Parental Stress and Attachment in Premature Infants in Their First Months of Life in Early Months of Life in Child Development and Early Care Centres.
Conditions: Premature Birth, Psychological Stress, Physiological Stress
Sponsor: Cardenal Herrera University

 
 
 
 
 
 
 
 
 
 
 
 
 
STUDY PROTOCOL PLAN AND STADISTICAL ANALYSIS PLAN (SAP)  
 
UNIVERSITY CEU CARDENAL HERRERA  
 
Principal investigator: Dr. Jose Miguel Soria 
 
TITLE: Parental stress, child development and attachment in premature children 
during their first 24 months of life cared for in Child Development and Early Care 
Centers. 
 
Research and Ethics Committee of CEU Cardenal Herrera University Number: CEEI24/544 
 
NCT ID: 
DATE: 12-20-2024 
 
 
 
 

 
STUDY PROTOCOL PLAN 
 
Objectives: 
 
The objectives of our study have been the following: 
 
To analyze the degree of perceived and physiological stress in mothers of premature 
children, as well as the possible effects that it can generate in the care of the child and, 
therefore, in the interaction and attachment patterns at 24 months of age. In such a way 
that preventive intervention programs can be designed that guarantee optimal interactive 
patterns between the neonate and its family. 
 
Specific objectives  
 
• Specifically analyze how parenting stress affects the development and formation of 
attachment in children born prematurely.  
• Examine how socioeconomic differences influence parents' stress level and, therefore, 
their interaction and attachment with their children.  
• Assess how parenting stress over time affects the perceived quality of life of families 
who attend Early Care, with a particular focus on how this influences the relationship and 
development of the child. 
• Analyze the influence on stress, depression and anxiety in mothers and fathers of 
premature children through biometric records and thermographic records in real time. 
• Determine by quantifying cortisol in hair as a biochemical marker that determines the 
level of chronic stress.  
• Determine the acute impact of stress on mothers and fathers of premature children, 
through the quantification of the level of cortisol and oxytocin in saliva. 
• Explore the association between child development and attachment at 24 months of 
age. 
 
 
 
 
 
 
 

 
Protocol 
 
Design, population and sample 
A prospective population-based cohort study will be conducted on premature births 
referred, after hospital discharge, to the Child Development and Early Care Centres of 
the Fundación Salud Infantil de Elche (Alicante, Spain). All mothers and fathers who give 
birth to a premature child and who are referred to the Child Development and Early Care 
Centre in the period between June 2024 and December 2025 will be invited to participate 
in the study. All participants who meet the following inclusion criteria will be invited to 
participate: 
• Children born under 37 weeks of gestation, admitted to the Neonatal Unit and who 
begin treatment at the Child Development and Early Care Centre of the Fundación Salud 
Infantil de Elche (Alicante, Spain). 
• Parents of legal age and who have sufficient cognitive capacity to understand the 
informed consent. 
 
Group 1: formed by mothers who had premature births before 37 weeks of gestation 
Group 2: formed by mothers who had births after 37 weeks of gestation 
 
Procedure 
After the child is admitted to the CDIAT, the parents will be informed about the study by 
the principal investigator of the study (Annex 1). If the parents decide to participate in the 
study, they will be called for a first interview (Annex 2) in which their participation in the 
study will be explained in more detail and they will be asked to sign the informed consent 
(Annex 3) to participate in the study. During this first visit, the parents provide information 
about their reproductive and sociodemographic history, data on the birth and the 
newborn (through the ad hoc interview generated for the study and the clinical report on 
admission). At the same time, information on stress is collected through the Spanish 
version of the Parenting Stress Index, PSI (Abidin, 1995; Spanish adaptation Brito de la 
Nuez, 2001) (Annex 4) and exosomatic records will be taken with the BIOPAC system 
and saliva and hair samples will be taken to determine oxytocin, serotonin and cortisol 
in both the father and the mother. On the other hand, information on family quality of life 
will be collected through the online Questionnaire on Family Quality of Life in Early 
Intervention (CVFAT) (Annex 5). Regarding the child, neonatal behavior will be assessed 
through the neonatal behavior assessment test (Brazelton and Nugent, 1997; Spanish 
adaptation Costa, Fornieles, Botet, Boatella and De Cáceres, 2014) (Annex 6) whenever 

