Book - Sex and internal secretions (1961) 16

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Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.
Section A Biologic Basis of Sex Cytologic and Genetic Basis of Sex | Role of Hormones in the Differentiation of Sex
Section B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduction Morphology of the Hypophysis Related to Its Function | Physiology of the Anterior Hypophysis in Relation to Reproduction
The Mammalian Testis | The Accessory Reproductive Glands of Mammals | The Mammalian Ovary | The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms | Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates | The Mammary Gland and Lactation | Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones | Nutritional Effects on Endocrine Secretions
Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy Biology of Spermatozoa | Biology of Eggs and Implantation | Histochemistry and Electron Microscopy of the Placenta | Gestation
Section E Physiology of Reproduction in Submammalian Vertebrates Endocrinology of Reproduction in Cold-blooded Vertebrates | Endocrinology of Reproduction in Birds
Section F Hormonal Regulation of Reproductive Behavior The Hormones and Mating Behavior | Gonadal Hormones and Social Behavior in Infrahuman Vertebrates | Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals | Sex Hormones and Other Variables in Human Eroticism | The Ontogenesis of Sexual Behavior in Man | Cultural Determinants of Sexual Behavior
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Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy

Gestation

M. X. Zarrow, Ph.D. Professor Of Zoology, Purdue University, Lafayette, Indiana


I. Introduction

Reproduction in the animal kingdom is accomiilished by a wide variety of methods, from simple budding and binary fission in the invertebrates to gestation in the mammal and the development of a new organ, the placenta. The development of viviparity, which covers millions of years of evolution, brought with it many new problems, and with each problem new factors came into play so that reproduction in the mammal is a highly co-ordinated series of events — a co-ordination that is both temporal and spatial, that requires certain events to occur in a proper sequential arrangement, and, above all, is dependent on the endocrine system.

It is obvious that the maintance of gestation in the mammal is a complex phenomenon. It involves directly or indirectly a major portion of the endocrine system with concomitant changes in the general metabolic state of the organism and in many of the enzymes present in the blood and the tissues. Finally, a new endocrine organ, the placenta, comes into being also to play its specific role in gestation.


II. Length of Gestation

The duration of gestation is highly variable and depends primarily on the species involved. In general, the longer the gestation, the more self-sufficient and mature are the young at the time of birth. It is obvious, however, that this is not true under all conditions. The young of the guinea pig are highly advanced at birth, although the length of gestation is approximately 69 days, whereas in the iH-iniate, with a gestation period of 6 to 9 months, depending on the species, the young are helpless at birth. A partial summary of gestation length and tlie litter size of a representative but not inclusive list of mammals is presented in Table 16.1.

The length of gestation appears to be rather constant for each species or at least within a strain. Even where the phenomenon of delayed implantation is a natural event the length of pregnancy remains constant, although the quiescent period may vary. It is, however, possible for delayed implantation to occur in a species where this does not ordinarily appear, which could lead to a marked increase in the duration of gestation. Thus, an increase of 1 to 7 days has been reported in the rat or mouse if mated while lactating (Pincus, 1936). Recently, Bruce and East (1956) examined the effect of concurrent lactation on the number and viability of the young and the length of pregnancy in the mouse. They observed a wide variation in the delay of implantation for every size of litter studied, but, in general, the delay tended to be longer for the larger suckling litters.

Smith, Albert and Wilson (1951) reported a 310-day pregnancy period in a human female. Gestation was confirmed early by pregnancy tests and a normal child with respect to body weight was born at 30 days after the expected parturition. Such phenomena seem to be rare in primates and no explanation is possible at the present time.

Although the lengths of the gestation periods are quite constant for a given strain, the length of gestation is inversely related to the litter size. This has been demonstrated in both a genetically pure strain and a heterogeneous strain of quinea pigs (Goy, Hoar and Young, 1957). An average gestation length of 69.9 days was obtained in the pure strain of guinea pigs with a litter size of 1, as compared with a gestation length of 65.3 days for a litter size of 6.

A sex difference has also been postulated in length of gestation. Although the difference is very small, e.g., only a fraction ■of a day in man, the difference is significant. Recently, McKeown and MacMahon (1956) concluded that pregnancy is longer in the cow, horse, and possibly the sheep and camel when the offspring are male, and longer in man and possibly the guinea pig when the offspring are female.


III. Normal Reproductive Potential

The reproductive potential in the primate is limited to the period from the menarche to the menopause. Hence, it is much shorter than the total life span of the female. Fertility studies as a function of age have l)ecn rather sparse for different species although it is generally agreed that fertility declines with age. A reproductive period considerably shorter than the life span of the animal has also been reported in certain strains of mice (Thung, Boot and Miihlbock, 1956) and in the rat (Ingram, Mandl and Zuckerman, 1958).

Although Slonaker (1928) showed that the rat may remain fertile for 22 months, it is known that the average size of successive litters in both rats and mice first rises to a maximum and then falls (King, 1924; Ingram, Mandl and Zuckerman, 1958). The latter have shown both a decrease in the number of fertile female rats with each successive litter and hence with age (Fig. 16.1) and a decline in the number of young with each successive litter (Fig. 16.2).

These results indicate that the reproductive potential of both the colony of rats and of the individual rat declines with age. Many factors may obviously be at work here, such as nutrition, size, and part played by the male. Ingram, Mandl and Zuckerman ( 1958 ) feel that none of the above factors is responsible for the decline in litter size and offer the following four possibilities: (1) the number of follicles which mature and ovulate declines with age, (2) the capacity of the ovum to be fertilized declines with age, (3) the number of fertilized ova that develop to term declines with age, and (4) the total number of available oocytes declines with age.

Evidence from the pig (Perry, 1954) and rat indicates that factors 2 and 3 are certainly involved. Inasmuch as the number of corpora lutea rises with age in the pig, the decline in size of litters can be


TABLE 16.1 Length uf gestation and litter size in various species of mammals



Species







No. of Young


Length of Gestation


Reference*^


Common name


Scientific name




Armadillo


Das y pus novemcinctus, L.


4


150 days


(1), (2)


Baboon


Papio hamadrys, L.


1


183 days


(1)


Baboon, chacma


Papio porcarius, B.


1


7 months


(1), (2)


Bat, common European


Vespertilio miirinus, L.


1


50 days


(1)


Bat, common pipistrello


Pipistrellus pipistrellus, S.


1


44 days


(3)


Bear, black


Euarctos americanus, P.


1-4


208 days


(1), (2>


Bush baby


Galago senegalensis, G.


1-2


4 months


(1)


Camel, batrachian


Cameliis bactrianus, L.


1


370-440 da.ys


(1)


Capuchin


Cebus apella, L.



160-170 davs


(1)


Cat


Felis catus, L.


3.8 (1-8)


56-65 days^


(4)


Cat, domestic


Felis catus, L.


4


63 days


(1)


Chimpanzee


Pan satyrus, L.


1


236.5 ± 13.3 davs


(1)


Chimpanzee


Pan satyrus, L.


1-2


226.8 ± 13.3" davs


(17)


Chinchilla


Chinchilla laniger, B.


1-4


105-111 days


(1), (2>


Chipmunk


Tanarios strialus, L.


3-5


31 davs


(1)


Cow


Bos taurus, L.


1-2


277-290 days


(1)


Cow (Jersey)


Bos taurus, L.


1-2


282.7 ± 5.4" days


(5)


Cow (Holstein-Friesian)


Bos taurus, L.


1-2


278-280 days-^


(6)


Coyote


Canis latrans, S.


5.7


60-(J5 days


(1)


Deer, Virginia


Odocoileus Virginian us, B.


2


7 months


(1)


Dog


Canis familiaris, L.


Multiple


58-63 days


(1)


Dog


Canis familiaris, L.


Multiple


61 davs


(4)


Echidna


Echidna acn.leata


1


16-28 days


(1)


Elephant



1


20 months


(8)


Elephant, Indian


Elephas maxim us, L.


1


607-641 days


(1)


Ferret


Mustela furo, L.


5-13


42 days


(1)


Ferret


Mustela furo, L.


5-13


42 days


(4)


Fox, red


Vulpes fulfa, D.


1-8


52 days


(1)


Goat


Capra hircxis, L.


1-2


21 weeks


(7)


Goat, domestic


Capra hircus, L.


1-3


146-151 days


(1)


Gopher, pocket


Geomys bursarius, S.


1-6



(1)


Ground squirrel, thirteen

Citellus tridecimlineatus, N.


5-13


28 days


(1>


lined






Guinea pig


Cavia porcellus, L.


1-6


67-68 davs


(1)


Guinea pig


Cavia porcellus, L.



65.3-70.5 days


(7)


Hamster, golden


Cricetus auratus, H.



16-19 days


(1)


Hamster, golden


Cricetus auratus, H.


5


10 days


(9)


Hare, snowshow


Lepus americanus, E.


1-7


38 days


(1)


Hedgehog, European


Erinoceus europaeus, L.


5


34-49 davs


(1)


Hippopotamus


Hippopotamus amphibius, L.


1


237 ± 12 days


(1)


Horse


Equus cabaUus, L.


1


330 davs


(1)


Hyena, spotted


Crocuta crocuta, E.


1-2


110 days


(1)


Kangaroo rat


Bettongia cuniculus, 0.


1


6 weeks


(1)


Lemur


Lemur macaco, L.


1-2


146 days


(1)


Lion


Felis leo, L.


2-6


105-113 davs


(1)


Macaque


Macaca mulatta, Z.


1


163.7 ± 8 days


(1), (10)


Macaque



1


24 weeks


(8)


Man


Homo sapiens, L.


1


280 ± 9.2 days


(1)


Marmoset


Hopale jacchus, L.


1-3


140-50 davs


(1)


Marten, pine


Martes americana, T.


3-5


220-265 days


(1^


Mink


Mustela vison, S.


4-10


39-76 days


(1)


Mink


Mustela vison


4


51 days'* (40-75)


(11), (12)


Mole, common American


Scalupus aquaticus, L.


2-5


6 weeks


(1)


Mouse


Mus musculus, L.



19-20 days


(4)


Mouse, field


Microtus pennsylvanicus , 0.


6-8



(1)


Mouse, house


Mus musculus, L.


4-5-7.5


19 days


(1)


Mouse, wild


Peromyscus maniculatus



23 davs


(13)


Mouse, wood


Peromyscus leucopus, L.


3-7


23 days


(1)


960


GESTATION


961


TABLE m.l— Continued



species







No. of Young


Length of Gestation


Reference"


Common name


Scientific name


Opossum, Australicaii


Trichosunis vulpecula, K.


1


16 days



Opossum, Virginia


Didclphis rir(/itiiana, K.


8-12


12.5-13 days


(1), (2), (8)


Otter


Lutra canadensis, S.


1-4


60 days


Pig, domestic


Sus scrofa, L.


4-12


112-115 days



Pig, wild


Sus cristatus, W.


4-6


4 months



Porcupine


ErclJiizan (lorsoliun, L.


1


16 weeks



Puma


Frlis ,„nr„ln,-, T.


1-4


90-93 days



Rabbit, domestic


Crycutaluyiis c/uiicidus. L.


Multiple


30-32 days



Rabbit, domestic


Crycotalagus cnnicuhis, L.


Multiple


31 (28-36 davs)



Racoon


Procyon lotor, L.


1-0


63 days



Rat


Rattus rattus, L.


(J. 1-9.2'

22 days



Rat


Rattus rattus, L.



21-23 days



Reindeer


Rangifer tarandus, L.


1-2


7-8 months



Rhinoceros



1


18 months



Rhinoceros, black


Rhinocerus bicornuis, L.


1


530-550 days



Seal, northern fur


CaUorhinns ursinus, L.


1


Almost 1 year



Sheep, bighorn


Ovis canadensis, L.


1


180 davs



Sheep, domestic


Ovis aries, L.


1-2


144-152 davs-^



Shrew, common


Sorex aranus, L.


6.45


13-19 davs



Shrew, short-tailed


Blarina brevicauda, S.


3-7


17-20 days



Skunk


Mephitis mephitis, S.


4-7


62 days



Squirrel, red


Sciurus hudsonicus, E.


3-()


40 days



Stoat


Mustela musleta



6 weeks'*


(14)


Tasmanian devil


Sarcophilus ursinus, K.


4


31 days



Vole


Microtus agrestis



20-22 days


(Ifi)


Weasel


Mustela nivalis


6-7


50 weeks'* (includes) lactation)


(15)


Whale, sperm


Fhyseter catadon, L.


1


1 year


(1)


Wolf, timber


Canis tycoon, S.


1-12


63 davs


(1)


Woodchuck


Marmot a nionox, L.


4.07


28 days


(1)


Zebu


Bos indicus, L.



285 davs


(1)


'- References. (1) Asdell, 1946. (2) Kenneth, 1947. (3) Deanesly and Warwick, 1939. (4) Farris, 1950. (5) Rollins, Laben and Mead, 1956. (6) Norton, 1956. (7) Goy, Hoar and Young, 1957. (8) Arey, 1946. (9) Selle, 1945. (10) Hartman, 1932. (11) Pearson and Enders, 1944. (12) Enders, 1952. (13) Svihia, 1932. (14) Deanesly, 1943. (15) Deanesly, 1944. (16) Chitty, 1957. (17) Peacock and Rogers, 1959.

  • " Standard deviation.

" Depends on the strain.

'* Excluding the quiescent period.



3l no. of litter


-1^


Fig. 16.1. Decline in litter size with birth of successive litters. • mean of 35 litters; EI mean of 14 litters; A mean of 4 litters. (From D. L. Ingram, A. M. Mandl and S. Zuckcinian. J. Endocrinol., 17, 280, 1958.)



1 2 3 4 5 6 7 8 9 10 11

Serial no. of litter

Fig. 16.2. Decline in number of fertile female rats with birth of successive litters. (From D. L, Ingram, A. M. Mandl and S. Zuckerman, J. Endocrinol., 17, 280, 1958.)


962


SPERM, OVA, AND PREGNANCY


attributed only to failure of fertilization or to fetal death. A similar increased incidence of embryonic death or failure of fertilization has been described in the aged rat, although it should be noted that the number of corpora lutea present at parturition in the rat is not an index of the number of ova released before conception because the corpora lutea in old age may persist for longer periods. Finally, a marked reduction in the number of oocytes with age has been shown in the rat, a drop from approximately 20,000 oocytes at age day 1 to approximately 2000 oocytes at age 250 to 300 days (Mandl and Zuckerman, 1951). In addition, Ingram (1958) showed that the litter size in rats declined markedly with the reduction in number of oocytes following graded doses of x-ray. This experiment tends to confirm the concept that the decline in fertility with age is due to a decline in the number of oocytes.

IV. Environment

A. CROWDING

The factors concerned in the growth and survival of a population under natural conditions may obviously involve reproduction. Variations in population level have been of great interest to the mammalogist and student of wildlife for many years. Decreased productivity in mammalian populations associated with increased density of the population has been considered a controlling factor in the regulation of wildlife population.

Experimental analysis by Christian and Lemunyan (1958) indicated that a number of factors are involved. These authors exl)osed mice to excessive crowding and noted the number of implantation sites, embryos.


and births. All the females became pregnant but only 3 of the 10 bore litters during the period of crowding or later (Table 16.2). It would seem that the crowded females were unable to maintain normal pregnancies and that the environmental situation interfered with the endocrine balance and resulted in a marked pregnancy wastage. The data reveal that in addition to a postimplantation loss, there was also a prc-implantation loss because the number of implantation scars in the uterus was markedly less in the crowded females. This could be due to a failure of the fertilized egg to implant or to a decrease in the number of available ova. Although direct data are not available, it is of interest to speculate as to whether this effect of crowding is mediated by way of the pituitaryadrenocortical axis.

B. BODY TEMPERATURE AND HYPOXIA

Disturbances in reproduction have been noted in mammals exposed to high temperatures or chronic hypoxia. It has been known for some time that women moving to the tropics show a high rate of abortion (Castellani and Chalmers, 1919). Recently Macfarlane, Pennyamt and Thrifte (1957) reported a 30 per cent reduction in human conception rates in the summer as compared with the winter in Australia. The same authors reported a marked degree of fetal resorption in rats exposed to a temperature of 35°C. This confirmed the previous observations of Sundstroem (1927) in rats and Oegle (1934) in mice where exposure to 31 °C. caused a reduction in litter size.

In a similar manner, disturbances have also been reported in reproduction following exposure to decreased oxygen tension.


TABLE 16.2 Productivity of mice crowded 10 pairs to a cage compared to their 10 control pairs Note that all of the females became pregnant but that the crowded females exhibited a marked intrauterine loss of young, reduction of implantation sites, reduction of litter size, and significant delay until the birth of first litters compared to the controls. Crowding produced a 75 per cent loss in the


number of young born.


(From J. J.


Christian


and C. D. Lemunyan, Endocrinology, 63


, 517, 1958.)



No. of Pairs


No. of Litters Born


Mean No. Days to Litter Birth


Mean No. Progeny per Litter ± S.E.


No. Females with Placental Scars


Mean No. Scars per Female


Crowded females

Isolated females


10 10


3 10


40 ± 1.0 26 ± 1.5


7.67 ± 0.33 9.00 ± 0.75


10 10


6.90 ± 1.37 11.00 ± 0.47


GESTATOON


963


Monge (1942) reported a lack of reproduction in the Spaniards for more than 50 years after residence in certain areas of Bolivia (14,000 feet or more above sea level). Many malformations have also been observed in the progency of mice, rats, and rabbits exposed to low atmosperic pressures. Exposure of mice on the 10th day of pregnancy for 2 hours to a 6 per cent oxygen-94 per cent nitrogen mixture at normal atmospheric pressure gave malformations in the young comparable with those found after exposure to a low atmospheric pressure which was equivalent to the above with respect to the number of oxygen molecules per unit of air (Curley and Ingalls, 1957). Although these malformations involved the ribs and vertebrae, it is conceivable that more extensive malformations could result in death of the fetuses leading to resorption or abortion of the young.

Vidovic (1952, 1956) made a very complete study of the effect of lowered body temperature on gestation in the rat using the technique of Giaja (1940) in which an hypoxic hypothermia is induced by cooling under reduced oxygen tension. The animal is placed in a sealed container which is surrounded by ice for a period of approximately 10 hours. Under these conditions a hypothermia of 3 to 4 hours' duration and body temperature of 14 to 18°C. can be induced. No deleterious effects were noted in the rats cooled on or before the 13th day of pregnancy. However, the induction of hypothermia after the 13th day resulted in a marked increase in the disturbance of gestation. These disturbances consisted of an increased number of resorbed fetuses, an increased ratio between stillborn and live young in that more stillborn occurred, a decreased body weight in the progency, and a delay in the onset of parturition. In addition, a marked increase in sensitivity to hypothermia was noted in the animals as pregnancy progressed. Courrier and Marois (1953) cooled pregnant rats by exposure to a temperature of 0°C. for 2 hours. Thereafter the rats were placed in cold water for 3 to 4 hours and a body temperature of 15.5 to 17°C. was obtained. Exposure to the above treatment on the 7th to the 11th day of preg


nancy had no effect on the fetuses or the pregnancy. Treatment on the 12th to the 18th day of pregnancy led to resorption and abortion of the young. The authors concluded that the degree of deleterious effects following exposure to cold varied with the length of the pregnancy.

Recently, Fernandez-Cano (1958a) exposed pregnant rats for 5 hours on 2 consecutive days to one of the following three experimental procedures: (1) an environmental temperature of 103°F. that led to an increase in body temperature to 104°F.; (2) an environmental temperature of 26°F. that led to a decrease in body temperature to 94°F.; and (3) barometric pressure of 410 mm. Hg. Both temperature changes led to a marked decrease in the number of implantations and, to a lesser extent, to some embryonic degeneration after implantation (Table 16.3). Although some deleterious action was seen before implantation, hyi^oxia was more harmful after implantation. Whereas these results are not in full agreement with Vidovic 's report, it must be remembered that Vidovic used a combination of cold and hypoxia to induce the effects that he observed. Adrenalectomy failed to increase embryonic degenerations in rats treated as above (Fernandez-Cano, 1958b). Inasmuch as adrenocorticotrophic hormone (ACTH) causes degeneration of the embryos in intact pregnant rats and not in adrenalectomized rats (Velardo, 1957 1 , it is apparent that these results are explainable on the basis of an increased release of adrenal corticoids due to the stressor and/or a direct action of the corticoids on the development of the embryo.

V. Maternal Hormone Levels during Gestation

Proof that certain hormones are necessary for a successful pregnancy came from evidence involving ablation of the source of the hormone and replacement therapy. This was followed by quantitative analyses of the concentration of the hormone in the blood and urine throughout gestation. The increasing concentrations of the hormones as pregnancy advances can be used as a second argument for the role of hormones in the development and maintenance of pregnancy (Zarrow, 1957). Changes of this


964


SPERM, OVA, AND PREGNANCY


TABLE 16.3

The effect of increase or decrease of body temperature and hypoxia on the pregnancy of the rat

(From L. Fernandez-Cano, Fertil. & Steril., 9, 45, 1958.)


Group


Control

High body temperature High body temperature High body temperature High body temperature Low body temperature . Low body temperature . Low body temperature . Low body temperature .

Hypoxia

Hypoxia

Hypoxia

Hypoxia


Days of Treatment


1-2 3-4 6-7

10-11 1-2 3-4 6-7

10-11 1-2 3-4 6-7

10-11


Total No. Corpora Lutea


166 98

117 95 89 93 98

100 91

103

108 97 94


Percentage of Degeneration


Before implantation


2.4 52 28

2

2 25 33

3

2.1 21.3 25.9


2.1


After implantation



12

3 14 10

5

4 13 12.1

2.9

3.7 25.7 65.9


Total degener


2.4 64 31 16 12 30 37 16

14.2 24.2 29.6 25.7 68.0


Means of Degeneration for Each Rat


0.2 8.3 4.6 1.9 1.3 3.5 4.5 2.0 1.6 3.1 3.8 3.1 8.0


Standard Error


0.11

1.6

2.6

0.5

1.8

1.0

1.4

0.2

0.5

0.4

0.3

1.0

1.4


Percentage against Control


>0.01 >0.01

>0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01


kind have been observed for such steroids as the estrogens, gestagens, and the 17a-hydroxycorticoids. In addition, certain nonsteroidal hormones such as the gonadotrophins human chorionic gonadotrophin (HCG) and pregnant mare's serum (PMS) and the polypeptide, relaxin, increase during gestation. Some evidence for a possible involvement of thyroxine, prolactin, and oxytocin will be included. The maximal concentration of these hormones in the blood of the female during pregnancy is given in Table 16.4.

