Introduction
Pathogenesis of preeclampsia (PE) is not fully explained. It is believed to be initiated by the placental ischemia. In PE, the invasion of the trophoblast is aberrant; the spiral arteries remodeling is impaired. Therefore they are narrowed and resistive. Placental hypoperfusion generates oxidative as well as endoplasmic reticulum stress, and the release of antiangiogenic factors into the circulation (soluble fms-like tyrosine kinase—sFlt-1, and soluble endoglin—sEng) [
1,
2]. The imbalance between pro and antiangiogenic factors leads to endothelial dysfunction [
1,
3]. Oxidative stress also induces the release of proinflammatory cytokines and chemokines [
2]. The systemic endothelial dysfunction, occurring in PE, is manifested in glomerular endotheliosis and proteinuria, hypertension, as well as coagulation abnormalities [
3].
A healthy pregnancy is a state of hyperactivity of HPA axis and of hypercortisolism [
4,
5]. Elevated cortisol (F) levels can be partially attributed to the estrogen-stimulated increase of corticosteroid-binding globulin (CBG). In normal pregnancy CBG levels rise with advancing gestation, with a corresponding increase of total plasma F, with a decline from 36 weeks onwards; this decline results in the rise of free F in maternal plasma [
5]. On the other hand, the placenta is an important source of CRH, which further stimulates the release of maternal ACTH. It leads to the elevation of F levels in the course of pregnancy [
4,
5]. F influences the hypothalamic CRH in a negative feedback loop, while the placental CRH is strongly stimulated by F in a mechanism of positive feedback loop [
5]. During normal labor, maternal CRH, ACTH and F levels increase in maternal plasma and then drop at 4 days postpartum [
4].
Lately, more and more is discussed the improper metabolism of glucocorticoids (GCs) in pregnancies with adverse outcomes [
6,
7]. F is metabolized mainly by the 11β-hydroxysteroid dehydrogenase (11β-HSD), of which two isoforms (type 1 and type 2) have been described. 11β-HSD2 inactivates F to cortisone (E) and is localized mainly in the kidneys and the placenta, while 11β-HSD1 regenerates E to F in the liver and the fat tissue. Both F and E are then catabolized by 5α- and 5β-reductase in the liver and converted into tetrahydro- and allo-tetrahydrometabolites [
8,
9].The disturbed activity of placental 11β-HSD2 in the course of PE was emphasized in last years [
6]. It has not been yet discovered whether the abnormal function of 11β-HSD2 occurs in PE only locally (in the placenta) or it affects the whole body of a pregnant patient. Most research groups focus solely on the placental 11β-HSD2 as the enzyme localized in the tissue, which directly influences the proper development of the fetus [
7]. Few available studies concerning the function of non-placental 11β-HSD2 in GH and PE are still inconclusive [
10‐
12].
There are limited data reporting levels of F and E metabolites in pregnant women [
13‐
15], some of which refers to spot urine only [
14]. Others include women in the first weeks of pregnancy [
15] or—like the study from 1980—concern only the tetrahydrometabolites of F and E assessed in samples from 6 patients [
16]. To our best knowledge, the only analysis of the function of 11β-HSD1 in GH and PE was published more than 20 years ago and comprised small study population [
10]. However, the recent data showed that some polymorphisms in
HSD11B1 gene, encoding 11β-HSD1, are linked to higher risk of developing PE [
17]. Moreover, some authors revealed the increased levels of 11β-HSD1 in decidua of PE women [
18]. These facts show the need for further research in the field of 11β-HSD function in pregnancy.
The aim of this study was to evaluate the overall maternal GC balance in pregnancy-related hypertension (both GH and PE) and to verify if the functions of primary enzymes involved in F metabolism: 11β-HSD1, 11β-HSD2, 5α and 5β-reductase are altered in hypertensive pregnancy. Plasma and urinary F and E, as well as their metabolites in urine, were determined and served as a basis for further indirect assessment of enzymes functions. The research question was whether the F conversion is impaired in the body of women suffering from GH and/or PE.
Discussion
The disturbed GC balance was reported in many medical conditions, inter alia the polycystic ovary syndrome [
32,
33], metabolic syndrome, insulin resistance [
25], cholestasis [
34] and mental disorders [
27]. They are usually associated with the abnormal activity of 11β-HSD, 5α and/or 5β-reductase. Here we present that the disturbed metabolism of F is also manifested in women suffering from pregnancy-related hypertension, including both PE and GH.