 
the child is at a corrected age of between 0 and 2 months and information on the child's 
development will be collected through the Bayley-III Infant Development Assessment 
Scales, BSID-III (Bayley, 2015) (Annex 7). Finally, the risk estimate will be taken through 
the Perinatal Risk Index, PERI (Scheiner and Sexton, 1991) (Annex 8). The estimated 
population n=30 and the control population will be made up of fathers and mothers 
(n=30) who had full-term births and gave birth to normotypical children (General 
University Hospital of Elche. 
 
Later, at 3, 6, 9, 12, 18 and 24 months of corrected age, the parents will be called 
again to collect information on the child's development, through the Bayley-III Child 
Development Assessment Scales, BSID-III (Bayley, 2015) (Annex 7) and on the parents' 
stress through the questionnaire of the Spanish version of the Parenting Stress Index, 
(PSI) (Annex 4), as well as exosomatic records will be taken with the BIOPAC system 
and saliva and hair samples for the determination of oxytocin, serotonin and cortisol in 
both the father and the mother. On the other hand, Information on family quality of life 
will be collected through the Family Quality of Life Questionnaire in Early Care (CVFAT) 
(Annex 5). 
 
At 24 months, information on attachment will also be collected through the 
Strange Situation (Ainsworth, Bleahr, Waters and Wall, 1978) (Annex 9). 
 
Families with their children will go to the Child Development and Early Care 
Center of the Children's Health Foundation of Elche (located at Calle Masamagrell, 4) 
for the entire process described above. For the collection of samples, health 
professionals from the Cardenal Herrera University (CEU, Elche) will take measurements 
and keep the samples, respecting the cold chains, in their laboratory. 
 
 
Primary outcomes  
 
During the study, data come from several sources of information such as: 
- Data derived from the interview (sociodemographic and neonatal and delivery 
status) 
- Data derived from the clinical report of hospital discharge (diagnosis, 
somatometry at discharge and estimation of the prognostic value) 
- Data derived from evaluation tests (questionnaires and observational tests for 

 
stress, development and neonatal behavior) 
- Data derived from medical tests (Saliva and hair test for cortisol in saliva and 
per, serotonin and oxytocin) 
- Data derived from biometric records using the BIOPAC system 
 
 
 
 
Description of the scales to be used: 
- Parenting Stress Index (PSI) created by Abidin, 1995 and adapted to Spanish 
by Brito de la Nuez, 2001. It evaluates the stress experienced in parenthood, through 
several subscales such as parental distress, dysfunctional parent-child interaction and 
whether the child is difficult based on how the parents perceive their children's behavior. 
- The Perinatal Risk Inventory designed by Scheiner and Sexton, 1991. It is a 
measurement instrument that offers information on the neonatal situation prior to 
discharge and that facilitates an estimate of the biological risk that the child may present 
from a very early age. It is made up of a total of 18 risk factors such as 1) Apgar score; 
2) electroencephalogram; 3) nonmetabolic seizures; 4) intraventricular hemorrhage; 5) 
hydrocephalus; 6) neurologic findings (not due to hydrocephalus or HIV); 7) gestational 
age at birth; 8) weight for gestational age; 9) dysmorphia; 10) assisted ventilation; 11) 
cranial growth (hospitalized children 6 weeks or more); 12) cranial growth (hospitalized 
children less than 3 weeks); 13) polycythemia; 14) meningitis; 15) hypoglycemia; 16) 
congenital infections; 17) hyperbilirubinemia; and 18) associated medical problems (not 
central nervous system). These factors are evaluated with a score of 0 to 3 for each of 
the aforementioned factors, and can range from a minimum of 0 points to a maximum of 
51. Taking into account the cohort criteria established by the authors (Muller-Nix et al., 
2004; Pierrehumbert, et al., 2003; Zaramella et al., 1996), the risk levels were determined 
as: 
 
a. Low Neonatal Risk: 0 to 6 points. 
b. Moderate Neonatal Risk: 7 to 9 points. 
c. High Neonatal Risk: 10 or more points. 
 