A. ESTROGENS

The fact that large amounts of estrogen are excreted in the urine of pregnant women and mares has been known for a long time. Additional data (reviewed by Newton, 1939) indicate that this phenomenon occurs in all species studied, such as the chimpanzee, the macaque, the cow, the pig, and the rat. In general, an increasing amount of estrogen is excreted as pregnancy progresses. The estrogenic material in the urine of the pregnant woman appears mostly in the form of estriol with lesser amounts of estrone and estradiol (Fig. 16.3) . The estriol concentration increases only slightly in the urine of women for the first 100 to 125 days of pregnancy, but thereafter it increases very rapidly until parturition. Newton (1939) discussed the possible


role of estrogen in pregnancy in great detail. He first asked whether the increased urinary concentration of estrogen indicates that this hormone is acting to a lesser degree as pregnancy advances or to a greater degree. He marshaled his facts pro and con and came to the conclusion that there is an increased production of estrogen throughout pregnancy and hence an increased activity of the hormone. In his analysis of the action of estrogen, five possibilities were suggested. (1.) Estrogen is involved in the growth of the uterus in pregnancy. (2) Estrogen is involved in the increased uterine contractility and sensitivity to oxytocin necessary for parturition. (3) Estrogen is concerned with the continued secretion of progesterone by way of the pituitary glancl or acting directly on the corpus luteum. (4) Estrogen synergizes with progesterone. (5) Estrogen stimulates mammary gland growth. A 6th possibility is that estrogen reverses the progesterone block (Csapo, 1956a). Several of these possibilities will be considered later in conjunction with progesterone, the maintenance of pregnancy, and jiarturition.

B. GESTAGENS

The significance of the role of progesterone during pregnancy stemmed from the historic work of Fraenkel who proved the validity of Gustav Born's suggestion that


GESTATION


965


TABLE 16.4 Maximal hormone levels in the blood during pregnancy


Hormone


Species


Type of Assay


Hormone Amt./ml. Plasma


Reference


Estriol


Man


Chem.


0.0914 Mg.


Aitkin and Preedy, 1957



Estriol


Man


Chem.


0.066 Mg •


Loraine, 1957



PJstrone


Man


Chem.


0.0647 Mg.


Aitkin and Preedy, 1957



E.strone


Man


Chem.


0.0305 Mg.


Loraine, 1957



]stnuli()l


Man


Chem.


0.0144 Mg.


Aitkin and Preedy, 1957



K.-^tradiol


Man


Chem.


0.0105 Mg

Loraine, 1957



( lestajfen


Rabbit


Biol.


10 Mg."


Zarrow and Neher, 1955



Cicstugen


Mouse


Biol.


SMg."


Forbes and Hooker, 1957



(Jostagen


Ewe


Biol.


12 Mg."


Neher and Zarrow, 1954



Progesterone ....


Ewe


Chem.


0.0033 Mg.


Short, 1957



Progesterone ....


Ewe (ovarian vein


Chem.


2Mg.


Edgar and Ronaldson, 1958



Progesterone ....


Cow


Chem.


0.0086 Mg.


Short, 1958b



Gestagen


Man


Biol.


2Mg."


Forbes, 1951



Gestagen


Man


Biol.


25Mg-"


Fujii, Hoshino, Aoki and Yao, 1956



Progesterone ....


Man


Chem.


0.239 Mg.


Oertel, Weiss and Eik-Nes, 1959



Prog(^stei'one ....


Man


Chem.


0.142 Mg.


Zander and Simmer, 1954



Progesterone . . . .


Sow


Chem.


0.0034 Mg.


Short, 1957



Progesterone . . . .


Goat


Chem.


0.0071 Mg.


Short, 1957



Rela.\in


Guinea pig


Biol.


0.5G.P.U.


Zarrow, 1947a



Rela.xin


Rabbit


Biol.


10 G.P.U."


Marder and Money, 1944



Relu.xin


Man


Biol.


2 G.P.U."


Zarrow, Holmstrom and Salhanick,


1955


Relaxin


Sow


Biol.


2G.P.U.


Hisaw and Zarrow, 1951



Rela.xin


Cow


Biol.


4 G.P.U.


Wada and Yuhara, 1955



Hydrocortisone. .


Man


Chem.


0.22 Mg.


Gemzell, 1953



Thyroxine


Man


Chem.


0.83 Mg.^


Peters, Man and Heinemann, 1948



STH


Rat Man Man Horse


Biol. Biol. Biol. Biol.


3.5-7-^ 120 I.U. 70 I.U. 50 I.U.


Conlopoulos and Simjjson, 1957 Haskiiis and Slierman. 1952 Wilson, Allien and Randall, 1949 Cole and Saunders, 1935



HOG



HCG



PMS





" Expressed as equivalents of progesterone.

Guinea pig units.

"■ Protein-bound iodine.

'Vg. equivalent of a purified bovine growth-promoting substance.


the corpus luteum is necessary for the maintenance of pregnancy. Fraenkel demonstrated at the turn of the century that the corpus luteum of the rabbit is essential for the maintenance of pregnancy in the rabbit (Fraenkel and Cohn, 1901; Fraenkel, 1903; Fraenkel, 1910). These observations were confirmed by Hammond and Marshall (1925.) who found that castration before the 20th day of pregnancy led to the termination of pregnancy in 24 hours. Castration later in pregnancy resulted in abortion approximately 2 days after the operation. In 1928, Corner showed that an extract of the corpus luteum could induce a progestational endometrium in the castrated rabbit. This was soon followed by the demonstration that this extract could induce implantation of the fertilized egg in the rabbit and maintain pregnancy in the castrated animal (Allen and Corner, 1929; 1930). Purification of the extract of the corpus luteum led to the chemical identification of the active substance by Butenandt, Westphal and Cobler in 1934, and in the following year Allen, Butenandt, Corner and Slotta (1935) agreed to the name pi^ogesterone for this hormone of the corpus luteum.


These events were soon followed by tlu' discovery that progesterone is excreted in the urine as tlie glucuronide of pregnanediol and i)regnanolone, metabolites of progesterone. Studies of urinary products of progesterone were immediately undertaken and a marked increase in urinary pregnancdiol was observed in the human female throughout pregnancy, especially in the second half (Fig. 16.3).

The discovery by Hooker and Forbes (1947) of a new assay for progesterone sensitive to a concentration of 0.3 /^g. per ml. led to many studies on the blood levels of this hormone during gestation. Subsequent studies revealed a lack of specificity for the assay (Zarrow, Neher, Lazo-Wasem and Salhanick, 1957; Zander, Forbes, von Miinstermann and Neher, 1958) and a discrepancy between the values obtained by chemical and biologic techniques. It is obvious that the bioassay data possess significance but a final evaluation can be made only when the identity of the compound or compounds measured in the blood of the animals by the Hooker-Forbes test has been established.


Fig. 16.3. Urinary excretion of estrogens and pregnanediol throughout gestation in the human being. (From E. Venning, Macy Foundation, Conferences on Gestation, 3, 1957.)


The concentration of gestagen in the blood of pregnant sheep (Neher and Zarrow, 1954) , women (Forbes, 1951 ; Schultz, 1953; Fujii, Hoshino, Aoki and Yao, 1956), rabbits (Zarrow and Neher, 1955), and mice (Forbes and Hooker, 1957) has been determined by the Hooker-Forbes test and expressed as /^g. equivalents of progesterone. The data obtained from pregnant women by the different investigators are in marked disagreement. Whereas both Forbes (1951) and Schultz (1953) failed to observe any significant rise in blood gestation of pregnant women throughout gestation, Fujii, Hoshino, Aoki and Yao (1956) obtained a conspicuous rise during this period. The data reported by Forbes (1951) indicate an extremely low level for protein-bound progesterone (0.5 /^g. per ml. plasma or less) and a maximum of 2 /xg. per ml. free progesterone (Fig. 16.4). The concentration of the hormone in the blood showed a series of irregular peaks throughout gestation and varied from less than 0.3 /xg. to 2 /xg. per ml. plasma. In general, these results were confirmed by Schultz (1953) who assayed the blood from 46 women at 6 to 17 weeks of pregnancy. Again the results failed to reveal any consistent change with the length of pregnancy. Both investigators (Forbes, 1951; Schultz, 1953) were led to question the importance of progesterone during gestation in the primate. Fujii, Hoshino, Aoki and Yao (1956), on the other hand, reported a significant increase in the level of circulating progesterone throughout gestation. Again these investigators used the Hooker-Forbes assay but indicated that the plasma was not treated in any way except for dilution before the assay. The results obtained by this latter group revealed a rise from a level of 6 /*g. progesterone per ml. plasma during the luteal phase of the cycle to a high of 25 /i,g. during: the last trimester of pregnancy (Fig. 16.5). The concentration showed a steady increase from the 4th to the 24th week of pregnancy,, and a plateau from the 24th week until term. A sharp drop occurred within 12 to 24 hours after parturition with zero values noted by 72 hours postpartum. Analysis of the urine for pregnanediol showed a rather good correlation between the two curves although the plasma levels rose sooner than the urinary pregnanediol.

The curve for the concentration of progesterone in the pregnant mouse is markedly different from those reported for other species (Forbes and Hooker, 1957). Again the Hooker-Forbes assay was used



Fig. 16.4. Free and bound ge.stagen in the plasma of the pregnant human female. (From




Fig. 16.5. Concentration of gestagen in the blood plasma and pregnanediol in the uterine of the pregnant human female. Gestagen levels were determined by the HookerForbes test. (From K. Fujii, K. Hoshino, I. Aoki and J. Yao, Bull. Tokyo Med. & Dent. Univ., 3, 225, 1956.)


as with the other species and the values expressed as activity equivalent to progesterone. The values for the bound action were consistently low and, in general, less than 1 fjig. per ml. plasma (Fig. 16.6). The concentration of the free hormone showed marked variations on the first day or so of pregnancy. Actually a variation from 1 fig. per ml. plasma to 8 fig. per ml. plasma was seen on day 0. This type of fluctuation has also been seen in the rabbit and is without explanation at the present time. However, such marked variations disappeared by the 4th day of pregnancy and the results became much more consistent. The average curve for the concentration of gestagen in the blood of the pregnant mouse showed two peaks, one the 7th to the 9th day and a second the 15th day. The concentration increased from 2 fxg. per ml. plasma the 4th day of gestation to an average of approximately 8 fig. the 7th day. This level was maintained until day 9 and fell thereafter with a second peak occurring on day 15 and an immediate drop on day 16. Thereafter the levels remained low throughout the remainder of pregnancy.

Although it may be assumed that the initial peak in the concentration of the gestagen is due to an increased activity



Fig. 16.6. Concentration of free and bound gestagen in the plasma of the pregnant mouse. Gestagen levels were determined by the Hooker-Forbes test. (From T. R. Forbes and C. W. Hooker, Endocrinology, 61, 281, 1957.)



on the part of the corpora lutea, an explanation of the second peak and the drop between the two peaks offers more difficulty. The latter may reflect a diminished luteal activity. This could be assumed on the grounds that the corpus luteum is the only source of gestagen during this period of gestation and that the luteal cells show cytologic signs of regressive changes, although the drop in serum progestogen antidates the cytologic changes by several days. An explanation for the second peak would probably involve increased secretory activity !)y the placenta. Progestational activity has l)een found in i:)lacental extracts and progesterone has been isolated from the placentae of human beings and mares (Salhanick, Noall, Zarrow^ and Samuels, 1952; Pearlman and Cerceo, 1952; Zander, 1954; Short, 1956). Thus, the drop in serum gestagen seen on day 10 could be due to loss in the activity of the corpora lutea and the second rise as a contribution from the placentas. It is of interest that the low levels on days 10 to 13 and between day 16 to term appear to have no counterpart in other species. The physiologic significance of this is still unknown and will require further work on additional species and on the mouse before an explanation is forthcoming. It is of interest that the concentration of gestagen in the blood dur


ing the first 12 days of pregnancy corresponds with the intensity of the response to progesterone exhibited by the endometrium during the same period (Atkinson and Hooker, 1945). This w^ould suggest that the serum gestagen levels reflect the physiologic state of the animal.

Serum gestagen levels in the rabbit reveal a curve of increasing concentration throughout pregnancy (Zarrow and Neher, 1955). Initial values of 0.3 to 1 yug. per ml. serum were noted at the time of mating, with a sharp rise beginning on the 4th day of gestation. The concentration rose to a level of 6 to 8 /xg. per ml. by the 12th day and thereafter showed only a slight rise to a maximal concentration of 8 to 10 /xg. per ml. serum at parturition (Fig. 16.7). No drop in serum hormone level was observable at parturition or 1 hour later. The first significant drop occurred at 6 to 12 hours postpartum when the gestagen level had decreased 50 per cent. It is of interest that the serum progestagen levels did not fall until after the conceptus had been expelled.

castrated the 12th, of gestation aborted following removal of the ovaries (Zarrow and Neher, 1955). In all instances the serum gestagen levels fell before the abortion. Figure 16.8 shows the


Pregnant rabbits 19th, or 24th day within 1 to 3 days


GESTATION


969


± 5


3


NORMAL PREGNANCY RABBIT 25 o RABBIT 26 • RABBIT 31 e - RABBIT 27 x RABBIT 30 ^



Fig. 16.7. Concentration of gestagen in the blood of the normal pregnant rabbit as determined by the Hooker-Forbes test. (From M. X. Zarrow and G. M. Neher, Endocrinology, 56, 1, 1955.)







Fig. 16.8. The effect of castration on serum progestogen levels and maintenance of gestathe rabbit. Gestagen levels were determined by the Hooker-Forbes test. (From M. X. and G. M. Xeher. Endocrinology, 56, 1, 1955.)


changes in serum gestagen levels before and after castration of a pregnant rabbit. The concentration increased from a level of 0.3 ing. per ml. at day to 10 /xg. per ml. on day 24 when the rabbit was castrated. A 60 per cent drop in serum gestagen level is seen 12 hours after castration with a fur


ther drop at the 36th hour, when the animal aborted.

Studies on the concentration of serum gestagen in the pregnant ewe (Neher and Zarrow, 1954) permit a comparison with the results obtained in the rabbit. Such a comparison is extremely valuable in view



Fig. 16.9. Concentration of gestagen in the blood of the pregnant ewe. Gestation levels were determined bv the Hooker-Forbes test. (From G. M. Neher and M. X. Zarrow, J. Endocrinol., 11,323,1954.)


of the fact that castration of the rabbit invariably leads to abortion whereas castration of the pregnant ewe does not do so if the ovaries are removed during the second half of pregnancy. Again the progesterone determinations were carried out on untreated serum and the samples assayed by the Hooker-Forbes technique using progesterone as a standard. An initial rise in the serum gestagen level occurred soon after mating and seemed to level off at a concentration of 6 ixg. per ml. approximately the 50th day of gestation (Fig. 16.9). Thereafter, the concentration remained unchanged for approximately 50 days, when a second rise to a level of 8 to 12 fj.g. occurred. These levels remained unchanged until at least 30 minutes after parturition was complete.

Castration at various times after the 66th day of pregnancy failed to influence the concentration of circulating gestagen or interfere with the pregnancy. The data in Figure 16.10 show a normal concentration of 8 to 10 fxg. gestagen from the 114th day of gestation to parturition although the animal was ovariectomized the 114th day. Pregnancy was normal in all castrated ewes and the expected drop in scrum gestagen was observed following parturition.

It can now be stated that the human being, the monkey, the ewe, the rabbit, the mouse, and probably the guinea pig (Herrick, 1928; Ford, Webster and Young, 1951) have met the problem of a second source of progesterone supply with varying degrees of success. In the ewe, placental replacement of the ovary as a source of progesterone can be considered as complete by approximately the 66th day of pregnancy. Castration at this time will neither interfere with the pregnancy nor with the concentration of the hormone in the blood. In the monkey, castration as early as the 25th day of gestation (Hartman, 1941) does not interfere with pregnancy and in the human being castration as early as the 41st day after the last menstrual period may not interfere with pregnancy (Melinkoff, 1950; Tulsky and Koff, 1957). One may conclude, therefore, that the placenta can adequately take on the role of the ovary in this regard. On the other hand, aspects of the situation in the human female are still puzzling, especially the blood gestagen values; but despite this ambiguity



Fig. 16.10. The effect of castration on gestagen levels in the pregnant ewe. Gestagen le\els were obtained by the Hooker-Forbes test. Note that castration failed to interfere with the pregnancy or the level of gestagen in the blood. (From G. M. Neher and M. X. Zarrow, J. Endocrinol., 11, 323, 1954.)


it might be concluded that here also the placenta has successfully replaced the ovary. In the rabbit, on the other hand, castration at any time during pregnancy vvill cause a decrease in the level of the circulating hormone and terminate the pregnancy. Hence, in this species, the placenta has failed to replace completely the ovary. The mouse is another instance in which castration leads to abortion so that one can assume a failure on the part of the placenta to replace the endocrine activity of the ovary. In this case, however, the second peak of circulating gestagen has been ascribed to the placenta and this presents the possibility of a partial replacement of the ovary by the placenta but a replacement that is not adequate since pregnancy is terminated by ovariectomy.

As indicated above, a marked discrepancy exists between the bioassays and the chemical determinations of gestagens in the blood and other tissues. The chemical determinations of progesterone invariably give results that are far lower than those obtained by bioassay methods. Edgar and Ronaldson (1958) found a maximal concentration of approximately 2 /xg. progesterone per ml. ovarian venous blood during


gestation in the ewe. This concentration was no higher than that seen in the ewe during a normal estrous cycle. The maximal level reached during the estrous cycle was maintained when pregnancy supervened and remained fairly constant until the last month of pregnancy. Thereafter the concentration fell and no progesterone was detectable at 15 days prepartum (Fig. 16.11). Inasmuch as no progesterone was found in the peripheral blood of the ewe, this poses again the following question: What was being measured in the peripheral blood by the bioassay procedure? In addition, a second question is posed by the earlier discussion on the need of the ovary in the maintenance of pregnancy as to the relative contributions of the ovary and the placenta to the concentration of this hormone in the body.

That the biologic methods are measuring more than progesterone is obvious from the many reports emphasizing the high levels obtained by bioassay and the low levels obtained by chemical techniques. In addition to the above data. Short (1957, 1958a, 1958b) reported the presence of progesterone in the peripheral blood of the pregnant


Fig. 16.11. The con«?ntiation of progesterone in the ovarian venous blood of the pregnant ewe. Progesterone was determined by chemical methods. (From D. G. Edgar and J. W. Ronaldson, J. Endocrinol., 16, 378, 1958.)


0.0098 ixg. per ml. plasma. It is of interest that the level remained constant from the 32nd to about the 256th day of pregnancy and then decreased several days before parturition. In the human being values of 0.17 to 0.44 fig. per ml. during the final trimester of pregnancy have recently been reported (Oertel, Weiss and Eik-Nes ( 1959 ) . Numerous investigators have suggested that the discrepancy between the chemical and biologic assays is due to the presence of unknown gestagens in the blood. This has been validated in part by the discovery of 2 metabolites in the blood of the pregnant human female (Zander, Forbes, Neher and Desaulles, 1957). They have been identified as 20a-hydroxypregn-4-en-3-one and 20^-hydroxypregn-4-en-3-one and have been shown to be active in both the Clauberg and Hooker-Forbes tests (Zander, Forbes, von IMiinstermann and Neher 1958 ) . The 20/?-epimer was twice as active as progesterone in the Hooker-Forbes test and the 20a-epimer one-fifth as active. It is likely that more unidentified gestagens occur in the blood and other tissues.


C. Sources of Gestagens

The second question asked above concerning the role of the placenta versus the ovary as a source of progesterone probably cannot be answered in a simple manner. Wide differences exist between species (1) in the need of the ovary for maintenance of pregnancy, (2) in the concentration of the hormone in peripheral blood, (3) in the activity of the placenta in secreting progesterone, and (4) in the presence of extraovarian and extraplacental sources of the hormone.

The presence of progesterone in the placenta of the human being has been confirmed (Salhanick, Noall, Zarrow and Samuels, 1952; Pearlman and Cerceo, 1952) and a high output of progesterone demonstrated. Zander and von ]\Iiinstermann (1956) and Pearlman (1957) independently reported the production of approximately 250 mg. progesterone into the peripheral circulation every 24 hours. This and other evidence tends to prove that the placenta is the major source of progesterone in the human species. However, with respect to other species, progesterone has been found only in the placenta of the mare (Short, 1957) although in amounts much less than in the human being. Placentas of the cow, ewe, sow, or bitch were all negative. Although the placenta of the mare contains progesterone and castration does not lead to abortion after day 200 of gestation, no progesterone was found in the peripheral blood or uterine vein blood. The ewe offers an even more intriguing problem inasmuch as (1) a discrepancy exists between the biologic and chemical values for progesterone in the peripheral blood, (2) the placentas contain no progesterone, and (3) no {progesterone is found in the uterine vein blood (Edgar, 1953). This has led to the conclusion that the maintenance of pregnancy in the ewe may be dependent on an extra-ovarian, extraplacental source of progesterone.

