Joti Sharma, MD (Host): Hello, everyone. Welcome back to another edition of our podcast, Neonatology Review, Isolette to Crib. I'm your host, Dr. Joti Sharma. The purpose of this podcast is to review high yield common topics in neonatology. While our focus is geared towards the neonatal perinatal boards, anyone learning or studying neonatology will find this podcast helpful.

For this episode, we once again have Dr. Sanjay Akangire back as our guest. As you know, he's one of our Neonatologists here at Children's Mercy in Kansas City. Tell everyone hi, Dr. Akangire!

Sanjay Akangire, MD: Hi, everyone. Happy to be here, as always.

Host: He is joining us today to talk about fetal lung development and surfactant physiology. We will discuss stages of lung development, physiology of fetal lung fluid, surfactant composition, production, and secretion. I think we should start with the stages of lung development, as this is a favorite for the neonatal boards.

Sanjay Akangire, MD: Sure, Dr. Sharma. So, once we become aware of the stages of lung development, lung physiology and even lung anomalies become easier to grasp. All right. Let's start with the stages of lung development first. As we all know, mnemonic helps us memorize things. So the mnemonic that I always remember to memorize stages of lung development is, Each Phase ComeS with Age.

So E stands for embryonic, P stands for pseudoglandular, C stands for canalicular, S stands for saccular and A stands for alveolar stage.

Host: That's a nice mnemonic. I was not aware of this one. I've seen others, but this is a great one, too. Having covered the stages of lung development, can you briefly go over the gestational ages at which these stages of lung development occur and the structural changes that are related to it?

Sanjay Akangire, MD: Sure, Dr. Sharma. So let's start with embryonic stage. So embryonic stage occurs at 3 to 6 gestational weeks in which trachea and asymmetric bronchi are formed. Lungs start developing during this stage.

Pseudoglandular stage occurs at 16 weeks in which branching of airway continues and non-respiratory bronchioles are developed. Amniotic fluid is secreted by bronchopulmonary epithelium.

Canalicular stage occurs at 16 to 26 gestational weeks. In this stage, respiratory bronchioles are formed unlike the previous stage where non-respiratory bronchioles are formed and gas exchanging units start developing during this stage. At the end of this stage, lung is considered viable.

Saccular stage occurs at 26 to 36 weeks of gestation. In this stage, alveolar ducts are formed, terminal bronchioles give rise to alveolar sacs and gas exchange via alveolar capillary membrane occurs.

Alveolar stage occurs at and beyond 36 weeks. In this stage, alveolar sacs give rise to mature alveoli. And gradually diameter of alveoli increases with age. Vessels and capillaries grow and mature with age.

Host: And the alveolar stage actually lasts up to about eight years or so. I think sometimes we don't realize that the lung continues to grow after birth. So this is a very nice summary you gave. I have to say one of the ways that I like to remember the stages of lung development based on the gestational age is what I call the rule of sixes.

As you see, embryonic is less than six weeks gestation. Pseudogranular is six to 16 weeks. Canalicular stage is 16 weeks to 26 weeks, the saccular stage is 26 to 36 weeks, and the alveolar stage is more than 36 weeks. And this is another good way to kind of just remember.

Moving on, can you go over some of the lung development abnormalities during each stage? Because I think this is important in our clinical practice.

Thanks.

Sanjay Akangire, MD: Sure, Dr. Sharma. So let's discuss that as it is very important from board standpoint as well. So during embryonic phase, laryngeal cleft, tracheoesophageal fistula, tracheal stenosis can occur.

During pseudoglandular phase, congenital diaphragmatic hernia or CDH, congenital pulmonary adenomatous malformation or CPAM, congenital lobar emphysema can occur.

During canalicular phase, alveolar capillary dysplasia, lung hypoplasia, and surfactant deficiency can occur.

During saccular phase, surfactant deficiency or lung hypoplasia can occur.

During alveolar phase, surfactant deficiency or persistent pulmonary hypertension or PPHN can occur.

Host: Thanks for covering that. This is actually a very high yield from the board standpoint. Before we move to discuss about the fetal lung fluid, I just want to highlight that the alveolar spaces are lined by type 1 and type 2 pneumocytes. Type 1 pneumocytes are derived actually from type 2 pneumocytes. And 90 percent of the alveolar surface is covered by type 1 cells.

Dr. Akangire, can you tell me the important functions of type 1 and type 2 pneumocytes?

Sanjay Akangire, MD: Sure, Dr. Sharma. So type 1 cells are important for gas exchange and type 2 cells have significant role in surfactant metabolism and secretion.

Host: That's correct. A very nice summary on the type 1 and type 2 pneumocytes. Let's move on to the fetal lung fluid. I think the important aspect is to go over the fetal lung fluid secretion and then its clearance.

Sanjay Akangire, MD: Absolutely Dr. Sharma. So fetal lungs are filled with fluid as gas exchange does not occur in fetal lungs. Let's discuss how fetal lungs are filled with fluid. The most important point to remember is that chloride ions are actively transported across pulmonary epithelial cells into the alveolar spaces, which is again a future airspace, and the fluid follows chloride ions, and that's how lungs are filled with fluid.

