J. Embryol. exp. Morph. Vol. 16, 1, pp. 1-16, August 1966
With 2 plates
Printed in Great Britain
Developmental effects of chronic maternal
audiovisual stress on the rat fetus
By WILLIAM F. GEBER 1
From the Department of Physiology and Pharmacology,
University of South Dakota School of Medicine
INTRODUCTION
A wide spectrum of agents and conditions has been imposed on the maternal
organism in an effort to learn more about possible factors in the development
of congenital malformations (Courrier & Marois, 1954; Ferm & Kilham, 1965;
Fraser, Walker & Trasler, 1957; Gillman, Gilbert & Gillman, 1948; Grabowski,
1963; Ingalls, Curley & Prindle, 1952; Kalter & Warkany, 1959; Russell, 1950;
Sikov & Noonan, 1958; Tuchmann-Duplessis & Mercier-Parot, 1960). The contribution of the genetic make-up of the organisms has received intensive study,
as have the effects of nutritional deficiencies, drugs, irradiation, anoxia, trauma
and virus infections. In the author's opinion, the most obvious and constant
source of potential danger to the developing fetus, the maternal organism itself,
has received relatively little experimental attention, although a number of
investigators have indicated that various types of maternal stress may contribute
to the production of abnormal behavior, metabolism and growth patterns of the
progeny (Calhoun, 1962; Grollman & Grollman, 1962; Harris & Harris, 1946;
Ibsen, 1928; Konstantinova, 1961; Malpas, 1937; Sontag, 1941; Spelt, 1948;
Stott, 1961; Zondek & Tamari, 1960). By contrast, other investigators (Warkany
& Kalter, 1962) have tended to believe that no significant fetal abnormalities resulted from emotional imbalances in pregnant humans or experimental animals.
Previous studies on sheep, dogs and rabbits (Geber, 1962) demonstrated
that the fetal cardiovascular system responds to a wide spectrum of maternal
stresses, sensory stimulations, and drug injections by a consistent and prolonged decrease in blood flow in the fetal brain and kidney. These results are
consistent with the following hypothesis: all sensory reception by the gravid
adult, whether consciously or subsconciously perceived, activates either directly
or indirectly the higher autonomic nervous system centers and the hypothalamicpituitary-adrenal axis. Subsequently, two additional interrelated responses
occur: (1) maternal autonomic neurohormones (epinephrine, norepinephrine)
and adrenal hormones are released, and (2) uterine blood flow is decreased
1
Author's address: Department of Pharmacology, Medical College of Georgia, Augusta,
Georgia, U.S.A.
I
JEEM l6
2
W. F. GEBER
directly by its nerve supply and indirectly by blood-borne neurohormones
released from other autonomic nerve endings. The passage of the maternal
neurohormones, pituitary and adrenal hormones through the placenta and into
the fetus causes a decrease in fetal blood flow. This and other metabolic effects,
coupled with the decrease in uterine blood flow, are postulated to affect fetal
development and metabolism adversely if they occur throughout the critical
periods of organogenesis.
The present study will attempt to define the effect of maternal auditory and
visual stresses on the developing fetus. The relationship of maternal age and
abnormal fetal development (Kuder & Johnson, 1944; MacMahon & Gordon,
1953) will also be evaluated.
Wilson (1961) in reviewing the parameters of experimental teratology has
commented that the mechanisms of action of the various teratogenic agents and
treatments have been relatively neglected. This state of affairs is understandable
because so wide a variety of compounds and treatments can induce similar
types of abnormal development. It is hoped that the present investigation may
indicate a possible common route of mechanism of action, namely decrease in
uterine and fetal blood flows, and imbalances in maternal hormone levels
which in turn influence fetal metabolism and organogenesis.
MATERIAL AND METHODS
Albino rats of the Sprague-Dawley strain were used in all the experiments.
All females were virgin animals and were 120-130 days old in the groups termed
'MA F and 280-320 days old in the group termed 'MA IF. The females of both
groups were selected to have a body weight within a 5 % range for all experiments. The males used for breeding were 130-150 days old. All animals were
maintained in the laboratory animal quarters for 3 weeks on Purina Rat Chow
prior to mating. Mating was carried out using one male to three females. The
first day of pregnancy was assumed to be the one on which the vaginal plug
was found. The pregnant females were divided at random, with the experimental
groups being placed in the stress chamber in the experimental room and the
control groups being transferred to another set of cages in the animal room to
to eliminate the effect of change of cage environment. The naturally occurring
ambient noise level in the control room was 64 dB with a frequency range of
20-75 c/s. The number of females per cage was the same for both control and
experimental groups for a particular experiment, i.e. twenty animals. Both
control and experimental cages were 10 x 12 x 36 in.
