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School of Hospitality, Culinary Arts and Meal Sciences, Örebro University, 702 81 Örebro, SwedenDepartment of Biosciences and Nutrition, Karolinska Institutet, 141 83 Huddinge, Sweden
Competence Centre on Reproductive Medicine and Biology, Tiigi 61b, 50410 Tartu, EstoniaDepartment of Paediatrics, School of Medicine, University of Granada, 18012 Granada, Spain
Competence Centre on Reproductive Medicine and Biology, Tiigi 61b, 50410 Tartu, EstoniaInstitute of Biomedicine, University of Tartu, Ravila 19, 50411 Tartu, Estonia
Folic acid supplements are commonly used by infertile women which leads to a positive folate status. However, the effect of folic acid supplements on pregnancy outcome in women with unexplained infertility has not been well investigated. This study evaluated folic acid supplement use and folate status in women with unexplained infertility in relation to IVF pregnancy outcome. In addition, use of folic acid supplements and folate status were compared between women with unexplained infertility and fertile, nonpregnant control women. Women with unexplained infertility used significantly more folic acid supplements and had higher median total folic acid intake from supplements compared with fertile control women (both P < 0.001). Women with unexplained infertility also had significantly higher median plasma folate and lower median plasma homocysteine concentrations than fertile women (both P < 0.001), but folic acid supplementation or folate status were not related to pregnancy outcome in women with unexplained infertility. In conclusion, folic acid supplementation or good folate status did not have a positive effect on pregnancy outcome following infertility treatment in women with unexplained infertility.
Folate is one of the B vitamins which has been suggested to be related to infertility. Folic acid is an artificial form of folate which is commonly used in dietary supplements. Folic acid supplementation has been shown to increase folate concentrations and decrease concentrations of the amino acid homocysteine in the blood. Folic acid supplementation is commonly used by infertile women, but the effect on pregnancy outcome in women with a diagnosis of unexplained infertility has not been thoroughly investigated. In the present study, folic acid supplement use and folate status (concentrations of folate and homocysteine) in women with unexplained infertility were evaluated in relation to pregnancy outcome. In addition, the use of folic acid supplements and folate status were compared between women with unexplained infertility and fertile control women. Our results showed that women with unexplained infertility used considerably more folic acid supplements and had higher total folic acid intake from supplements compared with fertile control women. Women with unexplained infertility had better blood folate and homocysteine concentrations than fertile women, but folic acid supplementation or folate status were not related to pregnancy outcome following the infertility treatment. In conclusion, high folic acid intake or good folate status did not increase the possibility of a birth of a healthy baby after infertility treatment in women with unexplained infertility.
). The standard treatment of infertility, IVF, is burdensome for affected couples and also demands considerable economic resources from the healthcare system (
). Folate plays an important role in DNA synthesis and epigenetic modification, as well as cell proliferation. Consequently, folate deficiency particularly affects highly proliferative cells (e.g. neural tube cells in the developing fetus), thus enhancing the risk of neural tube defects and several other birth defects (
), but there are also several micronutrients, including vitamins B2, B6 and B12, which are needed for folate metabolism. Insufficient concentrations of these vitamins impair metabolism, thereby causing functional folate deficiency accompanied by high concentrations of homocysteine (
Previous studies in infertile women have shown that preconceptional folic acid supplementation increases folate concentrations and decreases homocysteine concentrations in follicular fluid (
). Women of fertile age in Sweden do not receive adequate levels of folate through their diets. Therefore, the public health authorities recommend intake of 400 μg folic acid via supplements per day for women of reproductive age (
). However, despite information and the recommendations from the authorities, less than 50% of such women obtains folic acid supplements prior to conceiving (
Folic acid supplement use in women with unexplained infertility in relation to pregnancy outcome has not been previously studied. Women with unexplained infertility do not normally receive a proper diagnosis as a result of the fact that a uniform definition is lacking (
). Such women might also be subfertile rather than infertile. Additional knowledge of the role of folic acid supplementation in relation to pregnancy outcome could add further understanding as regards this group of infertile women. Therefore, this study investigated folic acid supplement use and folate status in women with unexplained infertility in relation to pregnancy outcome and in relation to folic acid supplement use and folate status in healthy, fertile, nonpregnant women.
Materials and methods
Study subjects and design
Eligible patients were recruited for the study at the Fertility Unit, Karolinska University Hospital from 2005 to 2007 and at the Centre for Reproduction at Uppsala University Hospital from 2008 to 2010. Women diagnosed with unexplained infertility who came for their first visit were asked to participate in the study. The control group was randomly selected from the same geographic area as the infertile women. The controls were healthy, proven fertile, nonpregnant and nonlactating women.
