Investigation of serum trimethylamine‐N‐oxide levels in missed abortion: A prospective study

The aim of our study was to investigate the relationship between missed abortion and serum trimethylamine N‐oxide (TMAO) levels.

Between 15% to 20% of all known pregnancies terminate spontaneously in the first and second trimesters.Missed abortion is defined as undetected intrauterine death of the embryo or fetus without expulsion of pregnancy products.It constitutes approximately 15% of clinically diagnosed pregnancies.Women experiencing a missed abortion may not be aware of this condition due to the lack of obvious symptoms. 1,2Traditionally, asymptomatic missed abortion is treated with surgical dilatation, curettage, or manual vacuum aspiration. 1 Surgical intervention, which has been widely applied all over the world in the last 50 years, is accepted as the standard treatment of missed abortion with a success rate of around 95%.However, surgery and hospitalization costs and complications associated with surgery and anesthesia remain a major unresolved issue. 2 Trimethylamine N-oxide (TMAO) is a small, colorless amine oxide composed of trimethylamine (TMA) produced by the metabolism of the intestinal microbiota from dietary precursors (choline, betaine, and L-carnitine). 3A part of TMA, which is taken orally and synthesized from molecules such as choline and carnitine in the intestine, is excreted with breath and feces.A total of 95% of TMA is oxidized to TMAO.Approximately 95% of TMAO is excreted in the urine within 24 h.TMAO is excreted in the urine by tubular secretion in the kidneys. 4Recently, TMAO has been identified as an independent risk factor for obesity, metabolic syndrome, fatty liver disease, cancer, polycystic ovary syndrome, and cardiovascular disease (CVD). 5nsidering the critical role of TMAO in endothelial dysfunction and vascular inflammation, it was thought that high circulating TMAO levels may contribute to the onset of pre-eclampsia and be associated with the severity of the disease. 5High TMAO levels have been shown to be associated with a higher risk of pre-eclampsia and also with increased systemic inflammation and endothelial dysfunction. 6gy et al., 7 investigated the relationship between TMAO levels in human follicular fluid (FF) and IVF pregnancy outcomes and found higher TMAO levels to be a negative indicator of oocyte fertilization and embryo quality.Considering the studies, it is thought that endothelial dysfunction and increasing systemic inflammation may be related to missed abortion.In the present study, we investigated the relationship between missed abortion and TMAO levels.

| Patients and study design
This was a single center prospective study conducted by evaluating pregnant women who applied to our clinic between March 2023 and May 2023.Ethical approval for the study was obtained from the Selcuk University Faculty of Medicine Local Ethics Committee.
A total of 56 pregnant women diagnosed with missed abortion and 73 healthy pregnant women were included in this study.The women included were between the ages of 18-45 years.Pregnant women in both the control and patient groups in the first and second trimesters were selected.Informed consent forms were obtained from all patients who agreed to participate in the study, after informing the patients about the study.
Patients with systemic diseases such as hypertension, diabetes, hematologic disease (e.g., antiphospholipid antibody syndrome), rheumatologic disease (e.g., systemic lupus erythematosus), patients with a history of teratogenic drug use or radiation, and patients who did not agree to participate in the study were excluded.

| TMAO analysis in human serum
After the venous blood samples were centrifuged, they were placed in 1.5 mL Eppendorf tubes and stored at −80°C until the study.
Trimethylamine-N-oxide with deuterium (d9-TMAO) was purchased from Cambridge Isotope Laboratories (catalog: DLM-4779-1), and all other reagents were purchased from Sigma Aldrich.Serum samples for TMAO analysis Phenomenex Luna (50 mm × 4,6-5 μm) C18 high performance liquid chromatography (HPLC) in ABSCIEX API 3200 high performance liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) device in Selcuk University Medical Faculty Medical Biochemistry Laboratory was studied using a colon.Two mobile phases were used during the analysis (HPLC grade water containing 0.1% formic acid and methanol containing 0.1% formic acid).
Serum samples stored at −80°C until the study day were thawed at room temperature and 250 μL was pipetted into empty Eppendorf tubes.Then, 100 μL of d9-TMAO, the isotope of TMAO, was added to the samples in Eppendorf as an internal standard.In order to separate the proteins in the serum, the samples were denaturized by adding alcohol to the samples with internal standard and they were vortexed for 30 s.Then, the supernatant was transferred to empty glass tubes by centrifugation at 13 000 g for 10 min.Liquids in glass tubes were evaporated under nitrogen gas at 28°C.Then, 250 μL of HPLC grade water was added as a solvent and vortexed.The dissolved samples were transferred to HPLC vials and their lids were closed.Results were obtained from the Analyst Software system of the LC-MS/MS instrument.The authors covered the laboratory kits from their own budget.No support was received from any institution or organization.