The stress chamber was constructed from sheet metal and measured 40 x
52 x 48 in. Three separate shelves were available inside and two cages were
placed on each shelf. The front of the chamber was a swinging door on which
were mounted several bells and buzzers. The back wall of the chamber and the
middle aisle of each shelf between two cages contained other horns, electric
Effects of maternal stress
3
gongs and rotating or moving objects. Six 10 in. speakers were mounted outside on the two sides of the chamber and opened to the interior through baffle
holes. The speaker system was used to send both pure tones from an Eico
Model 377 audio generator and 'white noise' from a Grason-Stadler noise
generator into the chamber. Various 'mobiles' of tinfoil mounted on revolving
timer motors connected to cycling timers were placed in the aisle also. Three
light bulbs (G.E. 40 W frosted) were mounted on the ceiling of each of the
three levels above the middle aisle; they were continually turned off and on
by means of a cycling circuit-breaker. All other audio and visual stimulation
apparatus was connected to individual cycling timers. All sound-generating
stimulators were continually recorded with a General Radio Noise Meter 1551 A.
It was determined that the decibel range of the stimulators was 74-94 and
the frequency range from 20 to 25000 c/s. Naturally occurring ambient noise
level in the experimental room was the same as in the control room, 64 dB with
a frequency range of 20-75 c/s. The effective daily stimulation time of the audio
stresses was arranged to occupy 10 % (6 min). of each hour of the day, with
relative quiet, i.e. ambient noise, existing throughout the remaining 90 %
(54 min) of each hour. Visual stimulation occupied approximately the same 10 %
of each hour of every 24 h period. Thus, both the auditory and visual stimulation program was continued uniformly throughout each day of the entire
pregnancy or until some other desired day (i.e. 16-20 days) of the pregnancy
was attained.
An exhaust blower was mounted on the chamber to aid in the circulation of
air and maintain temperature regulation. All four sides and the bottom of the
chamber contained 1 in. holes (twelve per side) to further aid in temperature
and air composition control. Temperature within the chamber was maintained
within a range of 26-28 °C.
Food and water were available for ad lib. feedings of both control and
experimental groups.
Both control and experimental females were killed at the end of either 16,
17, 18, 19, 20, 21 or 22 days of pregnancy. Fetuses were counted and uterine
distribution noted. They were then removed from the uterus using a dissecting
binocular microscope. Extreme care was exercised to prevent injury due to
handling. After gross, visual examination, the fetuses were placed in 10 %
formaldehyde for 2 weeks before weighing and additional examination for
abnormalities. After weighing and examination, the 18-22 day fetuses were
prepared for skeletal examination by a slight modification of the method of
Dawson (1926).
Maternal adrenals, kidneys and hearts of both control and experimental
groups were completely cleared of extraneous tissue and weighed following
2 weeks of hardening in formaldehyde.
All maternal organ weights and fetal weights were determined on a Mettler
balance (type H16, capacity 80 g).
4
W. F. GEBER
RESULTS
The results presented are a composite of eleven individual rat experiments
carried out in the stimulator over a period of 3 years. There was no detectable
overall seasonal variation except the rather marked decrease in pregnancy rates
during the hot summer months even though the animal quarters were adequately air-conditioned. It was determined that an average of 85 % of the
younger control females (MA I) and 60 % of the older control females (MA II)
were capable of producing litters. Those females of both control and experimental groups found to be non-pregnant were used for total body and organ
weight studies to be described later. The total number of fetuses obtained from
215 MA I control females was 2661, and from 218 MA I experimental females,
2219. The 186 MA II control females produced 1695 fetuses, and the 193 MA II
experimental females, 1356 fetuses.
Table 1. Effect of audiovisual stress on pregnancy,
litter size and resorption
Control
Percentage pregnant
Fetuses per litter
Resorptions per litter
MAI
MA II
85-0 + 4*
12-4 ±0-5
0-5±0-l
60-0 + 5
91 ±0-2
30±0-2
Experimental
MAI
51-3 ± 5 |
10-2±0-3f
20±0-3f
MA II
30-7 ±3f
7O±O-1J
2-3±0-2§
* Values are means ± S.E.
t Significant change from control {P = 001).
t Significant difference between MA I and MA II (P = < 001).