If such a conclusion is correct, and it must be added that the evidence is still tenuous, then the adrenal cortex must be considered as a possible source. Beall and Reichstein isolated a small amount of progesterone from the adrenal cortex in 1938 and Heehter, Zaffaroni, Jacobson, Levy, Jeanloz, Schenker and Pincus (1951) demonstrated from perfusion experiments that progesterone is an important intermediate metabolite in the synthesis of the adrenal corticoids. In addition, it has long been known that desoxycorticosterone possesses progesterone-like activity (Courrier, 1940) which is due to a conversion of the desoxycorticosterone molecule to a gestagen. This has been shown by experiments in vivo in the monkey (Zarrow, Hisaw and Bryans, 1950), rat, and rabbit (Lazo-Wasem and Zarrow, 1955), and by an incubation experiment with rat tissue (Lazo-Wasem and Zarrow, 1955). In addition, Zarrow and Lazo-Wasem reported the release of a gestagen from the adrenal cortex of the rat and rabbit following treatment with ACTH. The substance was obtained from the peripheral blood and measured by the Hooker-Forbes test, but it was not identified chemically. This was followed by the finding that pregnanediol is present in the urine of ovariectomized women, but not ovariectomized, adrenalectomized women (Klopper, Strong and Cook, 1957), and by the finding that progesterone is present in the adrenal venous blood of the cow, sow, and ewe (Balfour, Comline and Short, 1957). In all instances the concentration of progesterone in the adrenal venous blood was 10 to 100 times greater than the concentration in the arterial blood. Thus the total evidence that the adrenal cortex can secrete progesterone is more than adequate. The question remains as to whether the adrenal cortex contributes to the progesterone pool of the body during pregnancy and whether a species difference exists here.

D. Relaxin

The initial discovery by Hisaw (1926, 1929) of the presence of an active substance in the blood and ovaries responsible for relaxation of the pubic symphysis of the guinea pig has led in recent years to a consideration of this substance as a hormone of pregnancy (Hisaw and Zarrow, 1951). Some doubt as to the existence of relaxin was raised in the 1930's by investigators who were able to show that pubic relaxation in the guinea pig could be obtained with estrogen alone or estrogen and progesterone (de Fremery, Kober and Tausk, 1931; Courrier, 1931; Tapfer and Haslhofer, 1935; Dessau, 1935; Haterius and Fugo, 1939). This matter was resolved by the demonstration that pubic relaxation in the guinea pig following treatment with the steroids or relaxin differed in ( 1 ) time required for relaxation to occur, (2) histologic changes in the pubic ligament, and (3) treatment with estrogen and progesterone which induced the formation of relaxin (Zarrow, 1948; Talmage, 1947a, 1947b). Subsequent discoveries of additional biologic activities possessed by relaxin and further purification of the hormone has led to the conclusion that relaxin is an active substance in the body, and that it plays a significant role during parturition. The hormone has been found in the blood or other tissues of the dog, cat, rabbit, sheep, cow, rat, and man. The specific action of this hormone varies with the species involved. Still unsolved is the question as to whether the water-soluble extract obtained from the ovary and referred to as relaxin is a single substance or a group of active substances (Friedcn and Hisaw, 1933; Sher and Martin, 1956).

The concentration of relaxin in the blood increases as pregnancy progresses until a plateau is reached. This has been demonstrated in the rabbit (Marder and Money, 1944), guinea pig (Zarrow, 1947), cow (Wada and Yuhara, 1955), and human being (Zarrow, Holmstrom and Salhanick, 1955). Relaxin has also been found to increase in the ovary of the sow (Hisaw and Zarrow, 1949). In general, the shape of the curve for the concentration of relaxin in the blood as a function of the length of pregnancy has been more or less the same for all species studied. Figure 16.12 indicates that the concentration of relaxin in the blood of the pregnant rabbit rises from a level of 0.2 guinea pig unit (G.P.U.) per ml. for the first trimester of pregnancy, i.e.. until day 12, to a level of 10 G.P.U. per ml.


on day 24. This concentration was then maintained until parturition. After delivery of the young, the concentration of the hormone decreased 80 per cent in 6 hours. On the 3rd day postpartum no hormone could be detected.

As indicated above, the concentration of relaxin in the blood of the pregnant cow and human being showed approximately the same type of curve. In the cow the concentration rose gradually from a level of 1 G.P.U. per ml. to a maximum of approximately 4 G.P.U. at 6 months (Fig. 16.13). Thereafter the level remained unchanged until parturition, wdien the level dropped at a rate comparable to that seen in the rabbit. The curve for the concentration of relaxin in the blood serum of the pregnant woman followed the general pattern described above (Fig. 16.14). The concentration rose from a level of 0.2 G.P.U. per ml. the 6th week of i:)regnancy to a maximum of 2 G.P.U. the 36th week. Thereafter the level remained unchanged until delivery. Again the postpartum fall was precipitous and the hormone was not detectable at 24





Fig. 16.12. Concentration of relaxin in the blood of the rabbit during pregnancy. Parturition (P) occurred 32 days after mating. Guinea pig units (G.P.U.) of relaxin are plotted against days pregnant. (From S. N. Marder and W. L. Money, Endocrinology, 34, 115, 1944.)



Fig. 16.13. Concentration of I'elaxin in the blood of the cow during pregnancy. Partiuition is indicated by P. (From H. Wada and M. Yuhara, Jap. J. Zootech. Sc, 26, 12, 1955.)



Fig. 16.14. The concentration of relaxin in the blood serum of normal pregnant women. (From M. X. Zarrow, E. G. Holmstrom and H. A. Salhanick, Endocrinology. 15, 22. 1955.)


hours postpartum. Studies in the guinea pig revealed a marked rise in relaxin on day 21 of gestation to a maximal concentration of 0.5 G.P.U. per ml. serum on day 28 (Zarrow, 1948). Thereafter the level remained unchanged for approximately 4 weeks. Contrary to the results obtained in the rabbit, cow, and human being a drop in the concentration of the hormone in the pregnant guinea pig was noted before parturition. The concentration of relaxin fell to 0.33 G.P.U. per ml. on the 63rd day of gestation and then dropped to nondetectable levels within 48 hours postpartum.

Although no studies have been carried out on the blood levels of relaxin in the sow as a function of the length of pregnancy, analysis of the ovary for relaxin has revealed a situation comparable to that reported for the blood in other species. The concentration rose from 5 G.P.U. per gm. ovarian tissue during the luteal phase of the cycle to approximately 10,000 G.P.U. per gm. fresh ovarian tissue by the time a fetal length of 5 inches had been reached (Hisaw and Zarrow, 1949).


E. Sources of Relaxin

The ovaries, placentas, and uteri are possible sources of relaxin in different species. It seems from the extremely high concentration in the ovary of the sow during pregnancy that this organ is the major site of relaxin synthesis at this time. However, studies on other species indicate that both the placenta and uterus may be involved.

Treatment of castrated, ovariectomized rabbits with estradiol and progesterone stimulated the appearance of relaxin in the blood of the rabbit as indicated by the ability of the blood to induce relaxation of the pubic symphysis of estrogen-primed guinea pigs (Hisaw, Zarrow, Money, Talmage and Abramovitz, 1944). Similar experiments on castrated, hysterectomized rabbits failed to reveal the presence of the hormone in the blood of the treated animals. Treatment with estradiol alone also failed to stimulate the release of relaxin. It is obvious then that, if the bioassay is specific for relaxin, the uterus is a definite source of this hormone. Comparable results were also obtained in the guinea pig (Zarrow, 1948). Treatment with estradiol and progesterone caused pubic relaxation and the presence of relaxin in the l)lood after approximately 3 days of treatment with progesterone. In the absence of the uterus relaxin was not demonstrable in the blood.

The concentration of relaxin in the blood of the rabbit castrated the 14th day of i^regnancy and maintained with progesterone remained unaffected by removal of the ovaries, provided the pregnancy was maintained (Zarrow and Rosenberg, 1953). Figure 16.15 shows a typical curve for the relaxin content of the blood of such an animal. The concentration of the hormone rose between days 12 and 24 to a maximal concentration of 10 G.P.U. per ml. and was maintained till the time of normal parturition. It is of interest that in those instances in which the placentas were not maintained in good condition, the concentration of the hormone fell. Analysis of the reproductive tract revealed concentrations of 5 G.P.U. per gm. fresh ovarian tissue during pseudopregnancy and approximately 25 G.P.U. during the last trimester of gestation. The uterus contained 50 G.P.U. per gm. fresh tissue during pseudopregnancy and an equal concentration the first 24 days of pregnancy. The 26th day of pregnancy the concentration fell to 15 G.P.U. per gm. The highest concentration was in the placenta which contained from 200 to 350 G.P.U. per gm. Some evidence indicated that after treatment with estradiol minimal amounts of relaxin, i.e., 5 G.P.U. per gm., were present in the vaginal tissue (Table 16.5).

F. Adrenal Cortex

1. Hydrocortisone

Initial studies on the possible role of the adrenal cortex in gestation involved the determination of the two urinary metabolites of the gland, i.e., the 17-ketosteroids and the corticoids. Inasmuch as the 17-ketosteroids are believed to be associated with the androgenic activity of the adrenal cortex, bioassays for adrenogenic activity in the urine were carried out. Dingemanse, Borchart and Laqueur (1937) found no increase in urinary androgen by the 6th to the 8th month of pregnancy whereas Hain (1939) reported that pregnant women secreted even less androgen than nonjircgnant women. Pincus and Pearlman (1943) found no change in the urinary 17-ketosteroids of the pregnant and nonpregnant woman although Dobriner (1943), by the use of chromatograi)hic separation, showed a marked decrease in androsterone. Venning (1946) found no change in the urinary ketosteroids as measured by the antimony trichloride reagent described by Pincus (1943), but the ketosteroids measured by the Zimmerman reagent (dinitrobenzene) showed a significant rise in the latter part of pregnancy. The discrepancy between the two determinations can be explained by the fact that other ketonic substances besides 17-ketosteroids give a color in the Zimmerman reaction. These are the 20-ketosteroids and to a limited extent the 3-ketosteroids. V^enning (1946) believes most of this in



Fig. 16.15. Concentration of relaxin in the blood of a pregnant rabbit castrated the 14th day of gestation and maintained with 4 nig. progesterone daily until the 32nd day. Postmortem examination revealed 8 placentas and 2 dead fetuses. (From M. X. Zarrow and B. Rosenberg, Endocrinology, 53, 593, 1953.)


TABLE 16.5

Relaxin content of the blood serum and tissue of the reproductive tract of the rabbit

(From M. X. Zarrow and B. Rosenberg, Endocrinology, 53, 593, 1953.)



No. of Rabbits


Relaxin Concentration in G.P.U.


Treatment


Per ml. serum


Per gm fresh tissue



Ovary


Uterus


Placenta whole


Placenta fetal


Placenta maternal


Pseudopregnant

Chorionic gonadotrophin . . .


3

4 3 2 2

2

1


0.2-0.3 0.2 1.0 10.0 10.0 10.0 10.0


5 5 30 25 20 25 25


50 50 50 50 30 15


75 50 50

75


10 20 25



Pregnant 24 davs


250


Pregnant 25 days

Pregnant 26 days


350 200


Pregnant 28 days



crease in ketosteroid excretion during pregnancy is the result of increased output of the stereoisomers of pregnanolone:

Measurement of urinary glucocorticoids by the glycogen deposition test showed an initial increase in the first trimester of pregnancy in the human being. After the initial rise, the urinary excretion level returned to normal with a second increase the 140th to 160th day of pregnancy. Values of 200 to 300 /xg. equivalent of 17,hydroxy11-dehydrocorticosterone per 24 hours of urine were obtained at days 200 to 240. In most instances the urinary outj^ut fell several weeks before parturition. ' Analysis of the blood levels for 17ahydroxycorticosterone in the jiregnant wo


man confirmed the results obtained with the urine (Gemzell, 1953; Seeman, Varangot, Guiguet and Cedard, 1955). Gemzell ( 1953) reported a rise from approximately 5 /xg. per 100 ml. plasma to an average of approximately 22 fxg. per cent (Fig. 16.16). A further rise to 36 /xg. per cent was noted at the time of labor. This has been confirmed by McKay, Assali and Henley (1957) who found an average rise of approximately 40 /xg. per cent during labor lasting more than 6 hours. Although McKay, Assali and Henley reported values still well above normal on the 4th to 6th



Fig. 16.16. Correlation between the concentration of 17-hydroxycorticosteroids in the blood of pregnant women and the duration of pregnancy (in weeks). Conception at zero time. (From C. A. Gemzell, J. Clin. Endocrinol.,13, 898, 1953.)



The mechanism whereby labor induces a marked stimulation of the adrenal cortex is still obscure. It is possible that labor is a stressful state and the stress induced by both the pain and the muscular work act to stimulate the increased release of ACTH resulting in increased adrenocortical activity. Some confirmation of this may be obtained from the fact that significant in(■i'eas(> in }ilasma 17a-hydroxycorticoids is noted only if the labor lasts more than 6 hours.

Analysis of the rise in plasma levels of hydrocortisone during pregnancy has suggested that the phenomenon is not simply the result of an increased rate of secretion from the adrenal cortex, but rather the result of an increased retention and an alteration in the metabolism of the hormone < Cohen, Stiefel, Redely and Laidlaw, 1958).

2. Aldosterone

The isolation for aldosterone by Simi)son, Tait, Wettstein, Neher, von Euw, Schindler and Reichstein (1954) and its identification as the hormone regulating fluid and mineral metal)olism stimulated marked interest in the role of this hormone. Among the items of interest was its significance in pregnancy and in the toxemia of pregnancy. Early studies by Chart, Shipley and Gordon ( 1951 1 revealed the presence of a sodium retention factor in the urine that increased from a normal pregnancy value of 36 to 106 fxg. equivalent of desoxycorticosterone acetate (DOCA) per 24 hours to a maximum of 1008 |U.g. equivalent in pregnancy toxemia. These results were confirmed by Venning, Simpson and Singer (1954) and by Gordon, Chart, Hagedorn and Shipley (1954). In addition a slight increase in the sodium retaining factor was observed in gravid women as compared to nongravid women.

The discovery that the greater part of the aldosterone in urine is present in the conjugated fraction led to a repetition of the above work using both acid hydrolysis and incubation with /3-glucuronidase (Venning and Dyrenfurth, 1956; Venning, Primrose, Caligaris and Dyrenfurth, 1957). The results show little change in the excretion of free aldosterone throughout pregnancy, but the glucuronidase and acid-liydrolyzed


fractions increased markedly (Fig. 16.17). The urinary excretion values increased from a prepregnancy normal of 1 to 6 /xg. aldosterone (average for women was 3.8 ± 14 fig. per 24 hours; Venning, Dyrenfurth and Giroud, 1956) to approximately 25 /xg. per 24 hours. The first significant rise occurred about the third month of gestation and an increased concentration was obtained until after parturition, when there was a rapid fall to the nonpregnant values.


G. Thyroid Gland

Clinical data have long indicated a possible involvement of the thyroid gland in gestation (Salter, 1940). In regions where the iodine supply is low this is demonstrated by an enlargement of the thyroid during pregnancy. The formation of a goiter has been interpreted as evidence for an increased need for iodine during gestation. Scheringer (1930) and Bokelmann and Scheringer ( 1930) reported a rise in the iodine content of the blood of pregnant women during the first trimester of pregnancy with a peak at the seventh month. The increased concentration is maintained until shortly after parurition. In the goat, however, Leitch (1927) reported no change in serum iodine during gestation until just before parturition. Analysis of umbilical vein blood revealed values that were normal, i.e., lower than in the mother (Leipert, 1934). Increased thyroid secretion (Scheringer, 1931 ) and increased urinary excretion of iodine have been reported in pregnant women (Nakamura, 1932; 1933). However, Salter (1940) concluded in his review that no reliable evidence of increased thyroid hormone levels in the blood during jiregnancy is available.

Peters, Man and Heinemann (1948) reported a range of 4 to 8 fig. per cent of serum-precipitable iodine in the normal, nonpregnant woman with a rise to 8.3 fig. per cent (range 6 to 11.2 fig. per cent) in the pregnant woman (Fig. 16.18). It is of interest that the elevation in the proteinbound iodine (PBI) does not follow the course of changes in the basal metabolic rate. Whereas the former is already high by the second month of pregnancy the basal metabolic rate rises gradually after approximately the 4th month of pregnancy (Rowe and Boyd, 1932; Javert, 1940). No other sym})toms of hyperthyroidism are seen in pregnancy which leads to the question of the significance of the rise in protein-bound iodine. A somewhat comparable paradox exists in the guinea pig in which a rise in the rate of oxygen consumption during pregnancy is not accompanied by an increase in heart rate (Hoar and Young, 1957).

Recently, AVerner (1958) rcj^orted a decrease in the I^-^^ up-take after treatment with triiodothyronine in both the normal and pregnant woman. From this and other data he ruled out any abnormal pituitarythyroid relationship or marked secretion of thyroid-stimulating hormone (TSH) by the placenta and concluded that the increased PBI in pregnancy is due to an increased binding capacity of the serum protein.

Feldman ( 1958) failed to find any increase in the level of serum-hutanol-extracted iodine throughout pregnancy in the rat. Actually the values were consistently lower than in the controls and similarly the total amount of PBI in the thyroid of the pregnant rat was consistently lower. He did find an increase in the rate of excretion of V-\ a diminished up-take of I^^^ by the thyroid, and a decreased half-life for thyroxine in the pregnant rats. It is obvious that these results are quite dissimilar from those obtained in the pregnant women. One can only conclude at this time that pregnancy has an effect on iodine metabolism and a species difference exists.

H. Growth Hormone

Although it has been possible to demonstrate the presence of growth-promoting substance (STH) in the blood plasma, there are few data bearing on the identity of the substance and few ciuantitative measurements. Westman and Jacobsohn ( 1944) first showed the ]irescnce of a growth-]5romoting sub



Fig. 16.17. Urinary excretion of aldosterone throughout pregnancy in the human being. O, free fraction only; •, free and acid-hydrolyzed fraction; O, free, enzyme and acidhA'drolyzed. (From E. H. Venning and I. Dyrenfurth. J. Clin. Endocrinol,, 16, 426, 1956.)


stance in the blood by cross transfusion between a normal and hypophysectomized rat united in parabiosis. Gemzell, Heijkenskjold and Strom (1955), using the technique of adding exogenous growth hormone to the sample of blood, failed to find any growthjiromoting substance in 23-ml. equivalents of blood. However, retroplacental blood from human beings gave a positive response at a level of 7- to 15-ml. equivalents of plasma without the addition of exogenous STH. Increase in the width of the proximal tibial epiphysis of the rat was used as an end l)oint. A comparable concentration of 650 fxg. eciuivalent of the standard STH per 100 ml. plasma was also found in the blood from the umbilical cord.

Contopoulos and Simpson (1956, 1957) measured the STH of the plasma in the pregnant rat, using the tibial cartilage, tail length, and body weight increase. No significant increase in plasma STH was noted on the 5th day of pregnancy, however, a significant rise was observed by the 9th day. An estimated 3-fold increase in plasma STH during pregnancy was reported from calculations on both the tibial cartilage and the tail length tests. No changes were reported in the STH activity of the pituitary gland throughout pregnancy. Recently, the persistence of greater than normal amounts of growth-promoting activity was reported in the plasma of pregnant rats after hypophysectomy. Since the fetal pituitary probably does not contribute to the STH pool of the mother, at least in early pregnancy, it is likely that the placenta may be a source of the hormone.

I. Prolactin

Few data are available on the concentration of prolactin during gestation. This has been due, in part, to the minute amounts of the hormone present in the urine and blood and to the inadequacy of the available assays. Although Hoffmann ( 1936 ) failed to find any prolactin in the urine of women before parturition, Coppedge and Segaloff (1951) and Fujii and Schimizu (1958) reported measurable amounts of prolactin in the urine of pregnant women. Coppedge and Segaloff reported a gradual rise in the excretion of prolactin throughout pregnancy and a gradual decline following parturition even though lactation was maintained. The number of observations, however, was limited and the authors point out that the results were ecjuivocal. Fujii and Shimizu observed an initial drop in the prolactin output during the first month of pregnancy followed by a rise to approximately 32 P.C.U. (one pigeon crop sac unit is equivalent to 0.3 I.U.) per 24 hours during the second trimester of pregnancy in women. (Fig. 16.19). This was followed by a drop to approximately 10 P.C.U. per^24 hours between the 30th and 38th wrecks of pregnancy and a marked rise to 64 P.C.U. per 24 hours during the lactation period.


FiG. 16.18. The level of protein-bound iodine in the pregnant woman. (From J. P. Peters, E. B. Man and M. Heinemann, in The Normal and Pathologic Physiology oj Pregnancy, The Williams & Wilkins Co., 1948.)



Fig. 16.19. Urinary excretion of prolactin throughout gestation in the human being. One pigeon crop unit (P.C.U.) is equivalent to 0.3 I.U. (From K. Fujii and A. Shimizu, Bull. Tokyo Med. & Dental Univ., 5, 33, 1958.)


J. Placental Gonadotrophins

Placental gonadotrophins have been found in the monkey, chimpanzee, human being, mare, and rat (Hisaw and Astwood, 1942). The physiologic activities of these placental hormones differ among the three groups of niannnals and appear to represent divergent evolutionary steps in the adoption of pituitary function by the placenta. The physiologic properties of the placental gonadotrophins differ not only among themselves but also from the pituitary gonadotrophins. The gonadotrophin from the rat placenta (luteotrophin) has been shown to be leuto


trophic with the ability to maintain a functional corpus luteum in the hypophysectomized rat (Astwood and Greep, 1938). The hormone has no effect on follicular growth or ovulation. Its function appears to be that of maintaining the secretory activity of the corjius luteum in the rat from the 10th day of pregnancy to term.

The placental hormones of the human being (HCG) and the mare (PMS) have been studied in much greater detail. These two hormones differ markedly in both chemical and physiologic properties. The presence of HCG in the urine and the absence of P]\IS in the urine would alone indicate a marked difference in the size of the two molecules. Physiologically, PMS is highly active in producing follicular growth and some luteinization, whereas HCG has no effect on follicular growth but will induce ovulation and a delay in the onset of menstruation. This would indicate a luteotrophic action. Although chorionic gonadotrophin has been reported in the macaque (Hamlett, 1937) between the 18th and 25th day of pregnancy, and in the chimpanzee from the 25th to the 130th day of gestation (Zuckerman, 1935; Schultz and Snyder, 1935), little work has been done on the characterization and identification of these substances except in man and horse.



