*4.2. Homocysteine in PE*

The association of hyperhomocysteinemia and preeclampsia has initially been suggested by Decker et al. [131], and all authors have not confirmed it. However, the majority of evidence suggests a positive correlation.

Higher maternal plasma homocysteine concentrations in preeclamptic pregnancies as compared to normotensive were widely reported [93,125,130,132–137]. Overall, these di fferences seem to be present at all of the investigated time points across the gestation, starting from early pregnancy before 20 weeks [138–142] and in both severe and non-severe forms of PE. Comprehensively, this finding led to a conclusion that high homocysteine in early pregnancy constitutes a risk factor for PE. Therefore, attempts to introduce Hcy measurement into a screening test for PE to improve the prediction model, for example, by combining it with uterine artery Doppler test in the second trimester, resulted in being valuable [140]. However, there are also studies that rejected an association between elevated Hcy in early second trimester and subsequent PE [143–145]. This could result from di fferences in study designs, laboratory techniques, and disease definitions, among other reasons, which may be further investigated in a systematic review.

Still, most of the papers focus on the third trimester [93,130,132–135,137], where the di fferences between HHcys in severe and non-severe PE are marked. Pregnant women complicated with severe preeclampsia displayed significantly higher serum Hcy levels than with non-severe form [93,112,132,134,136,146]. Thus, homocysteine concentrations positively correlating with the clinical presentation of disease may constitute a marker of severity of preeclampsia.

Besides elevated Hcy concentrations in maternal plasma, the majority of authors analyse the possible association with either folate or vitamin B12 deficiencies, as well as NO pathways in preeclamptic patients. Here, contrary results are presented. Folate and vitamin B12 serum levels were described as unchanged [132,134,135] in PE when comparing to normotensive healthy women with uncomplicated pregnancies. However, opposite results indicating these vitamins deficiencies were also reported [135,146]. Nevertheless, further studies are needed to confirm whether the prescription of these vitamins could decrease serum homocysteine, thereby possibly reducing the risk of preeclampsia or (if it occurs) its severity.

Interesting association between HHcy and NO signalling pathway was reported. Homocysteine inhibits the expression and activity of dimethylamino dimethyl hydrolase (DDAH), which is the enzyme degrading ADMA to citrulline and dimethylamine [22,147–149]. It has been suggested that elevated ADMA is a mediator of endothelial dysfunction in hyperhomocysteinemia due to this metabolic relation [148,150]. HHcy, leading to accumulation of ADMA, may contribute to eNOS blockade and NO deficiency in the presence of general appropriate concentration of its synthase. The reduced release of NO by endothelial cells in HHcy was observed, suggesting the impairment of the eNOS pathway by DDAH inhibition [29].

The hypothesized mechanisms of disease linking homocysteine to preeclampsia are complex and still incompletely understood.

To date, vascular endothelial cell dysfunction that is provoked by an elevated level of homocysteine (Hcy) is suggested to be the most important connection. However, some authors questioned whether mild HHcy observed in PE, with Hcy values that are similar to those found in normotensive non-pregnan<sup>t</sup> women, can provoke damage of the vascular endothelium. They postulate that this damage can be mediated rather by oxidative stress, as endothelium of pregnan<sup>t</sup> women might be more vulnerable to oxidative injury [151]. Hyperhomocysteinemia is also associated with lesions in endothelial cells, due to vascular fibrosis, which results in alterations in coagulation system, enhanced platelet activation, and thrombogenesis—changes that are noted in preeclampsia [28–30].

On the other hand, metabolism in the kidney is the major route by which homocysteine is cleared from plasma. The association between Hcy and glomerular filtration rate (GFR) seems linear and it is present, even in the hyperfiltrating range [152,153]. Thus, this route of elimination may be affected by the already established preeclamptic changes in the kidney and secondarily lead to increased Hcy concentrations in plasma [154].

#### **5. Therapeutic Potential of NO Pathway during Pregnancy**

Enhancing the NO signalling in pregnancy is still thought to be an attractive option in both preeclampsia prevention in high-risk groups of women and treatment. There are attempts to prolong the pregnancy with the use of NOS substrates (L-arginine and L-citrulline), as well as NO-donors (glyceryl trinitrate, S-nitrosoglutathione, isosorbide mononitrate), natural derivatives, or vasodilators, such as sildenafil citrate [155–158]. NO donors and substrates have been also used for the managemen<sup>t</sup> of other pregnancy disorders, like recurrent abortions, treatment of preterm labour, and dysmenorrhea [155]. Unfortunately, the analysis of use of these substances in PE gives conflicting results.

