Objectives

The study aims to:

1. Determine whether the cord blood S100B protein concentration is significantly higher in neonates with prenatally diagnosed FGR compared to controls.
2. Evaluate the correlation between S100B protein concentration and fetal blood flow parameters in the UA, UtA, DV, and MCA, as assessed by ultrasound performed within 48 hours before delivery.
3. Evaluate the discriminatory performance of S100B concentration in relation to perinatal markers associated with fetal hypoxia (pH, BE, and lactate levels in umbilical artery blood).

Trial design

This single-center, prospective cohort study aims to assess the effectiveness of S100B protein concentration as a potential biomarker associated with perinatal hypoxic stress in neonates with FGR, compared to a control group without growth disorders.

Study setting

The study is being conducted at the Institute of Mother and Child in Warsaw, specifically within the Department of Obstetrics and Gynecology and the Department of Neonatology and Neonatal Intensive Care, both of which function as tertiary referral units. The center manages approximately 50 full-term newborns diagnosed with FGR annually. Recruitment commenced in June 2024 and was initially projected to continue for approximately 18 months, through December 2025, with the actual duration dependent on the number of FGR patients enrolled during that period. Complete data collection is expected to be finalized within three months of the end of recruitment (March 2026), with initial results anticipated in June 2026. At the time of protocol publication, the authors anticipated potential minor delays in patient recruitment due to a lower-than-expected incidence of pregnancies complicated by FGR at the study center, as well as a higher-than-anticipated number of exclusions. As of September 2025, approximately two-thirds of the targeted patient population have been enrolled in both the study and control groups.

Definitions

Fenton’s growth charts are used to assess birth weight percentiles [22]. The criteria for diagnosing FGR follow the 2020 recommendations of the Polish Society of Gynecologists and Obstetricians for late-onset growth restriction, which occurs after the 32nd week of pregnancy [23]. These guidelines are based on the Delphi Criteria for diagnosing early and late FGR [24].

Late FGR is diagnosed if any of the following criteria are detected during routine fetal ultrasound:

• Abdominal circumference (AC) below the 3rd percentile for the given gestational age.
• Estimated fetal weight (EFW) below the 3rd percentile for the given gestational age.

OR

• EFW or AC below the 10th percentile, along with at least one of the following:
  • Growth retardation by the 50th percentile on the growth curve.
  • Cerebroplacental index (CPR), defined as the ratio of the pulsatility indices (PI) in the MCA and UA, below the 5th percentile for the given gestational age.
  • UA PI above the 95th percentile for the given gestational age.

Eligibility criteria

Study procedures and data collection

Data will be collected through cord blood samples, ultrasound measurements, and clinical assessments.

For all patients who consent to participate in the study, an ultrasound examination will be performed within 48 hours before delivery. This ultrasound assessment includes:

1. a. Measurement of the fetus’s anthropometric dimensions and estimation of fetal weight.
2. b. Evaluation of blood flow using the pulsatility index (PI) in the UA, UtA, DV, and MCA.

After birth, in both the study and control groups:

1. A 1.5 ml blood sample will be collected from the clamped part of the umbilical cord:
   1. a. Immediate analysis: 0.5 ml of the cord blood will be sent immediately to the laboratory to determine pH, BE, and lactate levels in the umbilical cord blood analysis.
   2. b. S100B protein analysis: The remaining 1 ml of cord blood will be placed in a labeled tube (including the mother’s name, child’s gender, date of birth, and date of collection) and sent to the laboratory for centrifugation. Serum samples will be frozen at −20°C and stored. Once approximately 80 samples are collected, the serum will be thawed and analyzed for S100B protein concentration. Any remaining material after laboratory processing will be properly disposed of.
2. Data will be collected on gestational age, duration, and mode of delivery, along with any complications encountered during delivery. Additionally, details regarding the newborn’s condition after birth will be recorded, including gender, Apgar score, and anthropometric measurements. Observations made during hospitalization will also be documented, with particular attention to any concerning symptoms related to the CNS, such as muscle tone abnormalities or seizures.
3. A transfontanelle ultrasound examination will be performed to assess for any abnormalities in the newborn.

