Material and Methods: The study population consisted of 63 stillbirths, 28 neonatal deaths and 140 suspected SIDS. The victims were subjected to in-depth anatomopathological examination following appropriate guidelines. The protocol included, in particular, the histological evaluation on serial sections of the cardiorespiratory autonomic nervous system.

Results: A diagnosis of “unexplained death” was established for 217 of the 231 victims (59 stillbirths, 28 newborns and 130 SIDS). In a very high percentage of these deaths (84%) we observed one or more anomalies of the nuclei and/or structures of the brainstem and cerebellum related to vital functions.

A fundamental component of these deaths is neuropathology. In fact, pathogenesis of unexplained perinatal death and SIDS seems to privilege, in most cases, some nervous dysfunctions. Nevertheless, only a few studies in this field have adequately examined the neurological substrates, although even subtle abnormalities of structures of the autonomic nervous system can determine dysfunctions in the control of vital functions, leading to unexpected perinatal loss as well as infant death [7-10].

For every case, a complete clinical history, particularly referred to the mother in perinatal deaths, and including the death scene examination in infant deaths, was collected. Cases were number-coded and identifying data (name, address, etc.) were removed prior to analysis.

The study population consisted of 63 stillbirths, 28 neonatal deaths and 140 suspected SIDS.

The 63 fetuses, 36 males and 27 females, were aged from 22 to 41 gestational weeks, with a peak incidence at 36 to 38 weeks (62%). The weights of victims ranged from 352 to 3500 grams, being below the corresponding reference value in most cases.

The 28 newborns, 16 males and 12 females, died intra-partum or in the first week after birth.

Among the infant victims, 52 females and 88 males, sudden death occurred between 1 and 10 months of life, with the greatest incidence around the third month.

After an in-depth autopsy and histological examination, a diagnosis of “unexplained death” was established for 217 of the 231 victims, namely 59 stillbirths, 28 newborns and 130 infants, all clearly SIDS victims.

The remaining 14 cases (4 fetal and 10 infant deaths) (6%), were classified as “border-line”, since it was difficult to establish whether the pathological findings were sufficiently severe to have caused the death. In the infant deaths we observed: neoplastic lesions of the cardiac conduction system, i.e. Purkinje cell tumor and cardiac fibroma in 3 cases; encephalitic features (probably viral in etiology) in nuclei of the brainstem and moderate myocarditis in 4 cases; a case with focal necrosis of the tractus solitarii nucleus, a case with capillary hemangioendothelioma in the area postrema and a case with pneumonia associated to hypoglossal nucleus hypoplasia. In four stillbirths, abnormalities of the annexa were diagnosed (umbilical cord around the neck, head band of the umbilical cord, multifocal placental infarcts). Table 1 summarizes the case profiles of the study.

None of the mothers had any significant pathology. With reference to smoking habit, 81 mothers (35%) declared that they were active smokers (all of more than 3 cigarettes/day and all already before becoming pregnant) while 129 (56%) were non smokers. In 21 cases no information about smoking habit was available.

The focus of this study is the in-depth histological examination of the central autonomic nervous system. Hereafter, we will briefly describe the study protocol. More detailed information is available on the previous publications [12-14].

The brainstem and cerebellum, where the main vital centers are located, were processed and embedded in paraffin. Transverse serial sections were made at intervals of 30 μm. For each level, twelve 5 μm sections were obtained, two of which were routinely stained for histological examination using alternately hematoxylin-eosin and Klüver-Barrera stains. Two additional sections at each level were subjected to immunohistochemistry for the study of neurotransmitters (somatostatin and tyrosine hydroxylase (TH), indicative of catecholamine synthesis) and apoptosis. The remaining sections were saved and stained as deemed necessary for further investigations.