In vitro, nitric oxide synthase activity is inhibited by intracellular ADMA and is rescued by L-arginine [159]. Therefore, L-arginine supplementation in pregnancy could possibly overcome NOS blockage and its consequences. A study of intravenous infusion of l-arginine in pregnan<sup>t</sup> women showed a significant reduction in blood pressure—an effect that was greater in women with preeclampsia [157,160]. Additionally, a significant reduction in PE incidence was described in high risk women who received L-arginine and vitamins C and E (as antioxidants reducing oxidative stress implicated in the pathophysiology of PE) before 24 weeks of gestation, when comparing to placebo and only vitamin group, although the effects of L-arginine alone were not studied [161]. However, last year, meta-analysis showed that combined vitamin C and E supplementation has no influences on the occurrence of preeclampsia [162], although an interaction between these vitamins and L-arginine is not well explained. Supplementation of L-arginine for pregnan<sup>t</sup> women with chronic hypertension showed less need for antihypertensive drugs use, but no reduction in the incidence of superimposed preeclampsia [163]. Interestingly, women who go on to develop PE have been found to have higher, not lower, plasma L-arginine concentrations [129], which can counteract the raised ADMA values, but other vasoconstrictor effects may persist in women who are vulnerable to preeclampsia.

The Cochrane database systematic review, including six trials demonstrates, that there is insufficient evidence to draw reliable conclusions about whether NO donors and precursors prevent PE or its complications. Another 13 papers are still waiting to be assessed [164]. Conclusions from the review are limited mainly due to the fact that too few women have been studied. Adverse effects that were described during supplementation of NO donors included mainly headaches, often sufficiently severe to stop medication. However, more recent studies clearly show that isosorbid mononitrate and L-arginine are both effective in prevention of PE [165,166]. Besides the significant reduction of PE incidence in the treatment groups, there was also a reduction in FGR and improvement in foetal outcome, including less neonatal admissions to the intensive care unit.

An inorganic nitrate, NO3, with a capacity to increase the bioavailability of NO has been recently implemented in a clinical trial with beetroot juice as a norganic nitrate (NO3−) rich dietary supplement [167]. It was the first trial to investigate e ffects of nitrate supplementation on blood pressure in human pregnancy, according to the authors. The treatment group consisted of pregnan<sup>t</sup> women with chronic hypertension. In this group, the administration of NO3− donors significantly increased plasma and salivary nitrate/nitrite when compared with placebo, but there was no overall reduction in blood pressure. However, there was a highly significant correlation between changes in plasma nitrite and only lowering diastolic blood pressure in the nitrate-treated arm [168]

Sildenafil exerts its role by inducing vasodilatation, via inhibiting phosphodiesterase and thereby maintaining the availability of cGMP, the e ffector of NO activity within the cell [169]. In vivo studies of sildenafil citrate in rat models of preeclampsia have shown a significant reduction in the production of antiangiogenic molecules sFlt-1 and sEng [170], as well as an improvement in blood pressure, proteinuria, uteroplacental and foetal perfusion after treatment [171]. Lately, a randomized controlled trial to evaluate PE therapy with sildenafil showed that, when compared to controls (receiving a placebo), therapy with sildenafil resulted in four days prolongation of pregnancy [158].

Glyceryl trinitrate (GTN), which is commonly known as nitroglycerin, is a widely used organic nitrate in clinical practice, particularly for the treatment of angina pectoris. Transdermal nitroglycerin patches have been the focus of various studies, for both the prevention and managemen<sup>t</sup> of PE and related disorders. Studies of both forms: transdermal [172,173] and sublingual GTN [156] in preeclamptic patients consistently showed a significant reduction in blood pressure and resistance in the uterine artery without an adverse e ffect on the foetal Doppler parameters. Nonetheless, these studies have highlighted the potential use of GTN as an antihypertensive agen<sup>t</sup> in PE. However, it remains to be established whether GTN o ffers any competitive advantage over already existing treatment options. Certainly, the major disadvantage of organic nitrates, in general, and GTN, in particular, is the development of tolerance upon continuous dosing, necessitating the requirement of regular 'nitrate-free' intervals.
