Phytosterol nutritional supplement improves pregnancy and neonatal complications of gestational diabetes mellitus in a double-blind and placebo-controlled clinical study

Abstract

Gestational diabetes mellitus (GDM) is an increasingly serious health problem among pregnant women. Phytosterol-enriched spreads are known to reduce total cholesterol (TC) and low-density lipoprotein (LDL), but little is known about their effects on GDM. We aimed to examine the effect of the daily consumption of phytosterol-enriched spreads on both the maternal and neonatal outcomes of GDM patients. GDM patients during the third trimester of pregnancy were enrolled and assigned randomly to consume a regular spread or phytosterol-enriched spread daily until the end of their pregnancy. Maternal diabetic symptoms such as serum lipid profile, glucose and insulin metabolisms, as well as neonatal complications, were analyzed at the beginning and full-term. The daily consumption of the phytosterol-enriched spread exhibited significant beneficial effects on maternal diabetic symptoms, in terms of improved lipid compositions and glucose metabolism. Moreover, the incidence of neonatal complications was also significantly reduced by the phytosterol-enriched spread, in terms of birth weight, macrosomia, hypoglycemia, respiratory distress and Apgar scores. The daily consumption of a phytosterol-enriched spread is able to improve both the maternal and neonatal outcomes in GDM patients.

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Introduction

Gestational diabetes mellitus (GDM) is a disease commonly occurring during pregnancy, which usually manifests clinically as insulin resistance and glucose intolerance.¹ GDM-affected women often do not show any early sign of diabetic symptoms, but are only diagnosed with GDM as early as the second trimester during the pregnancy. It was estimated that almost one fifth of GDM patients suffer long-term risk of diabetes even after pregnancy.^2,3 Maternal diabetic symptoms of GDM include those similarly seen in patients of type 2 diabetes mellitus (T2DM), such as insulin resistance, increased serum levels of total cholesterol (TC) and low-density lipoproteins (LDL). In addition, GDM also presents with an elevated incidence of abnormal fetal development and neonatal complications, such as low birth weight, neonatal hypoglycemia and respiratory distress.^4,5

Phytosterols are able to compete with cholesterols for inclusion into mixed micelles in the gut due to their structural similarity, which in turn causes a reduced serum cholesterol level.⁶ Even though phytosterols were known to exert a cholesterol-lowering function as early as in the 1950s,^7,8 it was only nearly half a century later that their potential of controlling cholesterol levels was re-discovered.^9–12 In this context, many reports demonstrated the high solubility of phytosterols in margarine spreads, with even the addition of a small amount capable of greatly reducing serum cholesterols.^13–17 In addition, spreads enriched in phytosterols even exhibited beneficial roles in patients of T2DM,¹⁸ where it was able to lower TC and LDL levels and decrease the risk of cardiovascular disease in type 2 diabetes.

To date, there has been no study available on the effects of a phytosterol-enriched spread on pregnant GDM patients. Given the similarities in maternal symptoms of GDM with T2DM, we would like to employ the use of a phytosterol-enriched spread as a food supplement, and investigate its effect in improving maternal diabetic symptoms of GDM patients. We therefore designed this placebo-controlled and double-blind randomized clinical trial, with the aim to assess whether the daily consumption of a phytosterol-enriched spread was able to improve maternal diabetes and reduce neonatal complications among GDM patients.

Experimental

Ethical statements

The study was conducted following the guidelines laid down in the Declaration of Helsinki, and approved by the Ethics Committee of Cangzhou Central Hospital. All participants were fully aware of the potential risks of the GDM screen, and given written and informed consent forms before enrollment into the study.

Patient selection

This clinical study was conducted in Cangzhou Central Hospital, from January 2012 to December 2015. A total of 325 pregnant women carrying a singleton pregnancy between 26–35 years of age, and diagnosed with GDM at the onset of the third trimester (29 weeks) were recruited for the study. The GDM diagnostic criteria of the current study were according to the American Diabetes Association guidelines¹⁹ as follows: fasting plasma glucose (FPG) ≥ 95 mg dL⁻¹, 1 h oral glucose tolerance test (OGTT) ≥ 180 mg dL⁻¹ and 2 h OGTT ≥ 155 mg dL⁻¹. The exclusion criteria were: eclampsia, pre-eclampsia, hypo- and hyper-thyroidism, multiparity, urinary tract infection, maternal hypertension, liver, kidney or renal disease, patients with history of diabetes and/or requiring insulin therapy during the study, and patients who consumed margarine or butter during the 6 months prior to the enrollment.

