The study of hypovitaminosis D has aroused great interest in recent years, as it relates to numerous pathologies in both adults and children, and, especially, in neonates. Our study was conducted in a single tertiary center and enrolled 50 Caucasian neonates from the same geographical area, with similar socioeconomic status, feeding and environmental conditions. In contrast to other studies, we observed that low vitamin D levels alone are not associated with prematurity or related comorbidities [28, 31,32,33]. However, a field of study opens when low 25(OH) D and high PTH levels are associated with a greater incidence of LOS in PTs.
Levels of 25(OH) D in cord blood were lower in term infants than in PTs, however, there were no significant differences. These results might have been influenced by our sample size. The percentage of vitamin D deficiency among the global population remains similar to what has been described by other authors [34, 35]. In our study, we have detected a higher percentage of vitamin D deficiency among term infants than in PTs, but such does not have statistical significance.
McDonnell SL et al. have reported that higher target levels of vitamin D must be achieved in pregnant women and their neonates to prevent adverse outcomes . Despite having implemented vitamin D supplementation in PTs admitted to our neonatal unit, as shown elsewhere in this study, there were no significant differences regarding 25(OH) D serum levels in PTs at birth and at 28 days. Nevertheless, these levels increased significantly whereas PTH decreased also significantly at 4 months.
Vitamin D is responsible for increasing intestinal calcium absorption that feeds back the parathyroid gland to decrease PTH secretion. Exposure to comorbidities including hyaline membrane disease, NEC, patent ductus arteriosus and sepsis, among other pathologies, could explain the non-increase in vitamin D in PTs within the first 28 days of life. Consequently, PTs admitted to the NICU are at higher risk for vitamin D deficiency. On the other hand, defining hypovitaminosis D status in PTs and establishing a corresponding clinical significance is still a matter of controversy. To our knowledge, scarce is known about the PTH cut-off point related to 25 (OH) D in PTs [37, 38].
Maternal vitamin D stores have been the target of extensive scientific research, which has focused on their potential negative effects on maternal and neonatal health. Multiple observational studies have described that maternal hypovitaminosis D (maternal 25(OH) D serum levels < 20 ng/mL) is a crucial risk factor for neonatal adverse outcomes. Important differences in maternal and fetal outcomes have been reported when analysing pregnancy outcomes using serum biomarker 25(OH) D, with improved health in women who achieved circulating 25(OH) D serum levels of at least 40 ng/mL . Moreover, recent major evidence reports that an effect of vitamin D deficiency is linked to a risk of miscarriage and some authors advocate for the introduction of level measurement before pregnancy .
Up to date, there is no consensus on the normal range for 25(OH) D serum levels in PTs and term infants. Hollis et al.  have suggested that pregnant women should have 25(OH) D serum levels > 40 ng/mL for optimizing maternal and neonatal outcomes. There is growing evidence that the higher vitamin D levels are, the fewer perinatal comorbidities, cesarean sections and hypertensive disorders will develop . Mothers of both PTs and term infants showed no differences in blood pressure based on 25(OH) D serum levels.
Even though most prior studies highlight GA as a risk factor for vitamin D deficiency, we did not observe this in our study. Our findings, moreover, showed a correlation between maternal and neonatal 25(OH) D serum levels in PTs and term infants.
No mother received vitamin D supplementation during pregnancy, and vitamin D deficiency was prevalent in the recruited infants. Accordingly, previous studies have published a high prevalence of vitamin D deficiency in pregnant women [11, 40]. Unlike other studies, neither did we find higher 25(OH) D serum levels in term infants when compared to PTs nor even when participants in both groups had been born during the summer.
Vitamin D requirement during pregnancy is probably higher in the second and third trimester due to enhanced intestinal calcium absorption and fetal requirements . Currently, there is no consensus on this issue, and different authors highlight the importance of vitamin D supplementation in pregnancy even though in our environment it is not a common practice. As a result, vitamin D supplementation during pregnancy should be considered when our aim is to minimize the danger of neonatal birth infections and improve maternal outcomes .
Several maternal risk factors which contribute to low maternal/fetal 25(OH) D serum levels have been reported, but no clear pattern has been established for multi-ethnic populations. The winter season, obesity, a lower socioeconomic status including lifestyle factors (smoking) and medication pose a risk for lower maternal/fetal transfer of vitamin D. Notwithstanding, there is still scarce published research into the relationship between some of the maternal risk factors and neonatal 25(OH) D serum levels . In our study, factors such as the socioeconomic status, diet and lifestyle were similar for all the mothers. Parenteral nutrition could be considered a confounder factor related to the cumulative dosage of vitamin D. However, as parenteral nutrition is individualized since the first day of life, the amount of vitamin D administered to all the PTs is always the same (120 IU/day). The total amount of vitamin D was adjusted daily considering parenteral nutrition as well as feeding.