This phenomenon occurs during fetal life and is much needed for fetal lung development, and if there is less fluid, pulmonary hypoplasia occurs.

Host: Thanks for explaining this very important point.

Sanjay Akangire, MD: During the birth process, ENaC sodium channels on pulmonary epithelial cells are activated and start excreting sodium from alveolar sacs or air space into the interstitial space and fluid follows sodium. Hence, secretion of lung fluid occurs. This process is activated by epinephrine, glucocorticoid secretion, and exposure to oxygen during labor.

In general, one third of fetal lung fluid is absorbed prenatally, one third during labor, and one third after birth.

Host: Yes, I mean, this is actually very clear and extremely important concept for the boards. So in a nutshell, chloride channels are responsible for fetal lung fluid secretion and sodium channels are responsible for fetal lung fluid absorption. Okay, having covered that, let's switch gears to talk about surfactant. So I'd like you to go over the composition of surfactant.

Sanjay Akangire, MD: Sure, Dr. Sharma. So the primary role of surfactant is to decrease surface tension in the alveoli which improves gas exchange. Surfactant is primarily made of lipids, so the constituents of surfactant include dipalmitoylphosphatidylcholine, desaturated, or DPPC, which is 50%, phosphatidylcholine, monosaturated or PC, which is 20%, and surfactant proteins A, B, C, and D 8%, phosphatidylglycerol PG 8%, neutral lipids 8%, other phospholipids are 6% in the surfactant.

Host: The way I like to remember this is the most abundant is DPPC, which accounts for 50 percent of surfactant. Again, this is very high yield for the boards, and you explained it very well. Before getting into the properties of surfactant proteins, let's discuss how surfactant is produced and secreted in the alveolar sacs.

Sanjay Akangire, MD: Sure. Dr. Sharma. So I think that understanding this process will also help understand many diseases related to surfactant abnormalities. So the surfactant is produced in type 2 alveolar cells of the lung. Initially, it is synthesized in the endoplasmic reticulum and then transported to the Golgi apparatus for further modification. In type 2 alveolar cell, multivesicular bodies are formed by transport of SPB precursor protein, SPC and lipids into these bodies.

Joti Sharma, MD (Host): Dr. Akangire, I just want to make sure that when you say type 2 alveolar cells, you mean type 2 pneumocytes. They are pretty much the same thing, right?

Sanjay Akangire, MD: That is correct. So both mean the same thing. It's just two different names for those. So a proteolysis occurs and these bodies are stored in lamellar bodies. This SPB, SPC complex and lipids get into alveolar sub phase and meet SPA protein and multilayered tubular myelin is formed. This tubular myelin helps to reduce surface tension in the alveoli by forming a film. Surfactant remnants are endocytosed by type 2 alveolar cell and then recycled for another cycle of tubular myelin formation, and this continues. 90 to 95 percent of surfactant is recycled. Some of the surfactant remnants are also recycled by alveolar macrophages. 

Host: You have explained the pathway of surfactant formation and recycling very well, so thank you for that. I think that the role of surfactant protein may be important to discuss here as well.

Sanjay Akangire, MD: That is correct, Dr. Sharma. So the role of surfactant protein is also very important to know, especially from the board standpoint for neonatology. So, as discussed earlier, SPA assists in tubular myelin formation and has role in host defense, especially opsonization, phagocytosis, and bacterial lysis. SPB and SPC are very important in decreasing the surface tension in the alveoli by absorption of phospholipids,

as discussed earlier. SPD has a role in host defense and reduces viral infectivity. It also has antioxidant properties and maintains surfactant lipid homeostasis. Another important thing to add is that ABCA3 protein or ATP binding cassette member A3 helps in transport of lipids and formation of lamellar bodies for further surfactant synthesis.

Host: That's great, and I'm sure that sometimes things may not go as expected during the surfactant production, leading to various diseases. Can you discuss the effects of abnormal surfactant development?

Sanjay Akangire, MD: That is correct, Dr. Sharma. Sometimes things may not go as expected and also it is important to know abnormalities of surfactant production. Let's discuss this briefly. SPB deficiency is the most common surfactant protein deficiency on histology and electron microscopy studies of lung with SPB deficiency.

No laminar bodies or tubular myelin is seen and pulmonary alveolar proteinosis is seen. Treatment is lung transplantation for SPB deficiency. For SPC deficiency, infants can present as chronic lung disease or interstitial lung disease, or it can be present later in early childhood as well. ABCA3 deficiency leads to decreased transport of lipids and impaired lamellar bodies formation.

Histology and electron microscopy shows abnormally dense lamellar bodies.

Host: That's all great information and actually in clinical practice, the way that sometimes the surfactant deficiency SPB presents is even repeated doses of exogenous surfactant, it becomes ineffective. And I know now we do have genetic testing that has made it much easier for us to have an earlier diagnosis.

Let's talk about the important factors that stimulate surfactant production and that delays surfactant production.