Fig. 16.20. The relative time of appearance of placental gonadotropliins in the pregnant mare and the woman. (From E. T. Engle, in Sex and Internal Secretions, 2nd ed., The Williams & Wilkins Company, Baltimore, 1939.)


It is of some interest to note that the appearance of the placental gonadotrophins in the blood and urine of horse and man occurs at approximately the same relative time in pregnancy (Fig. 16.20). The role played by these hormones in gestation is still not clear, but it is significant that their appearance corresponds with the time of implantation of the blastocyst and their disappearance roughly with the time when ovariectomy no longer interferes with the maintenance of the pregnancy.

K. Human Chorionic Gonndotrophin (HCG)

The discovery of the presence of a gonadotrophic hormone in human pregnancy urine by Aschheim and Zondek (1927j was soon followed by a description of its biologic activity and quantitative determinations of its concentration in the urine throughout pregnancy (Ascheim and Zondek, 1928). Recently a number of investigators have determined the titer of chorionic gonadotrophin in the serum of pregnant women. These curves agree very well with the values obtained from the urine. Figure 16.21 is a typical curve for the concentration of chorionic gonadotrophin in the blood of pregnant women (Haskins and Sherman, 1952). A peak value of 120 I.U. per ml. of serum was obtained on the 62nd day after the last menses and a rapid decline was noted to a low of approximately 10 I.U. per ml. of serum on day 154. A subsequent rise to 20 I.U. was noted by day 200 and this was maintained until the end of pregnancy. These results are in excellent argeement with those reported by Wilson, Albert and Randall (1949) using the ovarian hyperemia test in the immature rat. These authors obtained a peak concentration of approximately 70 I.U. per ml. of serum on the 55th day after the last menses. A gradual decrease occurred thereafter to a low of approximately 20 I.U. per ml. of serum which remained unchanged from day 100 to parturition although the data indicate a slight rise towards the end of pregnancy.




Fig. 16.21. Concentration of human chorionic gonadotiophin in the blood of the normal pregnant woman. The hormone levels were determineti b>' the male frog test. (From A. L. Haskins and A. I. Sherman. J. CUn. Endocrinol., 12, 385, 1952.)


The significance of the excretion pattern and concentration of the hormone in the serum is still a matter of conjecture. Browne, Henry and Venning (1938) suggested that the peak level of chorionic gonadotrophin in the blood reflects an increased production and a physiologic need in order to maintain a functional corpus luteum during early pregnancy. Recent evidence has tended to confirm this opinion in that HCG has been found to be active in the maintenance of the secretory activity of the corjius luteum in the primate (Hisaw, 1944; Brown and Bradbury, 1947; Bryans, 1951 j. In addition, histologic studies reveal a direct proportion between the number of Langhans' cells and the amount of hormone excreted (Stewart, Sano and Montgomery, 1948; Wislocki, Dempsey and Fawcett, 1948) .

The possibility that the kidney plays a role in the changes in the concentration of HCG was investigated by Gastineau, Albert and Randall (1948) . The renal clearance was relatively constant throughout all stages of pregnancy although the urine and serum concentrations of the hormone varied as much as 20- fold. In addition, the mean, renal clearance found during pregnancy was not markedly different from that found in cases of hydatiform mole and testicular chorioma. Inasmuch as the renal elimination of the hormone remained constant, it was obvious that two possible explanations existed : these were (1) changes in the secretion rate, and (2) changes in extrarenal disposal of the hormone. Studies on the latter were contradictory. Whereas Friedman and Weinstein (1937) and Bradbury and Brown (1949) reported an excretion of 20 per cent and higher of HCG following the injection of HCG, Johnson, Albert and Wilson (1950) found an excretion of 5.8 per cent in pregnant women during the immediate postpartum period. Zondek and Sulman (1945) reported a 5 to 10 i)er cent elimination of HCG in the urine of animals. Thus Bradbury and Brown felt that there is relatively little destruction or utilization of the hormone in the body; Wilson, Albert and Randall ( 1949) believed that 94 per cent of the circulating hormone is affected by extrarenal factors and that the fluctuating character of hormonal level in serum or urine depends entirely on changes in rate of hormone production.

An analysis of the distribution of chorionic gonadotrophin in the mother and fetus led Bruner (1951) to conclude that the ratio of maternal blood to urinary gonadotrophin is not constant although the ratio of gonadotrophin in the chorion to maternal blood is constant. Consequently, she concluded that the concentration of gonadotrophin in the urine does not depend entirely on the rate of production of the hormone and that the method of gonadotrophin elimination changes during pregnancy. She also pointed out that a significant amount of chorionic gonadotrophin is found in the fetus and that this is due to the fact that, although the chorion releases the hormone into the maternal blood, secondarily some of it passes the placental barrier and enters the fetal system across the wall of the chorionic vesicle.

2. Equine Goyiadotrophin (PAIS)

The presence of a gonadotrophin in the blood of the pregnant mare was first described by Cole and Hart in 1930. The hormone appears in the blood about the 40th day of pregnancy and increases rapidly to a concentration of 50 to 100 rat units (R.U.) per ml. by the 60th day of pregnancy (Cole and Saunders, 1935). This concentration is maintained for approximately 40 to 65 days. By day 170 it has fallen to a nondetectable level (Fig. 16.22).

Catchpole and Lyons (1934) suggested that the placenta is the source of the gonadotrophin and indicated that the chorionic epithelium is the probable source. Cole and



Fk;. 16.22. Tlie concentration of i)rognant mare's serum in the blood of the mare throug out pregnancy. (From H. H. Cole and F. J. Saunders, Endocrinology. 19, 199, 1935.)


Goss (1943), on the other hand, concluded that the endometrial cups are the source of the hormone. Recent evidence tends to confirm the endometrial cups as the source of the hormone (Clegg, Boda and Cole, 1954). The endometrial cups form in the endometrium opposite the chorion in the area where the allantoic blood vessels fan out. The cups develop precisely at the time when the hormone is first obtained in the serum of the pregnant mares and desquamation of the enclometrial cups is complete at the time of the disappearance of the hormone from the maternal blood. Analyses of the cups for gonadotrophin content reveal a correlation between the concentration of the hormone in the maternal blood and the concentration in the endometrial cups. Finally, histochemical stains for glycoprotein indicate the presence of this substance only in the epithelial cells lining the uterine lumen and the uterine glands in the cup area ( for complete discussion of the subject see the chapter by Wislocki and Padykulal.


VI. Pregnancy Tests

The discovery of gonadotrophic activity in the urine of pregnant women by Aschheim and Zondek in 1927 led to introduction of the first valid test for pregnancy (Aschheim and Zondek, 1928). These investigators used the innnature mouse and reported the presence of corpora hemorrhagica as indicative of the presence of a gonadotrophin in the urine and a positive reaction for pregnancy. The Aschheim-Zondek test for pregnancy was the first successful test of its kind and has been used both as a qualitative and quantitative test. In the latter instance, a serial dilution of the urine is made in order to obtain the minimal effective dose.

It is not too surprising that many tests for pregnancy have been described. In general, all of the successful tests involve the detection of chorionic gonadotrophin in the urine, and to some extent in the blood. The changes that have appeared in the development of new pregnancy tests have been those concerned with the use of different species of animals, the rapidity with which the test could be completed, and convenience to the laboratory. Thus the Friedman test (Friedman, 1929; Friedman and Lapham, 1931 ) followed soon after the Aschheim-Zondek test and in turn was succeeded by several newer tests.

Ap])roximately five reliable tests are now available (Table 16.6). All are concerned with the detection of HCG and have an accuracy of 98 to 100 per cent. The AschheimZondek suffers from a time requirement of 96 hours and was largely supplanted by the Friedman test that used the isolated rabbit and required only 48 hours. Within recent years several new tests have been reported using the frog, toad, and immature rat. Frank and Berman (1941) first noted the occurrence of hyperemia in the ovary of the immature rat, following the injection of HCG. Albert (1949) reported excellent results with the use of this test in 1000 cases. Comparison of the rat hyperemia test with the Friedman test was on the whole very good and revealed the same order of accuracy for both tests. The Friedman test, however, will detect about 5 I.U. of HCG which would mean a concentration of 500 I.U. of HCG per 24-hour output of urine ( assuming a 24-hour urine output of 1500 ml.). Positive results in the rat test require a 24-hour output of 1000 I.U., indicating that the ovarian hyperemia test in the rat is about one-half as sensitive as the Friedman test. Nevertheless, the rat



TABLE 16.6 Pregnancy tests with an accuracy


oj 98 to 100 per cent


Animal


Sex


Observed End Point


Time


Reference


Immature mouse Isolated rabbit Xenopus laevis Bufo arenarum Immature rat


F F F M F


Corpora hemorrhagica Corpora hemorrhagica Extrusion of ova Extrusion of sperm Hyperema of ovary


hr.

96

48 8-12 2-4

4


Aschheim and Zondek, 1928 Friedman and Lapham, 1931 Shapiro and Zwarenstein, 1934a Galli-Mainini, 1947 Frank and Berman, 1941



test requires only 4 hours and a larger number of animals can be utilized, thus decreasing the error due to use of inadequate numbers of animals. Comparison of the rat hyperemia and the Friedman tests revealed that the former is slightly more accurate but a little less sensitive (Albert, 1949).

Within two years after the publication of the Friedman test for pregnancy, Shapiro and Zwarenstein (1934a, 1934b, 1935) and Bellerby (1934) reported the use of the African toad {Xenopus laevis, D) in the diagnosis of pregnancy. Again the test was based on the ability of HCG to induce the extrusion of ova by the frog following the injection of the urine into the dorsal lymph sac. Extrusion of the ova occurred in 6 to 15 hours and the test was shown to compare favorably with both the Aschheim-Zondek and Friedman tests, although it did not give tiie graded response seen with the A-Z test (Crew, 1939). Weisman and Coates (1944) found an accuracy of 98.9 per cent with the Xenopus test over a 5-year period during which 1000 clinical cases were examined.

Galli-Mainini (1947) first reported the use of the male batrachian in the diagnosis of pregnancy and Robbins, Parker and Bianco (1947) simultaneously reported the release of sperm by Xenopus following treatments with gonadotrophins. Galli-Mainini (1948» pointed out that this reaction is not restricted to a single toad, but would ]irobably be found in many frogs and toads. He added that care should be used to employ animals with a continuous spermatogenesis. This was immediately confirmed by reports from different countries using various species of frogs and toads endogenous to the areas. Immediate use of Rana pipiens was reported in the United States and this species became very popular in that country ( Wiltberger and :\Iiller, 1948) .

The advantages of the sperm-release test are the time requirements, simplicity, end point, and opportunity to use many animals. On the other hand, the reaction is all or none and shows no gradation in degree of reaction. In general, the urine is injected into the dorsal lymph sac and the cloaca aspirated for sperm 1 to 3 hours later. Although this is the most recent of the pregnancy tests, many reports have appeared and some evaluation as to accuracy may be attempted. Galli-IVIainini (1948) reported an accuracy of 98 to 100 per cent in a summary of more than 3000 tests and 100 per cent accuracy for negative results in more than 2000 controls. Robbins (1951 ) reported an accuracy of 89.5 per cent in the first trimester of pregnancy. Pollak (1950) indicated that as many as 20 per cent of the negative tests obtained in the summer were false. This suggested the existence of a refractory state at this season. Bromberg, Brzezinski, Rozin and Sulman (1951) reported on a comparison of several tests including 700 cases. An accuracy of 85 per cent was obtained with the male frog test, 99 per cent with the rat hyperemia test, 98.5 per cent with the Friedman test and 98 per cent with the Aschheim-Zondek test. The authors indicate that the 15 per cent failures to get a positive reaction in the frog could be due in part to the poor sensitivity of the animal which could only be overcome by concentrating and detoxifying the urine. Comparison of the minimal amounts of HCG to elicit a positive reaction are Vs I.U. for the rat hyperemia test, 1 I.U. for the Aschheim-Zondek and Hyla tests, 2 I.U. for the Rana and Bufo tests, and 5 I.U. for the Friedman test. Reinhart, Caplan and Shinowara (1951) reported an accuracy of 99 per cent with 840 urine specimens; only 3 false negatives were noted in 346 specimens from known pregnant women and no false positives noted in 125 nonpregnant women. The authors attribute the high degree of accuracy to standardization of the procedure by which extraneous factors were eliminated. These include ( 1 ) the use of 2 or more 30- to 40-gm. frogs for each test, (2) elimination of all animals suffering from red leg and other diseases, (3) adequate time for sperm release, (4) concentration of the urine, (5) maintenance of frogs at 15 to 22°C., and (6) during the summer the injection of an increased volume of urine and an increase to 4 hours in the period for sperm release. The maintenance of frogs in a hibernating state by keeping them in a refrigerator at 38°F. has been rei:)orted to insure a high degree of sensitivity regardless of the season (Allison, 1954). Although it is obvious that more data are needed, the present results are very promising for the "frog-sperm" test and if the seasonal effect can be eliminated, this test will be the equal of the other four. VII. Water and Electrolyte Balance The changes in the various components of the blood during pregnancy have been described in a number of species (Tables 16.7 and 16.8). It is generally agreed that a marked increase in the blood and plasma volume and a decrease in the relative amounts of erythrocytes and hemoglobin occur during the last trimester. Inasmuch as the increase in the plasma volume in man exceeds the concurrent increase in the total cell volume, the resultant hemodilution produces an anemia which has been described as the "physiologic anemia of jM-egnancy.


TABLE l(i.7 The average percentage of change in the constituents of the blood and in the extracellular fluid volume during

normal pregnancy in man


Extracellular space


Adams, 1954.


Bibb. 1941

Caton, Robv, Reid and Gibson, 2nd, "l949

Caton, Roby, Reid, Caswell. Maletskos, Fluharty and Gibson, 1951


Chesley, 1943

Dieckmann and Wegner,

1934a, b, c, d

Ferguson, 1950


Freis and Kenny, 1948

Friedman, Goodfriend, Berlin

and Goldstein, 1951

Ganguli, 1954


Gemzell, Robbe and Sjostrand, 1954


Hamilton and Higgins, 1949. .

Jarosova and Daum, 1951 ...

Lambiotte-Escoffier, Moore and Tavlor, Jr.. 1953

Lund, 1951

McLennan and Corey, 1950..

McLennan and Thouin, 1948. .

Miller, Keith and Rownetree, 1915

Mukherjee and Mukherjee, 1953

Roscoe and Donaldson, 1946.

Thompson, Hersheimer, Gibson and Evans, Jr., 1938 . . .

Tysoe and Lowenstein, 1950 .

White, 1950


59


25


15


Blood volume


49 None


45


23


30


59


32


Plasma volume


22


49


55


25


32


Total hemoglobin


20


59


20


Hematocrit


10


15


10


12


16


RBC

count


14


21


22


Gram % hemoglobin


25


15

25% in 50% of patients


10


Time of Determinat in Pregnancy


6th week antepartum 3rd trimester

3rd trimester


60 days antepar

tum 3rd trimester

3rd trimester


3rd trimester

At term

Throughout pregnancy

8th week antepartum

3 months antepartum

9th monlli

10th lunar month 3rd trimester 10th lunar month At term

3rd trimester

3rd trimester 3rd trimester

9th lunar month

At term

3rd trimester


GESTATION


989


TABLE 16.8


The


average percentage of cl


aiu/e in


the constituents


of the blood and


in the extracellular fluid



volume during norn


al pregnancy in


variouf


laboratory and domestic animals



Increase


Decrease



Animals





Total hemoglobin





Author



Blood


Pasma


Cell


Hema

RBC


Gram %




volume


volume


volume


tocrit


count


hemoglobin




%


%


%


%


%


%




Rat







40


20-40


Beard and Mvers, 1933



41




20


10 29


18 30 25


7 31 33


Bond, 1958 Newcomer, 1947 van Donk, Feldman and Steenbock, 1939


Rabbit


50





17


20


10-20 17


Salvesen, 1919

Zarrow and Zarrow, 1953



6


12






12


13


Horger and Zarrow, 1957


Sheep


25


22


10





Initial decrease, normal at term


Barcroft, Kenned}- and Mason, 1939


Cow


Slight


Slight








Reynolds, 1953


Comparable changes were observed in the blood constituents and plasma volumes of the rat and rabbit during the latter third of gestation. Although there is no increase in the total cell volume, the resultant "physiologic" anemia of pregnancy in the rabbit follows the same general pattern as that rei)orted in the human being (Horger and Zarrow, 1957).

A significant decrease in the erythrocyte number, hemoglobin concentration, and hematocrit, and an increase in the blood volume have been noted in the rat during pregnancy (Table 16.8). However, the increase in blood volume is correlated with an increase in body weight and the ratio of blood volume to body weight remains unchanged (Bond, 1948). Calculation of the total number of erythrocytes and grams of hemoglobin actually showed an increase in these constituents during gestation, indicating that the anemia of pregnancy in the rat is due to a hemodilution in which the blood volume increases proportionately faster than the number of erythrocytes.

Comparable results were also reported in the rabbit (Zarrow and Zarrow, 1953). A marked drop in the relative number of circulating erythrocytes and percentage of hemoglobin is seen invariably towards the end of gestation (Fig. 16.23). A marked fall in hematocrit occurs concomitantly with the fall in the two blood constituents along with an increase in the reticulocytes. The time of onset of the increase in reticulocytes is variable and seems to occur during the second trimester of gestation. Their number returns to normal before parturition in spite of the increasing severity of the anemia. A second rise in the reticulocytes is seen during the first week postpartum. All the other constituents return to normal values during the first or second week postpartum.



Fig. 16.23. Changes in the relative number of circulating erythrocytes, reticulocytes, percentage of hemoglobin, hematocrit, and nonprotein nitrogen of the blood of the rabbit during pregnancy and after parturition. (From M. X. Zarrow and I. G. Zarrow, Endocrinology, 52, 424, 1953.)




Disagreement exists as to whether there is a change in the volume of the extracellular fluid compartment during pregnancy in the human being. Whereas certain investigators have reported rather marked increases in the extracellular space (Chesley and Chesley, 1941; Chesley, 1943; Freis and Kenny, 1948; Caton, Roby, Reid and Gibson, 1949; Friedman, Goodfriend, Berlin and Goldstein, 1951; Jarosova and Damn, 1951), others have reported that the changes in this fluid compartment are proportional to changes in the body weight (LambiotteEscofiier, Moore and Taylor, 1953; Seitchik and Alper, 1954). The results obtained in the rabbit support the findings of the latter authors as no disproportionate increase in the thiocyanate space was observed during pregnancy in the rabbit. The slight increase that occurred during the last trimester of gestation was in good agreement with the fluid accumulation by the developing fetus. Similarly the increase in blood volume in the rat is correlated with increase in body weight.



Fig. 16.24. Changes in blood plasma and total erythrocyte volume in the ovariectomized rabbit treated with 1 mg. estradiol daily. (From L. M. Horger and M. X. Zarrow, Am. J. Physiol., 189, 407, 1957.)



Fig. 16.25. Changes in the blood plasma and total erythrocyte volume during pregnancy. (From L. M. Horger and M. X. Zarrow, Am. J. Physiol., 189, 407, 1957.)


Thus the anemia of pregnancy as observed in the rabbit and rat is very similar to that reported for man. It can be characterized as a normochromic and normocytic anemia. Although a decrease in the relative concentrations of hemoglobin and erythrocytes occurs, the total amounts of these components of the blood remain unchanged. Consequently, the anemia of pregnancy is due to a hemodilution.

The anemia induced by treatment with estradiol is similar to the anemia of pregnancy in many respects. Witten and Bradbury (1951) treated 16 women with 5 mg. estrone or 0.4 mg. estradiol dipropionate and noted an erythrocyte drop of 14.8 per cent, a hemoglobin drop of 8.5 per cent, a hematocrit drop of 15 per cent, and a blood volume increase. Treatment of the castrated rabbit with 1 mg. estradiol daily caused a 20 per cent decrease in both erythrocyte count and hemoglobin with no significant changes in total hemoglobin or number of erythrocytes. Estradiol also caused an increase in plasma and blood volume (Fig. 16.24) which was comparable to that seen during pregnancy (Fig. 16.25), but no significant change in cell voluiiic was obtained. The estradiol-induced anemia is both normochromic and normocytic and is caused by a htMnodilution. However, in addition to the ciianges in the blood and plasma volumes, estradiol induces a significant increase in the thiocyanate space. Furthermore, only the massive dosage of 1.0 mg. estradiol per day elicits an anemia comjKirable to that observed in pregnancy. This dosage level is probably toxic since there is a decrease in the body weight of most rabbits which received this treatment. Thus, in spite of the similarities of these anemias, it is likely that estrogen is not the sole etiologic agent in the anemia of pregnancy.

Progesterone alone at dosages of 4 mg. daily has little effect on the plasma volume or the thiocyanate space. This steroid does exert a significant influence on the action of estradiol on the blood and plasma volume, but it is to be noted that rather large dosages of estradiol were still needed to induce a significant hypervolemia and that the effect depends on the ratio of the concentration of the two hormones. The hypervolemia induced by the treatment with 4 mg. progesterone in combination with 0.1 mg. estradiol was greater than that caused by the estradiol alone, whereas the treatment with 4 mg. progesterone in combination with 1.0 mg. estradiol resulted in an inhibition of the estrogenic activity (Fig. 16.261. Thus progesterone may play a dual role in the water metabolism of the gravid female. In the presence of low titers of estrogen, progesterone augments its action which may be a means of insuring an adequate fluid retention to provide for the fluid requirements of the fetus. However, if the titers of the estrogens and possibly of other steroids affecting salt and water metabolism became excessively high, the progesterone may provide a protective measure by inhibiting the activity of these substances. This concept is in accord with reports describing the diuretic action of progesterone in the iiypophysectomized rat (Selye and Bassett, 1940) and the inhibition of the salt- and water-retaining action of DOCA and cortisone by progesterone (Landau, Bergenstal, Lugibihl and Kascht, 1955).


