L-Arginine supplementation in pregnancy: a systematic review of maternal and fetal outcomes

Abstract Background/aim of the study L-Arginine (L-Arg)/Nitric Oxide (NO) system is involved in the pathophysiology of relevant Obstetric conditions. This review aims at summarizing the effects of L-Arg supplementation in pregnancy looking at safety and efficacy. Methods We conducted a systematic review of the literature utilizing PubMed for studies published from inception to September 2022. The search included human and animal studies where L-Arg was supplemented pre-conceptionally or during pregnancy, by either oral or intravenous route. The main perinatal outcomes were focused. Results Among 1028 publications, 51 studies were eligible for inclusion, 25 were performed in women, and the remnant in animals. Compared to controls/placebo, the supplementation with L-Arg reduced the development of pre-eclampsia (four studies), decreased blood pressure, and reduced the need for antihypertensive drugs in women with Hypertensive Disorders of Pregnancy (HDP, eight studies). In women carrying growth retarded fetuses, L-Arg improved fetoplacental circulation, birth weight and neonatal outcomes (five studies), while in the case of threatened preterm birth, L-Arg reduced uterine contractions (two studies). In several animal species, L-Arg supplementation in pregnancy improved reproductive performance by increasing the litter number and size. Moreover, in pre-eclamptic and metabolic syndrome experimental models, maternal hypertension and fetal growth were improved. Conclusion L-Arg displays biological activities in pregnancies complicated by HDP and growth restriction, both in women and animal models. L-Arg administration is safe and could be a candidate as an intervention beneficial to maternal and fetal outcomes, at least in moderate clinical disorders.


Introduction
Hypertensive disorders of pregnancy (HDP) remain one of the major causes of pregnancy-related maternal and fetal morbidity and mortality, worldwide. Affected women are also at increased risk for cardiovascular disease later in life, independently from traditional cardiovascular risks [1]. Pre-eclampsia (PE) is the most severe HDP and represents one of the main causes of maternal death [2]. Being the most important reason for iatrogenic prematurity, PE is a major contributor to perinatal mortality and fetal growth restriction [3].
Placental insufficiency (or uteroplacental vascular insufficiency) is another important issue in pregnancy that compromises fetal growth and increases the risks of low birth weight, IUGR, pre-term birth, and stillbirth [4][5][6][7].
A recent systematic review and meta-analysis by Goto [8], conducted on human studies, confirmed a pathophysiological role of L-Arg in placental function and vascular compliance, on which pregnancy outcomes may be dependent [5] and reported the favorable effects of prenatal oral l-arginine on birth outcomes.
L-Arg has the potential to improve birth outcomes in pre-and peri-conceptional strategies, being also beneficial for pregnant women, their families, health professionals and policy makers. However, despite arginine being a topic studied by several researchers, there are still many unexplored areas in which it may play a role, such as the metabolic profile during pregnancy.
This systematic review aims to summarize the results of main human and animal studies supplementing L-Arginine either orally or intravenously during pregnancy in terms of pregnancy and perinatal outcomes, to finally underline the future research prospective of this semi-essential amino acid.

Literature search and data extraction
This systematic review followed the PRISMA guidelines [9].
A comprehensive literature search was conducted in PubMed (www.ncbi.nlm.nih.gov) for studies published from inception to September 2022, using as keywords: L-Arginine OR Arginine OR Nitric Oxide donor OR Arginine supplementation AND preeclampsia OR high-risk pregnancy OR fetal growth restriction OR hypertension OR hypertension in pregnancy OR perinatal outcomes). In addition to database searches, we performed a full-text review of studies included in meta-analyses investigating the effects of L-Arg supplementation in high-risk pregnancies on perinatal outcomes, selecting 4 more trials from two metanalysis [8,10].
This systematic review included both human and animal studies. We restricted our search to studies published in English. Review articles were excluded after a search of the reference lists.

