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Experimental studies on nicotine absorption during pregnancy
Experimental studies on nicotine absorption during pregnancy IV. The postmature
FREDERICK
R.
J.
neonate
BECKER*
EDWARD
CHLOE Durham,
KING R.
North
D.
LITTLE
Carolina
This report deals with 55 pregnant rats assigned randomly, 5 each, to 1 I study groups. Three groups were untreated controls delivered either spontaneously on the twenty-second day of gestation (term), or by cesarean section on the twenty-first and twenty-second day. Four groups were rendered postmature by prolonging gestation with chorionic gonadotropin and were delivered by section on days 21,22, 23, and 24. Four other experimental groups producing postmature young were delivered by section on these same days after having been treated chronically with nicotine throughout pregnancy. While gestation had been equally prolonged in the two experimental groups, hormone-induced postmaturity yielded young with many of the characteristics that have been ascribed to the postmaturity syndrome in man. However, nicotine-exposed young were underweight, virtually fetal in appearance, had narrower transverse head diameters, shorter vibrissae, shorter claws or none at all, and ossification in the long bones had not progressed as far as it had in term young of equal weights, Both groups of experimental animals would have delayed delivery even longer had sectioning not intervened. The nicotine-treated ofspring could have passed for premature and were somewhat comparable to the peculiar, small,malnourished infants with prolonged gestations described recently in the human literature.
R E c E N T L Y, we reported that when 3 or 5 mg. nicotine per kilogram were given to pregnant rats twice daily throughout pregnancy, immature, fetuslike young were delivered several days post term.l In our review of the human literature1 we noted that mothers who smoke heavily during pregnancy frequently abort or are delivered of light-weight premature babies. Postterm human neonates are generally described as heavier and somewhat more advanced than the normal term infant. Hence, our rat neonates exposed to
nicotine
fitted
neither
into
From the Department Laboratory of Perinatal Duke Medical Center,
the category
of
of Anatomy, Studies,
*Supported by grants-in-aid from American Medical Association Education and Research Foundation and from the Hoffmann-LaRoche Company. 1109
the prematurely delivered nor into that of the overly developed, postterm delivery. They were, paradoxically, immature postmature pups. The purpose of the present study was to produce experimental litters of postmature rat neonates in the absenceof nicotine treatment to determine whether postmaturity characteristics were, indeed, distinct from those features seen after delayed birth with nicotine. Physicians have been interested in and have argued about the duration of human pregnancy from the time of the ancient Hindus to modern times.zS8 While the average duration is 40 weeks, or 280 days, 71.4 percent of all pregnancies have been reported to occur 275 to 294 days beyond the last menses.Q
1110
Becker,
King,
and
Little
At present, there also seems to be general agreement that a premature infant is one weighing 2,500 grams or less, though much more caution seems to be exercised in expressing prematurity in terms of gestational age. It is even more difficult, currently, to gain a clear-cut definition of a postmature infant. While some physicians have described a postmaturity syndrome, others have denied its very existence. Nevertheless, it seems essential here to attempt to characterize postmaturity, since, inevitably, comparisons between the rat and the human being will have to be drawn later in this report. The reported incidence of presumably postmature human births varies between 2 and 12 per cent in the samplings recorded. While 294 days seems to be a widely accepted time for the upper limit of normal gestation, the times given for the onset of postmaturity vary with the reporting clinician. For example, Cliffordlo places the onset at 294 days; Eastman9 at 300; and McKiddiell somewhere between 285 and 304 days. The chief signs marking a postmaturity syndrome have been: (1) an increased tendency toward difficult labor; (2) signs of fetal distress; and (3) an increased rate of perinatal mortality. Taken without further comment, these signs are typical of many a difficult term delivery. In this instance, however, with continued growth in utero beyond term, the infant head increases in transverse diameter contributing to problems of pelvic disproportion. Delivery becomes prolonged and mechanically difficult. Fetal weight has increased likewise so that many babies, cIassed as postmature, weigh over 8 pounds. Not all have been large and chunky, however. Some long, thin, malnourished ones have been described, and even some small in size. The latter have been thought to be slower than normal in growth and maturation.12 Recently, Naeye13 reported on 16 newborn infants with gestations of over 42 weeks. Ten were retarded in growth by normal standards and certain organ weights were disproportionately small. The abnormalities resembled those encountered in undernutrition. The remaining 6 specimens were all oversized and
August 15, l!If% Am. J. Obst. & Gynec.
had symptoms associated with chronic prenatal hypoxia. Various signs of fetal distress have been reported to account for the hypoxic condition of many postmature infants. There has been the slowing of the fetal heart rate associated with the increase in head size and its pressure against soft tissue resistance.12 The staining of the fetal skin and nails with meconium has been offered as evidence of an interference with fetal oxygenation and the resultant parasympathetic response of the large bowel to hypoxic stress.14p I5 Oxygen saturation of venous cord blood has been reported to decline as pregnancy is prolonged. 14pI6 Others17-20 have not been able to substantiate the latter findings, and still others 21-22 have felt that cord blood samples taken at delivery indicate little about the normal or the hypoxic state of the infant prior to birth. Placental insufficiency, ascribable to such findings as fibrin deposition and the sludging and coagulation of placental blood in the intervillous spaces,Z3 reduced placental oxygen uptake,24 and calcium deposition,2 has been cited to show that an aging placenta may not sustain life satisfactorily beyond term and may explain the cyanosis of the postmature infant. Still, there are reports that postmaturity does not incur placental pathology beyond what one might wish to consider normal degenerative changes in term placentae.25 CliffordI” has probably given as fair a description of the postmature human infant as anyone. Many, but not all, are overweight. Some, but not all, display cyanosis or pallor, and even complete respiratory distress, atonia. The longer the sojourn in utero, the greater the degree of meconium passage and staining of the skin and nails. Prognosis for their survival generally has been poor. Many of our animal specimens in the category of delayed births present features similar to those that have been described for the postmature infant. This group of rats will be compared with a group of nicotine-treated specimens, quite different in appearance, but with gestation that has been equally prolonged.
Volume Number
101 8
Nicotine
Method Osborne-Mendel rats, 5 per group, were assigned randomly to 11 study groups when sperm were discovered in the vaginal lavage after overnight mating. There were three control groups of untreated females: one group being delivered spontaneously on the twenty-second day of gestation (term date) ; and two groups being delivered by cesarean section on the twenty-first and twenty-second days, respectively. Since untreated rats only rarely prolong delivery beyond term, we had to create postmature rats experimentally. Four experimental groups (in effect, postmature controls without nicotine) were given 25 rat units (R.U.) of a chorionic gonadotropin (Antuitrin-S) daily from the eighteenth day of gestation until one day prior to scheduled cesarean delivery on postcoital days 21, 22, 23, and 24, respectively. The 21 and 22 day specimens were to be compared with untreated controls of equal age; the 23 and 24 day animals, representing hormonally prolonged gestations, could be compared with 1 and 2 day control neonates or with the postdelivered nicotine groups of young. Four other experimental groups received 5.0 mg. nicotine per kilogram subcutaneously twice daily until delivered by cesarean sec-
Table I. Average
daily
maternal
food
consumption
absorption
and
postmature
neonate
tion on days 21, 22, 23, and 24. This was the same dosage that had previously produced the peculiar immature young after a considerable prolongation of gestati0n.l In general, the experimental plan followed that of our previously reported study1 except that daily weight records were kept through the day of cesarean section and special tallies were kept of food consumption for days 14 to 18, 18 to 21, and from the twenty-first day through the day of section. On the day of section, the pregnant mother was weighed, secured, and an abdominal incision was made under lidocaine. One fetus with placenta and membranes was placed under observation in a warming box where its general activity could be observed and its respiratory rate was recorded 5 minutes after delivery and then every 5 minutes for the first hour. The rest of the litter was delivered and notes were made on the amount and viscosity of amniotic fluid, presence or absence of meconium, skin color, skeletal deformities, and general activity level. Weight records were kept on the fetrrs, placentas and membranes, the llterus, and the empty maternal carcass. C!ertain gross observations were made on 5 fetuses randomly selected from each litter
according
to days of gestation
- ---
Grams Treatment I. Controls A. Spontaneous
B. Cesarean C. Cesarean II. Postmature A. Cesarean B. Cesarean C. Cesarean D. Cesarean III.
Day
delivery
section
No section. DeIivered on day 22
section section (An&&l-in-S)* section section section section
22 23 24
Nicotine (10 mg./Kg./day) A. Cesarean section B. Cesarean section C. Cesarean section D. Cesarean section “Dose
of
of 25 R.U.
from
day
21 223’ 24 18.
