Range of protein induced by vitamin K absence or antagonist-II levels in neonates at birth

Protein induced by vitamin K absence or antagonist-II (PIVKA-II) is avitamin K (VK) deficiency indicator in neonates. However, PIVKA-II detection frequency in neonatal blood at birth and the correlation between PIVKA-II and gestational age are unclear. We retrospectively analyzed infants admitted to our institution between June 1, 2018, and March 31, 2022, whose clinical and PIVKA-II data were available, and classified them into preterm and term infant groups. Overall incidence of PIVKA-II-positive cases (≥ 50 mAU/mL) was 42.8%, including 0.6% apparent VK deficiency (≥ 5000 mAU/mL), 3.1% experimental VK deficiency (1000–4999 mAU/mL), and 10.7% latent VK deficiency (200–999 mAU/mL) cases. Incidence of PIVKA-II-positive cases was significantly higher in the term group than in the preterm group (49.4% vs. 29.7%, p < 0.001). Gestational age correlated with PIVKA-II levels (r2 = 0.117, p < 0.0001). Median serum PIVKA-II levels and incidence of PIVKA-II-positive cases (≥ 50 mAU/mL, 16.4%) were lower at 5 days after birth than at birth, possibly reflecting the postnatal VK prophylaxis impact. Only one infant was diagnosed with VK deficiency bleeding (PIVKA-II levels, at birth: 10,567 mAU/mL; at day 5: 2418 mAU/mL). Thus, serum PIVKA-II levels after birth weakly correlated with gestational age. VK deficiency was more common in term infants than in preterm infants.

years, PIVKA-II in cord blood has been measured using ELISA.It was reported that 47% of term infants in the United States 7 , 16% of 683 infants (including 16 preterm infants) in Thailand 3 , and 66% of 141 infants (including 13 preterm infants) in Uganda 13 had detectable PIVKA-II levels (≥ 100 mAU/mL, > 150 mAU/mL, and ≥ 200 mAU/mL, respectively).The only study that measured PIVKA-II in cord blood using CLEIA included 75 preterm infants in India; PIVKA-II was detected in 49% of these infants (> 28 mAU/mL) 14 .Although it is widely known that cord blood can be used as a substitute for neonatal blood, it is not possible to collect a sufficient amount of cord blood in all cases 15 .Therefore, we believe that data on the range of PIVKA-II in neonatal blood at birth are more clinically useful.However, to date, no large-scale studies involving preterm infants have performed highly sensitive PIVKA-II measurements using neonatal blood.
This study aimed to clarify the frequency of PIVKA-II detection in neonatal blood immediately after birth and examine the correlation between PIVKA-II and gestational age.

Characteristics of the participants
A total of 2042 newborns were admitted to our institution between June 1, 2018, and March 31, 2022.Of these infants, 283 were excluded because of a lack of serum PIVKA-II data at birth.All the required data were available for the remaining 1759 infants.The clinical characteristics of the enrolled infants are demonstrated in Table 1.
Among all enrolled infants, only one was diagnosed with VK deficiency bleeding, with episodes of overt hematuria due to renal hemorrhage 2 days after birth.The case involved a female infant born as SGA at 33 weeks of gestation, with a birthweight of 1524 g (8.7 percentile of the mean value for Japanese newborns of the same gestational age 16 ), and Apgar scores of 6 and 8 at 1 min and 5 min, respectively.Her mother (38 years old) underwent duodenojejunal bypass surgery for superior mesenteric artery syndrome at the age of 32 years and had anorexia due to lower back pain for 1 week before delivery.Hematuria was observed in the infant 6 h after Table 1.Clinical characteristics of enrolled infants.Data are expressed as number (%), mean ± SD, or median [range] as applicable.SGA is defined as a birth weight less than the 10th percentile of the mean value for Japanese newborns of the same gestational age.SGA, small for gestational age; PIVKA-II, protein induced by vitamin K absence or antagonist-II.