§ Difference between control and experimental values (P = < 005).
Pregnancy, litter size and resorptions. Table 1 indicates the difference in the
number of pregnant animals found in the experimental series of both the MA I
and MA II females. It will be noted that in the MA II females the effects of the
stress were apparently more pronounced than in the MA I females. All uterine
horns were examined for any residual placental tissue, indicating resorptions.
In addition, Table 1 shows that the stressed females produced numerically
smaller litters than the control animals in both the MA I and MA II series.
There was no significant difference in the percentage decreases in numbers of
fetuses in the experimental females when MA I and MA II animals are compared.
The data on resorptions were obtained by counting the number of residual
placentae in the uterine horns of the control and experimental animals. The
total number of resorptions was then divided by the total number of litters.
There were approximately four times as many resorptions found in the experimental groups as were found in the controls in the MA I animals (Table 1).
In the MA II animals the control and experimental resorptions were approxi-
Effects of maternal stress
5
mately equal; however, the incidence of resorptions in MA II control animals
is four to six times as high as in the MA I controls.
Fetal developmental stage age-weight relationship
The effect of the maternal audiovisual stress on the relationship between
fetal age and weight can be clearly noted in Table 2. The mean weights of the
fetuses in the experimental litters are consistently lower than those in the control
groups. More important is the fact that there is a consistently wider range of
weights for each developmental stage in the experimental groups. No essential
difference exists between means or ranges when fetuses from MA I and MA II
females are compared.
Table 2. Relationship between fetal age and weight
Fetal
Control
(days)
16
17
18
19
20
21
22
Mean weight
Range
Mean weight
Range
Mean weight
Range
Mean weight
Range
Mean weight
Range
Mean weight
Range
Mean weight
Range
(g)
(g)
(g)
(g)
(g)
(g)
(g)
Experimental
MAI
MA II
MA I
MA II
0-231
(0165-0-259)
0-460
(0-419-O-510)
1076
(0-850-1-640)
1-876
(1-420-2-759)
3-869
(3-515-4-179)
4-950
(4-621-5-179)
5-840
(5-480-5-892)
0-226
(0-166-0-252)
0-470
(0-412-0-506)
1061
(0-858-1-613)
1-863
(1-467-2-877)
3-859
(3-504^-164)
4-965
(4-600-5-125)
5-844
(5-501-5-965)
0.205
(0-100-0-315)
0-407
(0-300-0-625)
0-970
(0-552-1-895)
1-673
(1050-3 005)
3-515
(2-012-4-613)
4-593
(3-021-5-739)
5-301
(3-422-6-501)
0-201
(0102-0-319)
0-400
(0-301-0-605)
0-960
(0-530-1-880)
1-689
(1042-3019)
3-505
(2-029^-573)
4-580
(3-010-5-723)
5-295
(3-398-6-509)
Estimation of fetal hypo- and hyperdevelopment
Further to emphasize the variations in weight distribution noted within the
control and experimental litters, Table 3 was constructed using the method of
McLaren & Michie (1961) to estimate hypo- and hyperdevelopment of an
individual fetus. Using this method for evaluating relative developmental size
within a given litter, it was found that the experimental groups of females produced more fetuses of aberrant size (weight), and the deviations were larger
than those measured in the control litters. Variation in growth patterns noted
in the experimental hypodeveloped fetuses included arrested growth in the
cephalo-caudal axis in some fetuses, while others exhibited a predominant
transverse hypodevelopment.
The size of the litter was not necessarily the determining factor in whether
6
W. F. GEBER
or not it would contain a hypodeveloped fetus. In the present series of experiments, hypodevelopment was as likely to appear in a small litter as in a large one.
Abnormal developmental variations
Table 4 indicates the numbers and types of abnormalities observed in experimental litters and in the control groups. It is obvious that the absolute numbers
are not large and are only significant from the standpoint that the control series
contained relatively few of these abnormalities. It is also necessary to point out
that the litters were not subjected to autopsy, which might have yielded additional information. It was noted that frequently an experimental hypo- or hyperdeveloped fetus had additional developmental abnormalities.