To investigate the relationship between folic acid supplement use, folate status and pregnancy outcome in women with unexplained infertility a longitudinal cohort study was performed. A prospective case–control study was used to compare the use of folic acid supplements and folate status between infertile and fertile women.
Written and oral informed consent was obtained from all participants. The study was approved by the regional Ethics Committee in Stockholm, Sweden (no. 2006/576-31/1, approved 3 July 2006).
Infertility diagnosis, treatment and pregnancy outcome
Diagnosis of infertility was based on the patient’s medical history, clinical investigation, including transvaginal ultrasonography, a standard set of tests that included hormone analyses, hysterosalpingo contrast sonography and semen analyses, as described previously (
). A diagnosis of unexplained infertility was chosen when no explanation for infertility was found, women had normal ovarian function and normal tubal passage (demonstrated by hysterosalpingo contrast sonography) and their partners had normal samples according to WHO criteria (
Data on assisted reproduction treatment were collected during one treatment cycle when an embryo transfer with one or a maximum of two embryo(s) transferred into the uterus was performed. Assisted reproduction treatments, including ovarian stimulation with clomiphene citrate (
Pregnancy was confirmed by a positive result in a urinary human chorionic gonadotrophin test (ColibriCheck, sensitivity ⩾25 mIU/ml; Colibri Medical, Helsingborg, Sweden). Gestational sacs were observed ultrasonographically 5 weeks after embryo transfer, corresponding to 7 weeks of pregnancy. Clinical pregnancy was defined as the presence of a gestational sac. Miscarriage was defined as pregnancy loss after the presence of a verified gestational sac, but before 20 weeks of gestation. Live birth was defined as delivery of a child after infertility treatment. There were no multiple pregnancies and therefore this was not included.
Questionnaire
A questionnaire, which was examined in two pilot tests, was used to assess general background and use of dietary supplements. Self-reported height and weight were transformed to body mass index. The question regarding use of dietary supplements included three options: yes, sometimes and no. A list of supplements, containing the 22 most commonly used dietary supplements was included. In addition, an open-ended question was included, where the participants could specify what kind of supplement(s) they used if it was not on the list. If taking a folic acid supplement, the participant could self-report the amount (μg) of folic acid ingested. As regards multivitamin supplements, the content of folic acid was approximated from the product information. When several folic acid-containing supplements were used, the total folic acid intake was calculated.
Folate and homocysteine analysis
Blood samples were taken to determine plasma folate and homocysteine concentrations. The blood samples were collected from the infertile women when they visited the Centre for Reproduction, in most cases on the day of oocyte retrieval. Blood samples from the controls were collected by a nurse at the research laboratory. Immediately after collection, the blood was centrifuged at 3000 g for 10 min and the plasma stored at −70°C until analysed. Folate was measured in a two-step competitive luminometric assay involving folate-binding protein and acridinium ester-labelled folate. Homocysteine was reduced through a series of reactions resulting in formation of nicotinamide adenine dinucleotide, which was measured at 450 nm, the result being in direct proportion to the concentration of homocysteine. Architect (Abbott) was used for both folate and homocysteine analyses. Blood samples showing haemolysis or plasma folate >90 nmol/l were excluded from the statistical analyses because of the risk of misleading values.
In a previous study, intake of folic acid at 250 μg/day for 4 weeks was shown to decrease plasma homocysteine concentrations significantly and to increase mean plasma folate concentrations to 22.5 nmol/l (
). Therefore, this plasma folate cut-off value was used to define folic acid supplement use: women with plasma folate concentrations of ⩾22.5 nmol/l were defined as folic acid supplement users and women with plasma folate concentrations <22.5 nmol/l were defined as nonusers. For assessment of pregnancy outcome in relation to folate status, women with unexplained infertility were divided into two groups according to plasma folate concentration: ⩾22.5 nmol/l and <22.5 nmol/l.
Statistical analysis
Statistical analyses were performed using IBM SPSS Statistics 20.0 (IBM Corporation, NY, USA). Variables are reported as mean ± SD or median and range. The Mann–Whitney U-test was used for comparisons between two groups. For comparisons including categorial variables, the chi-squared test or Fisher’s exact test (for less than five samples) were applied. Spearman’s analysis was used for correlation analyses. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by logistic regression analysis. Statistical significance was defined as P < 0.05. Answers to the open-ended question were categorized for analysis of folic acid supplement use. Sample size for the use of folic acid supplements was calculated using power of 80% and an alpha value of 0.05. On the basis of results of previous studies, approximately 80% of infertile women (
) use folic acid supplements. To confirm data for the Swedish situation, a sample size of 32 was needed.
