Neonatal Research

There’s a lot of activity in Lactoferrin research recently, this protein is very promising as prophylaxis against infections and perhaps also against necrotising enterocolitis.

First of all a study giving bovine lactoferrin to newborn piglets which showed a stimulatory effect on crypt cells in the developing intestine. Reznikov EA, Comstock SS, Yi C, Contractor N, Donovan SM: Dietary bovine lactoferrin increases intestinal cell proliferation in neonatal piglets. J Nutr 2014, 144(9):1401-1408. Exactly which cell type isn’t clear to me, but Paneth cells in the crypt have been suggested to be important in the pathophysiology of NEC. (McElroy SJ, Underwood MA, Sherman MP: Paneth Cells and Necrotizing Enterocolitis: A Novel Hypothesis for Disease Pathogenesis. Neonatology 2013, 103(1):10-20).

Another study from the same group, in the same model (in fact I think it is the same piglets) Comstock SS, Reznikov EA, Contractor N, Donovan SM: Dietary Bovine Lactoferrin Alters Mucosal and Systemic Immune Cell Responses in Neonatal Piglets. The Journal of Nutrition 2014, 144(4):525-532. Both spleen cells and mesenteric lymph node cells showed signs of enhanced immune responses with dietary bovine lactoferrin.

Colostrum, and mature breast milk contain significant amounts of lactoferrin. (in fact the piglets in the previous study were deprived of sow colostrum in order to do the study). Breast milk is almost always colonized with bacteria, very often bifidobacteria are present, and often lactobacilli also. In this study, which included both full term (34) and preterm (14) mother infant (human) pairs, the lactoferrin concentrations were similar to previous data, with around 7 mg/mL in colostrum dropping to 2.3 in mature milk, similar in term and preterm subjects. Mastromarino P, Capobianco D, Campagna G, Laforgia N, Drimaco P, Dileone A, Baldassarre ME: Correlation between lactoferrin and beneficial microbiota in breast milk and infant’s feces. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 2014, 27(5):1077-1086. They also showed bacterial DNA of lactobacilli in all of the breast milk, and bifidobacteria in all but one of the breast milk samples, with a higher concentration of lactobacilli in the preterm milk, and similar bifidobacteria concentrations. They also analyzed stool samples, and were able to find lactoferrin in the stools, and found lactobacilli and bifidobacteria in almost all the stools also.

The next study examined the effects of heat-treatment and ultraviolet light on the lactoferrin concentration of colostrum (also on what they call hospital milk, which is milk supplied by sick cows which cannot be sold, and may be contaminated with many bacteria). The heat treatment was to 63 degrees for 60 minutes, which is similar to many human milk bank standards I believe. Lactoferrin was measured by an ELISA, and I don’t know whether lactoferricin, or other active or potentially active parts of the molecule would react with the assay. Teixeira AGV, Bicalho MLS, Machado VS, Oikonomou G, Kacar C, Foditsch C, Young R, Knauer WA, Nydam DV, Bicalho RC: Heat and ultraviolet light treatment of colostrum and hospital milk: Effects on colostrum and hospital milk characteristics and calf health and growth parameters. The Veterinary Journal 2013, 197(2):175-181. The study shows a number of things : cows’ milk colostrum has much less lactoferrin than human (which was already known), about 0.3 mg/mL falling to around 0.2 in mature milk. Both heat treatment and ultraviolet treatment reduced the lactoferrin concentrations, and also the IgG concentrations. It’s interesting sometimes to read research form other fields in some detail, this study was performed in a farm which has 2800 cows, and they randomized over 280 animals into the 3 groups. They calculate the sample size based on daily weight gain, which is therefore the primary outcome variable, but they don’t use the term, and report that outcome in very little detail, right at the end of the results.

Also interestingly, low birth weight calves are at higher risk of infection than normal birth weight, being LCBW (low calf birth weight) less than 37 kg is associated with much more diarrhoea, and they also had lower serum IgG concentrations.

