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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2018  |  Volume : 61  |  Issue : 3  |  Page : 334-338
A study of spectrum of pulmonary pathology and expression of thyroid transcription factor-1 during neonatal period


1 Department of Pathology, NRS Medical College, Kolkata, West Bengal, India
2 Department of Pathology, IPGME and R, Kolkata, West Bengal, India
3 Department of Pathology, Midnapore Medical College, Midnapore, West Bengal, India
4 Department of Neonatology, IPGME and R, Kolkata, West Bengal, India

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Date of Web Publication13-Jul-2018
 

   Abstract 


Context: Neonatal period is the single most hazardous period of life. The major causes of neonatal death are prematurity and respiratory distress syndrome. We report a series of neonatal autopsies in our Neonatal Intensive Care Unit with special emphasis on pulmonary pathology. The spectrum of pathological changes in the lungs and thyroid transcription factor-1 (TTF-1) expression was studied in detail with reference to its spatial distribution. Aims: This study aims to analyze the causes of neonatal death with special attention to pulmonary pathology along with associated histopathological changes in lungs. We also evaluated the expression of TTF-1 at different levels of the airway. Materials and Methods: After taking consent and anthropometric measurements, autopsy was performed. Weights of all organs were taken, and histological sections were examined under hematoxylin and eosin stain. TTF-1 immunostaining was done on lung sections. Localization of TTF-1 was evaluated at the intrapulmonary level of terminal bronchioles (TBs), distal bronchioles, and alveoli. Results: We performed a series of 25 autopsies in neonates. In our series, most of the neonates were preterm (64%), had low birth weight (44%), and died within the first 7 days of life (80%). Majority (60%) of the neonates died due to pulmonary causes, followed by septicemia (24%), congenital anomalies (12%), and birth injury (4%). Among the respiratory causes, hyaline membrane disease (HMD) was diagnosed in maximum number of cases (32%), followed by pneumonia (12%) and pulmonary hemorrhage (12%). The TTF-1 expression in TBs, distal airways, and alveoli was significantly reduced or absent in cases of HMD compared to the control group. Conclusions: In this study, we observed that HMD is the most common cause of perinatal death among respiratory disorders, and in this disease, the expression of TTF-1 is significantly reduced in TBs, distal airways, and alveoli compared to the control group.

Keywords: Hyaline membrane disease, neonatal autopsy, thyroid transcription factor-1

How to cite this article:
Das I, Das RN, Paul B, Mandal B, Mukherjee S, Chatterjee U. A study of spectrum of pulmonary pathology and expression of thyroid transcription factor-1 during neonatal period. Indian J Pathol Microbiol 2018;61:334-8

How to cite this URL:
Das I, Das RN, Paul B, Mandal B, Mukherjee S, Chatterjee U. A study of spectrum of pulmonary pathology and expression of thyroid transcription factor-1 during neonatal period. Indian J Pathol Microbiol [serial online] 2018 [cited 2019 Dec 11];61:334-8. Available from: http://www.ijpmonline.org/text.asp?2018/61/3/334/236623





   Introduction Top


Over the last few years, neonatal mortality rates are reported to have declined considerably in many Western countries. In the developed countries, the perinatal mortality rate is <10/1000 live births as compared to the alarmingly high rate of 60–120/1000 live births in India. About 1.5 million perinatal deaths occur every year in our country.[1] Important causes of neonatal death are low birth weight (LBW), respiratory distress syndrome, birth injury, congenital anomalies, hemolytic disease of newborn, conditions of placenta and cord, diarrheal disease, and acute respiratory infections.[2]

Pulmonary pathology is the most common cause of death in preterm babies in most of the autopsy studies. The development of lung starts early during the embryonic life, and thyroid transcription factor-1 (TTF-1) plays a vital role in morphogenesis of lung and differentiation of pulmonary epithelial cells as well as in the transcription of surfactant proteins (SP-A, SP-B, and SP-C) and Clara cell secretory protein. Pulmonary surfactant is required for adaptation to air breathing after birth, reducing surface tension at the air–liquid interface to maintain lung volume during respiratory cycle.