Our preliminary study [
12] surprisingly revealed that, despite the literature data proving the diminished expression and function of placental 11β-HSD2 in PE [
35‐
38], the apparent activity of renal 11β-HSD2 is significantly increased in that condition. This finding was in contradiction to other results. Heilmann et al. [
11] reported higher UFF/UFE ratio in the group comprising GH and PE patients indicating decreased function of 11β-HSD2. Walker et al. [
10] presented no differences in UFF/UFE ratio between hypertensive pregnant women and controls. Our present study, conducted on a larger population and extended by the analysis of urinary metabolites of F and E, entirely confirms our previous observations. Patients with PE presented significantly lower values of UFF/UFE than controls, and this difference remained significant after adjustment for confounders (Table
3). One need to emphasize that this paper presents the results obtained using HPLC-MS/MS method [
23] instead of HPLC-FLD [
12], and the mass spectrometry is known as the most specific and accurate way of detection. However, independently of the method applied, we observed the same dependencies in urinary F and E. We suggest that the diminished function of placental 11β-HSD2 in PE, reported by others, might be a kind of compensatory mechanism limiting maternal F levels that reach the fetus. The reduced UFF/UFE ratio could be attributed to the substantial proteinuria in PE patients as some authors reported increased function of renal 11β-HSD2 in non-pregnant proteinuric patients [
39]. However, significantly lower UFF/UFE ratio also in usually non-proteinuric GH women suggests that other mechanisms should be considered to explain the enhanced 11β-HSD2 function in pregnancy-related hypertension. At the same time, CH group presented similar UFF/UFE ratios as controls, what confirms our previous conclusions [
12] that the enhanced function of renal 11β-HSD2 concerns only women with pregnancy-related hypertension (PE and GH).
We found that PE is associated with higher THFs
tot/THEs
tot ratio (Table
2) what might indicate the increased function of 11β-HSD1 in that condition. On the one hand, the THFs
tot/THE
tot is a non-specific parameter reflecting more precisely the overall balance between both 11β-HSD isoforms [
9,
13,
39] than the actual function of 11β-HSD1. It is widely used in the clinical practice in the diagnosis of apparent mineralocorticoid excess, as lack or diminished function of 11β-HSD2 implies much higher UFF/UFE ratio and also significantly increased THFs/THE [
24,
40]. The literature data, however, show that in case of the proper function of 11β-HSD2 (reflected in normal value of UFF/UFE), the THFs/THE ratio describes quite acceptably the function of 11β-HSD1 [
8,
28]. Our PE patients obtained not lower (as it could be supposed based on the lower UFF/UFE values) but higher THFs
tot/THEs
tot ratio as compared to controls (0.346 vs. 0.280). When analyzing not total but free metabolites of F and E (THF
free/THE
free ratio), one will get similar conclusions to those derived from UFF/UFE, as PE was associated with lower THF
free/THE
free (R = −0.214;
p = 0.034). Considering UFF/UFE, THF
free/THE
free and THFs
tot/THEs
tot we conclude that they reflect rather the augmented function of 11β-HSD2 than 11β-HSD1. The observed enhanced conjugation of F and THF in PE may further indicate the pursuit of woman’s body to the efficient removal of active GC and its major metabolite.
All mentioned observations may lead to the conclusion that the F metabolism is significantly intensified in PE. Firstly, the similar THFs
tot + THEs
tot indicates the comparable GC secretion (the equivalent activity of adrenal glands) in normotensive and preeclamptic pregnancy (380.1 vs. 394.2 µg/mmol Cr, respectively). However, plasma F concentration is markedly lower in PE (663 vs. 782 nmol/L), partially because of earlier GA at sampling, but also because of PE itself (Supplementary Table 3). Lower plasma F in PE suggests stronger GC metabolism in this group. The assumption of the enhanced F clearance could be strengthened by lower UFF/UFE in PE (increased 11β-HSD2 function) and significantly higher THF
tot/F
tot and alloTHF
tot/F
tot (increased functions of 5β- and 5α-reductase, respectively) (Table
3). Lastly, the parameter indicating the metabolic clearance of F: (THFs
tot + THEs
tot)/UFF is almost two times higher in PE when compared to controls (141.59 vs. 76.95). Notably, lower plasma F in PE with comparable GC secretion (reflected in the amounts of tetrahydrometabolites in urine) may suggest the blunted HPA response in this condition. Lower F production in PE was previously reported by Ho et al. [
5], who observed lower total and free F in maternal plasma in PE. They suggested it could result from the underactivity of maternal HPA axis.