Part of the efficacy of lactoferrin may be that it sequesters iron, so that it is no longer available for organisms that rely on iron to replicate, such as E Coli (although there are several other mechanisms also). If you then add more iron to the feed, does this overcome the benefits? A number of in vitro studies have had variable results, the latest took colostrum from mothers delivering at term, and tested bacterial growth effects of the milk with and without the addition of a human milk fortifier, used for preterm infants, that contains iron. They found no effect of the iron on Pseudomonas or Staph Aureus, but E Coli grew a little more actively when the fortifier with iron was used. Apparently other studies have had different effects, such as showing a direct bacteriostatic effect of breast milk, and showing more adverse effects of the iron. Campos LF, Repka JCD, Falcão MC: Effects of human milk fortifier with iron on the bacteriostatic properties of breast milk. Jornal de Pediatria 2013, 89(4):394-399.

Of course iron is also important as a catalyst for the production of reactive oxygen species, so another potential benefit of lactoferrin may be to reduce free iron, and therefore to reduce potential oxidative injury of the gut. This is suggested by the results of another study: Jegasothy H, Weerakkody R, Selby-Pham S, Bennett LE: In vitro heme and non-heme iron capture from hemoglobin, myoglobin and ferritin by bovine lactoferrin and implications for suppression of reactive oxygen species in vivo. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 2014, 27(6):1371-1382.

Finally a review article which discusses the potential benefit of lactoferrin supplementation. Michael Sherman, who has studied lactoferrin, including recombinant human lactoferrin relates the importance of ensuring that preterm babies get colostrum. Which I certainly agree with. The authors also state however that there is no lactoferrin preparation proved by the FDA for use in preterm infants, which is of course true, but they state in the abstract ‘regulatory burdens required to bring lactoferrin to the bedside may limit its availability’. When the list of trials completed and in progress at the end of the article are examined, I hope that is not true. If the data are confirmatory, we should have plenty of good data to be able to obtain approval. Sherman M, Miller M, Sherman J, Niklas V: Lactoferrin and necrotizing enterocolitis. Current Opinion in Pediatrics 2014, 26(2):146-150.

For those of you who might be interested in buying the book that I blogged about a few days ago, please note that you can get the book at a big discount, by contacting Saroj directly, using the link on her website.

Also, now I have had the chance to look at the book a bit more, there are sections after the former preterm babies stories that are very interesting, including several chapters written by Saroj about outcomes of very preterm babies, about how her ideas of quality of life etc have developed, and about impacts of prematurity on families. Also there is a great chapter by her long time collaborator Peter Rosenbaum. He reflects on views of disability and how our appreciation of impairments and the way they impact on individuals have changed over the last half century. Well worth reading, in addition to the ‘preemies’ voices’ stories and Saroj’s chapters.

I thought I’d blog about this article for two reasons.

Firstly, it points out the theoretical exposure to phthalates in patients in the NICU, which are enormous and potentially with multiple toxic effects. Exposures could possibly be as high as 160,000 times the limits of intakes that we really want for our babies, and this might lead to serious adverse effects. Those toxic effects include hepatic effects, adverse pulmonary impacts and reproductive (especially male) toxicities. Of course all those words, ‘possibly’ ‘theoretical’ ‘might’ are caveats that require investigation and more solid evidence, and also some assurance that alternatives are better. We need to be sure that alternative polymers that don’t require phthalate plasticizers are also safe, but if we can be reasonably sure it seems that it would be a good idea to make the change.

The second reason that I am writing this post is that I wanted to try out the new feature of the Nature group journals. I can now include a link so that anyone who follows it will be able to read the full text article if they wish. So give it a try, and, if it works, then, even if your institution does not have access to the Journal of Perinatology, or if you read from home, then you should be able to see the whole article. You won’t be able to download or print it, but having permanent access to the articles on-line will be helpful for many.

Here is the link:

http://rdcu.be/bKwS

Or rather the voices of adults who were born very preterm. Saroj Saigal is well known to many of us in neonatology, either by reputation, or, for the lucky ones among us, as a friend.

Her insights into quality of life, what it means to our patients, and how patients evaluate their own quality of life, have been extremely influential, and have had a great impact on how we practice, and how we talk to parents. Her studies of adaptation to adult life among former very preterm babies were also eye-opening, and showed how well her cohort was doing as they entered adulthood.

Her latest innovation is to gather the stories of former preterm babies, now they are adults, and publish them as a book. You can buy her book from a number of places, just follow the links under the bookstore tab on her web-site at www.preemievoicesbook.com.

Also on the home page of her web-site there is a 25 minute video, which has several of the book contributors (and a couple of mothers) telling some of their stories to the camera. I found it quietly moving, and encouraging, adults with and without disabilities talk about their lives, and about how they see their prematurity.

