Acta Pzdiatr Scand 64: 3 15-32 1 , 1975
HISTOLOGICAL A N D BIOCHEMICAL CHANGES IN NEONATAL THYROID TISSUES N . ETLING and J.CI. LARROCHE F r o m Unit; 30 I n b r r m , H6pitul drs Enfunts Muludrs, und C r n t r r de Rrchrrches Biologiques NPonutuIr.5, H6pitul Porr Royul, Puri;,, Fruncr
ABSTRACT. Etling, N. and Larroche, J. C. (Unite Inserm 30, Hopital des Enfants Malades, Centre de Recherches biologiques nbnatales, Hopital de Port Royal, Paris, Prance). Histoiogical and biochemical changes in neonatal thyroid tissues. Acta Paediatr Scand 64: 315, 1975.-The thyroid tissues of 17 infants who died between 3 hours and 46 days after birth were studied by histological and biochemical techniques. The morphological aspect and the iodine content of these tissues are not related to the gestational age of the neonates, but they are related to the survival time. There are dramatic events early after birth: desquamation of the epithelium and absence of colloid, low iodine content of tissue extract (less than 1 pg 1271per mg of protein) and low percentage of thyroglobulin (less than 10 %). 24 hours after birth, the vesicles fill with colloid and the epithelium is cuboidal; the iodine content of the protein increases (between 1 and 2 fig lZ7l per mg protein) as well as the thyroglobulin percentage (around 20%). One week after birth, there is a maximum of colloid and flat epithelium; the iodine content of the protein extract is much higher (more than 2 Fg lZ7lper mg protein) as is thyroglobulin percentage (up to 40 %). Our studies of thyroid tissues of neonates suggest that a leakage of colloid, iodine and thyroglobulin takes place in the perinatal period, this phenomenon being followed by their rapid repletion.
KEY WORDS: Newborn, thyroid tissue, colloid vesicle, thyroglobulin content, iodine content, gestational age
The fetal thyroid gland appears as a cellular mass, arranged in cords separated by vascular mesenchyme. Toward the third month ofgestation these cords acquire their follicular appearance, the central mass of colloid being surrounded by a simple cuboidal or low columnar epithelium; this “maturation” starts from the periphery and progresses toward the central part of the gland. During the second and third trimehter the pattern o f the gland does not vary much. Iodine metabolism has been studied in fetal thyroid glands from abortus either by in vitro incubation (18), organ culture ( 2 2 ) or ‘:r’I administration to the pregnant mother (26). The fetal thyroid gland begins to accumulate radioiodine by the IOth-12th week of gestation and synthesis of iodothyronine was shown to
take place at about the 17th week. It is well established that newborn sera present a transient increase of thyrotropin (TSH) in the early minutes of postnatal life (4, 8), and that the triiodothyronine (T3) and thyroxine (T4) contents present also an increase during the early hours of life (I). If there is no controversy on the embryologic development of the thyroid gland and the appearance of the colloid, as well as the dramatic changes of Serum TSH and hormones content that surround birth, there is no biochemical information on the thyroid tissue within the first days of life. Our purpose is to review the morphological features and biochemical correlations in neonatal thyroid tissues, and to relate the changes in the’tissues to the known blood modifications. Arta Prediarr Scand 64
3 16
N . Etling and J.Cl. Lurroche
Table I . Neonatal clinical data HMD=Hyaline Membrane Disease
Subject
Sex
(g)
Gest. age (weeks)
MON. no. I SCH, no. 2
M M
2 180 2 000
35 31
3h10 9h35
IZZ, no. 3 ST.L, no. 4 BER, no. 5 DEL, no. 6 BIL, no. 7 COU, no. 8 ROT, no. 9 JEA, no. 10
M F F F M M M M
I700 1860 I430
32 32 29 29 37 30 37 31
IIh30 16h20 17h 26h 31h 32h 34h 35h
MAX, no. I I HER, no. 12 ETI, no. 13 BOR, no. I4 TEP, no. 15
M F M M F
I 130 I240 97 5
3013 I 3013 I 29 29 26
36h 38h 2d 3d 12d
987 570.7 420 490 474
LAN, no. 16
M
3 650
36
13d
946.5
ALE. no. 17
M
2005
33
4Yd
Body weight
I 100
I910 I 180
2 300 1300 I660 1 180
Age at death
MATERIAL Seventeen thyroid glands of premature and full-term newborns were obtained from autopsies, performed within 10 hours (mean) after death; specimens are kept at 4°C before dissection. The gland is removed and weighed. Half is immediately stored in ice and rapidly frozen for biochemical studies; the other half is processed in paraffin for histological examination. Body weight, gestational age, length of life, sex, thyroid weight and presumed cause of death are shown in Tdbk 1 .