1111
consumed
o-7 Days
7-14 Days
14-18 Days
18-21 Days
22 Day
15.9 13.9 17.3
18.6 18.0 18.3
20.4 20.8 22.7
24.7 20.1 22.7
18.2
15.3 17.2 16.7 17.1
19.9 19.7 18.6 20.0
21.2 22.9 22.3 23.1
22.5 22.9 23.3 25.3
10.4 10.2 9.3 10.8
12.7 14.2 14.5 14.9
14.7 15.4 15.8 17.2
14.4 17.0 16.6 15.7
.--21-23 Days
__ 21-24 Days
17.9
13.9 15.9 14.8
14.9 12.6 17.1
1112
Becker, King, and Little Am.
whenever the litter size was greater than 5. Otherwise, all the young in the litter were studied. Individual birth weight and sex were recorded on these. Crown-rump length, transverse head diameter, length of claws and vibrissae, and zones of primary ossification in the radius and ulna were measured. In the case of control neonates delivered spontaneously, birth weight and number per litter were recorded. One third of this population was killed at birth; the remaining two thirds were killed on the first and second postnatal days of life, respectively, so that similar measurementscould be made. These last could be compared with measurements on experimental postmature fetuses removed from the uterus on the twenty-third and twenty-fourth postcoital days. Results
The mean daily food consumption for the various groups is shown in Table I. The third trimester is shown broken into two
August 15, 1968 J. Obst. & Gym-.
periods, days 14 to 18 and 18 to 21, because treatment with chorionic gonadotropin (Antuitrin-S) had begun on the eighteenth day for the control postmature group. This treatment appeared to have little effect upon food consumption, for these animals ate about as much as untreated controls, if not slightly more, through the twenty-first postcoital day. On the other hand, nicotine treatment seriously curtailed caloric intake from the start, though there were slight gains in amounts eaten daily asgestation progressed.All groups ate considerably less on the twenty-second day, the normal date for a term delivery. This decrement was most severe in the Antuitrin-S group, e.g., note the drop of 9 grams and compare it with the 4.6 gram drop for the untreated controls and the 2.1 gram drop in the nicotine group. Indeed, the longer mothers went beyond term, the more severe the decrement in daily food intake among the Antuitrin-S mothers. The effect of this reduction in food con-
Table II. Maternal weight gain according to days of gestation consumed 7-14 14-18 18-21 22 21-23 Days Days Days Day Days Grams
Treatment
Day of section
o-7 Days
21-24 Days
I. Controls
A. Spontaneous delivery
None
19.0 k7.6
Cesareansection
21
15.8 k4.1
C. Cesareansection
22
20.2 22.0
B.
31.4 24.9 26.2 23.0 22.8 26.6
31.2 25.9
36.2 23.0 27.2
28.2 A6.4 42.2 23.8 44.2 +8.8 35.6 r4.4
35.8 r4.5 40.0 27.8
51.2 26.9
7.0 23.0
35.4 22.6 41.6 k2.2
6.2 24.4
II. Postmature (Antthin-S) A.
Cesarean
B. Cesarean C. D.
Cesarean Cesarean
section section
21
15.8
22
22.9 25.4 23.4
23
18.8
section
24
k2.6 22.4 23.6
section
21
section
21.8 18.4 +12.1
27.0 27.5
49.0 26.3 41.6
t4.8 35.6 f2.6 39.8 k5.9
4.0 22.7 12.0 t3.2 (-5.2) (21.6)
111. Nicotine A.
Cesarean
B. Cesarean C. D.
Cesarean Cesarean
section section section
22 23 24
1.4
28.3 (-3.8) (f3.8) 2.0 26.4 (-2.2)
(r4.3j
15.4
12.6
14.2
k4.6 18.8 23.3
k3.6 20.2
+6.0 21.2
14.9
12.8
k2.0
10.4
+5.9 13.2
24.6
k2.5
16.0
13.8
10.4 k1.6 8.0
22.7
22.4
26.5
9.0 k1.8
20.6 96.3
Volume Number
101 8
Nicotine
sumed upon maternal weight gain is seen in Table II and Fig. 1. While the postmature group of mothers gained at about the same rate as control mothers until term, they always gained considerably more than nicotinetreated mothers. But, as they carried beyond term, their daily weight increments decreased progressively while mothers in the nicotine group continued to exhibit daily weight increments. Table III shows how the weight gains during the different periods of gestation were distributed between maternal and fetal compartments. The differences between initial weight and empty maternal carcass weight were always less for the nicotine group than ior control and postmature ( Antuitrin-S) groups. These differences held for the weights of the empty uterus, uterine content, and, as a result, for fetal weights. There was no difference in litter size between control and the Antuitrin-S groups. However, the number of fetuses per litter was significantly reduced in mothers treated with nicotine. The average birth weights are given in Table IV where it can be seen that they were significantly lower for the nicotine groups. The
Accumulated 140 -
Maternal Weight Gain in Rats During Prolonged Gestaiion
130-
-AlI O--4 ~..-a o-IO-
#’ I 7
Fig. 1. Maternal longed gestations.
I 14
Ant.!5 All Nicotine All Controls
I 1 t , i I 1 18 19 20 21 22 23 24 Days of Gestation
weight
gain
in
rats
with
pro-
absorption
and
postmature
neonate
1113
weight gain in Antuitrin-S postmatures continued in utero as term was passed but after the twenty-second day it was less than that of 1 and 2 day neonates delivered spontaneously by untreated control mothers. Young in the nicotine groups always had respiratory rates during the first hour that were considerably lower than those of control young. However, the older the gestational age at time of cesarean delivery, the higher the rate (Table V) . Young of nicotinetreated mothers were delivered at 23 and 24 days were always pink and healthy in appearance, though they seemed underdeveloped for their age. The postmaturc (4ntuitrin-S) young had respiratory rates equal to control rates at 21 and 22 days, but at 23 and 24 days these neonates were rya.notic and in respiratory distress with respiratory rates below those of even nicotine-treated young of the same age. The incidence of stillbirths was 14 per cent in the Antuitrin-S group in contrast to 3 per cent in the nicotine-treated group. No data are on hand for neonatal mortality beyond the first hour of life, but it was greatest at this time for the very small immature young of the 21 day deliveries in the nicotine group. It was also high for Antuitrin-S postmature young in respiratory distress. From the protocol notes we learned that placentae in control and Antuitrin-S groups at 21 days appeared normal, firmly attached to the uterine wall, and could not be stripped free easily. Amniotic fluid was variable in amount but usually scanty and somewhat viscous. Findings were similar for the 21 day nicotine group except that fluid was always abundant and watery. There was little change on the twenty-second day except that amniotic fluid viscosity increased in both control groups but not in the nicotine rats. In the 23 and 24 day Antuitrin-S specimens, amniotic fluid was extremely scanty and stringy; placentas exhibited yellowish, necrotic infarcts and detached readily from uterine walls; many fetuses were plastered with meconium; and uteri were stretched and hemorrhagic. In contrast, placentas in nicotine groups at 23 and 24 days were firmly adherent and nor-
1114
Becker, King, and Little
Table III.
