J. Embryol. exp. Morph. Vol. 17, 3, pp. 607-24, June 1967
617
Printed in Great Britain
Effects of position in the uterus on fetal mortality
and on response to trypan blue
By S I D N E Y L. B E C K
1
Department of Biology, The University of Toledo, Ohio
INTRODUCTION
In a polytocous species such as the mouse, the relative position in the uterus
that a fetus occupies may well have an effect upon the likelihood of normal
development or indeed survival (Trasler, 1960; Woollam & Millen, 1962). It
would seem desirable, therefore, especially since the mouse is so valuable a tool
in studies of development (Griineberg, 1963) and teratogenesis (Fave, 1964;
Woollam, 1966), to learn as much as possible about the contribution of the
uterine environment to embryonic development.
It has been suggested that fetuses implanted in the portion of the uterine horn
nearest the common uterus may have an increased tendency toward death or
deformity when their dams are made anoxic experimentally during gestation
(Woollam & Millen, 1962). These authors suggest that the variations in response
to anoxia might be accounted for by differences in blood supply or different
times of implantation in the different regions of the uterus. Trasler (1960),
studying the spontaneous occurrence of cleft palate in A/Jax mice, found a
significantly higher incidence of abnormality in the implantation site nearest the
ovary than in all other sites, regardless of the total number of fetuses in the
uterine horn. Reduced blood supply was suggested here as a possible factor also.
Still other investigations have shown that the embryo occupying the site nearest
the ovary weighed significantly less near term than those at other uterine sites
(Hashima, as cited by Trasler), and that runts in the random-bred TO stock of
mice occurred significantly more often at the ovarian site than elsewhere in the
uterus (McLaren & Michie 1960). The question of blood supply and its relation
to abnormalities in development was also investigated by Franklin & Brent
(1964), using the technique of vascular clamping of uterine blood vessels to
measure and alter blood flow to individual rat fetuses, and Geber (1962) has
suggested the importance of blood supply to development of certain domestic
animals.
The present investigation attempts to consider the matter of uterine position
and its possible effect on mortality on the normal mouse fetus, and after treat­
ment of dams with trypan blue.
1
Author's address: Department of Biology, University of Toledo, Ohio 43606, U.S.A.
618
S. L. B E C K
MATERIALS A N D
METHODS
The observations reported stem from experiments originally designed for
another purpose, and reported elsewhere (Beck, 1963, 1964). The data come
from brother-by-sister matings of Z R D C T - N , C57/BL6 and C57BL/10 mice,
as well as out-crosses of females from each of these strains with anophthalmic,
ZRDCT-An males. There are two bodies of data, both obtained by dissection
of litters at 18 days of gestation. In all cases the day upon which the copulation
plug was found was considered day 0. One group was untreated; the other was
injected intraperitoneally on the 7th, 8th and 9th days of gestation with 0-25 cc of
a 0-3 % solution of trypan blue in 0-9 % sodium chloride. Only those uterine
horns containing two or more implantations were considered in this analysis.
A further restriction placed on the data was that at least one implantation site in
each litter had to be occupied by a living fetus.
The implantation site nearest the common uterus was termed 'central' or
'cervical' (C), the one nearest the ovary was considered 'lateral' (L); those, if
any, between these were categorized 'intermediate' (/) in position. The implan­
tation sites were categorized as containing a fetus which was dead (D) or alive
(A). The dead fetuses were in varying stages of resorption; the living ones
included both normal and abnormal individuals. Most of the abnormal fetuses
were suffering microphthalmia or anophthalmia, although occasional cases of
exencephaly or other skeletal defects were noted.
RESULTS
(a) Extrinsic sources of variation
Owing to the presence of several extraneous variables it seemed desirable to
determine whether the confounding of sources of variation introduced serious
bias into the data.
