Int. J. Devl Neuroscience 53 (2016) 99–106
Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience
journal homepage: www.elsevier.com/locate/ijdevneu
Developmental neuropathology of brainstem respiratory centers in
unexplained stillbirth: What’s the meaning?
Anna M. Lavezzi a,∗ , Stefano Ferrero a,b , Luigi Matturri a , Luca Roncati c,d , Teresa Pusiol c
a
“Lino Rossi” Research Center for the Study and Prevention of Unexpected Perinatal Death and SIDS; Department of Biomedical, Surgical and Dental
Sciences, University of Milan, Italy
b
Division of Pathology, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy; Department of Biomedical, Surgical and Dental Sciences,
University of Milan, Milan, Italy
c
Institute of Pathology, Hospital of Rovereto (Trento), Italy
d
Department of Diagnostic and Clinical Medicine and of Public Health, Section of Pathology, University of Modena and Reggio Emilia, Policlinico Hospital,
Modena, Italy
a r t i c l e
i n f o
Article history:
Received 27 May 2016
Received in revised form 24 June 2016
Accepted 24 June 2016
Available online 28 July 2016
Keywords:
Brainstem
Developmental alterations
Maternal smoking
Respiratory network
Unexplained stillbirth
a b s t r a c t
Stillbirth is one of the most stressful life events affecting over 3 million pregnancies per year throughout
the world. An accurate autopsy of the stillborn fetus, including the placenta and umbilical cord examination, should be performed promptly after delivery. A thorough maternal history also should be taken,
including exposures to risk factors. In many cases a death cause, attributable to fetal, maternal, or placental
pathology, is clearly identified. However, in 50% or more of cases the cause remains unknown.
The purpose of this study is to highlight possible developmental alterations of the autonomic nervous
system in unexplained stillbirths to provide an explanation of the pathogenetic mechanism of their death.
We conducted a careful neuropathological study of the brainstem, where the main vital centers are
located, in 85 unexplained stillbirths and 52 age-matched controls died of known cause. Information on
the maternal lifestyle, including the smoking habit, was collected in all cases. Hypodevelopment of neuronal centers involved in breathing control, all connected together in a “respiratory network”, precisely
hypoplasia of the facial/parafacial complex, Kölliker-Fuse nucleus, pre-Bötzinger nucleus and intermediolateral nucleus, were frequently observed in unexplained deaths, significantly related to maternal
cigarette smoking.
We support the hypothesis of a strong action of maternal smoking during pregnancy on the development of brainstem respiratory nuclei and suggest an explanation of the high incidence of the respiratory
network alterations in unexplained fetal death, when breathing not represents a vital function.
© 2016 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Stillbirth is generally considered as the death of a baby before
or during delivery, or in any case before their first birthday (Fretts,
2009; Goldenberg et al., 2004). Each year about 24,000 babies are
stillborn in the United States and over 3 million throughout the
world (Macdorman and Gregory, 2015). Because of its apparent
randomness, and the lack of any warning, stillbirth cuts across
socio-economic classes, ethnicities, religions, and maternal age
∗ Corresponding author at: “Lino Rossi” Research Center for the Study and Prevention of Unexpected Perinatal Death, SIDS—Department of Biomedical, Surgical and
Dental Sciences, University of Milan, Via della Commenda 19, 20122 Milano, Italy.
E-mail address: [email protected] (A.M. Lavezzi).
http://dx.doi.org/10.1016/j.ijdevneu.2016.06.007
0736-5748/© 2016 ISDN. Published by Elsevier Ltd. All rights reserved.
groups. However, even if no woman is immune from stillbirth,
there are some maternal factors associated to the condition that
can increase the risk for stillbirth, including black race, age under
20 or over 35 years, obesity, nulliparity or a previous miscarriage
(McClure et al., 2009; Flenady et al., 2011; Yudkin et al., 1987;
Liu et al., 2014). A well-documented risk factor for unexplained
stillbirth is maternal cigarette smoking during pregnancy. A huge
number of works in the literature have, in fact, reported the harmful
effects exerted by the crossing of nicotine into the bloodstream of
the fetus (Lambers and Clark, 1996; Marufu et al., 2015; Raymond
et al., 1994; Wickström, 2007).