Study selection
After the primary records were retrieved from PubMed, duplicates were removed. The remaining records' titles and abstracts were screened, and irrelevant studies were excluded. Full texts of studies deemed relevant were obtained and reviewed in detail for eligibility according to the inclusion criteria. Reviews, Letters to the Editor, meeting pr ecis and other articles reporting studies that did not provide the primary data were excluded.
Publications were included in the final analysis if they met the following inclusion criteria: studies evaluating supplementation of L-Arg during pregnancy or it the pre-gestational and postnatal period. Studies with unclear treatment details or with a combination of several amino acids/supplements were excluded. At least one of the following outcomes was used as a primary or secondary endpoint: maternal hypertension, pre-eclampsia, fetal growth restriction/retardation, feto/placental hemodynamic, birthweight, perinatal outcomes. In case of human treatment, only randomized controlled trials and prospective or retrospective cohort studies were considered.
Studies were excluded according to the following criteria: narrative reviews, systematic reviews and meta-analyses, studies evaluating pathophysiology rather than clinical outcomes, studies published in other languages than English.

Analysis
The outcomes evaluated included maternal hypertensive disorders, pre-eclampsia, fetal growth restriction/retardation, and perinatal outcomes. We defined maternal hypertensive disorders as blood pressure values above 140/85 mmHg, pre-eclampsia as elevated blood pressure associated with proteinuria and/or the presence of kidney or liver function alterations, neurological signs, hemolysis or thrombocytopenia and/or fetal underdevelopment [11].
We also explored the association between maternal blood pressure levels during pregnancy and offspring outcomes whenever it was possible.

Flow chart
The literature search identified 1028 articles, of which 21 duplicate records were removed. The remaining 1007 records were screened and 75 of them matched the inclusion criteria. These studies were then assessed for eligibility: five narrative reviews, 10 systematic reviews and meta-analyses, two studies with unclear treatment strategies, three studies evaluating the pathophysiologic basis of disease, and four studies with unavailable full text were excluded. Thus, our review was restricted to 51 studies ( Figure 1).

Human studies
Doses and timing of administration Among the 25 human studies, L-Arg was administered orally in 16, intravenously in eight, and a mixed treatment was done in the remnant as reported in Table 1. Oral treatment doses ranged from 1 g/day [13] to 16 g/day [14,15], lasting from 8 to 10 days [15] to the entire duration of pregnancy [16]. Intravenous administrations were mainly utilized in the acute treatment of hypertension, PE or severe IUGR, doses ranging 15 g (in 500 ml of 5% glucose) [17] to 30 g (in 100 ml of saline) [18]. Treatments usually lasted one day, except for the study by Xiao et al. [19] which administered L-Arg for 7 days to women carrying IUGR fetuses.
L-Arginine and assisted reproductive technology L-Arg was administered in three studies to women undergoing assisted reproductive technology (ART) [14,15,20]; medications were taken orally in addition to gonadotrophin-releasing hormone analogue (GnRHa) and pure follicle stimulating hormone (pFSH) [14,15], or in addition to folate and vitamin E [20]. L-Arg led to less cycle cancelation, more oocytes and transferred embryos, increased plasma and follicular fluid nitrite/nitrate, as well as to Doppler flow improvement in one study [15]. A detrimental role of L-Arg was reported on embryo quality and pregnancy rate during controlled ovarian hyperstimulation cycles, due to the inverse correlation between follicular fluid concentration of nitrite/nitrate and embryo quality [14]. On the contrary, So [20] reported that, especially in cases of male infertility, both biochemical and clinical pregnancy rate were significantly increased by L-Arg (Table 2).
L-Arginine supplementation and fetal growth restriction L-Arg was administered in seven studies to women carrying IUGR fetuses: in three of them the administration occurred intravenously while in the remnant 4 it was oral. Overall, the studies showed that L-Arg infusion affects utero-placental circulation by lowering the uterine artery pulsatility index [18], improving birthweight [19,21,22], reducing placental apoptosis, and improved placental function and fetal development [17]. L-Arg also reduced the incidence of abnormal umbilical artery blood flow and uterine artery earlydiastolic notching [23], being not effective for severe vascular IUGR in possibly due to the severity of the   Insulin signaling and endothelial function In overweight/obese women, no responses to L-Arg were found in the 1st or 2nd trimesters. In the 1st trimester, insulin levels were significantly reduced in both groups after L-Arg infusion. Insulin levels during weeks 24-27 were suppressed only in normal-weight women after L-Arg infusion growth retardation (3rd percentile) and to the early course of gestation (28 weeks) [24] (Table 2).