Results
In total, 368 women were included in the study. There were 180 women with unexplained infertility in the study group and 188 fertile women in the control group. The age of women was (mean ± SD) 33.6 ± 3.7 years in the unexplained infertility group and 35.6 ± 4.3 years in the fertile control group. The body mass index of women with unexplained infertility was 23.1 ± 3.5 kg/m2 and of fertile women was 23.1 ± 3.3 kg/m2. A majority of women in both groups had an educational level of >12 years (65.0% and 63.1%, respectively).
Data on infertility treatments and pregnancy outcome were available in 180 women with unexplained infertility (Table 1).
Table 1Infertility treatment and pregnancy outcome in women with unexplained infertility.
Women with unexplained infertility used significantly more dietary supplements than fertile control women (P < 0.001), both in regard to all supplements containing folic acid and supplements containing folic acid only (P < 0.001 for both; Table 2). Supplements without folic acid were mainly used by fertile women (P = 0.005; Table 2). The median total folic acid daily intake from supplements was also notably higher in women with unexplained infertility than in fertile control women (P < 0.001; Table 2). Women with unexplained infertility had higher median plasma folate and lower median plasma homocysteine concentrations compared with those in fertile women (P < 0.001 for both; Figure 1). Both plasma folate and plasma homocysteine concentrations were associated with total folic acid daily intake (r = 0.444, P < 0.001 and r = −0.188, P < 0.001, respectively).
Table 2Supplement use and folic acid intake in women with unexplained infertility and fertile control women.
Unexplained infertility (n =180)
Fertile controls (n =188)
P-value
OR (95% CI)
Dietary supplement: all supplements
Daily
89 (49.4)
38 (20.2)
<0.001
3.861 (2.436–6.119)
Sometimes
62 (34.4)
72 (38.3)
NS
0.847 (0.553–1.295)
Total
151 (83.9)
110 (58.5)
<0.001
3.692 (2.257–6.039)
Dietary supplement: all supplements not containing folic acid
Daily
2 (1.1)
15 (8.0)
0.007
0.130 (0.029–0.575)
Sometimes
13 (7.2)
20 (10.6)
NS
0.654 (0.315–1.357)
Total
15 (8.3)
35 (18.6)
0.005
0.397 (0.209–0.756)
Folic acid supplement: all supplements containing folic acid
Figure 1Median plasma folate and plasma homocysteine values in unexplained infertile women and fertile control women. Boxes indicate medians and interquartile range; bars indicate 95% centiles; circles and asterisks indicate outliers (asteriks indicating extreme outliers). There were statistically significant differences in plasma folate and plasma homocysteine concentrations between the two groups (P < 0.001).
A plasma folate concentration of ⩾22.5 nmol/l was used as a biomarker of folic acid supplement intake. Use of this cut-off value resulted in 78 of 180 (43.3%) women with unexplained infertility and 29 of 188 (15.4%) fertile control women being indicated as using folic acid supplements (P < 0.001).
Pregnancy outcome and folic acid supplement use
As shown in Table 3, there were no statistically significant differences in pregnancy or live birth rates between women who used folic acid-containing supplements and those who did not. In Table 4, plasma homocysteine concentrations and pregnancy outcome are shown according to the plasma folate cut-off value. Due to some missing values (n = 13), 167 women were included in the analysis. There was a statistically significant difference in plasma homocysteine concentrations (P = 0.003), but not in pregnancy outcomes, between the groups.
Table 3Pregnancy outcome and questionnaire-based folic acid supplement use.
Folic acid supplement (n =137)
No folic acid supplement (n =42)
OR (95% CI)
Pregnancy
47 (34.3)
16 (38.1)
1.010 (0. 523–1.952)
Clinical pregnancy
45 (32.8)
15 (35.7)
1.003 (0. 515–1.953)
Miscarriage
12 (8.8)
2 (4.8)
–
Live birth
33 (24.1)
13 (31.0)
1.366 (0. 677–2.757)
Data are n (%).
– = NA.
There were no statistically significant differences between the two groups.
This study showed that women with unexplained infertility used significantly more folic acid supplements and had better folate status than fertile women, but did not show that this would have a positive effect on pregnancy outcome following the use of assisted reproduction.
A majority (84%) of women with unexplained infertility and almost 60% of fertile control women used dietary supplements, which is higher than presented in previous Swedish studies, where approximately 30% of Swedish women used dietary supplements (
). This study also found that 68% of women with unexplained infertility used folic acid supplements, which is similar to results reported previously: a Dutch study reported that 87% of women taking part in infertility treatment (
). In the present study, fertile women were somewhat older than the infertile women but they used fewer folic acid supplements, suggesting that folic acid intake is related to infertility rather than age.