Sanjay Akangire, MD: Sure, Dr. Sharma. So the factors that stimulate surfactant production are maternal PIH or pregnancy induced hypertension, chronic hypertension in mother, prolonged rupture of membranes, chorioamnionitis, intrauterine growth restriction, steroids, thyroid hormones, and methylxanthines or caffeine. And some key factors that delay surfactant production are maternal diabetes, male gender, C-section, insulin, Rh isoimmunization in mother, and obviously preterm birth. Another important aspect to keep in mind is that lower lecitin myelin ratio in amniotic fluid, especially less than two, increases the risk of RDS in newborns.

Host: Dr. Akangire, I think we covered a good amount of information on fetal lung development and surfactant physiology. Let's wrap up, and could you highlight some of the take home points?

Sanjay Akangire, MD: Sure, Dr. Sharma. So let's go over some key take home points. So the mnemonic for stages of lung development is Each Phase ComeS with Age and the stages are embryonic, three to six weeks gestation, pseudoglandular 6 to 16 weeks gestation, canalicular 16 to 26 weeks gestation, saccular 26 to 36 weeks gestation and alveolar stage happens after 36 weeks of gestation.

TE fistula or tracheal stenosis occurs due to abnormal events in embryonic stage. CDH or CPAM occurs due to abnormal events in pseudoglandular stage. Alveolar capillary dysplasia occurs due to abnormal events in canalicular stage. One third of fetal lung fluid is absorbed prenatally, one third during labor, and one third after birth.

Chloride channels are responsible for fetal lung fluid secretion and sodium or ENaC channels are responsible for fetal lung absorption. Surfactant consists of lipids manly, and primary lipid is dipalmitoylphosphatidylcholine, or DPPC. And again, this is desaturated. It is important to know the pathway of surfactant production and recycling to understand the diseases associated with abnormal surfactant production.

SPA has a role in surfactant tubular myelin formation and host defense. SPB is a primary surfactant protein and responsible for decreasing surface tension and its deficiency is the most common surfactant protein deficiency. ABCA3 protein deficiency leads to decreased transport of lipids and abnormal lamellar body formation and symptoms may be RDS initially.

Host: That's a great summary of the things that we covered in this podcast. I have a question for you. So do you have any questions on this topic for us to go over?

Sanjay Akangire, MD: Sure. As we did in our previous podcast, I have a few questions. So let's get started. So the question number one, so during a morning report, you are discussing stages of lung development and the timeline of gestational age with fellows and residents. At the end of what stage of lung development, the lung is considered viable. Saccular, B. Embryonic, C. Canalicular, D. Pseudoglandular, and E. Alveolar stage.

Host: Okay the to that is actually C, Canalicular.

Sanjay Akangire, MD: Correct, Dr. Sharma. And again, we discussed this a few times. Each Phase ComeS with Age. Again, E stands for embryonic three to six weeks gestational age P for pseudoglandular, six to 16 weeks gestational age, and canalicular 16 to 26 weeks gestational age, saccular 26 to 36 weeks gestational age, and alveolar more than 36 weeks.

And you also discussed the rule of six. So you, you just have to remember, six in each stage.

Host: Yeah, and also that during the canalicular phase, the surfactant starts to get produced. So that is the stage that we usually consider viability.

Sanjay Akangire, MD: That is correct, Dr. Sharma. So let's go to question number two. As we discussed, one third of fetal lung fluid is cleared prior to delivery, one third during delivery, and one third after delivery. Transfer of which electrolyte across pulmonary epithelial cells is responsible for fetal lung fluid clearance prior to delivery? So the choices are A. Potassium, B. Chloride, C. Bicarbonate, D. Sodium, and E. Calcium.

Host: I know this. It's either chloride or sodium, but chloride is secreted and sodium is absorbed. So the answer is D, sodium.

Sanjay Akangire, MD: That is correct, Dr. Sharma. So again, Sodium. So as we know, fetal lung fluid is important for lung growth. Chloride ions are responsible for fetal lung fluid secretion into the alveolar space. And sodium channels or ENaC channels, you know, are responsible for fetal lung fluid absorption. So the fluid is absorbed due to sodium channels and you are correct on this. 

So let's go to question number three. Which of the following has the highest amount in the surfactant? So the choices are A. Surfactant proteins. B. Phosphatidylcholine monounsaturated, C. Neutral lipids, D. Phosphatidylglycerol, and E. dipalmitoylphosphatidylcholine, desaturated or DPPC.

Host: We did cover this, and the answer is E, DPPC, desaturated.

Sanjay Akangire, MD: That is absolutely correct, Dr. Sharma. So very important point. DPPC is very important to remember. Surfactant is primarily made of lipids. Constituents of surfactant, as we know, the DPPC is 15%, phosphatidylcholine is 20%, surfactant proteins, 8%, phosphatidylglycerol, 8%, neutral lipids, 8%, and other phospholipids are 6%. 

Host: We have come to the end of our podcast. Thank you, Dr. Akangire, and hopefully this information will give you a better understanding of fetal lung development and surfactant physiology. Until next time, this is Neonatology Review, Isolette to Crib. I'm Dr. Joti Sharma. Thank you all for listening.