L-Arginine and hypertensive disorders of pregnancy
In 11 studies L-Arg was used to treat women with hypertensive disorders of pregnancy (HDP), chronic hypertension (CH), or preeclampsia (PE). Three studies administered L-Arg intravenously while seven orally and one either orally or intravenously when oral medication could not be taken [25]. Five studies included women diagnosed with PE [25][26][27][28][29], and treatments lasted from 2 days (acute treatment) [27] to 3 weeks [29]. L-Arg supplementation lowered systolic blood pressure (SBP), diastolic blood pressure (DPB), mean arterial pressure (MAP) and increased 24-h urinary excretion of NOx as well as plasma levels of L-citrulline [26]. Moreover, L-Arg markedly improved fetal growth [29] fetal well-being, neonatal outcome and prolonged pregnancy [28]. L-Arg also significantly improved blood pressure and kidney function the 10th day postpartum without hasten the recovery of women with PE [25]. An acute treatment of PE patients with gestational length varying from 28 to 36 weeks (4 g of L-Arg for 2 days orally) with L-Arg supplementation did not reduce mean diastolic blood pressure compared with placebo (Staff AC).
Three studies were conducted on women with HDP [30][31][32] and L-Arg was administered intravenously (20 g/day, for a maximum of 5 days). L-Arg infusion showed an acute hypotensive effect both on systolic and diastolic values [30,31], without affecting fetal movements, while prolonging pregnancy [31].
The remnant studies included women at high risk for PE [33,34] or suffering CH [16,35]. Oral prophylactic treatments started in the first trimester and lasted from 10 weeks to the entire duration of pregnancy (more than 30 weeks). Although L-Arg did not change BP a lower percentage of women required antihypertensive drugs [35] and improved uterine artery impedance [16]. PE was lesser developed and the incidence of superimposed PE indicating early delivery <34 weeks show a trend to be reduced [33][34][35]. L-Arg treatment was also associated with higher birthweight and less preterm births [34].
Two studies were conducted in women with threatened preterm birth (PTB), one using 3 g/day of orally since admission until delivery [28], while the other acutely infusing L-20 g/500 ml in 3 h of L-Arg [36]. Oral supplementation increased feto-placental blood flow while i.v. infusion reduced uterine contractions, increasing both serum growth hormone and nitrates levels.
Transgenerational/metabolic effects of L-Arg A single study evaluated L-Arg/NO system and its role in insulin signaling and endothelial function in pregnant women of different BMI categories [37]. NO availability was found impaired in overweight/obese women, and this deranged endothelial function and insulin regulation. L-Arg reduced insulin level in the first trimester, only in normal-weight in the second trimester.

