Symbolic Dynamics Analysis: a new methodology for foetal heart rate variability analysis

Cardiotocography (CTG) is a widespread foetal diagnostic methods. However, it lacks of objectivity and reproducibility since its dependence on observer's expertise. To overcome these limitations, more objective methods for CTG interpretation have been proposed. In particular, many developed techniques aim to assess the foetal heart rate variability (FHRV). Among them, some methodologies from nonlinear systems theory have been applied to the study of FHRV. All the techniques have proved to be helpful in specific cases. Nevertheless, none of them is more reliable than the others. Therefore, an in-depth study is necessary. The aim of this work is to deepen the FHRV analysis through the Symbolic Dynamics Analysis (SDA), a nonlinear technique already successfully employed for HRV analysis. Thanks to its simplicity of interpretation, it could be a useful tool for clinicians. We performed a literature study involving about 200 references on HRV and FHRV analysis; approximately 100 works were focused on non-linear techniques. Then, in order to compare linear and non-linear methods, we carried out a multiparametric study. 580 antepartum recordings of healthy fetuses were examined. Signals were processed using an updated software for CTG analysis and a new developed software for generating simulated CTG traces. Finally, statistical tests and regression analyses were carried out for estimating relationships among extracted indexes and other clinical information. Results confirm that none of the employed techniques is more reliable than the others. Moreover, in agreement with the literature, each analysis should take into account two relevant parameters, the foetal status and the week of gestation. Regarding the SDA, results show its promising capabilities in FHRV analysis. It allows recognizing foetal status, gestation week and global variability of FHR signals, even better than other methods. Nevertheless, further studies, which should involve even pathological cases, are necessary to establish its reliability.La Cardiotocografia (CTG) è una diffusa tecnica di diagnostica fetale. Nonostante ciò, la sua interpretazione soffre di forte variabilità intra- e inter- osservatore. Per superare tali limiti, sono stati proposti più oggettivi metodi di analisi. Particolare attenzione è stata rivolta alla variabilità della frequenza cardiaca fetale (FHRV). Nel presente lavoro abbiamo suddiviso le tecniche di analisi della FHRV in tradizionali, o lineari, e meno convenzionali, o non-lineari. Tutte si sono rivelate efficaci in casi specifici ma nessuna si è dimostrata più utile delle altre. Pertanto, abbiamo ritenuto necessario effettuare un’indagine più dettagliata. In particolare, scopo della tesi è stato approfondire una specifica metodologia non-lineare, la Symbolic Dynamics Analysis (SDA), data la sua notevole semplicità di interpretazione che la renderebbe un potenziale strumento di ausilio all’attività clinica. Sono stati esaminati all’incirca 200 riferimenti bibliografici sull’analisi di HRV e FHRV; di questi, circa 100 articoli specificamente incentrati sulle tecniche non-lineari. E’ stata condotta un’analisi multiparametrica su 580 tracciati CTG di feti sani per confrontare le metodologie adottate. Sono stati realizzati due software, uno per l’analisi dei segnali CTG reali e l’altro per la generazione di tracciati CTG simulati. Infine, sono state effettuate analisi statistiche e di regressione per esaminare le correlazioni tra indici calcolati e parametri di interesse clinico. I risultati dimostrano che nessuno degli indici calcolati risulta più vantaggioso rispetto agli altri. Inoltre, in accordo con la letteratura, lo stato del feto e le settimane di gestazione sono parametri di riferimento da tenere sempre in considerazione per ogni analisi effettuata. Riguardo la SDA, essa risulta utile all’analisi della FHRV, permettendo di distinguere – meglio o al pari di altre tecniche – lo stato del feto, la settimana di gestazione e la variabilità complessiva del segnale. Tuttavia, sono necessari ulteriori studi, che includano anche casi di feti patologici, per confermare queste evidenze

Fetal autonomic cardiac response during pregnancy and labour