Distribution gestational
at different
August 15, l!%lJ Am. J. Obst. & Gynec.
of mean maternal weight ages (weights in grams) * (
Treatment
Day
of section
/
in pregnant
rats being
( W!fht uterine contents
1 Azge young per litter
Final term weight
1 Weight
Initial weight
carcass
Weight of uterus
247.0 271.6
360.4 402.4
287.2 305.4
4.7 4.6
65.2 85.7
52.4 72.8
11 12
of
/
delivered
/ Litter weight
Control A B Postmature A B C D
21 22 23 24
239.4 272.6 257.0 248.0
369.0 409.0 382.0 369.6
293.0 312.2 281.9 275.0
4.2 5.1 4.7 5.1
63.7 90.1 88.3 82.8
51.7 74.6 71.4 67.3
10 12 11 10
A B c D
21 22 23 24
244.2 262.4 249.4 249.6
287.8 318.6 296.4 305.8
257.9 262.3 254.6 260.4
2.9 3.9 3.1 3.1
22.7 47.2 31.0 39.6
18.8 39.1 24.5 31.6
5 8 6 6
Nicotine
*Standard
errors
were small
Table IV. Weights
for all weight
of cesarean
values,
hence
means
section-delivered
young
Weight Treatment
Day
Controls Postmature
(Antuitrin-S)
)
*Weights
of nursing
young
Table V. Average
on first
respirations the first hour of life
21 22 23 24
29.4 56.9
in grams Day
related
22
(
to gestation
in days
Eq--23
/
Day
24
5.03+0.10
5.9OkO.13
6.95t0.12*
7.99ko.11*
5.29tO.08
6.13+0.10
6.3220.10
6.9M.13
3.OlkO.26
Nicotine
during
21
are reliable.
and
second
4.75+0.11 postnatal
day.
per minute
27.5 63.3 29.6 18.4
5.3 34.0 35.6 46.7
mal; amniotic fluid was still abundant, clear, and watery. Fig. 2 presents data on certain aspects of developmental progress. Except, perhaps, for claw growth, the postmature group (AS, in the figure) continued to develop within the uterus much like untreated fetuses (C) before term, and like 1 or 2 day control neonates. In some of the measurements, An-
All other
4.5820.46 weights
are at cesarean
4.7OkO.38 section
delivery.
tuitrin-S postmatures exceeded the values obtained for the 1 and 2 day control neonate; in others, they fell short. These differences were not significant but, if the postterm Antuitrin-S specimens were compared with term young, on the average they weighed heavier, were longer, had wider heads, longer claws and vibrissae, and ossification of the long bones had progressed further than. in the controls. Postterm cesarean section-delivered young of nicotine-treated mothers in no way resembled I- and 2-day-old rats. Neither did they often resemble term control young. On the average, they weighed less, had narrower heads, shorter claws or no nails at all, and vibrissal length and the ossification of long bones lagged behind control values. Fig. 3 contrasts the control term fetus (C22) with postmature Antuitrin-S fetuses (A-
Volume Number
Nicotine
101 8
absorption
and
postmature
1
[ F.Clow Length
-+T+ni-d
Day of Sectioning
Fig. 2. Maturational prolonged
by treatment
characteristics with nicotine
.35 / 21
of fetal rats with (N) or chorionic
23, A-24) and with a series of nicotine specimens designated by the letter (N) . The postmature Antuitrin-S specimens often exhibited clubbed hind feet with inversion at the ankle joint and humped backs. Comment All animals randomly scheduled for cesarearl section on the twenty-first through the twenty-third day were still carrying their young. This was not always true of those assigned for a 24 day delivery, some mothers having already delivered before we could get to them. Thus, the 24 day group formed a select category in the sense that not all animals assigned to it had an equal chance of being included in it. Would those animals comprising it show any physical differences peculiar to it and not seen with equal frequency in other age groups? The early deliveries which failed to carry to 24 days occurred in the nicotine-treated group only. Three were delivered late on the twenty-third
I 22
Related
neonate
1115
I
to
)
“‘--.._............._.... .N I 23 24
Day of Sectioning
normal gestations (CI gonadotropin (AS).
and
of gestations
day. Their average birth weight was 5.29 grams, slightly heavier than nicotine cesarean section deliveries on the twenty-third and twenty-fourth days. Otherwise there were no noticeable differences. Two delivering early on the twenty-fourth day weighed exactly the same as 24 day nicotine cesarean-delivered young (average of 4.70 grams). We have noted certain anomalies in 24 day AntuitrinS fetuses (clubbed feet and humped backs). These, undoubtedly, can be attributed to crowding in utero as growth continued beyond term. Otherwise, if the 24 day groups were select ones, the bias was minimal. The early spontaneous deliveries also demonstrated that the operative procedure of cesarean section was not effecting results significantly. It was possibIe that chorionic gonadotropin ( Antuitrin-S) might effect fetal growth directly so as to account for larger fetuses than normal. If it were given earlier in gestation than the eighteenth day, then
1116
Becker, King, and Little
August 15, I!368 Am. J. Obst. & Gynec.
__ Fig. 3. Comparison chorionic gonadotropin
of control (A)
specimens (C)
at the gestational
young delivered at term should be larger than normal. When such a check experiment was performed, Antuitrin-S young at term did not differ from untreated control young removed at 22 days. Fetal size, in and of itself, could be a factor which determined expulsion time. ReynoldP has pointed out that the myometrium adapts itself to the size of the conceptus, stretching
___
with fetal rats treated with nicotine
ages designated
(N)
or
by numerals.
