Hypoxic Status Is Associated With The Intensity Of Hypoxia Inducible Factor (Hif)-1α Expression In A Premature Placenta

Prematurity refers to live births before 37 weeks of gestation and associated with infant morbidity/mortality. Activation of HIF during the final pregnancy phase is believed to play a critical role in the pathogenesis of premature birth and other pregnancy disorders. This study aimed to analyze the relationship between hypoxicstatus and the intensity of HIF-1α expression in a premature placenta.Stored biological materials premature placenta (paraffin blocks) was used in this study. Thirtyone samples of placental hypoxia (H) and 28 samples of premature placental non-hypoxia (N) as controls, were selected non-random consecutively. Subsequently, immunohistochemistry was performed to analyze HIF-1α expression. TheChi-square testwas used to analyze the data and a p-value <0.05 was considered statistically significant.Moderate to strong intensity of HIF-1α expressionwas observed in 58% of hypoxic placenta samples, whereas most of nonhypoxic placental samples(86%) did not expressed or expressed weaklyHIF-1α.There was a significant correlation between the intensity of HIF-1α expression and placental hypoxia (p <0.05) and Odds Ratio (OR) value was 8.31 with a 95% confidence interval (2.32-29.77). The conclusion shows that hypoxic status is associated with intensity of hypoxia inducible factor (HIF)-1α expression in a premature placenta.

habits, increased levels of catecholamines in the mother's urine, and less than a year of pregnancy interval. The etiology of prematurity is still debatable.(Ahumada- Barrios and Alvarado, 2016) Premature birth is closely related to infant morbidity and mortality. (Dong et al., 2012;Sulistiarini and Berliana, 2016) Premature babies will be at risk of developing health problems such as respiratory distress syndrome, retinopathy, breastfeeding disorders, necrotizing enterocolitis, infections, cerebral palsy, and can even cause infant death. (Gopagondanahalli et al., 2016) Premature birth can also cause milder disorders such as behavioral disorders, learning and language difficulties, attention disorders and hyperactivity, resulting in poor quality of human resources of future generations. (Sulistiarini and Berliana, 2016) The incidence of preterm birth continues to increase worldwide and according to WHO data the number of premature births is estimated at 15 million every year, and nearly one million children die from complications of preterm birth. (Sulistiarini and Berliana, 2016) WHO notes that the rate of premature events in Indonesia in 2010 was 15.5 per 100 live births. (Sulistiarini and Berliana, 2016) In the United States it is noted that preterm birth rates continue to increase, and among infants born very prematurely, more than half have cognitive, sensory, and behavioral deficits, and around 5-10% experience motor deficits, including cerebral palsy. (Jellema et al., 2013;Ravarino et al., 2014) The state of hypoxia and tissue ischemia is likely to be the most common abnormality found in premature infant brain damage. (Ravarino et al., 2014) In carrying out the process of cell development and maintaining a state of homeostasis as well as functioning properly, cells need oxygen availability continuously. (Vadlapatla, Vadlapudi and Mitra, 2013) Oxygen plays an important role in regulating membrane transport, intracellular signaling processes, cellular endurance, and expression of various genes. When tissue oxygenation demand increases beyond the availability of oxygen in the vascular system, hypoxia can occur, (Vadlapatla, Vadlapudi and Mitra, 2013) leading