Timely recognition of fetal distress, during pregnancy and labour, in order to intervene adequately is of major importance to avoid neonatal morbidity and mortality. As discussed in chapter 1, the cardiotocogram (CTG) might be a useful screening test for fetal monitoring but it has insufficient specificity and requires additional diagnostic tests in case of suspected fetal compromise to avoid unnecessary operative deliveries. Potential additional techniques used in clinical practice are fetal scalp blood sampling (FBS) and ST-waveform analysis of the fetal electrocardiogram (ECG; STAN®). However, publications on these techniques provide limited support for the use of these methods in the presence of a non-reassuring CTG for reducing caesarean sections. In addition, these techniques are invasive and can therefore only be used during labour at the term or the near term period. Consequently, it is of great clinical importance that additional methods are developed that contribute to more reliable assessment of fetal condition. Preferably, this information is obtained non-invasively. Valuable additional information on the fetal condition can possibly be obtained by spectral analysis of fetal heart rate variability (HRV). The fetal heart rate fluctuates under the control of the autonomic part of the central nervous system. The autonomic cardiac modulation is discussed in chapter 2. The sympathetic and parasympathetic nervous systems typically operate on partly different timescales. Time-frequency analysis (spectral analysis) of fetal beat-to-beat HRV can hence quantify sympathetic and parasympathetic modulation and characterise autonomic cardiac control . The low frequency (LF) component of HRV is associated with both sympathetic and parasympathetic modulation while the high frequency (HF) component is associated with parasympathetic modulation alone2. Spectral estimates of HRV might indirectly reflect fetal wellbeing and increase insight in the human fetal autonomic cardiac response. In chapter 3, technical details for retrieving fetal beat-to-beat heart rate and its spectral estimates are provided. In this thesis spectral analysis of fetal HRV is investigated. The first objective is to study the value of spectral analysis of fetal HRV as a tool to assess fetal wellbeing during labour at term. The second objective is to monitor spectral estimates of fetal HRV, non-invasively, during gestation to increase insight in the development of human fetal autonomic cardiac control. Since Akselrod reported the relation between autonomic nervous system modulation and LF and HF peaks in frequency domain1, frequency analysis of RR interval fluctuations is widely performed . For human adults, standards for HRV measurement and physiological interpretation have been developed2. Although HRV parameters are reported to be highly prognostic in human adults in case of cardiac disease, little research is done towards the value of these parameters in assessing fetal distress in the human fetus, as shown in chapter 4. In this chapter, the literature about time-frequency analysis of human fetal HRV is reviewed in order to determine the value of spectral estimates for fetal surveillance. Articles that described spectral analysis of human fetal HRV and compared the energy in spectral bands with fetal bloodgas values were included. Only six studies met our inclusion criteria. One study found an initial increase in LF power during the first stage of fetal compromise, which was thought to point to stress-induced sympathetic hyperactivity3. Five out of six studies showed a decrease in LF power in case of fetal distress , , , , ,. This decrease in LF power in case of severe fetal compromise was thought to be the result of immaturity or decompensation of the fetal autonomic nervous system. These findings support the hypothesis that spectral analysis of fetal HRV might be a promising method for fetal surveillance. All studies included in the literature review used absolute values of LF and HF power. Although absolute LF and HF power of HRV provide useful information on autonomic modulation, especially when considering fetal autonomic development, LF and HF power may also be measured in normalised units. Normalised LF (LFn) and normalised HF power (HFn) of HRV represent the relative value of each power component in proportion to the total power2. Adrenergic stimulation can cause a sympathetically-modulated increase in fetal heart rate . A negative correlation however exists between heart rate and HRV . As a result, the sympathetic stimulation can decrease the total power of HRV and even the absolute LF power. When normalising the absolute LF (and HF) with respect to the total power, a shift in activity from HFn to LFn might become visible, revealing the expected underlying sympathetic activity. Thus, because changes in total power influence absolute spectral estimates in the same direction, normalised values of LF and HF power seem more suitable for fetal monitoring. In other words, normalised spectral estimates detect relative changes that are no longer masked by changes in total power2. LFn and HFn power are calculated by dividing LF and HF power, respectively, by total power and represent the controlled and balanced behaviour of the two branches of the autonomic nervous system2. In chapter 5 we hypothesised that the autonomic cardiovascular control is functional in fetuses at term, and that LFn power increases in case of distress due to increased sympathetic