and exerting greater powers of contraction the more it is distended. However well this law might hold for normal births, where large fetuses might deliver earlier than smaller ones, it did not apply to our postmatures when gestation was prolonged by either chorionic gonadotropin or nicotine. Nor did litter size appear to control the time of gestation nor the maturational state
Volume Number
101 8
of the individual. An old concept of mammalian reproductive physiology has been the tenet that large litters are associated with short gestations; small litters, with longer gestational times. 27 Our Ant&-in-S litters ran contrary to the first premise, even if the nicotine-treated litters fitted the second. Yet, Reynolds x has embroidered upon this concept by stating that “the larger the litter, the lrss mature the individuals at birth; and, conversely, the smaller the number, the larger and more mature the young born.” Our young certainly pointed up the reverse. Large Antuitrin-S-treated litters contained the larger, more mature fetuses; small nicotine litters contained the smaller, less mature young. While the food consumption of AntuitrinS mothers was virtually identical with that of control mothers throughout the normal stages of pregnancy, they ate less and gained less weight as they progressed beyond term (Fig. 1) . Such a weight loss late in pregnancy has been described among women who delay delivery abnormally.* Both caloric intake and weight gains were remarkably reduced in our nicotine-treated pregnant rats. No such noticeable reduction has been reported among women who smoke heavily during pregnancy. Our experiments certainly confirm the human findings that nicotine absorption throughout pregnancy can result in the production of an underweight infant, but not necessarily as a result of premature birth. While our nicotine-exposed rats present immature features, their birth has been abnormally prolo’nged. They are more representative of the peculiar postmature neonates recently described by Naeye. I3 Whether, as Naeye suggests, there is a problem of malnutrition involved is as yet unknown. What we observe in‘ our rat neonates might be more directly related to the secondary effects of maternal inanition than to a direct action of nicotine. It was difficult to know how to class these young of nicotine-treated mothers. Morphologically they looked more like premature young and contrasted markedly with the more mature and overweight young of Antuitrin-S mothers even though the births had
Nicotine
absorption
and
postmature
neonate
1117
been equally prolonged in both cases. In our various tables, data have been presented in terms of group averages. This is true for Fig. 2 also. Still, in all groups, at any gestational age, there was some disparity in individual sizes and weights. Some few controls were as large as the smaller postmature Antuitrin-S neonates; some of the larger nicotine specimens were as large as the smallest controls. Had we no foreknowledge of gestational ages it would have been difficult to determine, at times, which were term or pnstmature or which might be premature neonates. Were there any features that definitely pointed to postmaturity other than xceight differences ? All the young at each gestational age could be subdivided into weight classes separated by intervals of half a gram. If, now, all the measurements given in Fig. 2 should turn out to be proportionate to weight, then there would be no real way to discriminate between postmature, immature, or term neon&es if gestational age were unknown. When the foregoing was done, it was noted that crownrump length increased proportionately with body weight. Neither weight nor crownrump length alone could serve as a satisfactory index of postmaturity. It could not be said, for example, as has been said for the human infant, that the postterm infant was longer for its body weight than the term neonate. Other parameters, however, did tlurn out to be useful indices of postmaturity. e.g., Antuitrin-S postmature neonates always had head diameters proportionately wider than any other neonates in the same weight class. The extent of ossification in the radius and ulna was always proportionately greater, as were the vibrissa and claw lengths. Of all these features, only the extent of ossification in the long bones and vibrissa length held as postmaturity indices for the largest of the postterm nicotine-exposed young. In all other respects, all of these measurements for the majority of younp of’ nicotine-treated mothers were less than in other nennates of equal weight. Always transverse head diameters were disproportionatelv nar-
1118
Becker,
King,
and
August
Little
rower and claw lengths shorter; or, in some instances, there were no claws present at all. In general, then, for the bulk of nicotinetreated rats the physical signs were those of immaturity rather than postmaturity. There were other differences between these two groups of postmature young, as Table V on initial respiratory efforts indicates. The 23 and 24 day nicotine-treated young were pink and healthy in appearence, not cyanotic like their Antuitrin-S counterparts. Nor were they subject to respiratory distress and early neonatal death like the Antuitrin-S specimens, In part, this may have been due to their very immaturity. Placentas were normal and functional, not necrotic and infarcted. Uteri were not hemorrhagic. Amniotic fluid was clear and abundant, not scanty and meconium-stained as in Antuitrin-S births. All of these signs, except the prolonged gestation, suggested even more strongIy that these were fundamentally immature young with some slowing of the developmental processes. NesbitF has summarized the incidence of fetal mortality, i.e., the number of stillbirths as distinct from neonatal deaths for human postmature births. His figures, taken from several sources, range from 2 to 9 per cent as contrasted to 1 to 4 per cent for term deliveries. Our stillbirth rates were 14 per cent and 3 per cent for Antuitrin-S and nicotinetreated litters, respectively. It would probably have been higher for the Antuitrin-S group had we not kept the dosage to 25 R.U. purposely to assure reasonable survival at 24 days. COCO’~ quotes rates of 25.6 per cent in postmature rats receiving 40 to 100 R.U. of Antuitrin-S and 51.6 per cent when 200
15, 1968
Am. J. Obst. & Gynec.
R.U. was given. The rate was 84.6 per cent for rabbits given the high dose. Roux, Romney and Dinnersteinzg gave a figure of 60 per cent fetal mortality when 40 R.U. of Antuitrin-S was used to produce postmature rabbits. We have never seen a reduction in litter size before among pregnant rats given nicotine at the concentrations employed in this study. We had, only with much higher dosage levels, e.g., 15 to 20 mg. per kilogram. Others,30-32 however, have reported smaller litters than normal when pregnant rats or rabbits were exposed chronically to nicotine, but their animals had been exposed for several generations or had been treated in smoke -filled chambers where dosages were unknown and uncontrolled. If the relationship between developmental status and the duration of pregnancy varies because endogenous or exogenous agents can act to prolong or retard maturation, then in attempting to assess prematurity or postmaturity it may be necessary to look for other signs of maturity or immaturity, much as we have done here. Weight and time alone do not seem to be very satisfactory criteria. A foreshortened pregnancy may not be the only route to prematurity. Consider Naeye’s infants13 or our own nicotine-treated specimens in which morphologic characteristics were definitely immature, including low birth weights, yet the termination of pregnancy was abnormally prolonged. On the other hand, an equally prolonged gestation can result in infants exhibiting overdevelopment and all the cIassica1 signs of the much debated postmaturity syndrome.
REFERENCES
1.
2. 3. 4. 5.
Becker, R. F., Little, Chloe R. D., and King, J. Edward: AM. J. OBST. & GYNEC. 100: 957, 1968. Nesbitt, R. E. L.: Obst. & Gynec. Surv. 10: 311, 1955. La Coste, E.: Gym%. et. obst. ‘21: 489, 1930. Simpson, J. Y.: J. M. SC. London & Edinburgh 16: 50, 1853. Wahl, F. A.: Deutsch. med. Wchnschr. 63: 125, 1937.
6.
Reibold, G.: Zentralbl. GynLk. 62: 2594, 1938. 7. Joly, W. A.: J. Obst. & Gynaec. Brit. Emp. 35: 258, 1928. 8. Arnot, P. H.: West. J. Surg. 56: 115, 1942. 9. Eastman, N. J.: Williams Obstetrics, New York, 1950, Appleton-Century-Crofts, Inc., p. 1046. 10. Clifford, 3. H.: Am. J. Dis. Child. 82: 232, 1951.
Volume
101
Nwnber
8
11. McKiddie, J, M.: J. Obst. & Gynaec. Brit. Emp. 56: 386, 1949. 1”. Adair, F. L., editor: Obstetrics and Gynaecology, London, 1940, Hirschfeld Bras., Ltd., vol. I, p. 906. 13. Naeye, R. L.: Arch. Path. 84: 37, 1967. 14. Walker, J., and Turnbull, E. P. N.: Lancet 2: 312, 1953. 15. Clifford, S. H.: J. Pediat. 44: 1, 1954. 16. Barcroft, J.: J. Physiol. 83: 193, 1934-35. L. G.: Am. J. Pub. Health 45: 17. McKinney, 633, 1955. 18. Rooth, G., and Sjostedt, S.: Acta obst. et gynec. scandinav. 36: 374, 1957. 19. Minkowski, A.: Ann. paediat. Fenniae 3: Fascicle 2, 1957. “0. Bancroft-Livingston, G., and Neill, D. W.: J. Obst. & Gynaec. Brit. Emp. 64: 498, 1957. 21. Graham, B. D.: J. Pediat. 60: 882, 1962. 22. Brady, J. P., James, L. S., and Barker, M. P.: AM. J. OBST. & GYNEC. 84: 1, 1962.
