Clinical value of serum homocysteine, folate, and ultrasonography detection of yolk sac in predicting the outcome of threatened abortion

Abstract Background: To investigate the clinical value of serum homocysteine (Hcy), folate, and ultrasonography detection of yolk sac in predicting the outcome of threatened abortion.Methods: Ninety-eight pregnant women within 12 weeks of gestation were included in the study. Of these subjects, 29 presented with threatened abortion and were considered the case group (n=29). The other 69 subjects showed as a normal pregnancy were the control group (n=69). The case group was followed up with at 28 weeks of gestation, and further divided into inevitable abortion group (IA, n=9) and successful fetus protection group (SFP, n=20). The yolk sacs of the included cases were measured by ultrasonography. The serum Hcy and folate levels of the included cases were measured by an enzyme cycling assay and chemiluminescent immunoassay, respectively. The yolk sac visualization rate and serum Hcy and folate levels were compared between the two case groups and used as a predicting reference for the outcome of threatened abortion. Results: The yolk sac visualization rates detected by ultrasonography were 97.1% (67/69) for the control group and 69.0% (20/29) for the threatened abortion group, with a statistical difference (p<0.05). For the IA and SFP groups, the visualization rates were 22.2% (2/9) and 90.0% (18/20) respectively, with a statistical difference (p<0.05). The serum Hcy levels in the threatened abortion group were significant elevated compared with control group (6.1±2.0 μmol/L vs 4.5±1.3 μmol/L), with a statistical difference (p<0.05). However, the serum levels of folat in the threatened abortion group were significantly decreased compared with the control group (15.5±4.2 nmol/L vs 19.1±4.7 nmol/L), with a statistical difference (p<0.05). The serum levels of Hcy and folate were 7.5±1.7 μmol/L and 12.7±4.2 nmol/L for the IA group, which were statistically different compared to the SFP group (p<0.05). The predictive sensitivity for the outcome of threatened abortion based on serum Hcy and folate levels were 85.0% and 75.0%, with a predictive specificity of 66.7% and 88.9% respectively. Conclusion: Serum Hcy and folate levels were quite different in threatened abortion pregnant woman compared to the control group. Serum Hcy and folate level detection combined with ultrasonography detection of yolk sac can provide useful information for threatened abortions


Background
Threatened abortion refers to a small amount of vaginal bleeding within 12 weeks of pregnancy with or without lower abdominal pain and no pregnancy discharge [1,2]. Risk factors relevant to threatened abortions include advanced age, previous abortion, exposure to tobacco smoke, obesity, diabetes, thyroid problems, and drug or alcohol use [3]. About 80% of abortions occur in the first 12 weeks of pregnancy [4]. The threatened abortion rate is reported to be about 20% for all pregnant women, with 35% of those threatened abortion cases developing into inevitable abortions [5]. Early identification of high risk inevitable abortion patients and giving them proper treatment is important for the outcome of threatened abortions. Many studies have evaluated the serological markers for predicting the outcome of threatened abortion, such as serum CA125 [6][7][8], β-HCG, and E2 [9]. Recently, several studies have evaluated the serum Hcy

Introduction
Let F denote a eld and let V denote a vector space over F with nite positiv pair A, A * of diagonalizable F-linear maps on V, each of which acts on an eigen irreducible tridiagonal fashion. Such a pair is called a Leonard pair (see [13, De A, A * is said to be self-dual whenever there exists an automorphism of the endo swaps A and A * . In this case such an automorphism is unique, and called the d The literature contains many examples of self-dual Leonard pairs. For instanc ated with an irreducible module for the Terwilliger algebra of the hypercube (see Leonard pair of Krawtchouk type (see [10, De nition 6.1]); (iii) the Leonard pair a module for the Terwilliger algebra of a distance-regular graph that has a spin mo bra (see [1,Theorem], [3,Theorems 4.1,5.5]); (iv) an appropriately normalized t (see [11,Lemma 14.8]); (v) the Leonard pair consisting of any two of a modular L De nition 1.4]); (vi) the Leonard pair consisting of a pair of opposite generator bra, acting on an evaluation module (see [5,Proposition 9.2] [7,Proposition 8.7]). and folate levels between normal pregnant women and threatened abortion cases. They found that the serum Hcy and folate levels were quite different between the normal and threatened abortion cases [10,11]. In our present study, we investigate the clinical values of serum Hcy and folate, and ultrasonography detection of the yolk sac in predicting the outcome of threatened abortions.

Patients
Ninety-eight pregnant women within 12 weeks of gestation were included in the study. Of these cases, 29 subjects presented with threatened abortion and were considered as the case group (n=29). The other 69 subjects with normal pregnancy made up the control group (n=69). The threatened abortion case group was followed up with at 28 weeks of gestation, and further divided into inevitable abortion group (IA, n=9) and successful fetus protection group (SFP, n=20).
Informed consent: Informed consent has been obtained from all individuals included in this study.
Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and was approved by the Medical Ethics Committee of Zhangqiu Maternal and Child Health Care Hospital of Jinan City.