Although a comparison of the six mating types reveals heterogeneity among
treated litters (not among untreated) if the amount and distribution of mortality
are considered together (x = 59-6; D.F. = 25; P < 0-001), the bulk of the
heterogeneity is contributed by the cross of C57/10 x Z R D C T - A n ; a test of the
other five crosses shows no heterogeneity (x = 25-8; D.F. = 20; 0-20 > P >
0-10), and a comparison of C 57 BL/10 x Z R D C T - A n with all other crosses
combined shows a highly significant difference between the two groups (% = 35 - 5;
D.F. = 5; P < 0-001). Moreover, the source of the heterogeneity is the markedly
fewer fetal deaths in the C 57 BL/10 x ZRDCT-An matings than in the others,
and not in the distribution of deaths within the horn. There is no heterogeneity
between matings with respect to the distribution of mortality among positions
if this is considered separately. This can be made clear by setting the frequency of
mortality in the C position in each cross equal to unity and calculating the ratio
of mortality of either the L or / positions relative to the C position. One thereby
2
2
2
Effects of position in uterus
619
obtains values, in the order of the matings given in Table 1, of 0-89, 0-58, 1-0,
0-67, 1-77, 0-55 for the L:C ratio, and 0-82, 0-58, 1-02, 0-67, 1-82, 0-69, for the
I.C ratio. Since it is only heterogeneity of distribution of mortality among
positions and between crosses of which we seek a measure, a x analysis can be
performed considering only living or only dead fetuses for .each cross. In this
case the rows of the contingency table are crosses, and the columns are posi­
tions in the uterus; the numbers entered in each cell are either the number of liv­
ing (providing the best estimate in the untreated group) or the number of dead
(being a better comparison in the treated group) fetuses. In these analyses the
absence of heterogeneity is quite striking in both the treated (x = 2-9; D.F. =
12; P > 0-99) and the untreated (x = 3-4; D.F. = 12; P > 0-99) groups. One
is therefore justified in combining the data from all six mating types.
2
2
2
Table 1. Mortality at 18 days post coitus
Number of implantations
.
I
,
A
.
\
Alive
N o . of
Litters
Cross
C
I
Dead
,—
L
Mortality
A
A
c
c
/
L
C
/
L
Untreated
ZRDCT-N x ZRDCT-N
ZRDCT-N x ZRDCT-An
C57BL/6xC57BL/6
C 5 7 B L / 6 x ZRDCT-An
C57BL/10xC57BL/10
C57BL/10 x ZRDCT-An
7
6
8
6
5
5
37
Total
10
6
14
7
7
9
31
28
40
26
21
21
11
12
15
11
7
10
4
6
2
5
1
1
14
8
4
4
5
6
3
0
1
1
1
0
28-6
500
12-5
41-7
12-5
100
311
220
91
13-3
19-2
220
21-4
0
6-2
8-3
12-5
0
53 167
66
19
41
6
26-4
19-7
8-3
Treated
ZRDCT-N x ZRDCT-N
ZRDCT-N x ZRDCT-An
C57BL/6xC57BL/6
C57BL/6x ZRDCT-An
C57BL/10xC57BL/10
C57BL/10 x ZRDCT-An
Total
6
11
4
7
5
6
2
7
3
7
6
9
10
16
4
18
6
21
3
8
3
9
3
10
9
15
5
6
4
2
20
8
28 14
10
5
4
8
16 7
1
3
81-8
68-2
62-5
46-1
400
18-2
66-7
63-6
71-4
30-8
72-7
12-5
72-7
63-6
62-5
30-8
700
91
39
34
75
36
41
85
54-7
531
520
39
A comparison of the left and right horns of the uterus in which the implanta­
tions were described as being normal, abnormal or dead, and either C, / or L
positions in either the left or right horns, revealed no significant heterogeneity
between left and right in either the untreated (x — 12-3; D.F. = 8; 0-20 > P >
0-10) or treated (x = 13-1; D.F. = 8; 0-20 > P > 0-10) groups. One may
therefore pool the data from the left and right horns of the uterus.
Still another source of variation extrinsic to that with which we are concerned
and which could introduce a bias would be a correlation between the positions
within each uterine horn. There does not appear to be any association, however,
2
2
620
S. L. B E C K
between the likelihood of a fetus dying in the central position and its risk of death
in the lateral position in either the untreated (x = 2-2; D.F. = 1; 0-20 > P >
0-10) or the treated (% = 0-6; D.F. = 1; P > 0-50) groups.
2
2
(b) Effects of position on mortality and abnormality
.
Having shown that within horn correlations, left-right differences and mating
type differences do not bias the distribution of mortality throughout the uterus,
we may proceed to ask the questions of whether there is a correlation between
the position in the uterus which a fetus occupies and (a) its likelihood to survive
until term; and, among the survivors in treated litters, (b) its likelihood of
becoming abnormal in consequence of treatment.