modulation. During labour at term, ten acidaemic fetuses were compared with ten healthy fetuses. During the last 30 minutes of labour, acidaemic fetuses had significantly higher LFn power and lower HFn power than control fetuses, which points to increased sympathetic modulation. No differences in absolute LF or HF power were found between both groups. The observed differences in normalised spectral estimates of HRV were not observed earlier in labour. In conclusion, it seems that the autonomic nervous system of human fetuses at term responds adequately to severe stress during labour. Normalised spectral estimates of HRV might be able to discriminate between normal and abnormal fetal condition. Although we found significant differences in normalised spectral estimates between healthy and acidaemic fetuses, we wondered whether spectral power of HRV is also related to fetal distress in an earlier stage. The next step in chapter 6 was therefore, to investigate whether spectral estimates are related to fetal scalp blood pH during labour. Term fetuses during labour, in cephalic presentation, that underwent one or more scalp blood samples were studied. Beat-to-beat fetal heart rate segments, preceding the scalp blood measurement, were used to calculate spectral estimates. In total 39 FBS from 30 patients were studied. We found that normalised spectral estimates are related to fetal scalp blood pH while absolute spectral estimates are not related to fetal pH. It was further demonstrated that LFn power is negatively related and HFn power is positively related to fetal pH. These findings point to increased sympathetic and decreased parasympathetic cardiac modulation in human fetuses at term upon decrease of their pH value. This study confirms the hypothesis that normalised spectral values of fetal HRV are related to fetal distress in an early stage. Previous studies showed that absolute LF and HF power increase as pregnancy progresses, which is attributed to fetal autonomic maturation , . Since it is yet unclear how LFn and HFn evolve with progressing pregnancy, before using spectral analysis for fetal monitoring, it has to be determined whether gestational age has to be corrected for. In addition, fetal autonomic fluctuations, and thus spectral estimates of HRV, are influenced by fetal behavioural state . Since these states continue to change during labour , thorough understanding of the way in which these changes in state influence spectral power is necessary for the interpretation of spectral values during labour at term. Therefore, in chapter 7, we examined whether differences in spectral estimates exist between healthy near term and post term fetuses during labour. In case such differences do exist, they should be taken into consideration for fetal monitoring. The quiet and active sleep states were studied separately to determine the influence of fetal behavioural state on spectral estimates of HRV during labour around term. No significant differences in spectral estimates were found between near term and post term fetuses during active sleep. During quiet sleep, LFn power was lower and HF and HFn power were higher in post term compared to near term fetuses, no significant differences in LF power were observed between both groups. LF, HF and LFn power were higher and HFn power was lower during active sleep compared to quiet sleep in both groups. This seems to point to sympathetic predominance during the active state in fetuses around term. In addition, post term parasympathetic modulation during rest seems increased compared to near term. In conclusion, fetal behavioural state and gestational age cause a considerable variability in spectral estimates in fetuses during labour, around term, which should be taken into consideration when using spectral estimates for fetal monitoring. In chapters 4 to 6, spectral estimates of beat-to-beat fetal HRV were studied using fetal ECG recordings that were obtained directly from the fetal scalp during labour. However, the second objective of this thesis is to obtain spectral estimates non-invasively during gestation to increase insight in the development of human fetal autonomic cardiac control. The fetal ECG is also present on the maternal abdomen, although much smaller in amplitude and obscured by the maternal ECG and noise. Chapter 8 focused on non-invasive measurement of the fetal ECG from the maternal abdomen. These measurements allow for obtaining beat-to-beat fetal heart rate non-invasively. Therefore, this method can be used to obtain spectral estimates of fetal HRV throughout gestation. Although abdominal recording of the fetal ECG may offer valuable additional information, it is troubled by poor signal-to-noise ratios (SNR) during certain parts of pregnancy, e.g. during the immature period and during the vernix period. To increase the usability of abdominal fetal ECG recordings, an