to the stabilization of a transcription factor known as hypoxiainducible factor-1α (HIF-1 α). Hypoxia-inducible factor -1α is a subunit of HIF-1, regulating cellular homeostasis in response to hypoxic states. (Zaramellaet al., 2017) Hypoxia-inducible factor -1 was first discovered by Semenza, et al around 1991, in a study related to the erythropoietin gene (EPO). (Masoud and Li, 2015) In response to hypoxic states, the EPO gene is activated to transcribe in an effort to increase the production of red blood cells and oxygen-carrying capacity. (Vadlapatla, Vadlapudi and Mitra, 2013;Masoud and Li, 2015) This study found a DNA sequence that is important for the activation process of the EPO gene transcription called the hypoxia response element (HRE).
Another study found that the activation of transcription of various regulatory genes was initiated by the binding of a specific protein induced in a state of hypoxia to HRE. This protein is identified as HIF-1. (Masoud and Li, 2015) Normal developments of the placenta and fetus depend on the HIF1-α modulation and cellular defense against oxidative stress. (Horii et al., 2016) Placenta is responsible for distributing the feto-maternal circulation in terms of exchange of nutrients and metabolic waste. The welfare of the fetus in the womb depends on the placenta, that regulates the process of pregnancy and fetal growth and development in optimal conditions. (Salmani et al., 2014) A healthy placenta has a plasticity in adapting to environmental conditions thus facilitating the invasion of trophoblast and vascular remodeling. (Chakraborty, D. et al., 2016). The development and maturity of the placenta is a complex process and requires adequate blood supply so that the fetus can develop completely. In early pregnancy the placenta develops in an environment with low oxygen pressure (physiological hypoxia) in order to support placenta function. (Liu, Wang and Lin, 2014) All cells respond to hypoxic conditions by modifying gene expression.HIF is an important mediator in this process to facilitate placental vascularization. Additionally HIF activation during the late pregnancy phase is believed to play a critical role in the pathogenesis of IUGR, preeclampsia, premature birth or other pregnancy disorders. (Patel et al., 2010) In preeclampsia placenta, over expression of HIF-α was thought to be an indicator of hypoxia state response. Study by Liu et al.showed a decrease in the expression of hypoxia-inducible factor prolyl 4-hydroxylase-1 (PHD-1) resulting an increased expression of HIF-2α, not HIF-1α in placenta preeclampsia. Response to hypoxia would change the metabolism pattern of placenta cells so as to disrupt their growth. This study highlights the possibility that PHD and HIF could be potential targets for preeclampsia therapy. (Liu, Wang and Lin, 2014) Tal mentions that preeclampsia pathogenesis seems to be centered on the occurrence of placental hypoxia. This review also reports the increasing of cell-free HIF-1α RNA expression in maternal plasma, which suffered from preeclampsia and IUGR.
It can be thought that in preeclampsia, HIF-1α becomes a molecular link between placental hypoxia and its downstream mediators and it is possible that HIF-1α suppression or antagonism of its downstream mediators may serve as targets in preeclampsia therapy. (Tal, 2012) Futhermore, there have been no studies that analizing the relationship of hypoxia and HIF-1α expression in premature placentas.This study aims to compare the condition of cellular hypoxia by assessing the expression of HIF-1α, between the placenta of hypoxic premature infants and those without hypoxia.