It is universally recognized that the most important test in the
search for a possible death cause in stillbirth is gross and microscopic fetal autopsy, complete with placental, umbilical cord and
membrane examination, genetic analyses, and a detailed medical
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history evaluation (Faye-Petersen et al., 1999; Saller et al., 1995;
McPherson and Valdes-Dapena, 1998; Bove, 1997).
Known causes of stillbirth can generally be subdivided into three
broad categories, namely: 1) congenital fetal defects (including
genetic abnormalities), 2) placenta and/or umbilical cord pathologies and 3) maternal disorders (such as obesity, hypertension,
diabetes, etc.) (Bendon, 2001; Bukowski et al., 2011; Fatima et al.,
2014).
However, despite the progress in medical diagnostics and antenatal care over the last 30 years, after a careful investigation
one-half to two-thirds of stillbirths are still listed as deaths for
undeterminable reasons. Because these deaths cannot be attributed
to a specific identifiable cause, they are called “unexplained stillbirths” (Gardosi et al., 2005; Yudkin et al., 1987; Warland and
Mitchell, 2014). We have proposed that a sudden death during pregnancy, that remains unexplained after a thorough study,
should also be considered as a syndrome and referred to with
the acronym “SIUDS”, i.e., “Sudden Intrauterine Unexplained Death
Syndrome”, like “SIDS” for “Sudden Infant Death Syndrome”
(Matturri et al., 2014). This suggested definition is based on the
realization that several conditions simultaneously occurring may
contribute to a given stillbirth and on the presence in these unexpected deaths of common developmental abnormalities of the
autonomic nervous system, associated to the same risk factors
related to SIDS.
It is necessary to point out that the lack of uniform post-mortem
protocols applied worldwide for evaluating and classifying stillbirths has hindered significant studies in this field.
The “Lino Rossi” research Center of Milan University has
developed an investigative postmortem protocol that includes,
in particular, an in-depth examination of the autonomic nervous system (available at the webpage http://users.unimi.it/
centrolinorossi/en/guidelines.html), according to the Italian Law
31/2006 “Regulations for Diagnostic Post Mortem Investigation in
Victims of Sudden Infant Death Syndrome (SIDS) and Unexpected
Fetal Death.” This law states in particular that all stillbirths that died
suddenly without any apparent cause after 25 weeks of gestation
must be submitted to a depth diagnostic postmortem investigation.
Herein, we report the autonomic nervous system findings
obtained according to the above-mentioned protocol in a wide
group of stillbirths previously diagnosed as “unexplained” after a
routine fetal autopsy. Surprisingly, we frequently have observed
in the brainstem of these victims hypoplasia of neuronal centers
known to be involved in the autonomic control of breathing. So,
our main purpose was to provide an explanation of the high incidence of these developmental alterations, also suggesting how they
can lead to death in the womb, before breathing is yet required for
life.
2. Material and methods
We studied 85 stillbirths, aged from 25 to 40 gestational weeks,
sent to the “Lino Rossi” research Center of the Milan University from
hospitals and health Institutions of Italian regions, in accordance
with the abovementioned national Law 31/2006.
The fetuses had previously been submitted to a complete
autopsy, including examination of the placental disk, umbilical cord
and membranes, but without identifying a possible death cause. So,
they were classified as “unexplained stillbirths” or, more appropriately, “SIUDS”.