| Statistical analysis
All statistical analysis was performed using R Statistical Language (version 4.1.2;The R Foundation for Statistical Computing, Vienna, Austria; https:// www.r-proje ct.org).To check the normality of the data, Shapiro-Wilk's normality test and Q-Q plots were used.
Levene's test was used to assess the homogeneity of the variances.
Variables are presented as mean ± standard deviation (SD) or median with interquartiles (IQR, 25th percentile-75th percentile), as appropriate.An independent samples t-test and Mann-Whitney U test was run to determine if there was a statistically significant difference in age and TMAO values between healthy controls and patients with missed abortus.Hedges's g and rank biserial correlation (r rb ) values with 95% confidence intervals (CIs) were given as effect size (ES) for age and TMAO values, respectively.In addition, Spearman's rho correlation coefficients with 95% confidence intervals were calculated to examine the relationship between serum TMAO levels and age, and body mass index (BMI, calculated as weight in kilograms divided by the square of height in meters) in woman with missed abortus.A two-tailed P value less than 5% was considered statistically significant.An a priori power analysis was conducted using pwr packages in R 4.1.2(R Foundation for Statistical Computing, Vienna, Austria; https:// www.r-proje ct.org) to test the difference between healthy controls and patients with missed abortus using a two-tailed test, a large effect size (Cohen's d = 0.7), and an alpha of 0.05.Results showed that a minimum total sample of 110 participants with two equal sized groups of n = 55 was required to achieve a power of 0.95.

| RE SULTS
This study enrolled 129 participants, 56 (56.6%) of whom were women with missed abortus, and 73 (43.4%) who were healthy controls with a mean age 29.46 ± 6.48 years, ranging from 18 to 45 years.
The age and TMAO distribution of the groups, and BMI values of the patients with missed abortus are given in Table 1.

| DISCUSS ION
Changes in nutritional status have a potential impact on fertility by Intestinal microbiota plays an important role in regulating whole body metabolism and maintaining energy balance, as well as chronic diseases such as obesity and atherosclerosis. 9Trimethylamine-Noxide (TMAO) is a modern illustration of a metabolite that integrates food, microbiota, and host metabolism.The gut bacteria transforms dietary choline (found in dairy products and fish) and L-carnitine (found in red meat) into trimethylamine (TMA). 10,11The enzymes hepatic flavin monooxygenases (FMOs) 1 and 3 convert the gas TMA into TMAO.6][17][18] In contrast, no statistically significant difference between the BMI of missed abortions and healthy pregnancies was discovered in our investigation.
According to studies, circulating TMAO levels rise with aging. 19,20 our study, consistent with the literature, no statistically significant difference was detected between TMAO levels and age of the patients when both patient groups were compared.
Studies have shown that TMAO level is found to be high in FF of poor quality embryos, therefore high TMAO may have a negative predictive value in showing the quality of the embryo. 7Based on this study, it is expected that the TMAO level in missed abortion patients will be higher than in healthy pregnant women.Our study's findings, which showed that TMAO levels were greater in individuals who had missed abortions, are consistent with this condition.
Pre-eclampsia, intrauterine growth restriction, preterm labor, early rupture of membranes, late spontaneous abortion, and placental abruption are all major pregnancy complications associated with defective deep placentation. 21These complications are thought to arise from placental ischemia.In conclusion, it has been shown that poor placentation in early pregnancy causes uteroplacental insufficiency and perfusion dysfunction, together with hypoxia and placental ischemia. 22It has been determined that TMAO levels are high in these diseases.In our investigation, we discovered that patients with missed abortions had greater TMAO levels than pregnant women in good health.We believe that this situation is caused by possible placental ischemia.
We searched the studies in the literature, and found no study showing the relationship between missed abortion and TMAO.
This adds originality to our study.Although our study had limitations such as being conducted in a single center, having a small number of patients, and not being able to address all of the physiologic changes brought about by pregnancy, we believe that our study will contribute to the literature in terms of guiding future studies on this subject.