This study was done to analyze the causes of neonatal death in a tertiary care center with emphasis on the spectrum of pulmonary disorders along with special reference to expression of TTF-1. In addition, we have studied the spatial distribution of TTF-1 in different levels of lung parenchyma, which has not been elaborated in details earlier.


   Materials and Methods Top


A total of 25 autopsies were performed on neonates, who died in the Neonatal Intensive Care Unit (NICU) of our hospital over a period of 2 years. Consent was taken for every case.

The complete autopsy was performed after taking anthropometric measurements and weights of all the vital organs including lungs, liver, brain, kidney, heart, and spleen. Representative sections were taken from all vital organs. Two sections from the each lobe of the each lung were taken from hilum to the periphery.

Then, immunostaining with TTF-1 (monoclonal, DAKO) was performed, and immunohistochemical localization of TTF-1 was evaluated by light microscopy at three different levels of the airways including intrapulmonary bronchi (IB), intermediate airways, i.e., the so-called terminal bronchioles (TBs), and distal bronchioles and alveoli (DB, A). Immune expression was categorized as normal expression and reduced or absence of expression.

We divided our 25 cases into study and control groups. All the neonates who died due to respiratory pathology (e.g., hyaline membrane disease [HMD], pneumonia, and pulmonary hemorrhage) were included in the study group, whereas neonates died due to other causes (e.g., septicemia, congenital abnormalities, and birth injury) were included in the control group. Among 25 neonates, 15 were in the study group and 10 were in the control group. The spatial distribution of TTF-1 in the study and control groups was analyzed by Chi-square test.

We used the statistical data analysis Statistica version 6 (Tulsa, Oklahoma: StatSoft Inc., 2001) for statistical analysis.


   Results Top


Among the 25 autopsies, 15 (60%) neonates died due to respiratory pathology. Of these, 9 (36%) cases had HMD, 3 (12%) had pneumonia, and 3 (12%) had pulmonary hemorrhage. Among the rest, 6 (24%) had septicemia, 3 (12%) had congenital anomalies, and 1 (4%) died due to birth injury with intracranial hemorrhage.

Sixteen (64%) of these neonates were preterm (gestational age <37 weeks) and 9 (36%) cases were term (gestational age ≥37 weeks).

Three (12%) had birth weights of >2500 g, 7 (28%) had birth weights between 2000 and 2499 g, 4 (16%) had weights of 1500–1999 g, and 11 (44%) had birth weights of <1500 g.

The mean birth weight of the cases was 1827.4 g and of the control group was 1787.9 g. The mean birth weights for both the groups were below the cutoff mark of traditional LBW.

In the nine cases of HMD, TTF-1 expression in epithelial cells of IB, DBs, and alveoli was absent in all the nine cases and expression at the level of TBs was absent in six cases and reduced in three cases. In the three cases of pneumonia, TTF-1 expression in epithelial cells of IB and TBs was absent in all cases, but at the level of DBs and alveoli, it was absent in two cases and reduced in one case. In the three cases of pulmonary hemorrhage, TTF-1 expression in epithelial cells of IB was absent in all cases, but immune expression was absent in two cases and reduced in one case in TBs, DBs, and alveoli. Analyzing the distribution of TTF-1 expression in the control group, we observed that the expression was normal in the epithelial cells of TBs, DBs, and alveoli and absent in IB in one case of birth injury. In the three cases of congenital anomalies, expression was absent in IB in all the cases, normal in two cases, reduced in one case in TBs, and normal in all three cases in DBs and alveoli. Among the six septicemia cases, TTF-1 expression was absent in IB of all cases, normal in one case, reduced in three cases, and absent in two cases in TBs and normal in four cases and reduced in two cases in DBs and alveoli [Table 1].
Table 1: Expression of thyroid transcription factor-1 at different levels of lungs in case and control groups

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On analyzing the data, it was found that TTF-1 expression at the level of TB was significantly reduced in the study group (HMD, pneumonia, and pulmonary hemorrhage) compared to the control group (P = 0.041).