Such conclusion about diminished HPA response cannot be derived from the results for GH group. Plasma F in GH patients was similar to the values obtained for normotensive women (Supplementary Table 1), in spite of evidently enhanced F metabolism. Intensified F metabolism is manifested in significantly lower UFF/UFE values (increased 11β-HSD2 function), higher THF
tot/F
tot (increased 5β-reductase function) and almost two times higher (THFs
tot + THEs
tot)/UFF (strongly enhanced F clearance). Moreover, the GC secretion is substantially increased. All these facts lead to the conclusion that the enhanced metabolism of F in GH patients is compensated by higher F secretion due to HPA activity (Table
2). Our observations do not corroborate with the results of Walker et al. [
10] who showed no differences in 11β-HSD function between PE, GH and normotensive pregnant women. Such discrepancies may result from relatively small study population in the cited study (13 controls, 7 women with GH and 8 with PE). Moreover, it is worth noting that we calculated F and E metabolites ratio considering not only THF and THE but also allo-tetrahydrometabolites.
The results obtained for patients with CH are very similar to those for normotensive women. However, worth noting is the fact that the balance in functions of 5α and 5β-reductases is shifted towards 5β-reductase (lower alloTHF
tot/THF
tot values in CH, as presented in Tables
2 and
3). Surprisingly, this is in contradiction to results for non-pregnant hypertensive population presenting decreased activity of 5β-reductase [
8].
We are aware of limitations of our study. During the study, in 2016, the diagnostic criteria of PE changed [
41], and proteinuria is no longer obligatory for the diagnosis. We maintained the criteria with substantial proteinuria to ensure similar ones for all recruited patients, and for better comparability with previously published results. An important limitation is also the significantly earlier GA at sampling in PE group as compared to controls. Our study population comprised women who were admitted to hospital for routine tests or due to abnormalities arising during the course of pregnancy. Patients with PE, as a result of their rapidly deteriorating condition, were then usually enrolled to the hospital much earlier than other pregnant women. Secondly, the groups were poorly matched in regard to pre-pregnancy BMI. Moreover, women with PE were significantly more often nulliparous. The tendency to higher-pregnancy BMI among PE and GH patients, as well as the higher rates of nulliparity in PE but not in GH, were previously reported and these features are mentioned among risk factors of pregnancy-specific hypertension [
42]. Obesity and higher BMI are also linked to hypertension in general non-pregnant population [
43]. As those confounders could adversely affect the results, the multiple regression analyses were performed to confirm our observations.
Strengths of our research are the relatively large group of patients as compared to other published studies [
10] and thorough analysis of GC balance in pregnant women. We assessed the function of primary enzymes involved in F metabolism: 11β-HSD1, 11β-HSD2, 5α and 5β-reductase and the overall equilibrium between them. Additionally, the study group included both PE and GH patients, what brings the new insight into phenomena associated with pregnancy-related hypertension.
Our main findings include: markedly intensified F metabolism manifested in the increased function of renal 11β-HSD2, 5α and 5β-reductase in PE as well as the enhanced function of renal 11β-HSD2 and 5β-reductase in GH. The GC balance in PE is clearly shifted towards decreasing F concentration either due to the intensified conversion of F to E or enhanced production of tetrahydro- and allo-tetrahydrometabolites. The observed changes are similar, however, less marked in GH. Importantly, we suggest the blunted response of HPA axis in PE, what was not found in other hypertensive disorders of pregnancy. Further studies concerning non-pregnant women with a history of PE and GH are needed to assess whether the observed changes in GC balance are limited to gestation, or they remain in the body of affected women after pregnancy. Moreover, the comprehensive prospective study, including women with high risk of PE and GH, could bring the answer if any interference in GC balance during hypertensive pregnancy would be justified.
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