Randomization

Based on the inclusion and exclusion criteria, 49 patients were excluded, and 276 patients diagnosed with GDM were eligible to participate in the current clinical trial. They were randomized by using a permuted-block stratified to their baseline 1 h OGTT results, and evenly assigned to consume either a regular margarine spread as the placebo, or a phytosterol-enriched margarine spread as the intervention, with one serving (10 g per serving) at breakfast and the other one serving at dinner on a daily basis until full term. Each 10 g serving of the phytosterol-enriched margarine spread contains 2 g of phytosterols, as determined by a specialized laboratory certified by the Food and Drug Administration of China. Labels on the regular and phytosterol-enriched margarine spreads were removed to make the content blind to both the patients and investigators. Specific instructions of compliance were given to all patients: (1) they should conform to the prescribed therapeutic diets after their GDM diagnosis; (2) they should not consume any margarine spread except those issued by the study investigators. All patients were re-visited on a weekly basis to assess their compliance to the study instructions, as well as to issue the margarine spreads for the following week. 17 patients from the placebo group and 15 from the phytosterol intervention group dropped out from the study due to personal reasons, or emergency delivery at other hospitals, or non-compliance to the study instructions. All assessments were conducted at week 0 and full term by an investigator blind to the group assignment.

Assessments of the metabolic profile

10 mL of fasting blood samples were collected in the early morning with the overnight fasting status. Blood samples were collected in tubes with 0.1% EDTA and were centrifuged within 15 min of collection to separate the plasma. The plasma samples were stored at −80 °C until further analysis. Levels of triacylglycerol (TAG), TC, high-density lipoprotein (HDL) and LDL were determined using commercial assay kits (Biovision) following manufacturer's manuals. Plasma glucose levels were measured using a glucometer (Lifescan Surestep). Hemoglobin A1c and plasma insulin levels were determined by using commercial enzyme-linked immunosorbent assay kits for human hemoglobin A1c and insulin (Biovision) following manufacturer's manuals. Insulin resistance was collectively determined according to the quantitative insulin check index (QUICKI), homeostasis model of assessment of insulin resistance (HOMA-IR) and homeostasis model of assessment of beta cell function (HOMA-B) as previously described.²⁰

Measurements of anthropometrics

Body weight was measured after an overnight fast using a digital scale accurate to 0.1 kg, with participants in light clothing and without shoes. Body height was measured using a stadiometer accurate to 1 cm. The body mass index (BMI) was calculated by the formula: weight (kg)/square of height in m².

Statistics

Values were shown as mean ± SD. The normality of data distribution was assessed using the Kolmogorov–Smirnov goodness-of-fit test. The two-tailed student's T test was used to assess normally distributed data, whereas the Mann–Whitney test was used to assess non-normally distributed data. A P value less than 0.05 was considered as statistically significant. All statistical analyses were performed using the SPSS software 18.0 (SPSS Inc., USA).

Results and discussion

As shown in Fig. 1, a total of 325 pregnant women diagnosed with GDM in their third trimester of pregnancy were initially enrolled into the current study, among which 49 patients were excluded. The other 276 GDM patients were randomly assigned to the two treatment groups, with one group on a regular margarine spread as the placebo, and the other on a phytosterol-enriched spread as the intervention. All eligible patients were given specific instructions to consume two servings of either a regular margarine spread or phytosterol-enriched margarine spread every day until their full term. Labels on the regular and phytosterol-enriched margarine spreads were removed to make the content blind to both the patients and investigators. Patients were also instructed not to consume any margarine spread except those issued by the study investigators, and were re-visited every week to assess their compliance. 17 and 15 patients from the placebo group and the phytosterol intervention group, respectively, dropped out from the study due to personal reasons, or emergency delivery at other hospitals, or non-compliance to the study instructions. In the end 121 patients in the placebo group and 123 patients in the phytosterol group completed the trial, whose data were analyzed in this study.