Ultrasonography detection of yolk sac
The color Doppler ultrasound diagnostic system was purchased from General Electric Company (VOLUSON E10). An ultrasonic probe with 5 to 9MHz was used. The pregnant women were asked to lie flat on the examination bed and take the bladder lithotomy position. Coupling agent was evenly applied to the ultrasonic probe, a condom was put on the probe, and the probe was placed in the posterior fornix of the vagina. The uterus and bilateral appendages were scanned by ultrasound to observe the yolk sac and gestational sac, Figure 1.

Serum Hcy and folate detection
Three mL of peripheral venous blood was taken from the included pregnant women under fasting conditions. The blood was centrifuged for 10 min to obtain the serum, and then stored in a refrigerator for later use. The serum Hcy levels were determined by an enzyme cycling assay. A kit was purchased from Becton Dickinson, United States. The serum folate was examined by a chemiluminescence assay (Siemens, Col Ltd., ADVIA centaurXP)

Statistical analysis
Measured data (serum Hcy and folate levels) were recorded as s x ± , and comparisons were made by t-test. Counting data was showed by n (%), and comparisons were made by chi-square test. Statistical significance was determined when p<0.05.

Serum Hcy and folate detection
The serum Hcy levels in the threatened abortion group were significantly elevated compared with the control group (6.1±2.0 μmol/L vs 4.5±1.3 μmol/L), with a statistical difference (p<0.05), Figure 2. However, the serum levels of folate in the threatened abortion group were significant decreased compared with the control group (15.5±4.2 nmol/L vs 19.1±4.7 nmol/L), with a statistical difference (p<0.05), Table 3. The serum levels of Hcy and folate were 7.5±1.7 μmol/L and 12.7±4.2 nmol/L for the IA group, which were statistical different compared to the SFP group (p<0.05), Table 4.

Prediction value of serum Hcy and folate levels for the outcome of threatened abortion
The serum Hcy and folate levels were used to predict the outcomes of threatened abortion, Figure 3. The predictive sensitivity for the outcome of threatened abortion by serum Hcy and folate levels were 85.0% and 75.0%, with a predictive specificity of 66.7% and 88.9% respectively, Table 5.

Discussion
Epidemiology studies indicated that 20% of women are affected by threatened abortion during the pregnancy [12]. The outcome of threatened abortion is very concerning. Therefore, identifying high risk inevitable abortion patients and giving them proper treatment can improve the prognosis of threatened abortion. Due to its convenience and non-invasive nature, ultrasonography has become an important examination method in the diagnosis of early pregnancy [13][14][15]. The yolk sac is often used for evaluating the embryonic developmental status [16,17]. The yolk sac usually forms around 5 weeks of gestation, and then increases in size during the gestational weeks. The yolk sac is easily affected by teratogenic substances in the early stages of pregnancy. Abnormal development of the yolk sac usually manifests through a larger diameter and shape changes, which eventually leads to abortion. Xie et al. [18] evaluate the ability to predict pregnancy outcomes through the combination of ultrasound scanning of yolk sacs and serum CA125 examination in early threatened abortion patients. The authors found that this combination was of great value for predicting pregnancy outcomes. In our present study, we found that yolk sac visualization rates detected by ultrasonography were 22.2% (2/9) and 90.0% (18/20) for the IA and SFP groups respectively, with a statistical difference (p<0.05). The results indicated that the visualization rate in the inevitable abortion group was significantly lower than that of successful fetus protection group.
Hcy is a non-proteinogenic α-amino acid and considered as a risk factor related to heart attacks and strokes [19]. It has also been reported that hyperhomocysteinemia can also be associated with early  threatened abortion and neural tube defects [20]. Nasiri et al. have evaluated the serum levels of Hcy in threatened abortion cases, and found that the serum concentration of Hcy was significantly elevated and correlated with the poor pregnancy prognosis [21]. In our study, we found that the serum levels of Hcy and folate were 7.5±1.7 μmol/L and 12.7±4.2 nmol/L respectively for the IA group, which were statistically different compared to the SFP group (p<0.05). The serum Hcy levels were used as an outcome for threatened abortion prediction reference. The predictive sensitivity and specificity for the outcome of threatened abortion were 85.0%, 66.7% under the cut off value of 7.0 μmol/L. These results indicated that the greater serum Hcy levels the pregnant women had, the greater the possibility of development of an inevitable abortion. During pregnancy, insufficient intake of folate and vitamin B12 can lead to elevated blood Hcy levels. Hypercysteinemia in early pregnancy can inhibit the formation of chorion, reducing the number of chorionic vessels, affecting the blood perfusion of embryos, leading to poor implantation of embryos, and eventually leading to poor pregnancy outcomes. In our results, the serum folate levels in the IA group were significantly lower than the SFP group and further analysis indicated that serum folate levels can be used as an outcome predicting serological marker with the relative high sensitivity and specificity (75.0% and 88.9%).
In conclusion, serum homocysteine and folate were quite different in threatened abortion pregnant woman. Serum Hcy and folate detection combined with ultrasonography detection of yolk sac can provide useful information in threatened abortions. Serum Hcy and folate can be used as serological biomarkers for predicting the outcomes of the pregnant women with threatened abortions.