The main nuclei were analyzed in serial sections of the brainstem, namely the parafacial nucleus, the locus coeruleus and the parabrachial/Kölliker-Fuse complex in the pons and mesencephalon; the hypoglossus, the dorsal motor vagal, the tractus solitarius, the ambiguus, the pre-Bötzinger, the inferior olivary and the arcuate nuclei in the medulla oblongata. Fig. (1) shows the sampling of the brainstem (three specimens are obtained: the first, ponto-mesencephalic, includes the upper third of the pons and the adjacent portion of mesencephalon; the second extends from the rostral limit of the medulla oblongata to the adjacent caudal pons, and the third specimen takes as reference point, the obex and extends 2-3 mm above it and below it). Fig. (2) shows the histological sections corresponding to the three brainstem samples of Fig. (1) indicating the main nuclei and structures to be examined.

The accurate examination of the central autonomic nervous system, allowed us to identify even minute alterations of the nuclei and/or structures overseeing the vital functions in these pathologies.

In particular, in 84% of all the unexplained deaths, we highlighted structural and/or functional alterations, particularly of the brainstem and cerebellum. These alterations represent a common morphological substrate in both unexplained perinatal death and SIDS and, therefore, can be considered congenital in nature.

A common finding was hypoplasia of the arcuate nucleus, a chemoreceptoral component of the ventral surface of the medulla oblongata, which was present in over 50% of all the victims, albeit with different degrees of extension and severity. In perinatal deaths, in addition to hypoplasia of the arcuate nucleus, we observed hypodevelopment of the dendritic fibers with a decreased neuronal density in the pre-Bötzinger area, besides hypoplasia of the parabrachial/Kölliker-Fuse complex. The pre-Bötzinger nucleus has been physiologically defined as a group of neurons of the ventrolateral medulla, that are essential for generating the respiratory rhythm, as well as for modulating eupneic breathing [24,25]. Instead, the Kölliker-Fuse nucleus has an important function during intrauterine life, inhibiting the response of central and peripheral chemoreceptors (which are already fully formed and potentially functional) and therefore becomes any respiratory reflex. After birth, the Kölliker-Fuse abruptly reduces its inhibitory effects and becomes active as a respiratory center, able to coordinate the pulmonary motor responses to hematic oscillations of pO₂, pCO₂ and pH [18,21].

In addition, exclusively in unexplained stillbirths, we observed very frequently (in 70% of cases) the hypoplasia of the parafacial nucleus, consisting of “pre-inspiratory” neurons that periodically trigger the inspiratory neurons of the pre-Bötzinger complex [26,27]. Therefore, the function of hierarchical modulation of the breathing circuitry can be ascribed to the parafacial nucleus.

Among the functional alterations, we diagnosed altered expression of neurotransmitters in the brainstem, particularly of catecholamine in the locus coeruleus and somatostatin in the hypoglossus nucleus, besides defective apoptotic programs in the cerebellum, in the Purkinje and internal granular layers of the cerebellar cortex, as well as in the dentate nucleus [15-17].

The above reported findings indicate that both unexplained perinatal death and SIDS share common congenital anomalies of the central autonomic nervous system. Nevertheless, these alterations are wider and more severe in fetal deaths. In fact, whereas in SIDS victims the more frequent observation is hypoplasia of the arcuate nucleus, unexpected ante-partum deaths are characterized by hypodevelopment of various structures: hypoplasia of the arcuate nucleus, of the parabrachial/Kölliker-Fuse complex, of the pre-Bötzinger complex and of the parafacial nucleus. In particular, this last lesion represents a marker of unexpected ante-partum death. In fact it was found exclusively, and with a high frequency (70%), in fetal deaths.

The distinctive congenital anomaly that marks the intra-partum death was shown to be the hypoplasia of the parabrachial/Kölliker-Fuse complex.

All these alterations, responsible for serious vagal reflexes, can be interpreted as expressions of a disturbed development of the nervous system in pathophysiological response to a variety of mechanisms and, above all, to maternal cigarette smoking in pregnancy, as we have previously suggested [16,28,29].