Fig. 1 The study design in a flow chart.

General characteristics of the GDM patients in the two treatment groups are listed in Table 1. No significant differences were found between patients of the two groups, in terms of age at pregnancy (31.5 ± 5.2 versus 29.8 ± 6.4 years, P = 0.41), height (164 ± 16 versus 165 ± 13 cm, P = 0.56), gestation age at full term (39.8 ± 0.8 versus 40.2 ± 0.7 weeks, P = 0.62) or duration of spread consumption (9.9 ± 0.4 versus 10.1 ± 0.2 weeks, P = 0.47). We also measured the body weight for all patients, and could not observe any significant difference between the two groups either on week 0 (72.8 ± 8.1 versus 72.2 ± 7.3 kg, P = 0.33) or full term (74.1 ± 7.6 versus 73.9 ± 6.2 kg, P = 0.53). Next BMI was calculated for all patients, which as expected was also statistically similar at both week 0 (26.3 ± 5.1 versus 26.7 ± 3.8, P = 0.39) and full term (27.2 ± 3.9 vs. 27.8 ± 5.4, P = 0.21).

Table 1 General characteristics of the GDM patients in the two treatment groups

Characteristics	Regular (n = 121)	Phytosterol (n = 123)	P value
Values are mean ± SD.
Age at pregnancy (years)	31.5 ± 5.2	29.8 ± 6.4	0.41
Height (cm)	164 ± 16	165 ± 13	0.56
Weeks at full term	39.8 ± 0.8	40.2 ± 0.7	0.62
Weeks of spread consumption	9.9 ± 0.4	10.1 ± 0.2	0.47
Body weight at week 0 (kg)	72.8 ± 8.1	72.2 ± 7.3	0.33
Body weight at full term (kg)	74.1 ± 7.6	73.9 ± 6.2	0.53
BMI at week 0 (kg m^−2)	26.3 ± 5.1	26.7 ± 3.8	0.39
BMI at full term (kg m^−2)	27.2 ± 3.9	27.8 ± 5.4	0.21

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Blood samples were collected from patients of both treatment groups, at both week 0 and full term, in order to analyze their lipid composition (Table 2). It is of note that at week 0 no differences were found between any of the lipid composition parameters for the two treatment groups. In patients on the regular spread, TAG levels were found to be significantly elevated from 196.7 ± 71.6 mg dL⁻¹ at week 0 to 223.4 ± 76.1 mg dL⁻¹ at full term (P = 0.02). Over the same treatment period, TAG levels of patients in the phytosterol group were reduced from 193.5 ± 68.3 mg dL⁻¹ at week 0 to 181.3 ± 62.1 mg dL⁻¹ at full term, albeit to a statistically insignificant extent (P = 0.08). In a similar manner, the TC levels of patients receiving a regular spread were also increased, from 231.4 ± 52.1 mg dL⁻¹ to 251.9 ± 48.3 mg dL⁻¹ (P = 0.06), whereas those of patients in the phytosterol group were instead significantly down-regulated from 226.7 ± 61.3 mg dL⁻¹ to 179.7 ± 64.1 mg dL⁻¹ (P = 0.03). The LDL levels of patients in the regular spread group remained largely unchanged (105.6 ± 24.3 versus 116.8 ± 27.6 mg dL⁻¹, P = 0.39), but a significant reduction can be seen in patients receiving the phytosterol-enriched spread (103.7 ± 31.2 versus 82.8 ± 23.4 mg dL⁻¹, P = 0.02) over the entire study period. The levels of HDL, however, were significantly reduced from week 0 to full term in patients on the regular spread (67.5 ± 14.6 versus 60.3 ± 17.3 mg dL⁻¹, P = 0.04). In contrast, the HDL of patients on the phytosterol-enriched spread was greatly increased from 62.4 ± 16.7 mg dL⁻¹ at week 0 to 78.3 ± 16.5 mg dL⁻¹ at full term (P = 0.03). Finally, we calculated the ratio of TC/HDL, and found its trend over the study period to be quite different between the two treatment groups. The TC/HDL ratio of patients in the regular spread group was increased (2.8 ± 1.1 versus 3.3 ± 0.9, P = 0.05), whereas that of patients in the phytosterol group was significantly decreased (2.9 ± 1.1 versus 2.6 ± 0.8, P = 0.02).