- Neonatal Behavioral Assessment Score (NBAS) designed by Brazelton, 2014 
is an assessment technique that provides a score profile that allows us to detect a 
possible alteration or pathology, but at the same time, within normal parameters, detect 

 
both the potential or "strong points" and the problems or "weak points" of the newborn, 
as well as their peculiar ways of acting and reacting to environmental variables. 
Therefore, a profile of the child's behavioral characteristics is obtained, which can be 
considered as a first outline of their temperamental traits. It evaluates the newborn's 
repertoire of behaviors in 28 behavioral items that are valued according to a 9-point 
scale. The scale also includes an assessment of the neurological status in 18 reflex 
items, each with a 4-point graduation. In the second edition of the NBAS (Brazelton, 
1984) a series of 7 supplementary items were added with the intention of better capturing 
the degree of fragility and the quality of the behavior of high-risk children. These seven 
items attempt to summarize the quality of the child's response and the amount of 
stimulation that he/she needs from the examiner to organize his/her responses. The 
NBAS can be used without any type of adaptation in full-term children and can be applied 
until the end of the second month of life. By adding the supplementary items it can also 
be used in apparently healthy premature children (less than 37 weeks of gestation) and 
for them, depending on the degree of immaturity, the application is possible up to 48 
weeks post-conceptional age. 
- Bayley-III Infant Development Assessment Scales (Bayley, 2015). They are a 
set of three standardized assessment scales that evaluate cognitive, motor and language 
development in children aged 1 to 42 months. For our study, we will only use the motor 
scale, made up of two subscales, the gross motor scale and the fine motor scale. This 
motor scale is made up of 138 items (the gross motor scale is made up of 72 items and 
the fine motor scale is made up of 66 items) through which it evaluates the degree of 
body control, the coordination of large muscle masses and the manipulative ability of 
hands and fingers. The total administration time of the motor area for children aged 12 
months, as is the case in the sample, is between 15 and 20 minutes. - The Strange 
Situation is a standardized observational procedure, in which the child is exposed to two 
moments of separation from his/her attachment figure, two moments of reunion with the 
attachment figure and moments in which he/she interacts with an unknown person. 
Specifically, there are 8 episodes (3 minutes long) in which the level of stress caused in 
the child increases in intensity as the situation progresses (28). Its duration is estimated 
to be around 20 minutes. It is evaluated on a 7-point Likert-type scale, so that low scores 
indicate low frequency and low intensity. 
 
 
Determination and analysis of biomarkers: Cortisol and oxytocin 
- Determination of cortisol in hair, as a biomarker of chronic stress: To extract cortisol 

 
from hair, the sample is carefully sectioned into segment lengths that approximate the 
time period of interest (from the outermost part, if the hair were 3 cm long, the cortisol 
secreted proportionally by the individual in the last 3 months of life will be extracted). 
Next, an approximate average weight per sample of 150 mgrs will be selected. The 
samples will be individualized and placed in eppendorfs (one for each patient) and 
pulverized with scissors. After incubation in methanol for 36 hours, the supernatant 
portion that will already have the cortisol dissolved will be extracted. This solution will be 
evaporated to dryness, and then the pellet will be reconstituted with phosphate (PBS). A 
commercial kit will be used for the quantification of cortisol using the immunoassay 
technique (ELISA Victor X5). The ELISA test kit for the detection of cortisol in saliva 
(ELISA Kits › Cortisol ELISA Kits Human Cortisol Competitive ELISA Kit ,Invitrogen), the 
sensitivity of the test kit was 0.024 ng/ml, with a detection range of 0-30 ng/ml. The 
concentration of the hormone (picograms of cortisol/milligram of hair) in each of the 
samples was calculated from the standard curve. 
 
- Determination of cortisol in saliva, as an indicator of punctual stress. Three saliva 
samples will be collected from each subject, at 8:00, 15:00 and 23:00 hours. Before 
collecting the saliva, they will be told to rinse their mouth with cold water, without brushing 
their teeth. They should not eat or drink in the hour prior to taking the sample. 
Approximately 1 ml of saliva will be obtained, collected by direct expectoration into an 
ependor tube. The samples will be stored for 24 hours at 4ºC. The samples will then be 
centrifuged at 2,500 rpm/10 min and the supernatant will be stored at -20 °C until 
processed using an ELISA kit, as per the protocol indicated above. 
 