Animal studies
Doses and timing of administration In 23 out of 26 animal studies, L-Arg was administered orally, either dissolved in drinking water or introduced with the diet, whereas in the remnant 3 studies it was administered intravenously [38][39][40] (Table 3).
Studies focused on the effects of L-Arg on reproductive performances (five studies), fetal growth (16 studies), hyperinsulinemia and/or hypertensive disorders (four studies) while immune response was outcome of the remnant study.
L-Arginine supplementation and fetal growth Supplementation (mainly via drinking water or through diet) was done in undernourished sheep (six studies), pigs (eight studies), rats (two studies).
Generally, L-Arg increased birth weight and muscle weight as well as maturation [39,45,46] possibly through altered mTOR protein abundance [39]. No effect on the stillbirth rates was found in sows [47], while L-Arg supplementation enhanced fetal survival [48,49] in swine. In sheep L-Arg (and NCG) supplementation decreased IUGR by improving metabolic homeostasis and through the expression of fetal somatotropic axis genes [50,51]. Zhang et al. 2016 [52] confirmed the promotion of fetal-placental   Piglet birth weight and preweaning performance Late pregnancy supplementation with L-Arg had no effect on number of pigs born alive, piglet birth weight, or lactation performance Madsen et al.

2017
Growth performance, carcass composition, organ, and Semitendinosus muscle (STM) development ARG and CAR supplements were beneficial for muscle maturation Zhang et al. 2016 Maternal endocrine status; maternal, fetal, and placental antioxidation capability; and placental development Dietary supplementation of NCG and Arg influence maternal endocrine status, improve the maternal-fetalplacental antioxidation capability, and promote fetal and placental development Peine et al.

2018
Postnatal growth and development Arg supplementation during the last two-thirds of gestation can mitigate offspring, but not maternal negative consequences associated with restricted maternal nutrition Helmbrecht et al. 1996 PE, IURG, and renal glomerular capillary endothelial lesions L-Arg significantly lowered systolic blood pressure at late pregnancy, increased birthweight, decreased the degree of proteinuria and decreased the proportion of injured glomeruli. Bursztyn et al. 2003 Endothelial NO synthase in kidney L-Arg treatment significantly reduced blood pressure, serum creatinine, and excretion of NO metabolites. Expression of endothelial NO synthase in kidneys was doubled Sharkey et al.

2001
Hypertension and feto/placenta weight L-Arg prevented the elevation of Blood Pressure, particularly during the third trimester; avoid placenta overweight without effect of fetal growth Altun et al.

2008
Effect of oral L-Arg treatment on stressinduced hypertensive rats L-Arg supplementation decreased hypertension, proteinuria and ADMA levels Podjarny et al. 2001 Fetal growth and blood pressure L-Arg reversed insulin-induced hypertension and improved fetal weight not affecting plasma insulin/glucose De Costa et al.

2014
Parasitemia, corticosterone levels, NO production, fetal morphological measurements, heart, and placenta histology L-Arg supplementation decreased corticosterone and parasitemia, enhanced nitrite concentrations, fetal weight, length and placental weight. Cardiac tissue showed reduced amastigote burdens in the L-Arg group. The placental parasitism was similar between groups development through the improvement of antioxidation capability. Placental growth and vascularity were enhanced by L-Arg [53] also in pigs, although Li et al. [54] reported a decreased litter size in gilts.
Once supplemented late in pregnancy, L-Arg had no effect on piglet birth weight or lactation performance [55], being unable to mitigate consequences of restricted maternal nutrition [56] (Table 4).

L-Arg supplementation and hyperinsulinemia/hypertensive disorders
Three studies were conducted on murine models of chronic exogenous hyperinsulinemia, leading to hypertension and heart failure [57][58][59]. Another study used L-NAME in rats creating a model of preeclampsia [60].
L-Arg reversed the endothelial lesion due to the L-NAME exposure, and lowered blood pressure in late pregnancy [60], decreased the degree of proteinuria and the proportion of injured [58,60,61].
The supplementation with L-Arg increased birthweight, without any changes in the levels of plasma insulin or serum glucose [59,60], but stimulating NO system in the placenta [58] (Table 4).
These data, obtained in different experiment models of insulin-induced rat hypertension, suggest a direct reversal effect of L-Arg administration on hypertension and fetal weight changes, activating NO systems in placenta and kidney.