Table 2 Lipid composition of the GDM patients at week 0 and full term

Lipid composition	Regular (n = 121)			Phytosterol (n = 123)
	Week 0	Full term	P value	Week 0	Full term	P value
Values are mean ± SD, P values are an intragroup comparison for changes in parameters.
TAG (mg dL^−1)	196.7 ± 71.6	223.4 ± 76.1	0.02	193.5 ± 68.3	181.3 ± 62.1	0.08
TC (mg dL^−1)	231.4 ± 52.1	251.9 ± 48.3	0.06	226.7 ± 61.3	179.7 ± 64.1	0.03
LDL (mg dL^−1)	105.6 ± 24.3	116.8 ± 27.6	0.39	103.7 ± 31.2	82.8 ± 23.4	0.02
HDL (mg dL^−1)	67.5 ± 14.6	60.3 ± 17.3	0.04	62.4 ± 16.7	78.3 ± 16.5	0.03
TC/HDL ratio	2.8 ± 1.1	3.3 ± 0.9	0.05	2.9 ± 1.1	2.6 ± 0.8	0.02

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We next examined the serum glucose metabolic profiles of all GDM patients at both week 0 and full term (Table 3). The FPG levels of patients receiving the regular spread were essentially the same from the beginning to the end of the study (103.3 ± 4.2 versus 101.6 ± 4.9 mg dL⁻¹, P = 0.36), but they were significantly decreased in patients of the phytosterol group (102.4 ± 5.6 versus 92.1 ± 6.1 mg dL⁻¹, P = 0.03). The hemoglobin A1c levels displayed the exact same trend as FPG. The insulin levels exhibited a very distinct pattern of change between the two groups: they were slightly but significantly elevated in patients of the regular spread group (14.8 ± 3.7 versus 16.1 ± 5.2 mg dL⁻¹, P = 0.03), whereas markedly reduced in the phytosterol group (15.3 ± 4.2 versus 9.8 ± 5.0 mg dL⁻¹, P = 0.02). HOMA-IR and HOMA-B scores of the regular spread group were both slightly increased from week 0 to full term (HOMA-IR 3.7 ± 1.5 to 4.0 ± 1.9, P = 0.21; HOMA-B 52.1 ± 22.4 to 66.2 ± 23.8, P = 0.14), but in the phytosterol group they were both consistently decreased in a statistically significant manner (HOMA-IR 3.9 ± 1.6 to 1.7 ± 1.3, P = 0.03; HOMA-B 54.7 ± 19.6 to 41.7 ± 20.3, P = 0.02). Lastly, the QUICKI score exhibited the exact opposite trend to the two HOMA scores, with that of the regular spread group decreasing (0.41 ± 0.11 versus 0.27 ± 0.09, P = 0.05) while the phytosterol group significantly increasing (0.44 ± 0.08 versus 0.53 ± 0.07, P = 0.04).

Table 3 Serum glucose metabolism of the GDM patients at week 0 and full term

Glucose metabolism	Regular (n = 121)			Phytosterol (n = 123)
	Week 0	Full term	P value	Week 0	Full term	P value
Values are mean ± SD, P values are an intragroup comparison for changes in the parameters.
FPG (mg dL^−1)	103.3 ± 4.2	101.6 ± 4.9	0.36	102.4 ± 5.6	92.1 ± 6.1	0.03
Hemoglobin A1c (%)	6.8 ± 0.7	6.9 ± 0.5	0.42	6.9 ± 0.6	6.1 ± 0.7	0.04
Insulin (μIU mL^−1)	14.8 ± 3.7	16.1 ± 5.2	0.03	15.3 ± 4.2	9.8 ± 5.0	0.02
HOMA-IR	3.7 ± 1.5	4.0 ± 1.9	0.21	3.9 ± 1.6	1.7 ± 1.3	0.03
HOMA-B	52.1 ± 22.4	66.2 ± 23.8	0.14	54.7 ± 19.6	41.7 ± 20.3	0.02
QUICKI	0.41 ± 0.11	0.27 ± 0.09	0.05	0.44 ± 0.08	0.53 ± 0.07	0.04