For each case, a complete clinical history, with particular reference to the maternal lifestyle, was given. None of the mothers
of the 85 victims had any significant pathology. The mothers were
also asked for information about a smoking habit before and during
pregnancy. Forty-eight mothers (56%) claimed to be active smokers
before and during pregnancy, while 37 (44%) declared no history
of cigarette smoking. Since, as is well known (Shipton et al., 2009),
retrospective assessment of a mother’s smoking habit, mainly after
the death of a child, is sometimes unavoidable, the negative selfreports were verified by testing for cotinine, the main metabolite
of nicotine, that has a long half-life, in the hair of victims. In 4
stillbirths among the 37 mothers who denied a smoking habit the
cotinine-test was positive, thereby leading to a total of 52 (61%)
the cases with nicotine absorption in pregnancy and reducing the
actual number of non-smoking mothers to 33 (39%).
For every case, the material to be sent to the “Lino Rossi” Center
consists of the brain, heart (for the specific study of the cardiac conduction system), a lock of the victim’s hair and, when possible, lung
samples to evaluate the pulmonary maturation stage. A group of 52
fetuses who died of known causes, selected from a wide set of cases
previously collected at the “Lino Rossi” Research Center, were designated as “Controls”. The same material and information, including
the smoking habit, required for unexplained stillbirths was sent for
the control cases from the medical Institutions involved in application of Law 31, for the specific purpose of carrying out comparative
analyses.
The Controls were matched with the unexplained stillbirths for
gestational age, sex and area of origin. Table 1 summarizes the
SIUDS/Control case study, indicating the age-ranges, sex distribution and the death diagnoses.
Consent- Parents of all the infants included in the study provided
written informed consent to autopsy and related researches; study
approval was granted by the institutional review board of the Milan
University (Lino Rossi Research Center).
2.1. Protocol for the anatomopathological study of the brain
After fixation in 10% phosphate-buffered formalin, the brains
were processed and embedded in paraffin. Target of this study
was the in-depth microscopic study of the brainstem. Transverse
serial sections of the midbrain, pons, medulla oblongata and spinal
cord (rostral cervico-thoracic tract), where the main structures controlling the vital functions are located, were made at intervals of
60 ␮m. For each level, six-seven 5 ␮m sections were obtained, two
of which were stained using hematoxylin-eosin and Klüver-Barrera
for histological examination, while two sections were treated for
immunohistochemical detection of the neuronal nuclear antigen
(NeuN), a marker of neuronal functionality. The remaining sections were saved for further investigations and stained as deemed
necessary.
The routine histological evaluation of the brainstem was
performed on the locus coeruleus, Kölliker-Fuse nucleus, and
median raphé nucleus in the rostral pons/mesencephalon; on the
parafacial/facial complex, superior olivary complex, ambiguus, preBötzinger, inferior olivary, arcuate, obscurus, pallidus raphé nuclei
and solitary tract complex in the medulla oblongata; the intermediolateral nucleus in the spinal cord junction with the brainstem.
Many of these nuclei (and precisely the Kölliker–Fuse nucleus,
the facial/parafacial complex, the pre-Bötzinger nucleus and the
intermediolateral nucleus) are anatomically and functionally interconnected via interneuronal synapses, modulating one another,
within a network extending from the rostral spinal cord through the
medulla oblongata, pons, to the caudal pars of the midbrain. This
nervous complex is defined as “respiratory network” (RN), given
its primary involvement in the control of breathing (Cohen, 1979;
Bianchi et al., 1995; Viemari et al., 2003).
Fig. 1 presents a scheme of the human breathing control mechanism in perinatal life, proposed by one of the Authors in a
recent paper (Lavezzi, 2015), indicating the more representative
A.M. Lavezzi et al. / Int. J. Devl Neuroscience 53 (2016) 99–106
101
Table 1
SIUDS/control case study.