Also analyzing the TTF-1 expression in DBs and alveoli, it was observed that the TTF-1 expression was significantly reduced in the study group than the control group (P = 0.001). At the level of IB, there is no significant difference in TTF-1 expression between the study and control groups [Figure 1] and [Figure 2].
Figure 1: (a) Hyaline membrane disease; background shows congestion and interstitial edema (×10). Inset (×40) shows waxy hyaline membrane lining the alveolar wall. (b) Diminished thyroid transcription factor-1 expression in hyaline membrane disease at all levels of the respiratory tract (×10). (c) Pulmonary hemorrhage; diffuse intra-alveolar hemorrhage is seen (×10). (d) Variable thyroid transcription factor-1 expression in pulmonary hemorrhage with absent expression in intrapulmonary bronchi and distal airway. Reduced expression in terminal bronchiole (×10)

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Figure 2: (a) Bronchopneumonia: Septal widening, congestion, and inflammatory cell infiltration (×10). (b) Diminished thyroid transcription factor-1 expression in pneumonia (×10). (c) Normal pattern of expression of thyroid transcription factor-1 in control (×10). (d) Normal pattern of expression of thyroid transcription factor-1 in control (×40)

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We also analyzed the distribution of cases and controls according to gestational age; however, no significant association was found between gestational age and pulmonary pathology (Fisher's test; P = 0.53) [Table 2].
Table 2: Distribution of cases and controls according to gestational age

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   Discussion Top


Neonatal period is the single most hazardous period of life; never again in life is the individual confronted with more dramatic challenges than in the transition from dependent intrauterine existence to independent postnatal life. To determine the priorities in neonatal care, it is important to know the magnitude of each disease entity. It is important, therefore, to correlate the antenatal history, clinical profile, and cause of death. Hence, knowledge of the spectrum of common neonatal disorders in the Indian context is important as it would be of help in developing appropriate facilities and management protocols to improve neonatal survival.

Neonatal mortality is directly related to birth weight and gestational maturity of the infant. Among the preterm babies, respiratory distress syndrome is one of the major causes of death.[3]

Among the respiratory causes, HMD was diagnosed in 36% cases, pneumonia in 12%, and pulmonary hemorrhage in 12%. There are several reasons that may explain this increase of HMD incidence in our study. The first reason is that in our study all HMD cases are LBW neonates, with birth weight ranging from 846 g to 1478 g. Second, there is an increasing incidence of infants being delivered by elective cesarean section (CS), especially before onset labor.[4] Our study was, however, not designed to investigate an association between CS and HMD, but all our cases of HMD were delivered by CS. According to the WHO Bulletin 2006, neonatal deaths due to sepsis and pneumonia is 26% globally and 27% in Southeast Asia.[5],[6],[7] In our study, pulmonary hemorrhage was observed in 12% cases, whereas 6% was reported in 7th WHO Bulletin 2006[5] and 21% by Decosta et al.[8] According to Speer et al., at autopsy, the incidence of neonatal pneumonia ranges from 20% to 32% of live born, and in our study, it was 12%.[9] The results of our study could vary with others because of local ethnic factors, medical expertise of the NICU, and sample size.

Pulmonary pathology is the most common cause of death in LBW babies in most of the autopsy series. Miller found pulmonary pathology in 50% of cases in LWB babies.[10] In our study, 12 (48%) cases are LBW and died due to respiratory diseases.