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In addition to the maternal symptoms, we also summarized the neonatal outcome in Table 4. The average birth weight, incidences of low birth weight (<2.6 kg) and hypoglycemia were significantly reduced in the phytosterol group compared to the regular spread group. However, no significant differences can be observed in the case of macrosomia and respiratory distress. It is of note that both 1 and 5 min Apgar scores were found to be significantly lower in the phytosterol group than the regular spread group.

Table 4 Neonatal complications at full term of the GDM patients in the two treatment groups

Complications	Regular (n = 121)	Phytosterol (n = 123)	P value
Values are mean ± SD.
Birth weight (kg)	2.9 ± 0.6	3.3 ± 0.5	0.04
Low birth weight (<2.6 kg) (n)	13	6	0.04
Hypoglycemia (n)	8	2	0.02
Macrosomia (n)	4	2	0.43
Respiratory distress (n)	5	3	0.26
1 min Apgar score	9.6 ± 0.5	9.8 ± 0.4	0.02
5 min Apgar score	9.7 ± 0.4	10.1 ± 0.6	0.04

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Although insulin resistance and glucose intolerance are present in not just T2DM but also GDM, patients suffering from gestational diabetes often find themselves to be short of pharmaceutical interventions, because drugs that are approved for T2DM are not suitable during pregnancy. Therefore GDM patients are usually treated conservatively through dietary interventions that are often considered as the safe approach. In fact conservative dietary intervention was found to be surprisingly effective even in the clinical management of T2DM, as indicated by a recent study in which vitamin D supplementation was able to attenuate insulin resistance in T2DM patients.²¹ We speculated that since dietary interventions can be successful in treating T2DM with more severe symptoms, they could also be applied as therapies against GDM and may yield promising results. In this context, the clinical trial on T2DM conducted by Lee and colleagues demonstrated that consuming a phytosterol-enriched spread was able to lower TC and LDL levels in diabetic patients.¹⁸

In the current clinical trial, we hereby report that, consuming the phytosterol-enriched spread on a daily basis for GDM patients, from the onset of the third trimester during pregnancy, could markedly improve both maternal and neonatal outcomes. First of all, by assessing serum lipid biomarkers, such as TAG, TC, LDL and HDL, we found that the lipid compositions of patients were greatly improved by the phytosterol intervention over those of placebo, consistent with an earlier study in which a phytosterol-enriched spread was found to reduce the serum levels of TC and LDL in T2DM patients.¹⁸ Particularly, the ratio of TC/HDL was significantly reduced in GDM patients on phytosterol intervention. As a low TC/HDL ratio is a commonly used indicator of a healthy lipid composition, these data of ours are also in line with a beneficial effect of the phytosterol-enriched spread on improving the lipid composition.

Furthermore, we also found that serum glucose and insulin metabolism in GDM patients could be attenuated by phytosterol intervention, as indicated by the significantly reduced FPG and insulin levels. In addition, diagnostic index scores such as HOMA-IR and HOMA-B were found to be decreased, while QUICKI increased, all indicative of the significantly improved resistance to insulin. These above results consistently demonstrated that dietary intervention using the phytosterol-enriched spread could greatly improve the maternal outcome of GDM patients.

In addition to the maternal outcome, we also evaluated the incidence of neonatal complications of GDM patients in the current study, including low birth weight, hypoglycemia, macrosomia and respiratory distress. We found that significantly fewer infants with a low birth weight and hypoglycemia were born from GDM-affected women after phytosterol intervention, however incidences of macrosomia and respiratory distress were largely the same. This result suggested that neonatal complications were improved to a certain extent by phytosterol intervention, at least in the case of low birth weight and hypoglycemia.