Fig. 16.26. Changes in the blood volume of the ovariectomized rabbit treated with 0.1 mg. and 1.0 mg. estradiol daily and with a combination of the two estrogen treatments and 4 mg. progesterone. (From L. M. Horger and M. X. Zarrow, Am. J. Pliysiol., 189, 407, 1957.)

It is also of interest that no anemia was observed in animals treated with various combinations of estrogen and progesterone (Horger and Zarrow, 1957). Progesterone elicits an increase in the cell volume which api^roximates that of the plasma volume. Because no erythrocyte counts were made in this study, it is not possible to state whether this increase in the cell volume is caused by a macrocytosis or an increase in the number of erythrocytes. Vollmer and Gordon ( 1941 ) reported that progesterone caused an increase in the erythrocyte count of the rat but that the action was inconsistent. Hence it is possible that the increase in the cell volume is due to an enhancement of hematopoiesis by the progesterone. This possibility is not inconsistent with the absence of an increase in the reticulocyte count in response to these treatments since a reticulocytosis usually occurs only after an intense stimulation of the hematopoietic tissue such as by hemorrhage.

In view of the previous discussion, it is improbable that the anemia of pregnancy is due entirely to the interaction of estrogen and progesterone. These hormones appear to play an important role in the salt and water metabolism of the gravid female. Furthermore, it is noted that the cow exhibits a hypervolemia but no anemia during pregnancy (Reynolds, 1953) and that a similar condition is produced in the rabbit by the treatment with various combinations of these steroids. Hence the interaction of estrogen and progesterone may be responsible for this species difference.

Inasmuch as no antidiuretic hormone (ADH) could be detected in any of the plasma samples, it is apparent that the plasma titers of ADH did not rise above 10 fiV. per ml. during the experimental period. However, in view of the increased ability of the blood to inactivate ADH during pregnancy (McCartney, Vallach and Pottinger, 1952; Croxatto, Vera and Barnafi, 1953), there may be an increased rate of turnover of ADH during gestation. Consequently the data obtained in this study neither substantiate nor eliminate ADH as an etiologic agent in the anemia of pregnancy.

A number of investigators have attributed the hypervolemia of pregnancy to structural changes in the circulatory system. Burwell (1938) observed a marked similarity between the circulatory changes observed in pregnancy and those observed in a patient with an arteriovenous fistula. He noted that in both conditions there is an increase in the blood volume, cardiac output, pulse rate, pulse pressure, and an increased venous pressure near the opening of the fistula. He concluded that the changes in the circulation of the pregnant woman are caused by an arteriovenous leak through the placenta and the obstruction of the venous return by the enlarged uterus. Bickers (1942) correlated the intensity of the edema of the right or left leg with the location of the placenta in the uterus. The edema was observed to be consistently greater on the same side as the location of the placenta whereas the edema of the legs was equal when implantation occurred on the anterior or posterior wall of the uterus. However, when the uterus was lifted off the interior vena cava, there was no precipitous drop in the venous pressure in the femoral vein. Thus this study supports the arteriovenous shunt theory of Burwell.


One objection to this theory is that it i

does not account for the decrease in the i

blood volume during the 10th lunar month j

of pregnancy. However, it has been reported i

that during the latter part of pregnancy there is an increase in the resistance to the flow of blood through the placenta. This increase is due to the increased number of villi and to the anastomizing of the villi in the placenta. Since an increase in the peripheral resistance to blood flow results in a hemoconcentration, this would account for the decrease in the blood volume during the last lunar month of gestation (Kline, 1951; McGaughey, 1952).

Other objections to the arteriovenous shunt theory were reported by Kellar ( 1950) who found that blood flow through the placenta is sluggish rather than rapid as in an arteriovenous shunt. He also observed that the uterine venous blood is not exceedingly rich in oxygen as is the venous return of an arteriovenous aneuryism and lie concluded that, although the uterus is an area of decreased resistance to blood flow, the effect is not entirely due to the placenta. He suggested that thyroxine may be partially responsible for the expansion of the blood volume since mild thyrotoxicosis is common in pregnancy. This concept is supported by the observation that there is a tendency for vasodilation in the upper extremities during the latter months of gestation (Burt, 1950). Furthermore, the basal metabolic rate increases during this period (Sandiford and Wheeler, 1924; Rowe and Boyd, 1932). It is to be noted, however, that the changes in the blood flow in the extremities are closely correlated with the cardiovascular changes occurring during pregnancy but not with the changes in the hormonal levels in the blood (Herbert, Banner and Wakim, 1954 ) .

Since there is no disproportionate increase in the thiocyanate space of the rabbit during pregnancy, the increase in the blood volume can best be explained on the basis of cardiovascular changes during the latter part of gestation. However, in view of the previous discussion, it is extremely improbable that this hypervolemia is induced by any one factor. Rather, it is more probable that the condition is produced by a multiplicity of factors. On the basis of the previous reports, it is evident that the placenta, due to its similarity to an arteriovenous aneuryism, is partially responsible for the hypervolemia. The marked increase in the uterine size and vascularity during pregnancy (Barcroft and Rothschild, 1932) will also account for a considerable amount of the increase in the blood volume. In addition, the tendency for dilation of the peripheral blood vessels may account for another portion of the increase in the blood volume. Thus, in general, the hypervolemia of pregnancy can be attributed primarily to structural changes in the circulatory system.

It cannot be denied, however, that the cardiovascular system is influenced by the changes in the endocrine balance during gestation. It is well known that the placenta elaborates large amounts of sex steroids and corticoids. It has also been suggested that there is an increased production of thyroxine and ADH at this time. In addition, water-soluble extracts of the pregnant sow's ovaries have been shown to cause water retention and anemia in the rabbit (Zarrow and Zarrow, 1953). The resultant hormonal balance becomes somewhat precarious as the additional secretions of the glands tend to build up the blood titers of the sex steroids and other substances which influence water metabolism. It is possible that when a proper balance of these factors is maintained, the pregnancy is normal and the various requirements of the fetus are provided without disrupting the distribution of the body fluids outside of the vascular system. However, if the balance is not maintained, the animal tends to accumulate fluid, and edema and other pathologic complications result.


VIII. Plasma Proteins

It has long been known that the plasma proteins play a significant role in the fluid balance of the organism and as such are also involved in water balance during pregnancy (]\Iack, 1955). In addition, the plasma proteins are of importance in many other functions, such as heat and energy source and replacement of tissue in which function they act as a protein source whenever needed and form the metabolic pool. These proteins are synthesized in general in the liver and reticuloendothelial system and may be classified as albumins or globulins although many different entities of these two classifications are known to exist.


The maintenance and stabilization of blood volume and the equilibrium of fluid exchange between the extravascular and intravascular compartments is a function of the albumin fraction of special significance in i)regnancy, in addition to its other functions of acting as carrier for other substances and sui^jilying of nutrients. The regulation of blood volume by albumin depends on its osmotic action and is of much greater significance than the globulins. Approximately 4.6 gm. albumin and 3.17 gm. globulin per 100 ml. of plasma are found in the normal, nonpregnant woman. At least four types of glol)ulins are present in the plasma among which are found the lipoproteins, prothrombin, fibrinogen, antibodies, and several hormones.


Although both the plasma proteins and albumin drop during pregnancy, this does not necessarily indicate a drop in the total available albumin protein. An increase in the plasma volume compartment of 25 per cent as seen in pregnancy could easily result in an increase in the total amount of circulating protein. However, as the total circulating blood volume increases in pregnancy, the albumin fraction and yglobulin seem to be diluted whereas the other globulins become more concentrated. Nevertheless, the globulins cannot compensate for the albumin loss and the total protein decreases. Mack (1955) has listed several possible explanations for the above paradox : ( 1 ) the small albumin molecule may diffuse more freely into tissues and across placental membrane, and (2) albumin synthesis cannot keep pace with utilization.


Innumerable studies on the plasma i^-oteins of women during pregnancy have I'evealed markedly consistent changes in the albumin-globulin ratio of the plasma. The concentration of total protein and albumin decreases while the total globulin increases. The trend is apparent by the first trimester and continues throughout gestation. A return to the nonpregnant pattern is seen shortly after parturition. The total protein dropped 13 per cent and the albumin 26 per cent. The various globulin fractions showed a rise except for the y-globulin (Mack, 1955). As a result of these changes, the albumin-globulin ratio declines throughout pregnancy and shows the well known reversal (Fig. 16.27) and recovery to normal by 6 weeks postpartum.



Fig. 16.27. Progressive decreu.se in the albuminglobulin ratio of the plasma during pregnancy in women. (From H. C. Mack, The Plasma Proteins in Pregnancy, Charles C Thomas, Springfield, 111.. 1955.)



Fig. 16.28. Changes in the glomerular filtration rate throughout pregnancy in the woman. (From W. J. Dignam, P. Titus and N. S. Assali, Proc. Soc. Exper. Biol. & Med., 97, 512, 1958.)


Although it is obvious that the albumin fraction is important in maintaining the blood fluid compartment, the changed albumin-globulin ratio cannot solely account for the retention of water and edema present in pregnancy. Although it has been argued that the hypoalbuminemia through diminished colloid osmotic pressure is the cause of water retention in the tissues, the occurrence of the postpartum diuresis at the time when the albumin is lowest would tend to indicate some other mechanism (Dieckmann and Wegner, 1934a-d). Additional mechanisms, such as changes in the hormone level, especially the sex steroids and adrenal corticoids, may be responsible.

IX. Renal Function

Studies on renal function during pregnancy have resulted in contradictory reports. The earlier investigations failed to show any effect of pregnancy on renal function (Chesley and Chesley, 1941; Welsh, Wellen and Taylor, 1942; Dill, Isenhour, Cadden and Schaffer, 1942) , whereas recent studies indicate a marked change in renal function during gestation (Bucht, 1951; Dignam, Titus and Assali, 1958). Part of the explanation for the divergent results could be the type of patient studied, the periods when studied, and the types of controls. Dignam, Titus and Assali studied both the renal plasma flow and glomerular filtration rate in various patients throughout gestation and immediately following l^arturition. Care was taken to select individuals without any history of cardiovascular or renal disease. Both the renal plasma flow and the glomerular filtration rate (Fig. 16.28) were increased throughout gestation. The initial rise was extremely marked during the 1st and 2nd trimesters of pregnancy. A slight rise was noted during the 3rd trimester and a return to normal by 6 to 8 weeks postpartum.

Recently, de Alvarez (1958) reported a 50 to 60 per cent rise in the glomerular filtration rate and a 60 per cent rise in the renal plasma flow during the 1st trimester of pregnancy in the human being. This is in agreement with the findings of Dignam, Titus and Assali (1958). However, de Alvarez reported, in addition, a progressive decline in both the glomerular filtration rate and renal plasma flow during the 2nd and 3rd trimesters. The filtration factor (glomerular filtration rate divided by the renal plasma flow) remained low in the first 2 trimesters and increased in the last trimester. This is evidence for an increase in tubular resorption of water and electrolyte. It can only be concluded, therefore, that kidney function is altered during pregnancy, especially the 1st trimester. Results from investigations involving the 2nd and 3rd trimesters are contradictory. De Alvarez concludes that the changes in renal hemodynamics during pregnancy are mediated by the endocrine system because the alterations in renal function seem to be related to the sodium and water retention. If the changes are progressive throughout gestation, it would be possible to correlate the phenomenon with a number of hormones that increase during pregnancy. On the other hand, if the phenomenon is transient, i.e., only during the 1st trimester, then the phenomenon can only be correlated with HCG.

X. Enzymes

A. Histamixase

The presence of histaminase or diamine oxidase in tissues of the body has been known for some time. As yet the enzyme lias not been crystallized but is believed to he a flavoprotein (Swedin, 1943). The enzyme is not specific for histamine because it inactivates other diamines such as cadaverine and putrescine. Histaminase determinations, in general, are based on incubation of the test material with histamine dihydrochloride for a fixed period of time and the bioassay of the residual histamine carried out on an isolated strip of guinea pig intestine.

Histaminase has been found in the l)lasma of men and women with an increase (luring ])regnancy from a value of between 0.003 and 0.008 /^g. per ml. per hr. to a value of between 3.5 and 10 at parturition (Ahlmark, 1944, 1947). This has been confirmed by Swanberg (1950), who determined the histaminolytic activity in pei-iplK>ral blood throughout pregnancy (Fig. 16.29). A marked rise is observed from the 10th to the 20th week of pregnancy, and thereafter the concentration plateaus until after parturition.


Fig. 16.29. Tlie histaminase activity of the peripheral blood of the human female during pregnancy (•) and at parturition (®). (From H. Swanberg, Acta scandinav., Suppl. 79, 23, 1950.)

Both the maternal placenta and the decidual tissue have been identified as major sites for formation of the enzyme. Danforth and Gorham (1937) reported the presence of histaminase in the placenta of a series of patients at term. This was confirmed by Swanberg (1950) who, in addition, separated the placenta by a series of slices parallel to the surface of the organ and reported that the layer adjacent to the uterine wall, consisting of practically only the thin decidual membrane, contained a mean value of 614 /xg. per gm. per hr. of histaminase as compared to 38 for the fetal portion of the placenta. Confirmation of the concept that the maternal placenta is the main source of histaminolytic activity can be obtained from the finding of histaminase in decidual tissue of nonpregnant females and in the maternal placentas of animals. In cases in which maternal and fetal placentas can be separated easily, the maternal placenta contained from 14- to 100-fold the activity seen in the fetal placenta. Comparison of the histaminolytic activity in the decidual tissue of the sterile horn and the control pregnant horn of the uterus of a rabbit revealed 319 fig. per gm. per hr. and 222 fxg. per gm. per hr., respectively. Treatment with progesterone or induction of jiseudopregnancy caused a marked rise in the histaminase of the endometrium to upwards of 1000 fig. per gm. per hr. Nonetheless, histaminase was not observed in the blood plasma of the progesterone treated rabbits whereas progesterone treatment of two nonpregnant women caused a marked rise iti plasma histaminase.

The physiologic significance of histaminase is still unknown. A consideration of this problem must take into account not only the action of the enzyme and changes in its concentration under different physiologic conditions, but also the species problem. In regard to the latter point, the data are extremely inadequate. Only two species have been studied in any detail and these are the human being and the ral)bit. One can conclude from the available data that histaminase is produced by the maternal placenta, decidua, and uterine endometrium. It increases with pregnancy in these tissues and its concentration may be correlated with the progestational hormone. It increases in the blood of tlie human being, I'at, and guinea pig during pregnancy but nut in the cat or rabbit (Swanberg, 1950; Carlsten, 1950). The obvious hypothesis that histaminase })rotects the uterus from the stimulating action of histamine has not been confirmed. But it is somewhat jiai'adoxical to note that urinary histamine also increases during pregnancy. Kahlson, Rosengren and Westling (1958) reported a daily 24-hour excretion of 18 to 43 /xg. of histamine during the first 2 weeks of pregnancy in the human being. A marked increase was noted on the 15th day with a peak of 123 to 835 /xg. per 24 hr. at the peak of excretion which occurred 1 to 2 days before parturition. As yet no role can be attributed to this substance. It is of interest that the increased histaminase present during pregnancy can serve the role of protecting the uterus from the musclecontracting action of this substance. Because the amount of urinary histamine excreted is correlated with the number of young and no changes are apparent in the concentration of histamine in the tissues during pregnancy, it would seem that the excessive formation of histamine during the last trimester of pregnancy takes place in the uterus and its contents and the basic action of histaminase is protective.

It was shown recently that the excessive formation of histamine during the last trimester of pregnancy in the rat is due to an increase in the rate of histidine decarboxylase activity (Kahlson, Rosengren, Westling and White, 1958). Inasmuch as removal of the fetuses without other interference with the pregnancy abolishes the increased urinary histamine, it can l)e concluded that the site of formation is in the fetus. This histamine could escape into the maternal circulation and eventually be eliminated via the kidneys.

Roberts ( 1954) reported that aminoguanidine leads to a general disturbance of pregnancy in the rat; large doses tended to jiroduce death of the mother and smaller doses tended to kill all or part of the litters and some of the mothers. Again one could conclude a protective action on the part of histaminase dui'ing tlie latter i)art of i^regnancy.

B. Carbonic Anhydrase

Carbonic anhydrase was discovered by Aleldrum and Roughton in 1933 and soon shown to catalyze the following reaction, H,CO, z:± CO. + HoO. The enzyme was found to occur in many tissues and was generally located within the cell especially in cells possessing a secretory function. The discovery by Lutwak-Mann and Laser (1954) that carbonic anhydrase is present in tlu' uterine mucosa led to a thorougli study of the changes in the concentration of the enzyme and the factors controlling its presence (Lutwak-Mann, 1955; Lutwak-Mann and Adams, 1957). The enzyme has been found to be present in the reproductive tract of a wide variety of mammals. In general, the uterine endometrium, placenta, and Fallopian tubes are the main loci of activity although there are marked differences among different species. Carbonic anhydrase activity was found consistently in all the animals studied such as the rat, hamster, guinea pig, rabbit, pig, and ewe. No activity was noted in the uterine mucosa of the nonpregnant animal except the ewe and the rabbit. In several species, such as the cow, human being, and pig, carbonic anhydrase was also found in the Fallopian tube.

A marked rise in carbonic anhydrase of the endometrium of the rabbit was noted during the first trimester of pregnancy


(Fig. 16.30). The value rose from a prepregnancy level of 20 enzyme units (E.U.) per gm. of fresh tissue to a maximum of 100 E.U. per gm. at approximately the 8th day of pregnancy. This level was maintained until the 12th day and then declined to approximately the prepregnancy level by about the 20th day. Examination of the placentas at this time revealed marked activity, 68 E.U. per gm. of maternal placenta and 25 E.U. per gm. of fetal placenta. The curve for the concentration of carbonic anhydrase in the uterine mucosa during pscudopregnancy is essentially the same as that seen during pregnancy, although some minor differences exist.

It is obvious from the above data and from the evidence involving the increased concentration of carbonic anhydrase in the uterine mucosa following treatment with progesterone, that the enzyme is probably under the control of the luteoid hormone. Indeed, an excellent correlation has been shown between the degree of {progestational proliferation in the uterus and the concentration of carbonic anhydrase. In the ewe, however, the carbonic anhydrase of the uterus is independent of the ovary. A possible explanation for this discrepancy between the two species has been offered on the basis of differences in the blood level of progesterone. However, no explanation is forthcoming for the failure to maintain the carbonic anhydrase level throughout pregnancy in the rabbit, even though the circulating progesterone remains liigh.

The significance of this enzyme in the physiology of reproduction is still unknown. From the data on the rabbit, it miglit be inferred that the carbonic anhydrase contributes to the maintenance of bicarbonate in the blastocyst fluid. The universal presence of the enzyme in placental tissue could also lead to the assumption that carbonic anhydrase is involved in fetal metabolism. Lutwak-Mann (1955) indicates that the enzyme might be involved in the transmission of calcium across the placenta. ^^'hether carbonic anhydrase is essential for fetal (Icvelopment and successful pregnancy is still unanswered. Treatment with carbonic anhydrase inhibitors (Diamox) failed to affect adversely the pregnancy or fetuses in pregnant rats even though no enzyme acti\'ity was present either in the matei'nal blood or placenta.



Fig. 16.30. Carhonic anhydrase activity in the uteiu.s of the rabbit during pregnancy, i),seudopregnancy, large doses of gonadotrophin, and pregnant mare's serum (PMS). Pregnancy, • •; i^seudopregnancy, O O; gonadotrophin, D D: PMS, x" X. (From C. Lut \val<-Mann. .1. Kndocrinol.. 13, 26. 1955.)



XI. Factors in the Maintenance of Gestation

A. Thyroid Gland

Several recent reviews have pointed out that the extract role of the thyroid gland in reproductive physiology is still in need of elucidation (Peterson, Webster, Rayner and Young, 1952; Reineke and Soliman, 1953). Numerous investigations over the past half century have definitely indicated that the thyroid gland is involved in reproduction but the site and manner of action are still not well known. In addition, contradictory reports indicate that each species and even each strain may have to be studied independently (Alaqsood, 1952). Some evidence foi- the involvement of the thyroid gland in gestation has already been considered. The increase in FBI at the onset of jiregnancy and the incidence of miscarriage in the human female when the FBI fails to rise tend to involve the thyroid hormone in the maintenance of pregnancy. Habitual abortion in women is usually associated with t'ithcr hypo- or hyperthyroidism (Litzenberg, 1926). Litzenberg and Carey (1929 » I'eported that in 70 married women with low basal metabolic rates appi'oximately 45 per cent had one or more abortions or stillbirthsrtf one eliminates the sterile woman from the group, the figure for women showing abortion or stillborn rises to approximately 35 per cent. However, the results are still controversial both with regard to data obtained within a single species and from different species.


Hypothyroidism in the rat induced by the prolonged administration of thiouracil resulted in a resorption of the fetus in 100 per cent of the cases (Jones, Delfs and Foote, 1946). Rogers (1947) reported a reduction in litter size following sulfaguanidine and Krohn and White (1950) reported a reduction in litter size following thyroidectomy in the rat. Thyroidectomy early in pregnancy caused a resorption of the fetuses and if performed at a later stage in pregnancy resulted in the birth of stillborn young (Chu, 1945). Following the induction of pregnancy in thyroidectomized rabbits, either a resorption of the young or abortion or prolongation of gestation was noted and the newborn young were usually dead. Chu concluded that the thyroid hormone was concerned with the vitality and growth of the embryos during gestation. In the pig the average duration of pregnancy was 114 days for normal gilts and 124.5 days for thiouracil-treated animals. In addition, the controls farrowed an average of 8.67 pigs per litter compared with 3.25 per litter for the thiouracil-treated .sows ( Lucas, Brunstad and Fowler, 1958 ) . The difference was significant in both instances. Bruce and



Fig. 16.31. Tlie effect of tliyioid deficiency on litter size. O, 422 litters from tliyroid-defieient mice; •, 423 litters from normal control mothers. (From H. M. Bruce and H. A. Sloviter, J. Endocrinol., 15, 72. 1957.)