TTF-1 is a well-known growth factor for early lung development. Human TTF-1 protein has been reported to be detected in human fetal lung from 11 weeks of gestation, being localized in the nuclei of epithelial cells of the developing airway. It regulates the expression of surfactant-associated protein and Clara cell secretory protein. In general, the intensity of TTF-1 staining of terminal airways and cuboidal bronchiolar epithelium, especially those at the periphery of lobules and abutting septa, was more pronounced than in more proximal airways, especially in late gestational age. By 36 weeks of gestation, reduced labeling for TTF-1 has been observed in the conducting airways at all levels, whereas nuclei of Type II cells in respiratory airways and alveoli remained well stained.[11]

TTF-1 expression in TBs decreases as the gestational age progresses and it becomes restricted in the nonciliated cuboidal epithelium, especially those at the periphery of lobules and adjacent to peribronchial and perivascular connective tissue near term. In our study, it was observed that in the control group with gestational ages between 24 and 30 weeks, expression is normal (similar to normal lung development), but thereafter between 30 and 32 weeks it is reduced and near term (37 and 38 weeks) it becomes restricted to very few number of cells or absent at the periphery of lobules and adjacent to peribronchial and perivascular connective tissue. However, it does not show any statistically significant data because of the very small sample size which is a limitation of our study. In the study group, expression varies as the disease process reduced the TTF-1 expression. As among nine cases of HMD, four had gestational ages between 28 and 30 weeks, when normal TTF-1 expression in TBs is expected; however, we observed reduced or absent expression. The other three cases with gestational ages between 32 and 33 weeks had absent expression. TTF-1 expression in epithelial cells of alveoli and DBs was absent in all cases of HMD irrespective of gestational age; however, in the control group, it was normal, except in two cases where it is reduced.

From these observations, it is clear that in HMD, TTF-1 expression is absent in TBs, distal airways, and alveoli and reduced in IB. TTF-1 is required for lung morphogenesis and SP synthesis by Type II cells. Lack of TTF-1 leads to a reduction in the amount SP synthesized and can contribute to the development of HMD. However, the distribution of TTF-1 is not sufficient to explain the heterogeneity of gene expression of the SPs in the developing and postnatal lung. TTF-1 activates SP gene and CCSP promoters in concert with AP1, HNF family members, and other nuclear proteins that interact with the promoter elements of these genes.[12] In the present study, TTF-1 was not detected in Type I epithelial cells or in the ciliated cells in the conducting airways, suggesting that the differentiation of the subsets of cells from earlier progenitor cells is associated with the loss of expression of TTF-1.

In pneumonia, TTF-1 expression in epithelial cells of TBs was absent in all cases, whereas in DBs and alveoli, it was absent in two cases and reduced in one case. In pneumonia, activated neutrophils release, a variety of factors, such as leukotrienes, proteases, and platelet-activating factor, which contribute to local tissue damage, resulting in reduced or absent TTF-1 expression. Li et al. also observed similar results.[13]

In pulmonary hemorrhage, we found reduced or absent expression of TTF-1 in TBs and DBs and alveoli. Yeung proposed that pulmonary hemorrhage in the newborn occurs predominantly in LBW infants and is associated with many conditions that occur commonly in these infants, such as HMD, perinatal hypoxia, intrauterine growth retardation, kernicterus, hypothermia, aspiration, and infection.[14] He also established that pulmonary hemorrhage occurs as a complication of infection in the newborn infant. In our cases of pulmonary hemorrhage, the presence of underlying infection or HMD is also possible for which TTF-1 expression is absent or reduced. However, we could not diagnose it possibly because of severe hemorrhage obscuring the findings.

It is important to mention that none of the earlier studies in this context have tried to establish the pattern and distribution of TTF-1 in different levels of lung parenchyma. We found that TTF-1 expression in TB is significantly reduced in cases (HMD, pneumonia, and pulmonary hemorrhage) than the control group (P = 0.041) and also the TTF-1 expression in DBs and alveoli is also more significantly reduced in the same group (P = 0.001). Statistical analysis of TTF-1 expression in each of the individual disease condition in comparison to control group was not possible in our study because of the small number of cases in each individual group.