algorithm was developed that uses a priori knowledge on the physiology of the fetal heart to enhance the fetal ECG components in multi-lead abdominal fetal ECG recordings, before QRS-detection. Evaluation of the method on generated fetal ECG recordings with controlled SNR showed excellent results. The method for non-invasive fetal ECG and beat-to-beat heart rate detection presented in chapter 8 was used for analysis in chapter 9. The feasibility of this method in a longitudinal patient study was investigated. In addition, changes in spectral estimates of HRV during pregnancy were studied and related to fetal rest-activity state to study the development of fetal autonomic cardiac control. We found that approximately 3% of non-invasive fetal ECG recordings could be used for spectral analysis. Therefore, improvement of both equipment and algorithms is still needed to obtain more good-quality data. The percentage of successfully retrieved data depends on gestational age. Before 18 and between 30 and 34 weeks no good-quality beat-to-beat heart rate data were available. We found an increase in LF and HF power of fetal HRV with increasing gestational age, between 21 to 30 weeks of gestation. This increase in LF and HF power is probably due to increased sympathetic and parasympathetic modulation and might be a sign of autonomic development. Furthermore, we found sympathetic predominance during the active state compared to the quiet state in near term fetuses (34 to 41 weeks of gestation), comparable to the results observed during labour around term. During 34 to 41 weeks a (non-significant) decrease in LF and LFn power and a (non-significant) increase in HF and HFn power were observed. These non-significant changes in spectral estimates in near term fetuses might be associated with changes in fetal rest-activity state and increased parasympathetic modulation as pregnancy progresses. However, more research is needed to confirm this

IDENTIFICATION OF HYPOXIA-REGULATED MICRORNAs IN MATERNAL BLOOD AS EARLY PERIPHERAL BIOMARKERS FOR FETAL GROWTH RESTRICTION

Current tests available to diagnose fetal hypoxia in-utero lack sensitivity thus failing to identify many fetuses at risk. Emerging evidence suggests that microRNAs derived from the placenta circulate in the maternal blood during pregnancy and may be used as non-invasive biomarkers for pregnancy complications. With the intent to identify putative markers of fetal growth restriction(FGR) and new therapeutic druggable targets, we examined, in maternal blood samples, the expression of a group of microRNAs, known to be regulated by hypoxia. The expression of microRNAs was evaluated in maternal plasma samples collected from (1) women carrying a preterm FGR fetus(FGR group) or (2) women with an appropriately grown fetus matched at the same gestational age (Control group). To discriminate between early- and late-onset FGR, the study population was divided into two subgroups according to the gestational age at delivery. Four microRNAs were identified as possible candidate for the diagnosis of FGR: miR-16-5p, miR-103-3p, miR-107-3p, and miR-27b-3p that were upregulated in FGR blood samples before the 32nd week of gestation as compared to aged matched control group. By contrast, miRNA103-3p and miRNA107-3p analyzed between the 32nd and 37th week of gestation showed lower expression in the FGR group compared to aged matched controls. Notably, the expression of all miRNAs was increased through gestation in healthy control group. Our results showed that measurement of miRNAs in maternal blood may form the basis for a future diagnostic test to determine the degree of fetal hypoxia in FGR, thus allowing the start of appropriate therapeutic strategies in order to alleviate the burden of this disease

Characterization of uterine activity by electrohysterography

A growing number of pregnancies is complicated by miscarriage, preterm delivery, and birth defects, with consequent health problems later in life. It is therefore increasingly important to monitor the health status of mother and fetus, so as to permit timely medical intervention when acute health risks are detected. For timely recognition of complications, quantitative assessment of uterine activity can be fundamental during both pregnancy and delivery. During pregnancy, timely prediction of preterm delivery can improve the effectiveness of the required treatments. Unfortunately, the prognostic techniques employed in current obstetrical practice, namely, uterine contraction measurements using an elastic belt (external tocography), cervical change evaluation, and the use of biomarkers like fetal fibronectin, have been demonstrated to be inaccurate for the prediction of preterm delivery. In the last stage of pregnancy and during labor, contractions are routinely and constantly monitored. Especially when complications occur, e.g., when labor