Immune response and L-Arginine
In Trypanosoma cruzi infected pregnant Wistar rats, which allowed Chagas disease development [62], L-Arg decreased the levels of corticosterone and parasitemia and increased fetal and placental weigh, and reduced amastigote burdens. L-Arg supplementation might improve the host immune response during the acute phase (Table 4).

Safety and tolerability of dietary supplementation of L-Arginine
None of the human studies reported adverse events associated with the supplementation with L-Arg during pregnancy, neither for a long period of administration (3 months) [20] nor by using high doses as for the acute treatments (20 or 30 g/100 ml of saline for 1 day or 1 week) [18,19]. Among animal studies, only one study conducted on sows reported an increase in stillbirth rates, in the group receiving the combination of Arg and Ractopamine (Rac) from day 25 to 53 of gestation [47] (Table 4). However, it is unknown to which supplement such effect could be ascribed.

Discussion
This systematic review of the literature found human and animal studies over a large period of time, witnessing the still actual interest toward L-Arg administration for reproductive purposes.
Overall, the results demonstrated that L-Arg supplementation during pregnancy could be beneficial in several circumstances, especially on maternal hypertension and fetal growth, by reducing blood pressure levels, the onset of preeclampsia, and improving vascularity as well as placental function. However, it is worth emphasizing that many of the RCTs included were conducted more than 15 years ago and had weak power and a heterogeneous population. Also, no data are available on severest outcomes (i.e. stillbirth, placental abruption or severe IUGR), unlike other studies with NO donors [63,64] which however failed in demonstrating efficacy on the development of FGR, preterm delivery, and perinatal mortality and morbidity.
While pharmacological NO donors have been associated with poor efficacy (Cochrane), L-Arg which is the physiological substrate of endothelial NO synthase seems to show a better risk/benefit profile. Possibly, the production of peroxynitrites as a result of an excess NO bioavailability does not occur when administering the amino acid [65].
However, short-term supplementation of L-Arg, especially late in pregnancy, resulted insufficient to improve maternal hemodynamics and did not mitigate the effects of severe IUGRs [25]. This suggests that L-Arg should be initiated early and continued over the course of the entire pregnancy in order to positively affect blood pressure or placental vascular insufficiency, via the arginine-NO pathway [66].
These findings confirm what has been reported in a recent meta-analysis by Goto [8] (based upon solely 10 eligible articles) which concluded that the supplementation with L-Arg should be recommended in women with a history of poor pregnancy outcomes, both in those at high-risk of pre-eclampsia, as well as in those with already established HDP. However, we agree with the authors stating that more trials are required to provide stronger conclusions, since the small study effects.
The partial efficacy of L-Arg may be related to the splanchnic extraction and metabolism of arginine which often precluded its efficacy with possible degradation by arginase. Indeed, animal studies supplementing citrulline (direct NO donor without splanchnic degradation), reported an enhanced placental function and fetal growth in rat models of IUGR through the involvement of insulin-like growth factor 2 and angiogenic factors [67] and improved perinatal and postpartum maternal vascular function in a mouse model of preeclampsia [68]. Citrulline effectively raised fetal arginine availability, although it failed to increase the concentrations of essential amino acids in fetal plasma of murine models of IUGR [69]. Unfortunately, no human trials are available about Citrulline and/or combined supplementation.
Another systematic review [70] analyzed the role of Arginine synthesis and metabolism in pregnancy and provided evidence for the link between an impaired arginine metabolic pathway and the pathogenesis of compromised pregnancy and fetal programming. Interestingly, the Authors presented L-Arg supplementation as a potential reprogramming strategy during pregnancy, in order to prevent non-communicable diseases (NCDs) in the offspring. Many of the evidence supporting such an idea stay on the capacity of improving fetal growth, also in cases where placenta function is compromised. Accordingly, it has to be remembered that the Barker's observation correlating low than normal weight at birth with the later adult development of cardiovascular diseases was the milestone of epigenetic hypotheses [71].