Sloviter (1957) pointed out that part of the conflicting reports on the role of the thyroid in gestation might be due to the different methods used in producing a thyroid-deficient state. Surgical removal of the gland generally results in the loss of the parathyroids which may be also important in the maintenance of gestation (Krichesky, 1939), although adequate information is lacking. The use of antithyroidal substances offers more serious objections because these drugs not only pass through the placenta but they are nonspecific and interfere with other glands such as the adrenal cortex (Zarrow and Money, 1949; McCarthy, Corley and Zarrow, 1958), with nutrition, and with the general status of the animal. Consequently, Bruce and Sloviter preferred to establish a thyroidectomized state in mice by the use of radioactive iodine after establishing the dose necessary to induce total destruction of the thyroid without damage to the parathyroid or ganiete.s.

Although ( lorbman ( 1950 ) rei)orted a complete loss of reproductive activity in the mouse following treatment with P'*\ Bruce and Sloviter (1957) reported no effect on the ability of the mouse to conceive or bear young. This discrepancy could be due in part to the strain differences in the sensitivity of the ovary to the I^^^. Bruce and Sloviter (1957), however, noted a decrease in the average litter size of thyroid-deficient mice (Fig. 16.31 ). The data indicate a maximum of 6 young per litter in thyroid-deficient mice versus 10 young per litter for the normal mice. It is apparent that the entire curve for the litter size of thyroiddeficient mice is shifted toward a smaller size. This has also been observed in the rat following thyroidectomy (Nelson and Tobin, 1937). The thyroid-deficient mice also showed a prolongation of gestation as reported in rats, guinea pigs, and sows. Of the thyroid-deficient rats, 46 per cent showed a gestation period of more than 19 days whereas only 15 per cent of the normal controls showed a gestation period of more than 19 days whereas only 15 per cent of the normal controls showed a gestation period of more than 19 days (Table 16.9). Analysis of the data based on grouping according to litter size showed clearly an effect of litter size on length of gestation. The smaller litter size gave a higher inci(l(>nce of prolonged gestation.

Studies on oxygen consumption in the guinea i)ig revealed a slight but significant rise of 8 per cent at the end of gestation (Hoar and Young 1957). The increase in oxygen consumption is consistent but slight for the first 60 days of pregnancy after which the significant increase occurs (Fig. 16.32). The rise continued until 5 days postpartum and then fell rapidly. In a second set of experiments oxygen consumption was measured in control, thyroidectomized, and thyroxine-injected, pregnant guinea pigs. Measurements were taken at the time of mating and at parturition. In all three instances, an increase in the oxygen consumption was noted at parturition as compared with the values at the time of

TABLE 16.9

Ejfcct of thyroid-deficiency and litter size on length

of gestation in mice

(From H. M. Bruce and H. A. Sloviter, J. Endocrinol., 15, 72, 1957.)



Thyroid-deficient


Control


No. of

Young In

Litter


No. of pregnancies


> 19 days


No. of pregnancies


> 19 days


No.


Per cent


No.


Per

cent


1-5

6-9 10-14

Totals


36 40 20

96


24

13

7

44


67 33 35

46


16

38

28

82


7 3 2

12


44

7 8

15


mating (Fig. 16.33). Again the control guinea pigs showed a 7.9 per cent gain in oxygen consumption by the end of pregnancy, but both the thyroidectomized pregnant guinea pigs and the thyroxinc-treated guinea pigs also showed an increase in oxygen consumption of 11.9 and 16.2 per cent, respectively. The increase in oxygen consumption was not paralleled by increases in heart rate; actually the heart rate decreased in several instances. In addition, neither the weight of the thyroid gland nor the histology of the gland was changed during pregnancy. It is obvious then that an explanation for the rise in oxygen consumption during pregnancy may not involve the thyroid gland. On the basis of changes in its appearance. Hoar and Young (1957) suggested the possibility that the adrenal cortex is involved and that the increased oxygen consumption is due to an increased release of adrenal corticoids. More evidence is needed before this suggestion can be fully accepted.


Further work from the same laboratory has led to the concept that one locus of action of thyroxine during pregnancy is at parturition (Hoar, Goy and Young, 1957). These investigators used an inbred strain of guinea pigs that is characteristically hypothyroid and a genetically heterogeneous stock in which the level of thyroid activity is presumed to be higher. It had been previously shown that pregnancy wastage was high in the hypothyroid guinea pigs. Treatment with thyroxine reduced the percentage of stillborn from 40 to 13.6 in the hypothy




Fig. 16.32. Oxvgen consumption in the guinea pig during gestation. (From K. M. Hoar and W. C. Young, Am. J. Physiol., 190, 425, 1957.)



Fig. 16.33. Oxygen con.suinption in the pregnant guinea pig treated with thyroxine or thvroiclpctomized before mating. (From R. M. Hoar and W. C. Young, Am. J. PhysioL, 190, 425, 1957.)


roid guinea pig!>, i.e., to a level !?een in the untreated heterogeneous group. Treatment of the heterogeneous group with thyroxine not only failed to reduce the percentage of stillborn but actually increased the abortion rate particularly in the 2nd and 3rd trimesters. The most consistent result, however, was a decrease in length of gestation following treatment with thyroxine, and an increase following thyroidectomy. From these experiments it was concluded that the thyroid hormone facilitates parturition and need be present only late in gestation to exert its action.

It is apparent that in some species the thyroid hormone is involved directly in pregnancy. In the absence of the hormone, certain species tend to resorb or to abort; or if pregnancy is maintained gestation tends to be lengthened. This is probably due to an interference with the mechanism of parturition. In certain species such as the guinea pig only a parturitional problem has been demonstrated; in others an entire galaxy of symptoms may be present. Reduction in the size, number, and viability of the young give added emphasis to an essential role for thyroxine in the phenomenon of gestation.


B. Adrenal Cortex

Removal of the adrenal cortex without further treatment invariably leads to disturbances in rejM'oductive i)hysiology and the termination of pregnancy. Although the early results were controversial in that some investigators reported that adrenalectomy failed to affect gestation in the rat (Lewis, 1923; Ingle and Fisher, 1938), others reported that adrenalectomy led to abortion (Wyman, 1928; Dessau, 1937) or to some other disturbance of gestation (McKcown and Spurrel, 1940). Davis and Plotz (1954) adrenalectomized two groups of pregnant rats on the 4th to 6th and the 14th to 16th day of pregnancy. Abortion occurred in all 12 rats adrenalectomized during the first half of pregnancy whereas only 1 of the 12 adrenalectomized during the second half of pregnancy aborted. However, even in those adrenalectomized during the second half of gestation, an effect on jiregnancy was observed. A significantly higher incidence of stillborn and sickly young (14.4 per cent) and a marked decrease in the weight of the fetuses were noted (Table 16.10).

Early results indicated that extracts of the adrenal cortex could readily replace the absent adrenal gland and maintain successful pregnancies. Within recent years it has been demonstrated that many steroids such as cortisone and 9a-chlorohydrocortisone at 10 /xg. per day (Llaurado, 1955) permit fecundation and successful maintenance of pregnancy. Successful maintenance


GESTATION


1001


TABLE Ki.lO

Effects of adrenalectomy on the character of the litter,

and on fetal body weight and adrenal weight

(From M. E. Davis and E. J. Plotz,

Endocrinology, 54, 384, 1954.)



Pregnant Controls


Adrenalectomy 2nd Half of Pregnancy


Percentage versus Pregnant Controls


No. of litters ....


21


11



Dead and





"sickly" young. Vigorous voung . . .


5

182


13

78


<0.01


Fetal hodv weight





(gm.)


6.13


5.22


<0.01



(=b0.10)« 


(±0.30)"



Fetal adrenal





weight (mg.). . . .


0.49()


0.554


>0.3



(±0.017)"


(±0.041)« 



Fetal l)()dv weight/





fetal adrenal





weight X 1000. . .


12.35


9.42


<0.01



(0.34±)


(±0.31)"



Calculation of standard error of the mean:


S.E.


/ Ed-^

]/ nin


1)


of a pregnancy has also been reported in an adrenalectomized human female maintained on hydrocortisone 9a-fliiorohydrocortisone (Laidlaw, Cohen and Gornal, 1958). In this instance measurements of urine excretion of aldosterone revealed an increase to 4.4 fxg. per 24 hours during the last trimester of pregnancy and a postpartum value of 0.5 fig. Inasmuch as the value is only 1/10 of that seen in a normal pregnancy the authors concluded that the adrenal cortex of the mother is the major source of aldosterone during pregnancy and that a high output is not a major prerequisite for a normal pregnancy.

Treatment with either 0.9 per cent saline drinking water or with cortisone increased the number of successful pregnancies following adrenalectomy during the first half of gestation. Pregnancy was normal in 8 of 11 adrenalectomized rats (Davis and Plotz, 1954). Treatment with 2 mg. of cortisone acetate resulted in successful pregnancies in 13 of 14 rats adrenalectomized on the 4th to 6th day of gestation and 12 of 12 rats adrenalectomized on the 14th to 16th day of gestation. However, complete maintenance was not obtained. The body weight of the mothers and the weight of the fetuses were significantly lower than in the controls, and the number of stillborn and sickly young was increased.


A comparison of the pregnancy-maintenance activity in a number of adrenal corticoids indicated that a combination of a glucocorticoid and mineralocorticoid provides the best protection in the adrenalectomized rat (Cupps, 1955). Nulliparous rats were adrenalectomized, placed on treatment, and mated. Under these conditions the adrenalectomized controls and the rats treated with desoxycorticosterone acetate failed to become pregnant inasmuch as no implantation sites were obtained (Table

TABLE 16.11

Effect of adrenal steroids on reproduction in adrenalectomized female rats

(From P. T. Cupps, Endocrinology, 57, 1, 1955.)


Daily Treatment


Control

Cortisone acetate ^i mg

Cortisone acetate ^i mg

Cortisone acetate 1'^ mg

Cortisone acetate 2,4 mg

Hydrocortisone acetate Vi mg.. . ,

Cortisone acetate Vi mg. plus

Desoxycorticosterone acetate ^ mg

Desoxycorticosterone acetate ^^ mg

Desoxycorticosterone acetate ^ mg

Desoxycorl icosterone acflate 1 mg ,

Adrenalectomized control


No. of Rats


No. Born Alive

(average)


Implantation Sites (average)


7


8.2


11.0


6


3.5'>


5.6^


6


3.6*


6.2'^


5


3.5


8.6"


5


5.8


10.0


7


5.0


8.5"


5


9.0


9.6


4





5





4





5





Weight Change during Pregnancy (average)"


gm. 46.4

-30. S

-1.5«  17.2^ 12.8^ 30.7


44.6


"Weight change of mother from day of breeding to day after parturition. " Significant at 0.05 level. '= Significant at 0.01 level.


1002


SPERM, OVA, AND PREGNANCY


16.11). Treatment with 2.5 mg. cortisone acetate per day was partially effective in restoring reproductive capacity. Injections of 1.25 mg. hydrocortisone acetate per day gave results comparable with those obtained when cortisone was given, although the ratio of young born alive to implantation sites indicated that hydrocortisone acetate was more effective. It was definitely more effective than cortisone acetate in maintaining the body weight of the mother. However, reproduction was completely restored to normal in the adrenalectomized rat following treatment with desoxycorticosterone acetate and cortisone acetate.

Interference with gestation in the normal animal has been reported by several investigators following treatment with ACTH or adrenal corticoids (Courrier and Colonge, 1951; Robson and Sharaf, 1952; Velardo, 1957). This is taken to indicate that there is a finely balanced requirement for adrenocortical hormones during gestation ; and that suboptimal or supra-optimal amounts of the hormone interfere with pregnancy. Courrier and Colonge found that cortisone administered to intact rabbits in the second half of pregnancy interfered with gestation. Robson and Sharaf treated both pregnant rabbits and mice with ACTH and reported a marked effect on gestation. Abortion or resorption occurred in 8 of 9 mice and in 8 of 11 rabbits. Contamination by posterior pituitary hormones or gonadotrophins can be excluded. A subsequent experiment with cortisone also caused marked interference with pregnancy in the rabbit when 20 mg. were given ; 10 mg. were without effect. Administration of cortisone to castrated or hypophysectomized pregnant rabbits maintained with progesterone also caused damage to the pregnancy. Since the hormone was not acting by way of the ovary or pituitary gland, the authors felt that cortisone was acting directly on the uterus and the uterine contents.

In the rat, however, ]Meunier, Duluc and Mayer (1955) observed an effect on pregnancy only when cortisone acetate was injected at the time of mating. Rats injected with 10 to 25 mg. cortisone acetate daily for 5 to 6 days beginning on day 12 or day 14 of gestation had a normal pregnancy.


Velardo (1957) reinvestigated the problem in the rat and reported a marked reduction in litter size and an increase in the number of stillborn following ACTH treatment. Although quantitative differences appeared, a significant decrease in litter size w^as observed only when the hormone was given (1) before mating, (2) immediately after mating, or (3) between the 11th and 15th day after mating. However, the greatest effect was noted when the ACTH was administered immediately after mating. Surprisingly enough, litter size was markedly reduced only if adrenalectomy was performed on day 7 of gestation. Adrenalectomy on day 8 to 14 of gestation had no effect on live litter size. However, a total of 6, 9, and 13 stillbirths were obtained following adrenalectomy on days 8, 9, and 11. It is interesting that the number of stillbirths decreased from 21 following adrenalectomy on day 7 to none following adrenalectomy on day 14. It is apparent that the adverse effects of adrenalectomy on gestation decrease as pregnancy progresses. It is also apparent from these and other experiments that the action of ACTH is mediated by the adrenal cortex. From these results and others described above, it seems likely that the adrenal corticoids may be acting on the uterus.

Mayer and Duluc (1955) found that adrenalectomy of the I'at on the 14th to the 16th day of pregnancy led to variable results. In 17 pregnant adrenalectomized rats, gestation was terminated in 8, but no interference was observed in 9. The rats that failed to maintain pregnancy died witiiin 2 to 3 days. Again it would appear that delicate hormonal balances are involved. In a further investigation of this problem Aschkenasy-Lelu and Aschkenasy ( 1957) reported that a diet adequate in salt and proteins would prevent interference with pregnancy in rats adrenalectomized before mating. On a low protein diet, pregnancy could be maintained only in the intact rat (80 per cent) and then only if daily injections of progesterone were given. These authors believe that the role of the adrenal corticoids in pregnancy is concerned with stimulation of appetite and mobilization and degradation of proteins to amino acids. The latter action would permit the replacement of body protein in the absence of a normal jirotcin intake.


C. Pancreas

The impact of diabetes mellitus on the course of pregnancy has been of interest to the clinician for many years. In a recent review of the subject, Reis, DeCosta and Allweiss (1952) came to the conclusion that "the carefully controlled diabetic aborts no more frequently than the nondiabetic." On the other hand, it has been well known for many years that uncontrolled diabetes and pregnancy are basically incompatible (Eastman, 1946).

Studies in the rat have given controversial results with regard to the influence of insulin on pregnancy. Davis, Fugo and Lawrence (1947) reported that in the alloxan diabetic rat pregnancy was normal for the first 12 days. Thereafter death of the fetuses occurred followed by resorption. Sinden and Longwell (1949) and Levi and Weinberg (1949) reported no detrimental effect from diabetes on the course of i^regnancy. The latter group obtained 12 pregnancies from 25 rats made permanently diabetic with alloxan. Eleven of the 12 rats went to term and delivered normal fetuses and 1 died during pregnancy. Recently, Wells, Kim, Runge and Lazarow (1957) reported a 14 per cent loss in fetal weight, an increase in gestation length from a normal of 538 to 563 hours, and an increase in fetal or neonatal mortality in the pregnant rat made diabetic by pancreatectomy or treatment with alloxan.

In general, the clinical data indicate that uncontrolled diabetes has a detrimental effect on pregnancy, but that the abortion rate in the controlled diabetics approaches that seen in the normal" population. Since the crux of the matter seems to hinge on the severity of the diabetes, one might conclude that the effect of insulin is an indirect one by virtue of its action in maintaining a good metabolic state. The conflicting reports from animal experimentation may be due to the differences resulting from uncontrolled environmental and dietary factors.


D. Ovary: Progesterone, Estradiol, and Relaxin

Marshall and Jolly (1905) were probably the first to point out that ovariectomy during pregnancy leads to abortion or resorption of the fetuses in the rat. Subsequently, a number of investigators repeated these experiments and confirmed the findings in all species tested thus far, provided ovariectomy is performed before implantation. Removal of the ovaries after gestation is well under way, however, does not disturb the course of pregnancy in all species. The human being, monkey, horse, ewe, and cow are examples of species not dependent on the ovary for the maintenance of pregnancy once it has been well established. Species such as the rabbit and the rat require the presence of the ovary throughout pregnancy.

The importance of progesterone for i)regnancy was established by Allen and Corner (1929) who first showed that an extract of the corpus luteum will maintain pregnancy in the castrated rabbit. Identification of the active substance in the extract as progesterone led to the use of the hormone in many other species. Allen (1937) reported that crystalline progesterone was inferior to the crude luteal extract in the maintenance of pregnancy in the castrated rabbit. From these and other data, such as the enhancing action of estrogen on the progesterone-induced progestational reaction, he inferred that a combination of estrogen and progesterone should be superior to progesterone alone in the maintenance of pregnancy. However, he pointed out with proper caution that the dosages would have to be carefully regulated because estrogen could also antagonize progesterone. Although Robson (1936) failed to enhance the action of progesterone with estrone in the pregnant hypophysectomized rabbit, Pincus and Werthessen (1938) obtained enhancement with both the androgens and estrogen. Whereas the early work indicated that a pregnancy maintenance dose of progesterone varied from 0.5 to 2 mg. (Allen and Corner, 1930), later experimentation indicated that the dosage varied with the stage of pregnancy. An adequate dose of approximately 1 mg. progesterone in the early stages of pregnancy needs to be increased to 5 mg. in the later stages (Allen and Heckel, 1939; ComTier and Kehl, 1938a, b). These investigators also revealed that an optimal effect could be obtained by using a progesterone-estrogen combination in the ratio of 750 to 1. Chang (1951) transferred ova to nonovulated intact rabbits and noted that massive doses in the order of 25 mg. macrocrystalline progesterone injected for three times were required to obtain a 50 per cent maintenance of pregnancy. He also reported that under the conditions of his experiment an initially high dose was needed for the passage of the ova, implantation, and early maintenance. Since then, further experimentation, especially on other species, has revealed a significant role by estrogen in enhancing the pregnancy-maintaining action of progesterone.

A vast literature exists for the human being on the prevention of threatened abortion by progesterone which is beyond the scope of this review. Variation from negative results to excellent maintenance is reported. It is obvious that a great deal of variability exists here and, to some extent, this is explained by a need for more objective criteria in evaluating threatened abortion and the therapy (Guterman and Tulsky, 1949). It is obvious that if the

TABLE 16.12 Maintenance of pregnancy in the rat castrated on

the 12th day of gestation

(From J. Yochim and M. X. Zarrow, Fed. Proc,

18, 174, 1959.)



Progester







one







No.



Estradiol Daily


Implantation Site


No. of Fetuses


No. of

Fetuses

Alive


Preg

Rats


Daily dose


No. daily


nancy Index



mg.



Mg.






4





40


37


37


0.925


4


2


1



47


32


27


0.574


7


1


2



76


12


2


0.026


9


1.5


2



99


61


49


0.495


7


2


2



85


65


63


0.741


6


1


2


0.1


69


50


48


0.696


5


1.5


2


0.1


51


48


46


0.900


5


2


2


0.1


60


55


54


0.900


threatened abortion were the result of some disturbance other than progesterone, that progesterone therapy might be without success. Indirect evidence for the need for progesterone to maintain a successful pregnancy in the human being and for the lack of need for the corpus luteum once pregnancy is established has been presented by Tulsky and Koff (1957). Corpora lutea were removed from day 35 to day 77 of pregnancy in 14 women. Two of the women exhibited spontaneous abortion and a marked drop in pregnanediol excretion. The remaining 12 maintained a normal pregnancy and pregnanediol excretion. The data can be interpreted to indicate a need for progesterone during pregnancy and that this need can be met by a nonovarian source, i.e., the i)lacenta.


In both the rat and mouse, successful maintenance of pregnancy after castration has been obtained with progesterone or a combination of progesterone and estrogen. However, partial maintenance following castration can be obtained in the rat under special circumstances. Haterius (1936) removed all the fetuses except one and left all placentas intact. Under these conditions the remaining fetus was carried beyond term. Alexander, Fraser and Lee (1955) found that castration of the rat on the 9th day resulted in 100 per cent abortion, whereas 60 per cent of the fetuses were retained until term if castration was on the 17th day. Dosage of progesterone as high as 5 to 10 mg. daily following castration the 9th day gave only partial maintenance. It is possible that better results would have followed multiple daily injections. Yochim and Zarrow (1959) castrated rats on day 12 of gestation and obtained a pregnancy index (no. of fetuses alive at day 20 h- no. of implantation sites at day 12) of 0.741 when 2 mg. progesterone were gi^'en in two divided daily doses and 0.495 when 1.5 mg. progesterone was given (Table 16.12). However, the addition of 0.1 /^.g. estradiol daily markedly enhanced the action of the progesterone so that a pregnancy index of 0.9, i.e., equivalent to the normal controls, was obtained with 1.5 mg. progesterone.

Finally, Hall (1957) has indicated that relaxin synergizes with estradiol and progesterone in the maintenance of pregnancy in the castrated mouse. One nig. progesterone per day maintained pregnancy in 83 per cent of the mice castrated on day 14 of gestation, but 0.5 mg. maintained pregnancy in only 30 per cent of the animals. The addition of 1.5 ^g. estradiol per day was without effect. On the other hand, the addition of relaxin to the estradiol and 0.5 mg. progesterone gave pregnancy maintenance in over 80 per cent of the mice as compared with 30 per cent when progesterone alone was given.