Discussion
In this study, we observed that the incidences of cases with detectable PIVKA-II concentrations in neonatal serum soon after birth were 0.6% for ≥ 5000 mAU/mL, 3.1% for 1000-4999 mAU/mL, 10.7% for 200-999 mAU/mL, and 28.4% for 50-199 mAU/mL.In addition, serum PIVKA-II levels after birth weakly correlated with gestational age.
A study conducted in the US in 1998 using cord blood obtained from 156 term infants recorded PIVKA-II levels of ≥ 100 mAU/mL in 47%, > 2000 mAU/mL in 7%, and > 10,000 mAU/mL in 2% of the infants 7 .In a 2006 UK study of cord blood samples from 90 preterm infants (< 32 weeks), 23% and 1% of the infants had PIVKA-II levels of ≥ 200 and ≥ 1000 mAU/mL, respectively 6 .A 2010 study conducted in Thailand using cord blood (683 cases, including 16 preterm infants) reported that 16%, 8.6%, and 1.5% of the cases had PIVKA-II levels of > 150, ≥ 1000, and > 5000 mAU/mL, respectively 3 .In a 2015 study from Uganda based on 141 cord blood samples (including 13 preterm infants), PIVKA-II levels of ≥ 200 and ≥ 5000 mAU/mL were noted in 66% and www.nature.com/scientificreports/22% of samples, respectively.In this study, 8/13 (61.5%) preterm infants also had PIVKA-II levels ≥ 200 AU/ mL, and 3/13 (23%) had PIVKA-II levels ≥ 5000 mAU/mL 13 .A 2022 study from India that included 75 preterm infants with gestational age < 32 weeks reported that 49% of these infants had PIVKA-II levels > 28 mAU/mL, 2.7% had PIVKA-II levels ≥ 1000 mAU/mL, and none had PIVKA-II levels ≥ 5000 mAU/mL 14 .The strength of our study is that, to our knowledge, it enrolled the highest number of cases, including many preterm infants, as compared with previous reports.Our results are generally consistent with those of previous studies, except for a study from Uganda, which reported a substantially high incidence of abnormally high PIVKA-II levels.This difference observed in PIVKA-II levels between populations from Japan and Uganda (0.7% vs. 23% for ≥ 5000 mAU/mL) could be due to the differences in maternal nutritional status.Serum PIVKA-II levels after birth were weakly correlated with gestational age.Although studies on PIVKA-II that include sufficient numbers of preterm infants are limited, Bovill et al. reported that the PIVKA-II positivity rate increased with increasing gestational age (5.3% at < 34 weeks, 5.7% at 34-38 weeks, and 9.9% at ≥ 38 weeks).However, the difference was not statistically significant 12 .Despite the small number of preterm cases, Santorino et al. reported that preterm birth was an independent risk factor for high PIVKA-II levels 13 .VK deficiency has been reported in 23% of preterm infants in the UK (n = 90) 6 and 62% of preterm infants in Uganda (n = 13) 13 , which is substantially high compared to our study (8.1%, n = 592).However, the number of cases included in these studies was not sufficient.To our knowledge, our study is the first to measure PIVKA-II in neonates of all gestational ages and demonstrate that preterm infants are less prone to VK deficiency.
PIVKA-II levels in neonates can be influenced by external factors, such as VK deficiency, and internal factors, such as liver immaturity.We observed a significant correlation between gestational age and PIVKA-II levels in term infants but not in preterm infants.This suggests that some physiological changes in the intrauterine environment related to PIVKA-II production may occur around 37 weeks of gestation.According to a review comparing term pregnancy and prolonged pregnancy, microscopic changes, including aggregation of syncytiotrophoblast nuclei and reduced villous vascularity with concomitant impairment of trophoblast transport processes, were reported in placentas of prolonged pregnancy.These morphological changes are associated with increased evidence of oxidative stress, which could reflect placental aging or a reduction in placental function 17 .Whereas a study comparing oxidative stress markers between 37-39 weeks and ≥ 41 weeks placenta reported a significant increase in oxidative stress in the latter, suggesting placental aging or damage after 37 weeks' gestation 18 .In addition, in a study examining the expression of DNA damage markers by gestational age in the normal placenta, immunohistochemical expression of 8-OHdG, an oxidative damage marker, significantly increased with gestational age 19 .Thus, we speculate that the aforementioned decrease in placental function after 37 weeks of gestation causes decreased placental transfer of VK to the fetus and contributes to physiological VK shortage in term newborns that was observed in this study.In a study evaluating the placental transfer of vitamin K1 using maternal, fetal, and neonatal blood samples, the mean maternal-fetal gradient of endogenous vitamin K1 concentrations at mid-trimester (14-fold) was lower than those at term (18-fold), which is consistent with our hypothesis 20 .
This study had several limitations.First, because this was a single-center retrospective study in Japan, it may not be applicable to other countries where the nutritional status and genetic background of pregnant women differ.Second, as data regarding maternal background and detailed neonatal information, except those examined in previous studies, were not available, care should be taken when interpreting the results of this study.Third, since we were not able to extract long-term clinical outcomes, we could not confirm the correlation between PIVKA-II level at birth and the risk of subsequent bleeding symptoms.Thus, this study alone could not clarify the PIVKA-II threshold for the risk of developing VK deficiency bleeding.Hence, future prospective studies that include detailed maternal and neonatal information are required.