Conclusions

The overall results in our current study supported the potential clinical efficacy of the phytosterol-enriched spread in alleviating both maternal symptoms and neonatal complications of GDM patients. We believe that the factors contributing to these promising results of the current study include a stringent patient selection criterion. To elaborate, in selecting patients, we made it a prerequisite that the patients enrolled did not consume either margarine or butter recently, and several patients who did not comply with the study instructions and consumed spreads except those issued by the study investigators were excluded. In addition, the margarine spread is a traditional Chinese food, therefore many patients were in fact exposed to margarine and phytosterols for the first time, which might explain this initial strong clinical beneficial effect. Studies to investigate consumptions over a longer period, for example throughout the pregnancy, among a more diverse population (those with previous exposure to margarine) could shed a more comprehensive observation to fully evaluate the beneficial effects of a phytosterol-enriched spread on GDM patients.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This research is supported by the Department of Endocrinology and Clinical Test Room. The funding body has no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. We thank Yi Han for her help with the statistical evaluation and Yunna Zhang for her participation in the data collection.

References

1. K. Y. Lain and P. M. Catalano, Clin. Obstet. Gynecol., 2007, 50, 938–948.
2. D. S. Feig, B. Zinman, X. Wang and J. E. Hux, Can. Med. Assoc. J., 2008, 179, 229–234.
3. L. Bellamy, J. P. Casas, A. D. Hingorani and D. Williams, Lancet, 2009, 373, 1773–1779.
4. P. M. Catalano, J. P. Kirwan, S. Haugel-de Mouzon and J. King, J. Nutr., 2003, 133, 1674S–1683S.
5. J. Gardosi and A. Francis, Am. J. Obstet. Gynecol., 2009, 201, 25.e21–25.e27.
6. S. Rozner, A. Kogan, S. Mehta, P. Somasundaran, A. Aserin, N. Garti and M. F. Ottaviani, J. Phys. Chem. B, 2009, 113, 700–707.
7. O. J. Pollak, Circulation, 1953, 7, 702–706.
8. M. M. Best, C. H. Duncan, E. J. Van Loon and J. D. Wathen, Circulation, 1954, 10, 201–206.
9. P. J. Jones, D. E. MacDougall, F. Ntanios and C. A. Vanstone, Can. J. Physiol. Pharmacol., 1997, 75, 217–227.
10. P. J. Jones and F. Ntanios, Nutr. Rev., 1998, 56, 245–248.
11. P. J. Jones, F. Y. Ntanios, M. Raeini-Sarjaz and C. A. Vanstone, Am. J. Clin. Nutr., 1999, 69, 1144–1150.
12. M. A. Hallikainen, E. S. Sarkkinen and M. I. Uusitupa, J. Nutr., 2000, 130, 767–776.
13. T. A. Miettinen, P. Puska, H. Gylling, H. Vanhanen and E. Vartiainen, N. Engl. J. Med., 1995, 333, 1308–1312.
14. H. Gylling and T. A. Miettinen, J. Lipid Res., 1996, 37, 1776–1785.
15. J. A. Weststrate and G. W. Meijer, Eur. J. Clin. Nutr., 1998, 52, 334–343.
16. J. Plat, E. N. van Onselen, M. M. van Heugten and R. P. Mensink, Eur. J. Clin. Nutr., 2000, 54, 671–677.
17. F. Nigon, C. Serfaty-Lacrosniere, I. Beucler, D. Chauvois, C. Neveu, P. Giral, M. J. Chapman and E. Bruckert, Clin. Chem. Lab. Med., 2001, 39, 634–640.
18. Y. M. Lee, B. Haastert, W. Scherbaum and H. Hauner, Eur. J. Nutr., 2003, 42, 111–117.
19. A. American Diabetes, Diabetes Care, 2014, 37(Suppl 1), S81–S90.
20. D. R. Matthews, J. P. Hosker, A. S. Rudenski, B. A. Naylor, D. F. Treacher and R. C. Turner, Diabetologia, 1985, 28, 412–419.
21. D. Montero, G. Walther, C. D. Stehouwer, A. J. Houben, J. A. Beckman and A. Vinet, Obes. Rev., 2014, 15, 107–116.

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