblast in normal pregnancy may
Apoptosis in human chorionic villi and decidua
Figure 1. Apoptotic fragmentation of DNA in human chorionic villi. Villous tissues were obtained from pregnant women. Tissue DNA was
isolated, labelled at 3’-ends with [α-32P]ddATP and analysed by autoradiography (A). Radioactivity in the low mol. wt (ø15 kb) fragments
of DNA (mean 6 SEM) are shown in B. The amount of [α-32P]ddATP incorporated into the low mol. wt (ø15 kb) fragments of DNA in
each sample was compared with that in a sample from a normal pregnancy at 7 weeks gestation, which was designated arbitrarily as 100%.
*Significantly different from normal pregnancy, P , 0.05. N.P. 5 normal pregnancy; E.P. 5 ectopic pregnancy. In A, an arrow indicates the
point at which gels were cut for quantification of the high and low mol. wt DNA.
Figure 2. Apoptotic fragmentation of DNA in human decidual tissues. Decidual tissues were obtained from pregnant women. Tissue DNA
was isolated, labelled at 39-ends with [α-32P]ddATP, and analysed by autoradiography (A). Radioactivity in low mol. wt (ø15 kb) fragments
of DNA (mean 6 SEM) are shown in B. The amount of [α-32P]ddATP incorporated into the low mol. wt (ø15 kb) fragments of DNA in
each sample were compared with that in a sample from a normal pregnancy at 7 weeks gestation, which was designated arbitrarily as 100%.
*Significantly different from normal pregnancy, P , 0.05. N.P. 5 normal pregnancy; E.P. 5 ectopic pregnancy. In A, an arrow indicates the
point at which gels were cut for quantification of the high and low mol. wt DNA.
represent one aspect of the proliferation and degeneration of
the trophoblast during early pregnancy.
Tubal gestation traditionally has been considered to reflect
implantation within the tubal lumen. However, the fertilized
ovum does not remain on the surface, but soon penetrates the
epithelium and lies in the muscular wall, because the tube
lacks a submucosa (Pauerstein et al., 1986). Trophoblast
invades and erodes the subjacent musclaris, and maternal blood
vessels are opened at the same time. In some cases of ectopic
pregnancy, there is a decidual reaction in the form of a small
cell nest in the tube (Randall et al., 1987). This reaction,
however, is minimal compared with that in the endometrium.
In addition, the embryo or fetus is often absent. The mechanism
of failure of embryo development is still unknown. The decidua
is thought to provide nutrition for the developing embryo, and
to protect it from the immunological responses of the mother.
89
K.Kokawa, T.Shikone and R.Nakano
Figure 3. In-situ 3’-end-labelling of DNA in the sections of villous and decidual tissues. (A) Labelled cells were seen focally in the
cytotrophoblast in the normal pregnancy, not in the syncytiotrophoblast. (B) Labelled cells were predominant in the syncytiotrophoblast in
tubal pregnancy. (C) Small portion of the decidual tissue was labelled in normal pregnancy. (D) Few cells were labelled in the decidua in
tubal pregnancy. Arrows indicate some of the stained cells. The original magnification was 3200 for panels A and B and 3100 for panels
C and D.
Table I. The apoptotic index, determined in situ, in the villi and decidua in
normal and ectopic pregnancy
Cell components
Cytotrophoblast
Syncytiotrophoblast
Decidual cell
Apoptotic indexa
Normal pregnancy
Ectopic pregnancy
25.1 6 4.7
3.3 6 0.9
26.8 6 4.0
15.2 6 5.1
68.2 6 3.9b
7.7 6 2.7b
Results represent the means 6 SEM of the apoptotic index from three
different patients for normal and ectopic pregnancy.
a(The number of apoptotic cells/the total cell number)3100.
bP , 0.05, significantly different from corresponding values for normal
pregnancy.
We consider that the absence of the decidua may induce
apoptosis to occur in the syncytiotrophoblast, eliminating its
role in maintaining metabolic activity of the zygote and hence
causing stunting of embryo growth.