Smithberg and Runner (1956) induced ovulation and mating in prepubertal mice (age 30 to 35 days) and obtained 100 per cent implantation with 0.5 to 1 mg. progesterone daily and approximately 90 per cent successful pregnancies when 2 mg. progesterone were given. A comparison of the amount of progesterone required for maintenance of pregnancy in the normal and castrated prepubertal mouse is given in Figure 16.34. In an interesting application of the information available on the induction of ovulation and maintenance of pregnancy, Smithberg and Runner (1957) were able to obtain successful pregnancies in genetically sterile, obese mice.

Haterius (1936) observed that distortion of the fetus occurred following ovariectomy in the rat. This has been confirmed by Zeiner (1943) in the rat and by Courrier and Colonge (1950) in the rat and rabbit. It was noted that castration greatly compressed the fetuses and eventually caused death. Courrier and Colonge (1950) in very elegant experiments showed that removal of the rabbit fetus into the peritoneal cavity prevented the distortion and death which ordinarily followed castration. Frazer (1955) obtained similar results in the rat and concluded that fetal death after castration of the mother follows a rise in intrauterine pressure which is associated with an increased tone of the circular uterine muscle fibers. Consequently the increased survival of the extra-uterine fetuses following ovariectomy in the mother is the result of the removal of this pressure by the circular muscle of the uterus.


Fig. 16.34. Daily dose of progesterone required to maintain pregnancy in the normal and castrated prepubertal mouse. (From M. Smithberg and M. N. Runner. J. Exper. Zool., 133, 441, 1956.)

Many investigators have demonstrated that gestation can be prolonged by inhibiting parturition. Both the injection of large doses of progesterone or the formation of a new set of functional corpora lutea during pregnancy will prevent parturition. The injection of an ovulating dose of HCG on the 25th day of pregnancy in the rabbit delayed parturition for 15 days after the injection, i.e., until the 40th day of gestation (Snyder, 1934). The fetuses survived in utero for only 3 days and grew to greater than normal size during this period. The placentas persisted until day 41 of gestation. Comparable results were obtained following daily injections of progesterone into pregnant rabbits (Zarrow, 1947a). Haterius (1936) obtained prolongation of pregnancy in the castrated rat by removing all the fetuses except one, leaving all placentas intact. Recently a comparable experiment was performed in tlie rabbit with intact ovaries (Hafez, Zarrow and Pincus, 1959). In 2 of 10 rabbits, live fetuses were obtained l)y cesarean section on day 36. However, in 8 of the 10, delivery was delayed beyond day 36, although some degree of fetal resorption was present in all instances. Prolongation of pregnancy in the rat was obtained by the injection of prolactin (Meites and Shelesnyak, 1957), but only if the ovaries were present.


E. Pituitary Gland

In general, hypophysectomy before midpregnancy leads to resorption. This is especially true of the rat and mouse. On the other hand, hypophysectomy at midpregnancy or later does not interfere in the maintenance of gestation in these species (Pencharz and Long, 1933; Selye, Collip and Thompson, 1933a, b; Pencharz and Lyons, 1934 ) . In the dog, ferret, and rabbit, hypophysectomy leads to abortion (Aschner, 1912; McPhail, 1935a; White, 1932), whereas the results in the cat seem contradictory (Allan and Wiles, 1932; McPhail, 1935b) .

Hypophysectomy of the rhesus monkey does not always interfere with pregnancy. Smith (1954) obtained normal pregnancies in 10 of 20 hypophysectomized rhesus monkeys. The remaining animals aborted. Although more data are needed, it seems that the pituitary gland can be removed very early in gestation without disturbing the pregnancy. Whereas hypophysectomy before midterm invariably leads to abortion or resorption in the rat or mouse, 1 of the 4 monkeys hypophysectomized between the 29th and 34th day of gestation carried its young to term. Inasmuch as Hartman and Corner (1947) showed that the placenta secretes sufficient progesterone by the 25th day of gestation to maintain pregnancy, it is apparent that the placenta in the monkey is able to maintain its endocrine secretory activity independent of the pituitary and at a sufficiently high level to replace the ovary.

Little, Smith, Jessiman, Selenkow, van't Hoff, Eglin and Moore (1958) reported a successful pregnancy in the 37-year-old woman hypophysectomized the 25th week of pregnancy. The mother w^as maintained on thyroid, cortisone, and pitressin tannate replacement therapy. The excretion of chorionic gonadotropin and pregnandiol was not markedly different from that seen in normal gestation. Estrogen excretion was slightly reduced and the 17-hydroxy corticosteroids dropped to zero when cortisone therapy was discontinued. It would seem that this phase of adrenocortical activity was reduced and that ACTH or corticoidlike substances from the placenta were inadequate. No interference in aldosterone output was observed.

Hypophysectomy on the 10th day of gestation in mice terminated the pregnancy in only 3 of 19 animals (Gardner and Allen, 1942). Sixteen mice carried their litters to term although 7 of the 16 had a difficult and prolonged parturition. Body weight curves were normal and the corpora lutea appeared unaffected by the loss of the pituitary gland, indicating either the independence of the corpus luteum or the presence of a placental luteotrophin. Marked involution of the adrenal cortex was noted in all instances.

Simultaneous measurements of the concentration of cholesterol in the adrenal gland and ACTH in the pituitary of the rat revealed a drop in adrenal cholesterol and pituitary ACTH on the 15th day of gestation (Poulton and Reece, 1957). This was followed by a marked increase of both substances on the 21st day of pregnancy and a sharp drop at parturition. The authors concluded that a gradual increase occurs in the secretory activity of the adrenal cortex which reaches a peak on the 15th day of pregnancy in the rat. Thereafter the activity decreased until parturition when a marked increase was observed. The initial decrease in pituitary ACTH potency followed by an increase after day 15 is interpreted as an initial increase in ACTH release followed by a decreased release. The decrease in pituitary ACTH potency at parturition is compatible with the marked increase in adrenocortical activity at this time if the decreased pituitary ACTH activity is interpreted as indicative of ACTH release.

Maintenance of pregnancy in rats hyl')ophysectomized early in pregnancy was obtained with prolactin by Cutuly (1942), although Lyons, Simpson and Evans (1943) reported negative results with a purified prolactin. However, a partial maintenance of pregnancy was obtained with purified prolactin and estrone.


F. Placenta

The placenta is not only involved in the synthesis of hormones during pregnancy but also in the transfer of substances between mother and fetus. It is obvious that the transfer of substances is limited and the l^lacenta does offer a barrier. This problem bears not onlv on the matter of fetal nutrition, but also on the fetal environment and as such is important in the sexual development of the fetus (see chapter by Burns) .


The presence of estriol in the urine of newborn male infants has led to the conclusion that estrogens can pass through the placenta because of their low molecular weight (Diczfalusy, Tillinger and Westman, 1957). Studies on the transfer of estrogens across the placental barrier in the guinea pig with C^'^-labeled estradiol revealed an extremely rapid disappearance of radioactivity from the maternal blood following intravenous injection of the hormone into the mother, and the appearance of large amounts of water-so.luble radioactivity in the fetal plasma (Dancis, Money, Condon and Levitz, 1958). However, no estradiol was found in the fetal plasma. Replacement of fetal circulation with a perfusion system indicated that estradiol did not j^ass the placenta although estriol was readily transferred in both directions. These authors reported that the placenta was relatively impermeable to the water-soluble estrogens found in the urine, wliich are essentially glucuronides.

The discovery in 1927 of large amounts of estrogens and gonadotrophins in the blood and urine of pregnant w^omen led to the cjuestion as to whether the placenta is a gland of internal secretion. This can be answered with an uneciuivocal yes. Nevertheless, several questions are still unanswered: (1) the number of hormones produced by the placenta, (2) the quantities, and (3) the secretory activity of the placenta in different species.

Data on the presence of gonadotrophins in the placenta have already been discussed. At least three different types of gonadotrophins have been extracted from the placentas of the human being, mare, and rat. These have been defined physiologically and appear to be different in the three species. Cole and his co-workers have identified the endometrial cups as the source of PJVIS in the mare, whereas the elegant experiments of Stewart, Sano and Montgomery (1948) indicate that HCG in the human being is secreted by the Langhans cells. These investigators grew human placental cells in tissue culture and obtained ^ gonadotrophin in the culture. They also noted a direct correlation between the growth of the Langhans cells and the production of gonadotrophic hormone (see also the discussion of this subject in the chapter by Wislocki and Padykula).

The initial discovery of a progressive rise in the secretion of adrenal corticoids in pregnancy (Venning, 1946) has been confirmed by numerous investigators. Gemzell ( 1953) attributed the steady rise to a stimulation of the adrenal glands by excessive amounts of estrogen present during pregnancy and to hyperactivity of the fetal adrenals. The hypertrophy of the fetal adrenal cortex in the rat following adrenalectomy of the pregnant mother was first reported by Ingle and Fisher in 1938 and confirmed by Walaas and Walaas (1944), and Knobil and Briggs (1955). However, the 17-ketosteroid and corticoid level of fetal urine is very low (Day, 1948; Jailer and Knowlton, 1950) as are the 17-hydroxycorticosteroids in the blood of the newborn infant (Klein, Fortunato and Papados, 1953). ACTH-like activity has been found in extracts of the placenta (Jailer and Knowlton, 1950; Tarantino, 1951; Opsahl and Long, 1951) and corticoid activity has been found in the placenta of horses and human beings, as demonstrated by the glycogen deposition and growth-survival test in adrenalectomized rats (Johnson and Haines, 1952). Berliner, Jones and Salhanick (1956) isolated 17a-hydroxy corticoids from the human placenta.

Pincus (1956) reported that ACTH can stimulate steroidocorticogenesis in the perfused placenta. Using the ascorbic acid depletion test, Assali and Hamermesz (1954) assayed the blood in the intervillous space and the chorionic villous tissue for ACTH. Good activity was observed in the blood from the intervillous spaces and in the tissue of the chorionic villi. Corticotrophic activity was also obtained by Lundin and Holmdahl (1957) from placentas obtained at full term, but the activity was small compared with that obtained from the pituitary gland.

The possible role of the fetal pituitary was investigated by Knobil and Briggs (1955) who noted that hypophysectomy of the mother prevented the fetal adrenal weight increase observed following adrenalectomy of the pregnant mother. However, complete atrophy of the adrenal gland was not observed in the pregnant mother if the conceptus was present. It was concluded that ACTH can cross the placental barrier and that the fetus or placenta or both produce a sufficient amount of ACTH, to influence the maternal adrenal gland in the absence of the maternal hypophysis. It is still questionable, however, whether these sources, i.e., placenta and fetal pituitary, are of sufficient magnitude to account for the increased release of adrenal corticoids. Hofmann, Knobil and Caton (1954) showed that the ability of the hypophysectomized nonpregnant rat to secrete a water load is not greater than that of the hypophysectomized pregnant rat. Hence the contribution of the fetal pituitary or j^lacenta to the corticoid pool is not of sufficient magnitude to influence water balance.


As with the gonadotrophins, the increased amounts of estrogen ancl pregnanediol during pregnancy were thought to be derived from the placenta. In 1933, Selye, Collip and Thompson presented evidence to indicate that the placentas of rats jiroduce both estrogen and gestagen. Many physiologic data have been accumulated to prove this point, but completely convincing evidence was obtained only when these hormones were identified in placental extracts and in fluid perfused through the placenta. Diczfalusy and Lindkvist (1956) identified estradiol in the placenta and the presence of progesterone was described by Salhanick, Noall, Zarrow and Samuels (1952) and by Pearlman and Cerceo (1952).

Perfusion experiments on human placentas have revealed that this organ secretes a number of steroids (Pincus, 1956). These include progesterone, desoxycorticosterone Cortisol, and a number of unidentified steroids. Addition of ACTH to the perfusate had no effect on the concentration of Cortisol, but it did increase the concentration of the reduced corticosteroids, namely, the tetrahydro derivatives of cortisone and Cortisol. This was interpreted as a stimulation of the placenta by ACTH resulting in an increased release of the corticoid as demonstrated by the increase in the degradation products.

The identification of the placenta as a source of both sex steroids and certain gonadotrophins clarifies the manner by which jiregnancy can be maintained in certain species in the absence of the pituitary gland or ovary (see sections above on ovary and pituitary gland). Newton and Beck (1939) and others showed the hypophysectomy of the pregnant mouse does not precipitate abortion. Studies of the ovary reveal that, if the placentas are retained, the corpora lutea remain normal but removal of the placentas causes immediate degeneration of the corpora lutea (Deanesly and Newton, 1940). A comparable situation appears to exist in the rabbit and rat ; it is assumed, therefore, that the placenta takes over control of the corpus luteum in pregnancy in those species that require the ovary for successful gestation. In other species, such as man, sheep, cattle, and guinea pig, it seems that the placenta can supplant the ovary after pregnancy has progressed to a certain stage.


G. Pelvic Adaptation

The discovery that pelvic changes are under hormonal control in certain species was the result of extensive studies on pelvic adaptations associated with parturition (see reviews by Allen, Hisaw and Gardner, 1939; Hisaw and Zarrow, 1951). It has been argued that, in general, a narrow pelvis is present in mammals living in burrows. This would have the advantage of permitting an animal to turn within narrow confines, but a narrow pelvis would also interfere with the delivery of the young at parturition. As Hisaw pointed out in his extensive studies, this problem has been met by special adaptations on the part of different species. This has varied from a resorption of the cartilaginous pubic arch in the male and female mole iScalopiis aquaticus machrinus, Raf.) which is independent of the endocrine system (Hisaw and Zilley, 1927) to elongation of the pubic ligament which is directly under hormonal control (Hisaw and Zarrow, 1951).

The symphysis pubis of the pocket gopher, Geomys bursarius (Shaw), behaves as a female secondary sexual character so that a sex dimorphism exists in this species. The pubic cartilages ossify in both sexes and unite to form a complete pelvis with a rigid symphysis pubis. At this stage, the pelvis is too small for the passage of the young, but with the first estrus in the female, the pubic bones are gradually resorbed, leaving the pelvis open ventrally. The pelvis in the male remains intact (Hisaw, 1925). Treatment with estrogen alone can readily bring about the resorption of the pubic bones.

A third type of adaptive mechanism has been described in great detail in the guinea pig and led to the discovery of the hormone, relaxin. A sex dimorphism of the pelvis exists in the guinea pig, as in the pocket gopher, but in addition parturition is further facilitated by marked relaxation of the pubic ligaments and of the sacroiliac joint. Thus far extensive pelvic relaxation has been described in the guinea pig (Hisaw, 1926, 1929 », mouse (Gardner, 1936; Newton and Lits, 1938; Hall and Newton, 1946a), women (see review by Hisaw and Zarrow, 1951), and rhesus monkey (Straus, 1932; Hartman and Straus, 1939). No relaxation of the pubic symphysis has been reported in the ewe but a relaxation of the sacroiliac joint and an elongation of the sacrosciatic ligament was noted the 2nd to 3rd month of gestation. These changes increased as pregnancy progressed (Bassett and Phillips, 1955). Treatment with stilbestrol alone caused a marked loosening of the sacroiliac joint and the sacrosciatic ligament. The addition of relaxin to the treatment was without effect (Bassett and Phillips, 1954).

The role of relaxin in the relaxation of the pubic symphysis has been studied most extensively in the guinea pig and mouse. The work before 1950 was reviewed by Hisaw and Zarrow in 1951. The controversies (de Fremery, Kober and Tausk, 1931 ; Haterius and Fugo, 1939) as to whether such a hormone exists need not be discussed here, in detail, except to point out that the evidence supporting this opinion is more than adeciuate. Zarrow ( 1946, 1948) showed that pubic relaxation could be induced by estradiol alone, by a combination of estradiol and progesterone, or by relaxin in an estrogen primed animal (Table 16.13). The difference in the time required to induce relaxation, i.e., 23 days for estrogen alone, 13 days for estrogen and progesterone, and 6 hours for relaxin, and data indicating that progesterone caused the presence of relaxin in the blood of guinea pig only if a uterus was present led to the concept that pubic relaxation may be produced independ


TABLE 16.13

Relaxation of the symphysis pubis and relaxin content of blood, urine, and uteri of castrated and castrated,

hysterectomized guinea pigs after treatment with moderate doses of estradiol and progesterone

(From M. X. Zarrow, Endocrinology, 42, 129, 1948.)



Treatment, Daily


Average Relaxation Time


Relaxin Content


No. of Guinea Pigs


Estradiol


Progesterone


Total


After progesterone treatment


Blood serum


Urine


Uterus



MS

mg.



days


days


G.P.U./ml.


G.P.U./ml.


G.P.U./gm.


Castrated 9« 


10


1 from day


11


13.5

(13-14)


3.5


0.5


0.3


10


10


10


2 from day


11


13.0


3


0.5


0.5


10


10


10




(12-14)

23.7 (16.31)



Negative at 4 ml.


Negative at 5 ml.


Negative


Castrated, hys



terectomized 11


10


1 from day


11


23.7 (17-30)


13.7


Negative at 4 ml.


Negative at 8 ml.



10


10




25.6 (18-32)



Negative at 4 ml.


Negative at 4 ml.



One guinea pig not included in the table refiuired 22 days of treatment for pubic relaxation.


ently by estradiol (prolonged treatment) or relaxin (single injection). It is also possible to conclude that the action of progesterone is indirect and due to the formation of relaxin in the uterus (Zarrow, 1948; Hisaw, Zarrow, Money, Talmage and Abramovitz, 1944) . In the mouse, however, progesterone inhibits the action of relaxin on the pubic symphysis (Hall, 1949).

Further evidence that two hormones are involved in pubic relaxation was provided by histologic examination of the pubic ligament. Symphyseal relaxation following estrogen appeared to be due to a resorption of bone and a proliferation of loose fibrous connective tissue with an increase in mucoid alkaline phosphatase and water content (Talmage, 1947a, 1947b, 1950; Heringa and van der Meer, 1948). Relaxin produced a breakdown and splitting of the collagenous fibers into thin threads and a similar change was noted with progesterone (Talmage, 1947a, 1950).

Histochemical and biocliemical studies of the pubic symphysis have recently been reviewed (Frieden and Hisaw, 1933) and tend to show that relaxin produces specific changes. These include loss of metachromasia (Heringa and van der Meer, 1948) , accumulation of Evans blue m vivo, and increased solubility of the glycoproteins in the McManus-Hotchkiss reaction, all of wiiich indicate that a depolymerization of the ground substance and basement membrane glycoproteins had occurred (Perl and Catchpole, 1950) . Frieden and Hisaw (1951) found an increase in water content of the symphyseal tissue, but failed to find a decrease in the water-soluble hexose and hexoseamine following a single injection of relaxin. On the basis of a depolymerization of ground substance, a decrease should have occurred. However, repeated injections of relaxin led to a decrease in the insoluble hexoses and hexoseamines. In addition, consistent decreases in collagen content and trypsin-resistant protein content were noted. No hyaluronidase was found, but ^-glucuronidase was increased during relaxation. Gersh and Catchpole (1949) reported the presence of a collagenase from histochemical studies, but no confirmation has been forthcoming. Relaxin also has a protein


anabolic effect which occurs in the absence of pubic relaxation (Frieden, 1956). This action was demonstrated by the increased up-take of labeled glycine by the connective tissue proteins of the pubic symphysis. Recent experiments indicate that relaxin not only acts in conjunction with the female sex steroids but can also act alone (Brennan and Zarrow, 1959). However, it is apparent that the available data are still inadequate for a clear understanding of the mechanism of action of relaxin.

Relaxation of the pubic symphysis of the mouse has been studied in great detail by Hall. In a series of reports she showed that pubic relaxation occurs in the mouse during pregnancy and following treatment with estradiol and relaxin (Hall and Newton, 1946a, b). This was later confirmed by Kliman, Salhanick and Zarrow (1953). Contrary to the results reported following work on the guinea pig, progesterone not only failed to influence the effect of estrone on the pubic symphysis of the mouse, but progesterone also inhibited the action of relaxin. It was suggested that this inhibition is the result of an antagonism by progesterone on the action of relaxin and that a true species difference exists (Hall, 1949, 1955). Histologic studies revealed that changes in the pubic symphysis during pregnancy and after treatment with relaxin and estradiol are similar (Hall, 1947) . These changes consist of proliferation of articular hyaline cartilage, resorption of the medial ends of the pubes, lengthening of the pubic ligament by formation of new cartilage, and reversion of the cartilage to collagenous connective tissue. Hall (1956) suggested that estradiol causes a depolymerization of the mucopolysaccharides through enzymatic action resulting in a matrix sufficiently pliable to respond to the tensions set up by relaxin. Evidence presented in support of this concept was the loss of metachromasia and the increase in water. In addition, a two-step effect was seen with relaxin: (1) complete degradation of the matrix, and (2) the appearance of a gap in the cranial part of the cartilage produced by stretching of the symphyseal cleft. Some data in support of the latter part of this concept were presented by van der Meer (1954) who showed that inhihition of pelvic muscle tension inhibited relaxation in the guinea pig. In a similar type of experiment Crelin (1954) tied together the innominate bones of a mouse before pregnancy and obtained some dorsoventral displacement of the pubic symphysis but normal relaxation was inhibited.


H. Dilation of the Uterine Cervix

Dilation or softening of the uterine cervix in the pregnant woman at the time of labor has been known for a long time. This reaction has been used to determine whether delivery can be anticipated. Within recent years this phenomenon has been described in a number of animals and some analysis of the hormonal control of the reaction has been attempted.