Gestational weeks
Fetuses
25 < 28
28 < 31
M/F
M/F
SIUDS
(n.85)
Controlsa
(n.52)
31 < 34
34 < 37
M/F
37 < 40
M/F
M/F
n.6
3/3
n.9
4/5
n.11
5/6
n.28
11/17
n.31
16/15
n.3
1/2
n.6
3/3
n.10
4/6
n.15
8/7
n.18
9/9
SIUDS: sudden intrauterine unexplained death syndrome; M/F: males/females.
a
Death diagnosis in Controls were: placental/umbilical cord pathologies (cord accidents, abruption, utero/placental insufficiency, severe chorioamnionitis, premature ropture of membranes): n. 22 cases; infectious diseases (CMV, parvovirus B19, listeria, toxoplasmosis): n. 13 cases; preeclampsia/eclampsia: n.7 cases; congenital abnormalities
(cardiomyopathies, pulmonary dysplasia): n.8 cases; karyotype alterations (trisomy 21): n.2 cases.
Fig. 1. Schematic representation of the human breathing control mechanism in perinatal life. (A) Left to right, the steps of the respiratory control in human perinatal life.
(1) In the human fetus, episodic respiratory activity aimed at promoting lung development is generated by the intermediolateral nucleus (ILN) in the upper spinal cord. (2)
At the same time, during intrauterine life, the Kölliker–Fuse nucleus (KFN), located in the rostral pons, plays an important function by inhibiting the response of central and
peripheral chemoreceptors and therefore any respiratory reflex, while allowing the occasional breathing activity headed by the ILN. (3) The facial/parafacial complex (F/PFC),
in the caudal pons, starts working at birth, under the stimulation of the KFN, which drastically changes its function, giving rise to the first inspiratory act. The activity of the
F/PFC is called “pre-inspiratory” because it is limited to activating, in its turn, the proper inspiratory nucleus in the medulla oblongata: the pre-Bötzinger nucleus (pBN) (4),
responsible for starting postnatal breathing. (B) Brainstem schematic representation showing the localization of the RN components. (from: Lavezzi, Front. Neurol. 2015, 6,
220).
brainstem histological sections1 where the RN structures are well
analyzable, depicted in a chronological functional sequence, as
explained in the legend.
2.2. Neuropathology diagnostic criteria
All the above-listed nuclei and/or structures are examined in
serial histological sections throughout their extension. A diagnosis
of “hypoplasia” of a given nucleus is formulated when it shows a
significantly decreased number of neurons and/or decreased area
in transverse histological sections, compared to the mean values
obtained in age-matched controls. The morphometric evaluations
are quantitatively performed using an Image-Pro Plus Image Analyzer (Media Cybernetics, Silver Spring, MD).
All the histological examinations of the brain are carried out
by two independent and blinded observers and comparison of the
1
These selected histological sections, identified on the basis of over twenty-years
of research at the Lino Rossi Center in this field, are easily recognizable on the basis
of precise landmarks, that are the superior cerebellar peduncle decussation in the
rostral pons (for the Kölliker–Fuse nucleus analysis), medial nucleus of the superior
olivary complex in the caudal pons (for the facial/parafacial complex), dorsal accessory of the inferior olivary nucleus in the medulla oblongata (for the pre-Bötzinger
nucleus) and dorsal and ventral horns, adjacent to the central canal in the rostral
spinal cord (for the intermediolateral nucleus).
results performed employing K statistics (Kappa Index- KI) to evaluate the inter-observer reproducibility. The Landis and Koch system
(Landis and Koch, 1977) for the K interpretation is used, where
0–0.2 is slight agreement, 0.21–0.40 indicates fair agreement,
0.41–0.60 moderate agreement, 0.61–0.80 strong or substantial
agreement, and 0.81–1.00 indicates very strong or almost perfect
agreement (a value of 1.0 being perfect agreement). The application
of this method in the present study revealed a very satisfactory KI
(0.86).