Table 3. Distribution of abnormal fetal body weights
Control*
r
Percentage
deviation^
10
20
30
40
50
60
Experimental!
Hypodevelopment
Hyperdevelopment
A
A
Hypodevelopment
Hyperdevelopment
A
f
V
r
MA:I MA II Total MA I MA I I rotal M A ] [ MA II Total MA IMA II Total
5
3
4
1
0
0
4§
5
4
2
0
0
9
8
8
3
0
3
4
3
0
0
0
0
4
1
0
0
0
0
7
5
3
0
0
0
30
30
26
15
8
3
13
10
6
2
2
1
43
40
32
17
10
4
11
5
3
7
0
0
7
4
5
1
0
0
18
9
8
8
0
0
* 4880 fetuses.
t 3052 fetuses.
X Percentage difference in weight between the smallest or largest fetus as compared to next smallest
or next largest fetus in a given litter.
§ Number of fetuses exhibiting deviation.
Plates 1 and 2 (figs. A-H) illustrate a limited number of the abnormal
developmental patterns observed in the present study. Plate 1, fig. A illustrates
fetal hypodevelopment in the transverse axis, whereas Plate 1, fig. B shows
hypodevelopment in the cephalocaudal axis. Both these examples weighed
30% less than their,littermates shown with them. Plate 1, fig. C illustrates
multiple abnormalities in conjunction with hypodevelopment. Note the rudimentary tail, failure of midline closure, left hind-foot rotation, neck flexure,
and degenerate (or vestigial eyes). In Plate 1, fig. D the arrows indicate sublingual hematomas which are bilateral. The littermates were apparently free
of these localized hemorrhages. Plate 2, fig. E shows marked abdominal herniation in four of the fetuses. Note the dominance of the liver in the contents of
the sacculation. This is in contrast to the normal developmental omphalocele
which exists prior to this stage of maturation. Plate 2, fig. F illustrates a rather
Effects of maternal stress
7
small but well-defined interparietal hemorrhage and spina bifida. Plate 2, fig. G
shows a relatively extensive interparietal hemorrhage. Plate 2, fig. H illustrates
an interparietal meningocele of moderate size.
Table 4. Types and numbers of abnormal developmental variations
Number of variations observed
A
Control*
Experimental f
MA I MA II Total
1 Interparietal meningocele
2 Spina bifida
0
2
Thoracic level
1
Lumbar level
3 Cranial hematomas
1
Midline
0
Frontal
1
Parietal
4 Eye defects
0
Anophthalmia (bilateral)
0
(unilateral)
5 Tail defects
0
Complete absence
2
Curled, bent, etc.
6 Foot defects (hind)
0
Club foot (total rotation)
1
(partial rotation)
1
Foot defects (fore)
7 Neck hematomas
Unilateral
0
1
Bilateral
8 Ossification
0
Complete loss
2
Partial loss
2
Retardation
9 Abdominal hernia (omphalocele)
0
Intestine only
0
Intestine and liver
10 Fetal hydrops (general edema)
0
Total
* 4880 fetuses.
t 3052 fetuses.
% Number of fetuses exhibiting
MA I
MA II
Total
10
4
14
0
ot
0
0
2
1
6
3
4
4
10
7
0
0
0
1
0
1
5
10
20
3
9
4
8
19
24
0
0
0
0
2
3
0
0
2
3
1
1
1
3
8
15
10
13
18
28
0
0
0
0
1
1
4
12
3
2
8
3
6
20
6
0
0
0
1
2
7
0
0
2
7
2
4
0
2
6
2
52
60
60
37
67
98
89
127
158
0
0
0
0
0
0
22
2
9
1
4
6
3
13
15
579
9
abnormality.
Fetal distribution within uterine horn
Both MA I and MA II control and experimental groups were analyzed for
variations in the distribution of fetuses between the uterine horns. The MA I
control right horn carried an average of 6-4 + 0-4 while the left horn carried
8
W. F. GEBER
an average of 6 0 ± 0-5. In the MA I experimental groups the right horn averaged
5-2 + 0-4 while the left horn averaged 5-0 ± 0-2. The MA II control right horn
contained an average of 4-9 ± 0-2 and the left horn 4-2 ± 0-5. The MA II experimental right horn contained an average of 3-7 ± 0-4 and the left horn 3-3 ± 0-3.
This suggests that there is no anatomical or physiological difference between
the uterine horns which could account for the overall results.