The fetal somatotrophic stimulation by L-Arg is evident also in several animal species through the exploitation of the amino acid as a booster of NO availability in the placenta vasculature. In addition, we understand from rats that L-Arg display its effects also in cases where hypertension and growth restriction are sustained by chronic hyperinsulinemia allowing insulin resistance [57][58][59]. Indeed, besides cardiovascular benefits, serum glucose and free fatty acids concentration was reduced in overweight/obese females in fertile period [72] and type 2 diabetes [73]. Although reported only in a single study, the ability of L-Arg to activate endothelium-dependent vasodilation in obese pregnant women reducing circulating insulin levels suggests the possible employment of supplementation in such a condition, well characterized as being insulin-resistant [37]. Interestingly, among possible target of L-Arg administration there are also women with a reduced pre-pregnancy BMI. They showed low circulating Arginine levels which seem related to poor pregnancy outcome [79].
Finally, in ovine, swine and equines, L-Arg supplementation also improved the reproductive performances becoming one among the interventions able to increase animal production.
Among the studies included, in this review none reported serious adverse reactions to L-Arg supplementation thus confirming a previously reported safety profile in pregnancy [72]. Severe adverse events were reported only in a population of patients with a recent coronary heart disease over a long-term treatment (6 months) at high dose (9 g/day) [74]. Moreover, in a controlled study, L-Arg has been found safe in subjects ingesting 15-30 g/day for 90 days, with no impact on the intake of energy, protein, carbohydrates, vitamins, or minerals. However, it is worth mentioning that NO donor supplementation in pregnancy is still debated, as not all NO donors are considered safe in pregnancy. Sildenafil, for example, when administered for severe early-onset fetal growth retardation, not only did not reduce the risk of perinatal mortality or severe neonatal morbidity, but actually increased the risk of neonatal pulmonary hypertension [75].
Arginine is a non-essential amino acid whose intake from the diet (meats, dairy products, nuts, … ) has been estimated to be >4 g/day in western countries [76] while it seems much less available in people living in low resource settings [77]. For a lot of micronutrients, such as iron, iodine, calcium, Vitamin D, and so on, pregnancy represents a status of relative deficiency [78]. Thus, although we lack standard of Arg intake in pregnancy, it seems not unlikely that during gestation women require more intake due to both major needs (fetal growth) and to cover changes in eating behavior (i.e. less intake of meat).
Furthermore, L-Arg administered intravenously was associated with important fetal and maternal vascular effects [18,32]. However, the effects were not longlasting, and the clinical feasibility was poor allowing oral Arginine be preferred in clinical trials.
However, the evidence highlighted in this systematic review indicate that L-Arg displays biological activities supporting its potential as a "therapeutic agent." Moreover, neither human, nor animal ingestion of L-Arg have been associated to side effects and/or adverse reactions, at the given doses.
The "novelty" of this systematic review is that it examined not only the vascular but also the metabolic effect of arginine, including both animal and human studies. In particular, the L-NAME model was evaluated to target the well-consolidated, NO-mediated L-Arg action. The hyperinsulinemia animal model, on the other hand, allows for the first time to transfer the possible metabolic effects to pregnancy, which have not yet been well studied, contrary to non-pregnant conditions where various evidence of the metabolic impact of arginine is already available both in experimental [80,81] and human studies [81][82][83].
Nitric oxide (NO) is a key regulator of both maternal and fetal homeostasis during pregnancy nevertheless strategies involving supplementation with NO precursors, NO donors, natural derivatives or pharmacological modulators of the NO system need to be more evaluated and randomized trials are yet warranted. This review suggests that the supplementation of a certain kind of L-Arg (i.e. in intravenous form or in oral vial as salt free form at a dose of 3 g/day) may not simply be used as a replacement for a transitory deficiency. Instead, through its cardiovascular and metabolic effects, L-Arg could be candidate as an intervention beneficial to maternal and fetal outcomes, at least in moderate clinical disorders.

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