Critically ill patients have a high prevalence of vitamin D deficiency and low levels are associated with greater illness severity and morbidity . It is likely that PTs have a compromised vitamin D status at birth and during NICU hospitalization. Xiaonan et al.  have reported that vitamin D deficiency is a risk factor for BPD in extremely PTs. Cetinkaya et al.  have found that the lower the maternal/neonatal 25(OH) D serum levels, the higher the risk for BPD development. We determined BPD based on the definition suggested by Jobe AH and Bancalari . In contrast to other studies, we did not observe any relationship between 25(OH) D cord blood levels/28 days and risk for BPD. Joung KE et al.  have concluded that in extremely PTs neither cord blood nor the 36 weeks of corrected age for 25(OH) D serum levels are associated with BPD development. The lack of this relationship in our study may be accounted for the presence of a higher GA in our groups (median weeks’ GA 29.2 ± 2.45).
Recent studies in neonatal population have described improved outcomes including normalization of PTH at levels of vitamin D > 30 ng/mL. PTH is a major hormone in charge of bone resorption, and its serum levels may be a useful identification risk marker of secondary hyperparathyroidism and metabolic bone disease in extremely low birth weight neonates. In PTs, we had higher PTH levels at birth and these did not reach a normal range until 4 months. Our results indicate that the isolated determination of vitamin D does not define its deficiency or sufficiency and that the vitamin D-PTH relationship must be taken into consideration .
MMP-8 has been identified as a biomarker of neonatal sepsis . The activity of MMP-8 would increase in many pathophysiological conditions such as severe infection . Emerging evidence has supported the antimicrobial implications of vitamin D, as it enhances the innate immunity and induces the production of antimicrobial peptides that inhibit the growth of bacteria [48, 49]. Recently, Rosendahl et al.  have observed a positive correlation between 25(OH) D levels and MMP-8 in cord blood of healthy non-vitamin D deficient neonates. In our study, decreased serum levels of 25(OH) D neither correlated with MMP-8 levels nor with the incidence of early sepsis or LOS in PTs at any of the three-time points. However, we found a significant relationship between lower 25(OH) D cord blood levels and higher PTH with LOS in PTs (p < 0.031). In our PTs population, 25(OH) D levels in cord blood by itself did not represent an independent modifiable risk factor of lower morbidity related to sepsis.
The LOS incidence rate in PTs ranges between 20 and 38% in the first 120 days of life . Prevalence of LOS is rather high in our study population (48%), which might be due to the fact that PTs were selected consecutively so as to avoid selection bias. On the other hand, the total number of days of central catheter exposure and of parenteral nutrition is a known risk factor for sepsis whereas human breastmilk administration is protective. As shown in table 7, there were neither differences in the rates of breastmilk feeding nor in exposure to mechanical ventilation. On this statement, differences detected among days on parenteral nutrition in the PTs who developed sepsis were related to the slower advances of enteral feeding in this group, resulting also in a longer hospital stay. Vitamin D levels are decreased after 28 days in patients who developed LOS.
In contrast to the findings reported by Fort et al. , after vitamin D supplementation with 1000 IU/day in our cohort of PTs during NICU hospitalization, biochemical 25(OH) D deficiency did not decrease within the first 28 days, and it was not until 4 months when values reached > 30 ng/mL, which associated with an optimal physiological function. Similar results have been reported by Cho et al.  in this regard. It is pivotal to establish the optimal amount of vitamin D intake among hospitalized PTs to achieve adequate vitamin D levels and prevent adverse events. However, the role of vitamin D in neonatal immunomodulation and the timing/dosage of vitamin D supplementation are still unknown and warrant further research.
The limitations of the present study include the difficulty to collect a homogeneous sample of Caucasian PTs and healthy term infants with the same environmental conditions. The selection bias was avoided by establishing very strict inclusion criteria for PTs ≤ 32 weeks’ GA and /or ≤ 1500 g birth weight in a single Neonatology Unit, using the same protocol for all the patients. The estimation of the sample size, recruited consecutively, provided sufficient power to detect differences and associations.