Nicotine
absorption
and postmature
neonate
I I19
23. Flexner, L. B.: AM. J. OBST. & GYNEC. 55: 469, 1948. 24. Wong, H. N., and Hellman L. M.: Bull. Johns Hopkins Hosp. 73: 31, 1943. 25. Masters, M., and Clayton S. G.: J. Obst. & Gynaec. Brit. Emp. 47: 437, 1940. 26. Reynolds, S. M. R.: Medicine 53: 441, 1958. 27. Asdell, S. A.: Patterns of Mammalian Reproduction, Ithica, 1943, Comstock Publishing Company. 28. Coca, R. M.: Am. J. Physio!. 137: 143, 1942. 29. Roux, J. F., Romney, S. L., and Dinnerstein, A.: AM. J. OBST. & GYNEC. 90: 546, 1964. 30. Theines, C. H., and Behrend A.: J. Pharmacol. & Exper. Therap. 48: 317, 1933. 31. Essenberg, J. M., Schwind, J. V., and Patias, A. R.: J. Lab. & Clin. Med. 25: 708, 1940. 32. Schoeneck, F. J.: New York, J, Med. 41: 1945, 1941.
FREDERICK
R.
J.
neonate
BECKER*
EDWARD
CHLOE Durham,
KING R.
North
D.
LITTLE
Carolina
This report deals with 55 pregnant rats assigned randomly, 5 each, to 1 I study groups. Three groups were untreated controls delivered either spontaneously on the twenty-second day of gestation (term), or by cesarean section on the twenty-first and twenty-second day. Four groups were rendered postmature by prolonging gestation with chorionic gonadotropin and were delivered by section on days 21,22, 23, and 24. Four other experimental groups producing postmature young were delivered by section on these same days after having been treated chronically with nicotine throughout pregnancy. While gestation had been equally prolonged in the two experimental groups, hormone-induced postmaturity yielded young with many of the characteristics that have been ascribed to the postmaturity syndrome in man. However, nicotine-exposed young were underweight, virtually fetal in appearance, had narrower transverse head diameters, shorter vibrissae, shorter claws or none at all, and ossification in the long bones had not progressed as far as it had in term young of equal weights, Both groups of experimental animals would have delayed delivery even longer had sectioning not intervened. The nicotine-treated ofspring could have passed for premature and were somewhat comparable to the peculiar, small,malnourished infants with prolonged gestations described recently in the human literature.
R E c E N T L Y, we reported that when 3 or 5 mg. nicotine per kilogram were given to pregnant rats twice daily throughout pregnancy, immature, fetuslike young were delivered several days post term.l In our review of the human literature1 we noted that mothers who smoke heavily during pregnancy frequently abort or are delivered of light-weight premature babies. Postterm human neonates are generally described as heavier and somewhat more advanced than the normal term infant. Hence, our rat neonates exposed to
nicotine
fitted
neither
into
From the Department Laboratory of Perinatal Duke Medical Center,
the category
of
of Anatomy, Studies,
*Supported by grants-in-aid from American Medical Association Education and Research Foundation and from the Hoffmann-LaRoche Company. 1109
the prematurely delivered nor into that of the overly developed, postterm delivery. They were, paradoxically, immature postmature pups. The purpose of the present study was to produce experimental litters of postmature rat neonates in the absenceof nicotine treatment to determine whether postmaturity characteristics were, indeed, distinct from those features seen after delayed birth with nicotine. Physicians have been interested in and have argued about the duration of human pregnancy from the time of the ancient Hindus to modern times.zS8 While the average duration is 40 weeks, or 280 days, 71.4 percent of all pregnancies have been reported to occur 275 to 294 days beyond the last menses.Q
1110
Becker,
King,
and
Little
At present, there also seems to be general agreement that a premature infant is one weighing 2,500 grams or less, though much more caution seems to be exercised in expressing prematurity in terms of gestational age. It is even more difficult, currently, to gain a clear-cut definition of a postmature infant. While some physicians have described a postmaturity syndrome, others have denied its very existence. Nevertheless, it seems essential here to attempt to characterize postmaturity, since, inevitably, comparisons between the rat and the human being will have to be drawn later in this report. The reported incidence of presumably postmature human births varies between 2 and 12 per cent in the samplings recorded. While 294 days seems to be a widely accepted time for the upper limit of normal gestation, the times given for the onset of postmaturity vary with the reporting clinician. For example, Cliffordlo places the onset at 294 days; Eastman9 at 300; and McKiddiell somewhere between 285 and 304 days. The chief signs marking a postmaturity syndrome have been: (1) an increased tendency toward difficult labor; (2) signs of fetal distress; and (3) an increased rate of perinatal mortality. Taken without further comment, these signs are typical of many a difficult term delivery. In this instance, however, with continued growth in utero beyond term, the infant head increases in transverse diameter contributing to problems of pelvic disproportion. Delivery becomes prolonged and mechanically difficult. Fetal weight has increased likewise so that many babies, cIassed as postmature, weigh over 8 pounds. Not all have been large and chunky, however. Some long, thin, malnourished ones have been described, and even some small in size. The latter have been thought to be slower than normal in growth and maturation.12 Recently, Naeye13 reported on 16 newborn infants with gestations of over 42 weeks. Ten were retarded in growth by normal standards and certain organ weights were disproportionately small. The abnormalities resembled those encountered in undernutrition. The remaining 6 specimens were all oversized and
August 15, l!If% Am. J. Obst. & Gynec.
had symptoms associated with chronic prenatal hypoxia. Various signs of fetal distress have been reported to account for the hypoxic condition of many postmature infants. There has been the slowing of the fetal heart rate associated with the increase in head size and its pressure against soft tissue resistance.12 The staining of the fetal skin and nails with meconium has been offered as evidence of an interference with fetal oxygenation and the resultant parasympathetic response of the large bowel to hypoxic stress.14p I5 Oxygen saturation of venous cord blood has been reported to decline as pregnancy is prolonged. 14pI6 Others17-20 have not been able to substantiate the latter findings, and still others 21-22 have felt that cord blood samples taken at delivery indicate little about the normal or the hypoxic state of the infant prior to birth. Placental insufficiency, ascribable to such findings as fibrin deposition and the sludging and coagulation of placental blood in the intervillous spaces,Z3 reduced placental oxygen uptake,24 and calcium deposition,2 has been cited to show that an aging placenta may not sustain life satisfactorily beyond term and may explain the cyanosis of the postmature infant. Still, there are reports that postmaturity does not incur placental pathology beyond what one might wish to consider normal degenerative changes in term placentae.25 CliffordI” has probably given as fair a description of the postmature human infant as anyone. Many, but not all, are overweight. Some, but not all, display cyanosis or pallor, and even complete respiratory distress, atonia. The longer the sojourn in utero, the greater the degree of meconium passage and staining of the skin and nails. Prognosis for their survival generally has been poor. Many of our animal specimens in the category of delayed births present features similar to those that have been described for the postmature infant. This group of rats will be compared with a group of nicotine-treated specimens, quite different in appearance, but with gestation that has been equally prolonged.