Table 5. Effect of audiovisual stress on maternal body and organ weights
Experimental
Control
r
Relative
weight
(mg/100 g
body wt.)
Absolute
weight
(mg)
Body weight (mg)
255 ± 3 x 103
MAI
MA II
290±3xl0 3 t
Adrenals (mg)$
35 ±0-2
MAI
34 ± 0 1
MA II
Kidneys (mg)||
1079 ±6
MAI
1086 ±3
MA II
Heart (mg)H
907 ±4
MAI
MA II
986 ±3
Absolute
weight
(mg)
249±2xlO 3 *
272±3xlO 3 * t
—
13-70 ±005
ll-72±01t
34 ±0-4
33 ±0-2
Relative
weight
(mg/100 g
body wt.)
Percentage experimental difference
from control (based
on relative weight)
-
-2-3
-6-2
13-65 ±01§
12-15±0-l*t
-0-4
+ 4-2
423-5 ±1-9
374-5 ±l-3t
1029 ±3
1075 ± 5
412-3 ±1-4*
395-0±l-7*t
-2-6
+ 5-3
3560±l-5
3410+l-7t
867 ±3
1033 ±4
3480 ±1-1
3820±10*t
-2-2
+ 121
All values (except percentages) are means ± S.E.
* Significant change from control (P = < 001).
t Significant difference between MA I and MA II (P = < 001).
t Both adrenals averaged initially.
§ Not significant.
|| Both kidneys averaged initially.
Tf Ventricles only.
PLATE 1
Solid bar line in each figure represents one centimeter.
Fig. A. Predominantly transverse axis hypodevelopment is shown by fetus on left (22) as
compared to normal littermate on right. Fetal age, 20 days.
Fig. B. Cephalocaudal hypodevelopment in fetus on left (25) contrasts with normal littermate
on right. Fetal age, 20 days.
Fig. C. Fetus exhibits marked left hind-foot rotation (6), tail size reduction and right hindfoot rotation (7), vestigial eyes (8), and failure of closure of abdominal cavity without herniation (9). Note marked neck flexure. Fetal age, 20 days.
Fig. D. Two fetuses exhibit well-defined, localized neck hemorrhages (18, 19). Normal
littermate is at left. Fetal aee, 16 days.
J. Embryol. exp. Morph., Vol. 16, Part 1
W. F. GEBER
PLATE 1
facing p. 8
/. Embryol. exp. Morph., Vol. 16, Part 1
PLATE 2
Fig. E. Each fetus illustrates moderately severe omphaloceles consisting primarily of liver
(14, 15, 16, 17). Occasionally the intestine comprised the major portion of the contents.
Fetal age, 18 days.
Fig. F. Small interparietal hemorrhage (26) and spina bifida (25) in hyperdeveloped fetusNormal littermate on right. Fetal age, 17 days.
Fig. G. Large interparietal hemorrhage (28). Normal littermate on right. Fetal age, 16 days.
Fig. H. Interparietal meningocele (30). Fetal age, 19 days.
W. F. GEBER
facing p. 9
Effects of maternal stress
9
Maternal nutrition
The experimental animals were observed frequently throughout the day
for signs of failure to eat and drink properly, but none was seen. Within a
matter of hours after being put into the stress cages, the animals ate and
drank in what appeared to be a normal manner. However, since maternal
malnutrition of various degrees and types has been reported (Kruse, 1950) to be
capable of producing congenital malformations, total body weight and adrenal,
kidney, and heart weights of both pregnant and non-pregnant control and experimental groups of MA I and MA II animals were measured. Since the overall relationships were not affected by pregnancy, but were influenced by the age
of the animals at the time of mating, body weights as shown in Table 5 represent
those obtained on non-pregnant animals of both MA I and MA II groups.
A slight but significant difference in weight was noted in both experimental
groups with the difference being somewhat larger in the MA II groups. In all
probability this does not represent loss of body weight, but rather a failure
to gain the same amount as the controls, since all animals were still in their
growth phase. As previously mentioned, pregnancy did not alter this relationship.