Relaxation of the uterine cervix of the rat during pregnancy was first reported by de Vaal in 1946 and confirmed by Uyldert and de Vaal in 1947. Relaxation was measured by the insertion of a gauging pin into a cervix that had been removed and the diameter determined at the point where resistance is first felt. The measurements revealed a marked rise from approximately 3.5 mm. on the 17th day of pregnancy to 10 mm. at parturition. Recently, both Harkness and Harkness (1956) and Yochim and ZaiTow (1959) have taken in vitro measurements of the relaxation of the uterine cervix of the rat and observed marked relaxation during the latter part of gestation and at parturition. Yochim and Zarrow (1959) removed the cervix, suspended it from a rod and measured the stretch due to weights added at fixed intervals until the cervix broke. The amount of relaxation of the cervix was determined by the amount of stretch obtained with a weight of 50 gm. Under these conditions, the curve for relaxation of the cervix showed two sloi^es as pregnancy progressed (Fig. 16.35). The initial slope between day 12 and day 20 showed a rise of approximately 4 mm., with an extremely abrupt rise of 14 mm. on day 21. By 24 hours after parturition the degree of dilation had fallen to 3 mm. It is of interest that the curve for the tensile strength of the cervix (expressed in grams force necessary to tear 1 mg. cervical tissue in a rat weighing 100 gm.) was the opposite to that seen for cervical dilation. The tensile strength fell from approximately 50 gm. force to a low of 3 gm. at parturition and then rose during the postpartum period. The drop in tensile strength preceded the changes in the dilation of the cervix and was essentially completed 5 to 6 days before parturition or when the abrupt increase in dilatability of the cervix occurred.

Similar changes have been described in the dilatability of the cervix of the mouse (Steinetz, Beach and Kroc, 1957) with increased dilatability progressed beyond the 15th day (Fig. 16.36). The diameter of the cervix increased from a])proximately 2 mm. to about 5 mm. at delivery. It is apparent that the rate of the reaction, i.e., dilation, is much more rapid in the rat, although it is possible that the method of measurement is responsible for the differences.

The induction of cervical dilation by relaxin was reported by Graham and Dracy (1953) in the cow, and by Zarrow, Sikes and Neher (1954) in the sow and the heifer. Treatment with stilbestrol followed by relaxin caused a dilation of the uterine cervix of the gilt from % or % inch to 1 inch (Zarrow, Neher, Sikes, Brennan and Bullard, 1956). Measurements were made by the passage of aluminum rods, and, although the technique is not too exact, the differences are significant. Stilbestrol given alone or in combination with progesterone had no effect on the cervical dilation. On the other hand. Smith and Nalbandov (1958) have recently reported that estrogen treatment constricted the uterine cervix of the sow and that relaxin was without effect. A cue with respect to the mechanism of action of relaxin is given by the similarity of the action of relaxin on the pubic symphyseal ligament and the uterine cervix. In both instances, an increase in water content and a marked dei)olymerizatioii occurs.


Fig. 16.35. Dilation and ten.sile strength of the uterine cervLx of the rat during estrus, pregnancy, and 2 days postpartum. The dihition of the cervix in mm. of stretch per 50 gm. of added weight. The tensile strength is expressed in grams force necessary to tear 1 mg. cervical tissue in a rat weighing 100 gm. E = estrus; P = parturition. (From J. Yochim and M. X. Zarrow, Fed. Proc, 18, 174, 1959.)


Cullen and Harkness ( 1958) observed relaxation of the uterine cervix of the rat with estradiol alone, or with estradiol and progesterone, or with estradiol and relaxin, but maximal dilation was obtained only with a combination of estradiol, progesterone, and relaxin. In general Kroc, Steinetz and Beach (1959b) obtained comparable results in the rat. Estrogen alone caused some in crease in dilatability when 5 fxg. estradiol cyclopentylpropionate were given, and a decrease when 50 /Ag. were given. Progesterone had no consistent effect either alone or in estrogen-primed animals. Relaxin alone caused some softening of the cervix, but gave a maximal effect only when given with progesterone in estrogen-primed animals. Normal cervical dilation was also obtained in pregnant rats castrated the 15th day of gestation and maintained with progesterone, estradiol, and relaxin (Kroc, Steinetz and Beach, 1959; Yochim and Zarrow, 1959). Data on dilation of the uterine cervix of the mouse are rather sparse; nevertheless, softening of the cervix with relaxin has been reported (Kroc, Steinetz and Beach, 1959). It is not the purpose of this review to evaluate the data on cervical softening in the human female. The nature of the action of relaxin in the human female is controversial. Nevertheless, softening of the cervix following treatment with relaxin has been reported (Eichner, Waltner, Goodman and Post, 1956; Stone, Sedlis and Zuckerman, 1958) although McGaughey, Corey and Thornton (1958) reported no effect on the cervix following relaxin.



Fig. 16.36. Increased length of the pubic ligament, inciea.sed cervical dilatability, and increased responsiveness to oxytocin with the length of pregnanc.y in the mouse. L = lactating; NL = not lactating. (From B. G. Steinetz, V. L. Beach and R. L. Kroc, Endocrinology, 61, 271, 1957.)


XII. Uterine Myometrial Activity

The classical and well known description of uterine muscular activity has been more than adequately reviewed by Reynolds (1949). Since then Csapo and his colleagues have reported a series of elegant experiments involving the action of estrogen and progesterone on the uterine myometrium and have evolved the concept of "i)rogesterone block" in the control of uterine activity (1956a, 1956b). It has been shown that the ovarian steroid hormones regulate myometrial activity and that the uterine contractions are dependent on the relative amounts of the two hormones. Contractility is dependent basically on the concentration of the high energy phosphates which are maintained by estrogen w^iich in turn is involved in the synthesis of these substances (Csapo, 1950; Menkes and Csapo, 1952). Discovery of the staircase phenomenon in the uterine myometrium similar to that exhibited by cardiac muscle led to a marked difference between the action of estrogen and progesterone (Csapo and Corner, 1952 ) .


With decreasing freciuency of electrical stimidation in an isometric arrangement, tension decreased if the uterus was dominated by estrogen and increased if it was dominated by progesterone. Uteri from castrated rabbits were insensitive to the frequency of electrical stimulation. Thus estrogen induced a "positive staircase" response and progesterone a "negative staircase" response, although in the latter instance some estrogen is also present. These staircase responses have been used successfully as a measure of hormone domination and have been shown to be a function of the Na+ and K+ gradients across the myometrial cell membrane.

Uterine motility during estrus, the diestrum, and pregnancy has been described by many investigators in great detail (for a review see Reynolds, 1949). The diestrous uterus shows extremely slow, feeble, uncoordinated movements. The contractions may arise in any part of the uterus and extend in any direction. At estrus, the uterine contractions become rhythmic and sweep over


Fig. 16.37. Change from a positive to a negative staircase as the hormone dominance of the myometrium moves from the estrus to the progestational state after mating. X and O indicate the two strains of rabbits used. (From B. M. Schofield, J. Physiol., 138, 1, 1957.)


the uterine horn in a wave starting at the tubal end. Both amplitude and rate are increased. During pregnancy the uterus becomes relatively quiescent. In general this pattern of myometrial activity has been reproduced with both hormones, estradiol and progesterone.

Recently Schofield 11957), using the Csapo technique, has studied, in vivo, myometrial activity in the rabbit. In a series of experiments she was able to show in several strains of rabbits that, when mating occurs during estrus, the uterine myometrium is dominated by estrogen. Within 20 to 28


STAIRCASE Negative

Transient Positive


o


o o


o o


8x

OX


o







ox


X X X








o o o


ox

X



26 27 28 29 30 31 Day of pregnancy


32


Fig. 16.38. Change from negative through transient to positive staircase as the hormone dominance reverses at the end of pregnane}', indicating estrogen dominance. X and O indicate the two strains of rabbits used. (From B. M. Schofield, J. Physiol. 138, 1, 1957.)


hours after mating, the positive staircase effect passes through a transient effect to a negative effect indicating the development of progesterone dominance (Fig. 16.37j. This condition remained in effect throughout pregnancy until 24 hours before parturition when a reversion to estrogen domination was indicated by the positive staircase response ( Fig. 16.38) . Thus the progesteronedominated uterus is maintained throughout pregnancy and the uterus is nonreactive to oxytocin. Csapo (1956a) and others have shown that labor cannot be induced by oxytocin in the rabbit before day 30 of gestation, but 24 hours later, on removal of the progesterone block, 96 per cent of the rabbits delivered following treatment with oxytocin. He believes that the specific action of progesterone involves a blocking of the excitation-contraction coupling which is a consequence of the disturbed ionic balance in the myometrial cell. Thus a block is set up to the propagation of the contraction wave which can be removed only by a decrease in the level of progesterone.

The role of the water-soluble extract, relaxin in myometrial activity, is still uncertain. That an inhibition of estrogen-induced uterine contractions is obtained in certain species, such as the rat, mouse, and guinea pig, with relaxin preparations is un(luestionable. However, we still have not answered the questions as to w'hether this hormone plays a role in uterine contractions under normal physiologic conditions and whether the uterine contraction-inhibiting substance is relaxin or a contaminant of the relaxin extract.

Krantz, Bryant and Carr (1950) reported than an aqueous extract of the corpus luteum would produce an inhibition or decrease of uterine activity in the guinea pig and rabbit previously primed with estrone. This has been amply confirmed with both in vivo and in vitro preparations involving spontaneous contractions measured isometrically in the guinea pig (Felton, Frieden and Bryant, 1953; Wada and Yuhara, 1956; JMiller, Kisley and Murray, 1957) , rat (Sawyer, Frieden and Martin, 1953; Wada and Yuhara, 1956; Bloom, Paul and Wiqvist, 1958), and mouse (Kroc, Steinetz and Beach, 1959). However, Miller, Kisley and Murray (1957) failed to show any action of relaxin on uterine motility in the rabbit and the human being in vitro. Thus, the information on the rabbit is contradictory and a similar situation exists with regard to the human female for whom both positive and negative results have been reported following treatment with relaxin for threatened abortion (McGaughey, Corey and Thornton, 1958; Stone, Sedlis and Zuckerman, 1958; Eichner, Herman, Kritzer, Platock and Rubinstein, 1959). In briefly summarizing the action of relaxin on the uterine myometrium it should be pointed out that ( 1 ) relaxin inhibits uterine motility in an estrogen-primed animal, (2) the action may be species-limited, and (3) relaxin treatment docs not interfere with the action of pitocin.


XIII. Parturition

A. Progesterone

A number of theories have been suggested to explain the hormonal control of parturition. The most popular is that parturition is due to a decrease in the level of progesterone which allows oxytocin to exert its effect on the uterus. Evidence has already been presented indicating that pregnancy can be maintained in the castrated rabbit by an extract of corpora lutea, or progesterone, and even prolonged in rats (Nelson, Pfiffner and Haterius, 1930; Miklos, 1930), mice (Mandelstamm and Tschaikowsky, 1931), and rabbits (Zarrow, 1947a). Snyder (1934) and Koff and Davis (1937) prolonged gestation in rabbits by inducing the formation of new corpora lutea during the last trimester of pregnancy.

Knaus (1930) originally noted a marked antagonism between posterior pituitary extract and the corpus luteum hormone and Koff and Davis (1937) reported that in prolonged gestation induced by progesterone, posterior pituitary extract was ineffective until two days after the last injection. Csapo (1956a) performed a series of elegant experiments and concluded that progesterone blocks the uterine contractions, and that premature labor could not be induced with oxytocin before the 30th day of gestation in the rabbit except for a very small percentage of animals. This has been confirmed by Fuchs and Fuchs (1958).

Zarrow and Neher (1955) found the serum gestagen levels in the pregnant rabbit fell only after parturition was under way. Hence the problem arose as to how parturition could begin while a high blood concentration of gestagen was present. A partial answer was obtained in experiments by Csapo (1956b) and Schofield (1957) who showed that the progesterone-dominated uterus of the pregnant rabbit becomes estrogen-dominated and responsive to oxytocin 24 hours before parturition. Hence the concentration of progesterone in the serum is meaningless by itself and it could be theorized that the significant point is the ratio of estrogen to progesterone. Csapo (1956a), however, offered an alternative solution. He observed a local effect of placental progesterone on the myometrium so that the myometrium closest to the placenta is under a greater progesterone-dominance than that portion of the myometrium lying more distant. Hence the local level of progesterone would be the significant factor in the onset of parturition and not the systemic level.


B. Oxytocin

It is now generally believed that parturition is the result of the action of the posterior pituitary hormone on the myometrium of the uterus sensitized by estrogen. The development of this hypothesis followed from the well known fact that oxytocin produces uterine contractions and induces labor and delivery of the young. It is apparent, however, that a mass of contradictory data exist and the hypothesis is still in need of better evidence before it can be fully accepted (for review of early literature see Reynolds, 1949) .

Some of the evidence supporting the above hypothesis is the fact of the presence, to a limited degree, of a deficiency syndrome in parturition following removal of the posterior pituitary gland. The data, however, are still equivocal. Labor is apparently prolonged in the monkey (Smith, 1946) and guinea pig (Dey, Fisher and Ranson, 1941 ) after total hypophysectomy. Nevertheless, parturition will occur normally after removal of the pituitary gland in the rabbit (Robson, 1936), cat (Allen and Wiles, 1932), mouse (Gardner and Allen, 1942), and rat (Smith, 1932). Even where there is some indication of interference with labor, delivery occurs. However, the lack of consistent results and species differences may be due to the recent finding that the posterior pituitary hormones are synthesized in the hypothalamus and that removal of the posterior pituitary is only effective under limited conditions because the source of the hormone is still present. These experiments have also been criticized on the ground that the anterior pituitary was also removed and hence interference with many other hormones occurred.

Additional evidence in favor of a role for the neurohypophysis in the delivery of the young is the increase in uterine motility following stimuli that bring about release of the posterior pituitary hormones, and the lack of an effect on the uterus when release of the hormone is blocked.

Positive evidence for the release of oxytocin at the time of parturition is still lacking as are measurements of the concentration in the blood. Fitzpatrick (1957) takes the view that oxytocin is liberated as an essential part of normal parturition and cites the following evidence. (1) A superficial similarity exists between spontaneous labor and that induced by oxytocin. Harris (1955) also stresses the similarity in the uterine response to oxytocin and to electrical stimulation of the supraoptic hypophyseal nucleus. (2) Mechanical dilation of the uterus or cervix evokes an increase in uterine contractions presumably by way of a nervous reflex release of oxytocin (Ferguson, 1941). (3) Oxytocin is decreased in the posterior pituitary gland of the rat and the dog after labor (Dicker and Tyler, 1953).

Evidence from the attempts to measure the concentration of oxytocin in body fluids at the time of parturition is inadequate. The early reports of higher concentrations in the urine (Cockrill, Miller and Kurzrok, 1934) and blood (Bell and Morris, 1934; Bell and Robson, 1935) during parturition are questioned because of the inadequate methods of extraction and lack of specificity in the assay. Recently, Hawker and Robertson (1957, 1958) reinvestigated the problem and concluded that two oxytocic substances are present in the blood and hypothalamus of cats, cows, and rats and blood of women. However, they found that the concentration of oxytocin in the blood fell during labor from a high during pregnancy. It is apparent that this presents a paradoxical situation in view of the fact that the concentration of oxytocin is low at the time of parturition; a time when the hormone is supposedly exerting its greatest effect. The situation is further complicated by the presence of two oxytocic factors and the presence of an oxytocinase in the blood and l)lacenta (von Fekete, 1930; Page, 1946; Woodbury, Ahlquist, Abreu, Torpin and Watson, 1946; Hawker, 1956). Although more work is required on this problem and esi)ecially with regard to the specificity and concentration of the oxytocinase, there is some indication of a fall in enzyme level before parturition. Tyler (1955) reported a decrease in the blood level of the enzyme towards the end of pregnancy and Sawyer (1954) reported a decrease in oxytocinase activity in rat tissues at the end of pregnancy.

C. Relaxin

Recently, the discovery of the action of relaxin on the pubic symphysis, uterine cervix, and uterine motility has raised the question of the role of this hormone in parturition. Certainly in the species that normally show pubic relaxation, relaxin would appear to play a significant role. However, this phenomenon is a special adaptation and the question of cervical dilatability becomes more important because it seems to occur in all species examined thus far. It would seem that relaxin can induce cervical dilatability in conjunction with the sex steroids and that cervical dilation is a necessary event in parturition, but whether relaxin controls this event under physiologic conditions is still unknown and direct evidence is unavailable. It is also apparent in some species that relaxin can inhibit uterine contractions w'ithout interfering with the action of oxytocin. Kroc, Steinetz and Beach (1959) reported that relaxin actually restored responsiveness to oxytocin in mice treated with progesterone. Again the question is raised as to whether this is merely a good experiment or a part of the normal physiologic events.


In a general way the events leading to labor may be summarized as follows. As pregnancy approaches term, the uterus becomes subject to increasing pressure from within, due to a differential change in the growth rates of the fetus and the uterus (Woodbury, Hamilton and Torpin, 1938). Concurrently, a reversal from progesterone to estrogen domination occurs, which also contributes to an increase in uterine tonus. As intra-uterine tension increases, spontaneous contractions acquire a greater efficiency and forcefulness. Because the radius of curvature in the human uterus is greater at the fundus than at the cervix, and because the myometrium is thicker at the upper pole (by a factor of 2) the contractile force is stronger at the fundus than at the cervical end. This contractile gradient i^roduces a thrust toward the cervix.

Utilization of a type of strain gauge, the tokodynamometer, has afforded information on the rate and strength of contraction of the various parts of the parturient uterus simultaneously (Reynolds, Heard, Bruns and Hellman, 1948). These measurements have indicated that, during the first stage of labor, the fundus exerts strong contractions of rather long duration. The corpus exhibits less intense contractions, usually of shorter duration, which frequently diminish in force as labor advances. The lower uterine segment is almost inactive throughout the first stage of parturition. According to Reynolds (1949), both the fundus and the midportion contract at the same time, but the fundus remains contracted for a longer period of time than the corpus beneath, thus building up a force downward. If cervical dilation has not occurred, the three areas of the uterus will continue to contract. As cervical dilation begins, the contractions in the midportion of the uterus decrease in intensity and the contractions in the lower segment disappear. Cervical dilation has been observed only when there is a preponderance of rhythmic activity of the fundus over the rest of the uterus.

When amniotic fluid is lost after the rupture of the membranes, the absolute tension within the wall of the uterus is reduced so that the ratio of force between fundus and cervix is increased. Thus rupture of the membranes decreases the tension in the cervix more than the fundus and the net effect is an increased force from the fundus. This change tends to precipitate the parturition more rapidly.

As pregnancy nears term, both increased tonus of the myometrium and rapid growth of the fetus cause a rise in intra-uterine pressure. This rise results in a decrease of effective arterial blood pressure in the placenta. During this period also, thrombosis is observed in many of the venous sinuses of the placenta and many of the blood vessels become more or less obstructed by giant cells. During parturition, the systemic blood pressure of the mother rises with each contraction, but, due to the increased intrauterine pressure produced by the contractions, the effective maternal arterial blood pressure in the placenta decreases to zero. Thus maternal circulation is cut off from the fetus.

Measurements of intra-uterine pressure at term show that the human uterus contracts with a pressure wave which varies from 25 to 95 mm. Hg (Woodbury, Hamilton and Torpin, 1938). The uterine wall is subjected to an average tension of 500 gm. per cm.- and, during delivery of the head, may, with the aid of abdominal musculature, develop as much as 15 kg. force.

In animals giving birth to multiple young (rat and mouse) evacuation of the horn proceeds in an orderlv fashion beginning at the cervical end. As evacuation of the lowest implantation site starts, changes occur in the periods of contractions of segments of uterine artery near its entrance into the uterine wall (Knisely, 1934; Keiffer, 1919). Gradually the constriction phase becomes proportionately longer than the dilation phase until the arterial lumen is obliterated. The myometrium in the area of the constricting segments becofes more active and, after long intense local contractions of the uterine muscle, the fetuses and the placentas separate and are discharged through the dilated cervix. After evacuation, a relaxation of the contracted segment of uterus occurs and the process is repeated at the next implantation site.

Recently, Cross (1958) re-examined the problem of labor in the rabbit. He concluded that (1) oxytocin in physiologic amounts can induce labor that is comparable to the events normally seen, (2) oxytocin is released during a normal labor, and (3) oxytocin can induce delivery without supplementary mechanisms. He noted that straining movements involving reflex abdominal contractions initiated by distention of the vagina and cervix aided in expulsion of the fetus. It is also possible that this might cause reflexly an increased secretion of oxytocin. Other reflex mechanisms have been suggested, but evidence is inadequate. Cross cites a report by Kurdinowski published in 1904 in which the entire process of labor and delivery in an isolated full-term rabbit uterus perfused with Locke's solution is described. In these experiments orderly delivery of the viable fetuses was affected by the contractile efforts of the uterus and vagina in absence of any hormonal or nervous stimuli.


XIV. Conclusion

Although we have garnered much information, no major conclusions can be drawn at this time concerning gestation in the mammal. This is probably true because of the vastness of the subject and the lack of sufficient data, especially that of a comparative nature. It is probably fitting to close this chapter with the final statement written by Newton in the second edition of Sex and Internal Secretion, "It seems rather that the investigation of endocrine relationships during pregnancy is still in the exploratory stage and that the time is not ripe for systematization."

It is true that many data have been accumulated in the last two decades since the publication of the second edition of this book. It is also probably true that some systematization can now be started. But above all we need more data on different species in order to systematize fully the role of the various hormones and glands in pregnancy and to evaluate the metabolic and other changes that occur at this time.

XV. References

Adams, J. Q. 1954. Cardiovascular physiology in normal pregnancy: studies with the dye dilution technique. Am. J. Obst. & Gynec, 67, 741.

Ahl.mark, a. 1944. Studies on the histaminolytic jiower of i)lasma with special reference to pregnancy. Acta physiol. scandinav., 9, suppl. 28.

Ahlmark, a. 1947. The histaminolytic power of ]ilasma with special reference to pregnancy. In 17th International Physiological Congress, p. 127. Oxford.

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Aclcleiitluin

Several reports on an exteroceptive block to pregnancy in mice appeared since this manuscript was completed. In a series of three articles, Bruce (Nature, London, 184, 105, 1959; Science, 131, 1526, 1960; and J. Reprod. Fertil, 1, 96, 1960) has shown that exposure of newly mated, female mice to strange males caused an inhibition of pregnancy that ran as high as 80 per cent. Prior removal of the olfactory bulbs abolished the reaction. The pregnancy block in tliis instance consisted in a failure of the blastocysts to implant.