Volume Number
101 8
Nicotine
Method Osborne-Mendel rats, 5 per group, were assigned randomly to 11 study groups when sperm were discovered in the vaginal lavage after overnight mating. There were three control groups of untreated females: one group being delivered spontaneously on the twenty-second day of gestation (term date) ; and two groups being delivered by cesarean section on the twenty-first and twenty-second days, respectively. Since untreated rats only rarely prolong delivery beyond term, we had to create postmature rats experimentally. Four experimental groups (in effect, postmature controls without nicotine) were given 25 rat units (R.U.) of a chorionic gonadotropin (Antuitrin-S) daily from the eighteenth day of gestation until one day prior to scheduled cesarean delivery on postcoital days 21, 22, 23, and 24, respectively. The 21 and 22 day specimens were to be compared with untreated controls of equal age; the 23 and 24 day animals, representing hormonally prolonged gestations, could be compared with 1 and 2 day control neonates or with the postdelivered nicotine groups of young. Four other experimental groups received 5.0 mg. nicotine per kilogram subcutaneously twice daily until delivered by cesarean sec-
Table I. Average
daily
maternal
food
consumption
absorption
and
postmature
neonate
tion on days 21, 22, 23, and 24. This was the same dosage that had previously produced the peculiar immature young after a considerable prolongation of gestati0n.l In general, the experimental plan followed that of our previously reported study1 except that daily weight records were kept through the day of cesarean section and special tallies were kept of food consumption for days 14 to 18, 18 to 21, and from the twenty-first day through the day of section. On the day of section, the pregnant mother was weighed, secured, and an abdominal incision was made under lidocaine. One fetus with placenta and membranes was placed under observation in a warming box where its general activity could be observed and its respiratory rate was recorded 5 minutes after delivery and then every 5 minutes for the first hour. The rest of the litter was delivered and notes were made on the amount and viscosity of amniotic fluid, presence or absence of meconium, skin color, skeletal deformities, and general activity level. Weight records were kept on the fetrrs, placentas and membranes, the llterus, and the empty maternal carcass. C!ertain gross observations were made on 5 fetuses randomly selected from each litter
according
to days of gestation
- ---
Grams Treatment I. Controls A. Spontaneous
B. Cesarean C. Cesarean II. Postmature A. Cesarean B. Cesarean C. Cesarean D. Cesarean III.
Day
delivery
section
No section. DeIivered on day 22
section section (An&&l-in-S)* section section section section
22 23 24
Nicotine (10 mg./Kg./day) A. Cesarean section B. Cesarean section C. Cesarean section D. Cesarean section “Dose
of
of 25 R.U.
from
day
21 223’ 24 18.
1111
consumed
o-7 Days
7-14 Days
14-18 Days
18-21 Days
22 Day
15.9 13.9 17.3
18.6 18.0 18.3
20.4 20.8 22.7
24.7 20.1 22.7
18.2
15.3 17.2 16.7 17.1
19.9 19.7 18.6 20.0
21.2 22.9 22.3 23.1
22.5 22.9 23.3 25.3
10.4 10.2 9.3 10.8
12.7 14.2 14.5 14.9
14.7 15.4 15.8 17.2
14.4 17.0 16.6 15.7
.--21-23 Days
__ 21-24 Days
17.9
13.9 15.9 14.8
14.9 12.6 17.1
1112
Becker, King, and Little Am.
whenever the litter size was greater than 5. Otherwise, all the young in the litter were studied. Individual birth weight and sex were recorded on these. Crown-rump length, transverse head diameter, length of claws and vibrissae, and zones of primary ossification in the radius and ulna were measured. In the case of control neonates delivered spontaneously, birth weight and number per litter were recorded. One third of this population was killed at birth; the remaining two thirds were killed on the first and second postnatal days of life, respectively, so that similar measurementscould be made. These last could be compared with measurements on experimental postmature fetuses removed from the uterus on the twenty-third and twenty-fourth postcoital days. Results
The mean daily food consumption for the various groups is shown in Table I. The third trimester is shown broken into two
August 15, 1968 J. Obst. & Gym-.
periods, days 14 to 18 and 18 to 21, because treatment with chorionic gonadotropin (Antuitrin-S) had begun on the eighteenth day for the control postmature group. This treatment appeared to have little effect upon food consumption, for these animals ate about as much as untreated controls, if not slightly more, through the twenty-first postcoital day. On the other hand, nicotine treatment seriously curtailed caloric intake from the start, though there were slight gains in amounts eaten daily asgestation progressed.All groups ate considerably less on the twenty-second day, the normal date for a term delivery. This decrement was most severe in the Antuitrin-S group, e.g., note the drop of 9 grams and compare it with the 4.6 gram drop for the untreated controls and the 2.1 gram drop in the nicotine group. Indeed, the longer mothers went beyond term, the more severe the decrement in daily food intake among the Antuitrin-S mothers. The effect of this reduction in food con-
Table II. Maternal weight gain according to days of gestation consumed 7-14 14-18 18-21 22 21-23 Days Days Days Day Days Grams
Treatment
Day of section
o-7 Days
21-24 Days
I. Controls
A. Spontaneous delivery
None
19.0 k7.6
Cesareansection
21
15.8 k4.1
C. Cesareansection
22
20.2 22.0
B.
31.4 24.9 26.2 23.0 22.8 26.6
31.2 25.9
36.2 23.0 27.2
28.2 A6.4 42.2 23.8 44.2 +8.8 35.6 r4.4
35.8 r4.5 40.0 27.8
51.2 26.9
7.0 23.0
35.4 22.6 41.6 k2.2
6.2 24.4
II. Postmature (Antthin-S) A.
Cesarean
B. Cesarean C. D.
Cesarean Cesarean
section section
21
15.8
22
22.9 25.4 23.4
23
18.8
section
24
k2.6 22.4 23.6
section
21
section
21.8 18.4 +12.1
27.0 27.5
49.0 26.3 41.6
t4.8 35.6 f2.6 39.8 k5.9
4.0 22.7 12.0 t3.2 (-5.2) (21.6)
111. Nicotine A.
Cesarean
B. Cesarean C. D.
Cesarean Cesarean
section section section
22 23 24
1.4
28.3 (-3.8) (f3.8) 2.0 26.4 (-2.2)
(r4.3j
15.4
12.6
14.2
k4.6 18.8 23.3
k3.6 20.2
+6.0 21.2
14.9
12.8
k2.0
10.4
+5.9 13.2
24.6
k2.5
16.0
13.8
10.4 k1.6 8.0
22.7
22.4
26.5
9.0 k1.8
20.6 96.3
Volume Number
101 8
Nicotine
sumed upon maternal weight gain is seen in Table II and Fig. 1. While the postmature group of mothers gained at about the same rate as control mothers until term, they always gained considerably more than nicotinetreated mothers. But, as they carried beyond term, their daily weight increments decreased progressively while mothers in the nicotine group continued to exhibit daily weight increments. Table III shows how the weight gains during the different periods of gestation were distributed between maternal and fetal compartments. The differences between initial weight and empty maternal carcass weight were always less for the nicotine group than ior control and postmature ( Antuitrin-S) groups. These differences held for the weights of the empty uterus, uterine content, and, as a result, for fetal weights. There was no difference in litter size between control and the Antuitrin-S groups. However, the number of fetuses per litter was significantly reduced in mothers treated with nicotine. The average birth weights are given in Table IV where it can be seen that they were significantly lower for the nicotine groups. The
Accumulated 140 -
Maternal Weight Gain in Rats During Prolonged Gestaiion
130-
-AlI O--4 ~..-a o-IO-
#’ I 7
Fig. 1. Maternal longed gestations.
I 14
Ant.!5 All Nicotine All Controls
I 1 t , i I 1 18 19 20 21 22 23 24 Days of Gestation
weight
gain
in
rats
with
pro-
absorption
and
postmature
neonate
1113
weight gain in Antuitrin-S postmatures continued in utero as term was passed but after the twenty-second day it was less than that of 1 and 2 day neonates delivered spontaneously by untreated control mothers. Young in the nicotine groups always had respiratory rates during the first hour that were considerably lower than those of control young. However, the older the gestational age at time of cesarean delivery, the higher the rate (Table V) . Young of nicotinetreated mothers were delivered at 23 and 24 days were always pink and healthy in appearance, though they seemed underdeveloped for their age. The postmaturc (4ntuitrin-S) young had respiratory rates equal to control rates at 21 and 22 days, but at 23 and 24 days these neonates were rya.notic and in respiratory distress with respiratory rates below those of even nicotine-treated young of the same age. The incidence of stillbirths was 14 per cent in the Antuitrin-S group in contrast to 3 per cent in the nicotine-treated group. No data are on hand for neonatal mortality beyond the first hour of life, but it was greatest at this time for the very small immature young of the 21 day deliveries in the nicotine group. It was also high for Antuitrin-S postmature young in respiratory distress. From the protocol notes we learned that placentae in control and Antuitrin-S groups at 21 days appeared normal, firmly attached to the uterine wall, and could not be stripped free easily. Amniotic fluid was variable in amount but usually scanty and somewhat viscous. Findings were similar for the 21 day nicotine group except that fluid was always abundant and watery. There was little change on the twenty-second day except that amniotic fluid viscosity increased in both control groups but not in the nicotine rats. In the 23 and 24 day Antuitrin-S specimens, amniotic fluid was extremely scanty and stringy; placentas exhibited yellowish, necrotic infarcts and detached readily from uterine walls; many fetuses were plastered with meconium; and uteri were stretched and hemorrhagic. In contrast, placentas in nicotine groups at 23 and 24 days were firmly adherent and nor-
1114
Becker, King, and Little
Table III.