Unexpectedly, the intake of supplements containing folic acid alone was low in both groups; only 16 of 180 infertile women and none of fertile women chose this option. One reason is that multivitamin and multimineral supplements are the most commonly used dietary supplements (
) and individuals seem to prefer intake of many vitamins and minerals at the same time. The reason that none of the fertile women used supplements of folic acid alone may be because they were not pregnant. Therefore, incentives for the use of folic acid, which is strongly linked to pregnancy and avoidance of neural tube defects, are lacking (
). The results of previous studies have also suggested that lack of information and lack of knowledge concerning the advantages of folic acid intake have an impact on folic acid use in women (
). Furthermore, it is known that compliance concerning folic acid supplement intake is poor in the average female population. In a recent British study (
), it was reported that 31% of patients at an antenatal clinic who answered a questionnaire were folic acid users, whereas in a Swedish questionnaire-based study only 20% of antenatal clinic patients used folic acid preconceptionally (
The median total folic acid daily intake from supplements was within recommendations for women with unexplained infertility, but this was not so among the fertile control women, which is seen as better folate status in the infertile women. It has been shown that folic acid supplements increase serum folate concentrations more than dietary folate (
). However, only 43.3% of the infertile women and 15.4% of fertile women had adequate folic acid intake to achieve plasma folate concentrations ⩾22.5 nmol/l, suggesting that folic acid intake was not always on a daily basis in all women who stated this in the questionnaires. Others have also reported that data from questionnaires has indicated higher folic acid supplement use than intake verified by the plasma biomarker (
This study found that folic acid intake and folate status did not have a positive impact on pregnancy or live birth rates following the use of assisted reproduction treatment, which has also been shown previously in studies of infertile women (
). Quite unexpected was the fact that there were more miscarriages among folic acid users compared with nonusers, based on the data from the questionnaires, although this was statistically nonsignificant. Additionally, there was a nonsignificant trend towards a higher live birth rate in nonusers group compared with the folic acid supplement users. However, this difference became contradictory using the plasma folate cut-off of 22.5 nmol/l, which correlated with plasma homocysteine concentration. This finding confirms that it is likely that folic acid supplement intake was not on a daily basis as stated in the questionnaires.
As far as is known, there are no other studies on folic acid supplement use and pregnancy outcome in women with unexplained infertility compared with healthy, fertile, nonpregnant women. A strength of this investigation was that the study group was well defined, as only women with unexplained infertility were included. In addition, live birth was included in the pregnancy outcome following infertility treatment; very often, pregnancy outcome is measured only as pregnancy rate, and only a few investigators have reported live birth as an outcome, although a successful pregnancy outcome after infertility treatment is not reached before a live birth is achieved (
A limitation of this study is that using a questionnaire for collecting data on dietary supplement use can lead to some misreporting. It was assumed that women used dietary supplements on a daily basis when answering ‘yes’ to the question of supplement use. Moreover, self-reported amounts of folic acid intake can be considered to be information of limited value and some women also stated that they took folic acid supplements but they did not report the amount. Self-reported weight and height can also be subject to some degree of misreporting and lead to incorrect body mass index.
In conclusion, this study showed that women with unexplained infertility used significantly more folic acid supplements and had better folate status than fertile women, but could not show whether preconceptional folic acid intake or good folate status would have a positive effect on pregnancy outcome in infertility treatments in women with a diagnosis of unexplained infertility. In addition, daily folic acid supplement use was not as good as could be expected and did not correspond to the results of laboratory tests. Clearly, additional studies with larger study groups and with improved measurement methods are required to investigate further folic acid supplement use and its possible relation to IVF pregnancy outcome in women with unexplained infertility.
Acknowledgements
The study was supported by the Family Planning Foundation and Födelsefonden, Uppsala, Sweden, the Regional Research Foundation, Örebro, Sweden, a Pampers Scholarship, Sweden, an R and D grant from Praktikertjänst, Stockholm, Sweden, the EU-FP7 Eurostars programme (grant NOTED, EU41564), the EU-FP7 IAPP project (grant SARM, EU324509) and a Marie Curie postdoctoral fellowship (FP7, no. 329812, NutriOmics).
References
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Declaration: The authors report no financial or commercial conflicts of interest.
Footnotes
Tiina Murto is a scientist at the Department of Women’s and Children’s Health at Uppsala University, Sweden. Her research interest includes nutrition, female reproduction and genetics with special interest on folate and its metabolism in relation to unexplained infertility.
Tiina Murto is a scientist at the Department of Women’s and Children’s Health at Uppsala University, Sweden. Her research interest includes nutrition, female reproduction and genetics with special interest on folate and its metabolism in relation to unexplained infertility.
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