2.3. NeuN immunohistochestry
Representative sections from paraffin-embedded tissue blocks
were stained using commercially supplied mouse monoclonal antibodies against the neuronal nuclear antigen NeuN (Chemicon
International, MAB377). A standard avidin-biotin complex (ABC)
technique was used with peroxidase-diaminobenzadine to visualize and develop the antigen-antibody reaction. The antibody
dilution was 1:1500. Incubating solutions were boiled in 10 mM
citric acid at pH 6.5, in a microwave oven, for 5 min at high power,
then 5 min at 50% power, and finally cooled for 20 min. Sections
were lightly counterstained with Mayer’s hematoxylin. Negative
controls were prepared by replacing the primary antibody with
phosphate-buffered saline (PBS) in the incubation. In these procedures staining always failed to occur.
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2.3.1. Evaluation of the NeuN immunohistochemical results
Even if in health status the NeuN immunopositivity is diffused
in almost the entire brain, we specifically examined at the light
microscope the areas including the nuclei of interest.
For every structure, only cells with intense brown immunostaining were considered to be really positive. Moreover, also a weak
brown intensity was taken into account.
We quantified the scoring using a x 40 lens, as follow:
− = no positive cell (negativity)
−/+ = a number of cells with only weak positivity ≤30% per unit
area (weak positivity)
+ = a number of positive cells ≤30% per unit area (moderate positivity)
++ = a number of positive cells >30% per unit area (strong positivity)
The unit area was represented by square millimeteter (mm2 )
2.4. Protocol for the histological examination of the lungs
The stage of lung development in each case was evaluated
according to microscopic criteria, i.e. the presence of cartilaginous
bronchi up to the distal peripheral level and the radial alveolar
count (RAC). This last is a reliable index of lung maturation in
intrauterine life, closely related to the gestational age in weeks
(Emery and Mithal, 1960).
The RAC was evaluated in terminal lung units (distal portions
of parenchyma to the last respiratory bronchioles, identifiable by
an incomplete epithelial lining), examining at least 10 random histological fields for each case in order to estimate the number of
airspaces cut by a straight line drawn from the center of the most
peripheral bronchiole to the nearest connective tissue septum or
the pleura. Emery and Mithal had also provided the reference RAC
values at different ages, as reported in Table 2.
2.5. Statistical analysis
Quantitative data were expressed as means ± SEM. The significance of differences between different parameters in the groups
(SIUDS and Controls) were evaluated by Student-t test, chi-square
test or Fisher’s test. In cases of a skewed distribution, a nonparametric Whitney rank sum test was applied. One-way ANOVA is used
for quantifying and partitioning variance between groups. Statistics
was performed using SigmaPlot® statistical software (version 13,
Systat Software Inc, Chicago, IL). The selected level of significance
was p < 0.05, two-tailed.
3. Results
The histological examination performed on the brainstem of
85 SIUDS disclosed a marked hypodevelopment of one or more
structures making up the RN in 75% of cases. Hypoplasia of
the facial/parafacial complex in all its extension into the caudal
pons was observed in 51 SIUDS (60%), frequently associated to
hypoplasia of the pre-Bötzinger nucleus in the medulla oblongata. Hypoplasia of the Kölliker–Fuse nucleus was present in 40
SIUDS (47%), sometimes as the only alteration, and of the intermediolateral nucleus in 36 cases (42%). Figs. 2 and 3 illustrate
the cytoarchitecture of the two developmental alterations more
frequently observed (i.e. hypoplasia of the facial/parafacial complex and hypoplasia of the Kölliker–Fuse nucleus) in the specific
transversal histological sections.
Other developmental defects were the hypoplasia of the arcuate nucleus in the medulla oblongata, observed in 15 cases (13%),
almost always associated to hypoplasia of one or more raphè nuclei,
and hypoplasia of the pontine retrotrapezoid nucleus, detected in
8 cases (9%). Individual victims frequently displayed a combination
of all the above mentioned alterations.
A high correlation was evident between hypoplasia of the RN
components and the lack of NeuN expression. In fact, in 62 SIUDS
with various developmental morphological defects of respiratory
nuclei, only immunonegativity or, more rarely, weak positivity of
the NeuN protein was found.