Table 2. Abnormality at 18 days post coitus
Number of Offspring
Normal
N O . Of
Cross
litters
,
*
C
I
Abnormal
^
,
*
Abnormality %
*
,
*
,
L
C
I
L
C
I
L
31
28
36
24
16
20
11
12
13
11
6
10
0
0
2
1
1
0
0
0
4
2
5
1
0
0
2
0
1
0
0
0
14-3
14-3
14-3
0
0
0
100
7-7
23-8
4-8
0
0
13-3
0
14-3
0
49 155
63
4
12
3
7-6
7-2
8-7
Untreated
ZRDCT-N x ZRDCT-N
ZRDCT-N x ZRDCT-An
C57BL/6xC57BL/6
C57BL/6 x ZRDCT-An
C57BL/10xC57BL/10
C57BL/10 x ZRDCT-An
Total
7
6
8
6
5
5
37
10
6
12
6
6
9
Treated
ZRDCT-N x ZRDCT-N
ZRDCT-N x ZRDCT-An
C57BL/6xC57BL/6
C57BL/6 x ZRDCT-An
C57BL/10xC57BL/10
C57BL/10 x ZRDCT-An
Total
6
11
4
7
5
6
2
5
3
4
5
9
9
8
3
10
6
20
3
4
2
7
1
8
0
2
0
3
1
0
1
8
1
8
0
1
0
4
1
2
2
2
0
28-6
0
42-9
160
0
100
500
250
44-5
0
4-8
0
500
33-3
22-2
66-7
200
39
28
56
25
6
19
11
17-7
25-3
30-6
Mortality in untreated litters (Table 1) is not equally distributed throughout
the three regions of the uterine horn (x = 7-6; D.F. = 2 ; 0-05 > P > 0-02). A
considerably higher proportion of fetuses fail to survive in the most central
position (26-4 ± 4-2 %) than in the site nearest the ovary (8-3 ± 3-2 %) (t = 2-9;
D.F. = 142; P < 0-004). Indeed, the ovarian position is favored over all other
sites combined (t = 2-6; D.F. = 350; P < 0-02). Among treated litters mortality
is uniform throughout the uterus, suggesting a treatment-position interaction.
The excess of mortality over the spontaneous rate, that fraction of the total
which is caused by the treatment, seems different in the central (28-3 %) and
lateral (43-7 %) positions. This excess of mortality in response to the treatment
2
Effects of position in uterus
621
in the ovarian position is of borderline significance (Table 3), making interpretation difficult.
There is also a suggestion of a lower frequency of abnormality (Table 2) in
the central position (17-7 ± 6-5 %) than in the lateral (30-6 ± 7-7 %), but the
difference is not statistically significant. In those crosses in which spontaneous
abnormality occurs, this abnormality is uniformly distributed throughout the
uterus.
Table 3. Analysis for interaction between treatment and position
with respect to mortality.
n = number of implantations; x = number of dead and resorbed fetuses; p = percentage
mortality; 0 = percentage = arcsin.
Untreated
Central position
Lateral Position
nx
xt
px
0X
=
=
=
=
72
19
26-4
30-92
/73 = 72
x3 = 6
Pz = 8-3
03 = 16-74
Treated
w« =
*2 =
/», =
62 =
«4
x4
A
04
75
41
54-7
47-70
= 75
= 39
= 520
= 46-15
Analysis:
Interaction = / = 01-02-63 + 6i = 12-63.
Interaction variance = var. I = var. 0X + var. 02 + var. 03 + var.
=
82, ( 1 + 1 + 1 + 1 ) = 44-3.
Standard error of Interaction = S.E. I = var. I = 6-65.
Difference of means/S.E. diff. of means = I/S.E. = / = 1-90
Degrees of freedom = n1 + n2 + n +n — 4 = 290.
P = 0057.
CONCLUSIONS AND DISCUSSION
It has been shown, in three strains of mice and outcrosses of each, that the
fetus occupying the implantation site nearest the common uterus is much less
likely to survive than the one nearest the ovary. Indeed, the implantation nearest
the ovary occupies the most favored site. When the dams are treated with trypan
blue, mortality in excess of the spontaneous frequency appears greater in the
most ovarian position than elsewhere in the uterus, suggesting a treatmentposition interaction.
These observations are all consistent with the notion that the most cervical
implantation site is intrinsically poorer than any of the others. One possible
explanation is a poorer blood supply in this region of the uterine horn. The
differences in mortality in the different regions are not likely to be a direct consequence of differences in time of implantation because the mortality that is being
622
S. L. BECK
observed is occurring over a long interval. When the females were treated with
trypan blue, the region of the uterus which was poorest in the untreated state
actually appeared to afford some measure of protection against the treatment to
the fetus occupying it.