Many studies have demonstrated that apoptosis is affected
by various hormones (Thompson, 1994), such as follicle
stimulating hormone (FSH) and luteinizing hormone (LH) in
the ovary (Hsueh et al., 1994) and oestrogen and progestin in
90
the uterine endometrium (Pollard et al., 1987). HCG is a
hormone of major importance in early pregnancy. This hormone
has been shown to suppress apoptosis in the uterine epithelium
and the corpus luteum of rabbit in vivo (Nawaz et al., 1987;
Dharmarajan et al., 1994), and cultured preovulatory follicles
in vitro (Chun et al., 1994). In humans, we have previously
reported that there are significantly fewer apoptotic cells in
the corpus luteum in ectopic pregnancy than during the late
luteal phase (Shikone et al., 1996). Although apoptotic cell
death is marked in the endometrium during the late secretory
phase (Kokawa et al., 1996), in cases of ectopic pregnancy,
apoptosis was very inhibited in the decidua by comparison.
Therefore it is possible that HCG might be an inhibitor of
apoptosis in the decidua during pregnancy.
In the present study, apoptosis in the decidua in normal
pregnancy apparently occurred to a greater extent than in
ectopic pregnancy, despite the much higher HCG levels in
normal pregnancy and despite the fact that in intrauterine
pregnancy the HCG produced by syncytiotrophoblast is directly
adjacent to the decidua. This inconsistency might be due to
the difference in the implantation site between intrauterine
and ectopic pregnancies. In some rodents during embryo
Apoptosis in human chorionic villi and decidua
implantation, apoptotic cell death starts with the uterine
epithelial cells surrounding the embryo (Parr et al., 1987).
Another study demonstrated that tissue transglutaminase, an
enzyme synthesized by cells undergoing apoptosis, was present
at high levels in the antimesometrial region, whereas expression
of bcl-2 was detectable in the mesometrial region during
implantation of the embryo (Piacentini and Autuori, 1994).
These earlier reports lend support to our observations. We
speculate that decidual cells essentially escape apoptotic cell
death during preimplantation period, which they then undergo
just after embryo implantation has started. It is possible that
several molecules, including HCG, are implicated in the control
of apoptosis in the decidua during pregnancy.
Using molecular biochemical techniques, we have demonstrated in the present study that apoptosis occurs in a specific
population of cells in human chorionic villi and decidual tissue
during the first trimester of pregnancy. Moreover, the apoptotic
changes in the villi in ectopic pregnancy are more conspicuous
than those in normal pregnancy. Decidual cell apoptosis in
ectopic pregnancy occurred to a more limited extent than
that in normal pregnancy. These findings indicate that the
localization of the implantation site might affect the occurrence
of apoptosis and play a pivotal role in the development and
wastage of human embryos.
References
Arends, M.J., Morris, R.G. and Wyllie, A.H. (1990) Apoptosis: the role of
the endonuclease. Am. J. Pathol., 136, 593–608.
Arias-Stella, J. (1954) Atypical endometrial changes associated with the
presence of chorionic tissue. Arch. Pathol., 58, 112–128.
Chun, S.Y., Billig, H., Tilly, J.L. et al. (1994) Gonadotropin suppression of
apoptosis in cultured preovulatory follicles: mediatory role of endogenous
insulin-like growth factor I. Endocrinology, 135, 1845–1853.
Dharmarajan, A.M., Goodman, S.B., Tilly, K.I. and Tilly, J.L. (1994) Apoptosis
during functional corpus luteum regression: evidence of a role for chorionic
gonadotropin in promoting luteal cell survival. Endocr. J., 2, 295–303.
Gavrieli, Y., Sherman, Y. and Ben-Sasson, S.A. (1992) Identification of
programmed cell death in situ via specific labeling of nuclear DNA
fragmentation. J. Cell. Biol., 119, 493–501.
Glücksmann, A. (1951) Cell deaths in normal vertebrate ontogeny. Biol. Rev.
Camb. Philos. Soc., 26, 59–86.
Gompel, C. and Silverberg, S.G. (eds) (1994a) Pathology in Gynecology and
Obstetrics. J.B.Lippincott, Philadelphia, 448–512.
Gompel, C. and Silverberg, S.G. (eds) (1994b) Pathology in Gynecology and
Obstetrics. J.B.Lippincott, Philadelphia, 515–519.
Gu, Y., Jow, G.M., Moulton, B.C. et al. (1994) Apoptosis in decidual tissue
regression and reorganization. Endocrinology, 135, 1272–1279.