In Controls, the examination of the brainstem did not highlight
developmental alterations of the RN centers, except for two cases
(both died of severe chorioamnionitis) in which there was partial
hypoplasia of the Kölliker–Fuse nucleus. Arcuate nucleus hypoplasia was observed in 7 control cases (13%) and hypoplasia of the
obscurus raphe nucleus in 3 cases (6%).
The degree of maturation of the pulmonary parenchyma was
evaluated in order to highlight a possible correlation between
anomalies of the RN and lung hypodevelopment. In 28 SIUDS
with hypoplasia of the intermediolateral nucleus and/or hypoplasia of the Kölliker–Fuse nucleus, an interesting finding was the
lung hypoplasia, characterized by a markedly decreased RAC index
(almost always below 2.2) and frequent presence of cartilaginous
bronchi at peripheral level. The lung developmental stage was, on
the contrary, above the reference values in nearly all control cases.
Only in two fetuses, who died at the 36th and 38th gestational
week, respectively, a RAC value corresponding to 3.0 was obtained
(normal reference value for age = 3.6).
Finally, we related the neurodevelopmental defects observed
in the brainstem to the mother’s smoking habit. A significant
correlation was evident between maternal smoking and delayed
maturation of respiratory nuclei in SIUDS, compared to controls
(p < 0.01). In fact, in all SIUDS with a smoker mother, hypoplasia of
one or more components of the RN was highlighted.
Table 3 summarizes all the results.
4. Discussion
Stillbirth, occurring in particular in the last trimester of an
uncomplicated pregnancy, when the developing baby could survive outside the womb, is a very distressing event. Parents want to
know why their baby has died, so clinicians should be ready to call
for an autopsy in cases of stillbirth, also considering that the results
may be valuable in planning future pregnancies.
However, despite the recognized importance of a thorough
autopsy in cases of stillbirth so as to individuate the morphological
substrate of death, the rate of autopsies is very poor worldwide.
In addition, there is no uniform stillbirth post-mortem protocol in
use anywhere today, and every autopsy is done according to local
practice (Chichester, 2007; McPherson and Valdes-Dapena, 1998).
This state has been improved in Italy, thanks to Law 31/2006
that imposes, in cases of death of a fetus without any apparent
cause after the 25th week of gestation, an autopsy performed in
the referral centers of each Region following unified guidelines and,
when the diagnosis is not clarified (i.e. in cases of “unexplained stillbirth”), the autopsy samples, mandatorily including the brainstem,
must be sent to the “Lino Rossi” Research Center of Milan University
for further in-depth studies (Roncati et al., 2016a).
Here we illustrate how the thorough histological examination of
the brainstem may reveal specific developmental defects in these
deaths. In fact, in a wide range of SIUDS we very frequently observed
hypoplasia of neuronal centers that preside over respiratory function, namely of the Kölliker–Fuse nucleus and facial/parafacial
complex in the pons, the pre-Bötzinger nucleus in the medulla
oblongata and the intermediolateral nucleus in the upper spinal
cord, all components of the so-called “respiratory network” (RN).
These centers can coordinate each other through excitatory and/or
inhibitory connections, in relation to the need to control breath-
A.M. Lavezzi et al. / Int. J. Devl Neuroscience 53 (2016) 99–106
103
Table 2
Histological diagnostic criteria of lung hypoplasia in fetuses.
Criteria
Gestational weeks
RAC normal reference value
1) Radial alveolar count (RAC)a
minor than reference value for
gestational week
24–27
28–31
32–35
36–39
40
2.2±
2.6±
3.2 ± 0.9
3.6 ± 0.9
4.4±
2) presence of cartilaginous
bronchi up to the distal level
a
This parameter represents the mean number of alveoli transected by a perpendicular line drawn from the center of the most peripheral bronchiole (recognizable by not
being completely covered by epithelium) to the pleura or the nearest interlobular septum. Below, an histological picture showing the RAC method.