Regardless of the mechanism of action of trypan blue the results reported here
are consistent with the concept of a reduced blood supply in the most central
portion of the uterine horn. If the fetuses are being rendered hypoxic by the dye
(Gilbert & Gillman, 1954; Barber, 1957), then those fetuses occupying the
most central position would, through poorer blood supply, likely be more
anoxic already and might therefore be less susceptible to further anoxia caused
by the teratogen. The more favored positions, starting from higher base level,
might be more drastically affected by the treatment. If the dye is directly toxic to
the embryo (Barber & Geer, 1964; Turbow, 1966) or acts by way of entry into
the yolk sac (Ferm, 1956; Wilson, Beaudoin & Free, 1959) or indirectly by
alterations to the maternal physiology (Christie, 1964) or maternal serum proteins (Yamada, 1959; Beaudoin & Ferm, 1961), or even by entering and nutritionally damaging phagolysosomes of the embryonic yolk sac (Beck & Lloyd,
1966), then a poorer blood supply in the most central position might very well,
while causing a greater likelihood of spontaneous death, offer some protection
to the fetuses of treated dams by minimizing the contact between them and the
noxious principle of the teratogen. Whatever the mechanism of action of the
teratogen, it is apparent from these data that there is evidence for selection in
utero by position. Given an added stress, those fetuses having already survived
selection seem somewhat less likely to succumb than those not previously
selected.
In the present study the effects of the treatment appear greater in the most
ovarian position rather than in the most central, as was the case for Woollam &
Millen's (1962) experiments. That the central portion of the uterus is poorer in
the present study as well is seen from the data from untreated litters. The
untreated group is probably more closely comparable to Woollam & Millen's
anoxic dams than is the group treated with trypan blue. At first glance, the data
are apparently dissimilar to those of Trasler (1960) as well, in that in the present
report the untreated mice (the only kind Trasler reports) are poorer in the
central position, whereas in her report it is the ovarian position which is poorer.
However, the criterion of classification in the present communication is mortality
whereas she reports abnormality only. The present data allow no effect of time of
implantation to be seen because the mortality is occurring over a prolonged
interval, whereas differences in developmental stages of fetuses at a single
critical time during gestation caused by or related to their occupying different
sites in the uterus are likely to be important in explaining Trasler's findings.
Differences in the stages of development within a single litter of mice, if size
differences can be taken to be representative of differences in development, have
certainly been reported (Griineberg, 1943). That the observations reported here
Effects of position in uterus
623
may not agree in detail with other studies mentioned is of less importance than
the fact that they all provide reasonable demonstrations that the position which a
fetus occupies in the uterus can materially affect its chances of survival and
normal development. As such they are reasonably clear examples of the presence
of intra-uterine selection as an important force during development.
SUMMARY
In three strains of mice and an outcross of each to ZRDCT-An, the fetuses
occupying the implantation site nearest the common uterus are less likely to
survive than the ones nearest the ovary. When dams are treated with trypan blue,
mortality in excess of the spontaneous frequency seems greater in the ovarian
than in the cervical position. Both the heterogeneity of distribution of spontaneous mortality and the suggestion of treatment-position interaction show the
importance of intra-uterine selection upon survival of a developing organism.
None of the data are inconsistent with the hypothesis that one mechanism of
selection is through variations in blood supply in different regions of the
uterus.
RESUME
Effets de position dans Vuterus sur la mortalite foetale et
sur la reaction au bleu trypan
Dans trois races de Souris et dans les croisements de chacune de ces races avec
ZRDCT-An, les foetus implantes les plus pres de l'uterus commun ont moins
de chance de survie que les foetus situes pres de l'ovaire. Quand les femelles sont
traitees au bleu trypan, la mortalite supplemental par rapport a la frequence
spontanee, semble plus grande dans la position ovarienne que dans la position
cervicale. L'heterogeneite de distribution de la mortalite spontanee et l'indication
d'une interaction entre le traitement et la position mettent toutes deux en
evidence l'importance de la selection intra-uterine sur la survie d'un organisme
en voie de developpement. Aucun des resultats n'infirme l'hypothese qu'un des
mecanismes de selection residerait dans des variations du flux sanguin dans
differentes regions de l'uterus.
The author is indebted to Professor C. A. B. Smith, Weldon Professor of Biometry at
University College, London, for help with statistical matters, particularly the analysis for
interaction between position and treatment. This research was supported in part by U.S.P.H.S.
Fellowship 1, F11NB1345 V.S.N., and Research grant 7R 01 HD 02653-01 GEN.
624
S. L. BECK
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