   Conclusion Top


In this study, we observed that the majority of the neonatal death happened due to respiratory diseases, and among them, HMD is the most common one. Within this group, the expression of TTF-1 is significantly reduced in TBs, distal airways, and alveoli.

Acknowledgment

The authors are particularly thankful to our technical staff Janababu, Ashimda, and Raju who also actively helped in our work. Hence, they also owe special appreciation for their cooperation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Pryse-Davies J. The perinatal autopsy. Histopathol 1981;11:65-81.  Back to cited text no. 1
    
2.
Dubell GG, Stoll BJ. Respiratory tract disorders. In: Behrman RE, Kliegman RM, Jenson HB, editors. Nelson×3s Textbook of Paediatrics. 17th ed. India: Saunders Elsevier; 2004. p. 732.  Back to cited text no. 2
    
3.
Burri PH. Structural aspects of prenatal and postnatal development and growth of the lung. In: McDonald JA, editor. Lung Growth and Development. New York: Marcel Dekker; 1997. p. 1.  Back to cited text no. 3
    
4.
van den Berg A, van Elburg RM, van Geijn HP, Fetter WP. Neonatal respiratory morbidity following elective caesarean section in term infants. A 5-year retrospective study and a review of the literature. Eur J Obstet Gynecol Reprod Biol 2001;98:9-13.  Back to cited text no. 4
    
5.
Baqui AH, Darmstadt GL, Williams EK, Kumar V, Kiran TU, Panwar D, et al. Rates, timing and causes of neonatal deaths in rural India: Implications for neonatal health programmes. Bull World Health Organ 2006;84:706-13.  Back to cited text no. 5
    
6.
Khetarpal SK, Chandka RK. Hyaline membrane disease: Mortality and maternal and neonatal risk factors. Indian J Pediatr 1988;32:110.  Back to cited text no. 6
    
7.
Dalal SR, Jadhav MV, Deshmukh SD. Autopsy study of pediatric deaths. Indian J Pediatr 2002;69:23-5.  Back to cited text no. 7
    
8.
Decosta GF, Chincholikar M, Patil Y. Trends in neonatal lung pathology. Lancet 2006;342:71.  Back to cited text no. 8
    
9.
Speer M, Rosan RC, Rudolph AJ. Hemophilus influenzae infection in the neonate mimicking respiratory distress syndrome. J Pediatr 1978;93:295-6.  Back to cited text no. 9
    
10.
Miller HC. Analysis of fetal and neonatal deaths in 4117 consecutive births. Pediatrics 1950;5:184-92.  Back to cited text no. 10
    
11.
Stahlman MT, Gray ME, Whitsett JA. Expression of thyroid transcription factor-1(TTF-1) in fetal and neonatal human lung. J Histochem Cytochem 1996;44:673-8.  Back to cited text no. 11
    
12.
Clevidence DE, Overdier DG, Peterson RS, Porcella A, Ye H, Paulson KE, et al. Members of the HNF-3/forkhead family of transcription factors exhibit distinct cellular expression patterns in lung and regulate the surfactant protein B promoter. Dev Biol 1994;166:195-209.  Back to cited text no. 12
    
13.
Li Y, Du H, Qin Y, Roberts J, Cummings OW, Yan C, et al. Activation of the signal transducers and activators of the transcription 3 pathway in alveolar epithelial cells induces inflammation and adenocarcinomas in mouse lung. Cancer Res 2007;67:8494-503.  Back to cited text no. 13
    
14.
Yeung CY. Massive pulmonary hemorrhage in neonatal infection. Can Med Assoc J 1976;114:135-8.  Back to cited text no. 14
    

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Correspondence Address:
Ram Narayan Das
265, Purba Sinthee Bye Lane, Kolkata - 700 030, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_650_17

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