Study participation does not alter standard hospitalization procedures or duration. Furthermore, collecting a blood sample for the planned tests will not interfere with cord blood collection if the mother intends to bank it.

Criteria for discontinuing participation

If a full ultrasound flow assessment becomes unfeasible due to technical issues, advanced labor, or urgent delivery indications, it will be halted. However, any partial data collected will still be analyzed.

If an umbilical cord blood sample cannot be collected after birth, the patient will be excluded from the study. Nevertheless, data from excluded patients will be retained to support further analysis and ensure a comprehensive statistical evaluation.

In cases where consent is withdrawn, any data collected up to that point will be retained and used in the study.

Compliance

Before the study commenced, a meeting was held to familiarize the researchers and staff with the study protocol. The protocol was presented in detail, including the rationale behind the study. Additionally, schematic instructions were provided, covering patient qualification, the required tests for qualification, and the intervention procedures.

The medical records of participating patients are clearly labeled to ensure adherence to the study protocol. Each patient’s medical record includes a schematic outlining the inclusion and exclusion criteria.

Explanation for the choice of comparators

Biomarkers associated with the risk of hypoxia-related CNS injury should meet several key criteria:

• They should be detectable in easily accessible fluids such as blood, amniotic fluid, or urine, allowing for straightforward sample collection.
• They should identify patients at risk before symptoms emerge or clinical examinations, or other tests (e.g., ultrasound, MRI, or EEG) reveal any abnormalities.
• They should provide a quantitative measure of the severity of perinatal hypoxic stress.
• The tests should be simple, quick, cost-effective, and characterized by repeatability, sensitivity, and specificity [25].

To date, few markers have been thoroughly evaluated for each of these criteria. However, the S100B protein shows promise as a potential marker. It can be detected in blood, saliva, urine, and amniotic fluid, potentially allowing for the early identification of neonates at risk of adverse neurodevelopmental outcomes in a relatively quick and inexpensive manner. Nonetheless, additional prospective studies are needed.

Outcome measures

The study’s primary endpoint will be the concentration of S100B protein measured from a cord blood sample taken after birth in both the study and control groups.

Additional endpoints will include:

• The potential correlation between S100B concentration and pH, BE, and lactate levels, as determined in the cord blood gas analysis in both the study and control groups.
• The possible relationship between cord blood S100B protein concentration and blood flow parameters (PI) in the UA, UtA, DV, and MCA, as assessed by fetal ultrasound performed within 48 hours before delivery.

Participant timeline

For the primary endpoint data acquisition, the study period includes the fetal ultrasound examination conducted 48 hours before delivery and the delivery process itself. This encompasses umbilical cord clamping, collecting cord blood from the severed portion of the umbilical cord, and recording anthropometric measurements. Additional data that may lead to patient exclusion (e.g., diagnosis of intrauterine infection or neonatal anemia requiring a top-up transfusion in the control group) or that could impact the study results (e.g., transfontanelle ultrasound findings or abnormal neurological symptoms) will be collected until the child is discharged from the hospital. The overall study design and timeline are presented in Fig 1.

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Fig 1. Participant timeline according to the SPIRIT 2025 guidelines.