shows poor progress, quantitative assessment of uterine activity can guide the physician to choose a uterine contraction induction or augmentation, a cesarean section, or other therapies. Furthermore, monitoring the fetal heart response to the uterine activity (cardiotography) is widely used as a screening test for timely recognition of fetal distress (e.g. asphyxia). However, in current obstetrical practice, accurate quantitative assessment of the uterine contractions can be provided only invasively and during labor. The current golden standard for contraction monitoring, which is based on the direct internal uterine pressure (IUP) measurement by an intrauterine catheter, can be risky and its use is generally limited to very complicated deliveries. The contractile element of the uterus is the myometrium, which is composed of smooth muscle cells. Uterine contractions are caused by electrical activity in the form of action potentials (AP) that propagate through the myometrium cells. Electrohysterography is the measurement of the uterine electrical activity and can be performedby electrodes placed on the abdomen. Electrohysterographic (EHG) measurements are inexpensive and noninvasive. Moreover, it has been demonstrated that the noninvasively recorded EHG signal is representative of those APs that, by propagating from cell to cell, are the root cause of a uterine contraction. Therefore, in view of the limitation of current obstetrical practice, significant benefits could be expected from the introduction of EHG signal analysis for routine contraction monitoring. Previous studies highlighted the potential prognostic and diagnostic value of EHG signal analysis, but did not investigate the possibility of accurately estimating the IUP from noninvasive EHG recordings. Moreover, important issues like the effect of the tissues interposed between the uterus and the skin (volume conductor) on EHG recordings have not been studied. Besides, EHG signal interpretation has been typically based on single-channel measurements, while the use of multiple electrodes conveys additional information (e.g., distribution and dynamics of the electrical activation) that can possibly be predictive of delivery. In this thesis, we focus on the analysis of the EHG signal as an alternative to existing techniques for predicting preterm delivery and monitoring uterine contractions during both pregnancy and delivery. The main goal of this work is to contribute to the technical basis which is required for the introduction of electrohysterography in everyday clinical practice. A major part of this thesis investigates the possibility of using electrohysterography to replace invasive IUP measurements. A novel method for IUP estimation from EHG recordings is developed in the first part of this thesis. The estimates provided by the method are compared to the IUP invasively recorded on women during delivery and result in a root mean squared error (RMSE) with respect to the reference invasive IUP recording as low as 5 mmHg, which is comparable to the accuracy of the invasive golden standard. Another important objective of this thesis work is to contribute to the introduction of novel techniques for timely prediction of preterm delivery. As the spreading of electrical activity at the myometrium is the root cause of coordinated and effective contractions, i.e., contractions that are capable of pushing the fetus down into the birth canal ultimately leading to delivery, a multichannel analysis of the spatial propagation properties of the EHG signal could provide a fundamental contribution for predicting delivery. A thorough study of the EHG signal propagation properties is therefore carried out in this work. Parameters related to the EHG that are potentially predictive of delivery, such as the uterine area where the contraction originates (pacemaker area) or the distribution and dynamics of the EHG propagation vector, can be derived from the delay by which the signal is detected at multiple locations over the whole abdomen. To analyze the propagation of EHG signals on a large scale (cm), a method is designed for calculating the detection delay among the EHG signals recorded by by electrodes placed on the abdomen. Electrohysterographic (EHG) measurements are inexpensive and noninvasive. Moreover, it has been demonstrated that the noninvasively recorded EHG signal is representative of those APs that, by propagating from cell to cell, are the root cause of a uterine contraction. Therefore, in view of the limitation of current obstetrical practice, significant benefits could be expected from the introduction of EHG signal analysis for routine contraction monitoring. Previous studies highlighted the potential prognostic and diagnostic value of EHG signal analysis, but did not investigate the possibility of accurately estimating the IUP from noninvasive EHG