METHODS The lobe used for microscopy is fixed either in formalin or in Bouin's solutionand sections are stained by hematoxylin eosin and/or Masson's trichrome. The other lobe is kept at -20" and three extractions are performed with 0.14 M NaCl (200 mg wet tissue per ml). The extracts are centrifuged in a refrigerated MSE centrifuge at 8000g. Stable iodine ( I z 7 I ) is measured in the pellet solubilised into N NaOH and in the supernatant or soluble proteins of the extract. l2'I is determined by a semi-automatic micromethod with the Technicon Autoanalyser (12). Each sample is mineralised with sulfonitroperchloric mixture, and the catalytic effect of iodine o n the reduction of ceric ions by arsenous acid is used; using this method, amount of I t o 20 ng can be determined with a 5 % precision. Protein contents are estimated by U V readings at 260 and 280 nm (25). Acla Pzdiarr Scand 44
Weight thyroid (mg) 4 000 I 346 650 1 220.6
788.8 480.7 809.6 700 679.5 543.5
1 152
Histology colloid content
Presumed cause of death
0bs te t rical trauma Hemolytic disease of newborn HMD HMD HMD HMD HMD Pulmonarv hemorrhage HMD Hemolytic disease of newborn Second twin, HMD Congenital heart disease HMD HMD Prematurity; intraventricular hemorrhage Diabetic mother; hepatic necroses Respiratory distress syndrome I
0 0 0
+++ ++ ++ +++ +++ + + ++ ++ ++ ++++ ++++ ++++
0
The proteins of the extract are separated by 5% polyacrylamide gel electrophoresis, according to the technique of Barka (3) in a PleugerAcrylophor apparatus, at room temperature, 3 mA per gel during about I hour in a 0.02 M Tris-glycine buffer, at pH 8.5. The samples are stained with amido black 0.5% in 2 % acetic acid, and destained with acetic acid, until complete decoloration of the background. The optical densities of the stained proteins are recorded with a Gilford Spectrophotometer at 610 mm with a 10% precision. The results are compared with a reference thyroglobulin lyophilised. The thyroglobulin and other protein contents are measured by planimetry aDd/or weighing the peak surfaces and percentages are calculated.
RESULTS (A) Histological Examination
Morphology varies greatly and can be classified into 2 main types: Type I: thyroids free of colloid, with desquamation of the epithelium and desintegration of follicules (Fig. 1). Type 11: thyroids containing colloid. This type is classified into subgroups according to the number of colloid-filled vesicles, their size and the appearance of the epithelium.
Changes in neonatal thyroid
3 17
Fig. I. Thyroid gland from a 35-week newborn who died within 3 hours. The follicules are filled with desquamated cells. Absence of colloid (0). Fig. 2. Thyroid gland from a 31-week neonate dying at 36 hours. Vesicles are small, half of them contain colloid: others are empty or show a few desquamated cells ( + ). Fig. 3. Thyroid gland from a 30-week newborn who died at 32 hours. Large follicule,s containing colloid. The stroma is still abundant ( + + +).