Distribution gestational
at different
August 15, l!%lJ Am. J. Obst. & Gynec.
of mean maternal weight ages (weights in grams) * (
Treatment
Day
of section
/
in pregnant
rats being
( W!fht uterine contents
1 Azge young per litter
Final term weight
1 Weight
Initial weight
carcass
Weight of uterus
247.0 271.6
360.4 402.4
287.2 305.4
4.7 4.6
65.2 85.7
52.4 72.8
11 12
of
/
delivered
/ Litter weight
Control A B Postmature A B C D
21 22 23 24
239.4 272.6 257.0 248.0
369.0 409.0 382.0 369.6
293.0 312.2 281.9 275.0
4.2 5.1 4.7 5.1
63.7 90.1 88.3 82.8
51.7 74.6 71.4 67.3
10 12 11 10
A B c D
21 22 23 24
244.2 262.4 249.4 249.6
287.8 318.6 296.4 305.8
257.9 262.3 254.6 260.4
2.9 3.9 3.1 3.1
22.7 47.2 31.0 39.6
18.8 39.1 24.5 31.6
5 8 6 6
Nicotine
*Standard
errors
were small
Table IV. Weights
for all weight
of cesarean
values,
hence
means
section-delivered
young
Weight Treatment
Day
Controls Postmature
(Antuitrin-S)
)
*Weights
of nursing
young
Table V. Average
on first
respirations the first hour of life
21 22 23 24
29.4 56.9
in grams Day
related
22
(
to gestation
in days
Eq--23
/
Day
24
5.03+0.10
5.9OkO.13
6.95t0.12*
7.99ko.11*
5.29tO.08
6.13+0.10
6.3220.10
6.9M.13
3.OlkO.26
Nicotine
during
21
are reliable.
and
second
4.75+0.11 postnatal
day.
per minute
27.5 63.3 29.6 18.4
5.3 34.0 35.6 46.7
mal; amniotic fluid was still abundant, clear, and watery. Fig. 2 presents data on certain aspects of developmental progress. Except, perhaps, for claw growth, the postmature group (AS, in the figure) continued to develop within the uterus much like untreated fetuses (C) before term, and like 1 or 2 day control neonates. In some of the measurements, An-
All other
4.5820.46 weights
are at cesarean
4.7OkO.38 section
delivery.
tuitrin-S postmatures exceeded the values obtained for the 1 and 2 day control neonate; in others, they fell short. These differences were not significant but, if the postterm Antuitrin-S specimens were compared with term young, on the average they weighed heavier, were longer, had wider heads, longer claws and vibrissae, and ossification of the long bones had progressed further than. in the controls. Postterm cesarean section-delivered young of nicotine-treated mothers in no way resembled I- and 2-day-old rats. Neither did they often resemble term control young. On the average, they weighed less, had narrower heads, shorter claws or no nails at all, and vibrissal length and the ossification of long bones lagged behind control values. Fig. 3 contrasts the control term fetus (C22) with postmature Antuitrin-S fetuses (A-
Volume Number
Nicotine
101 8
absorption
and
postmature
1
[ F.Clow Length
-+T+ni-d
Day of Sectioning
Fig. 2. Maturational prolonged
by treatment
characteristics with nicotine
.35 / 21
of fetal rats with (N) or chorionic
23, A-24) and with a series of nicotine specimens designated by the letter (N) . The postmature Antuitrin-S specimens often exhibited clubbed hind feet with inversion at the ankle joint and humped backs. Comment All animals randomly scheduled for cesarearl section on the twenty-first through the twenty-third day were still carrying their young. This was not always true of those assigned for a 24 day delivery, some mothers having already delivered before we could get to them. Thus, the 24 day group formed a select category in the sense that not all animals assigned to it had an equal chance of being included in it. Would those animals comprising it show any physical differences peculiar to it and not seen with equal frequency in other age groups? The early deliveries which failed to carry to 24 days occurred in the nicotine-treated group only. Three were delivered late on the twenty-third
I 22
Related
neonate
1115
I
to
)
“‘--.._............._.... .N I 23 24
Day of Sectioning
normal gestations (CI gonadotropin (AS).
and
of gestations
day. Their average birth weight was 5.29 grams, slightly heavier than nicotine cesarean section deliveries on the twenty-third and twenty-fourth days. Otherwise there were no noticeable differences. Two delivering early on the twenty-fourth day weighed exactly the same as 24 day nicotine cesarean-delivered young (average of 4.70 grams). We have noted certain anomalies in 24 day AntuitrinS fetuses (clubbed feet and humped backs). These, undoubtedly, can be attributed to crowding in utero as growth continued beyond term. Otherwise, if the 24 day groups were select ones, the bias was minimal. The early spontaneous deliveries also demonstrated that the operative procedure of cesarean section was not effecting results significantly. It was possibIe that chorionic gonadotropin ( Antuitrin-S) might effect fetal growth directly so as to account for larger fetuses than normal. If it were given earlier in gestation than the eighteenth day, then
1116
Becker, King, and Little
August 15, I!368 Am. J. Obst. & Gynec.
__ Fig. 3. Comparison chorionic gonadotropin
of control (A)
specimens (C)
at the gestational
young delivered at term should be larger than normal. When such a check experiment was performed, Antuitrin-S young at term did not differ from untreated control young removed at 22 days. Fetal size, in and of itself, could be a factor which determined expulsion time. ReynoldP has pointed out that the myometrium adapts itself to the size of the conceptus, stretching
___
with fetal rats treated with nicotine
ages designated
(N)
or
by numerals.