Maternal organ weight
A number of studies involving various stress conditions have demonstrated
that the adrenal glands increase in weight following periods of high intensity
stimulation (Holland, 1958; Jones, Lloyd & Wyatt, 1953; Knobil & Briggs, 1954,
1955). In an attempt to evaluate the effect of the audiovisual stress imposed
on the maternal organism in both age-groups, the weights of the adrenals,
hearts and kidneys of both control and experimental animals were analyzed
(Table 5). Figures in the table represent randomly selected groups of 100
animals from both control and experimental MA I and MA II females. The
data indicate the organs of MA I experimental groups weighed slightly less than
the organs of the MA I control groups. In contrast, the adrenals, kidneys and
hearts of the MA II stressed females manifest definite increases in weight over
their respective controls.
DISCUSSION
At the present time it would appear that teratogenic influences fall into rather
definite but overlapping classes as follows: genetic, nutrition, infections, poisons
(drugs), other diverse environmental factors. It is virtually impossible to devise
an experimental procedure in which only one of these classes is effective at
one time, and it is certainly conceivable all may play relative roles under many
situations (Gruenwald, 1947; Warkany & Kalter, 1961).
Probably the least studied and understood is the very large class of environmental factors. Several of the virtually innumerable factors that can be placed
10
W. F. GEBER
in this class have been studied, but there have been comparatively few attempts
to analyze the effects of the more subtle types of environmental variation.
Recently Wilson (1964) has shown that a wide variety of teratogenic compounds and treatments can act not only in an additive manner, but actually in
a potentiating manner. Thus, audiovisual stress might be capable of exerting,
in addition to the effects demonstrated in the present study, a potentiating
effect on any number of chemical or other agents, although alone these factors
might be incapable of teratogenic effect at the doses used.
One important fact which emerges from all experimental studies is that teratogenic agents or influences do not induce a uniform amount or degree of abnormal
development even within a given litter. This fact in itself may well be a clue to
the common point or areas of action of many of these teratogenic agents.
As pointed out in a previous study (Geber, 1962), this variation may reflect
one or more of three possible maternal or fetal parameters which may be interdependent: (1) differences in placental orientation within the uterus, especially
with reference to the uterine blood supply, (2) differences in placental ability
partially to alter or detoxify a given compound, or (3) differences in susceptibility of various developing fetal organ tissues.
Reduced maternal and fetal blood flows as etiological factors
The relationship of the implanting and/or developing fetus to its uterine
environment is important for embryological events (Hashima, 1956; Ingalls
et al 1952; McLaren & Michie, 1961; Reed, 1936; Trasler, 1960). As previously
pointed out, the uterine and placental blood flows do not respond in the same
qualitative or quantitative manner to many of the maternal stresses used. The
fact that many litters were either destroyed or prevented from implanting in
the present study is postulated to be an index of severe reduction of blood
flow. Since the number of fetuses in the experimental litters was also reduced,
the disappearance of these two or three fetuses without a trace may indicate
certain areas of the uterus were selectively more affected, i.e. blood flow
decreased more, or implantation was not as effective in these areas and resulted
in complete loss of the fetus under the conditions of the maternal stresses early
in the pregnancy. When the blood flow reduction was slightly less, the fetuses
survived long enough to develop the membranes of the placenta with the result
that many more direct evidences of resorption were found in the experimental
groups at the time they were sacrificed. The study of Franklin & Brent (1964),
in which they physically blocked or reduced blood flow to individual early
fetuses for a very limited time, lends support to this concept.
The causative factors involved in the hypo- and hyperdeveloped fetuses,
and the other congenital malformations, are also very speculative. It certainly
is not unreasonable to assume that at least some part of these results may
also be due to reduced blood flows in both the maternal and fetal organism.
That these blood-flow alterations can occur and are prolonged in the late fetus
Effects of maternal stress
11
has been shown (Geber, 1962). Thus, a single maternal sensory stress of 1020 sec duration resulted in a decrease in fetal blood flow to the brain and
kidney for 3-20 min. In the present experimental situation, the audiovisual
stresses were continued throughout the entire pregnancy. It would seem reasonable to assume under this situation the fetal brain and kidney blood flow, and
other area blood flows, would be markedly affected. It may be argued that the
previous studies on late fetuses have no relevance to what may occur in the
younger, more rapidly developing and susceptible fetus as studied in the present
investigation. However, the findings of Franklin & Brent (1964) again tend to
support the fact that even a single, short-term reduction in blood flow to a young
fetus results in marked developmental deviations.