Study design
This observational-analytical cross-sectional study was conducted in the Histology Laboratory, Department of Histology, Faculty of Medicine, University of Indonesia, from July to December 2014.

Study samples
Using stored biological material (SBM) of placenta in preterm infantsacross sectional comparative formula was applied to determine the large samples. The sample were selected nonconsecutively, resulting 31 placenta hypoxia and 28 placenta non-hypoxia as controls (hypoxia based on assessment of blood gas analysis and / or pulse oxymetry of premature infants, if PO2<30 mmHg and / or O2saturation<60%).

Statistical analysis
Statistical Product and Service Solution (SPSS) statistical program version 16.0 was used to analyze the data and Chi-square test was used to analyze the relationship between hypoxia with the intensity of HIF-1α expression. A p-value <0.05 was considered statistically significant.

RESULTS AND DISCUSSION
HIF-1α protein expressed moderate to strong in 58% of hypoxic placentas, whereas 86% of non-hypoxic placental group had no or weak HIF-1α protein expression. This protein was observed in the syncytiotrophoblast and capillary endothelial cells nucleus. There was a significant relationship between hypoxic status and the intensity of HIF-1α protein expression (p <0.05) and Odds Ratio (OR) value was 8.31 with a 95% confidence interval (2.32-29.77). (Figure 1, table 1).
Our results showed an association between hypoxic status and HIF-1α intensity, where a hypoxic placenta has 8.31 times more likely express HIF-1α than non-hypoxic one.  *Chi-squaretest In the development of early pregnancy, hypoxia condition is needed for the process of cytotrophoblasts invasion and infiltration into the decidua, to maintain the feto-maternal circulation.Therefore, HIF-1α will be expressed in the early trimester placenta and decreased with the increasing gestational age. (Dong et al., 2012;Rath et al., 2016)However, if the hypoxia continues until the final trimester, it can cause pain and even death for both mother and fetus. (Rath et al., 2016)Hypoxic placenta releases several mediators into the maternal circulation, one of which plays an important role is HIF-1α, a transcription factor for oxygen-dependent genes, to express proteins expression of VEGF mRNA and also protein depends on the presence of oxygen and will be increased in hypoxic states.It is believed that a placenta with abnormality such as preeclampsia has hypoxia,leading to an increase in VEGF expression. However, it is still debatable whether this low VEGF level is a consequence of decreased production or precisely as a result of inhibition by inhibitor molecules, for example sFLT1 (antiangiogenic placenta) which actually increases in the circulation of the mother's preeclampsia. (Andraweera et al., 2012) Study by Abe et al.found that HIF-1α stabilization or its translocation into the nucleus is influenced by nitric oxide (NO), leading to an increased expression of VEGF and iNOS mRNAs.
Abe reported an increase in NO production in rat placenta during mid-pregnancy. Nitric oxide is a free radical in form of a water-soluble gas, which is a mediator of various cellular functions, including: smooth muscle relaxation, cell growth, and neurotransmission in the central nervous system. (Abe et al., 2013)NO regulates a number of cellular responses through cysteine Snitrosylation. S-nitrosylation process in Cys533 prevents the degradation process of HIF-1α resulting in the accumulation of these transcription factors. (Abe et al., 2013)Study by Kartini et al. showed that fetal blood vessels appeared wide in some premature placenta, both in the hypoxia and non-hypoxia groups. (Kartini et al., 2015)This widening of the fetal blood vessels may be affected by NO. This study concluded that the hypoxia that occureds in premature placenta causes an increase in the number of fetal blood vessels as compensation for impaired oxygen homeostasis. (Kartini et al., 2015)An increase in the number of fetal blood vessels may be due to the activation of VEGF triggered by hypoxic conditions. Further study is needed to analyze the possibility of an increase in levels of VEGF and NO in premature human placenta.
In this study we found that 14% of non-hypoxic placentas expressed HIF-1α moderate to strongly. Study by Kockar et al.stated that factors such as hormones and cytokines can induce the accumulation and activity of HIF-1α in normoxic conditions. (Kockar, 2012) The presence of HIF-1α in some non-hypoxic premature placenta is likely a part of the physiological process to achieve homeostasis due to stress on placental tissue.
Many studies analyze hypoxia in placenta preeclampsia, and in this study we also found the occurrence of hypoxia condition in premature placenta.Until now the incidence of preterm birth is unpredictable, so prevention depends only on the health conditions and behavior of mothers in maintaining pregnancy. However, this can not prevent preterm labor due to its spontaneous occurrence and association with the state of hypoxia in the placenta.Previous study found that the expression of HIF-1α at the gene and protein levels have increased in the premature placenta. (Dong et al., 2012)Thus, the results of our study are critical to initiatethedetectionof a molecular marker in preventing preterm birth, leading to the ultimate reduce of infant/child mortality and morbidity due to preterm birth complications. This study had limitations because no history of hypoxia has occurred in the placenta of premature infants due to the use of stored biological material.

CONCLUSIONS
In our study, hypoxic status is associated with intensity of hypoxia inducible factor (HIF)-1α expression in the premature placenta.Hypoxic circumstances affect HIF-1 levels, as a cellular response to low oxygen environments, and inducing gene transcription such as VEGF, NO, etc.A further study is required to analyze the expression of the HIF-1α gene and its hypoxia-related genes target, as a possibility of developing predictive marker in premature placenta, so the incidence of preterm birth can be reduce.

AUTHORS CONTRIBUTION
KK contributes to designing study methods, carrying out CPI processing, lab results analysis and statistical analysis; ME contributes to sample collection; ARP contributes to the supply of reagents for CPI; AAJ and SWJ contribute in designing study methods, lab results analysis and statistical analysis.

CONFLICTS OF INTEREST
There are no authors who declare a conflict of interest.

ACKNOWLEDGEMENT
Thank you to dr. Radiana D. Antarianto, M.Biomed, PhD, who helped analyze the expression of HIF-1α so that the examination was double blind. Thank you to the Research Council of the Faculty of Medicine, Universitas Trisakti, as well as those who have helped carry out this study.

CONCLUSION
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