recordings. Moreover, important issues like the effect of the tissues interposed between the uterus and the skin (volume conductor) on EHG recordings have not been studied. Besides, EHG signal interpretation has been typically based on single-channel measurements, while the use of multiple electrodes conveys additional information (e.g., distribution and dynamics of the electrical activation) that can possibly be predictive of delivery. In this thesis, we focus on the analysis of the EHG signal as an alternative to existing techniques for predicting preterm delivery and monitoring uterine contractions during both pregnancy and delivery. The main goal of this work is to contribute to the technical basis which is required for the introduction of electrohysterography in everyday clinical practice. A major part of this thesis investigates the possibility of using electrohysterography to replace invasive IUP measurements. A novel method for IUP estimation from EHG recordings is developed in the first part of this thesis. The estimates provided by the method are compared to the IUP invasively recorded on women during delivery and result in a root mean squared error (RMSE) with respect to the reference invasive IUP recording as low as 5 mmHg, which is comparable to the accuracy of the invasive golden standard. Another important objective of this thesis work is to contribute to the introduction of novel techniques for timely prediction of preterm delivery. As the spreading of electrical activity at the myometrium is the root cause of coordinated and effective contractions, i.e., contractions that are capable of pushing the fetus down into the birth canal ultimately leading to delivery, a multichannel analysis of the spatial propagation properties of the EHG signal could provide a fundamental contribution for predicting delivery. A thorough study of the EHG signal propagation properties is therefore carried out in this work. Parameters related to the EHG that are potentially predictive of delivery, such as the uterine area where the contraction originates (pacemaker area) or the distribution and dynamics of the EHG propagation vector, can be derived from the delay by which the signal is detected at multiple locations over the whole abdomen. To analyze the propagation of EHG signals on a large scale (cm), a method is designed for calculating the detection delay among the EHG signals recorded by multiple electrodes. Relative to existing interelectrode delay estimators, this method improves the accuracy of the delay estimates for interelectrode distances larger than 5-10 cm. The use of a large interelectrode distance aims at the assessment of the EHG propagation properties through the whole uterine muscle using a limited number of sensors. The method estimates values of velocity within the physiological range and highlights the upper part of the uterus as the most frequent (65%) pacemaker area during labor. Besides, our study suggests that more insight is needed on the effect that tissues interposed between uterus and skin (volume conductor) have on the EHG signal. With the aim of improving the current interpretation and measurement accuracy of EHG parameters with potential clinical relevance, such as the conduction velocity (CV), a volume conductor model for the EHG signal is introduced and validated. The intracellular AP at the myometrium is analytically modeled in the spatial domain by a 2-parameter exponential in the form of a Gamma variate function. The unknown atomical parameters of the volume conductor model are the thicknesses of the biological tissues interposed between the uterus and the abdominal surface. These model parameters can be measured by echography for validation. The EHG signal is recorded by an electrode matrix on women with contractions. In order to increase the spatial resolution of the EHG measurements and reduce the geometrical and electrical differences among the tissues below the recording locations, electrodes with a reduced surface and smaller interelectrode distance are needed relative to the previous studies on electrohysterography. The EHG signal is recorded, for the first time, by a 64-channel (8×8) high-density electrode grid, comprising 1 mm diameter electrodes with 4 mm interelectrode distance. The model parameters are estimated in the spatial frequency domain from the recorded EHG signal by a least mean square method. The model is validated by comparing the thickness of the biological tissues recorded by echography to the values estimated using the mathematical model. The agreement between the two measures (RMSE = 1 mm and correlation coefficient, R = 0.94) suggests the model to be representative of the underlying physiology. In the last part of this dissertation, the analysis of the EHG signal propagation focuses on the CV estimation of single APs. As on a large scale this parameter cannot be accurately