and exerting greater powers of contraction the more it is distended. However well this law might hold for normal births, where large fetuses might deliver earlier than smaller ones, it did not apply to our postmatures when gestation was prolonged by either chorionic gonadotropin or nicotine. Nor did litter size appear to control the time of gestation nor the maturational state
Volume Number
101 8
of the individual. An old concept of mammalian reproductive physiology has been the tenet that large litters are associated with short gestations; small litters, with longer gestational times. 27 Our Ant&-in-S litters ran contrary to the first premise, even if the nicotine-treated litters fitted the second. Yet, Reynolds x has embroidered upon this concept by stating that “the larger the litter, the lrss mature the individuals at birth; and, conversely, the smaller the number, the larger and more mature the young born.” Our young certainly pointed up the reverse. Large Antuitrin-S-treated litters contained the larger, more mature fetuses; small nicotine litters contained the smaller, less mature young. While the food consumption of AntuitrinS mothers was virtually identical with that of control mothers throughout the normal stages of pregnancy, they ate less and gained less weight as they progressed beyond term (Fig. 1) . Such a weight loss late in pregnancy has been described among women who delay delivery abnormally.* Both caloric intake and weight gains were remarkably reduced in our nicotine-treated pregnant rats. No such noticeable reduction has been reported among women who smoke heavily during pregnancy. Our experiments certainly confirm the human findings that nicotine absorption throughout pregnancy can result in the production of an underweight infant, but not necessarily as a result of premature birth. While our nicotine-exposed rats present immature features, their birth has been abnormally prolo’nged. They are more representative of the peculiar postmature neonates recently described by Naeye. I3 Whether, as Naeye suggests, there is a problem of malnutrition involved is as yet unknown. What we observe in‘ our rat neonates might be more directly related to the secondary effects of maternal inanition than to a direct action of nicotine. It was difficult to know how to class these young of nicotine-treated mothers. Morphologically they looked more like premature young and contrasted markedly with the more mature and overweight young of Antuitrin-S mothers even though the births had
Nicotine
absorption
and
postmature
neonate
1117
been equally prolonged in both cases. In our various tables, data have been presented in terms of group averages. This is true for Fig. 2 also. Still, in all groups, at any gestational age, there was some disparity in individual sizes and weights. Some few controls were as large as the smaller postmature Antuitrin-S neonates; some of the larger nicotine specimens were as large as the smallest controls. Had we no foreknowledge of gestational ages it would have been difficult to determine, at times, which were term or pnstmature or which might be premature neonates. Were there any features that definitely pointed to postmaturity other than xceight differences ? All the young at each gestational age could be subdivided into weight classes separated by intervals of half a gram. If, now, all the measurements given in Fig. 2 should turn out to be proportionate to weight, then there would be no real way to discriminate between postmature, immature, or term neon&es if gestational age were unknown. When the foregoing was done, it was noted that crownrump length increased proportionately with body weight. Neither weight nor crownrump length alone could serve as a satisfactory index of postmaturity. It could not be said, for example, as has been said for the human infant, that the postterm infant was longer for its body weight than the term neonate. Other parameters, however, did tlurn out to be useful indices of postmaturity. e.g., Antuitrin-S postmature neonates always had head diameters proportionately wider than any other neonates in the same weight class. The extent of ossification in the radius and ulna was always proportionately greater, as were the vibrissa and claw lengths. Of all these features, only the extent of ossification in the long bones and vibrissa length held as postmaturity indices for the largest of the postterm nicotine-exposed young. In all other respects, all of these measurements for the majority of younp of’ nicotine-treated mothers were less than in other nennates of equal weight. Always transverse head diameters were disproportionatelv nar-
1118
Becker,
King,
and
August
Little
rower and claw lengths shorter; or, in some instances, there were no claws present at all. In general, then, for the bulk of nicotinetreated rats the physical signs were those of immaturity rather than postmaturity. There were other differences between these two groups of postmature young, as Table V on initial respiratory efforts indicates. The 23 and 24 day nicotine-treated young were pink and healthy in appearence, not cyanotic like their Antuitrin-S counterparts. Nor were they subject to respiratory distress and early neonatal death like the Antuitrin-S specimens, In part, this may have been due to their very immaturity. Placentas were normal and functional, not necrotic and infarcted. Uteri were not hemorrhagic. Amniotic fluid was clear and abundant, not scanty and meconium-stained as in Antuitrin-S births. All of these signs, except the prolonged gestation, suggested even more strongIy that these were fundamentally immature young with some slowing of the developmental processes. NesbitF has summarized the incidence of fetal mortality, i.e., the number of stillbirths as distinct from neonatal deaths for human postmature births. His figures, taken from several sources, range from 2 to 9 per cent as contrasted to 1 to 4 per cent for term deliveries. Our stillbirth rates were 14 per cent and 3 per cent for Antuitrin-S and nicotinetreated litters, respectively. It would probably have been higher for the Antuitrin-S group had we not kept the dosage to 25 R.U. purposely to assure reasonable survival at 24 days. COCO’~ quotes rates of 25.6 per cent in postmature rats receiving 40 to 100 R.U. of Antuitrin-S and 51.6 per cent when 200
15, 1968
Am. J. Obst. & Gynec.
R.U. was given. The rate was 84.6 per cent for rabbits given the high dose. Roux, Romney and Dinnersteinzg gave a figure of 60 per cent fetal mortality when 40 R.U. of Antuitrin-S was used to produce postmature rabbits. We have never seen a reduction in litter size before among pregnant rats given nicotine at the concentrations employed in this study. We had, only with much higher dosage levels, e.g., 15 to 20 mg. per kilogram. Others,30-32 however, have reported smaller litters than normal when pregnant rats or rabbits were exposed chronically to nicotine, but their animals had been exposed for several generations or had been treated in smoke -filled chambers where dosages were unknown and uncontrolled. If the relationship between developmental status and the duration of pregnancy varies because endogenous or exogenous agents can act to prolong or retard maturation, then in attempting to assess prematurity or postmaturity it may be necessary to look for other signs of maturity or immaturity, much as we have done here. Weight and time alone do not seem to be very satisfactory criteria. A foreshortened pregnancy may not be the only route to prematurity. Consider Naeye’s infants13 or our own nicotine-treated specimens in which morphologic characteristics were definitely immature, including low birth weights, yet the termination of pregnancy was abnormally prolonged. On the other hand, an equally prolonged gestation can result in infants exhibiting overdevelopment and all the cIassica1 signs of the much debated postmaturity syndrome.
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11. McKiddie, J, M.: J. Obst. & Gynaec. Brit. Emp. 56: 386, 1949. 1”. Adair, F. L., editor: Obstetrics and Gynaecology, London, 1940, Hirschfeld Bras., Ltd., vol. I, p. 906. 13. Naeye, R. L.: Arch. Path. 84: 37, 1967. 14. Walker, J., and Turnbull, E. P. N.: Lancet 2: 312, 1953. 15. Clifford, S. H.: J. Pediat. 44: 1, 1954. 16. Barcroft, J.: J. Physiol. 83: 193, 1934-35. L. G.: Am. J. Pub. Health 45: 17. McKinney, 633, 1955. 18. Rooth, G., and Sjostedt, S.: Acta obst. et gynec. scandinav. 36: 374, 1957. 19. Minkowski, A.: Ann. paediat. Fenniae 3: Fascicle 2, 1957. “0. Bancroft-Livingston, G., and Neill, D. W.: J. Obst. & Gynaec. Brit. Emp. 64: 498, 1957. 21. Graham, B. D.: J. Pediat. 60: 882, 1962. 22. Brady, J. P., James, L. S., and Barker, M. P.: AM. J. OBST. & GYNEC. 84: 1, 1962.
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neonate
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23. Flexner, L. B.: AM. J. OBST. & GYNEC. 55: 469, 1948. 24. Wong, H. N., and Hellman L. M.: Bull. Johns Hopkins Hosp. 73: 31, 1943. 25. Masters, M., and Clayton S. G.: J. Obst. & Gynaec. Brit. Emp. 47: 437, 1940. 26. Reynolds, S. M. R.: Medicine 53: 441, 1958. 27. Asdell, S. A.: Patterns of Mammalian Reproduction, Ithica, 1943, Comstock Publishing Company. 28. Coca, R. M.: Am. J. Physio!. 137: 143, 1942. 29. Roux, J. F., Romney, S. L., and Dinnerstein, A.: AM. J. OBST. & GYNEC. 90: 546, 1964. 30. Theines, C. H., and Behrend A.: J. Pharmacol. & Exper. Therap. 48: 317, 1933. 31. Essenberg, J. M., Schwind, J. V., and Patias, A. R.: J. Lab. & Clin. Med. 25: 708, 1940. 32. Schoeneck, F. J.: New York, J, Med. 41: 1945, 1941.