Hsueh, A.J.W., Billig, H. and Tsafriri, A. (1994) Ovarian follicle atresia: a
hormonally controlled apoptotic process. Endocr. Rev., 15, 707–724.
Kerr, J.F.R., Wyllie, A.H. and Currie, A.R. (1972) Apoptosis: a basic biological
phenomenon with wide-ranging implications in tissue kinetics. Br. J.
Cancer, 26, 239–257.
Koh, E.A.T., Illingworth, P.J., Duncan, W.C. and Critchley, H.O.D. (1995)
Immunolocalization of bcl-2 protein in human endometrium in the menstrual
cycle and simulated early pregnancy. Hum. Reprod., 10, 1557–1562.
Kokawa, K., Shikone, T. and Nakano, R. (1996) Apoptosis in the human
uterine endometrium during the menstrual cycle. J. Clin. Endocrinol.
Metab., 81, 4144–4147.
Nawaz, S., Lynch, M.P., Galand, P. and Gerschenson, L.E. (1987) Hormonal
regulation of cell death in rabbit uterine epithelium. Am. J. Pathol., 127,
51–59.
Palumbo, A. and Yeh, J. (1994) In situ localization of apoptosis in the rat
ovary during follicular atresia. Biol. Reprod., 51, 888–895
Parr, E.L., Tung, H.N. and Parr, M.B. (1987) Apoptosis as the mode of uterine
epithelial cell death during embryo implantation in mice and rats. Biol.
Reprod., 36, 211–225.
Pauerstein, C.J., Croxatto, H.B., Eddy, C.A. et al. (1986) Anatomy and
pathology of tubal pregnancy. Obstet. Gynecol., 67, 301–308.
Piacentini, M. and Autuori, F. (1994) Immunohistochemical localization of
tissue transglutaminase and Bcl-2 in rat uterine tissues during embryo
implantation and post-partum involution. Differentiation, 57, 51–61.
Pollard, J.W., Pacey, J., Cheng, S.V.Y. and Jordan, E.G. (1987) Estrogens and
cell death in murine uterine luminal epithelium. Cell. Tissue Res., 249,
533–540.
Raff, M.C. (1992) Social controls on cell survival and cell death. Nature, 356,
397–400.
Randall, S., Buckley, C.H. and Fox, H. (1987) Placentation in the fallopian
tube. Int. J. Gynecol. Pathol., 6, 132–139.
Rotello, R.J., Hocker, M.B. and Gerschenson, L.E. (1989) Biochemical
evidence for programmed cell death in rabbit uterine epithelium. Am. J.
Pathol., 134 491–495.
Shikone, T., Billig, H. and Hsueh, A.J.W. (1994) Experimentally induced
cryptorchidism increases apoptosis in rat testis. Biol. Reprod., 51, 865–872.
Shikone, T., Yamoto, M., Kokawa, K. et al. (1996) Apoptosis of human
corpora lutea during cyclic luteal regression and early pregnancy. J. Clin.
Endocrinol. Metab., 81, 2376–2380.
Slayden, O.D. and Brenner, R.M. (1994) RU 486 action after estrogen priming
in the endometrium and oviducts of rhesus monkeys (Macaca mulatta). J.
Clin. Endocrinol. Metab., 78, 440–448.
Tabibzadeh, S., Zupi, E., Babaknia, A. et al. (1995) Site and menstrual cycledependent expression of proteins of the tumor necrosis factor (TNF)
receptor family, and BCL-2 oncoprotein and phase-specific production of
TNF α in human endometrium. Hum. Reprod., 10, 277–286.
Thompson, E.B. (1994) Apoptosis and steroid hormones. Mol. Endocrinol.,
18, 665–673.
Tilly, J.L. and Hsueh, A.J.W. (1993) Microscale autoradiographic method for
the qualitative and quantitative analysis of apoptotic DNA fragmentation.
J. Cell. Physiol., 154, 519–526.
Wyllie, AH. (1980) Glucocorticoid-induced thymocyte apoptosis is associated
with endogenous activation. Nature, 284, 555–556.
Zeleznik, A.J., Ihrig, L.L. and Bassett, S.G. (1989) Developmental expression
of Ca11/Mg11-dependent endonuclease activity in rat granulosa and luteal
cells. Endocrinology, 125, 2218–2220.
Received on June 9, 1997; accepted on October 9, 1997
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