Thanks again Saroj, you really make a difference.

In this study about the potential effects of α-tocopherol on IQ in ELBW infants, there are a number of problems: Starting with the title, Long-term alpha–Tocopherol supplements may improve mental development in extremely low birth weight infants. Acta Paediatrica. 2014. (It is by Kitajima and others, and published on-line in Acta.) A title should give some idea of the methodology that was used, this was not a prospective study which you need to argue causation, but a retrospective review. Also using the title to promote one interpretation of the results, which could easily be challenged, is not a good idea. The title states ‘may improve’, but the phrase ‘may be associated with’, would be much more appropriate.

The introduction refers to the study as a  retrospective cohort study, but in the sentence just before that they talk about the results of the study. Maybe I’m an old stick-in-the-mud, but I think the introduction is where you explain why you did the study, the methods is where you describe what you did, and the results are where you say what you found. (The discussion section is then where you make extravagant claims about the significance and implications of your findings, and end up with a section calling for further research).

If we try to figure this out, there were 579 ELBW survivors from an NICU over 12 years, ending in May 1998. 45% of them were followed up, the last follow up was at 8 years of age, which means that the study was finished 8 years ago.

Now what really confuses me are some of the other details of the report, the babies had vitamin E started at between 3 and 4 weeks of age; the authors talk about the infants being ‘divided into groups’ either getting no tocopherol, tocopherol for between 1 and 6 months, or for more than 6 months. It seems as I delve deeper that the babies who were ventilated for less than 4 weeks did not get it, those ventilated for more than four weeks ‘continued’ the tocopherol until discharge or until 4 months, and those who went home on oxygen had the tocopherol until the oxygen was discontinued. So the groups are very different, the long use babies had much more dexamethasone use, for example.

The results actually show that the IQ at school age was highest in the group who did not receive any tocopherol. Which isn’t exactly what they say in the introduction, abstract or title! Only by combining the two groups, those who received no tocopherol and the 1 to 6 month tocopherol, were the authors able to claim that the very prolonged tocopherol had higher IQ, but there are so many confounders that I really don’t think that any analysis of these data can be considered reliable; even if you do all the data manipulation that these authors do, leaving out high income families, infants who had very large amounts of dexamethasone etc. All you can say is that infants in the group who got no tocopherol had higher IQ than those who got 1 to 6 months, and those who got extremely prolonged supplementation had intermediate values. Whether this was due to the tocopherol or to other differences between the groups is open to question. In fact that pattern suggests to me a random effect.

Well obviously before clamping, after clamping there isn’t much flow! In this study with full term babies they were delivered and placed on the mother’s chest, and then an ultrasound probe was placed to measure umbilical cord flow.  (Boere I, et al. Umbilical blood flow patterns directly after birth before delayed cord clamping. Archives of disease in childhood Fetal and neonatal edition. 2014).

This is great study, really important in helping to understand what is actually happening during delayed clamping, and what probably happened in the past as we were evolving and didn’t have cord clamps, although exactly how the cord was managed in the distant past is unknown, was it bitten by the mother? left to dry?

The babies were all vaginal deliveries at term who breathed spontaneously.  The cord was clamped at a time determined by the midwife doing the delivery, the local standard was to wait at least one minute and after the cord stopped pulsating. Clamping occurred on average at 5 minutes after delivery, and the researcher checked to see if there was still pulsation palpable just before the cord was clamped.

Arterial flow often stopped before cord pulsation disappeared, and often continued longer than venous flow. Venous flow continued for an average of 4.5 minutes in babies in whom is stopped before cord clamping, and as long as over 8.5 minutes (that is the third quartile, so the maximum duration isn’t reported); some babies still had some pulsation when the cord was clamped, which was at 5.5 minutes and up to over 9.5 minutes.

The duration of flow was extremely variable, but the fact that venous flow often stopped first suggests that prolonged delays in cord clamping might sometimes decrease placental transfusion.

The infant’s breathing pattern affected flows, in particular vigorous crying reduced or even reversed venous flow, but large inspirations increased flow.

This technology doesn’t allow volume measurements, as you would need to be able to measure accurately the vessel diameters, which are probably themselves changing. A similar studies in preterms, and during resuscitation would be fascinating.