Subgroup +: few vesicles containing colloid at the periphery. The rest of the gland appears as a rather compact organ made up of many vesicles of varying size, well recognizable but without true lumen and without colloid. The epithelium is cuboidal high: however, there is very little or no desquamation at all (Fig. 9). Subgroup + +: increased number of colloid filled vesicles, mostly in the periphery. Few empty vesicles in the centre with still some solid buds. Subgroup +++: all vesicles are distended with colloid, the epithelium is still cuboidal (Fig. 3). Subgroup + + + + : represents the maximum colloid content we have observed, rather unformly distributed, with a flat epithelium (adult like feature). These morphological variations were related neither to birth weight or gestational age nor to sex. By contrast, they seem to be related to length of life.
Of these 17 infants, 5 died within 24 hours; 4 of them had neither normal vesicles nor colloid; the epithelium was vacuolated and desquamated into the lumen. However one (ST.L. no. 41, had mature vesicles filled with normal-looking colloid. 12 infants survived more than 24 hours. They all exhibited colloid in the vesicles, ranging from + to + + + +. We observed that from 15 hours to 6 days the vesicles open up, from the periphery to the centre, and progressively fill up with colloid. The epithelium, still rather high, is never vacuolated or detached from the basal membrane: it will flatten with survival. From 7 days on, the vesicles enlarge and fill with colloid: the epithelium flattens and resembles the adult type. Because of the rather small number of cases included in this parallel study of morphology and biochemistry, we surveyed the thyroid gland of 133 othe'r cases from the files of the department of pathology. Table 2 shows the Acra PiPdiatr Scand 64
N . Etling and J.Cl. Larroche
318
Table 2. Distribution qf’colloid f r e e L7esicles
(I) (2) (3) (4)
Age at death
No. of cases
No colloid
Absence of colloid (% cases)
6days
35 33 38 21
22 3 2 0
62.8 9 5.2 0
The differences between I and 2, I and 3 , 1 and 4 are highly significant: p
HISTOLOGICAL A N D BIOCHEMICAL CHANGES IN NEONATAL THYROID TISSUES N . ETLING and J.CI. LARROCHE F r o m Unit; 30 I n b r r m , H6pitul drs Enfunts Muludrs, und C r n t r r de Rrchrrches Biologiques NPonutuIr.5, H6pitul Porr Royul, Puri;,, Fruncr
ABSTRACT. Etling, N. and Larroche, J. C. (Unite Inserm 30, Hopital des Enfants Malades, Centre de Recherches biologiques nbnatales, Hopital de Port Royal, Paris, Prance). Histoiogical and biochemical changes in neonatal thyroid tissues. Acta Paediatr Scand 64: 315, 1975.-The thyroid tissues of 17 infants who died between 3 hours and 46 days after birth were studied by histological and biochemical techniques. The morphological aspect and the iodine content of these tissues are not related to the gestational age of the neonates, but they are related to the survival time. There are dramatic events early after birth: desquamation of the epithelium and absence of colloid, low iodine content of tissue extract (less than 1 pg 1271per mg of protein) and low percentage of thyroglobulin (less than 10 %). 24 hours after birth, the vesicles fill with colloid and the epithelium is cuboidal; the iodine content of the protein increases (between 1 and 2 fig lZ7l per mg protein) as well as the thyroglobulin percentage (around 20%). One week after birth, there is a maximum of colloid and flat epithelium; the iodine content of the protein extract is much higher (more than 2 Fg lZ7lper mg protein) as is thyroglobulin percentage (up to 40 %). Our studies of thyroid tissues of neonates suggest that a leakage of colloid, iodine and thyroglobulin takes place in the perinatal period, this phenomenon being followed by their rapid repletion.