Fig. 2. (A) Representative histological section of caudal pons showing the localization of the facial/parafacial complex; (B) normal cytoarchitecture of this nucleus; (C)
Hypoplasia of the Facial/Parafacial complex in a SIUDS case (36 gestational weeks). Klüver-Barrera staining. Magnification: (A): 0,5x; (B) and (C): 20x.
ing before and after birth. Based on experimental studies, we have
hypothesized a chronological functional sequence of action of these
nuclei (as depicted in Fig. 1). The intermediolateral nucleus has
the task of promoting the initial episodic breathing patterns in
early fetal developmental stages and the spread of these spontaneous bursts into the spinomedullary neuronal axis to the other
respiratory centers, even before the establishment of a synaptic
drive, through the release of neurotrophic signals, transmitted by
an extracellular ionic flux (Hanson and Landmesser, 2003; Ren and
Greer, 2003).
The intermediolateral nucleus then plays a leading role by
inducing the early rhythmic respiratory movements aimed, above
all, to promote lung development in utero and then to monitor the
respiratory output pattern in postnatal life.
During intrauterine life, at the same time, the Kölliker–Fuse
nucleus plays an important function by inhibiting any respiratory reflex, allowing only, at intervals, the occasional breathing
activity triggered by the intermediolateral nucleus (Damasceno
et al., 2014). At birth, the Kölliker–Fuse nucleus function changes
drastically, giving rise to the first inspiratory act through stimulation of the facial/parafacial complex which, in turn, triggers the
pre-Bötzinger nucleus, the effective activator of breathing (Cohen,
1979; Bianchi et al., 1995; Viemari et al., 2003).
Other alterations highlighted in this study in addition to
hypoplasia of the RN components, although with a lesser frequency,
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Fig. 3. (A) Representative histological section of rostral pons showing the localization of the Kölliker–Fuse nucleus; (B) normal cytoarchitecture of this nucleus; (C) Hypoplasia
of the Kölliker–Fuse nucleus in a SIUDS case (38 gestational weeks). Klüver-Barrera staining. Magnification: (A): 0,5x; (B) and (C): 20x.
Table 3
Pathological results from case series: individual victims frequently displayed a combination of these alterations. In all, 75% of SIUDS (n.64 cases) and 15% (n.8 cases) of
Controls showed developmental alterations of one or more respiratory nuclei.
Pathological Results
Neuropathology
Hypoplasia of brainstem nuclei
• Respiratory network
facial/parafacial complex
Kölliker-Fuse nucleus
pre-Bötzinger nucleus
intermediolateral nucleus
• Other nuclei
arcuate nucleus
raphè nuclei
retrotrapezoid nucleus
NeuN poor/negative immunoexpression
Lung hypoplasia (RAC)
Maternal smoking in pregnancy
a
SIUDS
(85 cases)
Controls
(52 cases)
51 (60%)
40 (47%)
22 (26%)
36 (42%)
–
2 (4%)
–
–
15 (18%)
11 (13%)
8 (9%)
62 (73%)
28 (33%)
52a (61%)
7 (13%)
3 (6%)
–
7 (13%)
2 (4%)
10a (19%)
In all 52 SIUDS cases, and in 3 of the 10 Controls with a smoking mother, developmental alterations of one or more respiratory nuclei were present.
were developmental defects of the arcuate, the retrotrapezoid and
the raphé nuclei, all involved to varying degrees in chemoreception,
a fundamental mechanism for an efficient uptake and delivery of
oxygen and removal of carbon dioxide (Wong-Riley and Liu, 2005;
Nattie, 1998; Feldman et al., 2003). Then chemoreception takes part
in the respiratory control system, preserving normoxic ventilator
parameters through a variety of responses.
The hypodevelopment of the respiratory nuclei in SIUDS was
also combined with a low viability of neurons, testified by the
immunonegativity of the NeuN antigen, a specific protein that
is only expressed in post-mitotic healthy neurons (Sarnat et al.,
1998). It has been reported that a decreased NeuN expression in
postembryonic life can be indicative of neuronal degeneration,
often resulting from a severe injury (Unal-Cevik et al., 2004).