- Determination of oxytocin in saliva. The treatment and obtaining of saliva as a biological 
sample will be identical to the protocol described for the determination of cortisol. The 
determination of oxytocin will be carried out using an ELISA kit for immunoassay 
(Oxytocin ELISA Kit (ab133050 ABCAM) 
 
Recording of biometric variables. 
The BIOPAC system is a device used to measure electrical signals in the human body. 
It is designed for research purposes, offering practicality and versatility by incorporating 
specialized software. This equipment allows understanding the generation and analysis 
of various medical conditions and their respective diagnoses. 
The system can measure different electrical signals from the body, such as 
electromyograms (EMG), electrocardiograms (ECG) and electroencephalograms (EEG). 

 
It is also capable of monitoring pulse, blood pressure, lung flow and volume, providing 
accurate measurements. It is useful for acquiring and analyzing electrical signals 
produced by the body. It is designed to record differential signals, which are then 
processed and analyzed with specialized software. 
The BIOPAC system can be used to analyze physiological signals related to mood 
changes. The system can measure various electrical signals in the body which can 
provide information about the body's response to different emotional states. 
For this study, the BIOPAC MP36R acquisition system  has been used. It is a research 
tool used in a variety of areas of physiology and science. This system features a four-
channel data acquisition unit with built-in universal amplifiers, capable of recording a 
wide range of physiological signals. The system also includes a stimulator, audio output, 
digital input and output lines, and supports air and gas analysis. The MP36R comes with 
AcKnowledge 5 software, which includes powerful automation and scoring tools. The 
software supports automated analysis for ECG, EEG, EMG, and many more. The system 
supports a wide range of signal-specific wired and wireless amplifiers. The MP36R is 
quick and easy to set up and is approved for human and animal research. When used in 
conjunction with AcKnowledge software, electrodes, transducers, and other system 
components, the MP36R forms part of a complete data acquisition and analysis system. 
The BIOPAC MP36R System will be used to collect data: 
The BIOPAC SS3LA Galvanic Response Transducer measures the galvanic response 
of the skin, also known as electrodermal activity (EDA). This transducer is used to record 
the electrical behavior of the skin, which varies according to the activity of the sweat 
glands. This measurement of electrodermal activity is essential for studying the activity 
of the autonomic nervous system, which controls involuntary body functions such as 
sweating and heart rate. 
The BIOPAC SS2LB 3-Lead ECG Cables are used for electrode connection in the 
context of electrocardiography, a technique that records the electrical activity of the 
heart. These cables allow the connection of electrodes in a three-lead format, which can 
improve the accuracy and quality of the recorded signals. Proper use of these cables 
can contribute to the diagnosis and monitoring of various cardiovascular conditions. 
The SS5LB Respiratory Band Transducer + Strap is a component of the BIOPAC system 
used to measure respiratory rate. The transducer attaches to a strap that is placed 
around the subject's chest, allowing chest movements to be detected during breathing. 
The strap ensures a secure and comfortable fit for the subject during the measurement 
process. This transducer is used in conjunction with other BIOPAC components, such 
as the EDA transducer, to assess physiological responses. Data obtained from the 

 
transducer provides information on respiratory patterns and their relationship to other 
physiological measurements. 
The temperature sensor is the SS6L, which is used to measure temperature at various 
body locations. The temperature sensor can be used to measure temperature at various 
locations on the body, such as the skin, mouth, or rectum. The sensor connects to the 
BIOPAC system via a cable, and temperature data can be viewed and analyzed using 
BIOPAC software. The sensor is designed to be accurate, reliable, and easy to use, 
allowing body temperature to be measured in a variety of environments. 
To use the BIOPAC System to collect physiological signals from the mother, the 
following steps will be followed: 
- Preparation: Verify that the sensors and transducers are correctly connected to 
the BIOPAC system. This involves connecting each sensor to the appropriate port on 
the BIOPAC hardware and ensuring that the sensors and transducers are firmly attached 
to the participant's body. 
- Calibration: Before beginning, the sensors are calibrated to ensure that they are 
accurately measuring the desired physiological signal. This may involve using a 
calibration device and following a specific calibration protocol provided by the 
manufacturer. 
- Data acquisition: This involves setting the software to record data at a specific 
sampling rate and specifying the duration of the recording which will be around 30 
minutes per patient. 
- Data analysis: The data is visualized, statistical measures are calculated, and 
patterns or trends in the data are identified. 
 