KEY WORDS: Newborn, thyroid tissue, colloid vesicle, thyroglobulin content, iodine content, gestational age
The fetal thyroid gland appears as a cellular mass, arranged in cords separated by vascular mesenchyme. Toward the third month ofgestation these cords acquire their follicular appearance, the central mass of colloid being surrounded by a simple cuboidal or low columnar epithelium; this “maturation” starts from the periphery and progresses toward the central part of the gland. During the second and third trimehter the pattern o f the gland does not vary much. Iodine metabolism has been studied in fetal thyroid glands from abortus either by in vitro incubation (18), organ culture ( 2 2 ) or ‘:r’I administration to the pregnant mother (26). The fetal thyroid gland begins to accumulate radioiodine by the IOth-12th week of gestation and synthesis of iodothyronine was shown to
take place at about the 17th week. It is well established that newborn sera present a transient increase of thyrotropin (TSH) in the early minutes of postnatal life (4, 8), and that the triiodothyronine (T3) and thyroxine (T4) contents present also an increase during the early hours of life (I). If there is no controversy on the embryologic development of the thyroid gland and the appearance of the colloid, as well as the dramatic changes of Serum TSH and hormones content that surround birth, there is no biochemical information on the thyroid tissue within the first days of life. Our purpose is to review the morphological features and biochemical correlations in neonatal thyroid tissues, and to relate the changes in the’tissues to the known blood modifications. Arta Prediarr Scand 64
3 16
N . Etling and J.Cl. Lurroche
Table I . Neonatal clinical data HMD=Hyaline Membrane Disease
Subject
Sex
(g)
Gest. age (weeks)
MON. no. I SCH, no. 2
M M
2 180 2 000
35 31
3h10 9h35
IZZ, no. 3 ST.L, no. 4 BER, no. 5 DEL, no. 6 BIL, no. 7 COU, no. 8 ROT, no. 9 JEA, no. 10
M F F F M M M M
I700 1860 I430
32 32 29 29 37 30 37 31
IIh30 16h20 17h 26h 31h 32h 34h 35h
MAX, no. I I HER, no. 12 ETI, no. 13 BOR, no. I4 TEP, no. 15
M F M M F
I 130 I240 97 5
3013 I 3013 I 29 29 26
36h 38h 2d 3d 12d
987 570.7 420 490 474
LAN, no. 16
M
3 650
36
13d
946.5
ALE. no. 17
M
2005
33
4Yd
Body weight
I 100
I910 I 180
2 300 1300 I660 1 180
Age at death
MATERIAL Seventeen thyroid glands of premature and full-term newborns were obtained from autopsies, performed within 10 hours (mean) after death; specimens are kept at 4°C before dissection. The gland is removed and weighed. Half is immediately stored in ice and rapidly frozen for biochemical studies; the other half is processed in paraffin for histological examination. Body weight, gestational age, length of life, sex, thyroid weight and presumed cause of death are shown in Tdbk 1 .
METHODS The lobe used for microscopy is fixed either in formalin or in Bouin's solutionand sections are stained by hematoxylin eosin and/or Masson's trichrome. The other lobe is kept at -20" and three extractions are performed with 0.14 M NaCl (200 mg wet tissue per ml). The extracts are centrifuged in a refrigerated MSE centrifuge at 8000g. Stable iodine ( I z 7 I ) is measured in the pellet solubilised into N NaOH and in the supernatant or soluble proteins of the extract. l2'I is determined by a semi-automatic micromethod with the Technicon Autoanalyser (12). Each sample is mineralised with sulfonitroperchloric mixture, and the catalytic effect of iodine o n the reduction of ceric ions by arsenous acid is used; using this method, amount of I t o 20 ng can be determined with a 5 % precision. Protein contents are estimated by U V readings at 260 and 280 nm (25). Acla Pzdiarr Scand 44
Weight thyroid (mg) 4 000 I 346 650 1 220.6
788.8 480.7 809.6 700 679.5 543.5
1 152
Histology colloid content
Presumed cause of death
0bs te t rical trauma Hemolytic disease of newborn HMD HMD HMD HMD HMD Pulmonarv hemorrhage HMD Hemolytic disease of newborn Second twin, HMD Congenital heart disease HMD HMD Prematurity; intraventricular hemorrhage Diabetic mother; hepatic necroses Respiratory distress syndrome I
0 0 0
+++ ++ ++ +++ +++ + + ++ ++ ++ ++++ ++++ ++++
0
The proteins of the extract are separated by 5% polyacrylamide gel electrophoresis, according to the technique of Barka (3) in a PleugerAcrylophor apparatus, at room temperature, 3 mA per gel during about I hour in a 0.02 M Tris-glycine buffer, at pH 8.5. The samples are stained with amido black 0.5% in 2 % acetic acid, and destained with acetic acid, until complete decoloration of the background. The optical densities of the stained proteins are recorded with a Gilford Spectrophotometer at 610 mm with a 10% precision. The results are compared with a reference thyroglobulin lyophilised. The thyroglobulin and other protein contents are measured by planimetry aDd/or weighing the peak surfaces and percentages are calculated.