A.M. Lavezzi et al. / Int. J. Devl Neuroscience 53 (2016) 99–106
From the analysis of the overall findings here obtained, at least
two inevitable questions arise:
1) why the developmental abnormalities of the brainstem respiratory centers are significantly increased in unexplained fetal
deaths, compared to controls, if breathing is not a vital function in
utero? We suggest the following possible explanation on the basis
of our experience: since, as well known, the fetal brain is particularly vulnerable to injuries in critical phases of its development, we
detain that the respiratory centers are more susceptible to environmental factor exposure than other neuronal structures and so
are more involved in the mechanism of intrauterine sudden death.
Given that, in our study, all cases with a smoker mother showed
these specific alterations, we are prone to believe that nicotine
represents the major triggering factor.
In cases of maternal smoking in pregnancy, carbon monoxide,
a gaseous combustion product of nicotine, may readily cross the
placenta and bind to fetal hemoglobin (Lambers and Clark, 1996;
Aubard and Magne, 2000; Prockop and Chichkova, 2007). The consequent carboxyhemoglobin is not able to release oxygen, causing
an altered physiological development of fetal organs and tissues,
especially those most susceptible to hypoxic damage, including
the brain. In addition, nicotine is one of the few lipid-soluble substances that, by passing through the blood-brain barrier by passive
diffusion, can act directly on the expression of genes checking the
developing nervous system, leading to hypoplasia of the main vital
centers (Lavezzi et al., 2005; Lichtensteiger et al., 1988; Gressens
et al., 2003).
However, we cannot exclude the involvement of other environmental risk factors, too, such as air pollution in the defective
brainstem development (Calderón-Garcidueñas et al., 2002; Genc
et al., 2012; Roncati et al., 2016b).
2) The second question concerns the indisputable link that we
observed between altered development and, obviously, function
of the respiratory centers and stillbirth. While death related to
improper breathing control is understandable in SIDS, it is reasonable to speculate whether breathing alterations can lead to death
during intrauterine life, when breathing is not yet a vital condition.
This is our own theory: in the last weeks of pregnancy, advancing
towards the time of birth, a general check of all the neuronal centers
essential for extra-uterine life occurs. Sudden unexpected late fetal
deaths could therefore be ascribed to a selective process of selfsuppression in presence of developmental alterations particularly
of the respiratory centers, preventing the even more serious and
stressful event for parents of a neonatal death.
5. Conclusions
The developmental defects of the brainstem nuclei involved in
the breathing control that we have identified in unexplained stillbirths can have serious consequences, ranging from pulmonary
hypodevelopment that can, however, be compatible with fetal life,
to deficits in essential autonomic functions, particularly respiratory
activity, that can lead to sudden death.
Therefore, a systematic evaluation of the brainstem, possibly performed by experienced, reliable pathologists, is extremely
important to highlight any possible pathogenic mechanism in
SIUDS.
A last consideration in terms of preventing stillbirth: the good
news is that even if many of the risk factors for unexplained stillbirth cannot be changed (such as race or a previous miscarriage),
many others, and in particular cigarette smoking, are modifiable.
Women planning a pregnancy should try to optimize their health,
at least taking care to refrain from smoking, knowing that nicotine
quickly passes in the fetal nervous system and first of all acts on
the nervous centers that will be essential for the life of their child.
105
Funding
This study was supported by the Italian Health’s Ministry in
accordance with the Law 31/2006 “Regulations for Diagnostic Post
Mortem Investigation in Victims of Sudden Infant Death Syndrome
(SIDS) and Unexpected Fetal Death”, and by the Convention with the
APSS – Provincia autonoma of Trento, Italy.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors thank Dr. Graziella Alfonsi for the excellent technical assistance and Ms. Mary Victoria Candace Pragnell, B.A. for
English revision of the manuscript.
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