Schedule of enrollment, data collection, assessments, and follow-up procedures in the prospective cohort study evaluating cord blood S100B protein concentration as a potential biomarker associated with fetal hypoxia in late fetal growth restriction. The timeline presents study procedures from screening and enrollment through baseline assessment, perinatal data collection, neonatal assessments, sample processing, data entry, and final statistical analysis. T0 indicates baseline assessment; T1–T5 represent subsequent study time points (T1: 48 hours after delivery, T2: birth, T3: hospital discharge or 2–14 days, T4: 3 months, T5: 6 months). X indicates the time point of assessment or data collection; arrows indicate ongoing procedures or follow-up periods.

https://doi.org/10.1371/journal.pone.0355156.g001

Sample size

The estimated minimum total sample size for the study is 120 patients, with 60 participants in each group. The planned sample size was determined for a two-sided comparison between two independent groups, assuming a significance level of 0.05, statistical power of 80%, and equal group sizes. The variability of the primary biomarker (S100B) was estimated based on data from a previously published reference study [12], which reported median values together with the 10th and 90th percentiles. As standard deviations were not provided, dispersion was approximated from the reported percentiles under an approximate normality assumption, following established methods for recovering variability from limited summary statistics [26,27]. In the reference study, the observed difference between group medians was 1.15. However, since group allocation in that study was performed retrospectively on the basis of cranial imaging findings (brain injury vs. no brain injury), a conservative approach was adopted in the present sample size estimation to minimize the risk of effect size overestimation. Therefore, the target detectable difference was defined as 50% of the previously reported median difference.

Based on these assumptions, the estimated required sample size was approximately 55 participants per group. Additionally, considering that up to 10% of collected samples could be non-diagnostic or otherwise non-evaluable, the planned recruitment target was increased to 60 participants per group (120 participants in total). Sample size calculations were performed using standard power analysis procedures implemented in G*Power software (version 3.1.9.4).

Sequence generation

Participants are assigned to the study group based on a diagnosis of FGR from routine fetal ultrasound and the absence of exclusion criteria. The control group comprises participants based on gestational age (≥37 + 0/7 weeks), no diagnosis of FGR on ultrasound, and the absence of exclusion criteria for this group.

Data collection

Patients’ data are collected using a password-protected electronic form, with access restricted to the researchers involved in the study.

The researchers are responsible for conducting blood tests on the umbilical cord blood and performing both fetal and postnatal transfontanelle ultrasound examinations.

Statistical methods

Descriptive statistics, including percentages for categorical variables and mean values, standard deviations, medians, and interquartile ranges for continuous variables, will be used to summarize the data. The Kolmogorov-Smirnov test, along with graphical analysis, will be employed to assess whether continuous variables conform to a normal distribution.

For analyzing the relationship between a quantitative variable and a qualitative variable (e.g., comparing S100B levels between the study and control groups), the Student’s t-test for independent samples will be used if the continuous variable is normally distributed. If the continuous variable does not meet the assumptions of normality, the Mann-Whitney U test will be applied instead.

To test hypotheses about the relationships between two continuous variables (e.g., S100B protein concentration versus flow rates), while accounting for confounding factors such as pregnancy duration and mode of delivery, either multivariate quantile regression or multivariate linear regression will be employed, depending on the distribution of the outcome variable.

Patient and public involvement

Patients and the public were not involved in the design of the study.

Ethics and dissemination

The study has received approval from the Bioethics Committee of the Institute of Mother and Child in Warsaw (Decision No. 13/2024, dated 21 March 2024). Any significant modifications to the protocol, including changes to the study’s purpose, design, population, inclusion or exclusion criteria, sample size, or procedures, will be documented as formal amendments in the study protocol and reported to the Bioethics Committee of the Institute of Mother and Child in Warsaw.

Participant recruitment is conducted by a designated member of the research team. Potential participants receive both oral and written information about the study and are allowed to ask questions. Written informed consent is obtained from each participant before enrollment.

All data collected from individual participants will be de-identified prior to analysis and data sharing. The de-identified dataset, together with the full study protocol, will be made available without time restrictions to researchers who submit a methodologically sound proposal, following publication of the study results.

Data monitoring is performed by the Science Department of the Institute of Mother and Child in Warsaw, the authors’ institutional affiliation, which receives periodic reports on study progress from the research team.

The study’s results will be published in a peer-reviewed journal related to neonatology, pediatrics, obstetrics, or perinatology.