Study design and patients
We retrospectively analyzed infants admitted to our hospital between June 1, 2018, and March 31, 2022, whose clinical and PIVKA-II data were available.To screen VK deficiency in neonates, we measured serum PIVKA-II levels at admission and 5 days after birth.According to the current Japanese guidelines 21 , the VK prophylaxis method in our hospital involves administering vitamin K2 (menatetrenone) within 24 h of birth (1 mg for infants with birthweight < 1500 g and 2 mg for infants with birthweight ≥ 1500 g) intravenously, if available, or orally.The second dose (2 mg) is administered at 4 days of age, intravenously or orally, in all cases 22,23 .Clinical data included gestational age, birth weight, sex, parity, delivery mode, Apgar scores, and SGA.
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee at Kobe University Graduate School of Medicine (IRB approval number: B220244, March 29, 2023).All parents provided written informed consent for the use of their children's personal medical data.
SGA was defined as having a birth weight less than the 10th percentile of the mean value for Japanese newborns of the same gestational age 16 .
We classified the patients into two groups according to their gestational age: preterm infants (< 37 gestational weeks) and term infants (≥ 37 gestational weeks).Clinical characteristics and serum PIVKA-II levels were compared between the two groups.Blood samples were collected within 2 h of admission and promptly centrifuged.Serum samples were stored at − 80 °C until use.With 20 µL of serum, we measured the PIVKA-II concentration using a two-step sandwich CLEIA with the LumipulsePresto PIVKAII-N kit (SEKISUI MEDICAL Inc., Japan).The data are expressed in arbitrary units, with 1 AU corresponding to 1 µg of purified prothrombin 24 .

Statistical analysis
Data are presented as the median [range], mean ± SD, or number (%) as applicable.The Mann-Whitney nonparametric rank test, chi-square test, and Fisher's exact test were used to compare data between the two groups as appropriate.Regression analysis was performed to linearly compare gestational age and serum PIVKA-II levels; regression equations and correlation coefficients (r 2 ) were calculated.Statistical significance was set at p < 0.05.Analyses were performed using GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA) and EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R 4.2.2 (R Foundation for Statistical Computing, Vienna, Austria).

Table 2 .
Clinical characteristics of infants with the follow-up values of PIVKA-II at 5 days after birth.Data are expressed as number (%), mean ± SD, or median [range] as applicable.SGA is defined as a birth weight less than the 10th percentile of the mean value for Japanese newborns of the same gestational age.SGA, small for gestational age; PIVKA-II, protein induced by vitamin K absence or antagonist-II.