Maternal adaptation to audiovisual stress
The degree of maternal adaptation to the stress under the present conditions is
certainly an extremely important and difficult question to answer. In the previous
studies mentioned above (Geber, 1962), anesthetized pregnant animals still
responded in such a manner to all sensory stimulations that fetal hemo-dynamics
were affected. This indicates that there is a functional connexion between the
sensory and the autonomic nervous systems despite the loss of consciousness.
Thus, it would appear to be reasonable to assume that when an animal is
conscious and 'accustomed' to audiovisual stress there may still be activation
of the autonomic nervous system and the hypothalamic-pituitary-adrenal axis
even though the animal no longer responds by overt movements.
With in a very few hours of being placed in the stimulator, experimental
animals were observed to be feeding and drinking in an apparently normal
manner. Subsequently, much the same phenomenon developed in reference to
the overt or superficial response of the experimental groups to the individual
audio and visual stresses as they occurred throughout the experimental period.
Generally, all animals responded more in the first few days in the stimulator than
they did later on, although some individuals never appeared to be particularly
bothered by the various stimulations at any time. This non-response group
was estimated to comprise 10 % of the total. On the other hand, another 10 %
always gave an exaggerated response to many of the stimulations. Many of
this latter group also exhibited aggressive fighting tendencies either spontaneously or in response to a stimulation. This characteristic appeared to
develop after 7-10 days in the stimulator. However, at no time during the
experimental period were convulsions, which might have been traumatic to
a litter, noted in response to the stresses.
Since approximately 80 % of the group of experimental animals appeared
gradually to adapt to the environment in which they were placed, in that they
no longer reacted overtly to the high density stress, it would seem logical to
question whether or not these particular animals were responding in any manner.
Two facts may indicate that they were responding although at too low a level
12
W. F. GEBER
to be easily observed. First, it was noted that very slight movements of the ears,
whiskers, etc., could be detected in many of the apparently unresponsive animals
each time an audio stimulation occurred while they were resting quietly. Secondly,
it has been reported (Geber, 1962) that anesthetized animals are still capable of
exhibiting cardiovascular responses, e.g. changes in blood flow, pressure and
heart rate, although obviously incapable of consciously directed motor activity.
Thus, gross overt movement is not the only indicator of generalized, internal
responses to stress.
Maternal body and organ weight as indicators of stress response
A comparison of the body weight in relation to the weight of the heart,
kidneys and adrenals in MA I and MA II control and experimental groups
revealed an age-dependent dichotomy in that the organs of the stressed younger
females manifest a decrease in weight, whereas the organs of the stressed older
females indicated that hypertrophy had occurred, particularly in the heart. This
would appear to indicate that the response to stress was either of a different type
in the younger females, or was not as severe. By contrast, a number of studies
have found the weight of the adrenals to be increased in response to other forms
of stress (Holland, 1958; Jones et al. 1953; Knobil & Briggs, 1954, 1955).
However, the present data does agree with other studies of a short-term nature
(Edelman, 1945).
The fact that there was an age-dependent dichotomy in the response of the
adrenals weakens the argument that a primary cause of the results was activation
of the pituitary-adrenal axis. If this were the case, it might be expected that both
age-groups would show the same organ-weight relationship since both produced
the same types, and to a large extent the same magnitude, of abnormal developmental problems. This raises the possibility that decreased placental and fetal
blood flow accounted for the major part of the fetal results since continued
alteration of blood supply to the placenta may well upset the maternal-fetal
membrane exchange balance and as a consequence adversely affect fetal cellular
anabolism and catabolism. Decreased blood flow in the developing fetal organs
and tissues would also be expected to disrupt normal developmental sequences.
This is not to imply that endocrine and metabolic imbalances, namely the primary one in the maternal organism and the secondary one in the fetus, were not
important in the present study. In all probability, these imbalances worked in
conjunction with the decreases in blood flow.
Fetal age and susceptibility to induction of developmental anomalies
It is obvious that the present study does not make it possible to determine
if certain periods during gestation are more or less susceptible to stress. On the
other hand, the fact that the stress was present throughout the entire pregnancy
cycle allowed an evaluation of stress on both early and late developmental
phases of the fetuses. However, other than in the case of complete elimination
Effects of maternal stress
13
of a litter (or prevention of implantation) and resorption, it was impossible
to determine whether or not some of the other results were due to cumulative
or very specific time-related effects.
