**4. Materials and Methods**

Patients hospitalized at the Department of Obstetrics and Pathology of Pregnancy at the Medical University of Lublin between 2019 and 2021 and between 32 and 36 weeks' gestation with a singleton pregnancy and late-onset FGR were selected for the research. Multiple pregnancies, the presence of any antenatal infections, a positive TORCH test result, treatment with antibiotics during pregnancy, any form of hypertension in pregnancy, pre-pregnancy and gestational diabetes, nephropathy, thyroid dysfunction and any other general diseases before pregnancy, the use of any drugs or stimulants, cigarette smoking, and fetuses with birth defects and chromosomal abnormalities were excluded from the study.

Thirty-six pregnant Caucasian women participated in the study, comprising 18 women with physiological pregnancy and eutrophic fetus (EFW > 10th percentile) (control group) and 18 women with late FGR identified after 32 weeks of pregnancy, according to Delphi consensus (study group) [3]. Placenta samples were successfully obtained from all eligible participants. Hadlock et al. devised a regression equation using the biparietal diameter, the length of the femur, and the head and belly circumferences to estimate the fetal weight during an ultrasound examination [44]. During one week before the birth, Doppler measurements of the umbilical artery free loop were taken using a Voluson E9 with RA4B 3D 4–8 MHz curvilinear probe (GE Healthcare, Hatfield, UK). Then, the pulsatility index (PI), resistance index (RI) and cerebroplacental ratio (CPR) were computed. PI = (S − D)/A and RI = (S − D)/S, where S represents the systolic peak, D represents the end-diastolic flow, and A represents the temporal average frequency. In contrast, the CPR is the ratio between

the PI of the middle cerebral artery (MCA) and the umbilical artery (UA) (PI MCA/PI UA) and reflects the distribution of cardiac output in favor of cerebral blood flow. It is one of the criteria with the highest predictive accuracy for perinatal outcomes [45]. In response to intrauterine hypoxia, fetal blood flow is redistributed to the brain, and the value of CPR reduces by 1. In cases of late-onset FGR, hypoxia tolerance is lower than in cases of early-onset FGR [46].

Using standardized medical records and patient interviews, smoking, age, weight, and body mass index (BMI) at the beginning of the first trimester, pregnancy weight increase, and TORCH were determined for the mothers. The BMI was computed by dividing body weight (kg) by height (m2). Moreover, data including information on infants: gestational age at delivery, gender and birth weight of the newborn, placental weight, body length, head circumference, and neonatal problems. The gestational age was calculated using the latest menstrual period and the first-trimester ultrasonography (based on crown–rump length (CRL)). Immediately after birth, placenta weight and the neonate birth weight, body length, and head circumference were measured using the proper measuring instruments.

Material for proteomic investigation consisted of pieces of normal placentas serving as controls and fragments of placentas obtained from mothers with FGR. Following the process, trained employees collected all samples. During the cesarean section, soon after childbirth, the placenta was put in sterile containers containing ice under aseptic circumstances. Those responsible for collecting specimens wore a sterile protective apron, face masks and sterile gloves to guarantee sterility throughout the sampling procedure. The placentae were collected and weighed. Four placenta samples measuring 1.0 × 1.0 × 1.5 cm were taken from each placenta (overall number of samples: 144), around 3 to 4 cm from the umbilical cord attachment point, from four separate quadrants of the placenta. To reduce the possibility of infection after cesarean delivery, only portions from the inner placenta were obtained for evaluation (risk of contamination). Each placenta sample was put in a sterile, labeled cryovial, frozen in liquid nitrogen, and kept at −80 degrees Celsius for future study.