derived, the propagation analysis is here carried out on a small scale (mm). Also for this analysis, the EHG signal is therefore recorded by a 3×3 cm2 high-density electrode grid containing 64 electrodes (8×8). A new method based on maximum likelihood estimation is then applied in two spatial dimensions to provide an accurate estimate of amplitude and direction of the AP CV. Simulation results prove the proposed method to be more robust to noise than the standard techniques used for other electrophysiological signals, leading to over 56% improvement of the RMS CV estimate accuracy. Furthermore, values of CV between 2 cm/s and 12 cm/s, which are in agreement with invasive and in-vitro measurements described in the literature, are obtained from real measurements on ten women in labor. In conclusion, this research provides a quantitative characterization of uterine contractions by EHG signal analysis. Based on an extensive validation, this thesis indicates that uterine contractions can be accurately monitored noninvasively by dedicated analysis of the EHG signal. Furthermore, our results open the way to new clinical studies and applications aimed at improving the understanding of the electrophysiological mechanisms leading to labor, possibly reducing the incidence of preterm delivery and improving the perinatal outcome

SOME PERINATAL ENDOCRINE AND MORPHOLOGICAL ASPECTS OF CANINE SPECIES

Despite the huge literature about dog medicine, the perinatal period of growth and development and possible perturbations of the health of fetuses and newborns are still scarcely investigated, but with a recent, increasing scientific interest. The present thesis was therefore aimed to investigate some perinatal aspects of canine perinatology. Firstly, the study focused on the presence of some growth and metabolic factors and protective substances in dog fetal fluids at term of normal pregnancy. About growth and metabolic factors, the first study showed that insulin-like growth factors I (IGF-I) were higher in amniotic (AM) than allantoic (AL) fluid and the effect of breed size, even if opposite, on both IGF-I and non-esterified fatty acids (NEFA) was evidenced. The findings suggested that AM IGF-I could be used as an indicator of growth potential in canine species, whereas AM NEFA could work as a marker of fat mobilization for an energy request. The second study on fetal fluids, aimed to assess AM and AL IgG and lysozyme concentrations, documented higher IgG levels in AM than AL fluid, as a consequence of a supposed direct fetal IgG production, whereas no significant differences were found in lysozyme values between the two fluids. Maternal parity was demonstrated to affect IgG concentrations. A second area of study was the assessment of newborns hair and nails usefulness as the newest non invasive matrices for a long-term retrospective investigation of the Hypothalamic-Pituitary-Adrenal axis activation, by cortisol (C) concentrations analysis. The study evidenced that C was detectable in both matrices, with significant higher values in premature puppies compared to term-born dead puppies or puppies dead within the first 30 days of age. Furthermore, because of the newborn puppy high susceptibility to bacterial infection, often followed by death, the fourth study investigated the bacterial involvement in canine neonatal mortality, demonstrating that bacterial infections, above all by E. coli, alone or in association with other bacteria, represent an important cause of neonatal losses. In addition, the study highlighted the importance of the antimicrobial susceptibility test in case of suspected neonatal bacterial infection for a more targeted therapy of surviving litter-mates and for a better management of further gestations in bitch with previous neonatal mortality. The last study was designed to investigate some aspects of skeletal development during the neonatal period in the attempt to provide further knowledge about the first month of skeletal growth in puppies, but also aimed to assess the possible use of some radiographic, morphometric, and anatomic parameters for the age estimation in newborn dogs. The study proved that the neonatal growth occurs gradually as the age progresses, and simultaneously in the body, limbs, and skull. The radiographic evaluation of the ossification centers appearance resulted a useful tool to estimate the neonatal age in puppies, even if during the first 14 days of age significant skeletal changes were not observed. In this respect, the radiographic and anatomic measures of the hindlimb long bones and skull length seem to provide better guarantees; specifically, the neurocranium width as well as tibial and femoral lengths resulted the most correlated measurements with the age. Thus, in the future radiographic and/or anatomical morphometry of limbs and skull could become the best tool for neonatal age estimation