In conclusion, it should be emphasized that the actual number, type or
percentage of congenital malformations is not the important result. Rather,
it is that abnormalities were produced by a method which uses no other mechanism^) except those already present and functioning in the animal. It is proposed that the experimental procedure merely produced an upset in the physiological balance of the nervous, vascular and endocrine systems to a point where
they became capable of producing complete or partial resorption, and a variety
of morphological changes in the fetus. It is an open question as to how much
additional alteration occurred in various fetal tissues which might manifest
itself post par turn.
SUMMARY
1. Pregnant Sprague-Dawley albino rats were subjected to controlled audiovisual stress throughout pregnancy. The total effective stress duration amounted
to 10 % (6 min) of every hour of the entire day.
2. Total litter resorption occurred in 40 and 50 % of the pregnancies in the
young and older females respectively. Litter size was reduced by an average of
two fetuses per litter in both age-groups. Visible resorptions per litter were
markedly increased in the young females and slightly reduced in the older
females.
3. Developmental abnormalities produced included interparietal meningocele, abdominal hernia (omphalocele), spina bifida, and defects of the eye,
tail, hind- and forefoot. Hematomas, varying in size and location, were found
throughout the cranial area. Localized hematomas were found in the sublingual
areas. Osteogenic effects, ranging from partial to complete inhibition, were
widespread.
4. There was a marked increase in number and degree of hypo- and hyperdeveloped fetuses over the control rate.
5. Total number of fetuses manifesting some type of developmental deviation amounted to 25 % of the experimental groups and 0-8 % of the control
groups.
6. Effects of the audiovisual stress on the maternal organism included slight
body-weight loss in both young and older females, decrease in the weight of
the adrenals, kidneys, and hearts of the younger females, and increase in organ
weight, especially the heart, in the older females.
7. The etiological factors postulated to be responsible for the fetal results
are (1) decreased uterine and fetal organ blood flow, and (2) imbalance of the
maternal autonomic neurohormone and hypothalamic-pituitary-adrenal
relationships.
14
W. F. GEBER
RESUME
Effets sur le developpement de foetus de rat d'aggression
audio-visuelles chronique appliquees a la mere
1. Des rattes albinos Sprague-Dawley ont ete soumises a des agressions
audiovisuelles precises pendant la gestation. La duree totale du stress actif etait
de 10 % de chaque periode de 24 heures et etait reparti egalement pendant les
24 heures.
2. Une resorption totale des portees s'est produite dans 40 et 50 % des cas,
suivant qu'il s'agissait de jeunes ou de vieilles femelles gestantes. Le nombre
des foetus par portee est reduit de 2 en moyenne dans chaque groupe d'age.
Le nombre des resorptions visibles augmente notablement chez les jeunes femelles
et diminue legerement chez les vieilles femelles.
3. Les anomalies de developpement comportent des meningoceles interparietaux, des hernies abdominales (omphaloceles), des spina bifida, des lesions
de l'oeil, de la queue, des pattes anterieures et posterieures. On trouve des
hematomes de dimensions et de localisation variees dans la region cranienne.
On trouve des hematomes localises dans la region sublinguale. Des lesions
osseuses allant de l'inhibition partielle a l'inhibition complete, sont largement
repandues.
4. II y a une augmentation importante dans le nombre et l'intensite des hypo
developpements et des hyper developpements, par rapport au foetus temoin.
5. Le nombre total des foetus manifestant certaines formes d'aberrations
dans le developpement se monte a 25 % dans les groupes experimentaux et
a 0,8 % dans les groupes temoins.
6. Les effets du stress audio-visuel sur l'organisme maternel comportent
une faible perte de poids total chez les jeunes aussi bien que chez les femelles
plus agees, une diminution de poids des surrenales, des reins, du coeur chez
les plus jeunes, une augmentation du poids des organes, particulierement du
coeur chez les plus agees.
7. Les facteurs etiologiques que Ton suppose etre responsables des effets
sur le foetus sont (1) une diminution du flux sanguin dans les organes uterins
et foetaux et (2) un desequilibre dans les relations entre les neuro-hormones
maternelles autonomes et le systeme hypothalamo-surrenalien.
The author wishes to express his deepest appreciation to John A. Richardson for invaluable
assistance in the technical and construction phases of the study, and to Patrick L. Mayclin
and Nancy L. Burns for significant contributions during several of the latter phases. This
study was supported by USPHS grant HE-6488 and the South Dakota Heart Association.
Effects of maternal stress
15
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{Manuscript received 14 October 1965)