Organic acids in the evaluation of cell mitochondrial function in underground pregnancy

Introduction

Miscarriage is one of the most acute problems in practical obstetrics; its occurrence rate is reported to be 20% [1]. Nearly every 5th pregnancy ends as a miscarriage, and this tendency does not decrease, despite numerous and highly effective diagnostic and treatment methods developed in recent years [2, 3]. Indeed, the missed abortion (MA) should be considered a "problem of the century". In 2006, the International Federation of Obstetricians and Gynecologists (FIGO) declared MA to be a modern epidemic. Currently, there is no single predictor for the development of MA. In 40–50% of pregnant women, the exact cause of MA is not established [4].

There is no doubt that endometrial dysfunction underlies MA. It is the ability of the endometrium to respond adequately to cyclic fluctuations in the level of steroid hormones in the blood that leads to the formation of the necessary ultrastructural changes in the endometrium to form the "implantation window" and ultimately provides implantation of a fertilized egg, the onset, and development of pregnancy [2, 5].

There is ample evidence to suggest that the pathogenesis of early reproductive losses is closely associated with oxidative stress, changes in the proteomic spectrum, impaired lipid metabolism, and decreased respiratory chain enzyme activity occurring in mitochondria [6, 7]. Although the pathophysiology underlying gestational complications is different, changes in the structure and function of mitochondria are in all cases a common terminal stage of cell death and can be a target for pharmacotherapy.

As an integral part of systems biology, metabolomics has numerous advantages over traditional laboratory studies. The study of organic acids (OAs) is of increasing scientific interest because OAs are important components of metabolism and play a significant role in the production of cellular energy. OAs mainly reflect mitochondrial function and fatty acid oxidation, which occurs during cellular respiration.

The study aimed to determine the prognostic significance of low molecular weight metabolites in women with MA.

Materials and Methods

A total of 100 women of reproductive age were included in the study; 79 of them had an ultrasound-verified MA, and 21 of them had advanced pregnancy who were admitted for an artificial abortion. The inclusion criteria were reproductive age and an MA, verified by ultrasound examination. Exclusion criteria were pelvic organ malformations, antiphospholipid syndrome, and systemic diseases.

Immunohistochemical studies of the endometrium and determination of OAs in blood serum and endometrium by high-performance liquid chromatography were performed. The authors analyzed statistically significant OAs in blood serum and endometrium associated with disorders of fatty acid and amino acid metabolism, as well as intermediate metabolites in the Krebs cycle.

Statistical analysis of the obtained data was performed using the STATISTICA® for Windows, Release 6.0 module package by StatSoft®Inc., USA (2003), serial number AXAR802D898511FA.

Results

The clinical and statistical analysis confirmed the comparability of the groups by age, social status, somatic and gynecological diseases. The diagnosis of MA on admission was made based on ultrasound examination findings (absence of an embryo with a fetal size of 25 mm or larger in the transabdominal scan and 18 mm or larger in the transvaginal scan, absence of heartbeat in an embryo with a parietal-coccygeal length of 5 mm or larger).

The mean age of patients with MA at the time of examination was 29.8±5.8 years old, a quarter of them were of late reproductive age (older than 35 years old).

The authors analyzed the levels of 11 low-molecular-weight metabolites in serum and endometrium that are substrates of the Krebs cycle and b-oxidation of fatty acids and play an important role in cellular energy production. Among the studied OAs, glycolic acid and adipic acid had no significant differences between the groups. Glycolic acid levels were 5.8±4.8 mmol/L in patients with MA and 4.8±3.2 mmol/L in patients with advanced pregnancy (p=0.102). Adipic acid levels were 0.10±0.3 mmol/L and 0.24±0.5 mmol/L, respectively (p=0.101). Statistically significant differences were revealed in the levels of lactic, pyruvic, hippuric, hydroxyphenyl lactic, 4-hydroxyphenylacetic and 4-hydroxyphenylpyruvic acids in blood serum (Table 1).

Three metabolites were identified that were detected only in women with MA in the serum, such as glutaric acid, 4-hydroxyphenylacetic acid, and homogentisic acid in the serum (Table 2).

The levels of lactic and pyruvic acids were also significantly different in the study of metabolites in the endometrium (Table 3).

4-hydroxyphenylacetic and methylmalonic acids were detected only in women with MA (Table 4).

Histological examination of the endometrium showed regressive changes combined with diffuse and focal lymphohistiocytic infiltration (96.2%) with an admixture of plasma cells, and in some cases, with the formation of lymphoid follicles. There were also small foci of stromal sclerosis (Figure 1 a, b) with sclerosed vessels (89.9%) and venous profusion with severe thrombosis (68.3%).

The immunohistochemical study revealed high expression of chronic endometritis markers HLA-DR, CD 16, and CD 20 in the endometrial stroma and decreased expression of VEGF and LIF (receptivity marker) in the epithelium and stroma. The authors found a direct correlation between VEGF expression and 4-hydroxyphenylacetic acid content (r=0.411), and CD16 and lactic acid content in the endometrium (r=0.523).

A binary logistic regression method was used to assess the nature and extent of the influence of various factors on the risk of recurrent MA. The analysis revealed the factors that had a statistically significant effect on the development of MA. Using the results of regression analysis, the authors built a reliable model (χ²=54.676 p<0.001).

Discussion

The comparison of the obtained results revealed statistically significant differences in the determination of lactic, pyruvic, and 4-hydroxyphenylacetic acids in blood serum and endometrium between the studied groups.

Lactic acid (lactate) is an early indicator of inadequate cellular O₂ perfusion and is used as a "quantitative" assessment of tissue hypoxia. Elevated lactate levels in women with MA compared with the controls (p=0.05) may indicate reduced tissue oxygenation, ischemia, and decreased mitochondrial function. Cellular lactate formation is influenced by the "redox state" of the cell. One such cellular redox reaction is the equilibrium between pyruvic acid (pyruvate) and lactate. Normally, the lactate/pyruvate ratio is 10:1. A shift in the lactate/pyruvate ratio inhibits mitochondrial ATP formation and leads to a shift in the cytoplasmic redox state of the cell: NADH accumulates and NAD+ decreases, which causes oxidative stress.

4-Hydroxyphenylacetic acid is an intermediate metabolite in the catabolism of the amino acid tyrosine, which is pre-aminated with a-ketoglutaric acid and splits into 4-hydroxyphenylacetic acid, hippuric acid, hydroxyphenyl-lactic acid and 4-hydroxyphenylpyruvic acid. Further, 4-hydroxyphenylacetic acid is oxidized to homogentisinic acid, the latter being oxidized to fumarylacetoacetate with subsequent conversion to fumarate, acetate, and release of water and carbon dioxide. Therefore, the determination of homogentisin and 4-hydroxyphenylacetic acid in women with MA may indicate impaired tyrosine metabolism, which may be a consequence of increased protein consumption and phenylalanine hydroxylation. Impaired tyrosine metabolism may be a risk factor for insulin resistance and decreased thyroid hormone production [6].

The revealed changes at the molecular level are highly correlated with histological and immunohistochemical changes in the endometrium in women with MA.

Conclusion

Thus, mitochondrial dysfunction, which is a universal mechanism of pathological processes, underlies the pathogenesis of MA. Mitochondrial dysfunction results in decreased ATP levels in the cell, increased production of reactive oxygen intermediates, and activation of cell death mechanisms. 4-hydroxyphenylacetic acid and changes in the lactate/pyruvate ratio in serum and endometrium are sensitive metabolites of mitochondrial function and reflect structural and functional inferiority of the endometrium and poor prognosis of the course and outcome of the subsequent pregnancy.