 
 

 
Statistical Analysis Plan (SAP) 
 
The Kolmogorov-Smirnov test will be used to assess the normality of the sample. 
Comparative analyses between groups will be performed by analysis of variance 
(ANOVA) or Kruskal-Wallis test. Associations of categorical variables will be 
analysed using the Chi-square test and Fisher's exact test. Intra-group 
assessment will be performed using the Wilcoxon test or Student's t-test for 
paired samples for continuous variables, and McNemar's test for categorical 
variables.  
 
The data will also be evaluated using ANOVA for repeated measures in order to 
simultaneously verify the influence of the two study groups (between-group 
effect), the two assessments (within-group effect) and to estimate the group × 
time interaction effect for each of the variables. The results will be analysed by 
intention-to-treat. The significance level will be 5%. IBM SPSS Statistics for 
Windows, version 29.0 (SPSS Inc., Chicago, IL, USA) will be used.  
 
Statistical analysis of the data collected will be performed by a researcher blinded 
for the intervention and for data collection. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
GENERAL STUDY INFORMATION AND INFORMED CONSENT 
 
UNIVERSITY CEU CARDENAL HERRERA  
 
Principal investigator: Dr. Jose Miguel Soria 
 
TITLE: Parental stress, child development and attachment in premature children 
during their first 24 months of life cared for in Child Development and Early Care 
Centers. 
 
 
Research and Ethics Committee of CEU Cardenal Herrera University Number: CEEI24/544 
 
NCT ID: 
DATE: 12-20-2024 
 
 
 

 
 
GENERAL STUDY INFORMATION 
 
TITLE: Parental stress, child development and attachment in premature children 
during their first 24 months of life cared for in Child Development and Early Care 
Centers. 
Mr. Jose Miguel Soria, Neurobiologist, principal investigator and researcher reports that: 
The study will analyze the association between the level of stress of mothers due to 
premature birth and attendance at a Child Development and Early Care Center and the 
development of the child and the patterns of interactions between mother-child 
 
The tests performed are simple and in no case involve difficulty, fatigue, danger, injury, 
pain or adverse reaction.  
 
They will be carried out by Biologist, Psychologists and physiotherapists. 
 
The general data of the subject will be collected (name, age, sex, physical variables and 
clinical history). The subject should come with comfortable clothes. The day that you were 
cited by the researcher, previous notice. Personal data is recognized in this study.  
 
The personal data are confidential, apply to the protection of personal data. The study will 
be carried out in accordance with the Declaration of Helsinki and in accordance with 
current Spanish legislation (Royal Decree 223/2004 and the Biomedical Research Act 2007) 
and any other thing that may be applicable. 
 
This study was approved by the Research and Ethics Committee of CEU Cardenal Herrera 
University (CEEI24/544). 
 
 
 
 

 
INFORMED CONSENT 
  
Mr/Mrs................................................................................................... with Number 
identification ....................................... .. freely and voluntarily, I DECLARE: 
 
That I have read the information contained in this document about the general information 
of the study. 
 
I have been informed that all tests are simple to perform and do not produce harmful effects 
on health. They will be carried out in appropriate facilities and will be carried out by 
qualified and specialized personnel. 
 
I have also been informed that, the data collected in this study will be treated confidentially, 
applying the current legislation on protection of personal data (Royal Decree 223/2004 and 
the Biomedical Research Act 2007) and any other applicable. 
 
Therefore, I give my consent, and I authorize Mr. Jose Miguel Soria, to carry out the 
detailed study in this document with the help of the necessary personnel with the 
appropriate qualification and specialization. 
 
In Elche, to ...... of .......................................... of 202 
 
SIGNED