RESULTS (A) Histological Examination
Morphology varies greatly and can be classified into 2 main types: Type I: thyroids free of colloid, with desquamation of the epithelium and desintegration of follicules (Fig. 1). Type 11: thyroids containing colloid. This type is classified into subgroups according to the number of colloid-filled vesicles, their size and the appearance of the epithelium.
Changes in neonatal thyroid
3 17
Fig. I. Thyroid gland from a 35-week newborn who died within 3 hours. The follicules are filled with desquamated cells. Absence of colloid (0). Fig. 2. Thyroid gland from a 31-week neonate dying at 36 hours. Vesicles are small, half of them contain colloid: others are empty or show a few desquamated cells ( + ). Fig. 3. Thyroid gland from a 30-week newborn who died at 32 hours. Large follicule,s containing colloid. The stroma is still abundant ( + + +).
Subgroup +: few vesicles containing colloid at the periphery. The rest of the gland appears as a rather compact organ made up of many vesicles of varying size, well recognizable but without true lumen and without colloid. The epithelium is cuboidal high: however, there is very little or no desquamation at all (Fig. 9). Subgroup + +: increased number of colloid filled vesicles, mostly in the periphery. Few empty vesicles in the centre with still some solid buds. Subgroup +++: all vesicles are distended with colloid, the epithelium is still cuboidal (Fig. 3). Subgroup + + + + : represents the maximum colloid content we have observed, rather unformly distributed, with a flat epithelium (adult like feature). These morphological variations were related neither to birth weight or gestational age nor to sex. By contrast, they seem to be related to length of life.
Of these 17 infants, 5 died within 24 hours; 4 of them had neither normal vesicles nor colloid; the epithelium was vacuolated and desquamated into the lumen. However one (ST.L. no. 41, had mature vesicles filled with normal-looking colloid. 12 infants survived more than 24 hours. They all exhibited colloid in the vesicles, ranging from + to + + + +. We observed that from 15 hours to 6 days the vesicles open up, from the periphery to the centre, and progressively fill up with colloid. The epithelium, still rather high, is never vacuolated or detached from the basal membrane: it will flatten with survival. From 7 days on, the vesicles enlarge and fill with colloid: the epithelium flattens and resembles the adult type. Because of the rather small number of cases included in this parallel study of morphology and biochemistry, we surveyed the thyroid gland of 133 othe'r cases from the files of the department of pathology. Table 2 shows the Acra PiPdiatr Scand 64
N . Etling and J.Cl. Larroche
318
Table 2. Distribution qf’colloid f r e e L7esicles
(I) (2) (3) (4)
Age at death
No. of cases
No colloid
Absence of colloid (% cases)
6days
35 33 38 21
22 3 2 0
62.8 9 5.2 0
The differences between I and 2, I and 3 , 1 and 4 are highly significant: p
