A large percentage of women in the childbearing period do not have sufficient folic acid through a regular diet [21–24]. So folate supplementation during pregnancy is routine in the prevention of fetal congenital abnormalities [25,26]. High doses of folic acid (4–5 mg/day) are recommended especially for the women having very high risk factors like infertility, habitual pregnancy loss, or a history of neural tube defect in the previous offspring [26]. A low intake of folate is related to different pregnancy complications such as miscarriage, placental abruption, and growth-restricted fetuses [27–29]. Reduced folate intake affects the developing fetus through decreased cell division, increased inflammatory cytokine production, disruption of methylation reactions, programmed cell death, and increased levels of oxidative stress. Low levels of folate increase neural tube defects (NTDs) in fetuses, and such fetuses are likely to miscarry. The activity of natural folic acid is reduced due to reduced absorption and availability of less active folates for metabolic processes. The absorption of natural folic acid is disrupted by many factors, so the high consumption of synthetic folic acid is the only option and preferable solution [30]. It has also been observed that the degradation and leaching of folic acid occur in cooking, which leads to the absorption of only 50% of the cooked folate, or folic acid supplementation may not lead to active metabolic transformations and may lead to inactive forms of folic acid, which is called the folate trap. This is more evident when there is deficiency in Vitamin B12. The level of folic acid is greatly reduced in the current study, which may have a direct effect on the incidence of abortion and threatened abortion. A decrease in maternal vitamin D during pregnancy has a direct effect on a higher risk of a variety of disturbing obstetric outcomes, including preeclampsia, a higher risk of primary cesarean delivery, and gestational diabetes mellitus (GDM) [31-33]. Furthermore, vitamin D deficiency during pregnancy is associated with intrauterine growth retardation and a range of adverse fetal and neonatal outcomes such as increased preterm delivery [34], miscarriage [35], decreased birth weight [36], and neonatal hypocalcemia [37]. Also noted in the scientific literature is that inadequate vitamin D status is related to impaired placental function, augmented placental inflammation, and other dangerous conditions associated with pregnancy. Vitamin D also plays a role in the regulation of immune and inflammatory responses during pregnancy [38].

The progesterone variations are often utilized as indicators during the estimation of the course and stability of pregnancy. A critical decline in the concentrations of progesterone is also known to adversely influence the outcome during pregnancy. Scientific research has been able to uncover an extraordinary association between immunological activity at the reproductive tract level and maternal progesterone, particularly after fetal antigens. These antigenic responses are governed by the interaction at the receptor level at the progesterone-endometrial site. There is also clinical experience that shows the appropriate supplementation with progesterone during early gestation can significantly lower the risk of threatened miscarriage [42]. A decline in progesterone level was observed in the abortion and threatened abortion groups compared with healthy pregnant women. Several studies have proven a relationship between oxidative stress and a wide range of pregnancy complications that can negatively impact fetal growth. These complications are usually caused by a shortage of nutrients and oxygen supply to the fetus and the resultant hypoplasia and placental dysfunction [43]. The placenta plays a vital role in the transfer of nutrients and oxygen from the mother to the embryonic growth. Interference with such transfer can result in hypoxia that is predominantly caused by oxidative stress. Impaired functional performance of the placenta can be caused by various contributing factors that ultimately result in pregnancy complications [44]. Numerous studies have linked such complications with increased oxidative stress that is sourced from placental or maternal tissues [45]. Oxidative stress is also believed to affect major cellular and molecular mechanisms that are vital in the course of disease progression, including abnormal angiogenesis and inflammation responses [46]. Oxidative imbalance results from an overproduction of ROS with an impaired antioxidant protective machinery. Such dysregulation is linked with embryonic developmental defects as well as with unexplained recurrent pregnancy loss and spontaneous abortion [43,47].

Spontaneous abortion is when a mother loses her pregnancy before 20 weeks of gestation and is the cause of an estimated half of miscarriage cases. Others are typically linked with structural abnormalities at birth, uterine abnormalities, infection, maternal disorder, or remain unexplained [45]. Placental tissues from early pregnancy loss have increased MDA and lipid peroxide levels in comparison with normal pregnancy tissues. Earlier research postulated that an overaccumulation of reactive oxygen species (ROS) might catalyze the abnormal and early advancement of the maternal placental blood flow. Additional findings suggest that oxidative pressure generates damaging effects on trophoblastic cells that might cause early pregnancy loss. The subsequent oxidative imbalance is typically attributed to an insufficient antioxidant defense mechanism that fails to salvage free radicals. Irrespective of inconsistencies within existing literature bases, a potential relationship between elevated ROS concentrations and antioxidant activity loss might form the basis for the miscarriage pathophysiology. Defective placental attachment, which may have its genesis in syncytiotrophoblast dysfunction, is blamed in the case of unexplained recurrent pregnancy loss [45]. Oxidative stress can negatively influence pregnancy by reducing antioxidant defense mechanisms within the body [43,49]. According to research findings, higher antioxidant concentrations can counterbalance excess reactive oxygen species (ROS) in women with recurrent miscarriages. Previous research findings have cited higher concentrations of lipoperoxide and lower values of beta-carotene and vitamin E and A in such women and have talked about the role of oxidative stress in the pathophysiology of recurrent pregnancy loss. Cp is an acute-phase reactant, and its expression has been shown to increase under hypoxic conditions, when compared to healthy subjects [50-52]. There is an increase in placental ceruloplasmin following hypoxia, indicating that placental ceruloplasmin, a protein with antioxidant properties, expression is clearly upregulated in threatened abortion [53].

A limitation of the current study is that the detailed assay description for MDA measurement is not available in the present manuscript version.

This study showed that low levels of folic acid, vitamin D, and progesterone, combined with high oxidative stress and ceruloplasmin levels, were associated with threatened abortion. Therefore, regular monitoring and proper supplementation of folic acid and VD levels are essential for reducing pregnancy complications, and antioxidant therapy will help mitigate oxidative stress and improve pregnancy outcomes.